Research ArticleThe Ethyl Acetate Extract of Gynura formosanaKitam Leaves Inhibited Cervical Cancer CellProliferation via Induction of Autophagy

Jing FanMa 123 Peng Fei Wei4 Chang Guo123 Yuan Peng Shi1

Yang Lv1 Long Xin Qiu 123 and Long PingWen 14

1Department of Biological Science and Technology School of Life Sciences Longyan University Longyan University China2Key Laboratory of Preventive Veterinary Medicine and Biotechnology (Longyan University) Fujian Province UniversityLongyan China3Fujian Provincial Key Laboratory for the Prevention and Control of Animal Infectious Diseases and BiotechnologyLongyan University Longyan Fujian 364012 China4Hefei National Laboratory for Physical Sciences at the Microscale School of Life SciencesUniversity of Science and Technology of China Hefei Anhui 230027 China

Correspondence should be addressed to Long Xin Qiu 1923015106qqcom and Long Ping Wen lpwenustceducn

Received 22 November 2017 Revised 16 March 2018 Accepted 2 April 2018 Published 24 May 2018

Academic Editor Gang Liu

Copyright copy 2018 Jing Fan Ma et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Gynura formosana Kitam belongs to the Compositae family and has been traditionally used for the prevention of cancer diabetesand inflammation in China Previous studies had indicated that the ethyl acetate extract ofGynura formosanaKitam leaves (EAEG)exhibited antioxidant and anti-inflammatory activity In this report we demonstrated that EAEG possessed potent anticanceractivity through autophagy-mediated inhibition of cell proliferation EAEG induced a strong cytostatic effect towards HeLa cellsand to a lesser extent HepG2 and MCF-7 cells This cytostatic effect of EAEG was not a consequence of increased apoptosis asneither DNA fragmentation nor change in protein expression level for a number of apoptosis-related genes including Bid Bax Bcl-2 and caspase-3 was observed after EAEG treatment and the apoptosis inhibitor Z-VAD-FMK did not inhibit the EAEG-elicitedcytostatic effect On the other hand EAEG induced autophagy in a dose-dependent fashion as shown by increased GFP punctaformation enhanced conversion of the microtubule-associated protein light chain LC3-I to LC3-II and downregulation of the p62protein Treating theHeLa cells with EAEG together with Chloroquine (CQ) further accelerated LC3 conversion and p62 clearanceindicating that EAEG induced complete autophagy flux Importantly the autophagy inhibitor 3-methyladenine (3MA) significantlyabrogated the cytostatic effect of EAEG strongly suggesting that EAEG inhibited HeLa cell proliferation through the induction ofautophagy rather than apoptosis Our results provided a novel and interesting mechanistic insight into the anticancer action ofEAEG supporting the traditional use of this plant for the treatment of the cancer

1 Introduction

Cervical cancer is the secondmost commonmalignant tumorand the fourth main cause of cancer deaths in womenworldwide [1 2]Over half amillion cases are diagnosed everyyear with the mortality rate of 9 in the world In China130000 cases are diagnosed with cervical cancer each yearand account for 28 of new cases globally The mortalityrate is 14 which is higher than that in other developing

countries [3 4] Cervical cancer could be traditionally treatedwith surgery and chemoradiotherapy However anticancerdrugs of such approach are highly toxic Therefore thedevelopment of new anticancer drugs with fewer side effectsis very necessary Plant-derived bioactive compounds areimportant resources for treating various forms of the cancer[5] Numerous studies have demonstrated that plant extractshad antiproliferative or antitumor effects on tumor cells [6ndash8]

HindawiBioMed Research InternationalVolume 2018 Article ID 4780612 10 pageshttpsdoiorg10115520184780612

2 BioMed Research International

Gynura formosana Kitam (family Compositae) is a fre-quently used vegetable in China It exhibited various bio-logical activities and had been used for the prevention ofcancer diabetes and inflammation in the Chinese traditionalsystem of medicine since ancient timesThe leaves of Gynuraformosana Kitam contained several medicinally relevantcomponents such as polyphenolics flavonoids alkaloidsterpenes and saponins [9] These substances might con-tribute to its biological activity Our previous studies hadexhibited that the ethyl acetate extract of Gynura formosanaKitam leaves (EAEG) had high quantity of polyphenolics andflavonoids and had antioxidant and anti-inflammatory effects[9] Recent studies showed that several bioactive compoundsextracted from plants mostly polyphenolics and flavonoidsplayed an important role in tumor cells inhibition effect[10 11] Distinctly EAEG a polyphenolic- and flavonoid-richextract is potentially relevant for preventing and treatingtumor cells However the anticancer effect of EAEG wasrarely reported Therefore in the present study we investi-gated the effect of EAEGon the proliferation of human tumorcells and its underlying mechanism

Autophagy as a cellular self-digestive process is also con-sidered an evolutionarily conserved and lysosome-dependentprocess in all mammalian cells which is associated with cellsurvival It helps cells obtain nutrients under deprivationconditions However more andmore studies [12] have shownthat despite its importance in maintaining cell homeostasisautophagy plays also a vital role in the regulation of cell deathActually continuous ldquoautophagyrdquo will commit suicide Soautophagy may play a protective role against cancer

2 Materials and Methods

21 Reagents All of cell culture reagents were purchasedfrom HyClone Laboratories (Logan UT USA) Trehalose(Tre) Z-VAD-FMK 3-methyladenine (3MA) and Chloro-quine (CQ) were purchased from Sigma-Aldrich (St LouisMO USA) LC3 antibody was purchased from Novus (Little-ton CO USA) and glyceraldehyde phosphate dehydrogenase(GAPDH) antibody from Millipore Bid Bax and Bcl-2antibodies were all obtained from Santa Cruz Biotechnology(Santa Cruz CA USA) Caspase-3 p62 PCNA and MCM7antibodies were purchased from Cell Signaling Technology(Danvers MA USA) 120573-Actin antibody was purchased fromSigma Chemical Co (St Louis MO USA)

22 Preparation of EAEG EAEG was prepared accordingto our previously published literature [9] Briefly 200 g ofGynura formosana Kitam leaves powder was firstly soakedin petroleum ether for 24 h and then the extract wasfiltered evaporated at 45∘C and dried in a freeze-dryerThe residue was extracted successively in chloroform andethyl acetate in the same way The yields of petroleumether chloroform and ethyl acetate extracts were 9923 and 32 respectively In our previous studies EAEGshowed the strongest 11-diphenyl-2-picrylhydrazyl radical22-diphenyl-1-(246-trinitrophenyl)hydrazyl (DPPH) and221015840-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS)radical scavenging activities in three extractsThe polyphenol

and flavonoid contents in EAEGwere 3412 plusmn 001mgg gallicacid equivalent and 4435 plusmn 002mgg catechin equivalentrespectively [9]

23 Cell Culture HeLa andHepG2 cells were purchased fromthe American Type Culture Collection (ATCC ManassasVA USA) MCF-7 cells were kindly provided by ProfessorLongping Wen (University of Science and Technology ofChina Hefei Anhui) HeLa cells stably expressing GFP-LC3 were constructed as previously described [13] All cellswere grown in DMEM supplemented with 10 FBS and 1penicillin-streptomycin in a humidified atmosphere of 5CO2at 37∘C

24 Cell Viability Assay Cell viability was determined by 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide(MTT) assay according to the manufacturerrsquos protocols(Sigma Chemical Co) In brief cells were cultured in 96-wellflat bottom plates overnight and then treated with differentconcentrations (0 15 30 45 60 75 90 and 105 120583gml) ofEAEG After incubation for 24 48 or 72 h 100 120583l MTTsolution (1mgml) was added to each well and the plates wereincubated at 37∘C for 4 h The supernatants were thenremoved and DMSO was added to each well to dissolve theMTT-formazan crystals formed by viable cells Finally theabsorbance values were determined by a microplate reader(Bio-Rad Hercules CA USA) at a wavelength of 570 nm

25 Detection of DNA Fragmentation by Agarose Gel Elec-trophoresis After treatment of HeLa cells with differentconcentrations (0 45 60 and 75120583gml) of EAEG for 24 or48 h the phenolchloroformisoamyl alcohol was used toextract DNA from cells that had been digested with pro-teinase K Cell lysate was collected incubated at 55∘C for 10 hand then treated with 40120583g120583l DNase-free RNase at 37∘Cfor 40min The DNA was precipitated with 4 volumes ofethanol and electrophoresed in 15 agarose gels and visu-alized by ethidium bromide staining using a Chemidox(BioRad Hercules CA)

26 Western Blot HeLa cells were trypsinized collectedby centrifugation and homogenized in ice-cold RIPA LysisBuffer (Beyotime Haimen China) Protein concentrationwas analyzed by Bradford Protein Assay Kit (BeyotimeHaimen China) 35 120583g of protein sample was loaded and sep-arated on an SDSPAGE gel and transferred to PVDF mem-brane (Millipore Billerica MA USA) The membrane wasincubated with Tris-buffered saline containing 01 Tween-20 and 5 nonfat dry milk followed by 2 h incubation atthe room temperature with antibody to Bax Bid Bcl-2caspase-3 120573-actin GAPDH p62 and LC3 respectively andthen with horseradish peroxidase-conjugated anti-mouseor anti-goat or anti-rabbit IgG (Sigma 1 2000) Detectionwas achieved using enhanced chemiluminescence (ECL) kit(Beyotime Haimen China)

27 Observation of GFP-LC3 Puncta HeLa cells stablyexpressing GFP-LC3 were incubated in 24-well plates andtreated with EAEG for 24 h GFP-LC3 puncta were observed

BioMed Research International 3

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EAEG (gml)1059075604530150

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)

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Figure 1 Cell viability comparison of HeLa (a) HepG2 (b) andMCF-7 (c) cells treated with different concentrations of EAEG (0ndash105 120583gml)for 24 48 and 72 h The data were expressed as mean plusmn SEM (119899 = 6) lowast119901 lt 005 and lowastlowast119901 lt 001 with respect to the control group EAEGethyl acetate extract of Gynura formosana Kitam leaves

under fluorescent microscopy (Olympus IX71 Tokyo Japan)All of photos were captured randomly

28 Statistical Analysis All of the results were expressedas mean plusmn standard error of mean (SEM) Statistical sig-nificances were analyzed by one-way analysis of variance(ANOVA) with Dunnettrsquos multiple comparison tests (SPSSversion 220 software SPSS Inc Chicago IL USA) 119901 lt 005was considered statistically significant

3 Results

31 EAEG Exhibited Cytostatic Effect in Tumor Cells Toinvestigate the cytostatic effect of EAEG on the tumor cells

HeLa cells (cervical cancer) HepG2 (liver cancer) andMCF-7 cells (breast cancer) were exposed to various concentrationsof EAEG for up to 3 days in vitro As shown in Figure 1EAEG within a dosage range of 0ndash105120583gml decreased thecell viability in a time- and dose-dependent manner in allthree cell lines Significant (119901 lt 005) viability-reducingeffect was observed at EAEG concentrations of 30 120583gml andthe time of 72 h in HeLa cells (Figure 1(a)) In comparisonsignificant decrease of cell viability inHepG2 andMCF-7 cellsstarted at EAEG concentration of 45120583gml at the time of 48 hand 72 h respectively (Figures 1(b) and 1(c)) Moreover theIC50

value of EAEG for HeLa HepG2 and MCF-7 cells was8147 10094 and 10476 120583gml at 24 h respectively (Figure 1)As HeLa cells possessed the lowest IC

50value they were

selected for subsequent studies

4 BioMed Research International

32 The Cytostatic Effect of EAEG Was Not due to EnhancedApoptosis The most likely explanation for the observedcytostatic effect of EAEG on tumor cells was the induction ofapoptotic cell death by EAEG To investigate this possibilitywe conductedDNA fragmentation (the hallmark of apoptoticcell death) assay and also assessed the expression level fora number of apoptosis-related proteins Treatment of HeLacells with EAEG at a range of concentrations up to 75120583gmlfor either 24 h or 48 h induced no DNA fragmentation(Figure 2(a)) and did not alter the expression level of Bid BaxBcl-2 and caspase-3 (Figures 2(b) and 2(c)) FurthermoreZ-VAD-FMK an apoptosis inhibitor [14] did not affect theviability-reducing effect of EAEG (Figures 2(d) and 2(e))Taken together these results indicated that the cytostaticeffect of EAEG in HeLa cells was apoptosis-independent

33 EAEG Induced Complete Autophagy Autophagy mod-ulation has been demonstrated to be a critical mediator ofanticancer efficacy for many drugs [15ndash17] Our results aboveshowed that EAEG did not cause apoptotic cell death inHeLa cells so we next investigated whether EAEG inducedautophagyWeusedGFP-LC3HeLa a cell line stably express-ing GFP-LC3 a fusion protein between green fluorescentprotein (GFP) and microtubule-associated light chain 3(LC3) protein GFP-LC3 protein accumulated to form greenfluorescence puncta on autophagosome membranes uponautophagy [13] Similar to trehalose the positive controlEAEG at the various concentrations induced significantpuncta formation while DMSO (EAEG solvent) did notindicating that EAEG elicited autophagy (Figure 3(a)) Inaddition EAEG treatment significantly enhanced the con-version of LC3-I to LC3-II and reduced the protein levelof p62 a common autophagic substrate with significant(119901 lt 001) changes observed at 60120583gml and above for 24 h(Figure 4(a)) lending further proof for the occurrence ofautophagy and also suggesting that the autophagy inducedby EAEG was complete with normal cargo degradation(Figure 3(b)) To further support the latter conclusion wetreated HeLa cells with 60 120583gml EAEG for 24 h in thepresence or absence of Chloroquine (CQ) a classical later-stage autophagy inhibitor Earlier literature showed that thedegradation of LC3-II would be impaired in cells treatedwith saturating dose of CQ (50mM) [18] Indeed a furtherincrease in the level of LC3-II was seen in HeLa cells treatedwith EAEG plus CQ as compared to HeLa cells treated withEAEG alone (Figure 3(c)) strongly suggesting that EAEGinduced complete autophagy with normal flux As would beexpected p62 degradation was retarded by CQ (Figure 3(c))

34 EAEG Induced Dose- and Time-Dependent AutophagyTo further characterize the autophagic response elicited byEAEG we performed dose- and time-dependent studiesusing LC3-ILC3-II conversion and p62 protein level as themeasures HeLa cells were treated with different concen-trations (0 40 50 60 70 and 80 120583gml) of EAEG for24 h or with 60 120583gml EAEG for different times (0 3 69 18 and 24 h) A dose-dependent conversion of LC3-I toLC3-II accompanied by a corresponding decrease in thelevel of p62 was observed following EAEG treatment in

HeLa cells with significant changes observed at 40120583gmland above (Figure 4(a)) The conversion of LC3-I to LC3-IIand the corresponding decrease in the level of p62 werealso time-dependent with significant change observed at 3 hand beyond following EAEG treatment in HeLa cells (Fig-ure 4(b))

35 The Autophagy Induced by EAEG Caused HeLa CellsDeath Did the autophagy induction contribute to the cyto-static effect elicited by EAEG To assess this possibility wemeasured cell viability after the treatment of HeLa cellswith EAEG plus 3MA a class III-PI3K autophagy inhibitorAs shown in Figure 5(a) 60 120583gml EAEG treatment for24 h reduced the cell viability to 6468 and this effectwas partially offset by the cotreatment with 5mM 3MA(Figure 5(a)) which inhibited significantly the autophagyinduced by EAEG (Figure 5(b)) Furthermore EAEG signif-icantly decreased the level of PCNA and MCM7 two widelyused cell proliferation markers [19] and this effect was alsopartially abrogated by 3MA (Figure 5(b)) We thus concludedthat EAEG elicited a cytostatic effect on cancer cells throughblocking autophagy-mediated cell proliferation rather thaninducing apoptosis

4 Discussion

Gynura formosana Kitam had been used traditionally forthe prevention of cancer diabetes and inflammation inChina Our previous results showed that EAEG containedpolyphenols and flavonoids and had antioxidant and anti-inflammatory activities [9]These collective results suggestedthat EAEG might present antitumor activity However therewas little evidence showing the antitumor effects of EAEG

The plant-derived compounds had served as a mainsource of drugs and aboutmore than 50of pharmaceuticalswere derived from natural compounds [20] To determine ifEAEG could affect the cells proliferation we first investigatedthe cytostatic activity of EAEG on HeLa HepG2 andMCF-7cells Our results showed that EAEG reduced the cell viabilityin time- and dose-dependent manners in three cell lines andwas more toxic to HeLa cells (Figure 1) which suggestedthat EAEG might have better chemotherapeutic potential tocervical cancer To elucidate the possiblemechanism involvedin the above cytostatic effects we assumed that HeLa cellswould undergo apoptotic cell death after EAEG treatmentHowever any DNA fragmentation could not been detectedin HeLa cells treated with even 75120583gml EAEG for 48 hWe further investigated the proapoptosis antiapoptosis andapoptosis-related proteins expression in HeLa cells treatedwith EAEG Bax and Bidwere both proapoptotic proteins andcould promote apoptotic proteins release However the anti-apoptotic protein Bcl-2 induced by various pathologic andphysiologic stimuli could suppress apoptotic proteins efflux[21ndash23] Caspase was a sensitive indicator of apoptosis [24]We evidenced in this study that EAEG could not induce theactivation of Bax Bid Bcl-2 and caspase-3 in HeLa cells byWestern blot analysis suggesting that apoptotic pathway wasnot triggered by EAEG treatment Most importantly the

BioMed Research International 5

48 Ｂ24 Ｂ

(a)

Bax

Bid

Bcl-2

Caspase 3

-actin

gml7560450

EAEG (gml)

BaxBid

Bcl-2Caspase 3

756045000

02

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Prot

ein

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ratio

(b)

gml

Bax

Bid

Bcl-2

Caspase 3

-actin

7560450

EAEG (gml)

BaxBid

Bcl-2Caspase 3

756045000

02

04

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10

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otei

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ControlZvadfmk (50M)

EAEG (gml)7560450

0

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EAEG (gml)7560450

0

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l via

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)

(e)

Figure 2 EAEG treatment did not induce apoptotic cell death in HeLa cells (a) DNA fragmentation analysis Lines a and e controls treatedfor 24 and 48 h respectively lines b and f 45 120583gml EAEG treated for 24 and 48 h respectively lines c and g 60 120583gml EAEG treated for 24and 48 h respectively lines d and h 75 120583gml EAEG treated for 24 and 48 h respectively line i DNAmarker ((b) and (c))The expression ofBid Bax Bcl-2 and caspase-3 in HeLa cells treated with different concentrations of EAEG (0 45 60 and 75120583gml) for 24 h (b) or 48 h (c)120573-Actin served as the loading control The relative protein120573-actin ratios were calculated using ImageJ ((d) and (e)) Cell viability of HeLacells treated withwithout Z-VAD-FMK (50120583M) and different concentrations of EAEG for 24 h (d) or 48 h (e) The data were expressed asmean plusmn SEM (119899 = 3) EAEG ethyl acetate extract of Gynura formosana Kitam leaves

6 BioMed Research International

Blank DMSO EAEG (45gml)

EAEG (60 gml) EAEG (75 gml) Tre

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P62

LC3-I

LC3-II

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EAEG (gml)

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7560450

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LC3-II

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evel

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CQEAEG + CQEAEGControl

P62GAPDHLC3-IILC3-I

lowastlowastlowastlowast

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lowast

(c)Figure 3 EAEG induced autophagy in HeLa cells (a) Analysis of GFP-LC3 puncta formation in GFP-LC3HeLa cells under fluorescencemicroscopy Cells were treated for 24 h with DMEM only (blank) EAEG solvent (DMSO) as control different concentrations (45 60 and75 120583gml) of EAEG or the positive control trehalose (Tre) (scale bar = 20120583m) (b)The expression analysis of the autophagy-related proteinsp62 and LC3 byWestern blot HeLa cells were treated for 24 h with different concentrations (0 45 60 and 75 120583gml) of EAEG (c)The relativeprotein levels of p62 and LC3-II in HeLa cells treated with 60 120583gml EAEG with or without CQ (50mM) for 24 h GAPDH served as theloading control The protein relative ratio was calculated using ImageJ The data were expressed as mean plusmn SEM (119899 = 3) lowast119901 lt 005 andlowastlowast119901 lt 001 with respect to the control group 119901 lt 001 with respect to EAEG treatment group EAEG ethyl acetate extract of Gynuraformosana Kitam leaves

BioMed Research International 7

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(b)

Figure 4The conversation of LC3-I to LC3-II Protein level of p62 was measured byWestern blot (a) HeLa cells were treated with EAEG at aconcentration of 0 40 50 60 70 or 80120583gml for 24 h (b)HeLa cells were treatedwith 60120583gml EAEG for 0 3 6 9 18 or 24 h GAPDH servedas the loading control The relative protein ratio was calculated using ImageJ The data were expressed as mean plusmn SEM (119899 = 3) lowast119901 lt 005lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001 with respect to the control group EAEG ethyl acetate extract of Gynura formosana Kitam leaves

apoptosis inhibitor Z-VAD-FMK did not affect the viability-reducing effect of EAEG (Figures 2(d) and 2(e)) This findingserved as a convincing evidence for nonapoptotic cell deathin EAEG treated HeLa cells

Autophagy was considered to be main way to inhibittumor cells proliferation when cells underwent nonapoptoticcell death [25 26] Increasing evidences had suggested thatautophagy could be induced by many natural products invarious anticancer therapies Triterpenoid B-group soyas-aponins could induce autophagy in colon cancer cells byinhibition of Akt signaling and enhanced ERK12 activity[27] Polyphenols extracts could induce autophagy whichwas advantageous to inhibit neurodegeneration and cancerprocess [28] Recent studies demonstrated that Emblicaofficinalis extracts could also induce autophagy and inhibithuman ovarian cancer cell proliferation [29] These reportssupported us to hypothesize that HeLa cells death caused byEAEG could involve autophagy program Autophagy wasa physiologically and pathologically regulated process andplayed an important role in the maintenance of intracellularhomeostasis in every eukaryotic cell [30ndash32] It started

with inclosing cytoplasmic contents in a vacuole calledautophagosome and then autophagosome fused with lyso-some to form autolysosomewhere substances inside could bedegraded and recycled [33] Autophagy could be initiated bysome autophagy-related proteins such as the microtubule-associated protein LC3 which was a reliable autophagicmarker [34] LC3 was a constitutively expressed protein inmammalian cells and could be processed to form cytoso-lic LC3-I During autophagy LC3-I was recruited to theautophagosome where LC3-II was generated by fusingLC3-I with phosphatidylethanolamine and then cellularautophagosome puncta including LC3-II were formed [35]Our results demonstrated that EAEG upregulated GFPpuncta formation (Figure 3(a)) and increased the conversionof LC3-I to LC3-II in HeLa cells (Figure 3(b)) suggest-ing the recruitment of LC3-II upon EAEG administrationFurthermore we analyzed the protein level of p62 anotherautophagicmarker [36] to study induction of autophagy p62could be trapped by LC3 incorporated into the autophago-some and then degraded via autophagy Correspondinglythe protein level of p62 should be decreased in the cells

8 BioMed Research International

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EAEG + 3MAEAEGControl

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Figure 5 EAEG induced HeLa cells undergoing autophagic cell death (a) Cell viability of HeLa cells treated with 60120583gml EAEG for 24 h inpresence or absence of 5mM 3MA (b) Western blot analysis of autophagy and proliferation proteins GAPDH served as the loading controlThe protein relative ratio was calculated using ImageJThe data were expressed as mean plusmn SEM (119899 = 3) lowastlowast119901 lt 001with respect to the controlgroup and 119901 lt 005 with respect to EAEG treatment group EAEG ethyl acetate extract of Gynura formosana Kitam leaves

because of autophagy Our results showed that the proteinlevel of p62 reduced significantly in a dosage-dependentman-ner after EAEG treatment (Figure 3(b)) indicating an appar-ent induction of autophagy However these results couldnot explain if EAEG could induce the complete autophagyflux For instance calcium phosphate precipitates (CPP)induced both autophagosome synthesis and reduction ofautophagy flux due to impaired autophagosome-lysosomefusion [37] Therefore it was very necessary to evaluate if theautophagy induced by EAEG was complete autophagy fluxincluding autophagosome formation and autophagosome-lysosome fusion Our results showed that the protein levels ofboth LC3-II and p62 strongly increased in HeLa cells treatedwith EAEG together with CQ compared to cells treatedwith EAEG alone (Figure 3(c)) These results confirmed thatEAEG could induce complete autophagy flux

To confirm autophagy intensity we estimated the con-version of LC3-I to LC3-II and protein level of p62 EAEGelicited dose- and time-dependent autophagic induction inHeLa cells through the conversion of LC3-I into LC3-IIand decrease of protein level of p62 (Figure 4) stronglysuggesting that EAEG had autophagic activity Additionallywe also demonstrated that 3MA could partially abrogateEAEG induced HeLa cells death the conversion of LC3-Ito LC3-II and decreasing level of PCNA and MCM7 Thepresented collective results indicated that EAEG mightinhibit HeLa cells proliferation by inducing autophagy

In summary our studies indicated that EAEG a naturalplant extract could be a potentially effective therapeutic agentfor treating cervical cancer Our experimental evidences alsosupported the traditional use of this plant for the treatment ofthe deadly disease cancerThe identification of such biological

BioMed Research International 9

compounds in this plant and the mechanism against tumorcells are our research targets in the future

Conflicts of Interest

The authors declare that there are no conflicts of interest

Acknowledgments

This project was funded by Minxi Public Service Platformfor deep processing of Characteristic Agricultural Prod-ucts (2016LY08) the Natural Science Foundation of FujianProvince (2018J01458) Longyan University (LC2016008) theTraining Program of Fujian Excellent Talents in University(MJR201558) and the National Natural Science Foundationof China (81601600)

References

[1] B J Monk ldquoInvasive Cervical Cancerrdquo in Clinical GynecologicOncology 2012 1119

[2] W Zheng Z Liu W Zhang and X Hu ldquomiR-31 functions asan oncogene in cervical cancerrdquo Archives of Gynecology andObstetrics vol 292 no 5 pp 1083ndash1089 2015

[3] S EWaggoner ldquoCervical cancerrdquoThe Lancet vol 361 no 9376pp 2217ndash2225 2003

[4] Y Chen HWangW Lin and P Shuai ldquoADAR1 overexpressionis associated with cervical cancer progression and angiogene-sisrdquo Diagnostic Pathology vol 12 no 12 2017

[5] S Sen R Chakraborty C Sridhar Y S R Reddy and BDe ldquoFree radicals antioxidants diseases and phytomedicinescurrent status and future prospectrdquo International Journal ofPharmaceutical Sciences Review and Research vol 3 no 1 pp91ndash100 2010

[6] C Liu J-Z Liao and P-Y Li ldquoTraditional Chinese herbalextracts inducing autophagy as a novel approach in therapy ofnonalcoholic fatty liver diseaserdquoWorld Journal of Gastroenterol-ogy vol 23 no 11 pp 1964ndash1973 2017

[7] F S Reis R T Lima P Morales I C F R Ferreira andM H Vasconcelos ldquoMethanolic extract of ganoderma luciduminduces autophagy of AGS human gastric tumor cellsrdquoMolecules vol 20 no 10 pp 17872ndash17882 2015

[8] N Yu Y Xiong and C Wang ldquoBu-Zhong-Yi-Qi Decoctionthe Water Extract of Chinese Traditional Herbal MedicineEnhances Cisplatin Cytotoxicity in A549DDP Cells throughInduction of Apoptosis and Autophagyrdquo BioMed ResearchInternational vol 2017 Article ID 3692797 9 pages 2017

[9] J Ma C Guo Y Pan D Lin L Qiu and L Wen ldquoAntioxidantand anti-inflammatory activities of ethyl acetate extract ofGynura formosana (Kitam) leavesrdquo Experimental and Thera-peutic Medicine vol 14 no 3 pp 2303ndash2309 2017

[10] Q Tian J Li X Xie et al ldquoStereospecific induction of nuclearfactor-120581B activation by isochamaejasminrdquo Molecular Pharma-cology vol 68 no 6 pp 1534ndash1542 2005

[11] W-K Liu F W K Cheung B P L Liu C Li W Ye and C-T Che ldquoInvolvement of p21 and FasL in induction of cell cyclearrest and apoptosis by neochamaejasmin a in human prostateLNCaP cancer cellsrdquo Journal of Natural Products vol 71 no 5pp 842ndash846 2008

[12] L Yang Y Liu M Wang et al ldquoCelastrus orbiculatus extracttriggers apoptosis and autophagy via PI3KAktmTOR inhibi-tion in human colorectal cancer cellsrdquo Oncology Letters vol 12no 5 pp 3771ndash3778 2016

[13] P-FWei L Zhang S K Nethi et al ldquoAccelerating the clearanceof mutant huntingtin protein aggregates through autophagyinduction by europium hydroxide nanorodsrdquo Biomaterials vol35 no 3 pp 899ndash907 2014

[14] J Yang Y Zhou X Cheng et al ldquoIsogambogenic acid inducesapoptosis-independent autophagic cell death in human non-small-cell lung carcinoma cellsrdquo Scientific Reports vol 5 pp 1ndash92015

[15] F Janku D J McConkey D S Hong and R KurzrockldquoAutophagy as a target for anticancer therapyrdquo Nature ReviewsClinical Oncology vol 8 no 9 pp 528ndash539 2011

[16] Z J Yang C E Chee S Huang and F A Sinicrope ldquoAutophagymodulation for cancer therapyrdquo Cancer Biology ampTherapy vol11 no 2 pp 169ndash176 2011

[17] Y Lao G Wan Z Liu et al ldquoThe natural compound oblongi-folin C inhibits autophagic flux and enhances antitumor efficacyof nutrient deprivationrdquo Autophagy vol 10 no 5 pp 736ndash7492014

[18] H-M Ni A Bockus A L Wozniak et al ldquoDissecting the dy-namic turnover of GFP-LC3 in the autolysosomerdquo Autophagyvol 7 no 2 pp 188ndash204 2011

[19] M Zhou L Yang M Shao et al ldquoEffects of ZearalenoneExposure on the TGF-1205731Smad3 Signaling Pathway and theExpression of Proliferation or Apoptosis Related Genes of Post-Weaning Giltsrdquo Toxins vol 10 no 2 p 49 2018

[20] D Xu Y LaoN Xu et al ldquoIdentification and characterization ofanticancer compounds targeting apoptosis and autophagy fromChinese native Garcinia speciesrdquo Planta Medica vol 81 no 1pp 79ndash89 2014

[21] D R Green and J C Reed ldquoMitochondria and apoptosisrdquoScience vol 281 no 5381 pp 1309ndash1312 1998

[22] C J Hawkins and D L Vaux ldquoAnalysis of the Role of bcl-2 inApoptosisrdquo Immunological Reviews vol 142 no 1 pp 127ndash1391994

[23] Y Tsujimoto ldquoStress-resistance conferred by high level of bcl-2120572 protein in human B lymphoblastoid cellrdquo Oncogene vol 4no 11 pp 1331ndash1336 1989

[24] D R Green ldquoApoptotic pathways paper wraps stone bluntsscissorsrdquo Cell vol 102 no 1 pp 1ndash4 2000

[25] S Tsolmon J Han and H Isoda ldquoInhibition of cell growthby Stellera chamaejasme extract is associated with induction ofautophagy and differentiation in chronic leukemia K562 cellsrdquoJournal of Bioscience and Bioengineering vol 110 no 2 pp 262ndash268 2010

[26] T Kanzawa Y Kondo H Ito S Kondo and I GermanoldquoInduction of autophagic cell death inmalignant glioma cells byarsenic trioxiderdquo Cancer Research vol 63 no 9 pp 2103ndash21082003

[27] A A EllingtonM A Berhow and KW Singletary ldquoInhibitionof Akt signaling and enhanced ERK12 activity are involved ininduction of macroautophagy by triterpenoid B-group soyas-aponins in colon cancer cellsrdquo Carcinogenesis vol 27 no 2 pp298ndash306 2006

[28] K Pallauf and G Rimbach ldquoAutophagy polyphenols andhealthy ageingrdquo Ageing Research Reviews vol 12 no 1 pp 237ndash252 2013

10 BioMed Research International

[29] A De A De C Papasian et al ldquoEmblica officinalis ExtractInduces Autophagy and Inhibits Human Ovarian CancerCell Proliferation Angiogenesis Growth of Mouse XenograftTumorsrdquo PLoS ONE vol 8 no 8 Article ID e72748 2013

[30] C CDenardin L AMMartinsMM Parisi et al ldquoAutophagyinduced by purple pitanga (Eugenia uniflora L) extract trig-gered a cooperative effect on inducing the hepatic stellate celldeathrdquo Cell Biology and Toxicology vol 33 no 2 pp 197ndash2062017

[31] D R Green L Galluzzi and G Kroemer ldquoMitochondriaand the autophagy-inflammation-cell death axis in organismalagingrdquo Science vol 333 no 6046 pp 1109ndash1112 2011

[32] B Levine N Mizushima and H W Virgin ldquoAutophagy inimmunity and inflammationrdquo Nature vol 469 no 7330 pp323ndash335 2011

[33] N Mizushima B Levine A M Cuervo and D J Klion-sky ldquoAutophagy fights disease through cellular self-digestionrdquoNature vol 451 no 7182 pp 1069ndash1075 2008

[34] V Thongrakard R Titone C Follo et al ldquoTurmeric toxicityin A431 epidermoid cancer cells associates with autophagydegradation of anti-apoptotic and anti-autophagic p53 mutantrdquoPhytotherapy Research vol 28 no 12 pp 1761ndash1769 2014

[35] A Kuma M Matsui and N Mizushima ldquoLC3 an autophago-some marker can be incorporated into protein aggregatesindependent of autophagy Caution in the interpretation of LC3localizationrdquo Autophagy vol 3 no 4 pp 323ndash328 2007

[36] M Komatsu S Waguri M Koike et al ldquoHomeostatic lev-els of p62 control cytoplasmic inclusion body formation inautophagy-deficient micerdquo Cell vol 131 no 6 pp 1149ndash11632007

[37] S SarkarVKorolchukMRennaAWinslow andDC Rubin-sztein ldquoMethodological considerations for assessing autophagymodulators A study with calcium phosphate precipitatesrdquoAutophagy vol 5 no 3 pp 307ndash313 2009

Hindawiwwwhindawicom

International Journal of

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Hindawiwwwhindawicom Volume 2018

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GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

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Hindawiwwwhindawicom Volume 2018

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Hindawiwwwhindawicom Volume 2018

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Submit your manuscripts atwwwhindawicom

BioMed Research International 3

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowast

24 Ｂ

48 Ｂ

72 Ｂ

EAEG (gml)1059075604530150

0

20

40

60

80

100

120C

ell v

iabi

lity

()

(a)

24 Ｂ

48 Ｂ

72 Ｂ

EAEG (gml)1059075604530150

0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

(b)

lowast lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

24 Ｂ

48 Ｂ

72 Ｂ

EAEG (gml)1059075604530150

0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

(c)

Figure 1 Cell viability comparison of HeLa (a) HepG2 (b) andMCF-7 (c) cells treated with different concentrations of EAEG (0ndash105 120583gml)for 24 48 and 72 h The data were expressed as mean plusmn SEM (119899 = 6) lowast119901 lt 005 and lowastlowast119901 lt 001 with respect to the control group EAEGethyl acetate extract of Gynura formosana Kitam leaves

under fluorescent microscopy (Olympus IX71 Tokyo Japan)All of photos were captured randomly

28 Statistical Analysis All of the results were expressedas mean plusmn standard error of mean (SEM) Statistical sig-nificances were analyzed by one-way analysis of variance(ANOVA) with Dunnettrsquos multiple comparison tests (SPSSversion 220 software SPSS Inc Chicago IL USA) 119901 lt 005was considered statistically significant

3 Results

31 EAEG Exhibited Cytostatic Effect in Tumor Cells Toinvestigate the cytostatic effect of EAEG on the tumor cells

HeLa cells (cervical cancer) HepG2 (liver cancer) andMCF-7 cells (breast cancer) were exposed to various concentrationsof EAEG for up to 3 days in vitro As shown in Figure 1EAEG within a dosage range of 0ndash105120583gml decreased thecell viability in a time- and dose-dependent manner in allthree cell lines Significant (119901 lt 005) viability-reducingeffect was observed at EAEG concentrations of 30 120583gml andthe time of 72 h in HeLa cells (Figure 1(a)) In comparisonsignificant decrease of cell viability inHepG2 andMCF-7 cellsstarted at EAEG concentration of 45120583gml at the time of 48 hand 72 h respectively (Figures 1(b) and 1(c)) Moreover theIC50

value of EAEG for HeLa HepG2 and MCF-7 cells was8147 10094 and 10476 120583gml at 24 h respectively (Figure 1)As HeLa cells possessed the lowest IC

50value they were

selected for subsequent studies

4 BioMed Research International

32 The Cytostatic Effect of EAEG Was Not due to EnhancedApoptosis The most likely explanation for the observedcytostatic effect of EAEG on tumor cells was the induction ofapoptotic cell death by EAEG To investigate this possibilitywe conductedDNA fragmentation (the hallmark of apoptoticcell death) assay and also assessed the expression level fora number of apoptosis-related proteins Treatment of HeLacells with EAEG at a range of concentrations up to 75120583gmlfor either 24 h or 48 h induced no DNA fragmentation(Figure 2(a)) and did not alter the expression level of Bid BaxBcl-2 and caspase-3 (Figures 2(b) and 2(c)) FurthermoreZ-VAD-FMK an apoptosis inhibitor [14] did not affect theviability-reducing effect of EAEG (Figures 2(d) and 2(e))Taken together these results indicated that the cytostaticeffect of EAEG in HeLa cells was apoptosis-independent

33 EAEG Induced Complete Autophagy Autophagy mod-ulation has been demonstrated to be a critical mediator ofanticancer efficacy for many drugs [15ndash17] Our results aboveshowed that EAEG did not cause apoptotic cell death inHeLa cells so we next investigated whether EAEG inducedautophagyWeusedGFP-LC3HeLa a cell line stably express-ing GFP-LC3 a fusion protein between green fluorescentprotein (GFP) and microtubule-associated light chain 3(LC3) protein GFP-LC3 protein accumulated to form greenfluorescence puncta on autophagosome membranes uponautophagy [13] Similar to trehalose the positive controlEAEG at the various concentrations induced significantpuncta formation while DMSO (EAEG solvent) did notindicating that EAEG elicited autophagy (Figure 3(a)) Inaddition EAEG treatment significantly enhanced the con-version of LC3-I to LC3-II and reduced the protein levelof p62 a common autophagic substrate with significant(119901 lt 001) changes observed at 60120583gml and above for 24 h(Figure 4(a)) lending further proof for the occurrence ofautophagy and also suggesting that the autophagy inducedby EAEG was complete with normal cargo degradation(Figure 3(b)) To further support the latter conclusion wetreated HeLa cells with 60 120583gml EAEG for 24 h in thepresence or absence of Chloroquine (CQ) a classical later-stage autophagy inhibitor Earlier literature showed that thedegradation of LC3-II would be impaired in cells treatedwith saturating dose of CQ (50mM) [18] Indeed a furtherincrease in the level of LC3-II was seen in HeLa cells treatedwith EAEG plus CQ as compared to HeLa cells treated withEAEG alone (Figure 3(c)) strongly suggesting that EAEGinduced complete autophagy with normal flux As would beexpected p62 degradation was retarded by CQ (Figure 3(c))

34 EAEG Induced Dose- and Time-Dependent AutophagyTo further characterize the autophagic response elicited byEAEG we performed dose- and time-dependent studiesusing LC3-ILC3-II conversion and p62 protein level as themeasures HeLa cells were treated with different concen-trations (0 40 50 60 70 and 80 120583gml) of EAEG for24 h or with 60 120583gml EAEG for different times (0 3 69 18 and 24 h) A dose-dependent conversion of LC3-I toLC3-II accompanied by a corresponding decrease in thelevel of p62 was observed following EAEG treatment in

HeLa cells with significant changes observed at 40120583gmland above (Figure 4(a)) The conversion of LC3-I to LC3-IIand the corresponding decrease in the level of p62 werealso time-dependent with significant change observed at 3 hand beyond following EAEG treatment in HeLa cells (Fig-ure 4(b))

35 The Autophagy Induced by EAEG Caused HeLa CellsDeath Did the autophagy induction contribute to the cyto-static effect elicited by EAEG To assess this possibility wemeasured cell viability after the treatment of HeLa cellswith EAEG plus 3MA a class III-PI3K autophagy inhibitorAs shown in Figure 5(a) 60 120583gml EAEG treatment for24 h reduced the cell viability to 6468 and this effectwas partially offset by the cotreatment with 5mM 3MA(Figure 5(a)) which inhibited significantly the autophagyinduced by EAEG (Figure 5(b)) Furthermore EAEG signif-icantly decreased the level of PCNA and MCM7 two widelyused cell proliferation markers [19] and this effect was alsopartially abrogated by 3MA (Figure 5(b)) We thus concludedthat EAEG elicited a cytostatic effect on cancer cells throughblocking autophagy-mediated cell proliferation rather thaninducing apoptosis

4 Discussion

Gynura formosana Kitam had been used traditionally forthe prevention of cancer diabetes and inflammation inChina Our previous results showed that EAEG containedpolyphenols and flavonoids and had antioxidant and anti-inflammatory activities [9]These collective results suggestedthat EAEG might present antitumor activity However therewas little evidence showing the antitumor effects of EAEG

The plant-derived compounds had served as a mainsource of drugs and aboutmore than 50of pharmaceuticalswere derived from natural compounds [20] To determine ifEAEG could affect the cells proliferation we first investigatedthe cytostatic activity of EAEG on HeLa HepG2 andMCF-7cells Our results showed that EAEG reduced the cell viabilityin time- and dose-dependent manners in three cell lines andwas more toxic to HeLa cells (Figure 1) which suggestedthat EAEG might have better chemotherapeutic potential tocervical cancer To elucidate the possiblemechanism involvedin the above cytostatic effects we assumed that HeLa cellswould undergo apoptotic cell death after EAEG treatmentHowever any DNA fragmentation could not been detectedin HeLa cells treated with even 75120583gml EAEG for 48 hWe further investigated the proapoptosis antiapoptosis andapoptosis-related proteins expression in HeLa cells treatedwith EAEG Bax and Bidwere both proapoptotic proteins andcould promote apoptotic proteins release However the anti-apoptotic protein Bcl-2 induced by various pathologic andphysiologic stimuli could suppress apoptotic proteins efflux[21ndash23] Caspase was a sensitive indicator of apoptosis [24]We evidenced in this study that EAEG could not induce theactivation of Bax Bid Bcl-2 and caspase-3 in HeLa cells byWestern blot analysis suggesting that apoptotic pathway wasnot triggered by EAEG treatment Most importantly the

BioMed Research International 5

48 Ｂ24 Ｂ

(a)

Bax

Bid

Bcl-2

Caspase 3

-actin

gml7560450

EAEG (gml)

BaxBid

Bcl-2Caspase 3

756045000

02

04

06

08

10

12

14

16

Prot

ein

-a

ctin

ratio

(b)

gml

Bax

Bid

Bcl-2

Caspase 3

-actin

7560450

EAEG (gml)

BaxBid

Bcl-2Caspase 3

756045000

02

04

06

08

10

12

14

16Pr

otei

n

-act

in ra

tio

(c)

ControlZvadfmk (50M)

EAEG (gml)7560450

0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

(d)

ControlZvadfmk (50M)

EAEG (gml)7560450

0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

(e)

Figure 2 EAEG treatment did not induce apoptotic cell death in HeLa cells (a) DNA fragmentation analysis Lines a and e controls treatedfor 24 and 48 h respectively lines b and f 45 120583gml EAEG treated for 24 and 48 h respectively lines c and g 60 120583gml EAEG treated for 24and 48 h respectively lines d and h 75 120583gml EAEG treated for 24 and 48 h respectively line i DNAmarker ((b) and (c))The expression ofBid Bax Bcl-2 and caspase-3 in HeLa cells treated with different concentrations of EAEG (0 45 60 and 75120583gml) for 24 h (b) or 48 h (c)120573-Actin served as the loading control The relative protein120573-actin ratios were calculated using ImageJ ((d) and (e)) Cell viability of HeLacells treated withwithout Z-VAD-FMK (50120583M) and different concentrations of EAEG for 24 h (d) or 48 h (e) The data were expressed asmean plusmn SEM (119899 = 3) EAEG ethyl acetate extract of Gynura formosana Kitam leaves

6 BioMed Research International

Blank DMSO EAEG (45gml)

EAEG (60 gml) EAEG (75 gml) Tre

(a)

gml

P62

LC3-I

LC3-II

GAPDH

756045006

05

04

03

02

01

00

Prot

ein

relat

ive l

evel

s

EAEG (gml)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

P62GAPDHLC3-IILC3-I

7560450

lowast

lowast

(b)

P62

LC3-I

LC3-II

GAPDH

EAEGCQ

minus

minusminus

minus

++

+ +06

05

04

03

02

01

00

Prot

ein

relat

ive l

evel

s

07

CQEAEG + CQEAEGControl

P62GAPDHLC3-IILC3-I

lowastlowastlowastlowast

lowastlowast

lowast

(c)Figure 3 EAEG induced autophagy in HeLa cells (a) Analysis of GFP-LC3 puncta formation in GFP-LC3HeLa cells under fluorescencemicroscopy Cells were treated for 24 h with DMEM only (blank) EAEG solvent (DMSO) as control different concentrations (45 60 and75 120583gml) of EAEG or the positive control trehalose (Tre) (scale bar = 20120583m) (b)The expression analysis of the autophagy-related proteinsp62 and LC3 byWestern blot HeLa cells were treated for 24 h with different concentrations (0 45 60 and 75 120583gml) of EAEG (c)The relativeprotein levels of p62 and LC3-II in HeLa cells treated with 60 120583gml EAEG with or without CQ (50mM) for 24 h GAPDH served as theloading control The protein relative ratio was calculated using ImageJ The data were expressed as mean plusmn SEM (119899 = 3) lowast119901 lt 005 andlowastlowast119901 lt 001 with respect to the control group 119901 lt 001 with respect to EAEG treatment group EAEG ethyl acetate extract of Gynuraformosana Kitam leaves

BioMed Research International 7

gml

P62

LC3-I

LC3-II

GAPDH

0 40 50 60 70 80

0 40 50 60 70 80

EAEG (gml)

00

01

02

03

04

05

06

Prot

ein

relat

ive l

evel

s

LC3-IILC3-IP62GAPDH

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowast

lowast

lowastlowastlowastlowast

(a)

P62

LC3-ILC3-II

GAPDH

0 3 6 9 18 24 h

0 3 6 9 18 24

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowast

lowastlowast

(hours)

00

01

02

03

04

05

06

Prot

ein

relat

ive l

evel

s

LC3-IILC3-IP62GAPDH

(b)

Figure 4The conversation of LC3-I to LC3-II Protein level of p62 was measured byWestern blot (a) HeLa cells were treated with EAEG at aconcentration of 0 40 50 60 70 or 80120583gml for 24 h (b)HeLa cells were treatedwith 60120583gml EAEG for 0 3 6 9 18 or 24 h GAPDH servedas the loading control The relative protein ratio was calculated using ImageJ The data were expressed as mean plusmn SEM (119899 = 3) lowast119901 lt 005lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001 with respect to the control group EAEG ethyl acetate extract of Gynura formosana Kitam leaves

apoptosis inhibitor Z-VAD-FMK did not affect the viability-reducing effect of EAEG (Figures 2(d) and 2(e)) This findingserved as a convincing evidence for nonapoptotic cell deathin EAEG treated HeLa cells

Autophagy was considered to be main way to inhibittumor cells proliferation when cells underwent nonapoptoticcell death [25 26] Increasing evidences had suggested thatautophagy could be induced by many natural products invarious anticancer therapies Triterpenoid B-group soyas-aponins could induce autophagy in colon cancer cells byinhibition of Akt signaling and enhanced ERK12 activity[27] Polyphenols extracts could induce autophagy whichwas advantageous to inhibit neurodegeneration and cancerprocess [28] Recent studies demonstrated that Emblicaofficinalis extracts could also induce autophagy and inhibithuman ovarian cancer cell proliferation [29] These reportssupported us to hypothesize that HeLa cells death caused byEAEG could involve autophagy program Autophagy wasa physiologically and pathologically regulated process andplayed an important role in the maintenance of intracellularhomeostasis in every eukaryotic cell [30ndash32] It started

with inclosing cytoplasmic contents in a vacuole calledautophagosome and then autophagosome fused with lyso-some to form autolysosomewhere substances inside could bedegraded and recycled [33] Autophagy could be initiated bysome autophagy-related proteins such as the microtubule-associated protein LC3 which was a reliable autophagicmarker [34] LC3 was a constitutively expressed protein inmammalian cells and could be processed to form cytoso-lic LC3-I During autophagy LC3-I was recruited to theautophagosome where LC3-II was generated by fusingLC3-I with phosphatidylethanolamine and then cellularautophagosome puncta including LC3-II were formed [35]Our results demonstrated that EAEG upregulated GFPpuncta formation (Figure 3(a)) and increased the conversionof LC3-I to LC3-II in HeLa cells (Figure 3(b)) suggest-ing the recruitment of LC3-II upon EAEG administrationFurthermore we analyzed the protein level of p62 anotherautophagicmarker [36] to study induction of autophagy p62could be trapped by LC3 incorporated into the autophago-some and then degraded via autophagy Correspondinglythe protein level of p62 should be decreased in the cells

8 BioMed Research International

lowastlowast

EAEG + 3MAEAEGControl0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

(a)

EAEG3MA

minus

minusminus +

+ +

LC3-I

LC3-II

PCNA

MCM7

GAPDH

LC3-IILC3-IPCNAGAPDHMCM7GAPDH

EAEG + 3MAEAEGControl

lowastlowast

lowastlowast

lowastlowast

00

01

02

03

04

05

06

07

08

Prot

ein

relat

ive l

evel

s

(b)

Figure 5 EAEG induced HeLa cells undergoing autophagic cell death (a) Cell viability of HeLa cells treated with 60120583gml EAEG for 24 h inpresence or absence of 5mM 3MA (b) Western blot analysis of autophagy and proliferation proteins GAPDH served as the loading controlThe protein relative ratio was calculated using ImageJThe data were expressed as mean plusmn SEM (119899 = 3) lowastlowast119901 lt 001with respect to the controlgroup and 119901 lt 005 with respect to EAEG treatment group EAEG ethyl acetate extract of Gynura formosana Kitam leaves

because of autophagy Our results showed that the proteinlevel of p62 reduced significantly in a dosage-dependentman-ner after EAEG treatment (Figure 3(b)) indicating an appar-ent induction of autophagy However these results couldnot explain if EAEG could induce the complete autophagyflux For instance calcium phosphate precipitates (CPP)induced both autophagosome synthesis and reduction ofautophagy flux due to impaired autophagosome-lysosomefusion [37] Therefore it was very necessary to evaluate if theautophagy induced by EAEG was complete autophagy fluxincluding autophagosome formation and autophagosome-lysosome fusion Our results showed that the protein levels ofboth LC3-II and p62 strongly increased in HeLa cells treatedwith EAEG together with CQ compared to cells treatedwith EAEG alone (Figure 3(c)) These results confirmed thatEAEG could induce complete autophagy flux

To confirm autophagy intensity we estimated the con-version of LC3-I to LC3-II and protein level of p62 EAEGelicited dose- and time-dependent autophagic induction inHeLa cells through the conversion of LC3-I into LC3-IIand decrease of protein level of p62 (Figure 4) stronglysuggesting that EAEG had autophagic activity Additionallywe also demonstrated that 3MA could partially abrogateEAEG induced HeLa cells death the conversion of LC3-Ito LC3-II and decreasing level of PCNA and MCM7 Thepresented collective results indicated that EAEG mightinhibit HeLa cells proliferation by inducing autophagy

In summary our studies indicated that EAEG a naturalplant extract could be a potentially effective therapeutic agentfor treating cervical cancer Our experimental evidences alsosupported the traditional use of this plant for the treatment ofthe deadly disease cancerThe identification of such biological

BioMed Research International 9

compounds in this plant and the mechanism against tumorcells are our research targets in the future

Conflicts of Interest

The authors declare that there are no conflicts of interest

Acknowledgments

This project was funded by Minxi Public Service Platformfor deep processing of Characteristic Agricultural Prod-ucts (2016LY08) the Natural Science Foundation of FujianProvince (2018J01458) Longyan University (LC2016008) theTraining Program of Fujian Excellent Talents in University(MJR201558) and the National Natural Science Foundationof China (81601600)

References

[1] B J Monk ldquoInvasive Cervical Cancerrdquo in Clinical GynecologicOncology 2012 1119

[2] W Zheng Z Liu W Zhang and X Hu ldquomiR-31 functions asan oncogene in cervical cancerrdquo Archives of Gynecology andObstetrics vol 292 no 5 pp 1083ndash1089 2015

[3] S EWaggoner ldquoCervical cancerrdquoThe Lancet vol 361 no 9376pp 2217ndash2225 2003

[4] Y Chen HWangW Lin and P Shuai ldquoADAR1 overexpressionis associated with cervical cancer progression and angiogene-sisrdquo Diagnostic Pathology vol 12 no 12 2017

[5] S Sen R Chakraborty C Sridhar Y S R Reddy and BDe ldquoFree radicals antioxidants diseases and phytomedicinescurrent status and future prospectrdquo International Journal ofPharmaceutical Sciences Review and Research vol 3 no 1 pp91ndash100 2010

[6] C Liu J-Z Liao and P-Y Li ldquoTraditional Chinese herbalextracts inducing autophagy as a novel approach in therapy ofnonalcoholic fatty liver diseaserdquoWorld Journal of Gastroenterol-ogy vol 23 no 11 pp 1964ndash1973 2017

[7] F S Reis R T Lima P Morales I C F R Ferreira andM H Vasconcelos ldquoMethanolic extract of ganoderma luciduminduces autophagy of AGS human gastric tumor cellsrdquoMolecules vol 20 no 10 pp 17872ndash17882 2015

[8] N Yu Y Xiong and C Wang ldquoBu-Zhong-Yi-Qi Decoctionthe Water Extract of Chinese Traditional Herbal MedicineEnhances Cisplatin Cytotoxicity in A549DDP Cells throughInduction of Apoptosis and Autophagyrdquo BioMed ResearchInternational vol 2017 Article ID 3692797 9 pages 2017

[9] J Ma C Guo Y Pan D Lin L Qiu and L Wen ldquoAntioxidantand anti-inflammatory activities of ethyl acetate extract ofGynura formosana (Kitam) leavesrdquo Experimental and Thera-peutic Medicine vol 14 no 3 pp 2303ndash2309 2017

[10] Q Tian J Li X Xie et al ldquoStereospecific induction of nuclearfactor-120581B activation by isochamaejasminrdquo Molecular Pharma-cology vol 68 no 6 pp 1534ndash1542 2005

[11] W-K Liu F W K Cheung B P L Liu C Li W Ye and C-T Che ldquoInvolvement of p21 and FasL in induction of cell cyclearrest and apoptosis by neochamaejasmin a in human prostateLNCaP cancer cellsrdquo Journal of Natural Products vol 71 no 5pp 842ndash846 2008

[12] L Yang Y Liu M Wang et al ldquoCelastrus orbiculatus extracttriggers apoptosis and autophagy via PI3KAktmTOR inhibi-tion in human colorectal cancer cellsrdquo Oncology Letters vol 12no 5 pp 3771ndash3778 2016

[13] P-FWei L Zhang S K Nethi et al ldquoAccelerating the clearanceof mutant huntingtin protein aggregates through autophagyinduction by europium hydroxide nanorodsrdquo Biomaterials vol35 no 3 pp 899ndash907 2014

[14] J Yang Y Zhou X Cheng et al ldquoIsogambogenic acid inducesapoptosis-independent autophagic cell death in human non-small-cell lung carcinoma cellsrdquo Scientific Reports vol 5 pp 1ndash92015

[15] F Janku D J McConkey D S Hong and R KurzrockldquoAutophagy as a target for anticancer therapyrdquo Nature ReviewsClinical Oncology vol 8 no 9 pp 528ndash539 2011

[16] Z J Yang C E Chee S Huang and F A Sinicrope ldquoAutophagymodulation for cancer therapyrdquo Cancer Biology ampTherapy vol11 no 2 pp 169ndash176 2011

[17] Y Lao G Wan Z Liu et al ldquoThe natural compound oblongi-folin C inhibits autophagic flux and enhances antitumor efficacyof nutrient deprivationrdquo Autophagy vol 10 no 5 pp 736ndash7492014

[18] H-M Ni A Bockus A L Wozniak et al ldquoDissecting the dy-namic turnover of GFP-LC3 in the autolysosomerdquo Autophagyvol 7 no 2 pp 188ndash204 2011

[19] M Zhou L Yang M Shao et al ldquoEffects of ZearalenoneExposure on the TGF-1205731Smad3 Signaling Pathway and theExpression of Proliferation or Apoptosis Related Genes of Post-Weaning Giltsrdquo Toxins vol 10 no 2 p 49 2018

[20] D Xu Y LaoN Xu et al ldquoIdentification and characterization ofanticancer compounds targeting apoptosis and autophagy fromChinese native Garcinia speciesrdquo Planta Medica vol 81 no 1pp 79ndash89 2014

[21] D R Green and J C Reed ldquoMitochondria and apoptosisrdquoScience vol 281 no 5381 pp 1309ndash1312 1998

[22] C J Hawkins and D L Vaux ldquoAnalysis of the Role of bcl-2 inApoptosisrdquo Immunological Reviews vol 142 no 1 pp 127ndash1391994

[23] Y Tsujimoto ldquoStress-resistance conferred by high level of bcl-2120572 protein in human B lymphoblastoid cellrdquo Oncogene vol 4no 11 pp 1331ndash1336 1989

[24] D R Green ldquoApoptotic pathways paper wraps stone bluntsscissorsrdquo Cell vol 102 no 1 pp 1ndash4 2000

[25] S Tsolmon J Han and H Isoda ldquoInhibition of cell growthby Stellera chamaejasme extract is associated with induction ofautophagy and differentiation in chronic leukemia K562 cellsrdquoJournal of Bioscience and Bioengineering vol 110 no 2 pp 262ndash268 2010

[26] T Kanzawa Y Kondo H Ito S Kondo and I GermanoldquoInduction of autophagic cell death inmalignant glioma cells byarsenic trioxiderdquo Cancer Research vol 63 no 9 pp 2103ndash21082003

[27] A A EllingtonM A Berhow and KW Singletary ldquoInhibitionof Akt signaling and enhanced ERK12 activity are involved ininduction of macroautophagy by triterpenoid B-group soyas-aponins in colon cancer cellsrdquo Carcinogenesis vol 27 no 2 pp298ndash306 2006

[28] K Pallauf and G Rimbach ldquoAutophagy polyphenols andhealthy ageingrdquo Ageing Research Reviews vol 12 no 1 pp 237ndash252 2013

10 BioMed Research International

[29] A De A De C Papasian et al ldquoEmblica officinalis ExtractInduces Autophagy and Inhibits Human Ovarian CancerCell Proliferation Angiogenesis Growth of Mouse XenograftTumorsrdquo PLoS ONE vol 8 no 8 Article ID e72748 2013

[30] C CDenardin L AMMartinsMM Parisi et al ldquoAutophagyinduced by purple pitanga (Eugenia uniflora L) extract trig-gered a cooperative effect on inducing the hepatic stellate celldeathrdquo Cell Biology and Toxicology vol 33 no 2 pp 197ndash2062017

[31] D R Green L Galluzzi and G Kroemer ldquoMitochondriaand the autophagy-inflammation-cell death axis in organismalagingrdquo Science vol 333 no 6046 pp 1109ndash1112 2011

[32] B Levine N Mizushima and H W Virgin ldquoAutophagy inimmunity and inflammationrdquo Nature vol 469 no 7330 pp323ndash335 2011

[33] N Mizushima B Levine A M Cuervo and D J Klion-sky ldquoAutophagy fights disease through cellular self-digestionrdquoNature vol 451 no 7182 pp 1069ndash1075 2008

[34] V Thongrakard R Titone C Follo et al ldquoTurmeric toxicityin A431 epidermoid cancer cells associates with autophagydegradation of anti-apoptotic and anti-autophagic p53 mutantrdquoPhytotherapy Research vol 28 no 12 pp 1761ndash1769 2014

[35] A Kuma M Matsui and N Mizushima ldquoLC3 an autophago-some marker can be incorporated into protein aggregatesindependent of autophagy Caution in the interpretation of LC3localizationrdquo Autophagy vol 3 no 4 pp 323ndash328 2007

[36] M Komatsu S Waguri M Koike et al ldquoHomeostatic lev-els of p62 control cytoplasmic inclusion body formation inautophagy-deficient micerdquo Cell vol 131 no 6 pp 1149ndash11632007

[37] S SarkarVKorolchukMRennaAWinslow andDC Rubin-sztein ldquoMethodological considerations for assessing autophagymodulators A study with calcium phosphate precipitatesrdquoAutophagy vol 5 no 3 pp 307ndash313 2009

Hindawiwwwhindawicom

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Submit your manuscripts atwwwhindawicom

4 BioMed Research International

32 The Cytostatic Effect of EAEG Was Not due to EnhancedApoptosis The most likely explanation for the observedcytostatic effect of EAEG on tumor cells was the induction ofapoptotic cell death by EAEG To investigate this possibilitywe conductedDNA fragmentation (the hallmark of apoptoticcell death) assay and also assessed the expression level fora number of apoptosis-related proteins Treatment of HeLacells with EAEG at a range of concentrations up to 75120583gmlfor either 24 h or 48 h induced no DNA fragmentation(Figure 2(a)) and did not alter the expression level of Bid BaxBcl-2 and caspase-3 (Figures 2(b) and 2(c)) FurthermoreZ-VAD-FMK an apoptosis inhibitor [14] did not affect theviability-reducing effect of EAEG (Figures 2(d) and 2(e))Taken together these results indicated that the cytostaticeffect of EAEG in HeLa cells was apoptosis-independent

33 EAEG Induced Complete Autophagy Autophagy mod-ulation has been demonstrated to be a critical mediator ofanticancer efficacy for many drugs [15ndash17] Our results aboveshowed that EAEG did not cause apoptotic cell death inHeLa cells so we next investigated whether EAEG inducedautophagyWeusedGFP-LC3HeLa a cell line stably express-ing GFP-LC3 a fusion protein between green fluorescentprotein (GFP) and microtubule-associated light chain 3(LC3) protein GFP-LC3 protein accumulated to form greenfluorescence puncta on autophagosome membranes uponautophagy [13] Similar to trehalose the positive controlEAEG at the various concentrations induced significantpuncta formation while DMSO (EAEG solvent) did notindicating that EAEG elicited autophagy (Figure 3(a)) Inaddition EAEG treatment significantly enhanced the con-version of LC3-I to LC3-II and reduced the protein levelof p62 a common autophagic substrate with significant(119901 lt 001) changes observed at 60120583gml and above for 24 h(Figure 4(a)) lending further proof for the occurrence ofautophagy and also suggesting that the autophagy inducedby EAEG was complete with normal cargo degradation(Figure 3(b)) To further support the latter conclusion wetreated HeLa cells with 60 120583gml EAEG for 24 h in thepresence or absence of Chloroquine (CQ) a classical later-stage autophagy inhibitor Earlier literature showed that thedegradation of LC3-II would be impaired in cells treatedwith saturating dose of CQ (50mM) [18] Indeed a furtherincrease in the level of LC3-II was seen in HeLa cells treatedwith EAEG plus CQ as compared to HeLa cells treated withEAEG alone (Figure 3(c)) strongly suggesting that EAEGinduced complete autophagy with normal flux As would beexpected p62 degradation was retarded by CQ (Figure 3(c))

34 EAEG Induced Dose- and Time-Dependent AutophagyTo further characterize the autophagic response elicited byEAEG we performed dose- and time-dependent studiesusing LC3-ILC3-II conversion and p62 protein level as themeasures HeLa cells were treated with different concen-trations (0 40 50 60 70 and 80 120583gml) of EAEG for24 h or with 60 120583gml EAEG for different times (0 3 69 18 and 24 h) A dose-dependent conversion of LC3-I toLC3-II accompanied by a corresponding decrease in thelevel of p62 was observed following EAEG treatment in

HeLa cells with significant changes observed at 40120583gmland above (Figure 4(a)) The conversion of LC3-I to LC3-IIand the corresponding decrease in the level of p62 werealso time-dependent with significant change observed at 3 hand beyond following EAEG treatment in HeLa cells (Fig-ure 4(b))

35 The Autophagy Induced by EAEG Caused HeLa CellsDeath Did the autophagy induction contribute to the cyto-static effect elicited by EAEG To assess this possibility wemeasured cell viability after the treatment of HeLa cellswith EAEG plus 3MA a class III-PI3K autophagy inhibitorAs shown in Figure 5(a) 60 120583gml EAEG treatment for24 h reduced the cell viability to 6468 and this effectwas partially offset by the cotreatment with 5mM 3MA(Figure 5(a)) which inhibited significantly the autophagyinduced by EAEG (Figure 5(b)) Furthermore EAEG signif-icantly decreased the level of PCNA and MCM7 two widelyused cell proliferation markers [19] and this effect was alsopartially abrogated by 3MA (Figure 5(b)) We thus concludedthat EAEG elicited a cytostatic effect on cancer cells throughblocking autophagy-mediated cell proliferation rather thaninducing apoptosis

4 Discussion

Gynura formosana Kitam had been used traditionally forthe prevention of cancer diabetes and inflammation inChina Our previous results showed that EAEG containedpolyphenols and flavonoids and had antioxidant and anti-inflammatory activities [9]These collective results suggestedthat EAEG might present antitumor activity However therewas little evidence showing the antitumor effects of EAEG

The plant-derived compounds had served as a mainsource of drugs and aboutmore than 50of pharmaceuticalswere derived from natural compounds [20] To determine ifEAEG could affect the cells proliferation we first investigatedthe cytostatic activity of EAEG on HeLa HepG2 andMCF-7cells Our results showed that EAEG reduced the cell viabilityin time- and dose-dependent manners in three cell lines andwas more toxic to HeLa cells (Figure 1) which suggestedthat EAEG might have better chemotherapeutic potential tocervical cancer To elucidate the possiblemechanism involvedin the above cytostatic effects we assumed that HeLa cellswould undergo apoptotic cell death after EAEG treatmentHowever any DNA fragmentation could not been detectedin HeLa cells treated with even 75120583gml EAEG for 48 hWe further investigated the proapoptosis antiapoptosis andapoptosis-related proteins expression in HeLa cells treatedwith EAEG Bax and Bidwere both proapoptotic proteins andcould promote apoptotic proteins release However the anti-apoptotic protein Bcl-2 induced by various pathologic andphysiologic stimuli could suppress apoptotic proteins efflux[21ndash23] Caspase was a sensitive indicator of apoptosis [24]We evidenced in this study that EAEG could not induce theactivation of Bax Bid Bcl-2 and caspase-3 in HeLa cells byWestern blot analysis suggesting that apoptotic pathway wasnot triggered by EAEG treatment Most importantly the

BioMed Research International 5

48 Ｂ24 Ｂ

(a)

Bax

Bid

Bcl-2

Caspase 3

-actin

gml7560450

EAEG (gml)

BaxBid

Bcl-2Caspase 3

756045000

02

04

06

08

10

12

14

16

Prot

ein

-a

ctin

ratio

(b)

gml

Bax

Bid

Bcl-2

Caspase 3

-actin

7560450

EAEG (gml)

BaxBid

Bcl-2Caspase 3

756045000

02

04

06

08

10

12

14

16Pr

otei

n

-act

in ra

tio

(c)

ControlZvadfmk (50M)

EAEG (gml)7560450

0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

(d)

ControlZvadfmk (50M)

EAEG (gml)7560450

0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

(e)

Figure 2 EAEG treatment did not induce apoptotic cell death in HeLa cells (a) DNA fragmentation analysis Lines a and e controls treatedfor 24 and 48 h respectively lines b and f 45 120583gml EAEG treated for 24 and 48 h respectively lines c and g 60 120583gml EAEG treated for 24and 48 h respectively lines d and h 75 120583gml EAEG treated for 24 and 48 h respectively line i DNAmarker ((b) and (c))The expression ofBid Bax Bcl-2 and caspase-3 in HeLa cells treated with different concentrations of EAEG (0 45 60 and 75120583gml) for 24 h (b) or 48 h (c)120573-Actin served as the loading control The relative protein120573-actin ratios were calculated using ImageJ ((d) and (e)) Cell viability of HeLacells treated withwithout Z-VAD-FMK (50120583M) and different concentrations of EAEG for 24 h (d) or 48 h (e) The data were expressed asmean plusmn SEM (119899 = 3) EAEG ethyl acetate extract of Gynura formosana Kitam leaves

6 BioMed Research International

Blank DMSO EAEG (45gml)

EAEG (60 gml) EAEG (75 gml) Tre

(a)

gml

P62

LC3-I

LC3-II

GAPDH

756045006

05

04

03

02

01

00

Prot

ein

relat

ive l

evel

s

EAEG (gml)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

P62GAPDHLC3-IILC3-I

7560450

lowast

lowast

(b)

P62

LC3-I

LC3-II

GAPDH

EAEGCQ

minus

minusminus

minus

++

+ +06

05

04

03

02

01

00

Prot

ein

relat

ive l

evel

s

07

CQEAEG + CQEAEGControl

P62GAPDHLC3-IILC3-I

lowastlowastlowastlowast

lowastlowast

lowast

(c)Figure 3 EAEG induced autophagy in HeLa cells (a) Analysis of GFP-LC3 puncta formation in GFP-LC3HeLa cells under fluorescencemicroscopy Cells were treated for 24 h with DMEM only (blank) EAEG solvent (DMSO) as control different concentrations (45 60 and75 120583gml) of EAEG or the positive control trehalose (Tre) (scale bar = 20120583m) (b)The expression analysis of the autophagy-related proteinsp62 and LC3 byWestern blot HeLa cells were treated for 24 h with different concentrations (0 45 60 and 75 120583gml) of EAEG (c)The relativeprotein levels of p62 and LC3-II in HeLa cells treated with 60 120583gml EAEG with or without CQ (50mM) for 24 h GAPDH served as theloading control The protein relative ratio was calculated using ImageJ The data were expressed as mean plusmn SEM (119899 = 3) lowast119901 lt 005 andlowastlowast119901 lt 001 with respect to the control group 119901 lt 001 with respect to EAEG treatment group EAEG ethyl acetate extract of Gynuraformosana Kitam leaves

BioMed Research International 7

gml

P62

LC3-I

LC3-II

GAPDH

0 40 50 60 70 80

0 40 50 60 70 80

EAEG (gml)

00

01

02

03

04

05

06

Prot

ein

relat

ive l

evel

s

LC3-IILC3-IP62GAPDH

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowast

lowast

lowastlowastlowastlowast

(a)

P62

LC3-ILC3-II

GAPDH

0 3 6 9 18 24 h

0 3 6 9 18 24

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowast

lowastlowast

(hours)

00

01

02

03

04

05

06

Prot

ein

relat

ive l

evel

s

LC3-IILC3-IP62GAPDH

(b)

Figure 4The conversation of LC3-I to LC3-II Protein level of p62 was measured byWestern blot (a) HeLa cells were treated with EAEG at aconcentration of 0 40 50 60 70 or 80120583gml for 24 h (b)HeLa cells were treatedwith 60120583gml EAEG for 0 3 6 9 18 or 24 h GAPDH servedas the loading control The relative protein ratio was calculated using ImageJ The data were expressed as mean plusmn SEM (119899 = 3) lowast119901 lt 005lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001 with respect to the control group EAEG ethyl acetate extract of Gynura formosana Kitam leaves

apoptosis inhibitor Z-VAD-FMK did not affect the viability-reducing effect of EAEG (Figures 2(d) and 2(e)) This findingserved as a convincing evidence for nonapoptotic cell deathin EAEG treated HeLa cells

Autophagy was considered to be main way to inhibittumor cells proliferation when cells underwent nonapoptoticcell death [25 26] Increasing evidences had suggested thatautophagy could be induced by many natural products invarious anticancer therapies Triterpenoid B-group soyas-aponins could induce autophagy in colon cancer cells byinhibition of Akt signaling and enhanced ERK12 activity[27] Polyphenols extracts could induce autophagy whichwas advantageous to inhibit neurodegeneration and cancerprocess [28] Recent studies demonstrated that Emblicaofficinalis extracts could also induce autophagy and inhibithuman ovarian cancer cell proliferation [29] These reportssupported us to hypothesize that HeLa cells death caused byEAEG could involve autophagy program Autophagy wasa physiologically and pathologically regulated process andplayed an important role in the maintenance of intracellularhomeostasis in every eukaryotic cell [30ndash32] It started

with inclosing cytoplasmic contents in a vacuole calledautophagosome and then autophagosome fused with lyso-some to form autolysosomewhere substances inside could bedegraded and recycled [33] Autophagy could be initiated bysome autophagy-related proteins such as the microtubule-associated protein LC3 which was a reliable autophagicmarker [34] LC3 was a constitutively expressed protein inmammalian cells and could be processed to form cytoso-lic LC3-I During autophagy LC3-I was recruited to theautophagosome where LC3-II was generated by fusingLC3-I with phosphatidylethanolamine and then cellularautophagosome puncta including LC3-II were formed [35]Our results demonstrated that EAEG upregulated GFPpuncta formation (Figure 3(a)) and increased the conversionof LC3-I to LC3-II in HeLa cells (Figure 3(b)) suggest-ing the recruitment of LC3-II upon EAEG administrationFurthermore we analyzed the protein level of p62 anotherautophagicmarker [36] to study induction of autophagy p62could be trapped by LC3 incorporated into the autophago-some and then degraded via autophagy Correspondinglythe protein level of p62 should be decreased in the cells

8 BioMed Research International

lowastlowast

EAEG + 3MAEAEGControl0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

(a)

EAEG3MA

minus

minusminus +

+ +

LC3-I

LC3-II

PCNA

MCM7

GAPDH

LC3-IILC3-IPCNAGAPDHMCM7GAPDH

EAEG + 3MAEAEGControl

lowastlowast

lowastlowast

lowastlowast

00

01

02

03

04

05

06

07

08

Prot

ein

relat

ive l

evel

s

(b)

Figure 5 EAEG induced HeLa cells undergoing autophagic cell death (a) Cell viability of HeLa cells treated with 60120583gml EAEG for 24 h inpresence or absence of 5mM 3MA (b) Western blot analysis of autophagy and proliferation proteins GAPDH served as the loading controlThe protein relative ratio was calculated using ImageJThe data were expressed as mean plusmn SEM (119899 = 3) lowastlowast119901 lt 001with respect to the controlgroup and 119901 lt 005 with respect to EAEG treatment group EAEG ethyl acetate extract of Gynura formosana Kitam leaves

because of autophagy Our results showed that the proteinlevel of p62 reduced significantly in a dosage-dependentman-ner after EAEG treatment (Figure 3(b)) indicating an appar-ent induction of autophagy However these results couldnot explain if EAEG could induce the complete autophagyflux For instance calcium phosphate precipitates (CPP)induced both autophagosome synthesis and reduction ofautophagy flux due to impaired autophagosome-lysosomefusion [37] Therefore it was very necessary to evaluate if theautophagy induced by EAEG was complete autophagy fluxincluding autophagosome formation and autophagosome-lysosome fusion Our results showed that the protein levels ofboth LC3-II and p62 strongly increased in HeLa cells treatedwith EAEG together with CQ compared to cells treatedwith EAEG alone (Figure 3(c)) These results confirmed thatEAEG could induce complete autophagy flux

To confirm autophagy intensity we estimated the con-version of LC3-I to LC3-II and protein level of p62 EAEGelicited dose- and time-dependent autophagic induction inHeLa cells through the conversion of LC3-I into LC3-IIand decrease of protein level of p62 (Figure 4) stronglysuggesting that EAEG had autophagic activity Additionallywe also demonstrated that 3MA could partially abrogateEAEG induced HeLa cells death the conversion of LC3-Ito LC3-II and decreasing level of PCNA and MCM7 Thepresented collective results indicated that EAEG mightinhibit HeLa cells proliferation by inducing autophagy

In summary our studies indicated that EAEG a naturalplant extract could be a potentially effective therapeutic agentfor treating cervical cancer Our experimental evidences alsosupported the traditional use of this plant for the treatment ofthe deadly disease cancerThe identification of such biological

BioMed Research International 9

compounds in this plant and the mechanism against tumorcells are our research targets in the future

Conflicts of Interest

The authors declare that there are no conflicts of interest

Acknowledgments

This project was funded by Minxi Public Service Platformfor deep processing of Characteristic Agricultural Prod-ucts (2016LY08) the Natural Science Foundation of FujianProvince (2018J01458) Longyan University (LC2016008) theTraining Program of Fujian Excellent Talents in University(MJR201558) and the National Natural Science Foundationof China (81601600)

References

[1] B J Monk ldquoInvasive Cervical Cancerrdquo in Clinical GynecologicOncology 2012 1119

[2] W Zheng Z Liu W Zhang and X Hu ldquomiR-31 functions asan oncogene in cervical cancerrdquo Archives of Gynecology andObstetrics vol 292 no 5 pp 1083ndash1089 2015

[3] S EWaggoner ldquoCervical cancerrdquoThe Lancet vol 361 no 9376pp 2217ndash2225 2003

[4] Y Chen HWangW Lin and P Shuai ldquoADAR1 overexpressionis associated with cervical cancer progression and angiogene-sisrdquo Diagnostic Pathology vol 12 no 12 2017

[5] S Sen R Chakraborty C Sridhar Y S R Reddy and BDe ldquoFree radicals antioxidants diseases and phytomedicinescurrent status and future prospectrdquo International Journal ofPharmaceutical Sciences Review and Research vol 3 no 1 pp91ndash100 2010

[6] C Liu J-Z Liao and P-Y Li ldquoTraditional Chinese herbalextracts inducing autophagy as a novel approach in therapy ofnonalcoholic fatty liver diseaserdquoWorld Journal of Gastroenterol-ogy vol 23 no 11 pp 1964ndash1973 2017

[7] F S Reis R T Lima P Morales I C F R Ferreira andM H Vasconcelos ldquoMethanolic extract of ganoderma luciduminduces autophagy of AGS human gastric tumor cellsrdquoMolecules vol 20 no 10 pp 17872ndash17882 2015

[8] N Yu Y Xiong and C Wang ldquoBu-Zhong-Yi-Qi Decoctionthe Water Extract of Chinese Traditional Herbal MedicineEnhances Cisplatin Cytotoxicity in A549DDP Cells throughInduction of Apoptosis and Autophagyrdquo BioMed ResearchInternational vol 2017 Article ID 3692797 9 pages 2017

[9] J Ma C Guo Y Pan D Lin L Qiu and L Wen ldquoAntioxidantand anti-inflammatory activities of ethyl acetate extract ofGynura formosana (Kitam) leavesrdquo Experimental and Thera-peutic Medicine vol 14 no 3 pp 2303ndash2309 2017

[10] Q Tian J Li X Xie et al ldquoStereospecific induction of nuclearfactor-120581B activation by isochamaejasminrdquo Molecular Pharma-cology vol 68 no 6 pp 1534ndash1542 2005

[11] W-K Liu F W K Cheung B P L Liu C Li W Ye and C-T Che ldquoInvolvement of p21 and FasL in induction of cell cyclearrest and apoptosis by neochamaejasmin a in human prostateLNCaP cancer cellsrdquo Journal of Natural Products vol 71 no 5pp 842ndash846 2008

[12] L Yang Y Liu M Wang et al ldquoCelastrus orbiculatus extracttriggers apoptosis and autophagy via PI3KAktmTOR inhibi-tion in human colorectal cancer cellsrdquo Oncology Letters vol 12no 5 pp 3771ndash3778 2016

[13] P-FWei L Zhang S K Nethi et al ldquoAccelerating the clearanceof mutant huntingtin protein aggregates through autophagyinduction by europium hydroxide nanorodsrdquo Biomaterials vol35 no 3 pp 899ndash907 2014

[14] J Yang Y Zhou X Cheng et al ldquoIsogambogenic acid inducesapoptosis-independent autophagic cell death in human non-small-cell lung carcinoma cellsrdquo Scientific Reports vol 5 pp 1ndash92015

[15] F Janku D J McConkey D S Hong and R KurzrockldquoAutophagy as a target for anticancer therapyrdquo Nature ReviewsClinical Oncology vol 8 no 9 pp 528ndash539 2011

[16] Z J Yang C E Chee S Huang and F A Sinicrope ldquoAutophagymodulation for cancer therapyrdquo Cancer Biology ampTherapy vol11 no 2 pp 169ndash176 2011

[17] Y Lao G Wan Z Liu et al ldquoThe natural compound oblongi-folin C inhibits autophagic flux and enhances antitumor efficacyof nutrient deprivationrdquo Autophagy vol 10 no 5 pp 736ndash7492014

[18] H-M Ni A Bockus A L Wozniak et al ldquoDissecting the dy-namic turnover of GFP-LC3 in the autolysosomerdquo Autophagyvol 7 no 2 pp 188ndash204 2011

[19] M Zhou L Yang M Shao et al ldquoEffects of ZearalenoneExposure on the TGF-1205731Smad3 Signaling Pathway and theExpression of Proliferation or Apoptosis Related Genes of Post-Weaning Giltsrdquo Toxins vol 10 no 2 p 49 2018

[20] D Xu Y LaoN Xu et al ldquoIdentification and characterization ofanticancer compounds targeting apoptosis and autophagy fromChinese native Garcinia speciesrdquo Planta Medica vol 81 no 1pp 79ndash89 2014

[21] D R Green and J C Reed ldquoMitochondria and apoptosisrdquoScience vol 281 no 5381 pp 1309ndash1312 1998

[22] C J Hawkins and D L Vaux ldquoAnalysis of the Role of bcl-2 inApoptosisrdquo Immunological Reviews vol 142 no 1 pp 127ndash1391994

[23] Y Tsujimoto ldquoStress-resistance conferred by high level of bcl-2120572 protein in human B lymphoblastoid cellrdquo Oncogene vol 4no 11 pp 1331ndash1336 1989

[24] D R Green ldquoApoptotic pathways paper wraps stone bluntsscissorsrdquo Cell vol 102 no 1 pp 1ndash4 2000

[25] S Tsolmon J Han and H Isoda ldquoInhibition of cell growthby Stellera chamaejasme extract is associated with induction ofautophagy and differentiation in chronic leukemia K562 cellsrdquoJournal of Bioscience and Bioengineering vol 110 no 2 pp 262ndash268 2010

[26] T Kanzawa Y Kondo H Ito S Kondo and I GermanoldquoInduction of autophagic cell death inmalignant glioma cells byarsenic trioxiderdquo Cancer Research vol 63 no 9 pp 2103ndash21082003

[27] A A EllingtonM A Berhow and KW Singletary ldquoInhibitionof Akt signaling and enhanced ERK12 activity are involved ininduction of macroautophagy by triterpenoid B-group soyas-aponins in colon cancer cellsrdquo Carcinogenesis vol 27 no 2 pp298ndash306 2006

[28] K Pallauf and G Rimbach ldquoAutophagy polyphenols andhealthy ageingrdquo Ageing Research Reviews vol 12 no 1 pp 237ndash252 2013

10 BioMed Research International

[29] A De A De C Papasian et al ldquoEmblica officinalis ExtractInduces Autophagy and Inhibits Human Ovarian CancerCell Proliferation Angiogenesis Growth of Mouse XenograftTumorsrdquo PLoS ONE vol 8 no 8 Article ID e72748 2013

[30] C CDenardin L AMMartinsMM Parisi et al ldquoAutophagyinduced by purple pitanga (Eugenia uniflora L) extract trig-gered a cooperative effect on inducing the hepatic stellate celldeathrdquo Cell Biology and Toxicology vol 33 no 2 pp 197ndash2062017

[31] D R Green L Galluzzi and G Kroemer ldquoMitochondriaand the autophagy-inflammation-cell death axis in organismalagingrdquo Science vol 333 no 6046 pp 1109ndash1112 2011

[32] B Levine N Mizushima and H W Virgin ldquoAutophagy inimmunity and inflammationrdquo Nature vol 469 no 7330 pp323ndash335 2011

[33] N Mizushima B Levine A M Cuervo and D J Klion-sky ldquoAutophagy fights disease through cellular self-digestionrdquoNature vol 451 no 7182 pp 1069ndash1075 2008

[34] V Thongrakard R Titone C Follo et al ldquoTurmeric toxicityin A431 epidermoid cancer cells associates with autophagydegradation of anti-apoptotic and anti-autophagic p53 mutantrdquoPhytotherapy Research vol 28 no 12 pp 1761ndash1769 2014

[35] A Kuma M Matsui and N Mizushima ldquoLC3 an autophago-some marker can be incorporated into protein aggregatesindependent of autophagy Caution in the interpretation of LC3localizationrdquo Autophagy vol 3 no 4 pp 323ndash328 2007

[36] M Komatsu S Waguri M Koike et al ldquoHomeostatic lev-els of p62 control cytoplasmic inclusion body formation inautophagy-deficient micerdquo Cell vol 131 no 6 pp 1149ndash11632007

[37] S SarkarVKorolchukMRennaAWinslow andDC Rubin-sztein ldquoMethodological considerations for assessing autophagymodulators A study with calcium phosphate precipitatesrdquoAutophagy vol 5 no 3 pp 307ndash313 2009

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

BioMed Research International 5

48 Ｂ24 Ｂ

(a)

Bax

Bid

Bcl-2

Caspase 3

-actin

gml7560450

EAEG (gml)

BaxBid

Bcl-2Caspase 3

756045000

02

04

06

08

10

12

14

16

Prot

ein

-a

ctin

ratio

(b)

gml

Bax

Bid

Bcl-2

Caspase 3

-actin

7560450

EAEG (gml)

BaxBid

Bcl-2Caspase 3

756045000

02

04

06

08

10

12

14

16Pr

otei

n

-act

in ra

tio

(c)

ControlZvadfmk (50M)

EAEG (gml)7560450

0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

(d)

ControlZvadfmk (50M)

EAEG (gml)7560450

0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

(e)

Figure 2 EAEG treatment did not induce apoptotic cell death in HeLa cells (a) DNA fragmentation analysis Lines a and e controls treatedfor 24 and 48 h respectively lines b and f 45 120583gml EAEG treated for 24 and 48 h respectively lines c and g 60 120583gml EAEG treated for 24and 48 h respectively lines d and h 75 120583gml EAEG treated for 24 and 48 h respectively line i DNAmarker ((b) and (c))The expression ofBid Bax Bcl-2 and caspase-3 in HeLa cells treated with different concentrations of EAEG (0 45 60 and 75120583gml) for 24 h (b) or 48 h (c)120573-Actin served as the loading control The relative protein120573-actin ratios were calculated using ImageJ ((d) and (e)) Cell viability of HeLacells treated withwithout Z-VAD-FMK (50120583M) and different concentrations of EAEG for 24 h (d) or 48 h (e) The data were expressed asmean plusmn SEM (119899 = 3) EAEG ethyl acetate extract of Gynura formosana Kitam leaves

6 BioMed Research International

Blank DMSO EAEG (45gml)

EAEG (60 gml) EAEG (75 gml) Tre

(a)

gml

P62

LC3-I

LC3-II

GAPDH

756045006

05

04

03

02

01

00

Prot

ein

relat

ive l

evel

s

EAEG (gml)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

P62GAPDHLC3-IILC3-I

7560450

lowast

lowast

(b)

P62

LC3-I

LC3-II

GAPDH

EAEGCQ

minus

minusminus

minus

++

+ +06

05

04

03

02

01

00

Prot

ein

relat

ive l

evel

s

07

CQEAEG + CQEAEGControl

P62GAPDHLC3-IILC3-I

lowastlowastlowastlowast

lowastlowast

lowast

(c)Figure 3 EAEG induced autophagy in HeLa cells (a) Analysis of GFP-LC3 puncta formation in GFP-LC3HeLa cells under fluorescencemicroscopy Cells were treated for 24 h with DMEM only (blank) EAEG solvent (DMSO) as control different concentrations (45 60 and75 120583gml) of EAEG or the positive control trehalose (Tre) (scale bar = 20120583m) (b)The expression analysis of the autophagy-related proteinsp62 and LC3 byWestern blot HeLa cells were treated for 24 h with different concentrations (0 45 60 and 75 120583gml) of EAEG (c)The relativeprotein levels of p62 and LC3-II in HeLa cells treated with 60 120583gml EAEG with or without CQ (50mM) for 24 h GAPDH served as theloading control The protein relative ratio was calculated using ImageJ The data were expressed as mean plusmn SEM (119899 = 3) lowast119901 lt 005 andlowastlowast119901 lt 001 with respect to the control group 119901 lt 001 with respect to EAEG treatment group EAEG ethyl acetate extract of Gynuraformosana Kitam leaves

BioMed Research International 7

gml

P62

LC3-I

LC3-II

GAPDH

0 40 50 60 70 80

0 40 50 60 70 80

EAEG (gml)

00

01

02

03

04

05

06

Prot

ein

relat

ive l

evel

s

LC3-IILC3-IP62GAPDH

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowast

lowast

lowastlowastlowastlowast

(a)

P62

LC3-ILC3-II

GAPDH

0 3 6 9 18 24 h

0 3 6 9 18 24

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowast

lowastlowast

(hours)

00

01

02

03

04

05

06

Prot

ein

relat

ive l

evel

s

LC3-IILC3-IP62GAPDH

(b)

Figure 4The conversation of LC3-I to LC3-II Protein level of p62 was measured byWestern blot (a) HeLa cells were treated with EAEG at aconcentration of 0 40 50 60 70 or 80120583gml for 24 h (b)HeLa cells were treatedwith 60120583gml EAEG for 0 3 6 9 18 or 24 h GAPDH servedas the loading control The relative protein ratio was calculated using ImageJ The data were expressed as mean plusmn SEM (119899 = 3) lowast119901 lt 005lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001 with respect to the control group EAEG ethyl acetate extract of Gynura formosana Kitam leaves

apoptosis inhibitor Z-VAD-FMK did not affect the viability-reducing effect of EAEG (Figures 2(d) and 2(e)) This findingserved as a convincing evidence for nonapoptotic cell deathin EAEG treated HeLa cells

Autophagy was considered to be main way to inhibittumor cells proliferation when cells underwent nonapoptoticcell death [25 26] Increasing evidences had suggested thatautophagy could be induced by many natural products invarious anticancer therapies Triterpenoid B-group soyas-aponins could induce autophagy in colon cancer cells byinhibition of Akt signaling and enhanced ERK12 activity[27] Polyphenols extracts could induce autophagy whichwas advantageous to inhibit neurodegeneration and cancerprocess [28] Recent studies demonstrated that Emblicaofficinalis extracts could also induce autophagy and inhibithuman ovarian cancer cell proliferation [29] These reportssupported us to hypothesize that HeLa cells death caused byEAEG could involve autophagy program Autophagy wasa physiologically and pathologically regulated process andplayed an important role in the maintenance of intracellularhomeostasis in every eukaryotic cell [30ndash32] It started

with inclosing cytoplasmic contents in a vacuole calledautophagosome and then autophagosome fused with lyso-some to form autolysosomewhere substances inside could bedegraded and recycled [33] Autophagy could be initiated bysome autophagy-related proteins such as the microtubule-associated protein LC3 which was a reliable autophagicmarker [34] LC3 was a constitutively expressed protein inmammalian cells and could be processed to form cytoso-lic LC3-I During autophagy LC3-I was recruited to theautophagosome where LC3-II was generated by fusingLC3-I with phosphatidylethanolamine and then cellularautophagosome puncta including LC3-II were formed [35]Our results demonstrated that EAEG upregulated GFPpuncta formation (Figure 3(a)) and increased the conversionof LC3-I to LC3-II in HeLa cells (Figure 3(b)) suggest-ing the recruitment of LC3-II upon EAEG administrationFurthermore we analyzed the protein level of p62 anotherautophagicmarker [36] to study induction of autophagy p62could be trapped by LC3 incorporated into the autophago-some and then degraded via autophagy Correspondinglythe protein level of p62 should be decreased in the cells

8 BioMed Research International

lowastlowast

EAEG + 3MAEAEGControl0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

(a)

EAEG3MA

minus

minusminus +

+ +

LC3-I

LC3-II

PCNA

MCM7

GAPDH

LC3-IILC3-IPCNAGAPDHMCM7GAPDH

EAEG + 3MAEAEGControl

lowastlowast

lowastlowast

lowastlowast

00

01

02

03

04

05

06

07

08

Prot

ein

relat

ive l

evel

s

(b)

Figure 5 EAEG induced HeLa cells undergoing autophagic cell death (a) Cell viability of HeLa cells treated with 60120583gml EAEG for 24 h inpresence or absence of 5mM 3MA (b) Western blot analysis of autophagy and proliferation proteins GAPDH served as the loading controlThe protein relative ratio was calculated using ImageJThe data were expressed as mean plusmn SEM (119899 = 3) lowastlowast119901 lt 001with respect to the controlgroup and 119901 lt 005 with respect to EAEG treatment group EAEG ethyl acetate extract of Gynura formosana Kitam leaves

because of autophagy Our results showed that the proteinlevel of p62 reduced significantly in a dosage-dependentman-ner after EAEG treatment (Figure 3(b)) indicating an appar-ent induction of autophagy However these results couldnot explain if EAEG could induce the complete autophagyflux For instance calcium phosphate precipitates (CPP)induced both autophagosome synthesis and reduction ofautophagy flux due to impaired autophagosome-lysosomefusion [37] Therefore it was very necessary to evaluate if theautophagy induced by EAEG was complete autophagy fluxincluding autophagosome formation and autophagosome-lysosome fusion Our results showed that the protein levels ofboth LC3-II and p62 strongly increased in HeLa cells treatedwith EAEG together with CQ compared to cells treatedwith EAEG alone (Figure 3(c)) These results confirmed thatEAEG could induce complete autophagy flux

To confirm autophagy intensity we estimated the con-version of LC3-I to LC3-II and protein level of p62 EAEGelicited dose- and time-dependent autophagic induction inHeLa cells through the conversion of LC3-I into LC3-IIand decrease of protein level of p62 (Figure 4) stronglysuggesting that EAEG had autophagic activity Additionallywe also demonstrated that 3MA could partially abrogateEAEG induced HeLa cells death the conversion of LC3-Ito LC3-II and decreasing level of PCNA and MCM7 Thepresented collective results indicated that EAEG mightinhibit HeLa cells proliferation by inducing autophagy

In summary our studies indicated that EAEG a naturalplant extract could be a potentially effective therapeutic agentfor treating cervical cancer Our experimental evidences alsosupported the traditional use of this plant for the treatment ofthe deadly disease cancerThe identification of such biological

BioMed Research International 9

compounds in this plant and the mechanism against tumorcells are our research targets in the future

Conflicts of Interest

The authors declare that there are no conflicts of interest

Acknowledgments

This project was funded by Minxi Public Service Platformfor deep processing of Characteristic Agricultural Prod-ucts (2016LY08) the Natural Science Foundation of FujianProvince (2018J01458) Longyan University (LC2016008) theTraining Program of Fujian Excellent Talents in University(MJR201558) and the National Natural Science Foundationof China (81601600)

References

[1] B J Monk ldquoInvasive Cervical Cancerrdquo in Clinical GynecologicOncology 2012 1119

[2] W Zheng Z Liu W Zhang and X Hu ldquomiR-31 functions asan oncogene in cervical cancerrdquo Archives of Gynecology andObstetrics vol 292 no 5 pp 1083ndash1089 2015

[3] S EWaggoner ldquoCervical cancerrdquoThe Lancet vol 361 no 9376pp 2217ndash2225 2003

[4] Y Chen HWangW Lin and P Shuai ldquoADAR1 overexpressionis associated with cervical cancer progression and angiogene-sisrdquo Diagnostic Pathology vol 12 no 12 2017

[5] S Sen R Chakraborty C Sridhar Y S R Reddy and BDe ldquoFree radicals antioxidants diseases and phytomedicinescurrent status and future prospectrdquo International Journal ofPharmaceutical Sciences Review and Research vol 3 no 1 pp91ndash100 2010

[6] C Liu J-Z Liao and P-Y Li ldquoTraditional Chinese herbalextracts inducing autophagy as a novel approach in therapy ofnonalcoholic fatty liver diseaserdquoWorld Journal of Gastroenterol-ogy vol 23 no 11 pp 1964ndash1973 2017

[7] F S Reis R T Lima P Morales I C F R Ferreira andM H Vasconcelos ldquoMethanolic extract of ganoderma luciduminduces autophagy of AGS human gastric tumor cellsrdquoMolecules vol 20 no 10 pp 17872ndash17882 2015

[8] N Yu Y Xiong and C Wang ldquoBu-Zhong-Yi-Qi Decoctionthe Water Extract of Chinese Traditional Herbal MedicineEnhances Cisplatin Cytotoxicity in A549DDP Cells throughInduction of Apoptosis and Autophagyrdquo BioMed ResearchInternational vol 2017 Article ID 3692797 9 pages 2017

[9] J Ma C Guo Y Pan D Lin L Qiu and L Wen ldquoAntioxidantand anti-inflammatory activities of ethyl acetate extract ofGynura formosana (Kitam) leavesrdquo Experimental and Thera-peutic Medicine vol 14 no 3 pp 2303ndash2309 2017

[10] Q Tian J Li X Xie et al ldquoStereospecific induction of nuclearfactor-120581B activation by isochamaejasminrdquo Molecular Pharma-cology vol 68 no 6 pp 1534ndash1542 2005

[11] W-K Liu F W K Cheung B P L Liu C Li W Ye and C-T Che ldquoInvolvement of p21 and FasL in induction of cell cyclearrest and apoptosis by neochamaejasmin a in human prostateLNCaP cancer cellsrdquo Journal of Natural Products vol 71 no 5pp 842ndash846 2008

[12] L Yang Y Liu M Wang et al ldquoCelastrus orbiculatus extracttriggers apoptosis and autophagy via PI3KAktmTOR inhibi-tion in human colorectal cancer cellsrdquo Oncology Letters vol 12no 5 pp 3771ndash3778 2016

[13] P-FWei L Zhang S K Nethi et al ldquoAccelerating the clearanceof mutant huntingtin protein aggregates through autophagyinduction by europium hydroxide nanorodsrdquo Biomaterials vol35 no 3 pp 899ndash907 2014

[14] J Yang Y Zhou X Cheng et al ldquoIsogambogenic acid inducesapoptosis-independent autophagic cell death in human non-small-cell lung carcinoma cellsrdquo Scientific Reports vol 5 pp 1ndash92015

[15] F Janku D J McConkey D S Hong and R KurzrockldquoAutophagy as a target for anticancer therapyrdquo Nature ReviewsClinical Oncology vol 8 no 9 pp 528ndash539 2011

[16] Z J Yang C E Chee S Huang and F A Sinicrope ldquoAutophagymodulation for cancer therapyrdquo Cancer Biology ampTherapy vol11 no 2 pp 169ndash176 2011

[17] Y Lao G Wan Z Liu et al ldquoThe natural compound oblongi-folin C inhibits autophagic flux and enhances antitumor efficacyof nutrient deprivationrdquo Autophagy vol 10 no 5 pp 736ndash7492014

[18] H-M Ni A Bockus A L Wozniak et al ldquoDissecting the dy-namic turnover of GFP-LC3 in the autolysosomerdquo Autophagyvol 7 no 2 pp 188ndash204 2011

[19] M Zhou L Yang M Shao et al ldquoEffects of ZearalenoneExposure on the TGF-1205731Smad3 Signaling Pathway and theExpression of Proliferation or Apoptosis Related Genes of Post-Weaning Giltsrdquo Toxins vol 10 no 2 p 49 2018

[20] D Xu Y LaoN Xu et al ldquoIdentification and characterization ofanticancer compounds targeting apoptosis and autophagy fromChinese native Garcinia speciesrdquo Planta Medica vol 81 no 1pp 79ndash89 2014

[21] D R Green and J C Reed ldquoMitochondria and apoptosisrdquoScience vol 281 no 5381 pp 1309ndash1312 1998

[22] C J Hawkins and D L Vaux ldquoAnalysis of the Role of bcl-2 inApoptosisrdquo Immunological Reviews vol 142 no 1 pp 127ndash1391994

[23] Y Tsujimoto ldquoStress-resistance conferred by high level of bcl-2120572 protein in human B lymphoblastoid cellrdquo Oncogene vol 4no 11 pp 1331ndash1336 1989

[24] D R Green ldquoApoptotic pathways paper wraps stone bluntsscissorsrdquo Cell vol 102 no 1 pp 1ndash4 2000

[25] S Tsolmon J Han and H Isoda ldquoInhibition of cell growthby Stellera chamaejasme extract is associated with induction ofautophagy and differentiation in chronic leukemia K562 cellsrdquoJournal of Bioscience and Bioengineering vol 110 no 2 pp 262ndash268 2010

[26] T Kanzawa Y Kondo H Ito S Kondo and I GermanoldquoInduction of autophagic cell death inmalignant glioma cells byarsenic trioxiderdquo Cancer Research vol 63 no 9 pp 2103ndash21082003

[27] A A EllingtonM A Berhow and KW Singletary ldquoInhibitionof Akt signaling and enhanced ERK12 activity are involved ininduction of macroautophagy by triterpenoid B-group soyas-aponins in colon cancer cellsrdquo Carcinogenesis vol 27 no 2 pp298ndash306 2006

[28] K Pallauf and G Rimbach ldquoAutophagy polyphenols andhealthy ageingrdquo Ageing Research Reviews vol 12 no 1 pp 237ndash252 2013

10 BioMed Research International

[29] A De A De C Papasian et al ldquoEmblica officinalis ExtractInduces Autophagy and Inhibits Human Ovarian CancerCell Proliferation Angiogenesis Growth of Mouse XenograftTumorsrdquo PLoS ONE vol 8 no 8 Article ID e72748 2013

[30] C CDenardin L AMMartinsMM Parisi et al ldquoAutophagyinduced by purple pitanga (Eugenia uniflora L) extract trig-gered a cooperative effect on inducing the hepatic stellate celldeathrdquo Cell Biology and Toxicology vol 33 no 2 pp 197ndash2062017

[31] D R Green L Galluzzi and G Kroemer ldquoMitochondriaand the autophagy-inflammation-cell death axis in organismalagingrdquo Science vol 333 no 6046 pp 1109ndash1112 2011

[32] B Levine N Mizushima and H W Virgin ldquoAutophagy inimmunity and inflammationrdquo Nature vol 469 no 7330 pp323ndash335 2011

[33] N Mizushima B Levine A M Cuervo and D J Klion-sky ldquoAutophagy fights disease through cellular self-digestionrdquoNature vol 451 no 7182 pp 1069ndash1075 2008

[34] V Thongrakard R Titone C Follo et al ldquoTurmeric toxicityin A431 epidermoid cancer cells associates with autophagydegradation of anti-apoptotic and anti-autophagic p53 mutantrdquoPhytotherapy Research vol 28 no 12 pp 1761ndash1769 2014

[35] A Kuma M Matsui and N Mizushima ldquoLC3 an autophago-some marker can be incorporated into protein aggregatesindependent of autophagy Caution in the interpretation of LC3localizationrdquo Autophagy vol 3 no 4 pp 323ndash328 2007

[36] M Komatsu S Waguri M Koike et al ldquoHomeostatic lev-els of p62 control cytoplasmic inclusion body formation inautophagy-deficient micerdquo Cell vol 131 no 6 pp 1149ndash11632007

[37] S SarkarVKorolchukMRennaAWinslow andDC Rubin-sztein ldquoMethodological considerations for assessing autophagymodulators A study with calcium phosphate precipitatesrdquoAutophagy vol 5 no 3 pp 307ndash313 2009

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

6 BioMed Research International

Blank DMSO EAEG (45gml)

EAEG (60 gml) EAEG (75 gml) Tre

(a)

gml

P62

LC3-I

LC3-II

GAPDH

756045006

05

04

03

02

01

00

Prot

ein

relat

ive l

evel

s

EAEG (gml)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

P62GAPDHLC3-IILC3-I

7560450

lowast

lowast

(b)

P62

LC3-I

LC3-II

GAPDH

EAEGCQ

minus

minusminus

minus

++

+ +06

05

04

03

02

01

00

Prot

ein

relat

ive l

evel

s

07

CQEAEG + CQEAEGControl

P62GAPDHLC3-IILC3-I

lowastlowastlowastlowast

lowastlowast

lowast

(c)Figure 3 EAEG induced autophagy in HeLa cells (a) Analysis of GFP-LC3 puncta formation in GFP-LC3HeLa cells under fluorescencemicroscopy Cells were treated for 24 h with DMEM only (blank) EAEG solvent (DMSO) as control different concentrations (45 60 and75 120583gml) of EAEG or the positive control trehalose (Tre) (scale bar = 20120583m) (b)The expression analysis of the autophagy-related proteinsp62 and LC3 byWestern blot HeLa cells were treated for 24 h with different concentrations (0 45 60 and 75 120583gml) of EAEG (c)The relativeprotein levels of p62 and LC3-II in HeLa cells treated with 60 120583gml EAEG with or without CQ (50mM) for 24 h GAPDH served as theloading control The protein relative ratio was calculated using ImageJ The data were expressed as mean plusmn SEM (119899 = 3) lowast119901 lt 005 andlowastlowast119901 lt 001 with respect to the control group 119901 lt 001 with respect to EAEG treatment group EAEG ethyl acetate extract of Gynuraformosana Kitam leaves

BioMed Research International 7

gml

P62

LC3-I

LC3-II

GAPDH

0 40 50 60 70 80

0 40 50 60 70 80

EAEG (gml)

00

01

02

03

04

05

06

Prot

ein

relat

ive l

evel

s

LC3-IILC3-IP62GAPDH

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowast

lowast

lowastlowastlowastlowast

(a)

P62

LC3-ILC3-II

GAPDH

0 3 6 9 18 24 h

0 3 6 9 18 24

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowast

lowastlowast

(hours)

00

01

02

03

04

05

06

Prot

ein

relat

ive l

evel

s

LC3-IILC3-IP62GAPDH

(b)

Figure 4The conversation of LC3-I to LC3-II Protein level of p62 was measured byWestern blot (a) HeLa cells were treated with EAEG at aconcentration of 0 40 50 60 70 or 80120583gml for 24 h (b)HeLa cells were treatedwith 60120583gml EAEG for 0 3 6 9 18 or 24 h GAPDH servedas the loading control The relative protein ratio was calculated using ImageJ The data were expressed as mean plusmn SEM (119899 = 3) lowast119901 lt 005lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001 with respect to the control group EAEG ethyl acetate extract of Gynura formosana Kitam leaves

apoptosis inhibitor Z-VAD-FMK did not affect the viability-reducing effect of EAEG (Figures 2(d) and 2(e)) This findingserved as a convincing evidence for nonapoptotic cell deathin EAEG treated HeLa cells

Autophagy was considered to be main way to inhibittumor cells proliferation when cells underwent nonapoptoticcell death [25 26] Increasing evidences had suggested thatautophagy could be induced by many natural products invarious anticancer therapies Triterpenoid B-group soyas-aponins could induce autophagy in colon cancer cells byinhibition of Akt signaling and enhanced ERK12 activity[27] Polyphenols extracts could induce autophagy whichwas advantageous to inhibit neurodegeneration and cancerprocess [28] Recent studies demonstrated that Emblicaofficinalis extracts could also induce autophagy and inhibithuman ovarian cancer cell proliferation [29] These reportssupported us to hypothesize that HeLa cells death caused byEAEG could involve autophagy program Autophagy wasa physiologically and pathologically regulated process andplayed an important role in the maintenance of intracellularhomeostasis in every eukaryotic cell [30ndash32] It started

with inclosing cytoplasmic contents in a vacuole calledautophagosome and then autophagosome fused with lyso-some to form autolysosomewhere substances inside could bedegraded and recycled [33] Autophagy could be initiated bysome autophagy-related proteins such as the microtubule-associated protein LC3 which was a reliable autophagicmarker [34] LC3 was a constitutively expressed protein inmammalian cells and could be processed to form cytoso-lic LC3-I During autophagy LC3-I was recruited to theautophagosome where LC3-II was generated by fusingLC3-I with phosphatidylethanolamine and then cellularautophagosome puncta including LC3-II were formed [35]Our results demonstrated that EAEG upregulated GFPpuncta formation (Figure 3(a)) and increased the conversionof LC3-I to LC3-II in HeLa cells (Figure 3(b)) suggest-ing the recruitment of LC3-II upon EAEG administrationFurthermore we analyzed the protein level of p62 anotherautophagicmarker [36] to study induction of autophagy p62could be trapped by LC3 incorporated into the autophago-some and then degraded via autophagy Correspondinglythe protein level of p62 should be decreased in the cells

8 BioMed Research International

lowastlowast

EAEG + 3MAEAEGControl0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

(a)

EAEG3MA

minus

minusminus +

+ +

LC3-I

LC3-II

PCNA

MCM7

GAPDH

LC3-IILC3-IPCNAGAPDHMCM7GAPDH

EAEG + 3MAEAEGControl

lowastlowast

lowastlowast

lowastlowast

00

01

02

03

04

05

06

07

08

Prot

ein

relat

ive l

evel

s

(b)

Figure 5 EAEG induced HeLa cells undergoing autophagic cell death (a) Cell viability of HeLa cells treated with 60120583gml EAEG for 24 h inpresence or absence of 5mM 3MA (b) Western blot analysis of autophagy and proliferation proteins GAPDH served as the loading controlThe protein relative ratio was calculated using ImageJThe data were expressed as mean plusmn SEM (119899 = 3) lowastlowast119901 lt 001with respect to the controlgroup and 119901 lt 005 with respect to EAEG treatment group EAEG ethyl acetate extract of Gynura formosana Kitam leaves

because of autophagy Our results showed that the proteinlevel of p62 reduced significantly in a dosage-dependentman-ner after EAEG treatment (Figure 3(b)) indicating an appar-ent induction of autophagy However these results couldnot explain if EAEG could induce the complete autophagyflux For instance calcium phosphate precipitates (CPP)induced both autophagosome synthesis and reduction ofautophagy flux due to impaired autophagosome-lysosomefusion [37] Therefore it was very necessary to evaluate if theautophagy induced by EAEG was complete autophagy fluxincluding autophagosome formation and autophagosome-lysosome fusion Our results showed that the protein levels ofboth LC3-II and p62 strongly increased in HeLa cells treatedwith EAEG together with CQ compared to cells treatedwith EAEG alone (Figure 3(c)) These results confirmed thatEAEG could induce complete autophagy flux

To confirm autophagy intensity we estimated the con-version of LC3-I to LC3-II and protein level of p62 EAEGelicited dose- and time-dependent autophagic induction inHeLa cells through the conversion of LC3-I into LC3-IIand decrease of protein level of p62 (Figure 4) stronglysuggesting that EAEG had autophagic activity Additionallywe also demonstrated that 3MA could partially abrogateEAEG induced HeLa cells death the conversion of LC3-Ito LC3-II and decreasing level of PCNA and MCM7 Thepresented collective results indicated that EAEG mightinhibit HeLa cells proliferation by inducing autophagy

In summary our studies indicated that EAEG a naturalplant extract could be a potentially effective therapeutic agentfor treating cervical cancer Our experimental evidences alsosupported the traditional use of this plant for the treatment ofthe deadly disease cancerThe identification of such biological

BioMed Research International 9

compounds in this plant and the mechanism against tumorcells are our research targets in the future

Conflicts of Interest

The authors declare that there are no conflicts of interest

Acknowledgments

This project was funded by Minxi Public Service Platformfor deep processing of Characteristic Agricultural Prod-ucts (2016LY08) the Natural Science Foundation of FujianProvince (2018J01458) Longyan University (LC2016008) theTraining Program of Fujian Excellent Talents in University(MJR201558) and the National Natural Science Foundationof China (81601600)

References

[1] B J Monk ldquoInvasive Cervical Cancerrdquo in Clinical GynecologicOncology 2012 1119

[2] W Zheng Z Liu W Zhang and X Hu ldquomiR-31 functions asan oncogene in cervical cancerrdquo Archives of Gynecology andObstetrics vol 292 no 5 pp 1083ndash1089 2015

[3] S EWaggoner ldquoCervical cancerrdquoThe Lancet vol 361 no 9376pp 2217ndash2225 2003

[4] Y Chen HWangW Lin and P Shuai ldquoADAR1 overexpressionis associated with cervical cancer progression and angiogene-sisrdquo Diagnostic Pathology vol 12 no 12 2017

[5] S Sen R Chakraborty C Sridhar Y S R Reddy and BDe ldquoFree radicals antioxidants diseases and phytomedicinescurrent status and future prospectrdquo International Journal ofPharmaceutical Sciences Review and Research vol 3 no 1 pp91ndash100 2010

[6] C Liu J-Z Liao and P-Y Li ldquoTraditional Chinese herbalextracts inducing autophagy as a novel approach in therapy ofnonalcoholic fatty liver diseaserdquoWorld Journal of Gastroenterol-ogy vol 23 no 11 pp 1964ndash1973 2017

[7] F S Reis R T Lima P Morales I C F R Ferreira andM H Vasconcelos ldquoMethanolic extract of ganoderma luciduminduces autophagy of AGS human gastric tumor cellsrdquoMolecules vol 20 no 10 pp 17872ndash17882 2015

[8] N Yu Y Xiong and C Wang ldquoBu-Zhong-Yi-Qi Decoctionthe Water Extract of Chinese Traditional Herbal MedicineEnhances Cisplatin Cytotoxicity in A549DDP Cells throughInduction of Apoptosis and Autophagyrdquo BioMed ResearchInternational vol 2017 Article ID 3692797 9 pages 2017

[9] J Ma C Guo Y Pan D Lin L Qiu and L Wen ldquoAntioxidantand anti-inflammatory activities of ethyl acetate extract ofGynura formosana (Kitam) leavesrdquo Experimental and Thera-peutic Medicine vol 14 no 3 pp 2303ndash2309 2017

[10] Q Tian J Li X Xie et al ldquoStereospecific induction of nuclearfactor-120581B activation by isochamaejasminrdquo Molecular Pharma-cology vol 68 no 6 pp 1534ndash1542 2005

[11] W-K Liu F W K Cheung B P L Liu C Li W Ye and C-T Che ldquoInvolvement of p21 and FasL in induction of cell cyclearrest and apoptosis by neochamaejasmin a in human prostateLNCaP cancer cellsrdquo Journal of Natural Products vol 71 no 5pp 842ndash846 2008

[12] L Yang Y Liu M Wang et al ldquoCelastrus orbiculatus extracttriggers apoptosis and autophagy via PI3KAktmTOR inhibi-tion in human colorectal cancer cellsrdquo Oncology Letters vol 12no 5 pp 3771ndash3778 2016

[13] P-FWei L Zhang S K Nethi et al ldquoAccelerating the clearanceof mutant huntingtin protein aggregates through autophagyinduction by europium hydroxide nanorodsrdquo Biomaterials vol35 no 3 pp 899ndash907 2014

[14] J Yang Y Zhou X Cheng et al ldquoIsogambogenic acid inducesapoptosis-independent autophagic cell death in human non-small-cell lung carcinoma cellsrdquo Scientific Reports vol 5 pp 1ndash92015

[15] F Janku D J McConkey D S Hong and R KurzrockldquoAutophagy as a target for anticancer therapyrdquo Nature ReviewsClinical Oncology vol 8 no 9 pp 528ndash539 2011

[16] Z J Yang C E Chee S Huang and F A Sinicrope ldquoAutophagymodulation for cancer therapyrdquo Cancer Biology ampTherapy vol11 no 2 pp 169ndash176 2011

[17] Y Lao G Wan Z Liu et al ldquoThe natural compound oblongi-folin C inhibits autophagic flux and enhances antitumor efficacyof nutrient deprivationrdquo Autophagy vol 10 no 5 pp 736ndash7492014

[18] H-M Ni A Bockus A L Wozniak et al ldquoDissecting the dy-namic turnover of GFP-LC3 in the autolysosomerdquo Autophagyvol 7 no 2 pp 188ndash204 2011

[19] M Zhou L Yang M Shao et al ldquoEffects of ZearalenoneExposure on the TGF-1205731Smad3 Signaling Pathway and theExpression of Proliferation or Apoptosis Related Genes of Post-Weaning Giltsrdquo Toxins vol 10 no 2 p 49 2018

[20] D Xu Y LaoN Xu et al ldquoIdentification and characterization ofanticancer compounds targeting apoptosis and autophagy fromChinese native Garcinia speciesrdquo Planta Medica vol 81 no 1pp 79ndash89 2014

[21] D R Green and J C Reed ldquoMitochondria and apoptosisrdquoScience vol 281 no 5381 pp 1309ndash1312 1998

[22] C J Hawkins and D L Vaux ldquoAnalysis of the Role of bcl-2 inApoptosisrdquo Immunological Reviews vol 142 no 1 pp 127ndash1391994

[23] Y Tsujimoto ldquoStress-resistance conferred by high level of bcl-2120572 protein in human B lymphoblastoid cellrdquo Oncogene vol 4no 11 pp 1331ndash1336 1989

[24] D R Green ldquoApoptotic pathways paper wraps stone bluntsscissorsrdquo Cell vol 102 no 1 pp 1ndash4 2000

[25] S Tsolmon J Han and H Isoda ldquoInhibition of cell growthby Stellera chamaejasme extract is associated with induction ofautophagy and differentiation in chronic leukemia K562 cellsrdquoJournal of Bioscience and Bioengineering vol 110 no 2 pp 262ndash268 2010

[26] T Kanzawa Y Kondo H Ito S Kondo and I GermanoldquoInduction of autophagic cell death inmalignant glioma cells byarsenic trioxiderdquo Cancer Research vol 63 no 9 pp 2103ndash21082003

[27] A A EllingtonM A Berhow and KW Singletary ldquoInhibitionof Akt signaling and enhanced ERK12 activity are involved ininduction of macroautophagy by triterpenoid B-group soyas-aponins in colon cancer cellsrdquo Carcinogenesis vol 27 no 2 pp298ndash306 2006

[28] K Pallauf and G Rimbach ldquoAutophagy polyphenols andhealthy ageingrdquo Ageing Research Reviews vol 12 no 1 pp 237ndash252 2013

10 BioMed Research International

[29] A De A De C Papasian et al ldquoEmblica officinalis ExtractInduces Autophagy and Inhibits Human Ovarian CancerCell Proliferation Angiogenesis Growth of Mouse XenograftTumorsrdquo PLoS ONE vol 8 no 8 Article ID e72748 2013

[30] C CDenardin L AMMartinsMM Parisi et al ldquoAutophagyinduced by purple pitanga (Eugenia uniflora L) extract trig-gered a cooperative effect on inducing the hepatic stellate celldeathrdquo Cell Biology and Toxicology vol 33 no 2 pp 197ndash2062017

[31] D R Green L Galluzzi and G Kroemer ldquoMitochondriaand the autophagy-inflammation-cell death axis in organismalagingrdquo Science vol 333 no 6046 pp 1109ndash1112 2011

[32] B Levine N Mizushima and H W Virgin ldquoAutophagy inimmunity and inflammationrdquo Nature vol 469 no 7330 pp323ndash335 2011

[33] N Mizushima B Levine A M Cuervo and D J Klion-sky ldquoAutophagy fights disease through cellular self-digestionrdquoNature vol 451 no 7182 pp 1069ndash1075 2008

[34] V Thongrakard R Titone C Follo et al ldquoTurmeric toxicityin A431 epidermoid cancer cells associates with autophagydegradation of anti-apoptotic and anti-autophagic p53 mutantrdquoPhytotherapy Research vol 28 no 12 pp 1761ndash1769 2014

[35] A Kuma M Matsui and N Mizushima ldquoLC3 an autophago-some marker can be incorporated into protein aggregatesindependent of autophagy Caution in the interpretation of LC3localizationrdquo Autophagy vol 3 no 4 pp 323ndash328 2007

[36] M Komatsu S Waguri M Koike et al ldquoHomeostatic lev-els of p62 control cytoplasmic inclusion body formation inautophagy-deficient micerdquo Cell vol 131 no 6 pp 1149ndash11632007

[37] S SarkarVKorolchukMRennaAWinslow andDC Rubin-sztein ldquoMethodological considerations for assessing autophagymodulators A study with calcium phosphate precipitatesrdquoAutophagy vol 5 no 3 pp 307ndash313 2009

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

BioMed Research International 7

gml

P62

LC3-I

LC3-II

GAPDH

0 40 50 60 70 80

0 40 50 60 70 80

EAEG (gml)

00

01

02

03

04

05

06

Prot

ein

relat

ive l

evel

s

LC3-IILC3-IP62GAPDH

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowast

lowast

lowastlowastlowastlowast

(a)

P62

LC3-ILC3-II

GAPDH

0 3 6 9 18 24 h

0 3 6 9 18 24

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowast

lowastlowast

(hours)

00

01

02

03

04

05

06

Prot

ein

relat

ive l

evel

s

LC3-IILC3-IP62GAPDH

(b)

Figure 4The conversation of LC3-I to LC3-II Protein level of p62 was measured byWestern blot (a) HeLa cells were treated with EAEG at aconcentration of 0 40 50 60 70 or 80120583gml for 24 h (b)HeLa cells were treatedwith 60120583gml EAEG for 0 3 6 9 18 or 24 h GAPDH servedas the loading control The relative protein ratio was calculated using ImageJ The data were expressed as mean plusmn SEM (119899 = 3) lowast119901 lt 005lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001 with respect to the control group EAEG ethyl acetate extract of Gynura formosana Kitam leaves

apoptosis inhibitor Z-VAD-FMK did not affect the viability-reducing effect of EAEG (Figures 2(d) and 2(e)) This findingserved as a convincing evidence for nonapoptotic cell deathin EAEG treated HeLa cells

Autophagy was considered to be main way to inhibittumor cells proliferation when cells underwent nonapoptoticcell death [25 26] Increasing evidences had suggested thatautophagy could be induced by many natural products invarious anticancer therapies Triterpenoid B-group soyas-aponins could induce autophagy in colon cancer cells byinhibition of Akt signaling and enhanced ERK12 activity[27] Polyphenols extracts could induce autophagy whichwas advantageous to inhibit neurodegeneration and cancerprocess [28] Recent studies demonstrated that Emblicaofficinalis extracts could also induce autophagy and inhibithuman ovarian cancer cell proliferation [29] These reportssupported us to hypothesize that HeLa cells death caused byEAEG could involve autophagy program Autophagy wasa physiologically and pathologically regulated process andplayed an important role in the maintenance of intracellularhomeostasis in every eukaryotic cell [30ndash32] It started

with inclosing cytoplasmic contents in a vacuole calledautophagosome and then autophagosome fused with lyso-some to form autolysosomewhere substances inside could bedegraded and recycled [33] Autophagy could be initiated bysome autophagy-related proteins such as the microtubule-associated protein LC3 which was a reliable autophagicmarker [34] LC3 was a constitutively expressed protein inmammalian cells and could be processed to form cytoso-lic LC3-I During autophagy LC3-I was recruited to theautophagosome where LC3-II was generated by fusingLC3-I with phosphatidylethanolamine and then cellularautophagosome puncta including LC3-II were formed [35]Our results demonstrated that EAEG upregulated GFPpuncta formation (Figure 3(a)) and increased the conversionof LC3-I to LC3-II in HeLa cells (Figure 3(b)) suggest-ing the recruitment of LC3-II upon EAEG administrationFurthermore we analyzed the protein level of p62 anotherautophagicmarker [36] to study induction of autophagy p62could be trapped by LC3 incorporated into the autophago-some and then degraded via autophagy Correspondinglythe protein level of p62 should be decreased in the cells

8 BioMed Research International

lowastlowast

EAEG + 3MAEAEGControl0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

(a)

EAEG3MA

minus

minusminus +

+ +

LC3-I

LC3-II

PCNA

MCM7

GAPDH

LC3-IILC3-IPCNAGAPDHMCM7GAPDH

EAEG + 3MAEAEGControl

lowastlowast

lowastlowast

lowastlowast

00

01

02

03

04

05

06

07

08

Prot

ein

relat

ive l

evel

s

(b)

Figure 5 EAEG induced HeLa cells undergoing autophagic cell death (a) Cell viability of HeLa cells treated with 60120583gml EAEG for 24 h inpresence or absence of 5mM 3MA (b) Western blot analysis of autophagy and proliferation proteins GAPDH served as the loading controlThe protein relative ratio was calculated using ImageJThe data were expressed as mean plusmn SEM (119899 = 3) lowastlowast119901 lt 001with respect to the controlgroup and 119901 lt 005 with respect to EAEG treatment group EAEG ethyl acetate extract of Gynura formosana Kitam leaves

because of autophagy Our results showed that the proteinlevel of p62 reduced significantly in a dosage-dependentman-ner after EAEG treatment (Figure 3(b)) indicating an appar-ent induction of autophagy However these results couldnot explain if EAEG could induce the complete autophagyflux For instance calcium phosphate precipitates (CPP)induced both autophagosome synthesis and reduction ofautophagy flux due to impaired autophagosome-lysosomefusion [37] Therefore it was very necessary to evaluate if theautophagy induced by EAEG was complete autophagy fluxincluding autophagosome formation and autophagosome-lysosome fusion Our results showed that the protein levels ofboth LC3-II and p62 strongly increased in HeLa cells treatedwith EAEG together with CQ compared to cells treatedwith EAEG alone (Figure 3(c)) These results confirmed thatEAEG could induce complete autophagy flux

To confirm autophagy intensity we estimated the con-version of LC3-I to LC3-II and protein level of p62 EAEGelicited dose- and time-dependent autophagic induction inHeLa cells through the conversion of LC3-I into LC3-IIand decrease of protein level of p62 (Figure 4) stronglysuggesting that EAEG had autophagic activity Additionallywe also demonstrated that 3MA could partially abrogateEAEG induced HeLa cells death the conversion of LC3-Ito LC3-II and decreasing level of PCNA and MCM7 Thepresented collective results indicated that EAEG mightinhibit HeLa cells proliferation by inducing autophagy

In summary our studies indicated that EAEG a naturalplant extract could be a potentially effective therapeutic agentfor treating cervical cancer Our experimental evidences alsosupported the traditional use of this plant for the treatment ofthe deadly disease cancerThe identification of such biological

BioMed Research International 9

compounds in this plant and the mechanism against tumorcells are our research targets in the future

Conflicts of Interest

The authors declare that there are no conflicts of interest

Acknowledgments

This project was funded by Minxi Public Service Platformfor deep processing of Characteristic Agricultural Prod-ucts (2016LY08) the Natural Science Foundation of FujianProvince (2018J01458) Longyan University (LC2016008) theTraining Program of Fujian Excellent Talents in University(MJR201558) and the National Natural Science Foundationof China (81601600)

References

[1] B J Monk ldquoInvasive Cervical Cancerrdquo in Clinical GynecologicOncology 2012 1119

[2] W Zheng Z Liu W Zhang and X Hu ldquomiR-31 functions asan oncogene in cervical cancerrdquo Archives of Gynecology andObstetrics vol 292 no 5 pp 1083ndash1089 2015

[3] S EWaggoner ldquoCervical cancerrdquoThe Lancet vol 361 no 9376pp 2217ndash2225 2003

[4] Y Chen HWangW Lin and P Shuai ldquoADAR1 overexpressionis associated with cervical cancer progression and angiogene-sisrdquo Diagnostic Pathology vol 12 no 12 2017

[5] S Sen R Chakraborty C Sridhar Y S R Reddy and BDe ldquoFree radicals antioxidants diseases and phytomedicinescurrent status and future prospectrdquo International Journal ofPharmaceutical Sciences Review and Research vol 3 no 1 pp91ndash100 2010

[6] C Liu J-Z Liao and P-Y Li ldquoTraditional Chinese herbalextracts inducing autophagy as a novel approach in therapy ofnonalcoholic fatty liver diseaserdquoWorld Journal of Gastroenterol-ogy vol 23 no 11 pp 1964ndash1973 2017

[7] F S Reis R T Lima P Morales I C F R Ferreira andM H Vasconcelos ldquoMethanolic extract of ganoderma luciduminduces autophagy of AGS human gastric tumor cellsrdquoMolecules vol 20 no 10 pp 17872ndash17882 2015

[8] N Yu Y Xiong and C Wang ldquoBu-Zhong-Yi-Qi Decoctionthe Water Extract of Chinese Traditional Herbal MedicineEnhances Cisplatin Cytotoxicity in A549DDP Cells throughInduction of Apoptosis and Autophagyrdquo BioMed ResearchInternational vol 2017 Article ID 3692797 9 pages 2017

[9] J Ma C Guo Y Pan D Lin L Qiu and L Wen ldquoAntioxidantand anti-inflammatory activities of ethyl acetate extract ofGynura formosana (Kitam) leavesrdquo Experimental and Thera-peutic Medicine vol 14 no 3 pp 2303ndash2309 2017

[10] Q Tian J Li X Xie et al ldquoStereospecific induction of nuclearfactor-120581B activation by isochamaejasminrdquo Molecular Pharma-cology vol 68 no 6 pp 1534ndash1542 2005

[11] W-K Liu F W K Cheung B P L Liu C Li W Ye and C-T Che ldquoInvolvement of p21 and FasL in induction of cell cyclearrest and apoptosis by neochamaejasmin a in human prostateLNCaP cancer cellsrdquo Journal of Natural Products vol 71 no 5pp 842ndash846 2008

[12] L Yang Y Liu M Wang et al ldquoCelastrus orbiculatus extracttriggers apoptosis and autophagy via PI3KAktmTOR inhibi-tion in human colorectal cancer cellsrdquo Oncology Letters vol 12no 5 pp 3771ndash3778 2016

[13] P-FWei L Zhang S K Nethi et al ldquoAccelerating the clearanceof mutant huntingtin protein aggregates through autophagyinduction by europium hydroxide nanorodsrdquo Biomaterials vol35 no 3 pp 899ndash907 2014

[14] J Yang Y Zhou X Cheng et al ldquoIsogambogenic acid inducesapoptosis-independent autophagic cell death in human non-small-cell lung carcinoma cellsrdquo Scientific Reports vol 5 pp 1ndash92015

[15] F Janku D J McConkey D S Hong and R KurzrockldquoAutophagy as a target for anticancer therapyrdquo Nature ReviewsClinical Oncology vol 8 no 9 pp 528ndash539 2011

[16] Z J Yang C E Chee S Huang and F A Sinicrope ldquoAutophagymodulation for cancer therapyrdquo Cancer Biology ampTherapy vol11 no 2 pp 169ndash176 2011

[17] Y Lao G Wan Z Liu et al ldquoThe natural compound oblongi-folin C inhibits autophagic flux and enhances antitumor efficacyof nutrient deprivationrdquo Autophagy vol 10 no 5 pp 736ndash7492014

[18] H-M Ni A Bockus A L Wozniak et al ldquoDissecting the dy-namic turnover of GFP-LC3 in the autolysosomerdquo Autophagyvol 7 no 2 pp 188ndash204 2011

[19] M Zhou L Yang M Shao et al ldquoEffects of ZearalenoneExposure on the TGF-1205731Smad3 Signaling Pathway and theExpression of Proliferation or Apoptosis Related Genes of Post-Weaning Giltsrdquo Toxins vol 10 no 2 p 49 2018

[20] D Xu Y LaoN Xu et al ldquoIdentification and characterization ofanticancer compounds targeting apoptosis and autophagy fromChinese native Garcinia speciesrdquo Planta Medica vol 81 no 1pp 79ndash89 2014

[21] D R Green and J C Reed ldquoMitochondria and apoptosisrdquoScience vol 281 no 5381 pp 1309ndash1312 1998

[22] C J Hawkins and D L Vaux ldquoAnalysis of the Role of bcl-2 inApoptosisrdquo Immunological Reviews vol 142 no 1 pp 127ndash1391994

[23] Y Tsujimoto ldquoStress-resistance conferred by high level of bcl-2120572 protein in human B lymphoblastoid cellrdquo Oncogene vol 4no 11 pp 1331ndash1336 1989

[24] D R Green ldquoApoptotic pathways paper wraps stone bluntsscissorsrdquo Cell vol 102 no 1 pp 1ndash4 2000

[25] S Tsolmon J Han and H Isoda ldquoInhibition of cell growthby Stellera chamaejasme extract is associated with induction ofautophagy and differentiation in chronic leukemia K562 cellsrdquoJournal of Bioscience and Bioengineering vol 110 no 2 pp 262ndash268 2010

[26] T Kanzawa Y Kondo H Ito S Kondo and I GermanoldquoInduction of autophagic cell death inmalignant glioma cells byarsenic trioxiderdquo Cancer Research vol 63 no 9 pp 2103ndash21082003

[27] A A EllingtonM A Berhow and KW Singletary ldquoInhibitionof Akt signaling and enhanced ERK12 activity are involved ininduction of macroautophagy by triterpenoid B-group soyas-aponins in colon cancer cellsrdquo Carcinogenesis vol 27 no 2 pp298ndash306 2006

[28] K Pallauf and G Rimbach ldquoAutophagy polyphenols andhealthy ageingrdquo Ageing Research Reviews vol 12 no 1 pp 237ndash252 2013

10 BioMed Research International

[29] A De A De C Papasian et al ldquoEmblica officinalis ExtractInduces Autophagy and Inhibits Human Ovarian CancerCell Proliferation Angiogenesis Growth of Mouse XenograftTumorsrdquo PLoS ONE vol 8 no 8 Article ID e72748 2013

[30] C CDenardin L AMMartinsMM Parisi et al ldquoAutophagyinduced by purple pitanga (Eugenia uniflora L) extract trig-gered a cooperative effect on inducing the hepatic stellate celldeathrdquo Cell Biology and Toxicology vol 33 no 2 pp 197ndash2062017

[31] D R Green L Galluzzi and G Kroemer ldquoMitochondriaand the autophagy-inflammation-cell death axis in organismalagingrdquo Science vol 333 no 6046 pp 1109ndash1112 2011

[32] B Levine N Mizushima and H W Virgin ldquoAutophagy inimmunity and inflammationrdquo Nature vol 469 no 7330 pp323ndash335 2011

[33] N Mizushima B Levine A M Cuervo and D J Klion-sky ldquoAutophagy fights disease through cellular self-digestionrdquoNature vol 451 no 7182 pp 1069ndash1075 2008

[34] V Thongrakard R Titone C Follo et al ldquoTurmeric toxicityin A431 epidermoid cancer cells associates with autophagydegradation of anti-apoptotic and anti-autophagic p53 mutantrdquoPhytotherapy Research vol 28 no 12 pp 1761ndash1769 2014

[35] A Kuma M Matsui and N Mizushima ldquoLC3 an autophago-some marker can be incorporated into protein aggregatesindependent of autophagy Caution in the interpretation of LC3localizationrdquo Autophagy vol 3 no 4 pp 323ndash328 2007

[36] M Komatsu S Waguri M Koike et al ldquoHomeostatic lev-els of p62 control cytoplasmic inclusion body formation inautophagy-deficient micerdquo Cell vol 131 no 6 pp 1149ndash11632007

[37] S SarkarVKorolchukMRennaAWinslow andDC Rubin-sztein ldquoMethodological considerations for assessing autophagymodulators A study with calcium phosphate precipitatesrdquoAutophagy vol 5 no 3 pp 307ndash313 2009

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

8 BioMed Research International

lowastlowast

EAEG + 3MAEAEGControl0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

(a)

EAEG3MA

minus

minusminus +

+ +

LC3-I

LC3-II

PCNA

MCM7

GAPDH

LC3-IILC3-IPCNAGAPDHMCM7GAPDH

EAEG + 3MAEAEGControl

lowastlowast

lowastlowast

lowastlowast

00

01

02

03

04

05

06

07

08

Prot

ein

relat

ive l

evel

s

(b)

Figure 5 EAEG induced HeLa cells undergoing autophagic cell death (a) Cell viability of HeLa cells treated with 60120583gml EAEG for 24 h inpresence or absence of 5mM 3MA (b) Western blot analysis of autophagy and proliferation proteins GAPDH served as the loading controlThe protein relative ratio was calculated using ImageJThe data were expressed as mean plusmn SEM (119899 = 3) lowastlowast119901 lt 001with respect to the controlgroup and 119901 lt 005 with respect to EAEG treatment group EAEG ethyl acetate extract of Gynura formosana Kitam leaves

because of autophagy Our results showed that the proteinlevel of p62 reduced significantly in a dosage-dependentman-ner after EAEG treatment (Figure 3(b)) indicating an appar-ent induction of autophagy However these results couldnot explain if EAEG could induce the complete autophagyflux For instance calcium phosphate precipitates (CPP)induced both autophagosome synthesis and reduction ofautophagy flux due to impaired autophagosome-lysosomefusion [37] Therefore it was very necessary to evaluate if theautophagy induced by EAEG was complete autophagy fluxincluding autophagosome formation and autophagosome-lysosome fusion Our results showed that the protein levels ofboth LC3-II and p62 strongly increased in HeLa cells treatedwith EAEG together with CQ compared to cells treatedwith EAEG alone (Figure 3(c)) These results confirmed thatEAEG could induce complete autophagy flux

To confirm autophagy intensity we estimated the con-version of LC3-I to LC3-II and protein level of p62 EAEGelicited dose- and time-dependent autophagic induction inHeLa cells through the conversion of LC3-I into LC3-IIand decrease of protein level of p62 (Figure 4) stronglysuggesting that EAEG had autophagic activity Additionallywe also demonstrated that 3MA could partially abrogateEAEG induced HeLa cells death the conversion of LC3-Ito LC3-II and decreasing level of PCNA and MCM7 Thepresented collective results indicated that EAEG mightinhibit HeLa cells proliferation by inducing autophagy

In summary our studies indicated that EAEG a naturalplant extract could be a potentially effective therapeutic agentfor treating cervical cancer Our experimental evidences alsosupported the traditional use of this plant for the treatment ofthe deadly disease cancerThe identification of such biological

BioMed Research International 9

compounds in this plant and the mechanism against tumorcells are our research targets in the future

Conflicts of Interest

The authors declare that there are no conflicts of interest

Acknowledgments

This project was funded by Minxi Public Service Platformfor deep processing of Characteristic Agricultural Prod-ucts (2016LY08) the Natural Science Foundation of FujianProvince (2018J01458) Longyan University (LC2016008) theTraining Program of Fujian Excellent Talents in University(MJR201558) and the National Natural Science Foundationof China (81601600)

References

[1] B J Monk ldquoInvasive Cervical Cancerrdquo in Clinical GynecologicOncology 2012 1119

[2] W Zheng Z Liu W Zhang and X Hu ldquomiR-31 functions asan oncogene in cervical cancerrdquo Archives of Gynecology andObstetrics vol 292 no 5 pp 1083ndash1089 2015

[3] S EWaggoner ldquoCervical cancerrdquoThe Lancet vol 361 no 9376pp 2217ndash2225 2003

[4] Y Chen HWangW Lin and P Shuai ldquoADAR1 overexpressionis associated with cervical cancer progression and angiogene-sisrdquo Diagnostic Pathology vol 12 no 12 2017

[5] S Sen R Chakraborty C Sridhar Y S R Reddy and BDe ldquoFree radicals antioxidants diseases and phytomedicinescurrent status and future prospectrdquo International Journal ofPharmaceutical Sciences Review and Research vol 3 no 1 pp91ndash100 2010

[6] C Liu J-Z Liao and P-Y Li ldquoTraditional Chinese herbalextracts inducing autophagy as a novel approach in therapy ofnonalcoholic fatty liver diseaserdquoWorld Journal of Gastroenterol-ogy vol 23 no 11 pp 1964ndash1973 2017

[7] F S Reis R T Lima P Morales I C F R Ferreira andM H Vasconcelos ldquoMethanolic extract of ganoderma luciduminduces autophagy of AGS human gastric tumor cellsrdquoMolecules vol 20 no 10 pp 17872ndash17882 2015

[8] N Yu Y Xiong and C Wang ldquoBu-Zhong-Yi-Qi Decoctionthe Water Extract of Chinese Traditional Herbal MedicineEnhances Cisplatin Cytotoxicity in A549DDP Cells throughInduction of Apoptosis and Autophagyrdquo BioMed ResearchInternational vol 2017 Article ID 3692797 9 pages 2017

[9] J Ma C Guo Y Pan D Lin L Qiu and L Wen ldquoAntioxidantand anti-inflammatory activities of ethyl acetate extract ofGynura formosana (Kitam) leavesrdquo Experimental and Thera-peutic Medicine vol 14 no 3 pp 2303ndash2309 2017

[10] Q Tian J Li X Xie et al ldquoStereospecific induction of nuclearfactor-120581B activation by isochamaejasminrdquo Molecular Pharma-cology vol 68 no 6 pp 1534ndash1542 2005

[11] W-K Liu F W K Cheung B P L Liu C Li W Ye and C-T Che ldquoInvolvement of p21 and FasL in induction of cell cyclearrest and apoptosis by neochamaejasmin a in human prostateLNCaP cancer cellsrdquo Journal of Natural Products vol 71 no 5pp 842ndash846 2008

[12] L Yang Y Liu M Wang et al ldquoCelastrus orbiculatus extracttriggers apoptosis and autophagy via PI3KAktmTOR inhibi-tion in human colorectal cancer cellsrdquo Oncology Letters vol 12no 5 pp 3771ndash3778 2016

[13] P-FWei L Zhang S K Nethi et al ldquoAccelerating the clearanceof mutant huntingtin protein aggregates through autophagyinduction by europium hydroxide nanorodsrdquo Biomaterials vol35 no 3 pp 899ndash907 2014

[14] J Yang Y Zhou X Cheng et al ldquoIsogambogenic acid inducesapoptosis-independent autophagic cell death in human non-small-cell lung carcinoma cellsrdquo Scientific Reports vol 5 pp 1ndash92015

[15] F Janku D J McConkey D S Hong and R KurzrockldquoAutophagy as a target for anticancer therapyrdquo Nature ReviewsClinical Oncology vol 8 no 9 pp 528ndash539 2011

[16] Z J Yang C E Chee S Huang and F A Sinicrope ldquoAutophagymodulation for cancer therapyrdquo Cancer Biology ampTherapy vol11 no 2 pp 169ndash176 2011

[17] Y Lao G Wan Z Liu et al ldquoThe natural compound oblongi-folin C inhibits autophagic flux and enhances antitumor efficacyof nutrient deprivationrdquo Autophagy vol 10 no 5 pp 736ndash7492014

[18] H-M Ni A Bockus A L Wozniak et al ldquoDissecting the dy-namic turnover of GFP-LC3 in the autolysosomerdquo Autophagyvol 7 no 2 pp 188ndash204 2011

[19] M Zhou L Yang M Shao et al ldquoEffects of ZearalenoneExposure on the TGF-1205731Smad3 Signaling Pathway and theExpression of Proliferation or Apoptosis Related Genes of Post-Weaning Giltsrdquo Toxins vol 10 no 2 p 49 2018

[20] D Xu Y LaoN Xu et al ldquoIdentification and characterization ofanticancer compounds targeting apoptosis and autophagy fromChinese native Garcinia speciesrdquo Planta Medica vol 81 no 1pp 79ndash89 2014

[21] D R Green and J C Reed ldquoMitochondria and apoptosisrdquoScience vol 281 no 5381 pp 1309ndash1312 1998

[22] C J Hawkins and D L Vaux ldquoAnalysis of the Role of bcl-2 inApoptosisrdquo Immunological Reviews vol 142 no 1 pp 127ndash1391994

[23] Y Tsujimoto ldquoStress-resistance conferred by high level of bcl-2120572 protein in human B lymphoblastoid cellrdquo Oncogene vol 4no 11 pp 1331ndash1336 1989

[24] D R Green ldquoApoptotic pathways paper wraps stone bluntsscissorsrdquo Cell vol 102 no 1 pp 1ndash4 2000

[25] S Tsolmon J Han and H Isoda ldquoInhibition of cell growthby Stellera chamaejasme extract is associated with induction ofautophagy and differentiation in chronic leukemia K562 cellsrdquoJournal of Bioscience and Bioengineering vol 110 no 2 pp 262ndash268 2010

[26] T Kanzawa Y Kondo H Ito S Kondo and I GermanoldquoInduction of autophagic cell death inmalignant glioma cells byarsenic trioxiderdquo Cancer Research vol 63 no 9 pp 2103ndash21082003

[27] A A EllingtonM A Berhow and KW Singletary ldquoInhibitionof Akt signaling and enhanced ERK12 activity are involved ininduction of macroautophagy by triterpenoid B-group soyas-aponins in colon cancer cellsrdquo Carcinogenesis vol 27 no 2 pp298ndash306 2006

[28] K Pallauf and G Rimbach ldquoAutophagy polyphenols andhealthy ageingrdquo Ageing Research Reviews vol 12 no 1 pp 237ndash252 2013

10 BioMed Research International

[29] A De A De C Papasian et al ldquoEmblica officinalis ExtractInduces Autophagy and Inhibits Human Ovarian CancerCell Proliferation Angiogenesis Growth of Mouse XenograftTumorsrdquo PLoS ONE vol 8 no 8 Article ID e72748 2013

[30] C CDenardin L AMMartinsMM Parisi et al ldquoAutophagyinduced by purple pitanga (Eugenia uniflora L) extract trig-gered a cooperative effect on inducing the hepatic stellate celldeathrdquo Cell Biology and Toxicology vol 33 no 2 pp 197ndash2062017

[31] D R Green L Galluzzi and G Kroemer ldquoMitochondriaand the autophagy-inflammation-cell death axis in organismalagingrdquo Science vol 333 no 6046 pp 1109ndash1112 2011

[32] B Levine N Mizushima and H W Virgin ldquoAutophagy inimmunity and inflammationrdquo Nature vol 469 no 7330 pp323ndash335 2011

[33] N Mizushima B Levine A M Cuervo and D J Klion-sky ldquoAutophagy fights disease through cellular self-digestionrdquoNature vol 451 no 7182 pp 1069ndash1075 2008

[34] V Thongrakard R Titone C Follo et al ldquoTurmeric toxicityin A431 epidermoid cancer cells associates with autophagydegradation of anti-apoptotic and anti-autophagic p53 mutantrdquoPhytotherapy Research vol 28 no 12 pp 1761ndash1769 2014

[35] A Kuma M Matsui and N Mizushima ldquoLC3 an autophago-some marker can be incorporated into protein aggregatesindependent of autophagy Caution in the interpretation of LC3localizationrdquo Autophagy vol 3 no 4 pp 323ndash328 2007

[36] M Komatsu S Waguri M Koike et al ldquoHomeostatic lev-els of p62 control cytoplasmic inclusion body formation inautophagy-deficient micerdquo Cell vol 131 no 6 pp 1149ndash11632007

[37] S SarkarVKorolchukMRennaAWinslow andDC Rubin-sztein ldquoMethodological considerations for assessing autophagymodulators A study with calcium phosphate precipitatesrdquoAutophagy vol 5 no 3 pp 307ndash313 2009

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

BioMed Research International 9

compounds in this plant and the mechanism against tumorcells are our research targets in the future

Conflicts of Interest

The authors declare that there are no conflicts of interest

Acknowledgments

This project was funded by Minxi Public Service Platformfor deep processing of Characteristic Agricultural Prod-ucts (2016LY08) the Natural Science Foundation of FujianProvince (2018J01458) Longyan University (LC2016008) theTraining Program of Fujian Excellent Talents in University(MJR201558) and the National Natural Science Foundationof China (81601600)

References

[1] B J Monk ldquoInvasive Cervical Cancerrdquo in Clinical GynecologicOncology 2012 1119

[2] W Zheng Z Liu W Zhang and X Hu ldquomiR-31 functions asan oncogene in cervical cancerrdquo Archives of Gynecology andObstetrics vol 292 no 5 pp 1083ndash1089 2015

[3] S EWaggoner ldquoCervical cancerrdquoThe Lancet vol 361 no 9376pp 2217ndash2225 2003

[4] Y Chen HWangW Lin and P Shuai ldquoADAR1 overexpressionis associated with cervical cancer progression and angiogene-sisrdquo Diagnostic Pathology vol 12 no 12 2017

[5] S Sen R Chakraborty C Sridhar Y S R Reddy and BDe ldquoFree radicals antioxidants diseases and phytomedicinescurrent status and future prospectrdquo International Journal ofPharmaceutical Sciences Review and Research vol 3 no 1 pp91ndash100 2010

[6] C Liu J-Z Liao and P-Y Li ldquoTraditional Chinese herbalextracts inducing autophagy as a novel approach in therapy ofnonalcoholic fatty liver diseaserdquoWorld Journal of Gastroenterol-ogy vol 23 no 11 pp 1964ndash1973 2017

[7] F S Reis R T Lima P Morales I C F R Ferreira andM H Vasconcelos ldquoMethanolic extract of ganoderma luciduminduces autophagy of AGS human gastric tumor cellsrdquoMolecules vol 20 no 10 pp 17872ndash17882 2015

[8] N Yu Y Xiong and C Wang ldquoBu-Zhong-Yi-Qi Decoctionthe Water Extract of Chinese Traditional Herbal MedicineEnhances Cisplatin Cytotoxicity in A549DDP Cells throughInduction of Apoptosis and Autophagyrdquo BioMed ResearchInternational vol 2017 Article ID 3692797 9 pages 2017

[9] J Ma C Guo Y Pan D Lin L Qiu and L Wen ldquoAntioxidantand anti-inflammatory activities of ethyl acetate extract ofGynura formosana (Kitam) leavesrdquo Experimental and Thera-peutic Medicine vol 14 no 3 pp 2303ndash2309 2017

[10] Q Tian J Li X Xie et al ldquoStereospecific induction of nuclearfactor-120581B activation by isochamaejasminrdquo Molecular Pharma-cology vol 68 no 6 pp 1534ndash1542 2005

[11] W-K Liu F W K Cheung B P L Liu C Li W Ye and C-T Che ldquoInvolvement of p21 and FasL in induction of cell cyclearrest and apoptosis by neochamaejasmin a in human prostateLNCaP cancer cellsrdquo Journal of Natural Products vol 71 no 5pp 842ndash846 2008

[12] L Yang Y Liu M Wang et al ldquoCelastrus orbiculatus extracttriggers apoptosis and autophagy via PI3KAktmTOR inhibi-tion in human colorectal cancer cellsrdquo Oncology Letters vol 12no 5 pp 3771ndash3778 2016

[13] P-FWei L Zhang S K Nethi et al ldquoAccelerating the clearanceof mutant huntingtin protein aggregates through autophagyinduction by europium hydroxide nanorodsrdquo Biomaterials vol35 no 3 pp 899ndash907 2014

[14] J Yang Y Zhou X Cheng et al ldquoIsogambogenic acid inducesapoptosis-independent autophagic cell death in human non-small-cell lung carcinoma cellsrdquo Scientific Reports vol 5 pp 1ndash92015

[15] F Janku D J McConkey D S Hong and R KurzrockldquoAutophagy as a target for anticancer therapyrdquo Nature ReviewsClinical Oncology vol 8 no 9 pp 528ndash539 2011

[16] Z J Yang C E Chee S Huang and F A Sinicrope ldquoAutophagymodulation for cancer therapyrdquo Cancer Biology ampTherapy vol11 no 2 pp 169ndash176 2011

[17] Y Lao G Wan Z Liu et al ldquoThe natural compound oblongi-folin C inhibits autophagic flux and enhances antitumor efficacyof nutrient deprivationrdquo Autophagy vol 10 no 5 pp 736ndash7492014

[18] H-M Ni A Bockus A L Wozniak et al ldquoDissecting the dy-namic turnover of GFP-LC3 in the autolysosomerdquo Autophagyvol 7 no 2 pp 188ndash204 2011

[19] M Zhou L Yang M Shao et al ldquoEffects of ZearalenoneExposure on the TGF-1205731Smad3 Signaling Pathway and theExpression of Proliferation or Apoptosis Related Genes of Post-Weaning Giltsrdquo Toxins vol 10 no 2 p 49 2018

[20] D Xu Y LaoN Xu et al ldquoIdentification and characterization ofanticancer compounds targeting apoptosis and autophagy fromChinese native Garcinia speciesrdquo Planta Medica vol 81 no 1pp 79ndash89 2014

[21] D R Green and J C Reed ldquoMitochondria and apoptosisrdquoScience vol 281 no 5381 pp 1309ndash1312 1998

[22] C J Hawkins and D L Vaux ldquoAnalysis of the Role of bcl-2 inApoptosisrdquo Immunological Reviews vol 142 no 1 pp 127ndash1391994

[23] Y Tsujimoto ldquoStress-resistance conferred by high level of bcl-2120572 protein in human B lymphoblastoid cellrdquo Oncogene vol 4no 11 pp 1331ndash1336 1989

[24] D R Green ldquoApoptotic pathways paper wraps stone bluntsscissorsrdquo Cell vol 102 no 1 pp 1ndash4 2000

[25] S Tsolmon J Han and H Isoda ldquoInhibition of cell growthby Stellera chamaejasme extract is associated with induction ofautophagy and differentiation in chronic leukemia K562 cellsrdquoJournal of Bioscience and Bioengineering vol 110 no 2 pp 262ndash268 2010

[26] T Kanzawa Y Kondo H Ito S Kondo and I GermanoldquoInduction of autophagic cell death inmalignant glioma cells byarsenic trioxiderdquo Cancer Research vol 63 no 9 pp 2103ndash21082003

[27] A A EllingtonM A Berhow and KW Singletary ldquoInhibitionof Akt signaling and enhanced ERK12 activity are involved ininduction of macroautophagy by triterpenoid B-group soyas-aponins in colon cancer cellsrdquo Carcinogenesis vol 27 no 2 pp298ndash306 2006

[28] K Pallauf and G Rimbach ldquoAutophagy polyphenols andhealthy ageingrdquo Ageing Research Reviews vol 12 no 1 pp 237ndash252 2013

10 BioMed Research International

[29] A De A De C Papasian et al ldquoEmblica officinalis ExtractInduces Autophagy and Inhibits Human Ovarian CancerCell Proliferation Angiogenesis Growth of Mouse XenograftTumorsrdquo PLoS ONE vol 8 no 8 Article ID e72748 2013

[30] C CDenardin L AMMartinsMM Parisi et al ldquoAutophagyinduced by purple pitanga (Eugenia uniflora L) extract trig-gered a cooperative effect on inducing the hepatic stellate celldeathrdquo Cell Biology and Toxicology vol 33 no 2 pp 197ndash2062017

[31] D R Green L Galluzzi and G Kroemer ldquoMitochondriaand the autophagy-inflammation-cell death axis in organismalagingrdquo Science vol 333 no 6046 pp 1109ndash1112 2011

[32] B Levine N Mizushima and H W Virgin ldquoAutophagy inimmunity and inflammationrdquo Nature vol 469 no 7330 pp323ndash335 2011

[33] N Mizushima B Levine A M Cuervo and D J Klion-sky ldquoAutophagy fights disease through cellular self-digestionrdquoNature vol 451 no 7182 pp 1069ndash1075 2008

[34] V Thongrakard R Titone C Follo et al ldquoTurmeric toxicityin A431 epidermoid cancer cells associates with autophagydegradation of anti-apoptotic and anti-autophagic p53 mutantrdquoPhytotherapy Research vol 28 no 12 pp 1761ndash1769 2014

[35] A Kuma M Matsui and N Mizushima ldquoLC3 an autophago-some marker can be incorporated into protein aggregatesindependent of autophagy Caution in the interpretation of LC3localizationrdquo Autophagy vol 3 no 4 pp 323ndash328 2007

[36] M Komatsu S Waguri M Koike et al ldquoHomeostatic lev-els of p62 control cytoplasmic inclusion body formation inautophagy-deficient micerdquo Cell vol 131 no 6 pp 1149ndash11632007

[37] S SarkarVKorolchukMRennaAWinslow andDC Rubin-sztein ldquoMethodological considerations for assessing autophagymodulators A study with calcium phosphate precipitatesrdquoAutophagy vol 5 no 3 pp 307ndash313 2009

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

10 BioMed Research International

[29] A De A De C Papasian et al ldquoEmblica officinalis ExtractInduces Autophagy and Inhibits Human Ovarian CancerCell Proliferation Angiogenesis Growth of Mouse XenograftTumorsrdquo PLoS ONE vol 8 no 8 Article ID e72748 2013

[30] C CDenardin L AMMartinsMM Parisi et al ldquoAutophagyinduced by purple pitanga (Eugenia uniflora L) extract trig-gered a cooperative effect on inducing the hepatic stellate celldeathrdquo Cell Biology and Toxicology vol 33 no 2 pp 197ndash2062017

[31] D R Green L Galluzzi and G Kroemer ldquoMitochondriaand the autophagy-inflammation-cell death axis in organismalagingrdquo Science vol 333 no 6046 pp 1109ndash1112 2011

[32] B Levine N Mizushima and H W Virgin ldquoAutophagy inimmunity and inflammationrdquo Nature vol 469 no 7330 pp323ndash335 2011

[33] N Mizushima B Levine A M Cuervo and D J Klion-sky ldquoAutophagy fights disease through cellular self-digestionrdquoNature vol 451 no 7182 pp 1069ndash1075 2008

[34] V Thongrakard R Titone C Follo et al ldquoTurmeric toxicityin A431 epidermoid cancer cells associates with autophagydegradation of anti-apoptotic and anti-autophagic p53 mutantrdquoPhytotherapy Research vol 28 no 12 pp 1761ndash1769 2014

[35] A Kuma M Matsui and N Mizushima ldquoLC3 an autophago-some marker can be incorporated into protein aggregatesindependent of autophagy Caution in the interpretation of LC3localizationrdquo Autophagy vol 3 no 4 pp 323ndash328 2007

[36] M Komatsu S Waguri M Koike et al ldquoHomeostatic lev-els of p62 control cytoplasmic inclusion body formation inautophagy-deficient micerdquo Cell vol 131 no 6 pp 1149ndash11632007

[37] S SarkarVKorolchukMRennaAWinslow andDC Rubin-sztein ldquoMethodological considerations for assessing autophagymodulators A study with calcium phosphate precipitatesrdquoAutophagy vol 5 no 3 pp 307ndash313 2009

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom