targeting tumor microenvironment: effects of chinese...
TRANSCRIPT
Research ArticleTargeting Tumor Microenvironment Effects of Chinese HerbalFormulae on Macrophage-Mediated Lung Cancer in Mice
Fei Xu12 Wenqiang Cui23 Zhengxiao Zhao12 Weiyi Gong12 YingWei12 Jiaqi Liu12
Mihui Li12 Qiuping Li12 Chen Yan12 Jian Qiu12 Baojun Liu12 and Jingcheng Dong12
1Department of Integrative Medicine Huashan Hospital Fudan University Shanghai China2Institutes of Integrative Medicine Fudan University Shanghai China3Department of Integrative Medicine and Neurobiology State Key Laboratory of Medical NeurobiologyInstitute of Acupuncture Research School of Basic Medical Science Fudan University Shanghai China
Correspondence should be addressed to Baojun Liu lbj825163com and Jingcheng Dong jcdong2004126com
Received 11 January 2017 Revised 11 April 2017 Accepted 4 May 2017 Published 28 May 2017
Academic Editor Valeria Sulsen
Copyright copy 2017 Fei Xu et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Our previous studies have shown that Qing-Re-Huo-Xue (QRHX) formulae had significant anti-inflammatory effects in chronicairway diseases such as asthma and chronic obstructive lung disease Here we examined the effects of QRHX on lung cancercell invasion and the potential associated mechanism(s) mainly polarization of macrophages in the tumor microenvironment Invivo QRHX both inhibited tumor growth and decreased the number of tumor-associated macrophages (TAMs) in mice with lungcancer Further study indicated that QRHX inhibited cancer-related inflammation in tumor by decreasing infiltration of TAMsand IL-6 and TNF-120572 production and meanwhile decreased arginase 1 (Arg-1) expression and increased inducible NO synthase(iNOS) expression QRHX could markedly inhibit CD31 and VEGF protein expression Additionally CXCL12CXCR4 expressionand JAK2STAT3 phosphorylation were reduced in QRHX treatment group Thus we draw that QRHX played a more importantrole in inhibiting tumor growth by regulating TAMs in mice which was found to be associated with the inhibition of inflammationand the CXCL12CXCR4JAK2STAT3 signaling pathway
1 Introduction
Lung cancer is the most common cause of cancer-relateddeaths in men and women globally and with the highest rateof morbidity and mortality [1] In 2014 about 15 millionnew patients were diagnosed worldwide and approximately16 million die of this disease every year [2] Non-small-celllung cancer (NSCLC) accounting for 80 of lung cancer hasa dismal five-year survival at 15 and even worse patientswith advanced NSCLC if left untreated have a mediansurvival of 4-5 months after diagnosis [2 3] During the pastdecades new treatments such asminimally invasive surgerytargeted therapy adjuvant chemotherapy and individualizedtherapy have been applied but have a less important effectin improving the overall 5-year survival especially in theadvanced stage [4] Nonetheless all above therapies havefocused on tumor cells
However uncontrolled growth in tumors invasion andmetastasis cannot be elucidated solely by aberrations incancer cells themselves Over the past few decades a majorparadigm shift happens to cancer therapy and a great deal ofeffort has been put forth to develop therapies that target thetumor microenvironment Accumulating evidences suggestthat the alterations that occur in the stroma around a tumorprove useful in antiangiogenesis antitumor metastasis andprognosis [5] Nontumoral cells including stromal cells(fibroblasts endothelium cells etc) and leukocytes are aprominent component of solid tumors [6] Tumor-associatedmacrophages (TAMs) an important component acquire adistinct tissue-specific phenotype in different microenviron-ments and have both anti- and protumor effect due to twodistinctly different polarization respectively referred to asldquoclassicalrdquo (or M1) and ldquoalternativerdquo (or M2) activation [7]In tumor microenvironment TAMs are primarily polarized
HindawiEvidence-Based Complementary and Alternative MedicineVolume 2017 Article ID 7187168 12 pageshttpsdoiorg10115520177187168
2 Evidence-Based Complementary and Alternative Medicine
toward a M2-like phenotype which have the ability topromote the growth and vascularization of tumors Collectiveevidences demonstrate that the dual roles of TAMs have beendemonstrated both in vitro and in vivo in different tumormodels [8] Moreover clinical studies make strong cases thatTAMs characterized by M2 phenotypes are poor predictorsof prognosis and progression in numerous malignancies [9]
TAMs also profoundly influence the effects of conven-tional treatment modalities (chemotherapy and radiothera-py) targeted drugs antiangiogenic agents and immunother-apy including checkpoint blockade [10] Therefore TAMsare essential for effective therapy and M2-like TAMs areconsidered to be potential target for adjuvant anticancer ther-apies In addition extensive studies have been carried out todeclare that approaches targeting M2-like TAMs have gainedencouraging resultsThus there is a growing appreciation thatskewing TAM polarization away from the M2- to M1-likephenotype is of great importance
Accumulated data indicates that traditional Chinesemedicine (TCM) plays a pivotal role in regulating tumormicroenvironment including remodeling immunosuppres-sive microenvironment hypoxia microenvironment angio-genesislymphangiogenesis and extracellular matrix [11]Qing-Re-Huo-Xue (QRHX) formulae consist of a 1 1mixture(ww) of Radix Paeoniae Rubra and Scutellaria baicalen-sis The above formula is frequently used in treatment ofchronic inflammatory diseases in the respiratory system andimmunocompromised diseases in TCM Increasing evidencerevealed that QRHX and its components extracts andderivative have the ability of anticancer and anti-inflam-mation [12ndash14] Nevertheless there are no reports whichconcerned the alleviated effects of QRHX on macrophage-mediated lung cancer In the present study we aimed toinvestigate the relevance between macrophage polarizationand the antitumor effect of QRHX in mice
2 Methods
21 Animal Male C57BL6J mice (5 weeks old) were pur-chased from Shanghai SLAC Co (Shanghai China) andhoused in separate stainless steel cages (six mice per cage) atconstant temperature (23∘C) with a 12 h lightdark cycle hadfree access to water and food All procedures of this studywere approved by the Fudan University Animal Care and UseCommittee (number 2015000518547)
22 Reagents The following antibodies were used VEGFabcam46160 and abcam1613 CXCR4 abcam124824 CXCL12abcam25117 p-JAK CST3717 CD31 abcam28364 inducibleNO synthase (iNOS) abcam15323 arginase 1 (Arg-1)CST385 CD11b abcam1211 CD206 abcam64693 andabcam8918 Alexa Fluor 488 ALEXA21202 and 594 andALEXA21207
23 QRHX Preparation Chemical Constituents IdentificationQRHX a two-herb Chinese medicinal formula is comprisedof Radix Paeoniae Rubra and Scutellaria baicalensis QRHXgranules (batch number 1211301) were prepared and supplied
by Jiangyin Tianjiang Pharmaceutical Co Ltd Briefly theircomponent herbswere admixed in the prescribed proportionwhich were soaked in distilled water (1 10 wv) for 2 hand extracted at 100∘C for twice (1 h each time) Then thedecoction was filtered and concentrated to the extract witha relative density 113 at 60∘C After spray drying the dryingpowder was blended thoroughly and made into 18ndash40 meshparticlesThegranuleswere stored at 4∘Canddissolved in dis-tilled water of double volume before use To ensure standard-ization and maintain interbatch reliability of QRHX chem-ical ingredients of QRHX were separated and identified byhigh-performance liquid chromatography quadrupole time-off light mass spectrometry ultraviolet (HPLUNGCANCER-QTOF MS-UV) Briefly 8 chemical components wererespectively identified asmajormaterial basis inQRHX(Sup-plementary Figure 1 and Table 1 in Supplementary Materialavailable online at httpsdoiorg10115520177187168)
24 Cell Culture Lewis lung cancer (LLC) cells lines werecultured in DMEM supplemented with 10 Hyclone FetalBovine Serum (FBS ThermoFisher Scientific Fremont CAUSA) in an atmosphere of 95 oxygen and 5 CO
2at 37∘C
The cells were grown in 75 cm2 culture flasks and harvestedin a solution of trypsin-EDTA at the logarithmic growthphase
25 Subcutaneous Models and Drugs Administration LLCcells were harvested by a brief treatment with trypsinEDTAand then resuspended in DMEM with 10 FBS Then cellswere washed with cold PBS by centrifugation and resus-pended in PBS to the concentration of 1times 107mL and kept onice before used The male mice were randomly divided intotwo groups (119899 = 12 each group) including NS and QRHXgroups Tumor cells (2times 106 cells in 02mLPBS)were injectedsubcutaneously into the right of the back After 10 days tumorsize was measured twice weekly by a digital caliper and wasestimated as (1198632 times 119889)2 where 119863 is the large diameter and119889 is the small diameter of the tumor Twenty-four hours afterestablishing model mice were administered by intragastric(ig) in 02mL volume for 24 consecutive days to differentgroups with QRHX and normal saline (NS) respectively andthen sacrificed at day 24 after injection
26 Enzyme-Linked Immunosorbent Assay (ELISA) Concen-trations of serum IL-6 and TNF-a were measured using anELISA The blood sample was stored at room temperaturefor 2 h centrifuged (5000 rpm) for 30min and then cryopre-served at minus80∘CThe concentrations of IL-6 and TNF-a weremeasured using a sandwichELISAkit (Multisciences China)
27 Flow Cytometric Analysis Tumor tissue was smearedpushed through 200 mesh screen twice and then resus-pended by PBS The suspension was treated with erythrocy-tolysin and then wash by PBS twice and finally suspended byPBS Cells were then fixed and stained with PerCP-Cyanine55-labeled anti-mouse CDD45 FITC labeled anti-mouseCD11b Antigen PE labeled anti-mouse F480 and AlexaFluor 647 labeled anti-mouse CD206 antibodies according
Evidence-Based Complementary and Alternative Medicine 3
(Days)
0
1
2
3
4
5
QRHXNS
Tum
or v
olum
e (cm
3)
10 13 17 20 24
(a)
Tum
or w
eigh
t (g)
0
1
2
3
4
QRHXNS
lowastlowast
(b)
Figure 1 QRHX inhibits growth of tumor in a subcutaneous mouse model C57BL6 mice were subcutaneously injected with LLC QRHXreduced tumor volume (a) and weight (b) The data represent means plusmn SEM (119899 ge 10) Compared with NS group lowastlowast119875 lt 001
to the manufacturerrsquos instructions followed by detection bya FACSCalibur instrument (BD Bioscience)
28 Western Blot Analysis Total protein was extracted fromthe cells using a RIPA kit (Beyotime China) Cell debriswas removed by microcentrifugation and supernatants werequickly frozenThe protein concentration was determined byBSA method And then the protein was electrophoresed ona polyacrylamide gel and transferred to a PVDF membraneNext the membranes was incubated 1 hndash15 h with 5 milkat room temperature and then incubated overnight at 4∘Cwith a 1 1000 or 1 500 dilution of corresponding primaryantibodies Blots were again washed three times with Tris-buffered salineTween 20 (TBST) and then incubated with a1 5000 dilution of HRP-conjugated secondary antibody for1 h at room temperature Blots were again washed three timeswith TBST and then developed by enhanced chemilumines-cence Band intensities were quantified using UN-SCAN-ITgel analysis software (version 6)
29 QT-PCR Total RNA was extracted with Trizol reagent(Ambion Thermo) and reverse transcriptionwas performedto obtain the cDNA using the PrimeScript RT Reagent Kit(Takara Japan) according to the manufacturerrsquos protocolThe primers used were synthesized by (Sangon BiotechChina) These queues were as follows iNOS 51015840-GTT-CTCAGCCCAACA ATACAAGA-31015840 (forward) and 51015840-GTG-GACGGGTCGATGTCAC-31015840 (reverse) IL-6 51015840-GATACC-ACTCCCAAC AGAC-31015840 (forward) and 51015840-CTTTTCTCA-TTTCCACGAT-31015840 (reverse) CCL22 51015840-AGGAAGGC TTG-GCTTTTAGG-31015840 (forward) and 51015840-TGGTACCTTGCA-GGCTCTCT-31015840 (reverse) TNF-120572 51015840-ACGGCATGGATC-TCAAAGAC-31015840 (forward) and 51015840-GTGGGTGAGGAGCAC-GTAGT-31015840 (reverse) GAPDH 51015840-AAATGGTGAAGGTCG-GTGTG-31015840 (forward) and 51015840-AGGTCAATGAAGGGGTCG-TT-31015840 (reverse) Quantitative real-time PCR (QT-PCR) was
The s
erum
conc
entr
atio
nsof
IL-6
TN
F-a (
pgm
L)
0
100
200
300
IL-6
NSQRHX
TNF-훼
lowastlowast
lowastlowast
Figure 2 QRHX suppresses cancer-related inflammation in lungcancer A subcutaneous mouse model was established and treatedwith QRHX or NS as described above Blood was collected fromeach mouse and the serum concentrations of the proinflammatorycytokines IL-6 and TNF-a were detected by ELISA Studentrsquos 119905-testwas used to determine the statistical significance lowastlowast119875 lt 001
performed using SYBR green reaction mixture in the ViiA7 Real Time PCR System (Bio-Rad MiniOpticon) Therelative expression levels were calculated using 2minusΔΔCtmethods
210 Immunofluorescence (IF) Cryostat sections were fixedand permeated CD11b andCD206 antibodies formice tumortissue were used followed by Alexa Fluor 488 (ALEXA) or594 (ALEXA)
211 Immunohistochemical Analysis IHC stain was per-formed using a two-step EnVisionHRP technique accordingto the manufacturersquos instruction For three slides cytoplasmstained with brown was scored as positive The expression ofCD206 CD31 and VEGF was quantitatively evaluated using
4 Evidence-Based Complementary and Alternative Medicine
M0 M2 M0M2 merged
NS
QRH
X
Figure 3 Immunofluorescence staining for CD11b and CD206 of tumor tissues in subcutaneous tumor Mice with subcutaneous tumor weredealt with NS and QRHX Images are at magnification of 200x The expression of M0 and TAMs (M2-like macrophage) was respectivelyanalyzed using an Alexa Fluor-549- and Alexa Fluor-488-conjugated secondary antibody
Olympus Cx31 microscope with Image-Pro Plus medicalimage analysis systemThedigital imageswere captured usinga digital camera (Canon A640) The positive area and OD ofCD206 and VEGF positive cells were evaluated by Image-J software and determined by measuring three randomlyselected microscopic fields for each slide The IHC index wasdefined as average integral optical density (AIOD) (AIOD =positive area times ODtotal area)
CD31 a marker factor in vascular endothelial cell waspositively correlated with microvascular density (MVD)Then we counted the MVD by detecting the expression ofCD31 antibody in tumor tissues by immunohistochemistrymethod Firstly at low power field (times40) three most intensetissue sections were selected each slice and then at high powerfield (times100) MVD counts of these areas were evaluatedFinally the mean microvessel counts of the three mostvascular areas were regarded as MVD
212 Statistical Analyses Data were from three independentexperiments and expressed as mean plusmn SEM Statistical anal-yses were performed by the one-way analysis of variance(ANOVA) for differences among different groups Aboutcomparison of two groups Studentrsquos 119905-test was used All
analyses were undertaken using GraphPad Prism6 119875 lt 005was considered statistically significant
3 Results
31 QRHX Inhibits Tumor Growth in a Subcutaneous MouseModel In order to explore the role of QRHX in tumorgrowth in vivo subcutaneous mouse model was establishedby subcutaneous injection of LLC in left extremity auxiliaryMice were sacrificed at the end of treatment As shown inFigure 1 the tumor volumes were performed on days 1013 17 20 and 24 In NS group the tumor volume grad-ually increased in a time-dependent manner (Figure 1(a))However treatment with QRHX significantly suppressed thetumor volume (Figure 1(a)) The final tumor weight on day24 after the start of treatment showed a significant decrease intheQRHX group comparedwithNS control (Figure 1(b)119875 lt001) These data suggested that QRHX could dramaticallyinhibit tumor growth in vivo
32 QRHX Suppresses Cancer-Related Inflammation in LungCancer Numerous studies have indicated that cancer-relatedinflammation promotes the development of tumor [15 16]
Evidence-Based Complementary and Alternative Medicine 5
DAPI CD206 Merge
NS
QRH
X
(a)
C
D20
6 po
sitiv
e cel
ls
0
10
20
30
40
50
lowastlowast
NS QRHX
(b)
Figure 4 Immunofluorescence staining for CD206 of tumor tissues in subcutaneous tumor Mice with subcutaneous tumor were dealt with thegroup of NS and QRHX Images are at magnification of 200x The expression of TAMs was respectively analyzed using an Alexa Fluor-488-conjugated secondary antibody The nuclei were stained with DAPI Compared with NS group lowastlowast119875 lt 001
In addition some proinflammatory cytokines such as IL-8IL-6 and TNF-a have been shown to ultimately facilitate cellinvasion and metastasis [15] Therefore we used an ELISAassay to examine serum levels of the two proinflammatorycytokines IL-6 and TNF-120572 in peripheral blood Serum IL-6 and TNF-120572 levels were lower in mice treated with QRHXcompared with those receiving NS treatment (Figure 2 both
119875 lt 001) These data demonstrated that QRHX decreasedthe production of proinflammatory cytokines
33 QRHX Reduces the Accumulation of TAMs in Lung Can-cer In order to ascertain the effects of the QRHX on TAMsM2-like macrophage phenotype we used flow cytometryIF and IHC assay to examine CD206 expression As shown
6 Evidence-Based Complementary and Alternative Medicine
NS
100 휇m
(a)
QRH
X
100 휇m
(b)
C
D20
6 po
sitiv
e cel
ls
0
20
40
60
80
100
NS QRHX
lowastlowast
(c)
Figure 5 Comparison of CD206+ macrophages infiltration between NS and QRHX groups Immunohistochemical staining of CD206+macrophages in lung cancer tissues Images are at magnification of 200x Data represent mean plusmn SEM 119899 = 4 Compared with NS grouplowastlowast119875 lt 001
in Figures 3ndash6 our results revealed that CD206 expressionwas dramatically expressed in tumor tissue Compared to NSgroup oral administration with QRHX led to a prominentinhibition in TAMs (Figures 3ndash6 119875 lt 001) These datademonstrated that QRHX had a more significant role inimpeding accumulating and reducing the number of TAMsin lung cancer
34 QRHX Alters Hallmarkers of M1 and M2 Macrophagein Lung Cancer To further confirm the role of QRHX inTAMs we analyzed the level of Arg-1 protein M2 markerand the mRNA expression of M1 marker iNOS After QRHXtreatment Arg-1 level decreased (Figure 7(a) 119875 lt 001) andiNOS level increased (Figure 7(b) 119875 lt 005) Accordinglythese results strongly supported the inhibitory effect ofQRHX on suppressing TAMs infiltration and regulating M2-like macrophage polarization
35 QRHX Inhibits Angiogenesis in Lung Cancer CD31endothelial cell surface antigen is a vascular endothelialmarker for MVD and TAMs have a positive effect on bloodvessels by inducing the production of various proangiogenic
genes [17] Therefore CD31 was evaluated in implantedtumors using IHC and WB MVD stained by anti-CD31 wasmeasured by counting tissue sections of central areas of thetumor As shown in Figure 8 QRHX treatment induced aremarkable reduction in MVD and CD31 protein (119875 lt 001)Furthermore IHC and WB demonstrated reduced vascularendothelial growth factor (VEGF) expression in the QRHXtreated groups relative to NS controls (Figure 8 119875 lt 001)Taken together these data clearly showed thatQRHXreducedangiogenesis in lung cancer
36 QRHX Blocks CXCL 12CXCR4JNK2STAT3 SignalingPathways To investigate the molecular mechanism underly-ing the formulae decreasing M2 macrophages we tested theeffects of inhibitors of signaling molecules CXCL12CXCR4and JNK2STAT3 are known to be important moleculesinvolved in M2 polarization [18 19] Compared to NS grouporal administration of QRHX dramatically attenuated theincreased expressions of CXCL12 and CXCR4 in tumortissue (Figure 9(a) 119875 lt 001) As shown in Figure 9(b)JAK2 and STAT3 activation was significantly suppressed byQRHX treatment (both 119875 lt 001) compared to NS group
Evidence-Based Complementary and Alternative Medicine 7
106
105
104
103
0
Com
p-YL
1-H
F4
80-R
-PE-
H
1061051041030
Comp-BL1-H CD11b-FITC-H
197
1M
800 K
600 K
400 K
200 K
0
SSC-
H
SSC-
H
Comp-RL1-H CD206-Alexa Flour 647-H1061051041030
106
105
104
103
0
Com
p-YL
1-H
F4
80-R
-PE-
H
Comp-BL1-H CD11b-FITC-H1061051041030
900
1M
800 K
600 K
400 K
200 K
0
SSC-
H
SSC-
H
Comp-RL1-H CD206-Alexa Flour 647-H1061051041030
(a)
0
10
20
30
NS
F480+CD11b+ F480+CD11b+CD206+
QRHX
p
ositi
ve ce
lls
lowastlowast
lowastlowast
(b)
Figure 6 QRHX decreases the number of TAMs in subcutaneous tumor Mice with subcutaneous tumors were dealt with NS and QRHXMacrophages and TAMs (M2-like subtype) in tumor tissue were measured by flow cytometry Data represent mean plusmn SEM 119899 = 4 Comparedwith NS group lowastlowast119875 lt 001
8 Evidence-Based Complementary and Alternative Medicine
훽-actin43 kDa
Arg-140 kDa
00
02
04
06
08
10
Arg-1
Relat
ive p
rote
in ex
pres
sion
lowastlowast
NSQRHX
(a)
NSQRHX
iNOS0
4
1
2
3
Relat
ive p
rote
in ex
pres
sion
lowast
(b)
Figure 7The expression of M2-related marker (Arg-1) and M1-related marker (iNOS) was detected by western blot and QT-PCR respectivelyMice with subcutaneous tumor were dealt with NS and QRHX Data were presented as means plusmn SEM (119899 = 3) Compared with NS grouplowast119875 lt 005 lowastlowast119875 lt 001
Taking together the data suggested that QRHX inhibitedtumor cell-TAMs interactions possibly through blockingCXCL12CXCR4JAK2STAT3 signaling pathways and thenregulated macrophages polarization
4 Discussion
In the present study our data demonstrated that QRHXplayed a more crucial role in inhibiting tumor growth bymodulating tumor microenvironment especially TAMsQRHX inhibited tumor cell-TAMs interactions via thesuppression of cancer-related inflammation and probablyblocking the response of macrophages to tumor signalsCXCL12CXCR4JAK2STAT3 axis
In the nineteenth century the association between cancerand inflammation was firstly put forward [20] Large numberof studies provided powerful evidence that chronic inflam-mation can promote tumor development progression andmetastasis as well as chemoresistance [16 21] Recent studieshave confirmed that paeoniflorin baicalein and wogoninimportant ingredients of QRHX had the potential to inhibitmany types of inflammation [22ndash24] Furthermore baicaleinand wogonin exerted obvious inhibitory effects on cancer aswell as macrophages and angiogenesis [24 25] Paeoniflorinone of major ingredients could reduce lung metastasis ofLLC through inhibiting the M2 activation [26] In additionthe cytokines produced by activated innate immune cells intumor microenvironment can stimulate tumorigenesis suchas IL-6 and TNF-120572 [27] Our results showed a remarkabledecrease in multiple proinflammatory cytokines such as
TNF-a and IL-6 both in serum and tumor tissue of subcu-taneous mouse model (Figure 2) suggesting the inhibitoryeffect of QRHX on cancer-related inflammation
Tumor microenvironment created by the tumor andmainly orchestrated by inflammatory cells contributes totumor escape growth progression and evolution towardmetastasis [28 29] Numerous studies in recent decadehave presented evidences that TCM have a good effect onregulating tumor microenvironment such as reversing theimmunosuppressive microenvironment [11] Macrophages abasic component of the innate immune system are infiltratedin virtually all malignancies TAMs M2-like polarized stylehave been regarded as a protumor inflammatory microen-vironment which links inflammation and cancer [15] Col-lective evidences demonstrate that TAMs have the abilityof enhancing tumor angiogenesis increasing migration andinvasion and suppressing the antitumor immune responsesIt is correlated with the prognosis of patients with malignanttumor such as lung cancer [30 31] Consistent with previousstudies in this study we detected that the fraction of TAMswas increased in tumor and QRHX inhibited tumor growthin the tumor mice model through decreasing accumulatingof TAMs and activation of M2
CXCR4 which is widely expressed on malignant cellsand binds to CXCL12 [32] plays an important role in hem-atopoiesis development and organization of the immunesystem by directly and indirectly mechanisms [33] Forexample in NSCLC CXCL12-CXCR4 axis is involved inmetastasis and associated with an unfavorable prognosis[34] in ovarian cancer it can control accumulation of
Evidence-Based Complementary and Alternative Medicine 9CD
31
NS QRHX
VEG
F
0
5
10
15
20
25
VEGFMVD
Den
sity
of V
EGF
MV
D co
unt
lowastlowast
lowastlowast
(a)
00
02
04
06
08
10
Relat
ive p
rote
in ex
pres
sion
CD31VEGF
NSQRHX
lowastlowastlowastlowast
VEGF
CD31
훽-actin43 kDa
100 kDa
43 kDa
(b)
Figure 8 QRHX inhibits angiogenesis in lung cancer Mice with subcutaneous tumors were dealt with NS and QRHX (a) The nucleus wasdyed as blue CD31 was dyed as brown MVD were performed at high power field (times200) (b) The VEGF and CD31 protein expression intumor tissues was detected by western blot Data was expressed as means plusmn SEM values (119899 = 4) Compared with NS group lowastlowast119875 lt 001
human MDSCs and is an independent prognostic factor fortumor progression [35] Several studies have reported thatCXCL12 plays an important role in monocyte recruitmentdifferentiation and function [18 36] In a mouse modelof lung cancer CXCL12 could recruit tumor-promotingmyeloid CD11b+ cells [19] Besides powerful evidencesindicate a role for CXCR4-CXCL12 axis in promotingmacrophages polarization toward theM2 phenotype [37 38]In addition M2 subpopulation is associated with angio-genic factors such as VEGF and CXCL12-CXCR4 axis canalso promote tumor vascularization [39] Excitedly ourresults showed QRHX treatment inhibited signaling fromtumor cells to macrophages through inducing a remark-able decrease in CXCL12 and CXCR4 Furthermore QRHX
inhibited angiogenesis likely through altering the tumormicroenvironment by targeting TAMs
In STAT family there are seven proteins STAT3 is one ofthem It is a key transcription factor transducing signals fromactivated receptors or intracellular kinases to the nucleus andcan be activated in tumor cells and immune cells [40] Intumor STAT3 could contribute to cancer development andprogression inhibit apoptosis of tumors and help tumorescape immune system by suppressing the immune response[41] Evidence indicated that when STAT3 binds to somereceptor it can be activated through Janus Kinases (JAKs)such as JAK2 [42] Accumulating evidence implicates theimportant role of JAK2STAT3 in tumor and macrophagepolarization [27] For instance IL-6 can contribute to tumor
10 Evidence-Based Complementary and Alternative Medicine
CXCR4
CXCL1210 kDa
훽-actin43 kDa
41 kDa
NSQRHX
00
05
10
15
Relat
ive p
rote
in ex
pres
sion
CXCL12 CXCR4
lowastlowastlowastlowast
(a)
p-JAK2
p-STAT3
훽-actin43 kDa
90 kDa
120 kDa
NSQRHX
p-JAK2 p-STAT300
05
10
15
Relat
ive p
rote
in ex
pres
sion
lowastlowast lowastlowast
(b)
Figure 9 QRXH regulates TAMs by inhibiting the CXCL12CXCR4JAK2STAT3 signaling pathways Mice with subcutaneous tumor weredealt with NS and QRHX Representative images of western blot and densitometry analysis showing the expressions of CXCL12 CXCR4p-JAK2 and p-STAT3 in tumor Compared with NS group lowastlowast119875 lt 001
cell survival and upregulate the antiapoptotic genes by driv-ing JAK2STAT3 signal [43] IFN- activates macrophageby JAK-STAT signaling pathway [42] What is more JAK2is associated with CXCR4 [39] Then we can draw thatTAMs in malignant tumors tend to M2 subtype possiblythrough CXCL12CXCR4JAK2STAT3 signaling pathwayQRHX treatment blocked the response of macrophages totumor signals by suppressing CXCL12CXCR4JAK2STAT3expression and induced a remarkable decrease of the recruit-ment of M2 macrophages suggesting the attenuation of M2subtype cells function by QRHX
In TCM theory TCM formulae have abundant medicinalmaterials and then regulate diseases through multitargetsandmultiways Although the chemical constituents of QRHXhave been separated and identified by HPLUNG CANCER-QTOF MS-UV method in our team (Supplementary Fig-ure 1 and Table 1) the ingredients are various and theirfunctions are enormous So there may be other mechanismsinvolved in macrophage polarization What is more there
are diverse types cells in tumor except for tumor cells andmacrophage Moreover it is worth mentioning that TAMswere not separated and extracted from tumor tissue CXCL12CXCR4 JAK2 and STAT3 can not only play significantrole in tumor cell or macrophage but also other cellssuch as T lymphocyte neutrophils tumor-associated fibrob-last and endothelial cells Therefore QRHX inhibited theCXCL12CXCR4JAK2STAT3 axis possibly through othercells Taken together further investigations are needed toidentify direct molecular targets of QRHX in macrophagesin the context of cancer
5 Conclusion
In conclusion data from this study revealed that QRHXcould suppress cancer progression by inhibiting the tumorpromotion of TAMs in subcutaneous mice model whichcould contribute to elucidating the underlying regulatorymode of QRHX on lung cancer treatment
Evidence-Based Complementary and Alternative Medicine 11
Abbreviations
ELISA Enzyme-linked immunosorbent assayFITC Fluorescein isothiocyanateTNF-120572 Tumor necrosis factor-120572IL-6 Interleukin-6iNOS Inducible nitric oxide synthaseVEGF Vascular endothelial growth factorQT-PCR Quantitative Polymerase Chain ReactionIF ImmunofluorescenceIHC ImmunohistochemicalTAMs Tumor-associated macrophagesArg-1 Arginase 1
Conflicts of Interest
The authors declare that there are no conflicts of interest
Authorsrsquo Contributions
Fei Xu and Wenqiang Cui contributed equally to this workFei Xu and Baojun Liu conceived and designed experimentsFei Xu and Wenqiang Cui analyzed data and wrote themanuscript Jingcheng Dong supervised the project Zhengx-iao Zhao and Ying Wei assisted in experimental design anddata evaluation Jiaqi Liu Mihui Li Qiuping Li Chen Yanand Jian Qiu performed the study All authors reviewed andapproved the manuscript
Acknowledgments
This study was supported by Natural Science Foundation ofChina (nos 81673916 and 81403148) Development Projectof Shanghai Peak Disciplines-Integrative Medicine (no20150407)
References
[1] I Ikonomidis C A Michalakeas J Parissis et al ldquoInflamma-tory markers in coronary artery diseaserdquo BioFactors vol 38 no5 pp 320ndash328 2012
[2] P Lee C C Leung M I Restrepo K Takahashi Y Song and JM Porcel ldquoYear in review 2015 lung cancer pleural diseasesrespiratory infections bronchiectasis and tuberculosis bron-choscopic intervention and imagingrdquo Respirology vol 21 no 5pp 961ndash967 2016
[3] S V Sharma DW Bell J Settleman and D A Haber ldquoEpider-mal growth factor receptor mutations in lung cancerrdquo NatureReviews Cancer vol 7 no 3 pp 169ndash181 2007
[4] D Leong R Rai B Nguyen A Lee and D Yip ldquoAdvancesin adjuvant systemic therapy for non-small-cell lung cancerrdquoWorld Journal of Clinical Oncology vol 5 no 4 pp 633ndash6452014
[5] M T Villanueva ldquoMicroenvironment the new midfielders inthe tumourmicroenvironmentrdquoNature reviews Cancer vol 14no 12 p 765 2014
[6] A Mantovani S Sozzani M Locati P Allavena and A SicaldquoMacrophage polarization tumor-associated macrophages as aparadigm for polarizedM2mononuclear phagocytesrdquoTrends inImmunology vol 23 no 11 pp 549ndash555 2002
[7] A Mantovani and M Locati ldquoTumor-associated macrophagesas a paradigm of macrophage plasticity diversity and polariza-tion lessons and open questionsrdquo Arteriosclerosis Thrombosisand Vascular Biology vol 33 no 7 pp 1478ndash1483 2013
[8] W Hu X Li C Zhang Y Yang J Jiang and C Wu ldquoTumor-associated macrophages in cancersrdquo Clinical and TranslationalOncology vol 18 no 3 pp 251ndash258 2016
[9] B Z Qian and J W Pollard ldquoMacrophage diversity enhancestumor progression and metastasisrdquo Cell vol 141 no 1 pp 39ndash51 2010
[10] A Mantovani and P Allavena ldquoThe interaction of anti-cancer therapies with tumor-associated macrophagesrdquo Journalof Experimental Medicine vol 212 no 4 pp 435ndash445 2015
[11] J Xu Z Song Q Guo and J Li ldquoSynergistic effect and molec-ular mechanisms of traditional Chinese medicine on regulatingtumor microenvironment and cancer cellsrdquo BioMed ResearchInternational vol 2016 Article ID 1490738 pp 1ndash14 2016
[12] J Wu J Xu E A Eksioglu et al ldquoIcariside II induces apoptosisof melanoma cells through the downregulation of survivalpathwaysrdquoNutrition and Cancer vol 65 no 1 pp 110ndash117 2013
[13] L Kong J Liu J Wang et al ldquoIcariin inhibits TNF-120572IFN-120574induced inflammatory response via inhibition of the substanceP and p38-MAPK signaling pathway in human keratinocytesrdquoInternational Immunopharmacology vol 29 no 2 pp 401ndash4072015
[14] JWu J Du C Xu et al ldquoIn vivo and in vitro anti-inflammatoryeffects of a novel derivative of icariinrdquo Immunopharmacologyand Immunotoxicology vol 33 no 1 pp 49ndash54 2010
[15] E M Conway L A Pikor S H Y Kung et al ldquoMacrophagesinflammation and lung cancerrdquoAmerican Journal of Respiratoryand Critical Care Medicine vol 193 no 2 pp 116ndash130 2016
[16] S Shalapour and M Karin ldquoImmunity inflammation andcancer an eternal fight between good and evilrdquo The Journal ofClinical Investigation vol 125 no 9 pp 3347ndash3355 2015
[17] L Bingle N J Brown and C E Lewis ldquoThe role of tumour-associated macrophages in tumour progression Implicationsfor new anticancer therapiesrdquo Journal of Pathology vol 196 no3 pp 254ndash265 2002
[18] D Kim J Kim J H Yoon et al ldquoCXCL12 secreted from adiposetissue recruits macrophages and induces insulin resistance inmicerdquo Diabetologia vol 57 no 7 pp 1456ndash1465 2014
[19] M C Schmid C J Avraamides P Foubert et al ldquoCombinedblockade of integrin-12057241205731 plus cytokines SDF-1120572 or IL-1120573potently inhibits tumor inflammation and growthrdquo CancerResearch vol 71 no 22 pp 6965ndash6975 2011
[20] F Balkwill and A Mantovani ldquoInflammation and cancer backto VirchowrdquoThe Lancet vol 357 no 9255 pp 539ndash545 2001
[21] B B Aggarwal S Shishodia S K Sandur M K Pandey andG Sethi ldquoInflammation and cancer how hot is the linkrdquoBiochemical Pharmacology vol 72 no 11 pp 1605ndash1621 2006
[22] W-L Jiang X-G Chen H-B Zhu Y-B Gao J-W Tian andF-H Fu ldquoPaeoniflorin inhibits systemic inflammation andimproves survival in experimental sepsisrdquo Basic and ClinicalPharmacology and Toxicology vol 105 no 1 pp 64ndash71 2009
[23] I D Kim and B J Ha ldquoThe effects of paeoniflorin on LPS-induced liver inflammatory reactionsrdquo Archives of PharmacalResearch vol 33 no 6 pp 959ndash966 2010
[24] G-W Fan Y Zhang X Jiang et al ldquoAnti-inflammatory activityof baicalein in LPS-stimulated RAW2647 macrophages viaestrogen receptor and NF-120581B-dependent pathwaysrdquo Inflamma-tion vol 36 no 6 pp 1584ndash1591 2013
12 Evidence-Based Complementary and Alternative Medicine
[25] M Li-Weber ldquoNew therapeutic aspects of flavones the anti-cancer properties of Scutellaria and its main active constituentsWogonin Baicalein and Baicalinrdquo Cancer Treatment Reviewsvol 35 no 1 pp 57ndash68 2009
[26] QWu G-L Chen Y-J Li Y Chen and F-Z Lin ldquoPaeoniflorininhibits macrophage-mediated lung cancer metastasisrdquoChineseJournal of Natural Medicines vol 13 no 12 pp 925ndash932 2015
[27] W Lin and M Karin ldquoA cytokine-mediated link betweeninnate immunity inflammation and cancerrdquo Journal of ClinicalInvestigation vol 117 no 5 pp 1175ndash1183 2007
[28] T L Whiteside ldquoThe tumor microenvironment and its role inpromoting tumor growthrdquo Oncogene vol 27 no 45 pp 5904ndash5912 2008
[29] G Lorusso and C Ruegg ldquoThe tumor microenvironment andits contribution to tumor evolution toward metastasisrdquo Histo-chemistry and Cell Biology vol 130 no 6 pp 1091ndash1103 2008
[30] L Ding G Liang Z Yao et al ldquoMetformin prevents cancermetastasis by inhibiting M2-like polarization of tumor associ-ated macrophagesrdquo Oncotarget vol 6 no 34 pp 36441ndash364552015
[31] A Yuan Y JHsiaoH Y Chen et al ldquoOpposite effects ofM1 andM2macrophage subtypes on lung cancer progressionrdquo ScientificReports vol 5 Article ID 14273 2015
[32] F Balkwill ldquoCancer and the chemokine networkrdquo NatureReviews Cancer vol 4 no 7 pp 540ndash550 2004
[33] J A Burger and T J Kipps ldquoCXCR4 a key receptor in thecrosstalk between tumor cells and their microenvironmentrdquoBlood vol 107 no 5 pp 1761ndash1767 2006
[34] R J PhillipsM D BurdickM Lutz J A BelperioM P Keaneand R M Strieter ldquoThe stromal derived factor-1CXCL12-CXCchemokine receptor 4 biological axis in non-small cell lungcancermetastasesrdquoAmerican Journal of Respiratory and CriticalCare Medicine vol 167 no 12 pp 1676ndash1686 2003
[35] N Obermajer R Muthuswamy K Odunsi R P Edwards andP Kalinski ldquoPGE-induced CXCL 12 production and CXCR4expression controls the accumulation of human MDSCs inovarian cancer environmentrdquo Cancer Research vol 71 no 24pp 7463ndash7470 2011
[36] L Sanchez-Martın A Estecha R Samaniego S Sanchez-Ramon M A Vega and P Sanchez-Mateos ldquoThe chemokineCXCL12 regulates monocyte-macrophage differentiation andRUNX3 expressionrdquo Blood vol 117 no 1 pp 88ndash97 2011
[37] K Beider H Bitner M Leiba et al ldquoMultiple myelomacells recruit tumor-supportive macrophages through theCXCR4CXCL12 axis and promote their polarization towardthe M2 phenotyperdquo Oncotarget vol 5 no 22 pp 11283ndash112962014
[38] J M Mota C A Leite and L E Souza ldquoPost-sepsis stateinduces tumor-associated macrophage accumulation throughCXCR4CXCL12 and favors tumor progression inmicerdquoCancerImmunology Research vol 4 no 4 pp 312ndash322 2016
[39] B A Teicher and S P Fricker ldquoCXCL12 (SDF-1)CXCR4 path-way in cancerrdquoClinical Cancer Research vol 16 no 11 pp 2927ndash2931 2010
[40] P C Heinrich I Behrmann G Muller-Newen F Schaper andL Graeve ldquoInterleukin-6-type cytokine signalling through thegp130JakSTAT pathwayrdquo Biochemical Journal vol 334 part 2pp 297ndash314 1998
[41] M Kortylewski M Kujawski T Wang et al ldquoInhibiting Stat3signaling in the hematopoietic system elicits multicomponentantitumor immunityrdquo Nature Medicine vol 11 no 12 pp 1314ndash1321 2005
[42] X Hu J Chen L Wang and L B Ivashkiv ldquoCrosstalk amongJak-STAT Toll-like receptor and ITAM-dependent pathways inmacrophage activationrdquo Journal of Leukocyte Biology vol 82no 2 pp 237ndash243 2007
[43] R Catlett-Falcone T H Landowski M M Oshiro et alldquoConstitutive activation of Stat3 signaling confers resistance toapoptosis in humanU266myeloma cellsrdquo Immunity vol 10 no1 pp 105ndash115 1999
Submit your manuscripts athttpswwwhindawicom
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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OncologyJournal of
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Oxidative Medicine and Cellular Longevity
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PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Research and TreatmentAIDS
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Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
2 Evidence-Based Complementary and Alternative Medicine
toward a M2-like phenotype which have the ability topromote the growth and vascularization of tumors Collectiveevidences demonstrate that the dual roles of TAMs have beendemonstrated both in vitro and in vivo in different tumormodels [8] Moreover clinical studies make strong cases thatTAMs characterized by M2 phenotypes are poor predictorsof prognosis and progression in numerous malignancies [9]
TAMs also profoundly influence the effects of conven-tional treatment modalities (chemotherapy and radiothera-py) targeted drugs antiangiogenic agents and immunother-apy including checkpoint blockade [10] Therefore TAMsare essential for effective therapy and M2-like TAMs areconsidered to be potential target for adjuvant anticancer ther-apies In addition extensive studies have been carried out todeclare that approaches targeting M2-like TAMs have gainedencouraging resultsThus there is a growing appreciation thatskewing TAM polarization away from the M2- to M1-likephenotype is of great importance
Accumulated data indicates that traditional Chinesemedicine (TCM) plays a pivotal role in regulating tumormicroenvironment including remodeling immunosuppres-sive microenvironment hypoxia microenvironment angio-genesislymphangiogenesis and extracellular matrix [11]Qing-Re-Huo-Xue (QRHX) formulae consist of a 1 1mixture(ww) of Radix Paeoniae Rubra and Scutellaria baicalen-sis The above formula is frequently used in treatment ofchronic inflammatory diseases in the respiratory system andimmunocompromised diseases in TCM Increasing evidencerevealed that QRHX and its components extracts andderivative have the ability of anticancer and anti-inflam-mation [12ndash14] Nevertheless there are no reports whichconcerned the alleviated effects of QRHX on macrophage-mediated lung cancer In the present study we aimed toinvestigate the relevance between macrophage polarizationand the antitumor effect of QRHX in mice
2 Methods
21 Animal Male C57BL6J mice (5 weeks old) were pur-chased from Shanghai SLAC Co (Shanghai China) andhoused in separate stainless steel cages (six mice per cage) atconstant temperature (23∘C) with a 12 h lightdark cycle hadfree access to water and food All procedures of this studywere approved by the Fudan University Animal Care and UseCommittee (number 2015000518547)
22 Reagents The following antibodies were used VEGFabcam46160 and abcam1613 CXCR4 abcam124824 CXCL12abcam25117 p-JAK CST3717 CD31 abcam28364 inducibleNO synthase (iNOS) abcam15323 arginase 1 (Arg-1)CST385 CD11b abcam1211 CD206 abcam64693 andabcam8918 Alexa Fluor 488 ALEXA21202 and 594 andALEXA21207
23 QRHX Preparation Chemical Constituents IdentificationQRHX a two-herb Chinese medicinal formula is comprisedof Radix Paeoniae Rubra and Scutellaria baicalensis QRHXgranules (batch number 1211301) were prepared and supplied
by Jiangyin Tianjiang Pharmaceutical Co Ltd Briefly theircomponent herbswere admixed in the prescribed proportionwhich were soaked in distilled water (1 10 wv) for 2 hand extracted at 100∘C for twice (1 h each time) Then thedecoction was filtered and concentrated to the extract witha relative density 113 at 60∘C After spray drying the dryingpowder was blended thoroughly and made into 18ndash40 meshparticlesThegranuleswere stored at 4∘Canddissolved in dis-tilled water of double volume before use To ensure standard-ization and maintain interbatch reliability of QRHX chem-ical ingredients of QRHX were separated and identified byhigh-performance liquid chromatography quadrupole time-off light mass spectrometry ultraviolet (HPLUNGCANCER-QTOF MS-UV) Briefly 8 chemical components wererespectively identified asmajormaterial basis inQRHX(Sup-plementary Figure 1 and Table 1 in Supplementary Materialavailable online at httpsdoiorg10115520177187168)
24 Cell Culture Lewis lung cancer (LLC) cells lines werecultured in DMEM supplemented with 10 Hyclone FetalBovine Serum (FBS ThermoFisher Scientific Fremont CAUSA) in an atmosphere of 95 oxygen and 5 CO
2at 37∘C
The cells were grown in 75 cm2 culture flasks and harvestedin a solution of trypsin-EDTA at the logarithmic growthphase
25 Subcutaneous Models and Drugs Administration LLCcells were harvested by a brief treatment with trypsinEDTAand then resuspended in DMEM with 10 FBS Then cellswere washed with cold PBS by centrifugation and resus-pended in PBS to the concentration of 1times 107mL and kept onice before used The male mice were randomly divided intotwo groups (119899 = 12 each group) including NS and QRHXgroups Tumor cells (2times 106 cells in 02mLPBS)were injectedsubcutaneously into the right of the back After 10 days tumorsize was measured twice weekly by a digital caliper and wasestimated as (1198632 times 119889)2 where 119863 is the large diameter and119889 is the small diameter of the tumor Twenty-four hours afterestablishing model mice were administered by intragastric(ig) in 02mL volume for 24 consecutive days to differentgroups with QRHX and normal saline (NS) respectively andthen sacrificed at day 24 after injection
26 Enzyme-Linked Immunosorbent Assay (ELISA) Concen-trations of serum IL-6 and TNF-a were measured using anELISA The blood sample was stored at room temperaturefor 2 h centrifuged (5000 rpm) for 30min and then cryopre-served at minus80∘CThe concentrations of IL-6 and TNF-a weremeasured using a sandwichELISAkit (Multisciences China)
27 Flow Cytometric Analysis Tumor tissue was smearedpushed through 200 mesh screen twice and then resus-pended by PBS The suspension was treated with erythrocy-tolysin and then wash by PBS twice and finally suspended byPBS Cells were then fixed and stained with PerCP-Cyanine55-labeled anti-mouse CDD45 FITC labeled anti-mouseCD11b Antigen PE labeled anti-mouse F480 and AlexaFluor 647 labeled anti-mouse CD206 antibodies according
Evidence-Based Complementary and Alternative Medicine 3
(Days)
0
1
2
3
4
5
QRHXNS
Tum
or v
olum
e (cm
3)
10 13 17 20 24
(a)
Tum
or w
eigh
t (g)
0
1
2
3
4
QRHXNS
lowastlowast
(b)
Figure 1 QRHX inhibits growth of tumor in a subcutaneous mouse model C57BL6 mice were subcutaneously injected with LLC QRHXreduced tumor volume (a) and weight (b) The data represent means plusmn SEM (119899 ge 10) Compared with NS group lowastlowast119875 lt 001
to the manufacturerrsquos instructions followed by detection bya FACSCalibur instrument (BD Bioscience)
28 Western Blot Analysis Total protein was extracted fromthe cells using a RIPA kit (Beyotime China) Cell debriswas removed by microcentrifugation and supernatants werequickly frozenThe protein concentration was determined byBSA method And then the protein was electrophoresed ona polyacrylamide gel and transferred to a PVDF membraneNext the membranes was incubated 1 hndash15 h with 5 milkat room temperature and then incubated overnight at 4∘Cwith a 1 1000 or 1 500 dilution of corresponding primaryantibodies Blots were again washed three times with Tris-buffered salineTween 20 (TBST) and then incubated with a1 5000 dilution of HRP-conjugated secondary antibody for1 h at room temperature Blots were again washed three timeswith TBST and then developed by enhanced chemilumines-cence Band intensities were quantified using UN-SCAN-ITgel analysis software (version 6)
29 QT-PCR Total RNA was extracted with Trizol reagent(Ambion Thermo) and reverse transcriptionwas performedto obtain the cDNA using the PrimeScript RT Reagent Kit(Takara Japan) according to the manufacturerrsquos protocolThe primers used were synthesized by (Sangon BiotechChina) These queues were as follows iNOS 51015840-GTT-CTCAGCCCAACA ATACAAGA-31015840 (forward) and 51015840-GTG-GACGGGTCGATGTCAC-31015840 (reverse) IL-6 51015840-GATACC-ACTCCCAAC AGAC-31015840 (forward) and 51015840-CTTTTCTCA-TTTCCACGAT-31015840 (reverse) CCL22 51015840-AGGAAGGC TTG-GCTTTTAGG-31015840 (forward) and 51015840-TGGTACCTTGCA-GGCTCTCT-31015840 (reverse) TNF-120572 51015840-ACGGCATGGATC-TCAAAGAC-31015840 (forward) and 51015840-GTGGGTGAGGAGCAC-GTAGT-31015840 (reverse) GAPDH 51015840-AAATGGTGAAGGTCG-GTGTG-31015840 (forward) and 51015840-AGGTCAATGAAGGGGTCG-TT-31015840 (reverse) Quantitative real-time PCR (QT-PCR) was
The s
erum
conc
entr
atio
nsof
IL-6
TN
F-a (
pgm
L)
0
100
200
300
IL-6
NSQRHX
TNF-훼
lowastlowast
lowastlowast
Figure 2 QRHX suppresses cancer-related inflammation in lungcancer A subcutaneous mouse model was established and treatedwith QRHX or NS as described above Blood was collected fromeach mouse and the serum concentrations of the proinflammatorycytokines IL-6 and TNF-a were detected by ELISA Studentrsquos 119905-testwas used to determine the statistical significance lowastlowast119875 lt 001
performed using SYBR green reaction mixture in the ViiA7 Real Time PCR System (Bio-Rad MiniOpticon) Therelative expression levels were calculated using 2minusΔΔCtmethods
210 Immunofluorescence (IF) Cryostat sections were fixedand permeated CD11b andCD206 antibodies formice tumortissue were used followed by Alexa Fluor 488 (ALEXA) or594 (ALEXA)
211 Immunohistochemical Analysis IHC stain was per-formed using a two-step EnVisionHRP technique accordingto the manufacturersquos instruction For three slides cytoplasmstained with brown was scored as positive The expression ofCD206 CD31 and VEGF was quantitatively evaluated using
4 Evidence-Based Complementary and Alternative Medicine
M0 M2 M0M2 merged
NS
QRH
X
Figure 3 Immunofluorescence staining for CD11b and CD206 of tumor tissues in subcutaneous tumor Mice with subcutaneous tumor weredealt with NS and QRHX Images are at magnification of 200x The expression of M0 and TAMs (M2-like macrophage) was respectivelyanalyzed using an Alexa Fluor-549- and Alexa Fluor-488-conjugated secondary antibody
Olympus Cx31 microscope with Image-Pro Plus medicalimage analysis systemThedigital imageswere captured usinga digital camera (Canon A640) The positive area and OD ofCD206 and VEGF positive cells were evaluated by Image-J software and determined by measuring three randomlyselected microscopic fields for each slide The IHC index wasdefined as average integral optical density (AIOD) (AIOD =positive area times ODtotal area)
CD31 a marker factor in vascular endothelial cell waspositively correlated with microvascular density (MVD)Then we counted the MVD by detecting the expression ofCD31 antibody in tumor tissues by immunohistochemistrymethod Firstly at low power field (times40) three most intensetissue sections were selected each slice and then at high powerfield (times100) MVD counts of these areas were evaluatedFinally the mean microvessel counts of the three mostvascular areas were regarded as MVD
212 Statistical Analyses Data were from three independentexperiments and expressed as mean plusmn SEM Statistical anal-yses were performed by the one-way analysis of variance(ANOVA) for differences among different groups Aboutcomparison of two groups Studentrsquos 119905-test was used All
analyses were undertaken using GraphPad Prism6 119875 lt 005was considered statistically significant
3 Results
31 QRHX Inhibits Tumor Growth in a Subcutaneous MouseModel In order to explore the role of QRHX in tumorgrowth in vivo subcutaneous mouse model was establishedby subcutaneous injection of LLC in left extremity auxiliaryMice were sacrificed at the end of treatment As shown inFigure 1 the tumor volumes were performed on days 1013 17 20 and 24 In NS group the tumor volume grad-ually increased in a time-dependent manner (Figure 1(a))However treatment with QRHX significantly suppressed thetumor volume (Figure 1(a)) The final tumor weight on day24 after the start of treatment showed a significant decrease intheQRHX group comparedwithNS control (Figure 1(b)119875 lt001) These data suggested that QRHX could dramaticallyinhibit tumor growth in vivo
32 QRHX Suppresses Cancer-Related Inflammation in LungCancer Numerous studies have indicated that cancer-relatedinflammation promotes the development of tumor [15 16]
Evidence-Based Complementary and Alternative Medicine 5
DAPI CD206 Merge
NS
QRH
X
(a)
C
D20
6 po
sitiv
e cel
ls
0
10
20
30
40
50
lowastlowast
NS QRHX
(b)
Figure 4 Immunofluorescence staining for CD206 of tumor tissues in subcutaneous tumor Mice with subcutaneous tumor were dealt with thegroup of NS and QRHX Images are at magnification of 200x The expression of TAMs was respectively analyzed using an Alexa Fluor-488-conjugated secondary antibody The nuclei were stained with DAPI Compared with NS group lowastlowast119875 lt 001
In addition some proinflammatory cytokines such as IL-8IL-6 and TNF-a have been shown to ultimately facilitate cellinvasion and metastasis [15] Therefore we used an ELISAassay to examine serum levels of the two proinflammatorycytokines IL-6 and TNF-120572 in peripheral blood Serum IL-6 and TNF-120572 levels were lower in mice treated with QRHXcompared with those receiving NS treatment (Figure 2 both
119875 lt 001) These data demonstrated that QRHX decreasedthe production of proinflammatory cytokines
33 QRHX Reduces the Accumulation of TAMs in Lung Can-cer In order to ascertain the effects of the QRHX on TAMsM2-like macrophage phenotype we used flow cytometryIF and IHC assay to examine CD206 expression As shown
6 Evidence-Based Complementary and Alternative Medicine
NS
100 휇m
(a)
QRH
X
100 휇m
(b)
C
D20
6 po
sitiv
e cel
ls
0
20
40
60
80
100
NS QRHX
lowastlowast
(c)
Figure 5 Comparison of CD206+ macrophages infiltration between NS and QRHX groups Immunohistochemical staining of CD206+macrophages in lung cancer tissues Images are at magnification of 200x Data represent mean plusmn SEM 119899 = 4 Compared with NS grouplowastlowast119875 lt 001
in Figures 3ndash6 our results revealed that CD206 expressionwas dramatically expressed in tumor tissue Compared to NSgroup oral administration with QRHX led to a prominentinhibition in TAMs (Figures 3ndash6 119875 lt 001) These datademonstrated that QRHX had a more significant role inimpeding accumulating and reducing the number of TAMsin lung cancer
34 QRHX Alters Hallmarkers of M1 and M2 Macrophagein Lung Cancer To further confirm the role of QRHX inTAMs we analyzed the level of Arg-1 protein M2 markerand the mRNA expression of M1 marker iNOS After QRHXtreatment Arg-1 level decreased (Figure 7(a) 119875 lt 001) andiNOS level increased (Figure 7(b) 119875 lt 005) Accordinglythese results strongly supported the inhibitory effect ofQRHX on suppressing TAMs infiltration and regulating M2-like macrophage polarization
35 QRHX Inhibits Angiogenesis in Lung Cancer CD31endothelial cell surface antigen is a vascular endothelialmarker for MVD and TAMs have a positive effect on bloodvessels by inducing the production of various proangiogenic
genes [17] Therefore CD31 was evaluated in implantedtumors using IHC and WB MVD stained by anti-CD31 wasmeasured by counting tissue sections of central areas of thetumor As shown in Figure 8 QRHX treatment induced aremarkable reduction in MVD and CD31 protein (119875 lt 001)Furthermore IHC and WB demonstrated reduced vascularendothelial growth factor (VEGF) expression in the QRHXtreated groups relative to NS controls (Figure 8 119875 lt 001)Taken together these data clearly showed thatQRHXreducedangiogenesis in lung cancer
36 QRHX Blocks CXCL 12CXCR4JNK2STAT3 SignalingPathways To investigate the molecular mechanism underly-ing the formulae decreasing M2 macrophages we tested theeffects of inhibitors of signaling molecules CXCL12CXCR4and JNK2STAT3 are known to be important moleculesinvolved in M2 polarization [18 19] Compared to NS grouporal administration of QRHX dramatically attenuated theincreased expressions of CXCL12 and CXCR4 in tumortissue (Figure 9(a) 119875 lt 001) As shown in Figure 9(b)JAK2 and STAT3 activation was significantly suppressed byQRHX treatment (both 119875 lt 001) compared to NS group
Evidence-Based Complementary and Alternative Medicine 7
106
105
104
103
0
Com
p-YL
1-H
F4
80-R
-PE-
H
1061051041030
Comp-BL1-H CD11b-FITC-H
197
1M
800 K
600 K
400 K
200 K
0
SSC-
H
SSC-
H
Comp-RL1-H CD206-Alexa Flour 647-H1061051041030
106
105
104
103
0
Com
p-YL
1-H
F4
80-R
-PE-
H
Comp-BL1-H CD11b-FITC-H1061051041030
900
1M
800 K
600 K
400 K
200 K
0
SSC-
H
SSC-
H
Comp-RL1-H CD206-Alexa Flour 647-H1061051041030
(a)
0
10
20
30
NS
F480+CD11b+ F480+CD11b+CD206+
QRHX
p
ositi
ve ce
lls
lowastlowast
lowastlowast
(b)
Figure 6 QRHX decreases the number of TAMs in subcutaneous tumor Mice with subcutaneous tumors were dealt with NS and QRHXMacrophages and TAMs (M2-like subtype) in tumor tissue were measured by flow cytometry Data represent mean plusmn SEM 119899 = 4 Comparedwith NS group lowastlowast119875 lt 001
8 Evidence-Based Complementary and Alternative Medicine
훽-actin43 kDa
Arg-140 kDa
00
02
04
06
08
10
Arg-1
Relat
ive p
rote
in ex
pres
sion
lowastlowast
NSQRHX
(a)
NSQRHX
iNOS0
4
1
2
3
Relat
ive p
rote
in ex
pres
sion
lowast
(b)
Figure 7The expression of M2-related marker (Arg-1) and M1-related marker (iNOS) was detected by western blot and QT-PCR respectivelyMice with subcutaneous tumor were dealt with NS and QRHX Data were presented as means plusmn SEM (119899 = 3) Compared with NS grouplowast119875 lt 005 lowastlowast119875 lt 001
Taking together the data suggested that QRHX inhibitedtumor cell-TAMs interactions possibly through blockingCXCL12CXCR4JAK2STAT3 signaling pathways and thenregulated macrophages polarization
4 Discussion
In the present study our data demonstrated that QRHXplayed a more crucial role in inhibiting tumor growth bymodulating tumor microenvironment especially TAMsQRHX inhibited tumor cell-TAMs interactions via thesuppression of cancer-related inflammation and probablyblocking the response of macrophages to tumor signalsCXCL12CXCR4JAK2STAT3 axis
In the nineteenth century the association between cancerand inflammation was firstly put forward [20] Large numberof studies provided powerful evidence that chronic inflam-mation can promote tumor development progression andmetastasis as well as chemoresistance [16 21] Recent studieshave confirmed that paeoniflorin baicalein and wogoninimportant ingredients of QRHX had the potential to inhibitmany types of inflammation [22ndash24] Furthermore baicaleinand wogonin exerted obvious inhibitory effects on cancer aswell as macrophages and angiogenesis [24 25] Paeoniflorinone of major ingredients could reduce lung metastasis ofLLC through inhibiting the M2 activation [26] In additionthe cytokines produced by activated innate immune cells intumor microenvironment can stimulate tumorigenesis suchas IL-6 and TNF-120572 [27] Our results showed a remarkabledecrease in multiple proinflammatory cytokines such as
TNF-a and IL-6 both in serum and tumor tissue of subcu-taneous mouse model (Figure 2) suggesting the inhibitoryeffect of QRHX on cancer-related inflammation
Tumor microenvironment created by the tumor andmainly orchestrated by inflammatory cells contributes totumor escape growth progression and evolution towardmetastasis [28 29] Numerous studies in recent decadehave presented evidences that TCM have a good effect onregulating tumor microenvironment such as reversing theimmunosuppressive microenvironment [11] Macrophages abasic component of the innate immune system are infiltratedin virtually all malignancies TAMs M2-like polarized stylehave been regarded as a protumor inflammatory microen-vironment which links inflammation and cancer [15] Col-lective evidences demonstrate that TAMs have the abilityof enhancing tumor angiogenesis increasing migration andinvasion and suppressing the antitumor immune responsesIt is correlated with the prognosis of patients with malignanttumor such as lung cancer [30 31] Consistent with previousstudies in this study we detected that the fraction of TAMswas increased in tumor and QRHX inhibited tumor growthin the tumor mice model through decreasing accumulatingof TAMs and activation of M2
CXCR4 which is widely expressed on malignant cellsand binds to CXCL12 [32] plays an important role in hem-atopoiesis development and organization of the immunesystem by directly and indirectly mechanisms [33] Forexample in NSCLC CXCL12-CXCR4 axis is involved inmetastasis and associated with an unfavorable prognosis[34] in ovarian cancer it can control accumulation of
Evidence-Based Complementary and Alternative Medicine 9CD
31
NS QRHX
VEG
F
0
5
10
15
20
25
VEGFMVD
Den
sity
of V
EGF
MV
D co
unt
lowastlowast
lowastlowast
(a)
00
02
04
06
08
10
Relat
ive p
rote
in ex
pres
sion
CD31VEGF
NSQRHX
lowastlowastlowastlowast
VEGF
CD31
훽-actin43 kDa
100 kDa
43 kDa
(b)
Figure 8 QRHX inhibits angiogenesis in lung cancer Mice with subcutaneous tumors were dealt with NS and QRHX (a) The nucleus wasdyed as blue CD31 was dyed as brown MVD were performed at high power field (times200) (b) The VEGF and CD31 protein expression intumor tissues was detected by western blot Data was expressed as means plusmn SEM values (119899 = 4) Compared with NS group lowastlowast119875 lt 001
human MDSCs and is an independent prognostic factor fortumor progression [35] Several studies have reported thatCXCL12 plays an important role in monocyte recruitmentdifferentiation and function [18 36] In a mouse modelof lung cancer CXCL12 could recruit tumor-promotingmyeloid CD11b+ cells [19] Besides powerful evidencesindicate a role for CXCR4-CXCL12 axis in promotingmacrophages polarization toward theM2 phenotype [37 38]In addition M2 subpopulation is associated with angio-genic factors such as VEGF and CXCL12-CXCR4 axis canalso promote tumor vascularization [39] Excitedly ourresults showed QRHX treatment inhibited signaling fromtumor cells to macrophages through inducing a remark-able decrease in CXCL12 and CXCR4 Furthermore QRHX
inhibited angiogenesis likely through altering the tumormicroenvironment by targeting TAMs
In STAT family there are seven proteins STAT3 is one ofthem It is a key transcription factor transducing signals fromactivated receptors or intracellular kinases to the nucleus andcan be activated in tumor cells and immune cells [40] Intumor STAT3 could contribute to cancer development andprogression inhibit apoptosis of tumors and help tumorescape immune system by suppressing the immune response[41] Evidence indicated that when STAT3 binds to somereceptor it can be activated through Janus Kinases (JAKs)such as JAK2 [42] Accumulating evidence implicates theimportant role of JAK2STAT3 in tumor and macrophagepolarization [27] For instance IL-6 can contribute to tumor
10 Evidence-Based Complementary and Alternative Medicine
CXCR4
CXCL1210 kDa
훽-actin43 kDa
41 kDa
NSQRHX
00
05
10
15
Relat
ive p
rote
in ex
pres
sion
CXCL12 CXCR4
lowastlowastlowastlowast
(a)
p-JAK2
p-STAT3
훽-actin43 kDa
90 kDa
120 kDa
NSQRHX
p-JAK2 p-STAT300
05
10
15
Relat
ive p
rote
in ex
pres
sion
lowastlowast lowastlowast
(b)
Figure 9 QRXH regulates TAMs by inhibiting the CXCL12CXCR4JAK2STAT3 signaling pathways Mice with subcutaneous tumor weredealt with NS and QRHX Representative images of western blot and densitometry analysis showing the expressions of CXCL12 CXCR4p-JAK2 and p-STAT3 in tumor Compared with NS group lowastlowast119875 lt 001
cell survival and upregulate the antiapoptotic genes by driv-ing JAK2STAT3 signal [43] IFN- activates macrophageby JAK-STAT signaling pathway [42] What is more JAK2is associated with CXCR4 [39] Then we can draw thatTAMs in malignant tumors tend to M2 subtype possiblythrough CXCL12CXCR4JAK2STAT3 signaling pathwayQRHX treatment blocked the response of macrophages totumor signals by suppressing CXCL12CXCR4JAK2STAT3expression and induced a remarkable decrease of the recruit-ment of M2 macrophages suggesting the attenuation of M2subtype cells function by QRHX
In TCM theory TCM formulae have abundant medicinalmaterials and then regulate diseases through multitargetsandmultiways Although the chemical constituents of QRHXhave been separated and identified by HPLUNG CANCER-QTOF MS-UV method in our team (Supplementary Fig-ure 1 and Table 1) the ingredients are various and theirfunctions are enormous So there may be other mechanismsinvolved in macrophage polarization What is more there
are diverse types cells in tumor except for tumor cells andmacrophage Moreover it is worth mentioning that TAMswere not separated and extracted from tumor tissue CXCL12CXCR4 JAK2 and STAT3 can not only play significantrole in tumor cell or macrophage but also other cellssuch as T lymphocyte neutrophils tumor-associated fibrob-last and endothelial cells Therefore QRHX inhibited theCXCL12CXCR4JAK2STAT3 axis possibly through othercells Taken together further investigations are needed toidentify direct molecular targets of QRHX in macrophagesin the context of cancer
5 Conclusion
In conclusion data from this study revealed that QRHXcould suppress cancer progression by inhibiting the tumorpromotion of TAMs in subcutaneous mice model whichcould contribute to elucidating the underlying regulatorymode of QRHX on lung cancer treatment
Evidence-Based Complementary and Alternative Medicine 11
Abbreviations
ELISA Enzyme-linked immunosorbent assayFITC Fluorescein isothiocyanateTNF-120572 Tumor necrosis factor-120572IL-6 Interleukin-6iNOS Inducible nitric oxide synthaseVEGF Vascular endothelial growth factorQT-PCR Quantitative Polymerase Chain ReactionIF ImmunofluorescenceIHC ImmunohistochemicalTAMs Tumor-associated macrophagesArg-1 Arginase 1
Conflicts of Interest
The authors declare that there are no conflicts of interest
Authorsrsquo Contributions
Fei Xu and Wenqiang Cui contributed equally to this workFei Xu and Baojun Liu conceived and designed experimentsFei Xu and Wenqiang Cui analyzed data and wrote themanuscript Jingcheng Dong supervised the project Zhengx-iao Zhao and Ying Wei assisted in experimental design anddata evaluation Jiaqi Liu Mihui Li Qiuping Li Chen Yanand Jian Qiu performed the study All authors reviewed andapproved the manuscript
Acknowledgments
This study was supported by Natural Science Foundation ofChina (nos 81673916 and 81403148) Development Projectof Shanghai Peak Disciplines-Integrative Medicine (no20150407)
References
[1] I Ikonomidis C A Michalakeas J Parissis et al ldquoInflamma-tory markers in coronary artery diseaserdquo BioFactors vol 38 no5 pp 320ndash328 2012
[2] P Lee C C Leung M I Restrepo K Takahashi Y Song and JM Porcel ldquoYear in review 2015 lung cancer pleural diseasesrespiratory infections bronchiectasis and tuberculosis bron-choscopic intervention and imagingrdquo Respirology vol 21 no 5pp 961ndash967 2016
[3] S V Sharma DW Bell J Settleman and D A Haber ldquoEpider-mal growth factor receptor mutations in lung cancerrdquo NatureReviews Cancer vol 7 no 3 pp 169ndash181 2007
[4] D Leong R Rai B Nguyen A Lee and D Yip ldquoAdvancesin adjuvant systemic therapy for non-small-cell lung cancerrdquoWorld Journal of Clinical Oncology vol 5 no 4 pp 633ndash6452014
[5] M T Villanueva ldquoMicroenvironment the new midfielders inthe tumourmicroenvironmentrdquoNature reviews Cancer vol 14no 12 p 765 2014
[6] A Mantovani S Sozzani M Locati P Allavena and A SicaldquoMacrophage polarization tumor-associated macrophages as aparadigm for polarizedM2mononuclear phagocytesrdquoTrends inImmunology vol 23 no 11 pp 549ndash555 2002
[7] A Mantovani and M Locati ldquoTumor-associated macrophagesas a paradigm of macrophage plasticity diversity and polariza-tion lessons and open questionsrdquo Arteriosclerosis Thrombosisand Vascular Biology vol 33 no 7 pp 1478ndash1483 2013
[8] W Hu X Li C Zhang Y Yang J Jiang and C Wu ldquoTumor-associated macrophages in cancersrdquo Clinical and TranslationalOncology vol 18 no 3 pp 251ndash258 2016
[9] B Z Qian and J W Pollard ldquoMacrophage diversity enhancestumor progression and metastasisrdquo Cell vol 141 no 1 pp 39ndash51 2010
[10] A Mantovani and P Allavena ldquoThe interaction of anti-cancer therapies with tumor-associated macrophagesrdquo Journalof Experimental Medicine vol 212 no 4 pp 435ndash445 2015
[11] J Xu Z Song Q Guo and J Li ldquoSynergistic effect and molec-ular mechanisms of traditional Chinese medicine on regulatingtumor microenvironment and cancer cellsrdquo BioMed ResearchInternational vol 2016 Article ID 1490738 pp 1ndash14 2016
[12] J Wu J Xu E A Eksioglu et al ldquoIcariside II induces apoptosisof melanoma cells through the downregulation of survivalpathwaysrdquoNutrition and Cancer vol 65 no 1 pp 110ndash117 2013
[13] L Kong J Liu J Wang et al ldquoIcariin inhibits TNF-120572IFN-120574induced inflammatory response via inhibition of the substanceP and p38-MAPK signaling pathway in human keratinocytesrdquoInternational Immunopharmacology vol 29 no 2 pp 401ndash4072015
[14] JWu J Du C Xu et al ldquoIn vivo and in vitro anti-inflammatoryeffects of a novel derivative of icariinrdquo Immunopharmacologyand Immunotoxicology vol 33 no 1 pp 49ndash54 2010
[15] E M Conway L A Pikor S H Y Kung et al ldquoMacrophagesinflammation and lung cancerrdquoAmerican Journal of Respiratoryand Critical Care Medicine vol 193 no 2 pp 116ndash130 2016
[16] S Shalapour and M Karin ldquoImmunity inflammation andcancer an eternal fight between good and evilrdquo The Journal ofClinical Investigation vol 125 no 9 pp 3347ndash3355 2015
[17] L Bingle N J Brown and C E Lewis ldquoThe role of tumour-associated macrophages in tumour progression Implicationsfor new anticancer therapiesrdquo Journal of Pathology vol 196 no3 pp 254ndash265 2002
[18] D Kim J Kim J H Yoon et al ldquoCXCL12 secreted from adiposetissue recruits macrophages and induces insulin resistance inmicerdquo Diabetologia vol 57 no 7 pp 1456ndash1465 2014
[19] M C Schmid C J Avraamides P Foubert et al ldquoCombinedblockade of integrin-12057241205731 plus cytokines SDF-1120572 or IL-1120573potently inhibits tumor inflammation and growthrdquo CancerResearch vol 71 no 22 pp 6965ndash6975 2011
[20] F Balkwill and A Mantovani ldquoInflammation and cancer backto VirchowrdquoThe Lancet vol 357 no 9255 pp 539ndash545 2001
[21] B B Aggarwal S Shishodia S K Sandur M K Pandey andG Sethi ldquoInflammation and cancer how hot is the linkrdquoBiochemical Pharmacology vol 72 no 11 pp 1605ndash1621 2006
[22] W-L Jiang X-G Chen H-B Zhu Y-B Gao J-W Tian andF-H Fu ldquoPaeoniflorin inhibits systemic inflammation andimproves survival in experimental sepsisrdquo Basic and ClinicalPharmacology and Toxicology vol 105 no 1 pp 64ndash71 2009
[23] I D Kim and B J Ha ldquoThe effects of paeoniflorin on LPS-induced liver inflammatory reactionsrdquo Archives of PharmacalResearch vol 33 no 6 pp 959ndash966 2010
[24] G-W Fan Y Zhang X Jiang et al ldquoAnti-inflammatory activityof baicalein in LPS-stimulated RAW2647 macrophages viaestrogen receptor and NF-120581B-dependent pathwaysrdquo Inflamma-tion vol 36 no 6 pp 1584ndash1591 2013
12 Evidence-Based Complementary and Alternative Medicine
[25] M Li-Weber ldquoNew therapeutic aspects of flavones the anti-cancer properties of Scutellaria and its main active constituentsWogonin Baicalein and Baicalinrdquo Cancer Treatment Reviewsvol 35 no 1 pp 57ndash68 2009
[26] QWu G-L Chen Y-J Li Y Chen and F-Z Lin ldquoPaeoniflorininhibits macrophage-mediated lung cancer metastasisrdquoChineseJournal of Natural Medicines vol 13 no 12 pp 925ndash932 2015
[27] W Lin and M Karin ldquoA cytokine-mediated link betweeninnate immunity inflammation and cancerrdquo Journal of ClinicalInvestigation vol 117 no 5 pp 1175ndash1183 2007
[28] T L Whiteside ldquoThe tumor microenvironment and its role inpromoting tumor growthrdquo Oncogene vol 27 no 45 pp 5904ndash5912 2008
[29] G Lorusso and C Ruegg ldquoThe tumor microenvironment andits contribution to tumor evolution toward metastasisrdquo Histo-chemistry and Cell Biology vol 130 no 6 pp 1091ndash1103 2008
[30] L Ding G Liang Z Yao et al ldquoMetformin prevents cancermetastasis by inhibiting M2-like polarization of tumor associ-ated macrophagesrdquo Oncotarget vol 6 no 34 pp 36441ndash364552015
[31] A Yuan Y JHsiaoH Y Chen et al ldquoOpposite effects ofM1 andM2macrophage subtypes on lung cancer progressionrdquo ScientificReports vol 5 Article ID 14273 2015
[32] F Balkwill ldquoCancer and the chemokine networkrdquo NatureReviews Cancer vol 4 no 7 pp 540ndash550 2004
[33] J A Burger and T J Kipps ldquoCXCR4 a key receptor in thecrosstalk between tumor cells and their microenvironmentrdquoBlood vol 107 no 5 pp 1761ndash1767 2006
[34] R J PhillipsM D BurdickM Lutz J A BelperioM P Keaneand R M Strieter ldquoThe stromal derived factor-1CXCL12-CXCchemokine receptor 4 biological axis in non-small cell lungcancermetastasesrdquoAmerican Journal of Respiratory and CriticalCare Medicine vol 167 no 12 pp 1676ndash1686 2003
[35] N Obermajer R Muthuswamy K Odunsi R P Edwards andP Kalinski ldquoPGE-induced CXCL 12 production and CXCR4expression controls the accumulation of human MDSCs inovarian cancer environmentrdquo Cancer Research vol 71 no 24pp 7463ndash7470 2011
[36] L Sanchez-Martın A Estecha R Samaniego S Sanchez-Ramon M A Vega and P Sanchez-Mateos ldquoThe chemokineCXCL12 regulates monocyte-macrophage differentiation andRUNX3 expressionrdquo Blood vol 117 no 1 pp 88ndash97 2011
[37] K Beider H Bitner M Leiba et al ldquoMultiple myelomacells recruit tumor-supportive macrophages through theCXCR4CXCL12 axis and promote their polarization towardthe M2 phenotyperdquo Oncotarget vol 5 no 22 pp 11283ndash112962014
[38] J M Mota C A Leite and L E Souza ldquoPost-sepsis stateinduces tumor-associated macrophage accumulation throughCXCR4CXCL12 and favors tumor progression inmicerdquoCancerImmunology Research vol 4 no 4 pp 312ndash322 2016
[39] B A Teicher and S P Fricker ldquoCXCL12 (SDF-1)CXCR4 path-way in cancerrdquoClinical Cancer Research vol 16 no 11 pp 2927ndash2931 2010
[40] P C Heinrich I Behrmann G Muller-Newen F Schaper andL Graeve ldquoInterleukin-6-type cytokine signalling through thegp130JakSTAT pathwayrdquo Biochemical Journal vol 334 part 2pp 297ndash314 1998
[41] M Kortylewski M Kujawski T Wang et al ldquoInhibiting Stat3signaling in the hematopoietic system elicits multicomponentantitumor immunityrdquo Nature Medicine vol 11 no 12 pp 1314ndash1321 2005
[42] X Hu J Chen L Wang and L B Ivashkiv ldquoCrosstalk amongJak-STAT Toll-like receptor and ITAM-dependent pathways inmacrophage activationrdquo Journal of Leukocyte Biology vol 82no 2 pp 237ndash243 2007
[43] R Catlett-Falcone T H Landowski M M Oshiro et alldquoConstitutive activation of Stat3 signaling confers resistance toapoptosis in humanU266myeloma cellsrdquo Immunity vol 10 no1 pp 105ndash115 1999
Submit your manuscripts athttpswwwhindawicom
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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OncologyJournal of
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Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
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Research and TreatmentAIDS
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 3
(Days)
0
1
2
3
4
5
QRHXNS
Tum
or v
olum
e (cm
3)
10 13 17 20 24
(a)
Tum
or w
eigh
t (g)
0
1
2
3
4
QRHXNS
lowastlowast
(b)
Figure 1 QRHX inhibits growth of tumor in a subcutaneous mouse model C57BL6 mice were subcutaneously injected with LLC QRHXreduced tumor volume (a) and weight (b) The data represent means plusmn SEM (119899 ge 10) Compared with NS group lowastlowast119875 lt 001
to the manufacturerrsquos instructions followed by detection bya FACSCalibur instrument (BD Bioscience)
28 Western Blot Analysis Total protein was extracted fromthe cells using a RIPA kit (Beyotime China) Cell debriswas removed by microcentrifugation and supernatants werequickly frozenThe protein concentration was determined byBSA method And then the protein was electrophoresed ona polyacrylamide gel and transferred to a PVDF membraneNext the membranes was incubated 1 hndash15 h with 5 milkat room temperature and then incubated overnight at 4∘Cwith a 1 1000 or 1 500 dilution of corresponding primaryantibodies Blots were again washed three times with Tris-buffered salineTween 20 (TBST) and then incubated with a1 5000 dilution of HRP-conjugated secondary antibody for1 h at room temperature Blots were again washed three timeswith TBST and then developed by enhanced chemilumines-cence Band intensities were quantified using UN-SCAN-ITgel analysis software (version 6)
29 QT-PCR Total RNA was extracted with Trizol reagent(Ambion Thermo) and reverse transcriptionwas performedto obtain the cDNA using the PrimeScript RT Reagent Kit(Takara Japan) according to the manufacturerrsquos protocolThe primers used were synthesized by (Sangon BiotechChina) These queues were as follows iNOS 51015840-GTT-CTCAGCCCAACA ATACAAGA-31015840 (forward) and 51015840-GTG-GACGGGTCGATGTCAC-31015840 (reverse) IL-6 51015840-GATACC-ACTCCCAAC AGAC-31015840 (forward) and 51015840-CTTTTCTCA-TTTCCACGAT-31015840 (reverse) CCL22 51015840-AGGAAGGC TTG-GCTTTTAGG-31015840 (forward) and 51015840-TGGTACCTTGCA-GGCTCTCT-31015840 (reverse) TNF-120572 51015840-ACGGCATGGATC-TCAAAGAC-31015840 (forward) and 51015840-GTGGGTGAGGAGCAC-GTAGT-31015840 (reverse) GAPDH 51015840-AAATGGTGAAGGTCG-GTGTG-31015840 (forward) and 51015840-AGGTCAATGAAGGGGTCG-TT-31015840 (reverse) Quantitative real-time PCR (QT-PCR) was
The s
erum
conc
entr
atio
nsof
IL-6
TN
F-a (
pgm
L)
0
100
200
300
IL-6
NSQRHX
TNF-훼
lowastlowast
lowastlowast
Figure 2 QRHX suppresses cancer-related inflammation in lungcancer A subcutaneous mouse model was established and treatedwith QRHX or NS as described above Blood was collected fromeach mouse and the serum concentrations of the proinflammatorycytokines IL-6 and TNF-a were detected by ELISA Studentrsquos 119905-testwas used to determine the statistical significance lowastlowast119875 lt 001
performed using SYBR green reaction mixture in the ViiA7 Real Time PCR System (Bio-Rad MiniOpticon) Therelative expression levels were calculated using 2minusΔΔCtmethods
210 Immunofluorescence (IF) Cryostat sections were fixedand permeated CD11b andCD206 antibodies formice tumortissue were used followed by Alexa Fluor 488 (ALEXA) or594 (ALEXA)
211 Immunohistochemical Analysis IHC stain was per-formed using a two-step EnVisionHRP technique accordingto the manufacturersquos instruction For three slides cytoplasmstained with brown was scored as positive The expression ofCD206 CD31 and VEGF was quantitatively evaluated using
4 Evidence-Based Complementary and Alternative Medicine
M0 M2 M0M2 merged
NS
QRH
X
Figure 3 Immunofluorescence staining for CD11b and CD206 of tumor tissues in subcutaneous tumor Mice with subcutaneous tumor weredealt with NS and QRHX Images are at magnification of 200x The expression of M0 and TAMs (M2-like macrophage) was respectivelyanalyzed using an Alexa Fluor-549- and Alexa Fluor-488-conjugated secondary antibody
Olympus Cx31 microscope with Image-Pro Plus medicalimage analysis systemThedigital imageswere captured usinga digital camera (Canon A640) The positive area and OD ofCD206 and VEGF positive cells were evaluated by Image-J software and determined by measuring three randomlyselected microscopic fields for each slide The IHC index wasdefined as average integral optical density (AIOD) (AIOD =positive area times ODtotal area)
CD31 a marker factor in vascular endothelial cell waspositively correlated with microvascular density (MVD)Then we counted the MVD by detecting the expression ofCD31 antibody in tumor tissues by immunohistochemistrymethod Firstly at low power field (times40) three most intensetissue sections were selected each slice and then at high powerfield (times100) MVD counts of these areas were evaluatedFinally the mean microvessel counts of the three mostvascular areas were regarded as MVD
212 Statistical Analyses Data were from three independentexperiments and expressed as mean plusmn SEM Statistical anal-yses were performed by the one-way analysis of variance(ANOVA) for differences among different groups Aboutcomparison of two groups Studentrsquos 119905-test was used All
analyses were undertaken using GraphPad Prism6 119875 lt 005was considered statistically significant
3 Results
31 QRHX Inhibits Tumor Growth in a Subcutaneous MouseModel In order to explore the role of QRHX in tumorgrowth in vivo subcutaneous mouse model was establishedby subcutaneous injection of LLC in left extremity auxiliaryMice were sacrificed at the end of treatment As shown inFigure 1 the tumor volumes were performed on days 1013 17 20 and 24 In NS group the tumor volume grad-ually increased in a time-dependent manner (Figure 1(a))However treatment with QRHX significantly suppressed thetumor volume (Figure 1(a)) The final tumor weight on day24 after the start of treatment showed a significant decrease intheQRHX group comparedwithNS control (Figure 1(b)119875 lt001) These data suggested that QRHX could dramaticallyinhibit tumor growth in vivo
32 QRHX Suppresses Cancer-Related Inflammation in LungCancer Numerous studies have indicated that cancer-relatedinflammation promotes the development of tumor [15 16]
Evidence-Based Complementary and Alternative Medicine 5
DAPI CD206 Merge
NS
QRH
X
(a)
C
D20
6 po
sitiv
e cel
ls
0
10
20
30
40
50
lowastlowast
NS QRHX
(b)
Figure 4 Immunofluorescence staining for CD206 of tumor tissues in subcutaneous tumor Mice with subcutaneous tumor were dealt with thegroup of NS and QRHX Images are at magnification of 200x The expression of TAMs was respectively analyzed using an Alexa Fluor-488-conjugated secondary antibody The nuclei were stained with DAPI Compared with NS group lowastlowast119875 lt 001
In addition some proinflammatory cytokines such as IL-8IL-6 and TNF-a have been shown to ultimately facilitate cellinvasion and metastasis [15] Therefore we used an ELISAassay to examine serum levels of the two proinflammatorycytokines IL-6 and TNF-120572 in peripheral blood Serum IL-6 and TNF-120572 levels were lower in mice treated with QRHXcompared with those receiving NS treatment (Figure 2 both
119875 lt 001) These data demonstrated that QRHX decreasedthe production of proinflammatory cytokines
33 QRHX Reduces the Accumulation of TAMs in Lung Can-cer In order to ascertain the effects of the QRHX on TAMsM2-like macrophage phenotype we used flow cytometryIF and IHC assay to examine CD206 expression As shown
6 Evidence-Based Complementary and Alternative Medicine
NS
100 휇m
(a)
QRH
X
100 휇m
(b)
C
D20
6 po
sitiv
e cel
ls
0
20
40
60
80
100
NS QRHX
lowastlowast
(c)
Figure 5 Comparison of CD206+ macrophages infiltration between NS and QRHX groups Immunohistochemical staining of CD206+macrophages in lung cancer tissues Images are at magnification of 200x Data represent mean plusmn SEM 119899 = 4 Compared with NS grouplowastlowast119875 lt 001
in Figures 3ndash6 our results revealed that CD206 expressionwas dramatically expressed in tumor tissue Compared to NSgroup oral administration with QRHX led to a prominentinhibition in TAMs (Figures 3ndash6 119875 lt 001) These datademonstrated that QRHX had a more significant role inimpeding accumulating and reducing the number of TAMsin lung cancer
34 QRHX Alters Hallmarkers of M1 and M2 Macrophagein Lung Cancer To further confirm the role of QRHX inTAMs we analyzed the level of Arg-1 protein M2 markerand the mRNA expression of M1 marker iNOS After QRHXtreatment Arg-1 level decreased (Figure 7(a) 119875 lt 001) andiNOS level increased (Figure 7(b) 119875 lt 005) Accordinglythese results strongly supported the inhibitory effect ofQRHX on suppressing TAMs infiltration and regulating M2-like macrophage polarization
35 QRHX Inhibits Angiogenesis in Lung Cancer CD31endothelial cell surface antigen is a vascular endothelialmarker for MVD and TAMs have a positive effect on bloodvessels by inducing the production of various proangiogenic
genes [17] Therefore CD31 was evaluated in implantedtumors using IHC and WB MVD stained by anti-CD31 wasmeasured by counting tissue sections of central areas of thetumor As shown in Figure 8 QRHX treatment induced aremarkable reduction in MVD and CD31 protein (119875 lt 001)Furthermore IHC and WB demonstrated reduced vascularendothelial growth factor (VEGF) expression in the QRHXtreated groups relative to NS controls (Figure 8 119875 lt 001)Taken together these data clearly showed thatQRHXreducedangiogenesis in lung cancer
36 QRHX Blocks CXCL 12CXCR4JNK2STAT3 SignalingPathways To investigate the molecular mechanism underly-ing the formulae decreasing M2 macrophages we tested theeffects of inhibitors of signaling molecules CXCL12CXCR4and JNK2STAT3 are known to be important moleculesinvolved in M2 polarization [18 19] Compared to NS grouporal administration of QRHX dramatically attenuated theincreased expressions of CXCL12 and CXCR4 in tumortissue (Figure 9(a) 119875 lt 001) As shown in Figure 9(b)JAK2 and STAT3 activation was significantly suppressed byQRHX treatment (both 119875 lt 001) compared to NS group
Evidence-Based Complementary and Alternative Medicine 7
106
105
104
103
0
Com
p-YL
1-H
F4
80-R
-PE-
H
1061051041030
Comp-BL1-H CD11b-FITC-H
197
1M
800 K
600 K
400 K
200 K
0
SSC-
H
SSC-
H
Comp-RL1-H CD206-Alexa Flour 647-H1061051041030
106
105
104
103
0
Com
p-YL
1-H
F4
80-R
-PE-
H
Comp-BL1-H CD11b-FITC-H1061051041030
900
1M
800 K
600 K
400 K
200 K
0
SSC-
H
SSC-
H
Comp-RL1-H CD206-Alexa Flour 647-H1061051041030
(a)
0
10
20
30
NS
F480+CD11b+ F480+CD11b+CD206+
QRHX
p
ositi
ve ce
lls
lowastlowast
lowastlowast
(b)
Figure 6 QRHX decreases the number of TAMs in subcutaneous tumor Mice with subcutaneous tumors were dealt with NS and QRHXMacrophages and TAMs (M2-like subtype) in tumor tissue were measured by flow cytometry Data represent mean plusmn SEM 119899 = 4 Comparedwith NS group lowastlowast119875 lt 001
8 Evidence-Based Complementary and Alternative Medicine
훽-actin43 kDa
Arg-140 kDa
00
02
04
06
08
10
Arg-1
Relat
ive p
rote
in ex
pres
sion
lowastlowast
NSQRHX
(a)
NSQRHX
iNOS0
4
1
2
3
Relat
ive p
rote
in ex
pres
sion
lowast
(b)
Figure 7The expression of M2-related marker (Arg-1) and M1-related marker (iNOS) was detected by western blot and QT-PCR respectivelyMice with subcutaneous tumor were dealt with NS and QRHX Data were presented as means plusmn SEM (119899 = 3) Compared with NS grouplowast119875 lt 005 lowastlowast119875 lt 001
Taking together the data suggested that QRHX inhibitedtumor cell-TAMs interactions possibly through blockingCXCL12CXCR4JAK2STAT3 signaling pathways and thenregulated macrophages polarization
4 Discussion
In the present study our data demonstrated that QRHXplayed a more crucial role in inhibiting tumor growth bymodulating tumor microenvironment especially TAMsQRHX inhibited tumor cell-TAMs interactions via thesuppression of cancer-related inflammation and probablyblocking the response of macrophages to tumor signalsCXCL12CXCR4JAK2STAT3 axis
In the nineteenth century the association between cancerand inflammation was firstly put forward [20] Large numberof studies provided powerful evidence that chronic inflam-mation can promote tumor development progression andmetastasis as well as chemoresistance [16 21] Recent studieshave confirmed that paeoniflorin baicalein and wogoninimportant ingredients of QRHX had the potential to inhibitmany types of inflammation [22ndash24] Furthermore baicaleinand wogonin exerted obvious inhibitory effects on cancer aswell as macrophages and angiogenesis [24 25] Paeoniflorinone of major ingredients could reduce lung metastasis ofLLC through inhibiting the M2 activation [26] In additionthe cytokines produced by activated innate immune cells intumor microenvironment can stimulate tumorigenesis suchas IL-6 and TNF-120572 [27] Our results showed a remarkabledecrease in multiple proinflammatory cytokines such as
TNF-a and IL-6 both in serum and tumor tissue of subcu-taneous mouse model (Figure 2) suggesting the inhibitoryeffect of QRHX on cancer-related inflammation
Tumor microenvironment created by the tumor andmainly orchestrated by inflammatory cells contributes totumor escape growth progression and evolution towardmetastasis [28 29] Numerous studies in recent decadehave presented evidences that TCM have a good effect onregulating tumor microenvironment such as reversing theimmunosuppressive microenvironment [11] Macrophages abasic component of the innate immune system are infiltratedin virtually all malignancies TAMs M2-like polarized stylehave been regarded as a protumor inflammatory microen-vironment which links inflammation and cancer [15] Col-lective evidences demonstrate that TAMs have the abilityof enhancing tumor angiogenesis increasing migration andinvasion and suppressing the antitumor immune responsesIt is correlated with the prognosis of patients with malignanttumor such as lung cancer [30 31] Consistent with previousstudies in this study we detected that the fraction of TAMswas increased in tumor and QRHX inhibited tumor growthin the tumor mice model through decreasing accumulatingof TAMs and activation of M2
CXCR4 which is widely expressed on malignant cellsand binds to CXCL12 [32] plays an important role in hem-atopoiesis development and organization of the immunesystem by directly and indirectly mechanisms [33] Forexample in NSCLC CXCL12-CXCR4 axis is involved inmetastasis and associated with an unfavorable prognosis[34] in ovarian cancer it can control accumulation of
Evidence-Based Complementary and Alternative Medicine 9CD
31
NS QRHX
VEG
F
0
5
10
15
20
25
VEGFMVD
Den
sity
of V
EGF
MV
D co
unt
lowastlowast
lowastlowast
(a)
00
02
04
06
08
10
Relat
ive p
rote
in ex
pres
sion
CD31VEGF
NSQRHX
lowastlowastlowastlowast
VEGF
CD31
훽-actin43 kDa
100 kDa
43 kDa
(b)
Figure 8 QRHX inhibits angiogenesis in lung cancer Mice with subcutaneous tumors were dealt with NS and QRHX (a) The nucleus wasdyed as blue CD31 was dyed as brown MVD were performed at high power field (times200) (b) The VEGF and CD31 protein expression intumor tissues was detected by western blot Data was expressed as means plusmn SEM values (119899 = 4) Compared with NS group lowastlowast119875 lt 001
human MDSCs and is an independent prognostic factor fortumor progression [35] Several studies have reported thatCXCL12 plays an important role in monocyte recruitmentdifferentiation and function [18 36] In a mouse modelof lung cancer CXCL12 could recruit tumor-promotingmyeloid CD11b+ cells [19] Besides powerful evidencesindicate a role for CXCR4-CXCL12 axis in promotingmacrophages polarization toward theM2 phenotype [37 38]In addition M2 subpopulation is associated with angio-genic factors such as VEGF and CXCL12-CXCR4 axis canalso promote tumor vascularization [39] Excitedly ourresults showed QRHX treatment inhibited signaling fromtumor cells to macrophages through inducing a remark-able decrease in CXCL12 and CXCR4 Furthermore QRHX
inhibited angiogenesis likely through altering the tumormicroenvironment by targeting TAMs
In STAT family there are seven proteins STAT3 is one ofthem It is a key transcription factor transducing signals fromactivated receptors or intracellular kinases to the nucleus andcan be activated in tumor cells and immune cells [40] Intumor STAT3 could contribute to cancer development andprogression inhibit apoptosis of tumors and help tumorescape immune system by suppressing the immune response[41] Evidence indicated that when STAT3 binds to somereceptor it can be activated through Janus Kinases (JAKs)such as JAK2 [42] Accumulating evidence implicates theimportant role of JAK2STAT3 in tumor and macrophagepolarization [27] For instance IL-6 can contribute to tumor
10 Evidence-Based Complementary and Alternative Medicine
CXCR4
CXCL1210 kDa
훽-actin43 kDa
41 kDa
NSQRHX
00
05
10
15
Relat
ive p
rote
in ex
pres
sion
CXCL12 CXCR4
lowastlowastlowastlowast
(a)
p-JAK2
p-STAT3
훽-actin43 kDa
90 kDa
120 kDa
NSQRHX
p-JAK2 p-STAT300
05
10
15
Relat
ive p
rote
in ex
pres
sion
lowastlowast lowastlowast
(b)
Figure 9 QRXH regulates TAMs by inhibiting the CXCL12CXCR4JAK2STAT3 signaling pathways Mice with subcutaneous tumor weredealt with NS and QRHX Representative images of western blot and densitometry analysis showing the expressions of CXCL12 CXCR4p-JAK2 and p-STAT3 in tumor Compared with NS group lowastlowast119875 lt 001
cell survival and upregulate the antiapoptotic genes by driv-ing JAK2STAT3 signal [43] IFN- activates macrophageby JAK-STAT signaling pathway [42] What is more JAK2is associated with CXCR4 [39] Then we can draw thatTAMs in malignant tumors tend to M2 subtype possiblythrough CXCL12CXCR4JAK2STAT3 signaling pathwayQRHX treatment blocked the response of macrophages totumor signals by suppressing CXCL12CXCR4JAK2STAT3expression and induced a remarkable decrease of the recruit-ment of M2 macrophages suggesting the attenuation of M2subtype cells function by QRHX
In TCM theory TCM formulae have abundant medicinalmaterials and then regulate diseases through multitargetsandmultiways Although the chemical constituents of QRHXhave been separated and identified by HPLUNG CANCER-QTOF MS-UV method in our team (Supplementary Fig-ure 1 and Table 1) the ingredients are various and theirfunctions are enormous So there may be other mechanismsinvolved in macrophage polarization What is more there
are diverse types cells in tumor except for tumor cells andmacrophage Moreover it is worth mentioning that TAMswere not separated and extracted from tumor tissue CXCL12CXCR4 JAK2 and STAT3 can not only play significantrole in tumor cell or macrophage but also other cellssuch as T lymphocyte neutrophils tumor-associated fibrob-last and endothelial cells Therefore QRHX inhibited theCXCL12CXCR4JAK2STAT3 axis possibly through othercells Taken together further investigations are needed toidentify direct molecular targets of QRHX in macrophagesin the context of cancer
5 Conclusion
In conclusion data from this study revealed that QRHXcould suppress cancer progression by inhibiting the tumorpromotion of TAMs in subcutaneous mice model whichcould contribute to elucidating the underlying regulatorymode of QRHX on lung cancer treatment
Evidence-Based Complementary and Alternative Medicine 11
Abbreviations
ELISA Enzyme-linked immunosorbent assayFITC Fluorescein isothiocyanateTNF-120572 Tumor necrosis factor-120572IL-6 Interleukin-6iNOS Inducible nitric oxide synthaseVEGF Vascular endothelial growth factorQT-PCR Quantitative Polymerase Chain ReactionIF ImmunofluorescenceIHC ImmunohistochemicalTAMs Tumor-associated macrophagesArg-1 Arginase 1
Conflicts of Interest
The authors declare that there are no conflicts of interest
Authorsrsquo Contributions
Fei Xu and Wenqiang Cui contributed equally to this workFei Xu and Baojun Liu conceived and designed experimentsFei Xu and Wenqiang Cui analyzed data and wrote themanuscript Jingcheng Dong supervised the project Zhengx-iao Zhao and Ying Wei assisted in experimental design anddata evaluation Jiaqi Liu Mihui Li Qiuping Li Chen Yanand Jian Qiu performed the study All authors reviewed andapproved the manuscript
Acknowledgments
This study was supported by Natural Science Foundation ofChina (nos 81673916 and 81403148) Development Projectof Shanghai Peak Disciplines-Integrative Medicine (no20150407)
References
[1] I Ikonomidis C A Michalakeas J Parissis et al ldquoInflamma-tory markers in coronary artery diseaserdquo BioFactors vol 38 no5 pp 320ndash328 2012
[2] P Lee C C Leung M I Restrepo K Takahashi Y Song and JM Porcel ldquoYear in review 2015 lung cancer pleural diseasesrespiratory infections bronchiectasis and tuberculosis bron-choscopic intervention and imagingrdquo Respirology vol 21 no 5pp 961ndash967 2016
[3] S V Sharma DW Bell J Settleman and D A Haber ldquoEpider-mal growth factor receptor mutations in lung cancerrdquo NatureReviews Cancer vol 7 no 3 pp 169ndash181 2007
[4] D Leong R Rai B Nguyen A Lee and D Yip ldquoAdvancesin adjuvant systemic therapy for non-small-cell lung cancerrdquoWorld Journal of Clinical Oncology vol 5 no 4 pp 633ndash6452014
[5] M T Villanueva ldquoMicroenvironment the new midfielders inthe tumourmicroenvironmentrdquoNature reviews Cancer vol 14no 12 p 765 2014
[6] A Mantovani S Sozzani M Locati P Allavena and A SicaldquoMacrophage polarization tumor-associated macrophages as aparadigm for polarizedM2mononuclear phagocytesrdquoTrends inImmunology vol 23 no 11 pp 549ndash555 2002
[7] A Mantovani and M Locati ldquoTumor-associated macrophagesas a paradigm of macrophage plasticity diversity and polariza-tion lessons and open questionsrdquo Arteriosclerosis Thrombosisand Vascular Biology vol 33 no 7 pp 1478ndash1483 2013
[8] W Hu X Li C Zhang Y Yang J Jiang and C Wu ldquoTumor-associated macrophages in cancersrdquo Clinical and TranslationalOncology vol 18 no 3 pp 251ndash258 2016
[9] B Z Qian and J W Pollard ldquoMacrophage diversity enhancestumor progression and metastasisrdquo Cell vol 141 no 1 pp 39ndash51 2010
[10] A Mantovani and P Allavena ldquoThe interaction of anti-cancer therapies with tumor-associated macrophagesrdquo Journalof Experimental Medicine vol 212 no 4 pp 435ndash445 2015
[11] J Xu Z Song Q Guo and J Li ldquoSynergistic effect and molec-ular mechanisms of traditional Chinese medicine on regulatingtumor microenvironment and cancer cellsrdquo BioMed ResearchInternational vol 2016 Article ID 1490738 pp 1ndash14 2016
[12] J Wu J Xu E A Eksioglu et al ldquoIcariside II induces apoptosisof melanoma cells through the downregulation of survivalpathwaysrdquoNutrition and Cancer vol 65 no 1 pp 110ndash117 2013
[13] L Kong J Liu J Wang et al ldquoIcariin inhibits TNF-120572IFN-120574induced inflammatory response via inhibition of the substanceP and p38-MAPK signaling pathway in human keratinocytesrdquoInternational Immunopharmacology vol 29 no 2 pp 401ndash4072015
[14] JWu J Du C Xu et al ldquoIn vivo and in vitro anti-inflammatoryeffects of a novel derivative of icariinrdquo Immunopharmacologyand Immunotoxicology vol 33 no 1 pp 49ndash54 2010
[15] E M Conway L A Pikor S H Y Kung et al ldquoMacrophagesinflammation and lung cancerrdquoAmerican Journal of Respiratoryand Critical Care Medicine vol 193 no 2 pp 116ndash130 2016
[16] S Shalapour and M Karin ldquoImmunity inflammation andcancer an eternal fight between good and evilrdquo The Journal ofClinical Investigation vol 125 no 9 pp 3347ndash3355 2015
[17] L Bingle N J Brown and C E Lewis ldquoThe role of tumour-associated macrophages in tumour progression Implicationsfor new anticancer therapiesrdquo Journal of Pathology vol 196 no3 pp 254ndash265 2002
[18] D Kim J Kim J H Yoon et al ldquoCXCL12 secreted from adiposetissue recruits macrophages and induces insulin resistance inmicerdquo Diabetologia vol 57 no 7 pp 1456ndash1465 2014
[19] M C Schmid C J Avraamides P Foubert et al ldquoCombinedblockade of integrin-12057241205731 plus cytokines SDF-1120572 or IL-1120573potently inhibits tumor inflammation and growthrdquo CancerResearch vol 71 no 22 pp 6965ndash6975 2011
[20] F Balkwill and A Mantovani ldquoInflammation and cancer backto VirchowrdquoThe Lancet vol 357 no 9255 pp 539ndash545 2001
[21] B B Aggarwal S Shishodia S K Sandur M K Pandey andG Sethi ldquoInflammation and cancer how hot is the linkrdquoBiochemical Pharmacology vol 72 no 11 pp 1605ndash1621 2006
[22] W-L Jiang X-G Chen H-B Zhu Y-B Gao J-W Tian andF-H Fu ldquoPaeoniflorin inhibits systemic inflammation andimproves survival in experimental sepsisrdquo Basic and ClinicalPharmacology and Toxicology vol 105 no 1 pp 64ndash71 2009
[23] I D Kim and B J Ha ldquoThe effects of paeoniflorin on LPS-induced liver inflammatory reactionsrdquo Archives of PharmacalResearch vol 33 no 6 pp 959ndash966 2010
[24] G-W Fan Y Zhang X Jiang et al ldquoAnti-inflammatory activityof baicalein in LPS-stimulated RAW2647 macrophages viaestrogen receptor and NF-120581B-dependent pathwaysrdquo Inflamma-tion vol 36 no 6 pp 1584ndash1591 2013
12 Evidence-Based Complementary and Alternative Medicine
[25] M Li-Weber ldquoNew therapeutic aspects of flavones the anti-cancer properties of Scutellaria and its main active constituentsWogonin Baicalein and Baicalinrdquo Cancer Treatment Reviewsvol 35 no 1 pp 57ndash68 2009
[26] QWu G-L Chen Y-J Li Y Chen and F-Z Lin ldquoPaeoniflorininhibits macrophage-mediated lung cancer metastasisrdquoChineseJournal of Natural Medicines vol 13 no 12 pp 925ndash932 2015
[27] W Lin and M Karin ldquoA cytokine-mediated link betweeninnate immunity inflammation and cancerrdquo Journal of ClinicalInvestigation vol 117 no 5 pp 1175ndash1183 2007
[28] T L Whiteside ldquoThe tumor microenvironment and its role inpromoting tumor growthrdquo Oncogene vol 27 no 45 pp 5904ndash5912 2008
[29] G Lorusso and C Ruegg ldquoThe tumor microenvironment andits contribution to tumor evolution toward metastasisrdquo Histo-chemistry and Cell Biology vol 130 no 6 pp 1091ndash1103 2008
[30] L Ding G Liang Z Yao et al ldquoMetformin prevents cancermetastasis by inhibiting M2-like polarization of tumor associ-ated macrophagesrdquo Oncotarget vol 6 no 34 pp 36441ndash364552015
[31] A Yuan Y JHsiaoH Y Chen et al ldquoOpposite effects ofM1 andM2macrophage subtypes on lung cancer progressionrdquo ScientificReports vol 5 Article ID 14273 2015
[32] F Balkwill ldquoCancer and the chemokine networkrdquo NatureReviews Cancer vol 4 no 7 pp 540ndash550 2004
[33] J A Burger and T J Kipps ldquoCXCR4 a key receptor in thecrosstalk between tumor cells and their microenvironmentrdquoBlood vol 107 no 5 pp 1761ndash1767 2006
[34] R J PhillipsM D BurdickM Lutz J A BelperioM P Keaneand R M Strieter ldquoThe stromal derived factor-1CXCL12-CXCchemokine receptor 4 biological axis in non-small cell lungcancermetastasesrdquoAmerican Journal of Respiratory and CriticalCare Medicine vol 167 no 12 pp 1676ndash1686 2003
[35] N Obermajer R Muthuswamy K Odunsi R P Edwards andP Kalinski ldquoPGE-induced CXCL 12 production and CXCR4expression controls the accumulation of human MDSCs inovarian cancer environmentrdquo Cancer Research vol 71 no 24pp 7463ndash7470 2011
[36] L Sanchez-Martın A Estecha R Samaniego S Sanchez-Ramon M A Vega and P Sanchez-Mateos ldquoThe chemokineCXCL12 regulates monocyte-macrophage differentiation andRUNX3 expressionrdquo Blood vol 117 no 1 pp 88ndash97 2011
[37] K Beider H Bitner M Leiba et al ldquoMultiple myelomacells recruit tumor-supportive macrophages through theCXCR4CXCL12 axis and promote their polarization towardthe M2 phenotyperdquo Oncotarget vol 5 no 22 pp 11283ndash112962014
[38] J M Mota C A Leite and L E Souza ldquoPost-sepsis stateinduces tumor-associated macrophage accumulation throughCXCR4CXCL12 and favors tumor progression inmicerdquoCancerImmunology Research vol 4 no 4 pp 312ndash322 2016
[39] B A Teicher and S P Fricker ldquoCXCL12 (SDF-1)CXCR4 path-way in cancerrdquoClinical Cancer Research vol 16 no 11 pp 2927ndash2931 2010
[40] P C Heinrich I Behrmann G Muller-Newen F Schaper andL Graeve ldquoInterleukin-6-type cytokine signalling through thegp130JakSTAT pathwayrdquo Biochemical Journal vol 334 part 2pp 297ndash314 1998
[41] M Kortylewski M Kujawski T Wang et al ldquoInhibiting Stat3signaling in the hematopoietic system elicits multicomponentantitumor immunityrdquo Nature Medicine vol 11 no 12 pp 1314ndash1321 2005
[42] X Hu J Chen L Wang and L B Ivashkiv ldquoCrosstalk amongJak-STAT Toll-like receptor and ITAM-dependent pathways inmacrophage activationrdquo Journal of Leukocyte Biology vol 82no 2 pp 237ndash243 2007
[43] R Catlett-Falcone T H Landowski M M Oshiro et alldquoConstitutive activation of Stat3 signaling confers resistance toapoptosis in humanU266myeloma cellsrdquo Immunity vol 10 no1 pp 105ndash115 1999
Submit your manuscripts athttpswwwhindawicom
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
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Computational and Mathematical Methods in Medicine
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Research and TreatmentAIDS
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Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
4 Evidence-Based Complementary and Alternative Medicine
M0 M2 M0M2 merged
NS
QRH
X
Figure 3 Immunofluorescence staining for CD11b and CD206 of tumor tissues in subcutaneous tumor Mice with subcutaneous tumor weredealt with NS and QRHX Images are at magnification of 200x The expression of M0 and TAMs (M2-like macrophage) was respectivelyanalyzed using an Alexa Fluor-549- and Alexa Fluor-488-conjugated secondary antibody
Olympus Cx31 microscope with Image-Pro Plus medicalimage analysis systemThedigital imageswere captured usinga digital camera (Canon A640) The positive area and OD ofCD206 and VEGF positive cells were evaluated by Image-J software and determined by measuring three randomlyselected microscopic fields for each slide The IHC index wasdefined as average integral optical density (AIOD) (AIOD =positive area times ODtotal area)
CD31 a marker factor in vascular endothelial cell waspositively correlated with microvascular density (MVD)Then we counted the MVD by detecting the expression ofCD31 antibody in tumor tissues by immunohistochemistrymethod Firstly at low power field (times40) three most intensetissue sections were selected each slice and then at high powerfield (times100) MVD counts of these areas were evaluatedFinally the mean microvessel counts of the three mostvascular areas were regarded as MVD
212 Statistical Analyses Data were from three independentexperiments and expressed as mean plusmn SEM Statistical anal-yses were performed by the one-way analysis of variance(ANOVA) for differences among different groups Aboutcomparison of two groups Studentrsquos 119905-test was used All
analyses were undertaken using GraphPad Prism6 119875 lt 005was considered statistically significant
3 Results
31 QRHX Inhibits Tumor Growth in a Subcutaneous MouseModel In order to explore the role of QRHX in tumorgrowth in vivo subcutaneous mouse model was establishedby subcutaneous injection of LLC in left extremity auxiliaryMice were sacrificed at the end of treatment As shown inFigure 1 the tumor volumes were performed on days 1013 17 20 and 24 In NS group the tumor volume grad-ually increased in a time-dependent manner (Figure 1(a))However treatment with QRHX significantly suppressed thetumor volume (Figure 1(a)) The final tumor weight on day24 after the start of treatment showed a significant decrease intheQRHX group comparedwithNS control (Figure 1(b)119875 lt001) These data suggested that QRHX could dramaticallyinhibit tumor growth in vivo
32 QRHX Suppresses Cancer-Related Inflammation in LungCancer Numerous studies have indicated that cancer-relatedinflammation promotes the development of tumor [15 16]
Evidence-Based Complementary and Alternative Medicine 5
DAPI CD206 Merge
NS
QRH
X
(a)
C
D20
6 po
sitiv
e cel
ls
0
10
20
30
40
50
lowastlowast
NS QRHX
(b)
Figure 4 Immunofluorescence staining for CD206 of tumor tissues in subcutaneous tumor Mice with subcutaneous tumor were dealt with thegroup of NS and QRHX Images are at magnification of 200x The expression of TAMs was respectively analyzed using an Alexa Fluor-488-conjugated secondary antibody The nuclei were stained with DAPI Compared with NS group lowastlowast119875 lt 001
In addition some proinflammatory cytokines such as IL-8IL-6 and TNF-a have been shown to ultimately facilitate cellinvasion and metastasis [15] Therefore we used an ELISAassay to examine serum levels of the two proinflammatorycytokines IL-6 and TNF-120572 in peripheral blood Serum IL-6 and TNF-120572 levels were lower in mice treated with QRHXcompared with those receiving NS treatment (Figure 2 both
119875 lt 001) These data demonstrated that QRHX decreasedthe production of proinflammatory cytokines
33 QRHX Reduces the Accumulation of TAMs in Lung Can-cer In order to ascertain the effects of the QRHX on TAMsM2-like macrophage phenotype we used flow cytometryIF and IHC assay to examine CD206 expression As shown
6 Evidence-Based Complementary and Alternative Medicine
NS
100 휇m
(a)
QRH
X
100 휇m
(b)
C
D20
6 po
sitiv
e cel
ls
0
20
40
60
80
100
NS QRHX
lowastlowast
(c)
Figure 5 Comparison of CD206+ macrophages infiltration between NS and QRHX groups Immunohistochemical staining of CD206+macrophages in lung cancer tissues Images are at magnification of 200x Data represent mean plusmn SEM 119899 = 4 Compared with NS grouplowastlowast119875 lt 001
in Figures 3ndash6 our results revealed that CD206 expressionwas dramatically expressed in tumor tissue Compared to NSgroup oral administration with QRHX led to a prominentinhibition in TAMs (Figures 3ndash6 119875 lt 001) These datademonstrated that QRHX had a more significant role inimpeding accumulating and reducing the number of TAMsin lung cancer
34 QRHX Alters Hallmarkers of M1 and M2 Macrophagein Lung Cancer To further confirm the role of QRHX inTAMs we analyzed the level of Arg-1 protein M2 markerand the mRNA expression of M1 marker iNOS After QRHXtreatment Arg-1 level decreased (Figure 7(a) 119875 lt 001) andiNOS level increased (Figure 7(b) 119875 lt 005) Accordinglythese results strongly supported the inhibitory effect ofQRHX on suppressing TAMs infiltration and regulating M2-like macrophage polarization
35 QRHX Inhibits Angiogenesis in Lung Cancer CD31endothelial cell surface antigen is a vascular endothelialmarker for MVD and TAMs have a positive effect on bloodvessels by inducing the production of various proangiogenic
genes [17] Therefore CD31 was evaluated in implantedtumors using IHC and WB MVD stained by anti-CD31 wasmeasured by counting tissue sections of central areas of thetumor As shown in Figure 8 QRHX treatment induced aremarkable reduction in MVD and CD31 protein (119875 lt 001)Furthermore IHC and WB demonstrated reduced vascularendothelial growth factor (VEGF) expression in the QRHXtreated groups relative to NS controls (Figure 8 119875 lt 001)Taken together these data clearly showed thatQRHXreducedangiogenesis in lung cancer
36 QRHX Blocks CXCL 12CXCR4JNK2STAT3 SignalingPathways To investigate the molecular mechanism underly-ing the formulae decreasing M2 macrophages we tested theeffects of inhibitors of signaling molecules CXCL12CXCR4and JNK2STAT3 are known to be important moleculesinvolved in M2 polarization [18 19] Compared to NS grouporal administration of QRHX dramatically attenuated theincreased expressions of CXCL12 and CXCR4 in tumortissue (Figure 9(a) 119875 lt 001) As shown in Figure 9(b)JAK2 and STAT3 activation was significantly suppressed byQRHX treatment (both 119875 lt 001) compared to NS group
Evidence-Based Complementary and Alternative Medicine 7
106
105
104
103
0
Com
p-YL
1-H
F4
80-R
-PE-
H
1061051041030
Comp-BL1-H CD11b-FITC-H
197
1M
800 K
600 K
400 K
200 K
0
SSC-
H
SSC-
H
Comp-RL1-H CD206-Alexa Flour 647-H1061051041030
106
105
104
103
0
Com
p-YL
1-H
F4
80-R
-PE-
H
Comp-BL1-H CD11b-FITC-H1061051041030
900
1M
800 K
600 K
400 K
200 K
0
SSC-
H
SSC-
H
Comp-RL1-H CD206-Alexa Flour 647-H1061051041030
(a)
0
10
20
30
NS
F480+CD11b+ F480+CD11b+CD206+
QRHX
p
ositi
ve ce
lls
lowastlowast
lowastlowast
(b)
Figure 6 QRHX decreases the number of TAMs in subcutaneous tumor Mice with subcutaneous tumors were dealt with NS and QRHXMacrophages and TAMs (M2-like subtype) in tumor tissue were measured by flow cytometry Data represent mean plusmn SEM 119899 = 4 Comparedwith NS group lowastlowast119875 lt 001
8 Evidence-Based Complementary and Alternative Medicine
훽-actin43 kDa
Arg-140 kDa
00
02
04
06
08
10
Arg-1
Relat
ive p
rote
in ex
pres
sion
lowastlowast
NSQRHX
(a)
NSQRHX
iNOS0
4
1
2
3
Relat
ive p
rote
in ex
pres
sion
lowast
(b)
Figure 7The expression of M2-related marker (Arg-1) and M1-related marker (iNOS) was detected by western blot and QT-PCR respectivelyMice with subcutaneous tumor were dealt with NS and QRHX Data were presented as means plusmn SEM (119899 = 3) Compared with NS grouplowast119875 lt 005 lowastlowast119875 lt 001
Taking together the data suggested that QRHX inhibitedtumor cell-TAMs interactions possibly through blockingCXCL12CXCR4JAK2STAT3 signaling pathways and thenregulated macrophages polarization
4 Discussion
In the present study our data demonstrated that QRHXplayed a more crucial role in inhibiting tumor growth bymodulating tumor microenvironment especially TAMsQRHX inhibited tumor cell-TAMs interactions via thesuppression of cancer-related inflammation and probablyblocking the response of macrophages to tumor signalsCXCL12CXCR4JAK2STAT3 axis
In the nineteenth century the association between cancerand inflammation was firstly put forward [20] Large numberof studies provided powerful evidence that chronic inflam-mation can promote tumor development progression andmetastasis as well as chemoresistance [16 21] Recent studieshave confirmed that paeoniflorin baicalein and wogoninimportant ingredients of QRHX had the potential to inhibitmany types of inflammation [22ndash24] Furthermore baicaleinand wogonin exerted obvious inhibitory effects on cancer aswell as macrophages and angiogenesis [24 25] Paeoniflorinone of major ingredients could reduce lung metastasis ofLLC through inhibiting the M2 activation [26] In additionthe cytokines produced by activated innate immune cells intumor microenvironment can stimulate tumorigenesis suchas IL-6 and TNF-120572 [27] Our results showed a remarkabledecrease in multiple proinflammatory cytokines such as
TNF-a and IL-6 both in serum and tumor tissue of subcu-taneous mouse model (Figure 2) suggesting the inhibitoryeffect of QRHX on cancer-related inflammation
Tumor microenvironment created by the tumor andmainly orchestrated by inflammatory cells contributes totumor escape growth progression and evolution towardmetastasis [28 29] Numerous studies in recent decadehave presented evidences that TCM have a good effect onregulating tumor microenvironment such as reversing theimmunosuppressive microenvironment [11] Macrophages abasic component of the innate immune system are infiltratedin virtually all malignancies TAMs M2-like polarized stylehave been regarded as a protumor inflammatory microen-vironment which links inflammation and cancer [15] Col-lective evidences demonstrate that TAMs have the abilityof enhancing tumor angiogenesis increasing migration andinvasion and suppressing the antitumor immune responsesIt is correlated with the prognosis of patients with malignanttumor such as lung cancer [30 31] Consistent with previousstudies in this study we detected that the fraction of TAMswas increased in tumor and QRHX inhibited tumor growthin the tumor mice model through decreasing accumulatingof TAMs and activation of M2
CXCR4 which is widely expressed on malignant cellsand binds to CXCL12 [32] plays an important role in hem-atopoiesis development and organization of the immunesystem by directly and indirectly mechanisms [33] Forexample in NSCLC CXCL12-CXCR4 axis is involved inmetastasis and associated with an unfavorable prognosis[34] in ovarian cancer it can control accumulation of
Evidence-Based Complementary and Alternative Medicine 9CD
31
NS QRHX
VEG
F
0
5
10
15
20
25
VEGFMVD
Den
sity
of V
EGF
MV
D co
unt
lowastlowast
lowastlowast
(a)
00
02
04
06
08
10
Relat
ive p
rote
in ex
pres
sion
CD31VEGF
NSQRHX
lowastlowastlowastlowast
VEGF
CD31
훽-actin43 kDa
100 kDa
43 kDa
(b)
Figure 8 QRHX inhibits angiogenesis in lung cancer Mice with subcutaneous tumors were dealt with NS and QRHX (a) The nucleus wasdyed as blue CD31 was dyed as brown MVD were performed at high power field (times200) (b) The VEGF and CD31 protein expression intumor tissues was detected by western blot Data was expressed as means plusmn SEM values (119899 = 4) Compared with NS group lowastlowast119875 lt 001
human MDSCs and is an independent prognostic factor fortumor progression [35] Several studies have reported thatCXCL12 plays an important role in monocyte recruitmentdifferentiation and function [18 36] In a mouse modelof lung cancer CXCL12 could recruit tumor-promotingmyeloid CD11b+ cells [19] Besides powerful evidencesindicate a role for CXCR4-CXCL12 axis in promotingmacrophages polarization toward theM2 phenotype [37 38]In addition M2 subpopulation is associated with angio-genic factors such as VEGF and CXCL12-CXCR4 axis canalso promote tumor vascularization [39] Excitedly ourresults showed QRHX treatment inhibited signaling fromtumor cells to macrophages through inducing a remark-able decrease in CXCL12 and CXCR4 Furthermore QRHX
inhibited angiogenesis likely through altering the tumormicroenvironment by targeting TAMs
In STAT family there are seven proteins STAT3 is one ofthem It is a key transcription factor transducing signals fromactivated receptors or intracellular kinases to the nucleus andcan be activated in tumor cells and immune cells [40] Intumor STAT3 could contribute to cancer development andprogression inhibit apoptosis of tumors and help tumorescape immune system by suppressing the immune response[41] Evidence indicated that when STAT3 binds to somereceptor it can be activated through Janus Kinases (JAKs)such as JAK2 [42] Accumulating evidence implicates theimportant role of JAK2STAT3 in tumor and macrophagepolarization [27] For instance IL-6 can contribute to tumor
10 Evidence-Based Complementary and Alternative Medicine
CXCR4
CXCL1210 kDa
훽-actin43 kDa
41 kDa
NSQRHX
00
05
10
15
Relat
ive p
rote
in ex
pres
sion
CXCL12 CXCR4
lowastlowastlowastlowast
(a)
p-JAK2
p-STAT3
훽-actin43 kDa
90 kDa
120 kDa
NSQRHX
p-JAK2 p-STAT300
05
10
15
Relat
ive p
rote
in ex
pres
sion
lowastlowast lowastlowast
(b)
Figure 9 QRXH regulates TAMs by inhibiting the CXCL12CXCR4JAK2STAT3 signaling pathways Mice with subcutaneous tumor weredealt with NS and QRHX Representative images of western blot and densitometry analysis showing the expressions of CXCL12 CXCR4p-JAK2 and p-STAT3 in tumor Compared with NS group lowastlowast119875 lt 001
cell survival and upregulate the antiapoptotic genes by driv-ing JAK2STAT3 signal [43] IFN- activates macrophageby JAK-STAT signaling pathway [42] What is more JAK2is associated with CXCR4 [39] Then we can draw thatTAMs in malignant tumors tend to M2 subtype possiblythrough CXCL12CXCR4JAK2STAT3 signaling pathwayQRHX treatment blocked the response of macrophages totumor signals by suppressing CXCL12CXCR4JAK2STAT3expression and induced a remarkable decrease of the recruit-ment of M2 macrophages suggesting the attenuation of M2subtype cells function by QRHX
In TCM theory TCM formulae have abundant medicinalmaterials and then regulate diseases through multitargetsandmultiways Although the chemical constituents of QRHXhave been separated and identified by HPLUNG CANCER-QTOF MS-UV method in our team (Supplementary Fig-ure 1 and Table 1) the ingredients are various and theirfunctions are enormous So there may be other mechanismsinvolved in macrophage polarization What is more there
are diverse types cells in tumor except for tumor cells andmacrophage Moreover it is worth mentioning that TAMswere not separated and extracted from tumor tissue CXCL12CXCR4 JAK2 and STAT3 can not only play significantrole in tumor cell or macrophage but also other cellssuch as T lymphocyte neutrophils tumor-associated fibrob-last and endothelial cells Therefore QRHX inhibited theCXCL12CXCR4JAK2STAT3 axis possibly through othercells Taken together further investigations are needed toidentify direct molecular targets of QRHX in macrophagesin the context of cancer
5 Conclusion
In conclusion data from this study revealed that QRHXcould suppress cancer progression by inhibiting the tumorpromotion of TAMs in subcutaneous mice model whichcould contribute to elucidating the underlying regulatorymode of QRHX on lung cancer treatment
Evidence-Based Complementary and Alternative Medicine 11
Abbreviations
ELISA Enzyme-linked immunosorbent assayFITC Fluorescein isothiocyanateTNF-120572 Tumor necrosis factor-120572IL-6 Interleukin-6iNOS Inducible nitric oxide synthaseVEGF Vascular endothelial growth factorQT-PCR Quantitative Polymerase Chain ReactionIF ImmunofluorescenceIHC ImmunohistochemicalTAMs Tumor-associated macrophagesArg-1 Arginase 1
Conflicts of Interest
The authors declare that there are no conflicts of interest
Authorsrsquo Contributions
Fei Xu and Wenqiang Cui contributed equally to this workFei Xu and Baojun Liu conceived and designed experimentsFei Xu and Wenqiang Cui analyzed data and wrote themanuscript Jingcheng Dong supervised the project Zhengx-iao Zhao and Ying Wei assisted in experimental design anddata evaluation Jiaqi Liu Mihui Li Qiuping Li Chen Yanand Jian Qiu performed the study All authors reviewed andapproved the manuscript
Acknowledgments
This study was supported by Natural Science Foundation ofChina (nos 81673916 and 81403148) Development Projectof Shanghai Peak Disciplines-Integrative Medicine (no20150407)
References
[1] I Ikonomidis C A Michalakeas J Parissis et al ldquoInflamma-tory markers in coronary artery diseaserdquo BioFactors vol 38 no5 pp 320ndash328 2012
[2] P Lee C C Leung M I Restrepo K Takahashi Y Song and JM Porcel ldquoYear in review 2015 lung cancer pleural diseasesrespiratory infections bronchiectasis and tuberculosis bron-choscopic intervention and imagingrdquo Respirology vol 21 no 5pp 961ndash967 2016
[3] S V Sharma DW Bell J Settleman and D A Haber ldquoEpider-mal growth factor receptor mutations in lung cancerrdquo NatureReviews Cancer vol 7 no 3 pp 169ndash181 2007
[4] D Leong R Rai B Nguyen A Lee and D Yip ldquoAdvancesin adjuvant systemic therapy for non-small-cell lung cancerrdquoWorld Journal of Clinical Oncology vol 5 no 4 pp 633ndash6452014
[5] M T Villanueva ldquoMicroenvironment the new midfielders inthe tumourmicroenvironmentrdquoNature reviews Cancer vol 14no 12 p 765 2014
[6] A Mantovani S Sozzani M Locati P Allavena and A SicaldquoMacrophage polarization tumor-associated macrophages as aparadigm for polarizedM2mononuclear phagocytesrdquoTrends inImmunology vol 23 no 11 pp 549ndash555 2002
[7] A Mantovani and M Locati ldquoTumor-associated macrophagesas a paradigm of macrophage plasticity diversity and polariza-tion lessons and open questionsrdquo Arteriosclerosis Thrombosisand Vascular Biology vol 33 no 7 pp 1478ndash1483 2013
[8] W Hu X Li C Zhang Y Yang J Jiang and C Wu ldquoTumor-associated macrophages in cancersrdquo Clinical and TranslationalOncology vol 18 no 3 pp 251ndash258 2016
[9] B Z Qian and J W Pollard ldquoMacrophage diversity enhancestumor progression and metastasisrdquo Cell vol 141 no 1 pp 39ndash51 2010
[10] A Mantovani and P Allavena ldquoThe interaction of anti-cancer therapies with tumor-associated macrophagesrdquo Journalof Experimental Medicine vol 212 no 4 pp 435ndash445 2015
[11] J Xu Z Song Q Guo and J Li ldquoSynergistic effect and molec-ular mechanisms of traditional Chinese medicine on regulatingtumor microenvironment and cancer cellsrdquo BioMed ResearchInternational vol 2016 Article ID 1490738 pp 1ndash14 2016
[12] J Wu J Xu E A Eksioglu et al ldquoIcariside II induces apoptosisof melanoma cells through the downregulation of survivalpathwaysrdquoNutrition and Cancer vol 65 no 1 pp 110ndash117 2013
[13] L Kong J Liu J Wang et al ldquoIcariin inhibits TNF-120572IFN-120574induced inflammatory response via inhibition of the substanceP and p38-MAPK signaling pathway in human keratinocytesrdquoInternational Immunopharmacology vol 29 no 2 pp 401ndash4072015
[14] JWu J Du C Xu et al ldquoIn vivo and in vitro anti-inflammatoryeffects of a novel derivative of icariinrdquo Immunopharmacologyand Immunotoxicology vol 33 no 1 pp 49ndash54 2010
[15] E M Conway L A Pikor S H Y Kung et al ldquoMacrophagesinflammation and lung cancerrdquoAmerican Journal of Respiratoryand Critical Care Medicine vol 193 no 2 pp 116ndash130 2016
[16] S Shalapour and M Karin ldquoImmunity inflammation andcancer an eternal fight between good and evilrdquo The Journal ofClinical Investigation vol 125 no 9 pp 3347ndash3355 2015
[17] L Bingle N J Brown and C E Lewis ldquoThe role of tumour-associated macrophages in tumour progression Implicationsfor new anticancer therapiesrdquo Journal of Pathology vol 196 no3 pp 254ndash265 2002
[18] D Kim J Kim J H Yoon et al ldquoCXCL12 secreted from adiposetissue recruits macrophages and induces insulin resistance inmicerdquo Diabetologia vol 57 no 7 pp 1456ndash1465 2014
[19] M C Schmid C J Avraamides P Foubert et al ldquoCombinedblockade of integrin-12057241205731 plus cytokines SDF-1120572 or IL-1120573potently inhibits tumor inflammation and growthrdquo CancerResearch vol 71 no 22 pp 6965ndash6975 2011
[20] F Balkwill and A Mantovani ldquoInflammation and cancer backto VirchowrdquoThe Lancet vol 357 no 9255 pp 539ndash545 2001
[21] B B Aggarwal S Shishodia S K Sandur M K Pandey andG Sethi ldquoInflammation and cancer how hot is the linkrdquoBiochemical Pharmacology vol 72 no 11 pp 1605ndash1621 2006
[22] W-L Jiang X-G Chen H-B Zhu Y-B Gao J-W Tian andF-H Fu ldquoPaeoniflorin inhibits systemic inflammation andimproves survival in experimental sepsisrdquo Basic and ClinicalPharmacology and Toxicology vol 105 no 1 pp 64ndash71 2009
[23] I D Kim and B J Ha ldquoThe effects of paeoniflorin on LPS-induced liver inflammatory reactionsrdquo Archives of PharmacalResearch vol 33 no 6 pp 959ndash966 2010
[24] G-W Fan Y Zhang X Jiang et al ldquoAnti-inflammatory activityof baicalein in LPS-stimulated RAW2647 macrophages viaestrogen receptor and NF-120581B-dependent pathwaysrdquo Inflamma-tion vol 36 no 6 pp 1584ndash1591 2013
12 Evidence-Based Complementary and Alternative Medicine
[25] M Li-Weber ldquoNew therapeutic aspects of flavones the anti-cancer properties of Scutellaria and its main active constituentsWogonin Baicalein and Baicalinrdquo Cancer Treatment Reviewsvol 35 no 1 pp 57ndash68 2009
[26] QWu G-L Chen Y-J Li Y Chen and F-Z Lin ldquoPaeoniflorininhibits macrophage-mediated lung cancer metastasisrdquoChineseJournal of Natural Medicines vol 13 no 12 pp 925ndash932 2015
[27] W Lin and M Karin ldquoA cytokine-mediated link betweeninnate immunity inflammation and cancerrdquo Journal of ClinicalInvestigation vol 117 no 5 pp 1175ndash1183 2007
[28] T L Whiteside ldquoThe tumor microenvironment and its role inpromoting tumor growthrdquo Oncogene vol 27 no 45 pp 5904ndash5912 2008
[29] G Lorusso and C Ruegg ldquoThe tumor microenvironment andits contribution to tumor evolution toward metastasisrdquo Histo-chemistry and Cell Biology vol 130 no 6 pp 1091ndash1103 2008
[30] L Ding G Liang Z Yao et al ldquoMetformin prevents cancermetastasis by inhibiting M2-like polarization of tumor associ-ated macrophagesrdquo Oncotarget vol 6 no 34 pp 36441ndash364552015
[31] A Yuan Y JHsiaoH Y Chen et al ldquoOpposite effects ofM1 andM2macrophage subtypes on lung cancer progressionrdquo ScientificReports vol 5 Article ID 14273 2015
[32] F Balkwill ldquoCancer and the chemokine networkrdquo NatureReviews Cancer vol 4 no 7 pp 540ndash550 2004
[33] J A Burger and T J Kipps ldquoCXCR4 a key receptor in thecrosstalk between tumor cells and their microenvironmentrdquoBlood vol 107 no 5 pp 1761ndash1767 2006
[34] R J PhillipsM D BurdickM Lutz J A BelperioM P Keaneand R M Strieter ldquoThe stromal derived factor-1CXCL12-CXCchemokine receptor 4 biological axis in non-small cell lungcancermetastasesrdquoAmerican Journal of Respiratory and CriticalCare Medicine vol 167 no 12 pp 1676ndash1686 2003
[35] N Obermajer R Muthuswamy K Odunsi R P Edwards andP Kalinski ldquoPGE-induced CXCL 12 production and CXCR4expression controls the accumulation of human MDSCs inovarian cancer environmentrdquo Cancer Research vol 71 no 24pp 7463ndash7470 2011
[36] L Sanchez-Martın A Estecha R Samaniego S Sanchez-Ramon M A Vega and P Sanchez-Mateos ldquoThe chemokineCXCL12 regulates monocyte-macrophage differentiation andRUNX3 expressionrdquo Blood vol 117 no 1 pp 88ndash97 2011
[37] K Beider H Bitner M Leiba et al ldquoMultiple myelomacells recruit tumor-supportive macrophages through theCXCR4CXCL12 axis and promote their polarization towardthe M2 phenotyperdquo Oncotarget vol 5 no 22 pp 11283ndash112962014
[38] J M Mota C A Leite and L E Souza ldquoPost-sepsis stateinduces tumor-associated macrophage accumulation throughCXCR4CXCL12 and favors tumor progression inmicerdquoCancerImmunology Research vol 4 no 4 pp 312ndash322 2016
[39] B A Teicher and S P Fricker ldquoCXCL12 (SDF-1)CXCR4 path-way in cancerrdquoClinical Cancer Research vol 16 no 11 pp 2927ndash2931 2010
[40] P C Heinrich I Behrmann G Muller-Newen F Schaper andL Graeve ldquoInterleukin-6-type cytokine signalling through thegp130JakSTAT pathwayrdquo Biochemical Journal vol 334 part 2pp 297ndash314 1998
[41] M Kortylewski M Kujawski T Wang et al ldquoInhibiting Stat3signaling in the hematopoietic system elicits multicomponentantitumor immunityrdquo Nature Medicine vol 11 no 12 pp 1314ndash1321 2005
[42] X Hu J Chen L Wang and L B Ivashkiv ldquoCrosstalk amongJak-STAT Toll-like receptor and ITAM-dependent pathways inmacrophage activationrdquo Journal of Leukocyte Biology vol 82no 2 pp 237ndash243 2007
[43] R Catlett-Falcone T H Landowski M M Oshiro et alldquoConstitutive activation of Stat3 signaling confers resistance toapoptosis in humanU266myeloma cellsrdquo Immunity vol 10 no1 pp 105ndash115 1999
Submit your manuscripts athttpswwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
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Diabetes ResearchJournal of
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 5
DAPI CD206 Merge
NS
QRH
X
(a)
C
D20
6 po
sitiv
e cel
ls
0
10
20
30
40
50
lowastlowast
NS QRHX
(b)
Figure 4 Immunofluorescence staining for CD206 of tumor tissues in subcutaneous tumor Mice with subcutaneous tumor were dealt with thegroup of NS and QRHX Images are at magnification of 200x The expression of TAMs was respectively analyzed using an Alexa Fluor-488-conjugated secondary antibody The nuclei were stained with DAPI Compared with NS group lowastlowast119875 lt 001
In addition some proinflammatory cytokines such as IL-8IL-6 and TNF-a have been shown to ultimately facilitate cellinvasion and metastasis [15] Therefore we used an ELISAassay to examine serum levels of the two proinflammatorycytokines IL-6 and TNF-120572 in peripheral blood Serum IL-6 and TNF-120572 levels were lower in mice treated with QRHXcompared with those receiving NS treatment (Figure 2 both
119875 lt 001) These data demonstrated that QRHX decreasedthe production of proinflammatory cytokines
33 QRHX Reduces the Accumulation of TAMs in Lung Can-cer In order to ascertain the effects of the QRHX on TAMsM2-like macrophage phenotype we used flow cytometryIF and IHC assay to examine CD206 expression As shown
6 Evidence-Based Complementary and Alternative Medicine
NS
100 휇m
(a)
QRH
X
100 휇m
(b)
C
D20
6 po
sitiv
e cel
ls
0
20
40
60
80
100
NS QRHX
lowastlowast
(c)
Figure 5 Comparison of CD206+ macrophages infiltration between NS and QRHX groups Immunohistochemical staining of CD206+macrophages in lung cancer tissues Images are at magnification of 200x Data represent mean plusmn SEM 119899 = 4 Compared with NS grouplowastlowast119875 lt 001
in Figures 3ndash6 our results revealed that CD206 expressionwas dramatically expressed in tumor tissue Compared to NSgroup oral administration with QRHX led to a prominentinhibition in TAMs (Figures 3ndash6 119875 lt 001) These datademonstrated that QRHX had a more significant role inimpeding accumulating and reducing the number of TAMsin lung cancer
34 QRHX Alters Hallmarkers of M1 and M2 Macrophagein Lung Cancer To further confirm the role of QRHX inTAMs we analyzed the level of Arg-1 protein M2 markerand the mRNA expression of M1 marker iNOS After QRHXtreatment Arg-1 level decreased (Figure 7(a) 119875 lt 001) andiNOS level increased (Figure 7(b) 119875 lt 005) Accordinglythese results strongly supported the inhibitory effect ofQRHX on suppressing TAMs infiltration and regulating M2-like macrophage polarization
35 QRHX Inhibits Angiogenesis in Lung Cancer CD31endothelial cell surface antigen is a vascular endothelialmarker for MVD and TAMs have a positive effect on bloodvessels by inducing the production of various proangiogenic
genes [17] Therefore CD31 was evaluated in implantedtumors using IHC and WB MVD stained by anti-CD31 wasmeasured by counting tissue sections of central areas of thetumor As shown in Figure 8 QRHX treatment induced aremarkable reduction in MVD and CD31 protein (119875 lt 001)Furthermore IHC and WB demonstrated reduced vascularendothelial growth factor (VEGF) expression in the QRHXtreated groups relative to NS controls (Figure 8 119875 lt 001)Taken together these data clearly showed thatQRHXreducedangiogenesis in lung cancer
36 QRHX Blocks CXCL 12CXCR4JNK2STAT3 SignalingPathways To investigate the molecular mechanism underly-ing the formulae decreasing M2 macrophages we tested theeffects of inhibitors of signaling molecules CXCL12CXCR4and JNK2STAT3 are known to be important moleculesinvolved in M2 polarization [18 19] Compared to NS grouporal administration of QRHX dramatically attenuated theincreased expressions of CXCL12 and CXCR4 in tumortissue (Figure 9(a) 119875 lt 001) As shown in Figure 9(b)JAK2 and STAT3 activation was significantly suppressed byQRHX treatment (both 119875 lt 001) compared to NS group
Evidence-Based Complementary and Alternative Medicine 7
106
105
104
103
0
Com
p-YL
1-H
F4
80-R
-PE-
H
1061051041030
Comp-BL1-H CD11b-FITC-H
197
1M
800 K
600 K
400 K
200 K
0
SSC-
H
SSC-
H
Comp-RL1-H CD206-Alexa Flour 647-H1061051041030
106
105
104
103
0
Com
p-YL
1-H
F4
80-R
-PE-
H
Comp-BL1-H CD11b-FITC-H1061051041030
900
1M
800 K
600 K
400 K
200 K
0
SSC-
H
SSC-
H
Comp-RL1-H CD206-Alexa Flour 647-H1061051041030
(a)
0
10
20
30
NS
F480+CD11b+ F480+CD11b+CD206+
QRHX
p
ositi
ve ce
lls
lowastlowast
lowastlowast
(b)
Figure 6 QRHX decreases the number of TAMs in subcutaneous tumor Mice with subcutaneous tumors were dealt with NS and QRHXMacrophages and TAMs (M2-like subtype) in tumor tissue were measured by flow cytometry Data represent mean plusmn SEM 119899 = 4 Comparedwith NS group lowastlowast119875 lt 001
8 Evidence-Based Complementary and Alternative Medicine
훽-actin43 kDa
Arg-140 kDa
00
02
04
06
08
10
Arg-1
Relat
ive p
rote
in ex
pres
sion
lowastlowast
NSQRHX
(a)
NSQRHX
iNOS0
4
1
2
3
Relat
ive p
rote
in ex
pres
sion
lowast
(b)
Figure 7The expression of M2-related marker (Arg-1) and M1-related marker (iNOS) was detected by western blot and QT-PCR respectivelyMice with subcutaneous tumor were dealt with NS and QRHX Data were presented as means plusmn SEM (119899 = 3) Compared with NS grouplowast119875 lt 005 lowastlowast119875 lt 001
Taking together the data suggested that QRHX inhibitedtumor cell-TAMs interactions possibly through blockingCXCL12CXCR4JAK2STAT3 signaling pathways and thenregulated macrophages polarization
4 Discussion
In the present study our data demonstrated that QRHXplayed a more crucial role in inhibiting tumor growth bymodulating tumor microenvironment especially TAMsQRHX inhibited tumor cell-TAMs interactions via thesuppression of cancer-related inflammation and probablyblocking the response of macrophages to tumor signalsCXCL12CXCR4JAK2STAT3 axis
In the nineteenth century the association between cancerand inflammation was firstly put forward [20] Large numberof studies provided powerful evidence that chronic inflam-mation can promote tumor development progression andmetastasis as well as chemoresistance [16 21] Recent studieshave confirmed that paeoniflorin baicalein and wogoninimportant ingredients of QRHX had the potential to inhibitmany types of inflammation [22ndash24] Furthermore baicaleinand wogonin exerted obvious inhibitory effects on cancer aswell as macrophages and angiogenesis [24 25] Paeoniflorinone of major ingredients could reduce lung metastasis ofLLC through inhibiting the M2 activation [26] In additionthe cytokines produced by activated innate immune cells intumor microenvironment can stimulate tumorigenesis suchas IL-6 and TNF-120572 [27] Our results showed a remarkabledecrease in multiple proinflammatory cytokines such as
TNF-a and IL-6 both in serum and tumor tissue of subcu-taneous mouse model (Figure 2) suggesting the inhibitoryeffect of QRHX on cancer-related inflammation
Tumor microenvironment created by the tumor andmainly orchestrated by inflammatory cells contributes totumor escape growth progression and evolution towardmetastasis [28 29] Numerous studies in recent decadehave presented evidences that TCM have a good effect onregulating tumor microenvironment such as reversing theimmunosuppressive microenvironment [11] Macrophages abasic component of the innate immune system are infiltratedin virtually all malignancies TAMs M2-like polarized stylehave been regarded as a protumor inflammatory microen-vironment which links inflammation and cancer [15] Col-lective evidences demonstrate that TAMs have the abilityof enhancing tumor angiogenesis increasing migration andinvasion and suppressing the antitumor immune responsesIt is correlated with the prognosis of patients with malignanttumor such as lung cancer [30 31] Consistent with previousstudies in this study we detected that the fraction of TAMswas increased in tumor and QRHX inhibited tumor growthin the tumor mice model through decreasing accumulatingof TAMs and activation of M2
CXCR4 which is widely expressed on malignant cellsand binds to CXCL12 [32] plays an important role in hem-atopoiesis development and organization of the immunesystem by directly and indirectly mechanisms [33] Forexample in NSCLC CXCL12-CXCR4 axis is involved inmetastasis and associated with an unfavorable prognosis[34] in ovarian cancer it can control accumulation of
Evidence-Based Complementary and Alternative Medicine 9CD
31
NS QRHX
VEG
F
0
5
10
15
20
25
VEGFMVD
Den
sity
of V
EGF
MV
D co
unt
lowastlowast
lowastlowast
(a)
00
02
04
06
08
10
Relat
ive p
rote
in ex
pres
sion
CD31VEGF
NSQRHX
lowastlowastlowastlowast
VEGF
CD31
훽-actin43 kDa
100 kDa
43 kDa
(b)
Figure 8 QRHX inhibits angiogenesis in lung cancer Mice with subcutaneous tumors were dealt with NS and QRHX (a) The nucleus wasdyed as blue CD31 was dyed as brown MVD were performed at high power field (times200) (b) The VEGF and CD31 protein expression intumor tissues was detected by western blot Data was expressed as means plusmn SEM values (119899 = 4) Compared with NS group lowastlowast119875 lt 001
human MDSCs and is an independent prognostic factor fortumor progression [35] Several studies have reported thatCXCL12 plays an important role in monocyte recruitmentdifferentiation and function [18 36] In a mouse modelof lung cancer CXCL12 could recruit tumor-promotingmyeloid CD11b+ cells [19] Besides powerful evidencesindicate a role for CXCR4-CXCL12 axis in promotingmacrophages polarization toward theM2 phenotype [37 38]In addition M2 subpopulation is associated with angio-genic factors such as VEGF and CXCL12-CXCR4 axis canalso promote tumor vascularization [39] Excitedly ourresults showed QRHX treatment inhibited signaling fromtumor cells to macrophages through inducing a remark-able decrease in CXCL12 and CXCR4 Furthermore QRHX
inhibited angiogenesis likely through altering the tumormicroenvironment by targeting TAMs
In STAT family there are seven proteins STAT3 is one ofthem It is a key transcription factor transducing signals fromactivated receptors or intracellular kinases to the nucleus andcan be activated in tumor cells and immune cells [40] Intumor STAT3 could contribute to cancer development andprogression inhibit apoptosis of tumors and help tumorescape immune system by suppressing the immune response[41] Evidence indicated that when STAT3 binds to somereceptor it can be activated through Janus Kinases (JAKs)such as JAK2 [42] Accumulating evidence implicates theimportant role of JAK2STAT3 in tumor and macrophagepolarization [27] For instance IL-6 can contribute to tumor
10 Evidence-Based Complementary and Alternative Medicine
CXCR4
CXCL1210 kDa
훽-actin43 kDa
41 kDa
NSQRHX
00
05
10
15
Relat
ive p
rote
in ex
pres
sion
CXCL12 CXCR4
lowastlowastlowastlowast
(a)
p-JAK2
p-STAT3
훽-actin43 kDa
90 kDa
120 kDa
NSQRHX
p-JAK2 p-STAT300
05
10
15
Relat
ive p
rote
in ex
pres
sion
lowastlowast lowastlowast
(b)
Figure 9 QRXH regulates TAMs by inhibiting the CXCL12CXCR4JAK2STAT3 signaling pathways Mice with subcutaneous tumor weredealt with NS and QRHX Representative images of western blot and densitometry analysis showing the expressions of CXCL12 CXCR4p-JAK2 and p-STAT3 in tumor Compared with NS group lowastlowast119875 lt 001
cell survival and upregulate the antiapoptotic genes by driv-ing JAK2STAT3 signal [43] IFN- activates macrophageby JAK-STAT signaling pathway [42] What is more JAK2is associated with CXCR4 [39] Then we can draw thatTAMs in malignant tumors tend to M2 subtype possiblythrough CXCL12CXCR4JAK2STAT3 signaling pathwayQRHX treatment blocked the response of macrophages totumor signals by suppressing CXCL12CXCR4JAK2STAT3expression and induced a remarkable decrease of the recruit-ment of M2 macrophages suggesting the attenuation of M2subtype cells function by QRHX
In TCM theory TCM formulae have abundant medicinalmaterials and then regulate diseases through multitargetsandmultiways Although the chemical constituents of QRHXhave been separated and identified by HPLUNG CANCER-QTOF MS-UV method in our team (Supplementary Fig-ure 1 and Table 1) the ingredients are various and theirfunctions are enormous So there may be other mechanismsinvolved in macrophage polarization What is more there
are diverse types cells in tumor except for tumor cells andmacrophage Moreover it is worth mentioning that TAMswere not separated and extracted from tumor tissue CXCL12CXCR4 JAK2 and STAT3 can not only play significantrole in tumor cell or macrophage but also other cellssuch as T lymphocyte neutrophils tumor-associated fibrob-last and endothelial cells Therefore QRHX inhibited theCXCL12CXCR4JAK2STAT3 axis possibly through othercells Taken together further investigations are needed toidentify direct molecular targets of QRHX in macrophagesin the context of cancer
5 Conclusion
In conclusion data from this study revealed that QRHXcould suppress cancer progression by inhibiting the tumorpromotion of TAMs in subcutaneous mice model whichcould contribute to elucidating the underlying regulatorymode of QRHX on lung cancer treatment
Evidence-Based Complementary and Alternative Medicine 11
Abbreviations
ELISA Enzyme-linked immunosorbent assayFITC Fluorescein isothiocyanateTNF-120572 Tumor necrosis factor-120572IL-6 Interleukin-6iNOS Inducible nitric oxide synthaseVEGF Vascular endothelial growth factorQT-PCR Quantitative Polymerase Chain ReactionIF ImmunofluorescenceIHC ImmunohistochemicalTAMs Tumor-associated macrophagesArg-1 Arginase 1
Conflicts of Interest
The authors declare that there are no conflicts of interest
Authorsrsquo Contributions
Fei Xu and Wenqiang Cui contributed equally to this workFei Xu and Baojun Liu conceived and designed experimentsFei Xu and Wenqiang Cui analyzed data and wrote themanuscript Jingcheng Dong supervised the project Zhengx-iao Zhao and Ying Wei assisted in experimental design anddata evaluation Jiaqi Liu Mihui Li Qiuping Li Chen Yanand Jian Qiu performed the study All authors reviewed andapproved the manuscript
Acknowledgments
This study was supported by Natural Science Foundation ofChina (nos 81673916 and 81403148) Development Projectof Shanghai Peak Disciplines-Integrative Medicine (no20150407)
References
[1] I Ikonomidis C A Michalakeas J Parissis et al ldquoInflamma-tory markers in coronary artery diseaserdquo BioFactors vol 38 no5 pp 320ndash328 2012
[2] P Lee C C Leung M I Restrepo K Takahashi Y Song and JM Porcel ldquoYear in review 2015 lung cancer pleural diseasesrespiratory infections bronchiectasis and tuberculosis bron-choscopic intervention and imagingrdquo Respirology vol 21 no 5pp 961ndash967 2016
[3] S V Sharma DW Bell J Settleman and D A Haber ldquoEpider-mal growth factor receptor mutations in lung cancerrdquo NatureReviews Cancer vol 7 no 3 pp 169ndash181 2007
[4] D Leong R Rai B Nguyen A Lee and D Yip ldquoAdvancesin adjuvant systemic therapy for non-small-cell lung cancerrdquoWorld Journal of Clinical Oncology vol 5 no 4 pp 633ndash6452014
[5] M T Villanueva ldquoMicroenvironment the new midfielders inthe tumourmicroenvironmentrdquoNature reviews Cancer vol 14no 12 p 765 2014
[6] A Mantovani S Sozzani M Locati P Allavena and A SicaldquoMacrophage polarization tumor-associated macrophages as aparadigm for polarizedM2mononuclear phagocytesrdquoTrends inImmunology vol 23 no 11 pp 549ndash555 2002
[7] A Mantovani and M Locati ldquoTumor-associated macrophagesas a paradigm of macrophage plasticity diversity and polariza-tion lessons and open questionsrdquo Arteriosclerosis Thrombosisand Vascular Biology vol 33 no 7 pp 1478ndash1483 2013
[8] W Hu X Li C Zhang Y Yang J Jiang and C Wu ldquoTumor-associated macrophages in cancersrdquo Clinical and TranslationalOncology vol 18 no 3 pp 251ndash258 2016
[9] B Z Qian and J W Pollard ldquoMacrophage diversity enhancestumor progression and metastasisrdquo Cell vol 141 no 1 pp 39ndash51 2010
[10] A Mantovani and P Allavena ldquoThe interaction of anti-cancer therapies with tumor-associated macrophagesrdquo Journalof Experimental Medicine vol 212 no 4 pp 435ndash445 2015
[11] J Xu Z Song Q Guo and J Li ldquoSynergistic effect and molec-ular mechanisms of traditional Chinese medicine on regulatingtumor microenvironment and cancer cellsrdquo BioMed ResearchInternational vol 2016 Article ID 1490738 pp 1ndash14 2016
[12] J Wu J Xu E A Eksioglu et al ldquoIcariside II induces apoptosisof melanoma cells through the downregulation of survivalpathwaysrdquoNutrition and Cancer vol 65 no 1 pp 110ndash117 2013
[13] L Kong J Liu J Wang et al ldquoIcariin inhibits TNF-120572IFN-120574induced inflammatory response via inhibition of the substanceP and p38-MAPK signaling pathway in human keratinocytesrdquoInternational Immunopharmacology vol 29 no 2 pp 401ndash4072015
[14] JWu J Du C Xu et al ldquoIn vivo and in vitro anti-inflammatoryeffects of a novel derivative of icariinrdquo Immunopharmacologyand Immunotoxicology vol 33 no 1 pp 49ndash54 2010
[15] E M Conway L A Pikor S H Y Kung et al ldquoMacrophagesinflammation and lung cancerrdquoAmerican Journal of Respiratoryand Critical Care Medicine vol 193 no 2 pp 116ndash130 2016
[16] S Shalapour and M Karin ldquoImmunity inflammation andcancer an eternal fight between good and evilrdquo The Journal ofClinical Investigation vol 125 no 9 pp 3347ndash3355 2015
[17] L Bingle N J Brown and C E Lewis ldquoThe role of tumour-associated macrophages in tumour progression Implicationsfor new anticancer therapiesrdquo Journal of Pathology vol 196 no3 pp 254ndash265 2002
[18] D Kim J Kim J H Yoon et al ldquoCXCL12 secreted from adiposetissue recruits macrophages and induces insulin resistance inmicerdquo Diabetologia vol 57 no 7 pp 1456ndash1465 2014
[19] M C Schmid C J Avraamides P Foubert et al ldquoCombinedblockade of integrin-12057241205731 plus cytokines SDF-1120572 or IL-1120573potently inhibits tumor inflammation and growthrdquo CancerResearch vol 71 no 22 pp 6965ndash6975 2011
[20] F Balkwill and A Mantovani ldquoInflammation and cancer backto VirchowrdquoThe Lancet vol 357 no 9255 pp 539ndash545 2001
[21] B B Aggarwal S Shishodia S K Sandur M K Pandey andG Sethi ldquoInflammation and cancer how hot is the linkrdquoBiochemical Pharmacology vol 72 no 11 pp 1605ndash1621 2006
[22] W-L Jiang X-G Chen H-B Zhu Y-B Gao J-W Tian andF-H Fu ldquoPaeoniflorin inhibits systemic inflammation andimproves survival in experimental sepsisrdquo Basic and ClinicalPharmacology and Toxicology vol 105 no 1 pp 64ndash71 2009
[23] I D Kim and B J Ha ldquoThe effects of paeoniflorin on LPS-induced liver inflammatory reactionsrdquo Archives of PharmacalResearch vol 33 no 6 pp 959ndash966 2010
[24] G-W Fan Y Zhang X Jiang et al ldquoAnti-inflammatory activityof baicalein in LPS-stimulated RAW2647 macrophages viaestrogen receptor and NF-120581B-dependent pathwaysrdquo Inflamma-tion vol 36 no 6 pp 1584ndash1591 2013
12 Evidence-Based Complementary and Alternative Medicine
[25] M Li-Weber ldquoNew therapeutic aspects of flavones the anti-cancer properties of Scutellaria and its main active constituentsWogonin Baicalein and Baicalinrdquo Cancer Treatment Reviewsvol 35 no 1 pp 57ndash68 2009
[26] QWu G-L Chen Y-J Li Y Chen and F-Z Lin ldquoPaeoniflorininhibits macrophage-mediated lung cancer metastasisrdquoChineseJournal of Natural Medicines vol 13 no 12 pp 925ndash932 2015
[27] W Lin and M Karin ldquoA cytokine-mediated link betweeninnate immunity inflammation and cancerrdquo Journal of ClinicalInvestigation vol 117 no 5 pp 1175ndash1183 2007
[28] T L Whiteside ldquoThe tumor microenvironment and its role inpromoting tumor growthrdquo Oncogene vol 27 no 45 pp 5904ndash5912 2008
[29] G Lorusso and C Ruegg ldquoThe tumor microenvironment andits contribution to tumor evolution toward metastasisrdquo Histo-chemistry and Cell Biology vol 130 no 6 pp 1091ndash1103 2008
[30] L Ding G Liang Z Yao et al ldquoMetformin prevents cancermetastasis by inhibiting M2-like polarization of tumor associ-ated macrophagesrdquo Oncotarget vol 6 no 34 pp 36441ndash364552015
[31] A Yuan Y JHsiaoH Y Chen et al ldquoOpposite effects ofM1 andM2macrophage subtypes on lung cancer progressionrdquo ScientificReports vol 5 Article ID 14273 2015
[32] F Balkwill ldquoCancer and the chemokine networkrdquo NatureReviews Cancer vol 4 no 7 pp 540ndash550 2004
[33] J A Burger and T J Kipps ldquoCXCR4 a key receptor in thecrosstalk between tumor cells and their microenvironmentrdquoBlood vol 107 no 5 pp 1761ndash1767 2006
[34] R J PhillipsM D BurdickM Lutz J A BelperioM P Keaneand R M Strieter ldquoThe stromal derived factor-1CXCL12-CXCchemokine receptor 4 biological axis in non-small cell lungcancermetastasesrdquoAmerican Journal of Respiratory and CriticalCare Medicine vol 167 no 12 pp 1676ndash1686 2003
[35] N Obermajer R Muthuswamy K Odunsi R P Edwards andP Kalinski ldquoPGE-induced CXCL 12 production and CXCR4expression controls the accumulation of human MDSCs inovarian cancer environmentrdquo Cancer Research vol 71 no 24pp 7463ndash7470 2011
[36] L Sanchez-Martın A Estecha R Samaniego S Sanchez-Ramon M A Vega and P Sanchez-Mateos ldquoThe chemokineCXCL12 regulates monocyte-macrophage differentiation andRUNX3 expressionrdquo Blood vol 117 no 1 pp 88ndash97 2011
[37] K Beider H Bitner M Leiba et al ldquoMultiple myelomacells recruit tumor-supportive macrophages through theCXCR4CXCL12 axis and promote their polarization towardthe M2 phenotyperdquo Oncotarget vol 5 no 22 pp 11283ndash112962014
[38] J M Mota C A Leite and L E Souza ldquoPost-sepsis stateinduces tumor-associated macrophage accumulation throughCXCR4CXCL12 and favors tumor progression inmicerdquoCancerImmunology Research vol 4 no 4 pp 312ndash322 2016
[39] B A Teicher and S P Fricker ldquoCXCL12 (SDF-1)CXCR4 path-way in cancerrdquoClinical Cancer Research vol 16 no 11 pp 2927ndash2931 2010
[40] P C Heinrich I Behrmann G Muller-Newen F Schaper andL Graeve ldquoInterleukin-6-type cytokine signalling through thegp130JakSTAT pathwayrdquo Biochemical Journal vol 334 part 2pp 297ndash314 1998
[41] M Kortylewski M Kujawski T Wang et al ldquoInhibiting Stat3signaling in the hematopoietic system elicits multicomponentantitumor immunityrdquo Nature Medicine vol 11 no 12 pp 1314ndash1321 2005
[42] X Hu J Chen L Wang and L B Ivashkiv ldquoCrosstalk amongJak-STAT Toll-like receptor and ITAM-dependent pathways inmacrophage activationrdquo Journal of Leukocyte Biology vol 82no 2 pp 237ndash243 2007
[43] R Catlett-Falcone T H Landowski M M Oshiro et alldquoConstitutive activation of Stat3 signaling confers resistance toapoptosis in humanU266myeloma cellsrdquo Immunity vol 10 no1 pp 105ndash115 1999
Submit your manuscripts athttpswwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
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Diabetes ResearchJournal of
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Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
6 Evidence-Based Complementary and Alternative Medicine
NS
100 휇m
(a)
QRH
X
100 휇m
(b)
C
D20
6 po
sitiv
e cel
ls
0
20
40
60
80
100
NS QRHX
lowastlowast
(c)
Figure 5 Comparison of CD206+ macrophages infiltration between NS and QRHX groups Immunohistochemical staining of CD206+macrophages in lung cancer tissues Images are at magnification of 200x Data represent mean plusmn SEM 119899 = 4 Compared with NS grouplowastlowast119875 lt 001
in Figures 3ndash6 our results revealed that CD206 expressionwas dramatically expressed in tumor tissue Compared to NSgroup oral administration with QRHX led to a prominentinhibition in TAMs (Figures 3ndash6 119875 lt 001) These datademonstrated that QRHX had a more significant role inimpeding accumulating and reducing the number of TAMsin lung cancer
34 QRHX Alters Hallmarkers of M1 and M2 Macrophagein Lung Cancer To further confirm the role of QRHX inTAMs we analyzed the level of Arg-1 protein M2 markerand the mRNA expression of M1 marker iNOS After QRHXtreatment Arg-1 level decreased (Figure 7(a) 119875 lt 001) andiNOS level increased (Figure 7(b) 119875 lt 005) Accordinglythese results strongly supported the inhibitory effect ofQRHX on suppressing TAMs infiltration and regulating M2-like macrophage polarization
35 QRHX Inhibits Angiogenesis in Lung Cancer CD31endothelial cell surface antigen is a vascular endothelialmarker for MVD and TAMs have a positive effect on bloodvessels by inducing the production of various proangiogenic
genes [17] Therefore CD31 was evaluated in implantedtumors using IHC and WB MVD stained by anti-CD31 wasmeasured by counting tissue sections of central areas of thetumor As shown in Figure 8 QRHX treatment induced aremarkable reduction in MVD and CD31 protein (119875 lt 001)Furthermore IHC and WB demonstrated reduced vascularendothelial growth factor (VEGF) expression in the QRHXtreated groups relative to NS controls (Figure 8 119875 lt 001)Taken together these data clearly showed thatQRHXreducedangiogenesis in lung cancer
36 QRHX Blocks CXCL 12CXCR4JNK2STAT3 SignalingPathways To investigate the molecular mechanism underly-ing the formulae decreasing M2 macrophages we tested theeffects of inhibitors of signaling molecules CXCL12CXCR4and JNK2STAT3 are known to be important moleculesinvolved in M2 polarization [18 19] Compared to NS grouporal administration of QRHX dramatically attenuated theincreased expressions of CXCL12 and CXCR4 in tumortissue (Figure 9(a) 119875 lt 001) As shown in Figure 9(b)JAK2 and STAT3 activation was significantly suppressed byQRHX treatment (both 119875 lt 001) compared to NS group
Evidence-Based Complementary and Alternative Medicine 7
106
105
104
103
0
Com
p-YL
1-H
F4
80-R
-PE-
H
1061051041030
Comp-BL1-H CD11b-FITC-H
197
1M
800 K
600 K
400 K
200 K
0
SSC-
H
SSC-
H
Comp-RL1-H CD206-Alexa Flour 647-H1061051041030
106
105
104
103
0
Com
p-YL
1-H
F4
80-R
-PE-
H
Comp-BL1-H CD11b-FITC-H1061051041030
900
1M
800 K
600 K
400 K
200 K
0
SSC-
H
SSC-
H
Comp-RL1-H CD206-Alexa Flour 647-H1061051041030
(a)
0
10
20
30
NS
F480+CD11b+ F480+CD11b+CD206+
QRHX
p
ositi
ve ce
lls
lowastlowast
lowastlowast
(b)
Figure 6 QRHX decreases the number of TAMs in subcutaneous tumor Mice with subcutaneous tumors were dealt with NS and QRHXMacrophages and TAMs (M2-like subtype) in tumor tissue were measured by flow cytometry Data represent mean plusmn SEM 119899 = 4 Comparedwith NS group lowastlowast119875 lt 001
8 Evidence-Based Complementary and Alternative Medicine
훽-actin43 kDa
Arg-140 kDa
00
02
04
06
08
10
Arg-1
Relat
ive p
rote
in ex
pres
sion
lowastlowast
NSQRHX
(a)
NSQRHX
iNOS0
4
1
2
3
Relat
ive p
rote
in ex
pres
sion
lowast
(b)
Figure 7The expression of M2-related marker (Arg-1) and M1-related marker (iNOS) was detected by western blot and QT-PCR respectivelyMice with subcutaneous tumor were dealt with NS and QRHX Data were presented as means plusmn SEM (119899 = 3) Compared with NS grouplowast119875 lt 005 lowastlowast119875 lt 001
Taking together the data suggested that QRHX inhibitedtumor cell-TAMs interactions possibly through blockingCXCL12CXCR4JAK2STAT3 signaling pathways and thenregulated macrophages polarization
4 Discussion
In the present study our data demonstrated that QRHXplayed a more crucial role in inhibiting tumor growth bymodulating tumor microenvironment especially TAMsQRHX inhibited tumor cell-TAMs interactions via thesuppression of cancer-related inflammation and probablyblocking the response of macrophages to tumor signalsCXCL12CXCR4JAK2STAT3 axis
In the nineteenth century the association between cancerand inflammation was firstly put forward [20] Large numberof studies provided powerful evidence that chronic inflam-mation can promote tumor development progression andmetastasis as well as chemoresistance [16 21] Recent studieshave confirmed that paeoniflorin baicalein and wogoninimportant ingredients of QRHX had the potential to inhibitmany types of inflammation [22ndash24] Furthermore baicaleinand wogonin exerted obvious inhibitory effects on cancer aswell as macrophages and angiogenesis [24 25] Paeoniflorinone of major ingredients could reduce lung metastasis ofLLC through inhibiting the M2 activation [26] In additionthe cytokines produced by activated innate immune cells intumor microenvironment can stimulate tumorigenesis suchas IL-6 and TNF-120572 [27] Our results showed a remarkabledecrease in multiple proinflammatory cytokines such as
TNF-a and IL-6 both in serum and tumor tissue of subcu-taneous mouse model (Figure 2) suggesting the inhibitoryeffect of QRHX on cancer-related inflammation
Tumor microenvironment created by the tumor andmainly orchestrated by inflammatory cells contributes totumor escape growth progression and evolution towardmetastasis [28 29] Numerous studies in recent decadehave presented evidences that TCM have a good effect onregulating tumor microenvironment such as reversing theimmunosuppressive microenvironment [11] Macrophages abasic component of the innate immune system are infiltratedin virtually all malignancies TAMs M2-like polarized stylehave been regarded as a protumor inflammatory microen-vironment which links inflammation and cancer [15] Col-lective evidences demonstrate that TAMs have the abilityof enhancing tumor angiogenesis increasing migration andinvasion and suppressing the antitumor immune responsesIt is correlated with the prognosis of patients with malignanttumor such as lung cancer [30 31] Consistent with previousstudies in this study we detected that the fraction of TAMswas increased in tumor and QRHX inhibited tumor growthin the tumor mice model through decreasing accumulatingof TAMs and activation of M2
CXCR4 which is widely expressed on malignant cellsand binds to CXCL12 [32] plays an important role in hem-atopoiesis development and organization of the immunesystem by directly and indirectly mechanisms [33] Forexample in NSCLC CXCL12-CXCR4 axis is involved inmetastasis and associated with an unfavorable prognosis[34] in ovarian cancer it can control accumulation of
Evidence-Based Complementary and Alternative Medicine 9CD
31
NS QRHX
VEG
F
0
5
10
15
20
25
VEGFMVD
Den
sity
of V
EGF
MV
D co
unt
lowastlowast
lowastlowast
(a)
00
02
04
06
08
10
Relat
ive p
rote
in ex
pres
sion
CD31VEGF
NSQRHX
lowastlowastlowastlowast
VEGF
CD31
훽-actin43 kDa
100 kDa
43 kDa
(b)
Figure 8 QRHX inhibits angiogenesis in lung cancer Mice with subcutaneous tumors were dealt with NS and QRHX (a) The nucleus wasdyed as blue CD31 was dyed as brown MVD were performed at high power field (times200) (b) The VEGF and CD31 protein expression intumor tissues was detected by western blot Data was expressed as means plusmn SEM values (119899 = 4) Compared with NS group lowastlowast119875 lt 001
human MDSCs and is an independent prognostic factor fortumor progression [35] Several studies have reported thatCXCL12 plays an important role in monocyte recruitmentdifferentiation and function [18 36] In a mouse modelof lung cancer CXCL12 could recruit tumor-promotingmyeloid CD11b+ cells [19] Besides powerful evidencesindicate a role for CXCR4-CXCL12 axis in promotingmacrophages polarization toward theM2 phenotype [37 38]In addition M2 subpopulation is associated with angio-genic factors such as VEGF and CXCL12-CXCR4 axis canalso promote tumor vascularization [39] Excitedly ourresults showed QRHX treatment inhibited signaling fromtumor cells to macrophages through inducing a remark-able decrease in CXCL12 and CXCR4 Furthermore QRHX
inhibited angiogenesis likely through altering the tumormicroenvironment by targeting TAMs
In STAT family there are seven proteins STAT3 is one ofthem It is a key transcription factor transducing signals fromactivated receptors or intracellular kinases to the nucleus andcan be activated in tumor cells and immune cells [40] Intumor STAT3 could contribute to cancer development andprogression inhibit apoptosis of tumors and help tumorescape immune system by suppressing the immune response[41] Evidence indicated that when STAT3 binds to somereceptor it can be activated through Janus Kinases (JAKs)such as JAK2 [42] Accumulating evidence implicates theimportant role of JAK2STAT3 in tumor and macrophagepolarization [27] For instance IL-6 can contribute to tumor
10 Evidence-Based Complementary and Alternative Medicine
CXCR4
CXCL1210 kDa
훽-actin43 kDa
41 kDa
NSQRHX
00
05
10
15
Relat
ive p
rote
in ex
pres
sion
CXCL12 CXCR4
lowastlowastlowastlowast
(a)
p-JAK2
p-STAT3
훽-actin43 kDa
90 kDa
120 kDa
NSQRHX
p-JAK2 p-STAT300
05
10
15
Relat
ive p
rote
in ex
pres
sion
lowastlowast lowastlowast
(b)
Figure 9 QRXH regulates TAMs by inhibiting the CXCL12CXCR4JAK2STAT3 signaling pathways Mice with subcutaneous tumor weredealt with NS and QRHX Representative images of western blot and densitometry analysis showing the expressions of CXCL12 CXCR4p-JAK2 and p-STAT3 in tumor Compared with NS group lowastlowast119875 lt 001
cell survival and upregulate the antiapoptotic genes by driv-ing JAK2STAT3 signal [43] IFN- activates macrophageby JAK-STAT signaling pathway [42] What is more JAK2is associated with CXCR4 [39] Then we can draw thatTAMs in malignant tumors tend to M2 subtype possiblythrough CXCL12CXCR4JAK2STAT3 signaling pathwayQRHX treatment blocked the response of macrophages totumor signals by suppressing CXCL12CXCR4JAK2STAT3expression and induced a remarkable decrease of the recruit-ment of M2 macrophages suggesting the attenuation of M2subtype cells function by QRHX
In TCM theory TCM formulae have abundant medicinalmaterials and then regulate diseases through multitargetsandmultiways Although the chemical constituents of QRHXhave been separated and identified by HPLUNG CANCER-QTOF MS-UV method in our team (Supplementary Fig-ure 1 and Table 1) the ingredients are various and theirfunctions are enormous So there may be other mechanismsinvolved in macrophage polarization What is more there
are diverse types cells in tumor except for tumor cells andmacrophage Moreover it is worth mentioning that TAMswere not separated and extracted from tumor tissue CXCL12CXCR4 JAK2 and STAT3 can not only play significantrole in tumor cell or macrophage but also other cellssuch as T lymphocyte neutrophils tumor-associated fibrob-last and endothelial cells Therefore QRHX inhibited theCXCL12CXCR4JAK2STAT3 axis possibly through othercells Taken together further investigations are needed toidentify direct molecular targets of QRHX in macrophagesin the context of cancer
5 Conclusion
In conclusion data from this study revealed that QRHXcould suppress cancer progression by inhibiting the tumorpromotion of TAMs in subcutaneous mice model whichcould contribute to elucidating the underlying regulatorymode of QRHX on lung cancer treatment
Evidence-Based Complementary and Alternative Medicine 11
Abbreviations
ELISA Enzyme-linked immunosorbent assayFITC Fluorescein isothiocyanateTNF-120572 Tumor necrosis factor-120572IL-6 Interleukin-6iNOS Inducible nitric oxide synthaseVEGF Vascular endothelial growth factorQT-PCR Quantitative Polymerase Chain ReactionIF ImmunofluorescenceIHC ImmunohistochemicalTAMs Tumor-associated macrophagesArg-1 Arginase 1
Conflicts of Interest
The authors declare that there are no conflicts of interest
Authorsrsquo Contributions
Fei Xu and Wenqiang Cui contributed equally to this workFei Xu and Baojun Liu conceived and designed experimentsFei Xu and Wenqiang Cui analyzed data and wrote themanuscript Jingcheng Dong supervised the project Zhengx-iao Zhao and Ying Wei assisted in experimental design anddata evaluation Jiaqi Liu Mihui Li Qiuping Li Chen Yanand Jian Qiu performed the study All authors reviewed andapproved the manuscript
Acknowledgments
This study was supported by Natural Science Foundation ofChina (nos 81673916 and 81403148) Development Projectof Shanghai Peak Disciplines-Integrative Medicine (no20150407)
References
[1] I Ikonomidis C A Michalakeas J Parissis et al ldquoInflamma-tory markers in coronary artery diseaserdquo BioFactors vol 38 no5 pp 320ndash328 2012
[2] P Lee C C Leung M I Restrepo K Takahashi Y Song and JM Porcel ldquoYear in review 2015 lung cancer pleural diseasesrespiratory infections bronchiectasis and tuberculosis bron-choscopic intervention and imagingrdquo Respirology vol 21 no 5pp 961ndash967 2016
[3] S V Sharma DW Bell J Settleman and D A Haber ldquoEpider-mal growth factor receptor mutations in lung cancerrdquo NatureReviews Cancer vol 7 no 3 pp 169ndash181 2007
[4] D Leong R Rai B Nguyen A Lee and D Yip ldquoAdvancesin adjuvant systemic therapy for non-small-cell lung cancerrdquoWorld Journal of Clinical Oncology vol 5 no 4 pp 633ndash6452014
[5] M T Villanueva ldquoMicroenvironment the new midfielders inthe tumourmicroenvironmentrdquoNature reviews Cancer vol 14no 12 p 765 2014
[6] A Mantovani S Sozzani M Locati P Allavena and A SicaldquoMacrophage polarization tumor-associated macrophages as aparadigm for polarizedM2mononuclear phagocytesrdquoTrends inImmunology vol 23 no 11 pp 549ndash555 2002
[7] A Mantovani and M Locati ldquoTumor-associated macrophagesas a paradigm of macrophage plasticity diversity and polariza-tion lessons and open questionsrdquo Arteriosclerosis Thrombosisand Vascular Biology vol 33 no 7 pp 1478ndash1483 2013
[8] W Hu X Li C Zhang Y Yang J Jiang and C Wu ldquoTumor-associated macrophages in cancersrdquo Clinical and TranslationalOncology vol 18 no 3 pp 251ndash258 2016
[9] B Z Qian and J W Pollard ldquoMacrophage diversity enhancestumor progression and metastasisrdquo Cell vol 141 no 1 pp 39ndash51 2010
[10] A Mantovani and P Allavena ldquoThe interaction of anti-cancer therapies with tumor-associated macrophagesrdquo Journalof Experimental Medicine vol 212 no 4 pp 435ndash445 2015
[11] J Xu Z Song Q Guo and J Li ldquoSynergistic effect and molec-ular mechanisms of traditional Chinese medicine on regulatingtumor microenvironment and cancer cellsrdquo BioMed ResearchInternational vol 2016 Article ID 1490738 pp 1ndash14 2016
[12] J Wu J Xu E A Eksioglu et al ldquoIcariside II induces apoptosisof melanoma cells through the downregulation of survivalpathwaysrdquoNutrition and Cancer vol 65 no 1 pp 110ndash117 2013
[13] L Kong J Liu J Wang et al ldquoIcariin inhibits TNF-120572IFN-120574induced inflammatory response via inhibition of the substanceP and p38-MAPK signaling pathway in human keratinocytesrdquoInternational Immunopharmacology vol 29 no 2 pp 401ndash4072015
[14] JWu J Du C Xu et al ldquoIn vivo and in vitro anti-inflammatoryeffects of a novel derivative of icariinrdquo Immunopharmacologyand Immunotoxicology vol 33 no 1 pp 49ndash54 2010
[15] E M Conway L A Pikor S H Y Kung et al ldquoMacrophagesinflammation and lung cancerrdquoAmerican Journal of Respiratoryand Critical Care Medicine vol 193 no 2 pp 116ndash130 2016
[16] S Shalapour and M Karin ldquoImmunity inflammation andcancer an eternal fight between good and evilrdquo The Journal ofClinical Investigation vol 125 no 9 pp 3347ndash3355 2015
[17] L Bingle N J Brown and C E Lewis ldquoThe role of tumour-associated macrophages in tumour progression Implicationsfor new anticancer therapiesrdquo Journal of Pathology vol 196 no3 pp 254ndash265 2002
[18] D Kim J Kim J H Yoon et al ldquoCXCL12 secreted from adiposetissue recruits macrophages and induces insulin resistance inmicerdquo Diabetologia vol 57 no 7 pp 1456ndash1465 2014
[19] M C Schmid C J Avraamides P Foubert et al ldquoCombinedblockade of integrin-12057241205731 plus cytokines SDF-1120572 or IL-1120573potently inhibits tumor inflammation and growthrdquo CancerResearch vol 71 no 22 pp 6965ndash6975 2011
[20] F Balkwill and A Mantovani ldquoInflammation and cancer backto VirchowrdquoThe Lancet vol 357 no 9255 pp 539ndash545 2001
[21] B B Aggarwal S Shishodia S K Sandur M K Pandey andG Sethi ldquoInflammation and cancer how hot is the linkrdquoBiochemical Pharmacology vol 72 no 11 pp 1605ndash1621 2006
[22] W-L Jiang X-G Chen H-B Zhu Y-B Gao J-W Tian andF-H Fu ldquoPaeoniflorin inhibits systemic inflammation andimproves survival in experimental sepsisrdquo Basic and ClinicalPharmacology and Toxicology vol 105 no 1 pp 64ndash71 2009
[23] I D Kim and B J Ha ldquoThe effects of paeoniflorin on LPS-induced liver inflammatory reactionsrdquo Archives of PharmacalResearch vol 33 no 6 pp 959ndash966 2010
[24] G-W Fan Y Zhang X Jiang et al ldquoAnti-inflammatory activityof baicalein in LPS-stimulated RAW2647 macrophages viaestrogen receptor and NF-120581B-dependent pathwaysrdquo Inflamma-tion vol 36 no 6 pp 1584ndash1591 2013
12 Evidence-Based Complementary and Alternative Medicine
[25] M Li-Weber ldquoNew therapeutic aspects of flavones the anti-cancer properties of Scutellaria and its main active constituentsWogonin Baicalein and Baicalinrdquo Cancer Treatment Reviewsvol 35 no 1 pp 57ndash68 2009
[26] QWu G-L Chen Y-J Li Y Chen and F-Z Lin ldquoPaeoniflorininhibits macrophage-mediated lung cancer metastasisrdquoChineseJournal of Natural Medicines vol 13 no 12 pp 925ndash932 2015
[27] W Lin and M Karin ldquoA cytokine-mediated link betweeninnate immunity inflammation and cancerrdquo Journal of ClinicalInvestigation vol 117 no 5 pp 1175ndash1183 2007
[28] T L Whiteside ldquoThe tumor microenvironment and its role inpromoting tumor growthrdquo Oncogene vol 27 no 45 pp 5904ndash5912 2008
[29] G Lorusso and C Ruegg ldquoThe tumor microenvironment andits contribution to tumor evolution toward metastasisrdquo Histo-chemistry and Cell Biology vol 130 no 6 pp 1091ndash1103 2008
[30] L Ding G Liang Z Yao et al ldquoMetformin prevents cancermetastasis by inhibiting M2-like polarization of tumor associ-ated macrophagesrdquo Oncotarget vol 6 no 34 pp 36441ndash364552015
[31] A Yuan Y JHsiaoH Y Chen et al ldquoOpposite effects ofM1 andM2macrophage subtypes on lung cancer progressionrdquo ScientificReports vol 5 Article ID 14273 2015
[32] F Balkwill ldquoCancer and the chemokine networkrdquo NatureReviews Cancer vol 4 no 7 pp 540ndash550 2004
[33] J A Burger and T J Kipps ldquoCXCR4 a key receptor in thecrosstalk between tumor cells and their microenvironmentrdquoBlood vol 107 no 5 pp 1761ndash1767 2006
[34] R J PhillipsM D BurdickM Lutz J A BelperioM P Keaneand R M Strieter ldquoThe stromal derived factor-1CXCL12-CXCchemokine receptor 4 biological axis in non-small cell lungcancermetastasesrdquoAmerican Journal of Respiratory and CriticalCare Medicine vol 167 no 12 pp 1676ndash1686 2003
[35] N Obermajer R Muthuswamy K Odunsi R P Edwards andP Kalinski ldquoPGE-induced CXCL 12 production and CXCR4expression controls the accumulation of human MDSCs inovarian cancer environmentrdquo Cancer Research vol 71 no 24pp 7463ndash7470 2011
[36] L Sanchez-Martın A Estecha R Samaniego S Sanchez-Ramon M A Vega and P Sanchez-Mateos ldquoThe chemokineCXCL12 regulates monocyte-macrophage differentiation andRUNX3 expressionrdquo Blood vol 117 no 1 pp 88ndash97 2011
[37] K Beider H Bitner M Leiba et al ldquoMultiple myelomacells recruit tumor-supportive macrophages through theCXCR4CXCL12 axis and promote their polarization towardthe M2 phenotyperdquo Oncotarget vol 5 no 22 pp 11283ndash112962014
[38] J M Mota C A Leite and L E Souza ldquoPost-sepsis stateinduces tumor-associated macrophage accumulation throughCXCR4CXCL12 and favors tumor progression inmicerdquoCancerImmunology Research vol 4 no 4 pp 312ndash322 2016
[39] B A Teicher and S P Fricker ldquoCXCL12 (SDF-1)CXCR4 path-way in cancerrdquoClinical Cancer Research vol 16 no 11 pp 2927ndash2931 2010
[40] P C Heinrich I Behrmann G Muller-Newen F Schaper andL Graeve ldquoInterleukin-6-type cytokine signalling through thegp130JakSTAT pathwayrdquo Biochemical Journal vol 334 part 2pp 297ndash314 1998
[41] M Kortylewski M Kujawski T Wang et al ldquoInhibiting Stat3signaling in the hematopoietic system elicits multicomponentantitumor immunityrdquo Nature Medicine vol 11 no 12 pp 1314ndash1321 2005
[42] X Hu J Chen L Wang and L B Ivashkiv ldquoCrosstalk amongJak-STAT Toll-like receptor and ITAM-dependent pathways inmacrophage activationrdquo Journal of Leukocyte Biology vol 82no 2 pp 237ndash243 2007
[43] R Catlett-Falcone T H Landowski M M Oshiro et alldquoConstitutive activation of Stat3 signaling confers resistance toapoptosis in humanU266myeloma cellsrdquo Immunity vol 10 no1 pp 105ndash115 1999
Submit your manuscripts athttpswwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 7
106
105
104
103
0
Com
p-YL
1-H
F4
80-R
-PE-
H
1061051041030
Comp-BL1-H CD11b-FITC-H
197
1M
800 K
600 K
400 K
200 K
0
SSC-
H
SSC-
H
Comp-RL1-H CD206-Alexa Flour 647-H1061051041030
106
105
104
103
0
Com
p-YL
1-H
F4
80-R
-PE-
H
Comp-BL1-H CD11b-FITC-H1061051041030
900
1M
800 K
600 K
400 K
200 K
0
SSC-
H
SSC-
H
Comp-RL1-H CD206-Alexa Flour 647-H1061051041030
(a)
0
10
20
30
NS
F480+CD11b+ F480+CD11b+CD206+
QRHX
p
ositi
ve ce
lls
lowastlowast
lowastlowast
(b)
Figure 6 QRHX decreases the number of TAMs in subcutaneous tumor Mice with subcutaneous tumors were dealt with NS and QRHXMacrophages and TAMs (M2-like subtype) in tumor tissue were measured by flow cytometry Data represent mean plusmn SEM 119899 = 4 Comparedwith NS group lowastlowast119875 lt 001
8 Evidence-Based Complementary and Alternative Medicine
훽-actin43 kDa
Arg-140 kDa
00
02
04
06
08
10
Arg-1
Relat
ive p
rote
in ex
pres
sion
lowastlowast
NSQRHX
(a)
NSQRHX
iNOS0
4
1
2
3
Relat
ive p
rote
in ex
pres
sion
lowast
(b)
Figure 7The expression of M2-related marker (Arg-1) and M1-related marker (iNOS) was detected by western blot and QT-PCR respectivelyMice with subcutaneous tumor were dealt with NS and QRHX Data were presented as means plusmn SEM (119899 = 3) Compared with NS grouplowast119875 lt 005 lowastlowast119875 lt 001
Taking together the data suggested that QRHX inhibitedtumor cell-TAMs interactions possibly through blockingCXCL12CXCR4JAK2STAT3 signaling pathways and thenregulated macrophages polarization
4 Discussion
In the present study our data demonstrated that QRHXplayed a more crucial role in inhibiting tumor growth bymodulating tumor microenvironment especially TAMsQRHX inhibited tumor cell-TAMs interactions via thesuppression of cancer-related inflammation and probablyblocking the response of macrophages to tumor signalsCXCL12CXCR4JAK2STAT3 axis
In the nineteenth century the association between cancerand inflammation was firstly put forward [20] Large numberof studies provided powerful evidence that chronic inflam-mation can promote tumor development progression andmetastasis as well as chemoresistance [16 21] Recent studieshave confirmed that paeoniflorin baicalein and wogoninimportant ingredients of QRHX had the potential to inhibitmany types of inflammation [22ndash24] Furthermore baicaleinand wogonin exerted obvious inhibitory effects on cancer aswell as macrophages and angiogenesis [24 25] Paeoniflorinone of major ingredients could reduce lung metastasis ofLLC through inhibiting the M2 activation [26] In additionthe cytokines produced by activated innate immune cells intumor microenvironment can stimulate tumorigenesis suchas IL-6 and TNF-120572 [27] Our results showed a remarkabledecrease in multiple proinflammatory cytokines such as
TNF-a and IL-6 both in serum and tumor tissue of subcu-taneous mouse model (Figure 2) suggesting the inhibitoryeffect of QRHX on cancer-related inflammation
Tumor microenvironment created by the tumor andmainly orchestrated by inflammatory cells contributes totumor escape growth progression and evolution towardmetastasis [28 29] Numerous studies in recent decadehave presented evidences that TCM have a good effect onregulating tumor microenvironment such as reversing theimmunosuppressive microenvironment [11] Macrophages abasic component of the innate immune system are infiltratedin virtually all malignancies TAMs M2-like polarized stylehave been regarded as a protumor inflammatory microen-vironment which links inflammation and cancer [15] Col-lective evidences demonstrate that TAMs have the abilityof enhancing tumor angiogenesis increasing migration andinvasion and suppressing the antitumor immune responsesIt is correlated with the prognosis of patients with malignanttumor such as lung cancer [30 31] Consistent with previousstudies in this study we detected that the fraction of TAMswas increased in tumor and QRHX inhibited tumor growthin the tumor mice model through decreasing accumulatingof TAMs and activation of M2
CXCR4 which is widely expressed on malignant cellsand binds to CXCL12 [32] plays an important role in hem-atopoiesis development and organization of the immunesystem by directly and indirectly mechanisms [33] Forexample in NSCLC CXCL12-CXCR4 axis is involved inmetastasis and associated with an unfavorable prognosis[34] in ovarian cancer it can control accumulation of
Evidence-Based Complementary and Alternative Medicine 9CD
31
NS QRHX
VEG
F
0
5
10
15
20
25
VEGFMVD
Den
sity
of V
EGF
MV
D co
unt
lowastlowast
lowastlowast
(a)
00
02
04
06
08
10
Relat
ive p
rote
in ex
pres
sion
CD31VEGF
NSQRHX
lowastlowastlowastlowast
VEGF
CD31
훽-actin43 kDa
100 kDa
43 kDa
(b)
Figure 8 QRHX inhibits angiogenesis in lung cancer Mice with subcutaneous tumors were dealt with NS and QRHX (a) The nucleus wasdyed as blue CD31 was dyed as brown MVD were performed at high power field (times200) (b) The VEGF and CD31 protein expression intumor tissues was detected by western blot Data was expressed as means plusmn SEM values (119899 = 4) Compared with NS group lowastlowast119875 lt 001
human MDSCs and is an independent prognostic factor fortumor progression [35] Several studies have reported thatCXCL12 plays an important role in monocyte recruitmentdifferentiation and function [18 36] In a mouse modelof lung cancer CXCL12 could recruit tumor-promotingmyeloid CD11b+ cells [19] Besides powerful evidencesindicate a role for CXCR4-CXCL12 axis in promotingmacrophages polarization toward theM2 phenotype [37 38]In addition M2 subpopulation is associated with angio-genic factors such as VEGF and CXCL12-CXCR4 axis canalso promote tumor vascularization [39] Excitedly ourresults showed QRHX treatment inhibited signaling fromtumor cells to macrophages through inducing a remark-able decrease in CXCL12 and CXCR4 Furthermore QRHX
inhibited angiogenesis likely through altering the tumormicroenvironment by targeting TAMs
In STAT family there are seven proteins STAT3 is one ofthem It is a key transcription factor transducing signals fromactivated receptors or intracellular kinases to the nucleus andcan be activated in tumor cells and immune cells [40] Intumor STAT3 could contribute to cancer development andprogression inhibit apoptosis of tumors and help tumorescape immune system by suppressing the immune response[41] Evidence indicated that when STAT3 binds to somereceptor it can be activated through Janus Kinases (JAKs)such as JAK2 [42] Accumulating evidence implicates theimportant role of JAK2STAT3 in tumor and macrophagepolarization [27] For instance IL-6 can contribute to tumor
10 Evidence-Based Complementary and Alternative Medicine
CXCR4
CXCL1210 kDa
훽-actin43 kDa
41 kDa
NSQRHX
00
05
10
15
Relat
ive p
rote
in ex
pres
sion
CXCL12 CXCR4
lowastlowastlowastlowast
(a)
p-JAK2
p-STAT3
훽-actin43 kDa
90 kDa
120 kDa
NSQRHX
p-JAK2 p-STAT300
05
10
15
Relat
ive p
rote
in ex
pres
sion
lowastlowast lowastlowast
(b)
Figure 9 QRXH regulates TAMs by inhibiting the CXCL12CXCR4JAK2STAT3 signaling pathways Mice with subcutaneous tumor weredealt with NS and QRHX Representative images of western blot and densitometry analysis showing the expressions of CXCL12 CXCR4p-JAK2 and p-STAT3 in tumor Compared with NS group lowastlowast119875 lt 001
cell survival and upregulate the antiapoptotic genes by driv-ing JAK2STAT3 signal [43] IFN- activates macrophageby JAK-STAT signaling pathway [42] What is more JAK2is associated with CXCR4 [39] Then we can draw thatTAMs in malignant tumors tend to M2 subtype possiblythrough CXCL12CXCR4JAK2STAT3 signaling pathwayQRHX treatment blocked the response of macrophages totumor signals by suppressing CXCL12CXCR4JAK2STAT3expression and induced a remarkable decrease of the recruit-ment of M2 macrophages suggesting the attenuation of M2subtype cells function by QRHX
In TCM theory TCM formulae have abundant medicinalmaterials and then regulate diseases through multitargetsandmultiways Although the chemical constituents of QRHXhave been separated and identified by HPLUNG CANCER-QTOF MS-UV method in our team (Supplementary Fig-ure 1 and Table 1) the ingredients are various and theirfunctions are enormous So there may be other mechanismsinvolved in macrophage polarization What is more there
are diverse types cells in tumor except for tumor cells andmacrophage Moreover it is worth mentioning that TAMswere not separated and extracted from tumor tissue CXCL12CXCR4 JAK2 and STAT3 can not only play significantrole in tumor cell or macrophage but also other cellssuch as T lymphocyte neutrophils tumor-associated fibrob-last and endothelial cells Therefore QRHX inhibited theCXCL12CXCR4JAK2STAT3 axis possibly through othercells Taken together further investigations are needed toidentify direct molecular targets of QRHX in macrophagesin the context of cancer
5 Conclusion
In conclusion data from this study revealed that QRHXcould suppress cancer progression by inhibiting the tumorpromotion of TAMs in subcutaneous mice model whichcould contribute to elucidating the underlying regulatorymode of QRHX on lung cancer treatment
Evidence-Based Complementary and Alternative Medicine 11
Abbreviations
ELISA Enzyme-linked immunosorbent assayFITC Fluorescein isothiocyanateTNF-120572 Tumor necrosis factor-120572IL-6 Interleukin-6iNOS Inducible nitric oxide synthaseVEGF Vascular endothelial growth factorQT-PCR Quantitative Polymerase Chain ReactionIF ImmunofluorescenceIHC ImmunohistochemicalTAMs Tumor-associated macrophagesArg-1 Arginase 1
Conflicts of Interest
The authors declare that there are no conflicts of interest
Authorsrsquo Contributions
Fei Xu and Wenqiang Cui contributed equally to this workFei Xu and Baojun Liu conceived and designed experimentsFei Xu and Wenqiang Cui analyzed data and wrote themanuscript Jingcheng Dong supervised the project Zhengx-iao Zhao and Ying Wei assisted in experimental design anddata evaluation Jiaqi Liu Mihui Li Qiuping Li Chen Yanand Jian Qiu performed the study All authors reviewed andapproved the manuscript
Acknowledgments
This study was supported by Natural Science Foundation ofChina (nos 81673916 and 81403148) Development Projectof Shanghai Peak Disciplines-Integrative Medicine (no20150407)
References
[1] I Ikonomidis C A Michalakeas J Parissis et al ldquoInflamma-tory markers in coronary artery diseaserdquo BioFactors vol 38 no5 pp 320ndash328 2012
[2] P Lee C C Leung M I Restrepo K Takahashi Y Song and JM Porcel ldquoYear in review 2015 lung cancer pleural diseasesrespiratory infections bronchiectasis and tuberculosis bron-choscopic intervention and imagingrdquo Respirology vol 21 no 5pp 961ndash967 2016
[3] S V Sharma DW Bell J Settleman and D A Haber ldquoEpider-mal growth factor receptor mutations in lung cancerrdquo NatureReviews Cancer vol 7 no 3 pp 169ndash181 2007
[4] D Leong R Rai B Nguyen A Lee and D Yip ldquoAdvancesin adjuvant systemic therapy for non-small-cell lung cancerrdquoWorld Journal of Clinical Oncology vol 5 no 4 pp 633ndash6452014
[5] M T Villanueva ldquoMicroenvironment the new midfielders inthe tumourmicroenvironmentrdquoNature reviews Cancer vol 14no 12 p 765 2014
[6] A Mantovani S Sozzani M Locati P Allavena and A SicaldquoMacrophage polarization tumor-associated macrophages as aparadigm for polarizedM2mononuclear phagocytesrdquoTrends inImmunology vol 23 no 11 pp 549ndash555 2002
[7] A Mantovani and M Locati ldquoTumor-associated macrophagesas a paradigm of macrophage plasticity diversity and polariza-tion lessons and open questionsrdquo Arteriosclerosis Thrombosisand Vascular Biology vol 33 no 7 pp 1478ndash1483 2013
[8] W Hu X Li C Zhang Y Yang J Jiang and C Wu ldquoTumor-associated macrophages in cancersrdquo Clinical and TranslationalOncology vol 18 no 3 pp 251ndash258 2016
[9] B Z Qian and J W Pollard ldquoMacrophage diversity enhancestumor progression and metastasisrdquo Cell vol 141 no 1 pp 39ndash51 2010
[10] A Mantovani and P Allavena ldquoThe interaction of anti-cancer therapies with tumor-associated macrophagesrdquo Journalof Experimental Medicine vol 212 no 4 pp 435ndash445 2015
[11] J Xu Z Song Q Guo and J Li ldquoSynergistic effect and molec-ular mechanisms of traditional Chinese medicine on regulatingtumor microenvironment and cancer cellsrdquo BioMed ResearchInternational vol 2016 Article ID 1490738 pp 1ndash14 2016
[12] J Wu J Xu E A Eksioglu et al ldquoIcariside II induces apoptosisof melanoma cells through the downregulation of survivalpathwaysrdquoNutrition and Cancer vol 65 no 1 pp 110ndash117 2013
[13] L Kong J Liu J Wang et al ldquoIcariin inhibits TNF-120572IFN-120574induced inflammatory response via inhibition of the substanceP and p38-MAPK signaling pathway in human keratinocytesrdquoInternational Immunopharmacology vol 29 no 2 pp 401ndash4072015
[14] JWu J Du C Xu et al ldquoIn vivo and in vitro anti-inflammatoryeffects of a novel derivative of icariinrdquo Immunopharmacologyand Immunotoxicology vol 33 no 1 pp 49ndash54 2010
[15] E M Conway L A Pikor S H Y Kung et al ldquoMacrophagesinflammation and lung cancerrdquoAmerican Journal of Respiratoryand Critical Care Medicine vol 193 no 2 pp 116ndash130 2016
[16] S Shalapour and M Karin ldquoImmunity inflammation andcancer an eternal fight between good and evilrdquo The Journal ofClinical Investigation vol 125 no 9 pp 3347ndash3355 2015
[17] L Bingle N J Brown and C E Lewis ldquoThe role of tumour-associated macrophages in tumour progression Implicationsfor new anticancer therapiesrdquo Journal of Pathology vol 196 no3 pp 254ndash265 2002
[18] D Kim J Kim J H Yoon et al ldquoCXCL12 secreted from adiposetissue recruits macrophages and induces insulin resistance inmicerdquo Diabetologia vol 57 no 7 pp 1456ndash1465 2014
[19] M C Schmid C J Avraamides P Foubert et al ldquoCombinedblockade of integrin-12057241205731 plus cytokines SDF-1120572 or IL-1120573potently inhibits tumor inflammation and growthrdquo CancerResearch vol 71 no 22 pp 6965ndash6975 2011
[20] F Balkwill and A Mantovani ldquoInflammation and cancer backto VirchowrdquoThe Lancet vol 357 no 9255 pp 539ndash545 2001
[21] B B Aggarwal S Shishodia S K Sandur M K Pandey andG Sethi ldquoInflammation and cancer how hot is the linkrdquoBiochemical Pharmacology vol 72 no 11 pp 1605ndash1621 2006
[22] W-L Jiang X-G Chen H-B Zhu Y-B Gao J-W Tian andF-H Fu ldquoPaeoniflorin inhibits systemic inflammation andimproves survival in experimental sepsisrdquo Basic and ClinicalPharmacology and Toxicology vol 105 no 1 pp 64ndash71 2009
[23] I D Kim and B J Ha ldquoThe effects of paeoniflorin on LPS-induced liver inflammatory reactionsrdquo Archives of PharmacalResearch vol 33 no 6 pp 959ndash966 2010
[24] G-W Fan Y Zhang X Jiang et al ldquoAnti-inflammatory activityof baicalein in LPS-stimulated RAW2647 macrophages viaestrogen receptor and NF-120581B-dependent pathwaysrdquo Inflamma-tion vol 36 no 6 pp 1584ndash1591 2013
12 Evidence-Based Complementary and Alternative Medicine
[25] M Li-Weber ldquoNew therapeutic aspects of flavones the anti-cancer properties of Scutellaria and its main active constituentsWogonin Baicalein and Baicalinrdquo Cancer Treatment Reviewsvol 35 no 1 pp 57ndash68 2009
[26] QWu G-L Chen Y-J Li Y Chen and F-Z Lin ldquoPaeoniflorininhibits macrophage-mediated lung cancer metastasisrdquoChineseJournal of Natural Medicines vol 13 no 12 pp 925ndash932 2015
[27] W Lin and M Karin ldquoA cytokine-mediated link betweeninnate immunity inflammation and cancerrdquo Journal of ClinicalInvestigation vol 117 no 5 pp 1175ndash1183 2007
[28] T L Whiteside ldquoThe tumor microenvironment and its role inpromoting tumor growthrdquo Oncogene vol 27 no 45 pp 5904ndash5912 2008
[29] G Lorusso and C Ruegg ldquoThe tumor microenvironment andits contribution to tumor evolution toward metastasisrdquo Histo-chemistry and Cell Biology vol 130 no 6 pp 1091ndash1103 2008
[30] L Ding G Liang Z Yao et al ldquoMetformin prevents cancermetastasis by inhibiting M2-like polarization of tumor associ-ated macrophagesrdquo Oncotarget vol 6 no 34 pp 36441ndash364552015
[31] A Yuan Y JHsiaoH Y Chen et al ldquoOpposite effects ofM1 andM2macrophage subtypes on lung cancer progressionrdquo ScientificReports vol 5 Article ID 14273 2015
[32] F Balkwill ldquoCancer and the chemokine networkrdquo NatureReviews Cancer vol 4 no 7 pp 540ndash550 2004
[33] J A Burger and T J Kipps ldquoCXCR4 a key receptor in thecrosstalk between tumor cells and their microenvironmentrdquoBlood vol 107 no 5 pp 1761ndash1767 2006
[34] R J PhillipsM D BurdickM Lutz J A BelperioM P Keaneand R M Strieter ldquoThe stromal derived factor-1CXCL12-CXCchemokine receptor 4 biological axis in non-small cell lungcancermetastasesrdquoAmerican Journal of Respiratory and CriticalCare Medicine vol 167 no 12 pp 1676ndash1686 2003
[35] N Obermajer R Muthuswamy K Odunsi R P Edwards andP Kalinski ldquoPGE-induced CXCL 12 production and CXCR4expression controls the accumulation of human MDSCs inovarian cancer environmentrdquo Cancer Research vol 71 no 24pp 7463ndash7470 2011
[36] L Sanchez-Martın A Estecha R Samaniego S Sanchez-Ramon M A Vega and P Sanchez-Mateos ldquoThe chemokineCXCL12 regulates monocyte-macrophage differentiation andRUNX3 expressionrdquo Blood vol 117 no 1 pp 88ndash97 2011
[37] K Beider H Bitner M Leiba et al ldquoMultiple myelomacells recruit tumor-supportive macrophages through theCXCR4CXCL12 axis and promote their polarization towardthe M2 phenotyperdquo Oncotarget vol 5 no 22 pp 11283ndash112962014
[38] J M Mota C A Leite and L E Souza ldquoPost-sepsis stateinduces tumor-associated macrophage accumulation throughCXCR4CXCL12 and favors tumor progression inmicerdquoCancerImmunology Research vol 4 no 4 pp 312ndash322 2016
[39] B A Teicher and S P Fricker ldquoCXCL12 (SDF-1)CXCR4 path-way in cancerrdquoClinical Cancer Research vol 16 no 11 pp 2927ndash2931 2010
[40] P C Heinrich I Behrmann G Muller-Newen F Schaper andL Graeve ldquoInterleukin-6-type cytokine signalling through thegp130JakSTAT pathwayrdquo Biochemical Journal vol 334 part 2pp 297ndash314 1998
[41] M Kortylewski M Kujawski T Wang et al ldquoInhibiting Stat3signaling in the hematopoietic system elicits multicomponentantitumor immunityrdquo Nature Medicine vol 11 no 12 pp 1314ndash1321 2005
[42] X Hu J Chen L Wang and L B Ivashkiv ldquoCrosstalk amongJak-STAT Toll-like receptor and ITAM-dependent pathways inmacrophage activationrdquo Journal of Leukocyte Biology vol 82no 2 pp 237ndash243 2007
[43] R Catlett-Falcone T H Landowski M M Oshiro et alldquoConstitutive activation of Stat3 signaling confers resistance toapoptosis in humanU266myeloma cellsrdquo Immunity vol 10 no1 pp 105ndash115 1999
Submit your manuscripts athttpswwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
8 Evidence-Based Complementary and Alternative Medicine
훽-actin43 kDa
Arg-140 kDa
00
02
04
06
08
10
Arg-1
Relat
ive p
rote
in ex
pres
sion
lowastlowast
NSQRHX
(a)
NSQRHX
iNOS0
4
1
2
3
Relat
ive p
rote
in ex
pres
sion
lowast
(b)
Figure 7The expression of M2-related marker (Arg-1) and M1-related marker (iNOS) was detected by western blot and QT-PCR respectivelyMice with subcutaneous tumor were dealt with NS and QRHX Data were presented as means plusmn SEM (119899 = 3) Compared with NS grouplowast119875 lt 005 lowastlowast119875 lt 001
Taking together the data suggested that QRHX inhibitedtumor cell-TAMs interactions possibly through blockingCXCL12CXCR4JAK2STAT3 signaling pathways and thenregulated macrophages polarization
4 Discussion
In the present study our data demonstrated that QRHXplayed a more crucial role in inhibiting tumor growth bymodulating tumor microenvironment especially TAMsQRHX inhibited tumor cell-TAMs interactions via thesuppression of cancer-related inflammation and probablyblocking the response of macrophages to tumor signalsCXCL12CXCR4JAK2STAT3 axis
In the nineteenth century the association between cancerand inflammation was firstly put forward [20] Large numberof studies provided powerful evidence that chronic inflam-mation can promote tumor development progression andmetastasis as well as chemoresistance [16 21] Recent studieshave confirmed that paeoniflorin baicalein and wogoninimportant ingredients of QRHX had the potential to inhibitmany types of inflammation [22ndash24] Furthermore baicaleinand wogonin exerted obvious inhibitory effects on cancer aswell as macrophages and angiogenesis [24 25] Paeoniflorinone of major ingredients could reduce lung metastasis ofLLC through inhibiting the M2 activation [26] In additionthe cytokines produced by activated innate immune cells intumor microenvironment can stimulate tumorigenesis suchas IL-6 and TNF-120572 [27] Our results showed a remarkabledecrease in multiple proinflammatory cytokines such as
TNF-a and IL-6 both in serum and tumor tissue of subcu-taneous mouse model (Figure 2) suggesting the inhibitoryeffect of QRHX on cancer-related inflammation
Tumor microenvironment created by the tumor andmainly orchestrated by inflammatory cells contributes totumor escape growth progression and evolution towardmetastasis [28 29] Numerous studies in recent decadehave presented evidences that TCM have a good effect onregulating tumor microenvironment such as reversing theimmunosuppressive microenvironment [11] Macrophages abasic component of the innate immune system are infiltratedin virtually all malignancies TAMs M2-like polarized stylehave been regarded as a protumor inflammatory microen-vironment which links inflammation and cancer [15] Col-lective evidences demonstrate that TAMs have the abilityof enhancing tumor angiogenesis increasing migration andinvasion and suppressing the antitumor immune responsesIt is correlated with the prognosis of patients with malignanttumor such as lung cancer [30 31] Consistent with previousstudies in this study we detected that the fraction of TAMswas increased in tumor and QRHX inhibited tumor growthin the tumor mice model through decreasing accumulatingof TAMs and activation of M2
CXCR4 which is widely expressed on malignant cellsand binds to CXCL12 [32] plays an important role in hem-atopoiesis development and organization of the immunesystem by directly and indirectly mechanisms [33] Forexample in NSCLC CXCL12-CXCR4 axis is involved inmetastasis and associated with an unfavorable prognosis[34] in ovarian cancer it can control accumulation of
Evidence-Based Complementary and Alternative Medicine 9CD
31
NS QRHX
VEG
F
0
5
10
15
20
25
VEGFMVD
Den
sity
of V
EGF
MV
D co
unt
lowastlowast
lowastlowast
(a)
00
02
04
06
08
10
Relat
ive p
rote
in ex
pres
sion
CD31VEGF
NSQRHX
lowastlowastlowastlowast
VEGF
CD31
훽-actin43 kDa
100 kDa
43 kDa
(b)
Figure 8 QRHX inhibits angiogenesis in lung cancer Mice with subcutaneous tumors were dealt with NS and QRHX (a) The nucleus wasdyed as blue CD31 was dyed as brown MVD were performed at high power field (times200) (b) The VEGF and CD31 protein expression intumor tissues was detected by western blot Data was expressed as means plusmn SEM values (119899 = 4) Compared with NS group lowastlowast119875 lt 001
human MDSCs and is an independent prognostic factor fortumor progression [35] Several studies have reported thatCXCL12 plays an important role in monocyte recruitmentdifferentiation and function [18 36] In a mouse modelof lung cancer CXCL12 could recruit tumor-promotingmyeloid CD11b+ cells [19] Besides powerful evidencesindicate a role for CXCR4-CXCL12 axis in promotingmacrophages polarization toward theM2 phenotype [37 38]In addition M2 subpopulation is associated with angio-genic factors such as VEGF and CXCL12-CXCR4 axis canalso promote tumor vascularization [39] Excitedly ourresults showed QRHX treatment inhibited signaling fromtumor cells to macrophages through inducing a remark-able decrease in CXCL12 and CXCR4 Furthermore QRHX
inhibited angiogenesis likely through altering the tumormicroenvironment by targeting TAMs
In STAT family there are seven proteins STAT3 is one ofthem It is a key transcription factor transducing signals fromactivated receptors or intracellular kinases to the nucleus andcan be activated in tumor cells and immune cells [40] Intumor STAT3 could contribute to cancer development andprogression inhibit apoptosis of tumors and help tumorescape immune system by suppressing the immune response[41] Evidence indicated that when STAT3 binds to somereceptor it can be activated through Janus Kinases (JAKs)such as JAK2 [42] Accumulating evidence implicates theimportant role of JAK2STAT3 in tumor and macrophagepolarization [27] For instance IL-6 can contribute to tumor
10 Evidence-Based Complementary and Alternative Medicine
CXCR4
CXCL1210 kDa
훽-actin43 kDa
41 kDa
NSQRHX
00
05
10
15
Relat
ive p
rote
in ex
pres
sion
CXCL12 CXCR4
lowastlowastlowastlowast
(a)
p-JAK2
p-STAT3
훽-actin43 kDa
90 kDa
120 kDa
NSQRHX
p-JAK2 p-STAT300
05
10
15
Relat
ive p
rote
in ex
pres
sion
lowastlowast lowastlowast
(b)
Figure 9 QRXH regulates TAMs by inhibiting the CXCL12CXCR4JAK2STAT3 signaling pathways Mice with subcutaneous tumor weredealt with NS and QRHX Representative images of western blot and densitometry analysis showing the expressions of CXCL12 CXCR4p-JAK2 and p-STAT3 in tumor Compared with NS group lowastlowast119875 lt 001
cell survival and upregulate the antiapoptotic genes by driv-ing JAK2STAT3 signal [43] IFN- activates macrophageby JAK-STAT signaling pathway [42] What is more JAK2is associated with CXCR4 [39] Then we can draw thatTAMs in malignant tumors tend to M2 subtype possiblythrough CXCL12CXCR4JAK2STAT3 signaling pathwayQRHX treatment blocked the response of macrophages totumor signals by suppressing CXCL12CXCR4JAK2STAT3expression and induced a remarkable decrease of the recruit-ment of M2 macrophages suggesting the attenuation of M2subtype cells function by QRHX
In TCM theory TCM formulae have abundant medicinalmaterials and then regulate diseases through multitargetsandmultiways Although the chemical constituents of QRHXhave been separated and identified by HPLUNG CANCER-QTOF MS-UV method in our team (Supplementary Fig-ure 1 and Table 1) the ingredients are various and theirfunctions are enormous So there may be other mechanismsinvolved in macrophage polarization What is more there
are diverse types cells in tumor except for tumor cells andmacrophage Moreover it is worth mentioning that TAMswere not separated and extracted from tumor tissue CXCL12CXCR4 JAK2 and STAT3 can not only play significantrole in tumor cell or macrophage but also other cellssuch as T lymphocyte neutrophils tumor-associated fibrob-last and endothelial cells Therefore QRHX inhibited theCXCL12CXCR4JAK2STAT3 axis possibly through othercells Taken together further investigations are needed toidentify direct molecular targets of QRHX in macrophagesin the context of cancer
5 Conclusion
In conclusion data from this study revealed that QRHXcould suppress cancer progression by inhibiting the tumorpromotion of TAMs in subcutaneous mice model whichcould contribute to elucidating the underlying regulatorymode of QRHX on lung cancer treatment
Evidence-Based Complementary and Alternative Medicine 11
Abbreviations
ELISA Enzyme-linked immunosorbent assayFITC Fluorescein isothiocyanateTNF-120572 Tumor necrosis factor-120572IL-6 Interleukin-6iNOS Inducible nitric oxide synthaseVEGF Vascular endothelial growth factorQT-PCR Quantitative Polymerase Chain ReactionIF ImmunofluorescenceIHC ImmunohistochemicalTAMs Tumor-associated macrophagesArg-1 Arginase 1
Conflicts of Interest
The authors declare that there are no conflicts of interest
Authorsrsquo Contributions
Fei Xu and Wenqiang Cui contributed equally to this workFei Xu and Baojun Liu conceived and designed experimentsFei Xu and Wenqiang Cui analyzed data and wrote themanuscript Jingcheng Dong supervised the project Zhengx-iao Zhao and Ying Wei assisted in experimental design anddata evaluation Jiaqi Liu Mihui Li Qiuping Li Chen Yanand Jian Qiu performed the study All authors reviewed andapproved the manuscript
Acknowledgments
This study was supported by Natural Science Foundation ofChina (nos 81673916 and 81403148) Development Projectof Shanghai Peak Disciplines-Integrative Medicine (no20150407)
References
[1] I Ikonomidis C A Michalakeas J Parissis et al ldquoInflamma-tory markers in coronary artery diseaserdquo BioFactors vol 38 no5 pp 320ndash328 2012
[2] P Lee C C Leung M I Restrepo K Takahashi Y Song and JM Porcel ldquoYear in review 2015 lung cancer pleural diseasesrespiratory infections bronchiectasis and tuberculosis bron-choscopic intervention and imagingrdquo Respirology vol 21 no 5pp 961ndash967 2016
[3] S V Sharma DW Bell J Settleman and D A Haber ldquoEpider-mal growth factor receptor mutations in lung cancerrdquo NatureReviews Cancer vol 7 no 3 pp 169ndash181 2007
[4] D Leong R Rai B Nguyen A Lee and D Yip ldquoAdvancesin adjuvant systemic therapy for non-small-cell lung cancerrdquoWorld Journal of Clinical Oncology vol 5 no 4 pp 633ndash6452014
[5] M T Villanueva ldquoMicroenvironment the new midfielders inthe tumourmicroenvironmentrdquoNature reviews Cancer vol 14no 12 p 765 2014
[6] A Mantovani S Sozzani M Locati P Allavena and A SicaldquoMacrophage polarization tumor-associated macrophages as aparadigm for polarizedM2mononuclear phagocytesrdquoTrends inImmunology vol 23 no 11 pp 549ndash555 2002
[7] A Mantovani and M Locati ldquoTumor-associated macrophagesas a paradigm of macrophage plasticity diversity and polariza-tion lessons and open questionsrdquo Arteriosclerosis Thrombosisand Vascular Biology vol 33 no 7 pp 1478ndash1483 2013
[8] W Hu X Li C Zhang Y Yang J Jiang and C Wu ldquoTumor-associated macrophages in cancersrdquo Clinical and TranslationalOncology vol 18 no 3 pp 251ndash258 2016
[9] B Z Qian and J W Pollard ldquoMacrophage diversity enhancestumor progression and metastasisrdquo Cell vol 141 no 1 pp 39ndash51 2010
[10] A Mantovani and P Allavena ldquoThe interaction of anti-cancer therapies with tumor-associated macrophagesrdquo Journalof Experimental Medicine vol 212 no 4 pp 435ndash445 2015
[11] J Xu Z Song Q Guo and J Li ldquoSynergistic effect and molec-ular mechanisms of traditional Chinese medicine on regulatingtumor microenvironment and cancer cellsrdquo BioMed ResearchInternational vol 2016 Article ID 1490738 pp 1ndash14 2016
[12] J Wu J Xu E A Eksioglu et al ldquoIcariside II induces apoptosisof melanoma cells through the downregulation of survivalpathwaysrdquoNutrition and Cancer vol 65 no 1 pp 110ndash117 2013
[13] L Kong J Liu J Wang et al ldquoIcariin inhibits TNF-120572IFN-120574induced inflammatory response via inhibition of the substanceP and p38-MAPK signaling pathway in human keratinocytesrdquoInternational Immunopharmacology vol 29 no 2 pp 401ndash4072015
[14] JWu J Du C Xu et al ldquoIn vivo and in vitro anti-inflammatoryeffects of a novel derivative of icariinrdquo Immunopharmacologyand Immunotoxicology vol 33 no 1 pp 49ndash54 2010
[15] E M Conway L A Pikor S H Y Kung et al ldquoMacrophagesinflammation and lung cancerrdquoAmerican Journal of Respiratoryand Critical Care Medicine vol 193 no 2 pp 116ndash130 2016
[16] S Shalapour and M Karin ldquoImmunity inflammation andcancer an eternal fight between good and evilrdquo The Journal ofClinical Investigation vol 125 no 9 pp 3347ndash3355 2015
[17] L Bingle N J Brown and C E Lewis ldquoThe role of tumour-associated macrophages in tumour progression Implicationsfor new anticancer therapiesrdquo Journal of Pathology vol 196 no3 pp 254ndash265 2002
[18] D Kim J Kim J H Yoon et al ldquoCXCL12 secreted from adiposetissue recruits macrophages and induces insulin resistance inmicerdquo Diabetologia vol 57 no 7 pp 1456ndash1465 2014
[19] M C Schmid C J Avraamides P Foubert et al ldquoCombinedblockade of integrin-12057241205731 plus cytokines SDF-1120572 or IL-1120573potently inhibits tumor inflammation and growthrdquo CancerResearch vol 71 no 22 pp 6965ndash6975 2011
[20] F Balkwill and A Mantovani ldquoInflammation and cancer backto VirchowrdquoThe Lancet vol 357 no 9255 pp 539ndash545 2001
[21] B B Aggarwal S Shishodia S K Sandur M K Pandey andG Sethi ldquoInflammation and cancer how hot is the linkrdquoBiochemical Pharmacology vol 72 no 11 pp 1605ndash1621 2006
[22] W-L Jiang X-G Chen H-B Zhu Y-B Gao J-W Tian andF-H Fu ldquoPaeoniflorin inhibits systemic inflammation andimproves survival in experimental sepsisrdquo Basic and ClinicalPharmacology and Toxicology vol 105 no 1 pp 64ndash71 2009
[23] I D Kim and B J Ha ldquoThe effects of paeoniflorin on LPS-induced liver inflammatory reactionsrdquo Archives of PharmacalResearch vol 33 no 6 pp 959ndash966 2010
[24] G-W Fan Y Zhang X Jiang et al ldquoAnti-inflammatory activityof baicalein in LPS-stimulated RAW2647 macrophages viaestrogen receptor and NF-120581B-dependent pathwaysrdquo Inflamma-tion vol 36 no 6 pp 1584ndash1591 2013
12 Evidence-Based Complementary and Alternative Medicine
[25] M Li-Weber ldquoNew therapeutic aspects of flavones the anti-cancer properties of Scutellaria and its main active constituentsWogonin Baicalein and Baicalinrdquo Cancer Treatment Reviewsvol 35 no 1 pp 57ndash68 2009
[26] QWu G-L Chen Y-J Li Y Chen and F-Z Lin ldquoPaeoniflorininhibits macrophage-mediated lung cancer metastasisrdquoChineseJournal of Natural Medicines vol 13 no 12 pp 925ndash932 2015
[27] W Lin and M Karin ldquoA cytokine-mediated link betweeninnate immunity inflammation and cancerrdquo Journal of ClinicalInvestigation vol 117 no 5 pp 1175ndash1183 2007
[28] T L Whiteside ldquoThe tumor microenvironment and its role inpromoting tumor growthrdquo Oncogene vol 27 no 45 pp 5904ndash5912 2008
[29] G Lorusso and C Ruegg ldquoThe tumor microenvironment andits contribution to tumor evolution toward metastasisrdquo Histo-chemistry and Cell Biology vol 130 no 6 pp 1091ndash1103 2008
[30] L Ding G Liang Z Yao et al ldquoMetformin prevents cancermetastasis by inhibiting M2-like polarization of tumor associ-ated macrophagesrdquo Oncotarget vol 6 no 34 pp 36441ndash364552015
[31] A Yuan Y JHsiaoH Y Chen et al ldquoOpposite effects ofM1 andM2macrophage subtypes on lung cancer progressionrdquo ScientificReports vol 5 Article ID 14273 2015
[32] F Balkwill ldquoCancer and the chemokine networkrdquo NatureReviews Cancer vol 4 no 7 pp 540ndash550 2004
[33] J A Burger and T J Kipps ldquoCXCR4 a key receptor in thecrosstalk between tumor cells and their microenvironmentrdquoBlood vol 107 no 5 pp 1761ndash1767 2006
[34] R J PhillipsM D BurdickM Lutz J A BelperioM P Keaneand R M Strieter ldquoThe stromal derived factor-1CXCL12-CXCchemokine receptor 4 biological axis in non-small cell lungcancermetastasesrdquoAmerican Journal of Respiratory and CriticalCare Medicine vol 167 no 12 pp 1676ndash1686 2003
[35] N Obermajer R Muthuswamy K Odunsi R P Edwards andP Kalinski ldquoPGE-induced CXCL 12 production and CXCR4expression controls the accumulation of human MDSCs inovarian cancer environmentrdquo Cancer Research vol 71 no 24pp 7463ndash7470 2011
[36] L Sanchez-Martın A Estecha R Samaniego S Sanchez-Ramon M A Vega and P Sanchez-Mateos ldquoThe chemokineCXCL12 regulates monocyte-macrophage differentiation andRUNX3 expressionrdquo Blood vol 117 no 1 pp 88ndash97 2011
[37] K Beider H Bitner M Leiba et al ldquoMultiple myelomacells recruit tumor-supportive macrophages through theCXCR4CXCL12 axis and promote their polarization towardthe M2 phenotyperdquo Oncotarget vol 5 no 22 pp 11283ndash112962014
[38] J M Mota C A Leite and L E Souza ldquoPost-sepsis stateinduces tumor-associated macrophage accumulation throughCXCR4CXCL12 and favors tumor progression inmicerdquoCancerImmunology Research vol 4 no 4 pp 312ndash322 2016
[39] B A Teicher and S P Fricker ldquoCXCL12 (SDF-1)CXCR4 path-way in cancerrdquoClinical Cancer Research vol 16 no 11 pp 2927ndash2931 2010
[40] P C Heinrich I Behrmann G Muller-Newen F Schaper andL Graeve ldquoInterleukin-6-type cytokine signalling through thegp130JakSTAT pathwayrdquo Biochemical Journal vol 334 part 2pp 297ndash314 1998
[41] M Kortylewski M Kujawski T Wang et al ldquoInhibiting Stat3signaling in the hematopoietic system elicits multicomponentantitumor immunityrdquo Nature Medicine vol 11 no 12 pp 1314ndash1321 2005
[42] X Hu J Chen L Wang and L B Ivashkiv ldquoCrosstalk amongJak-STAT Toll-like receptor and ITAM-dependent pathways inmacrophage activationrdquo Journal of Leukocyte Biology vol 82no 2 pp 237ndash243 2007
[43] R Catlett-Falcone T H Landowski M M Oshiro et alldquoConstitutive activation of Stat3 signaling confers resistance toapoptosis in humanU266myeloma cellsrdquo Immunity vol 10 no1 pp 105ndash115 1999
Submit your manuscripts athttpswwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 9CD
31
NS QRHX
VEG
F
0
5
10
15
20
25
VEGFMVD
Den
sity
of V
EGF
MV
D co
unt
lowastlowast
lowastlowast
(a)
00
02
04
06
08
10
Relat
ive p
rote
in ex
pres
sion
CD31VEGF
NSQRHX
lowastlowastlowastlowast
VEGF
CD31
훽-actin43 kDa
100 kDa
43 kDa
(b)
Figure 8 QRHX inhibits angiogenesis in lung cancer Mice with subcutaneous tumors were dealt with NS and QRHX (a) The nucleus wasdyed as blue CD31 was dyed as brown MVD were performed at high power field (times200) (b) The VEGF and CD31 protein expression intumor tissues was detected by western blot Data was expressed as means plusmn SEM values (119899 = 4) Compared with NS group lowastlowast119875 lt 001
human MDSCs and is an independent prognostic factor fortumor progression [35] Several studies have reported thatCXCL12 plays an important role in monocyte recruitmentdifferentiation and function [18 36] In a mouse modelof lung cancer CXCL12 could recruit tumor-promotingmyeloid CD11b+ cells [19] Besides powerful evidencesindicate a role for CXCR4-CXCL12 axis in promotingmacrophages polarization toward theM2 phenotype [37 38]In addition M2 subpopulation is associated with angio-genic factors such as VEGF and CXCL12-CXCR4 axis canalso promote tumor vascularization [39] Excitedly ourresults showed QRHX treatment inhibited signaling fromtumor cells to macrophages through inducing a remark-able decrease in CXCL12 and CXCR4 Furthermore QRHX
inhibited angiogenesis likely through altering the tumormicroenvironment by targeting TAMs
In STAT family there are seven proteins STAT3 is one ofthem It is a key transcription factor transducing signals fromactivated receptors or intracellular kinases to the nucleus andcan be activated in tumor cells and immune cells [40] Intumor STAT3 could contribute to cancer development andprogression inhibit apoptosis of tumors and help tumorescape immune system by suppressing the immune response[41] Evidence indicated that when STAT3 binds to somereceptor it can be activated through Janus Kinases (JAKs)such as JAK2 [42] Accumulating evidence implicates theimportant role of JAK2STAT3 in tumor and macrophagepolarization [27] For instance IL-6 can contribute to tumor
10 Evidence-Based Complementary and Alternative Medicine
CXCR4
CXCL1210 kDa
훽-actin43 kDa
41 kDa
NSQRHX
00
05
10
15
Relat
ive p
rote
in ex
pres
sion
CXCL12 CXCR4
lowastlowastlowastlowast
(a)
p-JAK2
p-STAT3
훽-actin43 kDa
90 kDa
120 kDa
NSQRHX
p-JAK2 p-STAT300
05
10
15
Relat
ive p
rote
in ex
pres
sion
lowastlowast lowastlowast
(b)
Figure 9 QRXH regulates TAMs by inhibiting the CXCL12CXCR4JAK2STAT3 signaling pathways Mice with subcutaneous tumor weredealt with NS and QRHX Representative images of western blot and densitometry analysis showing the expressions of CXCL12 CXCR4p-JAK2 and p-STAT3 in tumor Compared with NS group lowastlowast119875 lt 001
cell survival and upregulate the antiapoptotic genes by driv-ing JAK2STAT3 signal [43] IFN- activates macrophageby JAK-STAT signaling pathway [42] What is more JAK2is associated with CXCR4 [39] Then we can draw thatTAMs in malignant tumors tend to M2 subtype possiblythrough CXCL12CXCR4JAK2STAT3 signaling pathwayQRHX treatment blocked the response of macrophages totumor signals by suppressing CXCL12CXCR4JAK2STAT3expression and induced a remarkable decrease of the recruit-ment of M2 macrophages suggesting the attenuation of M2subtype cells function by QRHX
In TCM theory TCM formulae have abundant medicinalmaterials and then regulate diseases through multitargetsandmultiways Although the chemical constituents of QRHXhave been separated and identified by HPLUNG CANCER-QTOF MS-UV method in our team (Supplementary Fig-ure 1 and Table 1) the ingredients are various and theirfunctions are enormous So there may be other mechanismsinvolved in macrophage polarization What is more there
are diverse types cells in tumor except for tumor cells andmacrophage Moreover it is worth mentioning that TAMswere not separated and extracted from tumor tissue CXCL12CXCR4 JAK2 and STAT3 can not only play significantrole in tumor cell or macrophage but also other cellssuch as T lymphocyte neutrophils tumor-associated fibrob-last and endothelial cells Therefore QRHX inhibited theCXCL12CXCR4JAK2STAT3 axis possibly through othercells Taken together further investigations are needed toidentify direct molecular targets of QRHX in macrophagesin the context of cancer
5 Conclusion
In conclusion data from this study revealed that QRHXcould suppress cancer progression by inhibiting the tumorpromotion of TAMs in subcutaneous mice model whichcould contribute to elucidating the underlying regulatorymode of QRHX on lung cancer treatment
Evidence-Based Complementary and Alternative Medicine 11
Abbreviations
ELISA Enzyme-linked immunosorbent assayFITC Fluorescein isothiocyanateTNF-120572 Tumor necrosis factor-120572IL-6 Interleukin-6iNOS Inducible nitric oxide synthaseVEGF Vascular endothelial growth factorQT-PCR Quantitative Polymerase Chain ReactionIF ImmunofluorescenceIHC ImmunohistochemicalTAMs Tumor-associated macrophagesArg-1 Arginase 1
Conflicts of Interest
The authors declare that there are no conflicts of interest
Authorsrsquo Contributions
Fei Xu and Wenqiang Cui contributed equally to this workFei Xu and Baojun Liu conceived and designed experimentsFei Xu and Wenqiang Cui analyzed data and wrote themanuscript Jingcheng Dong supervised the project Zhengx-iao Zhao and Ying Wei assisted in experimental design anddata evaluation Jiaqi Liu Mihui Li Qiuping Li Chen Yanand Jian Qiu performed the study All authors reviewed andapproved the manuscript
Acknowledgments
This study was supported by Natural Science Foundation ofChina (nos 81673916 and 81403148) Development Projectof Shanghai Peak Disciplines-Integrative Medicine (no20150407)
References
[1] I Ikonomidis C A Michalakeas J Parissis et al ldquoInflamma-tory markers in coronary artery diseaserdquo BioFactors vol 38 no5 pp 320ndash328 2012
[2] P Lee C C Leung M I Restrepo K Takahashi Y Song and JM Porcel ldquoYear in review 2015 lung cancer pleural diseasesrespiratory infections bronchiectasis and tuberculosis bron-choscopic intervention and imagingrdquo Respirology vol 21 no 5pp 961ndash967 2016
[3] S V Sharma DW Bell J Settleman and D A Haber ldquoEpider-mal growth factor receptor mutations in lung cancerrdquo NatureReviews Cancer vol 7 no 3 pp 169ndash181 2007
[4] D Leong R Rai B Nguyen A Lee and D Yip ldquoAdvancesin adjuvant systemic therapy for non-small-cell lung cancerrdquoWorld Journal of Clinical Oncology vol 5 no 4 pp 633ndash6452014
[5] M T Villanueva ldquoMicroenvironment the new midfielders inthe tumourmicroenvironmentrdquoNature reviews Cancer vol 14no 12 p 765 2014
[6] A Mantovani S Sozzani M Locati P Allavena and A SicaldquoMacrophage polarization tumor-associated macrophages as aparadigm for polarizedM2mononuclear phagocytesrdquoTrends inImmunology vol 23 no 11 pp 549ndash555 2002
[7] A Mantovani and M Locati ldquoTumor-associated macrophagesas a paradigm of macrophage plasticity diversity and polariza-tion lessons and open questionsrdquo Arteriosclerosis Thrombosisand Vascular Biology vol 33 no 7 pp 1478ndash1483 2013
[8] W Hu X Li C Zhang Y Yang J Jiang and C Wu ldquoTumor-associated macrophages in cancersrdquo Clinical and TranslationalOncology vol 18 no 3 pp 251ndash258 2016
[9] B Z Qian and J W Pollard ldquoMacrophage diversity enhancestumor progression and metastasisrdquo Cell vol 141 no 1 pp 39ndash51 2010
[10] A Mantovani and P Allavena ldquoThe interaction of anti-cancer therapies with tumor-associated macrophagesrdquo Journalof Experimental Medicine vol 212 no 4 pp 435ndash445 2015
[11] J Xu Z Song Q Guo and J Li ldquoSynergistic effect and molec-ular mechanisms of traditional Chinese medicine on regulatingtumor microenvironment and cancer cellsrdquo BioMed ResearchInternational vol 2016 Article ID 1490738 pp 1ndash14 2016
[12] J Wu J Xu E A Eksioglu et al ldquoIcariside II induces apoptosisof melanoma cells through the downregulation of survivalpathwaysrdquoNutrition and Cancer vol 65 no 1 pp 110ndash117 2013
[13] L Kong J Liu J Wang et al ldquoIcariin inhibits TNF-120572IFN-120574induced inflammatory response via inhibition of the substanceP and p38-MAPK signaling pathway in human keratinocytesrdquoInternational Immunopharmacology vol 29 no 2 pp 401ndash4072015
[14] JWu J Du C Xu et al ldquoIn vivo and in vitro anti-inflammatoryeffects of a novel derivative of icariinrdquo Immunopharmacologyand Immunotoxicology vol 33 no 1 pp 49ndash54 2010
[15] E M Conway L A Pikor S H Y Kung et al ldquoMacrophagesinflammation and lung cancerrdquoAmerican Journal of Respiratoryand Critical Care Medicine vol 193 no 2 pp 116ndash130 2016
[16] S Shalapour and M Karin ldquoImmunity inflammation andcancer an eternal fight between good and evilrdquo The Journal ofClinical Investigation vol 125 no 9 pp 3347ndash3355 2015
[17] L Bingle N J Brown and C E Lewis ldquoThe role of tumour-associated macrophages in tumour progression Implicationsfor new anticancer therapiesrdquo Journal of Pathology vol 196 no3 pp 254ndash265 2002
[18] D Kim J Kim J H Yoon et al ldquoCXCL12 secreted from adiposetissue recruits macrophages and induces insulin resistance inmicerdquo Diabetologia vol 57 no 7 pp 1456ndash1465 2014
[19] M C Schmid C J Avraamides P Foubert et al ldquoCombinedblockade of integrin-12057241205731 plus cytokines SDF-1120572 or IL-1120573potently inhibits tumor inflammation and growthrdquo CancerResearch vol 71 no 22 pp 6965ndash6975 2011
[20] F Balkwill and A Mantovani ldquoInflammation and cancer backto VirchowrdquoThe Lancet vol 357 no 9255 pp 539ndash545 2001
[21] B B Aggarwal S Shishodia S K Sandur M K Pandey andG Sethi ldquoInflammation and cancer how hot is the linkrdquoBiochemical Pharmacology vol 72 no 11 pp 1605ndash1621 2006
[22] W-L Jiang X-G Chen H-B Zhu Y-B Gao J-W Tian andF-H Fu ldquoPaeoniflorin inhibits systemic inflammation andimproves survival in experimental sepsisrdquo Basic and ClinicalPharmacology and Toxicology vol 105 no 1 pp 64ndash71 2009
[23] I D Kim and B J Ha ldquoThe effects of paeoniflorin on LPS-induced liver inflammatory reactionsrdquo Archives of PharmacalResearch vol 33 no 6 pp 959ndash966 2010
[24] G-W Fan Y Zhang X Jiang et al ldquoAnti-inflammatory activityof baicalein in LPS-stimulated RAW2647 macrophages viaestrogen receptor and NF-120581B-dependent pathwaysrdquo Inflamma-tion vol 36 no 6 pp 1584ndash1591 2013
12 Evidence-Based Complementary and Alternative Medicine
[25] M Li-Weber ldquoNew therapeutic aspects of flavones the anti-cancer properties of Scutellaria and its main active constituentsWogonin Baicalein and Baicalinrdquo Cancer Treatment Reviewsvol 35 no 1 pp 57ndash68 2009
[26] QWu G-L Chen Y-J Li Y Chen and F-Z Lin ldquoPaeoniflorininhibits macrophage-mediated lung cancer metastasisrdquoChineseJournal of Natural Medicines vol 13 no 12 pp 925ndash932 2015
[27] W Lin and M Karin ldquoA cytokine-mediated link betweeninnate immunity inflammation and cancerrdquo Journal of ClinicalInvestigation vol 117 no 5 pp 1175ndash1183 2007
[28] T L Whiteside ldquoThe tumor microenvironment and its role inpromoting tumor growthrdquo Oncogene vol 27 no 45 pp 5904ndash5912 2008
[29] G Lorusso and C Ruegg ldquoThe tumor microenvironment andits contribution to tumor evolution toward metastasisrdquo Histo-chemistry and Cell Biology vol 130 no 6 pp 1091ndash1103 2008
[30] L Ding G Liang Z Yao et al ldquoMetformin prevents cancermetastasis by inhibiting M2-like polarization of tumor associ-ated macrophagesrdquo Oncotarget vol 6 no 34 pp 36441ndash364552015
[31] A Yuan Y JHsiaoH Y Chen et al ldquoOpposite effects ofM1 andM2macrophage subtypes on lung cancer progressionrdquo ScientificReports vol 5 Article ID 14273 2015
[32] F Balkwill ldquoCancer and the chemokine networkrdquo NatureReviews Cancer vol 4 no 7 pp 540ndash550 2004
[33] J A Burger and T J Kipps ldquoCXCR4 a key receptor in thecrosstalk between tumor cells and their microenvironmentrdquoBlood vol 107 no 5 pp 1761ndash1767 2006
[34] R J PhillipsM D BurdickM Lutz J A BelperioM P Keaneand R M Strieter ldquoThe stromal derived factor-1CXCL12-CXCchemokine receptor 4 biological axis in non-small cell lungcancermetastasesrdquoAmerican Journal of Respiratory and CriticalCare Medicine vol 167 no 12 pp 1676ndash1686 2003
[35] N Obermajer R Muthuswamy K Odunsi R P Edwards andP Kalinski ldquoPGE-induced CXCL 12 production and CXCR4expression controls the accumulation of human MDSCs inovarian cancer environmentrdquo Cancer Research vol 71 no 24pp 7463ndash7470 2011
[36] L Sanchez-Martın A Estecha R Samaniego S Sanchez-Ramon M A Vega and P Sanchez-Mateos ldquoThe chemokineCXCL12 regulates monocyte-macrophage differentiation andRUNX3 expressionrdquo Blood vol 117 no 1 pp 88ndash97 2011
[37] K Beider H Bitner M Leiba et al ldquoMultiple myelomacells recruit tumor-supportive macrophages through theCXCR4CXCL12 axis and promote their polarization towardthe M2 phenotyperdquo Oncotarget vol 5 no 22 pp 11283ndash112962014
[38] J M Mota C A Leite and L E Souza ldquoPost-sepsis stateinduces tumor-associated macrophage accumulation throughCXCR4CXCL12 and favors tumor progression inmicerdquoCancerImmunology Research vol 4 no 4 pp 312ndash322 2016
[39] B A Teicher and S P Fricker ldquoCXCL12 (SDF-1)CXCR4 path-way in cancerrdquoClinical Cancer Research vol 16 no 11 pp 2927ndash2931 2010
[40] P C Heinrich I Behrmann G Muller-Newen F Schaper andL Graeve ldquoInterleukin-6-type cytokine signalling through thegp130JakSTAT pathwayrdquo Biochemical Journal vol 334 part 2pp 297ndash314 1998
[41] M Kortylewski M Kujawski T Wang et al ldquoInhibiting Stat3signaling in the hematopoietic system elicits multicomponentantitumor immunityrdquo Nature Medicine vol 11 no 12 pp 1314ndash1321 2005
[42] X Hu J Chen L Wang and L B Ivashkiv ldquoCrosstalk amongJak-STAT Toll-like receptor and ITAM-dependent pathways inmacrophage activationrdquo Journal of Leukocyte Biology vol 82no 2 pp 237ndash243 2007
[43] R Catlett-Falcone T H Landowski M M Oshiro et alldquoConstitutive activation of Stat3 signaling confers resistance toapoptosis in humanU266myeloma cellsrdquo Immunity vol 10 no1 pp 105ndash115 1999
Submit your manuscripts athttpswwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
10 Evidence-Based Complementary and Alternative Medicine
CXCR4
CXCL1210 kDa
훽-actin43 kDa
41 kDa
NSQRHX
00
05
10
15
Relat
ive p
rote
in ex
pres
sion
CXCL12 CXCR4
lowastlowastlowastlowast
(a)
p-JAK2
p-STAT3
훽-actin43 kDa
90 kDa
120 kDa
NSQRHX
p-JAK2 p-STAT300
05
10
15
Relat
ive p
rote
in ex
pres
sion
lowastlowast lowastlowast
(b)
Figure 9 QRXH regulates TAMs by inhibiting the CXCL12CXCR4JAK2STAT3 signaling pathways Mice with subcutaneous tumor weredealt with NS and QRHX Representative images of western blot and densitometry analysis showing the expressions of CXCL12 CXCR4p-JAK2 and p-STAT3 in tumor Compared with NS group lowastlowast119875 lt 001
cell survival and upregulate the antiapoptotic genes by driv-ing JAK2STAT3 signal [43] IFN- activates macrophageby JAK-STAT signaling pathway [42] What is more JAK2is associated with CXCR4 [39] Then we can draw thatTAMs in malignant tumors tend to M2 subtype possiblythrough CXCL12CXCR4JAK2STAT3 signaling pathwayQRHX treatment blocked the response of macrophages totumor signals by suppressing CXCL12CXCR4JAK2STAT3expression and induced a remarkable decrease of the recruit-ment of M2 macrophages suggesting the attenuation of M2subtype cells function by QRHX
In TCM theory TCM formulae have abundant medicinalmaterials and then regulate diseases through multitargetsandmultiways Although the chemical constituents of QRHXhave been separated and identified by HPLUNG CANCER-QTOF MS-UV method in our team (Supplementary Fig-ure 1 and Table 1) the ingredients are various and theirfunctions are enormous So there may be other mechanismsinvolved in macrophage polarization What is more there
are diverse types cells in tumor except for tumor cells andmacrophage Moreover it is worth mentioning that TAMswere not separated and extracted from tumor tissue CXCL12CXCR4 JAK2 and STAT3 can not only play significantrole in tumor cell or macrophage but also other cellssuch as T lymphocyte neutrophils tumor-associated fibrob-last and endothelial cells Therefore QRHX inhibited theCXCL12CXCR4JAK2STAT3 axis possibly through othercells Taken together further investigations are needed toidentify direct molecular targets of QRHX in macrophagesin the context of cancer
5 Conclusion
In conclusion data from this study revealed that QRHXcould suppress cancer progression by inhibiting the tumorpromotion of TAMs in subcutaneous mice model whichcould contribute to elucidating the underlying regulatorymode of QRHX on lung cancer treatment
Evidence-Based Complementary and Alternative Medicine 11
Abbreviations
ELISA Enzyme-linked immunosorbent assayFITC Fluorescein isothiocyanateTNF-120572 Tumor necrosis factor-120572IL-6 Interleukin-6iNOS Inducible nitric oxide synthaseVEGF Vascular endothelial growth factorQT-PCR Quantitative Polymerase Chain ReactionIF ImmunofluorescenceIHC ImmunohistochemicalTAMs Tumor-associated macrophagesArg-1 Arginase 1
Conflicts of Interest
The authors declare that there are no conflicts of interest
Authorsrsquo Contributions
Fei Xu and Wenqiang Cui contributed equally to this workFei Xu and Baojun Liu conceived and designed experimentsFei Xu and Wenqiang Cui analyzed data and wrote themanuscript Jingcheng Dong supervised the project Zhengx-iao Zhao and Ying Wei assisted in experimental design anddata evaluation Jiaqi Liu Mihui Li Qiuping Li Chen Yanand Jian Qiu performed the study All authors reviewed andapproved the manuscript
Acknowledgments
This study was supported by Natural Science Foundation ofChina (nos 81673916 and 81403148) Development Projectof Shanghai Peak Disciplines-Integrative Medicine (no20150407)
References
[1] I Ikonomidis C A Michalakeas J Parissis et al ldquoInflamma-tory markers in coronary artery diseaserdquo BioFactors vol 38 no5 pp 320ndash328 2012
[2] P Lee C C Leung M I Restrepo K Takahashi Y Song and JM Porcel ldquoYear in review 2015 lung cancer pleural diseasesrespiratory infections bronchiectasis and tuberculosis bron-choscopic intervention and imagingrdquo Respirology vol 21 no 5pp 961ndash967 2016
[3] S V Sharma DW Bell J Settleman and D A Haber ldquoEpider-mal growth factor receptor mutations in lung cancerrdquo NatureReviews Cancer vol 7 no 3 pp 169ndash181 2007
[4] D Leong R Rai B Nguyen A Lee and D Yip ldquoAdvancesin adjuvant systemic therapy for non-small-cell lung cancerrdquoWorld Journal of Clinical Oncology vol 5 no 4 pp 633ndash6452014
[5] M T Villanueva ldquoMicroenvironment the new midfielders inthe tumourmicroenvironmentrdquoNature reviews Cancer vol 14no 12 p 765 2014
[6] A Mantovani S Sozzani M Locati P Allavena and A SicaldquoMacrophage polarization tumor-associated macrophages as aparadigm for polarizedM2mononuclear phagocytesrdquoTrends inImmunology vol 23 no 11 pp 549ndash555 2002
[7] A Mantovani and M Locati ldquoTumor-associated macrophagesas a paradigm of macrophage plasticity diversity and polariza-tion lessons and open questionsrdquo Arteriosclerosis Thrombosisand Vascular Biology vol 33 no 7 pp 1478ndash1483 2013
[8] W Hu X Li C Zhang Y Yang J Jiang and C Wu ldquoTumor-associated macrophages in cancersrdquo Clinical and TranslationalOncology vol 18 no 3 pp 251ndash258 2016
[9] B Z Qian and J W Pollard ldquoMacrophage diversity enhancestumor progression and metastasisrdquo Cell vol 141 no 1 pp 39ndash51 2010
[10] A Mantovani and P Allavena ldquoThe interaction of anti-cancer therapies with tumor-associated macrophagesrdquo Journalof Experimental Medicine vol 212 no 4 pp 435ndash445 2015
[11] J Xu Z Song Q Guo and J Li ldquoSynergistic effect and molec-ular mechanisms of traditional Chinese medicine on regulatingtumor microenvironment and cancer cellsrdquo BioMed ResearchInternational vol 2016 Article ID 1490738 pp 1ndash14 2016
[12] J Wu J Xu E A Eksioglu et al ldquoIcariside II induces apoptosisof melanoma cells through the downregulation of survivalpathwaysrdquoNutrition and Cancer vol 65 no 1 pp 110ndash117 2013
[13] L Kong J Liu J Wang et al ldquoIcariin inhibits TNF-120572IFN-120574induced inflammatory response via inhibition of the substanceP and p38-MAPK signaling pathway in human keratinocytesrdquoInternational Immunopharmacology vol 29 no 2 pp 401ndash4072015
[14] JWu J Du C Xu et al ldquoIn vivo and in vitro anti-inflammatoryeffects of a novel derivative of icariinrdquo Immunopharmacologyand Immunotoxicology vol 33 no 1 pp 49ndash54 2010
[15] E M Conway L A Pikor S H Y Kung et al ldquoMacrophagesinflammation and lung cancerrdquoAmerican Journal of Respiratoryand Critical Care Medicine vol 193 no 2 pp 116ndash130 2016
[16] S Shalapour and M Karin ldquoImmunity inflammation andcancer an eternal fight between good and evilrdquo The Journal ofClinical Investigation vol 125 no 9 pp 3347ndash3355 2015
[17] L Bingle N J Brown and C E Lewis ldquoThe role of tumour-associated macrophages in tumour progression Implicationsfor new anticancer therapiesrdquo Journal of Pathology vol 196 no3 pp 254ndash265 2002
[18] D Kim J Kim J H Yoon et al ldquoCXCL12 secreted from adiposetissue recruits macrophages and induces insulin resistance inmicerdquo Diabetologia vol 57 no 7 pp 1456ndash1465 2014
[19] M C Schmid C J Avraamides P Foubert et al ldquoCombinedblockade of integrin-12057241205731 plus cytokines SDF-1120572 or IL-1120573potently inhibits tumor inflammation and growthrdquo CancerResearch vol 71 no 22 pp 6965ndash6975 2011
[20] F Balkwill and A Mantovani ldquoInflammation and cancer backto VirchowrdquoThe Lancet vol 357 no 9255 pp 539ndash545 2001
[21] B B Aggarwal S Shishodia S K Sandur M K Pandey andG Sethi ldquoInflammation and cancer how hot is the linkrdquoBiochemical Pharmacology vol 72 no 11 pp 1605ndash1621 2006
[22] W-L Jiang X-G Chen H-B Zhu Y-B Gao J-W Tian andF-H Fu ldquoPaeoniflorin inhibits systemic inflammation andimproves survival in experimental sepsisrdquo Basic and ClinicalPharmacology and Toxicology vol 105 no 1 pp 64ndash71 2009
[23] I D Kim and B J Ha ldquoThe effects of paeoniflorin on LPS-induced liver inflammatory reactionsrdquo Archives of PharmacalResearch vol 33 no 6 pp 959ndash966 2010
[24] G-W Fan Y Zhang X Jiang et al ldquoAnti-inflammatory activityof baicalein in LPS-stimulated RAW2647 macrophages viaestrogen receptor and NF-120581B-dependent pathwaysrdquo Inflamma-tion vol 36 no 6 pp 1584ndash1591 2013
12 Evidence-Based Complementary and Alternative Medicine
[25] M Li-Weber ldquoNew therapeutic aspects of flavones the anti-cancer properties of Scutellaria and its main active constituentsWogonin Baicalein and Baicalinrdquo Cancer Treatment Reviewsvol 35 no 1 pp 57ndash68 2009
[26] QWu G-L Chen Y-J Li Y Chen and F-Z Lin ldquoPaeoniflorininhibits macrophage-mediated lung cancer metastasisrdquoChineseJournal of Natural Medicines vol 13 no 12 pp 925ndash932 2015
[27] W Lin and M Karin ldquoA cytokine-mediated link betweeninnate immunity inflammation and cancerrdquo Journal of ClinicalInvestigation vol 117 no 5 pp 1175ndash1183 2007
[28] T L Whiteside ldquoThe tumor microenvironment and its role inpromoting tumor growthrdquo Oncogene vol 27 no 45 pp 5904ndash5912 2008
[29] G Lorusso and C Ruegg ldquoThe tumor microenvironment andits contribution to tumor evolution toward metastasisrdquo Histo-chemistry and Cell Biology vol 130 no 6 pp 1091ndash1103 2008
[30] L Ding G Liang Z Yao et al ldquoMetformin prevents cancermetastasis by inhibiting M2-like polarization of tumor associ-ated macrophagesrdquo Oncotarget vol 6 no 34 pp 36441ndash364552015
[31] A Yuan Y JHsiaoH Y Chen et al ldquoOpposite effects ofM1 andM2macrophage subtypes on lung cancer progressionrdquo ScientificReports vol 5 Article ID 14273 2015
[32] F Balkwill ldquoCancer and the chemokine networkrdquo NatureReviews Cancer vol 4 no 7 pp 540ndash550 2004
[33] J A Burger and T J Kipps ldquoCXCR4 a key receptor in thecrosstalk between tumor cells and their microenvironmentrdquoBlood vol 107 no 5 pp 1761ndash1767 2006
[34] R J PhillipsM D BurdickM Lutz J A BelperioM P Keaneand R M Strieter ldquoThe stromal derived factor-1CXCL12-CXCchemokine receptor 4 biological axis in non-small cell lungcancermetastasesrdquoAmerican Journal of Respiratory and CriticalCare Medicine vol 167 no 12 pp 1676ndash1686 2003
[35] N Obermajer R Muthuswamy K Odunsi R P Edwards andP Kalinski ldquoPGE-induced CXCL 12 production and CXCR4expression controls the accumulation of human MDSCs inovarian cancer environmentrdquo Cancer Research vol 71 no 24pp 7463ndash7470 2011
[36] L Sanchez-Martın A Estecha R Samaniego S Sanchez-Ramon M A Vega and P Sanchez-Mateos ldquoThe chemokineCXCL12 regulates monocyte-macrophage differentiation andRUNX3 expressionrdquo Blood vol 117 no 1 pp 88ndash97 2011
[37] K Beider H Bitner M Leiba et al ldquoMultiple myelomacells recruit tumor-supportive macrophages through theCXCR4CXCL12 axis and promote their polarization towardthe M2 phenotyperdquo Oncotarget vol 5 no 22 pp 11283ndash112962014
[38] J M Mota C A Leite and L E Souza ldquoPost-sepsis stateinduces tumor-associated macrophage accumulation throughCXCR4CXCL12 and favors tumor progression inmicerdquoCancerImmunology Research vol 4 no 4 pp 312ndash322 2016
[39] B A Teicher and S P Fricker ldquoCXCL12 (SDF-1)CXCR4 path-way in cancerrdquoClinical Cancer Research vol 16 no 11 pp 2927ndash2931 2010
[40] P C Heinrich I Behrmann G Muller-Newen F Schaper andL Graeve ldquoInterleukin-6-type cytokine signalling through thegp130JakSTAT pathwayrdquo Biochemical Journal vol 334 part 2pp 297ndash314 1998
[41] M Kortylewski M Kujawski T Wang et al ldquoInhibiting Stat3signaling in the hematopoietic system elicits multicomponentantitumor immunityrdquo Nature Medicine vol 11 no 12 pp 1314ndash1321 2005
[42] X Hu J Chen L Wang and L B Ivashkiv ldquoCrosstalk amongJak-STAT Toll-like receptor and ITAM-dependent pathways inmacrophage activationrdquo Journal of Leukocyte Biology vol 82no 2 pp 237ndash243 2007
[43] R Catlett-Falcone T H Landowski M M Oshiro et alldquoConstitutive activation of Stat3 signaling confers resistance toapoptosis in humanU266myeloma cellsrdquo Immunity vol 10 no1 pp 105ndash115 1999
Submit your manuscripts athttpswwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 11
Abbreviations
ELISA Enzyme-linked immunosorbent assayFITC Fluorescein isothiocyanateTNF-120572 Tumor necrosis factor-120572IL-6 Interleukin-6iNOS Inducible nitric oxide synthaseVEGF Vascular endothelial growth factorQT-PCR Quantitative Polymerase Chain ReactionIF ImmunofluorescenceIHC ImmunohistochemicalTAMs Tumor-associated macrophagesArg-1 Arginase 1
Conflicts of Interest
The authors declare that there are no conflicts of interest
Authorsrsquo Contributions
Fei Xu and Wenqiang Cui contributed equally to this workFei Xu and Baojun Liu conceived and designed experimentsFei Xu and Wenqiang Cui analyzed data and wrote themanuscript Jingcheng Dong supervised the project Zhengx-iao Zhao and Ying Wei assisted in experimental design anddata evaluation Jiaqi Liu Mihui Li Qiuping Li Chen Yanand Jian Qiu performed the study All authors reviewed andapproved the manuscript
Acknowledgments
This study was supported by Natural Science Foundation ofChina (nos 81673916 and 81403148) Development Projectof Shanghai Peak Disciplines-Integrative Medicine (no20150407)
References
[1] I Ikonomidis C A Michalakeas J Parissis et al ldquoInflamma-tory markers in coronary artery diseaserdquo BioFactors vol 38 no5 pp 320ndash328 2012
[2] P Lee C C Leung M I Restrepo K Takahashi Y Song and JM Porcel ldquoYear in review 2015 lung cancer pleural diseasesrespiratory infections bronchiectasis and tuberculosis bron-choscopic intervention and imagingrdquo Respirology vol 21 no 5pp 961ndash967 2016
[3] S V Sharma DW Bell J Settleman and D A Haber ldquoEpider-mal growth factor receptor mutations in lung cancerrdquo NatureReviews Cancer vol 7 no 3 pp 169ndash181 2007
[4] D Leong R Rai B Nguyen A Lee and D Yip ldquoAdvancesin adjuvant systemic therapy for non-small-cell lung cancerrdquoWorld Journal of Clinical Oncology vol 5 no 4 pp 633ndash6452014
[5] M T Villanueva ldquoMicroenvironment the new midfielders inthe tumourmicroenvironmentrdquoNature reviews Cancer vol 14no 12 p 765 2014
[6] A Mantovani S Sozzani M Locati P Allavena and A SicaldquoMacrophage polarization tumor-associated macrophages as aparadigm for polarizedM2mononuclear phagocytesrdquoTrends inImmunology vol 23 no 11 pp 549ndash555 2002
[7] A Mantovani and M Locati ldquoTumor-associated macrophagesas a paradigm of macrophage plasticity diversity and polariza-tion lessons and open questionsrdquo Arteriosclerosis Thrombosisand Vascular Biology vol 33 no 7 pp 1478ndash1483 2013
[8] W Hu X Li C Zhang Y Yang J Jiang and C Wu ldquoTumor-associated macrophages in cancersrdquo Clinical and TranslationalOncology vol 18 no 3 pp 251ndash258 2016
[9] B Z Qian and J W Pollard ldquoMacrophage diversity enhancestumor progression and metastasisrdquo Cell vol 141 no 1 pp 39ndash51 2010
[10] A Mantovani and P Allavena ldquoThe interaction of anti-cancer therapies with tumor-associated macrophagesrdquo Journalof Experimental Medicine vol 212 no 4 pp 435ndash445 2015
[11] J Xu Z Song Q Guo and J Li ldquoSynergistic effect and molec-ular mechanisms of traditional Chinese medicine on regulatingtumor microenvironment and cancer cellsrdquo BioMed ResearchInternational vol 2016 Article ID 1490738 pp 1ndash14 2016
[12] J Wu J Xu E A Eksioglu et al ldquoIcariside II induces apoptosisof melanoma cells through the downregulation of survivalpathwaysrdquoNutrition and Cancer vol 65 no 1 pp 110ndash117 2013
[13] L Kong J Liu J Wang et al ldquoIcariin inhibits TNF-120572IFN-120574induced inflammatory response via inhibition of the substanceP and p38-MAPK signaling pathway in human keratinocytesrdquoInternational Immunopharmacology vol 29 no 2 pp 401ndash4072015
[14] JWu J Du C Xu et al ldquoIn vivo and in vitro anti-inflammatoryeffects of a novel derivative of icariinrdquo Immunopharmacologyand Immunotoxicology vol 33 no 1 pp 49ndash54 2010
[15] E M Conway L A Pikor S H Y Kung et al ldquoMacrophagesinflammation and lung cancerrdquoAmerican Journal of Respiratoryand Critical Care Medicine vol 193 no 2 pp 116ndash130 2016
[16] S Shalapour and M Karin ldquoImmunity inflammation andcancer an eternal fight between good and evilrdquo The Journal ofClinical Investigation vol 125 no 9 pp 3347ndash3355 2015
[17] L Bingle N J Brown and C E Lewis ldquoThe role of tumour-associated macrophages in tumour progression Implicationsfor new anticancer therapiesrdquo Journal of Pathology vol 196 no3 pp 254ndash265 2002
[18] D Kim J Kim J H Yoon et al ldquoCXCL12 secreted from adiposetissue recruits macrophages and induces insulin resistance inmicerdquo Diabetologia vol 57 no 7 pp 1456ndash1465 2014
[19] M C Schmid C J Avraamides P Foubert et al ldquoCombinedblockade of integrin-12057241205731 plus cytokines SDF-1120572 or IL-1120573potently inhibits tumor inflammation and growthrdquo CancerResearch vol 71 no 22 pp 6965ndash6975 2011
[20] F Balkwill and A Mantovani ldquoInflammation and cancer backto VirchowrdquoThe Lancet vol 357 no 9255 pp 539ndash545 2001
[21] B B Aggarwal S Shishodia S K Sandur M K Pandey andG Sethi ldquoInflammation and cancer how hot is the linkrdquoBiochemical Pharmacology vol 72 no 11 pp 1605ndash1621 2006
[22] W-L Jiang X-G Chen H-B Zhu Y-B Gao J-W Tian andF-H Fu ldquoPaeoniflorin inhibits systemic inflammation andimproves survival in experimental sepsisrdquo Basic and ClinicalPharmacology and Toxicology vol 105 no 1 pp 64ndash71 2009
[23] I D Kim and B J Ha ldquoThe effects of paeoniflorin on LPS-induced liver inflammatory reactionsrdquo Archives of PharmacalResearch vol 33 no 6 pp 959ndash966 2010
[24] G-W Fan Y Zhang X Jiang et al ldquoAnti-inflammatory activityof baicalein in LPS-stimulated RAW2647 macrophages viaestrogen receptor and NF-120581B-dependent pathwaysrdquo Inflamma-tion vol 36 no 6 pp 1584ndash1591 2013
12 Evidence-Based Complementary and Alternative Medicine
[25] M Li-Weber ldquoNew therapeutic aspects of flavones the anti-cancer properties of Scutellaria and its main active constituentsWogonin Baicalein and Baicalinrdquo Cancer Treatment Reviewsvol 35 no 1 pp 57ndash68 2009
[26] QWu G-L Chen Y-J Li Y Chen and F-Z Lin ldquoPaeoniflorininhibits macrophage-mediated lung cancer metastasisrdquoChineseJournal of Natural Medicines vol 13 no 12 pp 925ndash932 2015
[27] W Lin and M Karin ldquoA cytokine-mediated link betweeninnate immunity inflammation and cancerrdquo Journal of ClinicalInvestigation vol 117 no 5 pp 1175ndash1183 2007
[28] T L Whiteside ldquoThe tumor microenvironment and its role inpromoting tumor growthrdquo Oncogene vol 27 no 45 pp 5904ndash5912 2008
[29] G Lorusso and C Ruegg ldquoThe tumor microenvironment andits contribution to tumor evolution toward metastasisrdquo Histo-chemistry and Cell Biology vol 130 no 6 pp 1091ndash1103 2008
[30] L Ding G Liang Z Yao et al ldquoMetformin prevents cancermetastasis by inhibiting M2-like polarization of tumor associ-ated macrophagesrdquo Oncotarget vol 6 no 34 pp 36441ndash364552015
[31] A Yuan Y JHsiaoH Y Chen et al ldquoOpposite effects ofM1 andM2macrophage subtypes on lung cancer progressionrdquo ScientificReports vol 5 Article ID 14273 2015
[32] F Balkwill ldquoCancer and the chemokine networkrdquo NatureReviews Cancer vol 4 no 7 pp 540ndash550 2004
[33] J A Burger and T J Kipps ldquoCXCR4 a key receptor in thecrosstalk between tumor cells and their microenvironmentrdquoBlood vol 107 no 5 pp 1761ndash1767 2006
[34] R J PhillipsM D BurdickM Lutz J A BelperioM P Keaneand R M Strieter ldquoThe stromal derived factor-1CXCL12-CXCchemokine receptor 4 biological axis in non-small cell lungcancermetastasesrdquoAmerican Journal of Respiratory and CriticalCare Medicine vol 167 no 12 pp 1676ndash1686 2003
[35] N Obermajer R Muthuswamy K Odunsi R P Edwards andP Kalinski ldquoPGE-induced CXCL 12 production and CXCR4expression controls the accumulation of human MDSCs inovarian cancer environmentrdquo Cancer Research vol 71 no 24pp 7463ndash7470 2011
[36] L Sanchez-Martın A Estecha R Samaniego S Sanchez-Ramon M A Vega and P Sanchez-Mateos ldquoThe chemokineCXCL12 regulates monocyte-macrophage differentiation andRUNX3 expressionrdquo Blood vol 117 no 1 pp 88ndash97 2011
[37] K Beider H Bitner M Leiba et al ldquoMultiple myelomacells recruit tumor-supportive macrophages through theCXCR4CXCL12 axis and promote their polarization towardthe M2 phenotyperdquo Oncotarget vol 5 no 22 pp 11283ndash112962014
[38] J M Mota C A Leite and L E Souza ldquoPost-sepsis stateinduces tumor-associated macrophage accumulation throughCXCR4CXCL12 and favors tumor progression inmicerdquoCancerImmunology Research vol 4 no 4 pp 312ndash322 2016
[39] B A Teicher and S P Fricker ldquoCXCL12 (SDF-1)CXCR4 path-way in cancerrdquoClinical Cancer Research vol 16 no 11 pp 2927ndash2931 2010
[40] P C Heinrich I Behrmann G Muller-Newen F Schaper andL Graeve ldquoInterleukin-6-type cytokine signalling through thegp130JakSTAT pathwayrdquo Biochemical Journal vol 334 part 2pp 297ndash314 1998
[41] M Kortylewski M Kujawski T Wang et al ldquoInhibiting Stat3signaling in the hematopoietic system elicits multicomponentantitumor immunityrdquo Nature Medicine vol 11 no 12 pp 1314ndash1321 2005
[42] X Hu J Chen L Wang and L B Ivashkiv ldquoCrosstalk amongJak-STAT Toll-like receptor and ITAM-dependent pathways inmacrophage activationrdquo Journal of Leukocyte Biology vol 82no 2 pp 237ndash243 2007
[43] R Catlett-Falcone T H Landowski M M Oshiro et alldquoConstitutive activation of Stat3 signaling confers resistance toapoptosis in humanU266myeloma cellsrdquo Immunity vol 10 no1 pp 105ndash115 1999
Submit your manuscripts athttpswwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
12 Evidence-Based Complementary and Alternative Medicine
[25] M Li-Weber ldquoNew therapeutic aspects of flavones the anti-cancer properties of Scutellaria and its main active constituentsWogonin Baicalein and Baicalinrdquo Cancer Treatment Reviewsvol 35 no 1 pp 57ndash68 2009
[26] QWu G-L Chen Y-J Li Y Chen and F-Z Lin ldquoPaeoniflorininhibits macrophage-mediated lung cancer metastasisrdquoChineseJournal of Natural Medicines vol 13 no 12 pp 925ndash932 2015
[27] W Lin and M Karin ldquoA cytokine-mediated link betweeninnate immunity inflammation and cancerrdquo Journal of ClinicalInvestigation vol 117 no 5 pp 1175ndash1183 2007
[28] T L Whiteside ldquoThe tumor microenvironment and its role inpromoting tumor growthrdquo Oncogene vol 27 no 45 pp 5904ndash5912 2008
[29] G Lorusso and C Ruegg ldquoThe tumor microenvironment andits contribution to tumor evolution toward metastasisrdquo Histo-chemistry and Cell Biology vol 130 no 6 pp 1091ndash1103 2008
[30] L Ding G Liang Z Yao et al ldquoMetformin prevents cancermetastasis by inhibiting M2-like polarization of tumor associ-ated macrophagesrdquo Oncotarget vol 6 no 34 pp 36441ndash364552015
[31] A Yuan Y JHsiaoH Y Chen et al ldquoOpposite effects ofM1 andM2macrophage subtypes on lung cancer progressionrdquo ScientificReports vol 5 Article ID 14273 2015
[32] F Balkwill ldquoCancer and the chemokine networkrdquo NatureReviews Cancer vol 4 no 7 pp 540ndash550 2004
[33] J A Burger and T J Kipps ldquoCXCR4 a key receptor in thecrosstalk between tumor cells and their microenvironmentrdquoBlood vol 107 no 5 pp 1761ndash1767 2006
[34] R J PhillipsM D BurdickM Lutz J A BelperioM P Keaneand R M Strieter ldquoThe stromal derived factor-1CXCL12-CXCchemokine receptor 4 biological axis in non-small cell lungcancermetastasesrdquoAmerican Journal of Respiratory and CriticalCare Medicine vol 167 no 12 pp 1676ndash1686 2003
[35] N Obermajer R Muthuswamy K Odunsi R P Edwards andP Kalinski ldquoPGE-induced CXCL 12 production and CXCR4expression controls the accumulation of human MDSCs inovarian cancer environmentrdquo Cancer Research vol 71 no 24pp 7463ndash7470 2011
[36] L Sanchez-Martın A Estecha R Samaniego S Sanchez-Ramon M A Vega and P Sanchez-Mateos ldquoThe chemokineCXCL12 regulates monocyte-macrophage differentiation andRUNX3 expressionrdquo Blood vol 117 no 1 pp 88ndash97 2011
[37] K Beider H Bitner M Leiba et al ldquoMultiple myelomacells recruit tumor-supportive macrophages through theCXCR4CXCL12 axis and promote their polarization towardthe M2 phenotyperdquo Oncotarget vol 5 no 22 pp 11283ndash112962014
[38] J M Mota C A Leite and L E Souza ldquoPost-sepsis stateinduces tumor-associated macrophage accumulation throughCXCR4CXCL12 and favors tumor progression inmicerdquoCancerImmunology Research vol 4 no 4 pp 312ndash322 2016
[39] B A Teicher and S P Fricker ldquoCXCL12 (SDF-1)CXCR4 path-way in cancerrdquoClinical Cancer Research vol 16 no 11 pp 2927ndash2931 2010
[40] P C Heinrich I Behrmann G Muller-Newen F Schaper andL Graeve ldquoInterleukin-6-type cytokine signalling through thegp130JakSTAT pathwayrdquo Biochemical Journal vol 334 part 2pp 297ndash314 1998
[41] M Kortylewski M Kujawski T Wang et al ldquoInhibiting Stat3signaling in the hematopoietic system elicits multicomponentantitumor immunityrdquo Nature Medicine vol 11 no 12 pp 1314ndash1321 2005
[42] X Hu J Chen L Wang and L B Ivashkiv ldquoCrosstalk amongJak-STAT Toll-like receptor and ITAM-dependent pathways inmacrophage activationrdquo Journal of Leukocyte Biology vol 82no 2 pp 237ndash243 2007
[43] R Catlett-Falcone T H Landowski M M Oshiro et alldquoConstitutive activation of Stat3 signaling confers resistance toapoptosis in humanU266myeloma cellsrdquo Immunity vol 10 no1 pp 105ndash115 1999
Submit your manuscripts athttpswwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
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Stem CellsInternational
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
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Behavioural Neurology
EndocrinologyInternational Journal of
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BioMed Research International
OncologyJournal of
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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ObesityJournal of
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom