dihydromyricetin activates amp-activated protein kinase and p38

Research Article

Dihydromyricetin Activates AMP-Activated Protein Kinaseand P38MAPK Exerting Antitumor Potential inOsteosarcoma

Zhiqiang Zhao1, Jun-qiang Yin1, Man-si Wu2, Guohui Song1, Xian-biao Xie1, Changye Zou1, Qinglian Tang1,Yuanzhong Wu2, Jinchang Lu1, Yongqian Wang1, Jin Wang1, Tiebang Kang2, Qiang Jia3, and Jingnan Shen1

AbstractNumerous patientswithosteosarcoma either are not sensitive to chemotherapyor developdrug resistance

to current chemotherapy regimens. Therefore, it is necessary to develop several potentially useful therapeutic

agents. Dihydromyricetin is the major flavonoid component derived from Ampelopsis grossedentata, which

has a long history of use in food and medicine. The present study examined the antitumor activity both in

vitro and in vivowithout noticeable side effects and the underlyingmechanismof action of dihydromyricetin

in osteosarcoma cells. We found that dihydromyricetin induced increased p21 expression and G2–M cell-

cycle arrest, caused DNA damage, activated ATM–CHK2–H2AX signaling pathways, and induced apoptosis

in osteosarcoma cells as well as decreasing the sphere formation capability by downregulating Sox2

expression. Mechanistic analysis showed that the antitumor potential of dihydromyricetin may be due

to the activation of AMPKa and p38MAPK, as the activating AMPKa led to the inactivation of GSK3b in

osteosarcoma cells. Moreover, GSK3b deletion or GSK3b inhibition by LiCl treatment resulted in increased

p21 expression and reduced Sox2 expression in osteosarcoma cells. Taken together, our results strongly

indicate that the antitumor potential of dihydromyricetin is correlated with P38MAPK and the AMPKa–GSK3b–Sox2 signaling pathway. Finally, immunohistochemical analysis indicated that some patients had a

lower p-AMPK expression after chemotherapy, which supports that the combination of dihydromyricetin

and chemotherapy drug will be beneficial for patients with osteosarcoma. In conclusion, our results are the

first to suggest that dihydromyricetin may be a therapeutic candidate for the treatment of osteosarcoma.

Cancer Prev Res; 7(9); 927–38. �2014 AACR.

IntroductionOsteosarcoma is the most common primary malignant

bone tumor in childhood and adolescence (1).The clinicaloutcome of patients with osteosarcoma can be improvedwith chemotherapy, and the 5-year survival rate has reached60% to 70% (2). However, there is currently a need toidentify effective agents for the treatment of this deadlydisease and to develop new therapeutic strategies with lesssevere side effects, because numerous patients with osteo-

sarcoma are either not sensitive to chemotherapyor developdrug resistance with current chemotherapy regimens.

Ampelopsis grossedentata, a vine plant in South China, is apopular and multipurpose traditional Chinese medicinalherb and has a long history of being used as food andmedicine (3). Dihydromyricetin, a 2,3-dihydroflavonolcompound, is the main bioactive component extractedfrom Ampelopsis grossedentata, is one kind of flavonoids thathas many biologic effects, including antialcohol intoxica-tion, reducing blood pressure, antibacterial, antioxidant,and antitumor properties (4–6). Recently, it has beenshown in some cancer cells that dihydromyricetin possessesantitumor effects, such as antiproliferation, cell-cycle arrest,induction of apoptosis, and increased sensitivity to chemo-therapeutic drugs (7, 8). Moreover, dihydromyricetin hasshown potential in ameliorating chemotherapy-inducedside effects (9). However, very little is known about itseffects on osteosarcoma, and the underlying mechanismsof dihydromyricetin’s anticancer effects are still underinvestigation.

AMP-activated protein kinase (AMPK), a serine/threo-nine protein kinase and a member of the Snf1/AMPKprotein kinase family, is a metabolic checkpoint proteindownstream of the LKB1 tumor suppressor and integrates

Authors' Affiliations: 1Department ofMusculoskeletalOncology, The FirstAffiliated Hospital of Sun Yat-Sen University; 2State Key Laboratoryof Oncology in South China, Sun Yat-Sen University Cancer Center,Guangzhou; and 3The Institute of Biology, Guizhou Academy of Sciences,Guiyang, China

Z. Zhao, J. Yin, and M. Wu contributed equally to this article.

Corresponding Authors: Jingnan Shen, Department of MusculoskeletalOncology, The First Affiliated Hospital, Sun Yat-Sen University, Guangz-hou, 510080, China. Phone: 86-020-87335039; Fax: 86-20-87332150;E-mail: [email protected]; and Qiang Jia, The Institute of Biology,Guizhou Academy of Sciences, Guiyang, 550001 China. Phone: 86-851-3804942; Fax: 86-851-3838181; E-mail: [email protected]

doi: 10.1158/1940-6207.CAPR-14-0067

�2014 American Association for Cancer Research.

CancerPreventionResearch

www.aacrjournals.org 927

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Published OnlineFirst June 3, 2014; DOI: 10.1158/1940-6207.CAPR-14-0067

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growth factor receptor signaling with cellular energy status.AMPK is activated by metabolic stresses and xenobioticcompounds that cause a cellular energy imbalance (10).Evidence suggesting that AMPK can inhibit cell-cycle pro-gression in human hepatocellular carcinoma cells (11), andthat AMPK activation requires the presence of LKB1, led usto hypothesize that AMPK activators might be useful in theprevention and/or treatment of cancer. It is possible thatAMPK has many downstream targets whose phosphoryla-tion mediates dramatic changes in cell metabolism, cellgrowth, and other functions. 5-Aminoimidazole-4-carbox-amide riboside (AICAR) and metformin are pharmacolog-ically active, potent AMPK activators and have become thefocus of much research in carcinogenesis due to theirregulation of various signaling pathways, such as the inhi-bition of mTOR signaling and blocking of the growth ofglioblastoma cells that express the activated EGFR mutant,aswell as their ability to control the levels of p53, p21, cyclinD1, and caspases (12, 13). In addition, metformin has beenfound to be an effective antitumor agent due to induction ofDNA damage and apoptosis in osteosarcoma (14).

The p38MAPK and JNK protein kinases affect a variety ofintracellular responses, such as inflammation, cell-cycleregulation, cell death, development, differentiation, senes-cence, and tumorigenesis; as such, these kinases have beenexploited for the development of therapeutics to treat avariety of different diseases, including cancer (15, 16).Constitutive activation of JNK or p38MAPK has been impli-cated in the induction ofmany forms of neuronal apoptosisin response to a variety of cellular injuries (17). Moreover,p38MAPK phosphorylation by anandamide treatment sub-sequently activated caspase-3, leading to apoptosis in oste-osarcoma cells (18).

In this study, we have investigated the antitumor activityof dihydromyricetin in osteosarcoma and examined itseffects on cell-cycle progression, the induction of DNAdamage and apoptosis, and sphere formation. Furthermore,we have investigated the changes in AMPK/GSK3b/Sox2and p38MAPK cell signaling in osteosarcoma cells treatedwith dihydromyricetin. This study is the first to demonstratethe effect of dihydromyricetin on osteosarcoma cells andhas identified the mechanism of its action, through activat-ing AMPK and p38MAPK signaling pathways, which mayhelp guide the clinical use of dihydromyricetin.

Materials and MethodsChemicals and reagents

Dihydromyricetin was prepared from Ampelopsis grosse-dentata using the chromatographic method. The variablelevels for extracting dihydromyricetin were 74% ethanolconsistency, a temperature of 65�Cwith aheating timeof 94minutes, and a 1:35 ratio of Ampelopsis grossedentata towater. The purity of the dihydromyricetin was shown to behigher than 98%, based on reversed-phase HPLC analysis.The compoundwasdissolved inDMSO. In addition, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide(MTT), adriamycin, AICAR, andmetforminwere purchasedfrom Sigma-Aldrich.

Cell cultureThe human osteosarcoma cell lines U2OS, MG63, Saos2,

HOS, and 143B were obtained from the American TypeCulture Collection (ATCC). U2OS/MTX cells, a methotrex-ate-resistant derivative of the U2OS human osteosarcomacell line, were provided by Dr. M. Serra (Istituti OrtopediciRizzoli, Bologna, Italy). The ZOS and ZOS-M cell lines havebeen described previously (19). The cells were cytogeneti-cally tested and authenticated before being frozen. The cellswere grown inDMEM supplemented with 10% fetal bovineserum (Invitrogen) at 37�C with 5% CO2. All cell-basedexperiments were performed on cells thatwere in culture for4 weeks or less.

Plasmids and antibodiesConstitutively active GSK3b and SOX2/GFP plasmids

were provided by Tiebang Kang (State Key Laboratory ofOncology in South China, Sun YatSen University CancerCenter, Guangzhou, China, 510060). Antibodies againstGSK3b, phospho-Ser9-GSK3b, Sox2, PARP, caspase-3,cleaved caspase-3, p38MAPK, phospho-p38MAPK (Thr180/Tyr182), AMPKa, phospho-AMPKa (Thr172), and theDNA Damage Antibody Sampler Kit were obtained fromCell Signaling Technology; thep65andp21antibodieswerepurchased from Santa Cruz Biotechnology.

Cell-cycle analysesCells were treated with dihydromyricetin for 48 hours

and were subsequently collected and analyzed using aCytomics FC 500 instrument (Beckman Coulter) equippedwith CXP software after propidium iodide staining. TheModFit LT 3.1 Trial cell-cycle analysis software was used todetermine the percentage of cells in the different phases ofthe cell cycle.

Hoechst 33258 stainingAfter the cells were treated with or without dihydromyr-

icetin for 24 hours, the cells were washed twice with PBS,fixed with 4% paraformaldehyde for 20 minutes, andwashed twice with ice-cold PBS. Then, the cells were stainedwith the DNA-specific dye Hoechst 33258 (10 mg/L),washed twice, and observed in random microscopic fieldsusing a fluorescence microscope with the standard excita-tion filters (Leica DMIRB).

Sphere formation assaySphere formation assay was carried out as previously

described (20). Briefly, 2,000 cells were plated in triplicatein 6-well ultra-low attachment plates (Corning) in DMEM/F12 (Invitrogen) supplemented with N2 medium (Invitro-gen), 10 ng/mL human EGF (PeproTech), and 10 ng/mLhuman bFGF (PeproTech) and treated with or withoutdihydromyricetin for approximately 2 weeks. Spheres werecounted in each plate using an inverted phase-contrastmicroscope.

Caspase-3 activity assayTo assess the cell viability after the indicated treatments,

caspase-3 activity assays were performed according to

Zhao et al.

Cancer Prev Res; 7(9) September 2014 Cancer Prevention Research928




the manufacturer’s instructions (Calbiochem). Caspase-3activity was measured at 405 nm using a microtiter platereader, as recommended in themanufacturer’s instructions.

Immunofluorescence analysisU2OS cells were plated on culture slides (Costar) and

were treated with or without dihydromyricetin. After 48hours, the samples were rinsedwith PBS and fixed using 4%paraformaldehyde for 15minutes at room temperature. Theslideswere thenwashedwith 0.1%NP40/PBS and extractedwith buffer containing 0.5% Triton X-100 for 5 minutes.The cells were then blocked with 5% goat serum andincubated with primary antibodies overnight. After threewashes with PBS, the samples were incubated with second-ary antibody at room temperature for 1 hour. Cells werethen counterstained with Hoechst 33342 at room temper-ature for 5 minutes to visualize the nuclear DNA, and theslides were examined using an Olympus confocal imagingsystem (Olympus FV100).

RNA extraction and quantitative real-time PCRTotal cellular RNA was extracted using the TRIzol

reagent (Invitrogen) according to the manufacturer’sinstructions. RNA was reverse transcribed to producecDNA using the Thermo Scientific Maxima First StrandcDNA Synthesis Kit (Thermo Scientific). Real-time PCRamplification was performed using Platinum SYBR GreenqPCR SuperMix-UDG with ROX (Invitrogen) on Hard-Shell PCR Plates (Bio-Rad).

Western blot analysisThe procedures have been described previously (21).

Equal amounts of protein were resolved on a 10% sodiumdodecyl sulfate polyacrylamide gel (SDS-PAGE) and trans-ferred to a polyvinylidene difluoride (PVDF) membrane(Millipore). Membranes were blocked for 1 hour with 5%non-fat dry milk (Bio-Rad) in Trisbuffered saline withTween20 (TBST) and incubatedwith primary antibody over-night at 4�C. Membranes were washed with TBST andincubated with horseradish peroxidase–conjugated second-ary antibody. Proteins were visualized using the enhancedchemiluminescence system (Pierce).

Cell viability assayThe osteosarcoma cell lines were seeded in 96-well plates

at a density of 3,000 cells per well. The cells were treatedwith different concentrations of dihydromyricetin. After theindicated incubation times at 37�C, 20 mL of MTT (5 mg/mL)was added, and the plates were incubated for 4 hours at37�C. Then, themediumwas removed and200mLofDMSOwas added and mixed thoroughly. The absorbance wassubsequently measured using a Bio-Rad Microplate Reader(wavelength, 490 nm). All data are the average of threeindependent experiments.

Proteome profiler arrayThe proteome profiler array (R&D Systems) was per-

formed according to the manufacturer’s protocol. Briefly,the cell lysates were incubated with activated array mem-

branes overnight at 4�C. Each array membrane was washedthree times with 1� wash buffer and incubated with thediluted antibody cocktail for 2 hours at room temperature.The arraymembraneswere thenwashed three timeswith1�wash buffer and further incubated with streptavidin–horse-radish peroxidase for 30minutes at room temperature. Eacharraymembranewas thenwashed three timeswith 1�washbuffer, and finally, the membranes were exposed to X-rayfilm following chemiluminescent detection.

TUNEL staining assayApoptosis of the tumor tissues from the animal experi-

ments was determined using a TUNEL Assay Kit (RocheApplied Science) as described previously (22). All sampleswere visualized using diaminobenzidine (DAB; Dako), andthe nuclei were counterstained with hematoxylin.

Animal experimentsTheanimal studywasapprovedby the InstitutionalReview

Board of the Sun Yat-Sen University. Athymic nude (nu/nu)mice, 5 to6weeks of age,were purchased fromShanghai SlacLaboratory Animal Company Limited. U2OS/MTX and ZOScells (1�106 cells in 200 mL of PBS) were subcutaneouslyinjected near the scapula of the nude mice. After 9 days, themice were randomly separated into the appropriate groups.The mice bearing the U2OS/MTX cells were separated intothree groups. Thefirst group, the control,was treatedwith thevehicle. The other two groupswere treatedwith dihydromyr-icetin (dose1, 150 mg/kg; or dose2, 300 mg/kg) every day.After 7 days, the mice bearing the ZOS cells were separatedinto four groups. Thefirst group, the control,was treatedwiththe vehicle. Two groups of animals were treated with dihy-dromyricetin (150 mg/kg, every day) or adriamycin (6 mg/kg, once per week), and the last group was treated withdihydromyricetin (100 mg/kg, every day) in combinationwith adriamycin (6 mg/kg, once per week). All the groupsreceived the drug through intraperitoneal injection. Theresulting tumors were measured with a caliper every 2 days,and the tumor volume was calculated using the formula V¼1/2 (width2 � length). The weights of the mice were alsorecorded. At the end of the experiment, the animals weresacrificed using cervical dislocation, and the tumor weightswere measured after careful resection.

For the orthotopic model of osteosarcoma, 143Bcells were slowly injected following the procedure asdescribed previously (23). Twelve days after injection,the mice were randomly separated into treatment groups(n ¼ 6). Mice were treated with the vehicle or dihydromyr-icetin (dose1,150 mg/kg; or dose2, 300 mg/kg) by intra-peritoneal injection every day. The length and width of thetumors (D1 and D2) were measured with a caliper every 3days, and the tumor volume was calculated using theformula V ¼ 4/3p[1/4(D1 þ D2)]

2. Finally, the mice wereanesthetized using chloral hydrate during X-ray scans.

Immunohistochemical stainingImmunohistochemical (IHC) staining was performed

as described previously (20). The SOX2 primary antibody

Antitumor Potential of Dihydromyricetin in Osteosarcoma

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(rabbit monoclonal, clone D6D9; Cell Signaling Tech-nology) was diluted at the ratio of 1:50 and theslides were incubated with anti-SOX2 antibody overnightat 4�C.

The clinical samples were from 24 patients with osteo-sarcoma, including biopsy sample (before standard neoad-juvant chemotherapy) and operation samples (afterstandard neoadjuvant chemotherapy). The p-AMPK prima-ry antibody (rabbit monoclonal; Cell Signaling Technolo-gy) was diluted at the ratio of 1:100 and IHC staining wasperformed as described. Patient sample study was approvedby the Institutional Review Board of the Sun Yat-SenUniversity.

Statistical analysisAn unpaired Student t test or an analysis of variance

with a Bonferroni post hoc test was used for statisticalsignificance.

ResultsEffect of dihydromyricetin on cell-cycle progression ofosteosarcoma cells

Dihydromyricetin (molecular weight, 320.25 Da), themajor bioactive constituent of Rattan Tea, is an importanttype of flavonol, and its chemical structure is shownin Fig. 1A. To test the effect of dihydromyricetin onosteosarcoma cells, U2OS, MG63, ZOS, 143B, Saos2,HOS, and U2OS/MTX were treated with different dosesof dihydromyricetin, and MTT assays were performed todetermine the IC50 (Fig. 1B). Moreover, the amount ofcells that survived significantly decreased after treatmentwith dihydromyricetin, as compared with the untreatedcells (Fig. 1C). We examined the effect of dihydromyr-icetin on cell-cycle progression in U2OS cells, whichrevealed that dihydromyricetin could induce cell-cycleG2–M arrest, and the percentage of cells in G2–Mincreased from 13.755% to 43.287% after dihydromyr-icetin treatment (Fig. 1D). P21 is a potent inhibitor ofcell-cycle progression; we assessed the level of P21 fol-lowing dihydromyricetin treatment and found that dihy-dromyricetin increased the levels of p21 protein and RNA(Fig. 1E and 1F). CDKN1A is regulated by many tran-scription factors, including p53, a well-known tumorsuppressor, and was not significantly increased by dihy-dromyricetin exposure, suggesting that P21 increase was aP53-independent mechanism (data not shown). Theseresults indicate that dihydromyricetin could induce G2–Marrest in human osteosarcoma cells.

Dihydromyricetin induces DNA damage and apoptosisin osteosarcoma cells

Our study has showed that dihydromyricetin can inducea G2 arrest and increased P21 expression in a p53-indepen-dent manner in human osteosarcoma cells. To furtherinvestigate the molecular mechanisms behind this arrest,we examined the expression of P-ATM, P-CHK2, gH2AX inU2OS and ZOS cells. Previous studies have demonstratedthat a G2 cell-cycle arrest is frequently the result of

DNA damage. Moreover, different molecular mechanismsinvolved in DNA damage checkpoints at different phasesand ATM–CHK2–H2AX signaling pathways play importantroles (24, 25). As shown in Fig. 2A, the phosphorylationlevels of ataxia telangiectasia mutated (ATM; Ser1981),CHK2 (Thr68), and H2AX (Ser139) in U2OS and ZOStreated with 0 to 60 mmol/L dihydromyricetin significant-ly increased in a dose-dependent manner. These resultsindicate that dihydromyricetin treatment induced notonly a G2–M arrest in osteosarcoma cells, but also DNAdamage.

Cell-cycle arrest allows cells to repair the damaged DNAto maintain genomic stability in eukaryotic cells; a failureto repair the DNA can result in cell death or apoptosis (26).Therefore, we sought to determine whether treatmentwith dihydromyricetin could induce apoptosis in osteosar-coma cells. As shown in Fig. 2B, treatment with dihydro-myricetin resulted in a reduction in cell viability asmeasured by the MTT assay. Hoechst 33258 staining wasused to detectmorphologic characteristics of apoptosis afterU2OS, U2OS/MTX, and ZOS cells were exposed to 0 to 60mmol/L dihydromyricetin for 24 hours. The number ofapoptotic cells increased gradually in a dose-dependentmanner, and the cells displayed a reduction of the cellularvolume; brightly stained, condensed or fragmented nuclei;and the appearance of apoptotic bodies (Fig. 2C). U2OSand ZOS cells were treated with dihydromyricetin for 24hours, and immunoblot analysis was used to measure thecleavage of caspase-3 cleavage, amarker of apoptosis, whichresults in the cleavage of another protein, PARP (Fig. 2D). Inaddition, caspase-3 activity assays were also performedusing in dihydromyricetin-treatedU2OS andZOS cells (Fig.2E). Taken together, these results indicate that dihydromyr-icetin induces DNA damage and causes apoptosis in oste-osarcoma cells.

Dihydromyricetin exerts antitumor activity in vivoThe study of DNA damage and apoptosis in tumor cells

will not only help us to understand regulatorymechanisms,but also provide a potential avenue for the development oftumor therapies. Thus, the in vivo antitumor ability ofdihydromyricetin was investigated by the nude mousexenograft model using U2OS/MTX cells. The mice wererandomly separated into three groups (Control, Dose 1,and Dose 2). The Dose 1 group received 150 mg/kg ofdihydromyricetin every day, and the Dose 2 group received300 mg/kg of dihydromyricetin every day. At the termina-tion of the study, the mean volumes of the tumors were1,957mm3 for the control group, 1,238mm3 for theDose 1group, and 834.6 mm3 for the Dose 2 group (Fig. 3A). Theaverage tumor weights were 1.73 g for the control group,1.15 g for the Dose 1 group, and 0.816 g for the Dose 2group (Fig. 3B). In addition, the average body weight of themice did not significantly differ between the two dihydro-myricetin treatment groups and the control group (Fig. 3C),and no obvious side effects were observed in the importantorgans (heart, livers, and kidney), as detected by hematox-ylin and eosin (H&E) staining (Supplementary Fig. S1). To

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further evaluate the antitumor effect of dihydromyricetin,we used an orthotopic model in which the 143B osteo-sarcoma cells were injected. As shown in Fig. 3D, on day33, the mean volume of the tumors in the control groupwas 2,317 mm3, whereas the mean volumes were 1,613mm3 and 1,399 mm3 for the Dose 1 and Dose 2 groups,respectively. X-ray tests and H&E staining also showedthat bone destruction and periosteal reactions around thetibia were more obvious in the control group (Supple-mentary Fig. S2). Moreover, the levels of in vivo apoptosisfollowing dihydromyricetin treatment were analyzedusing the TUNEL assay, and these results also confirmedthe in vitro results that dihydromyricetin could induceapoptosis in osteosarcoma cells (Fig. 3E and 3F). Ourresults demonstrate that dihydromyricetin possesses anti-tumor properties and can induce apoptosis in humanosteosarcoma cells in vivo.

Dihydromyricetin decreases the osteosphereformation by downregulating Sox2 in humanosteosarcoma cells

Osteosphere culture was used to isolate and expandosteosarcoma stem cells in a serum-free suspension.Recent studies demonstrated that some inhibitors hadthe potential of targeting osteosarcoma stem cells, such assalinomycin (20). Thus, to test whether dihydromyricetincould target osteosarcoma stem cells, we determined theability of the control and dihydromyricetin-treated cellsto form osteospheres. As shown in Fig. 4A, U2OS, U2OS/MTX, and 143B cells treated with dihydromyricetin asindicated have reduced ability to form osteospheres. Sox2is a transcription factor of the high-mobility group(HMG) domain family that has a critical role in embry-onic development and in maintaining pluripotencyand self-renewal of osteosarcoma stem cells (20, 27).

OH

OH

OH

OH

OH

HO O

O

Chemical structure

IC50

(mm

ol/L

)

605550454035302520151050

U2OS

MG63

ZOS14

3BSao

S2

HOSU2O

S/MTX

DMSO 15 mmol/L 60 mmol/L30 mmol/L

U2OS

MG63

ZOS

DMSO DHM 15 mmol/L

DHM 30 mmol/L DHM 60 mmol/L

Cel

l nu

mb

erC

ell n

um

ber

Cel

l nu

mb

erC

ell n

um

ber

G1 = 52.711%

G1 = 32.402% G1 = 20.962%

G1 = 49.628%

G2 = 13.755%

G2 = 35.955% G2 = 43.287%

G2 = 21.927%

S = 33.514%

S = 31.643% S = 35.747%

S = 28.466%

DNA content DNA content

DNA contentDNA content

1,080 1,140

900

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180

00 32 64 96 128 0 32 64 96 128

0 32 64 96 1280 32 64 96 128

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DHM (mmol/L)

P21

mR

NA

exp

ress

ion

P21

mR

NA

exp

ress

ion

6

4

2

0

P21

P21

GAPDH

GAPDH

GAPDH

GAPDH

P21

P21

0 15 30 60

U2OS/MTX

MG63

ZOS

U2OS0 15 30 60

A B C

DE

F

Figure 1. Effect of dihydromyricetin on cell-cycle progression of osteosarcoma cells. A, chemical structure of dihydromyricetin. B, cells were seeded in 96-wellplates and after 24 hours were treated with a range of concentrations of dihydromyricetin for 24 hours. The viable cells were measured by the MTT assay andthe IC50 was calculated. C, cells were seeded in 6-well plates and were subsequently treated with DMSO or dihydromyricetin as indicated. The panels showcolony assays stained with crystal violet 4 days later. D, representative cell-cycle analysis by flow cytometry of U2OS cells treated with DMSO ordihydromyricetin as indicated for 24 hours and the length of each cell-cycle phase was calculated. E, the graph shows levels of P21mRNA relative to GAPDHdeterminedbyquantitativeRT-PCR. The indicatedcell lineswere incubatedwithDMSOordihydromyricetin for 24hoursbeforeRNAwasprepared. F,Westernblot analysis for P21 in the indicated cell lines incubated with DMSO or dihydromyricetin for 24 hours before protein lysates were prepared.






Therefore, we investigated whether dihydromyricetincould affect the expression of Sox2. We found that Sox2mRNA and protein were downregulated in dihydromyr-icetin-treated cells (Fig. 4B and C). As expected, theexpression of Sox2 in the nuclei was also reduced asdetected by immunofluorescence (Fig. 4D). To furtherconfirm the role of Sox2 in the dihydromyricetin-inducedsuppression of osteosphere formation, U2OS cells weretransfected with a Sox2-expressing plasmid or an emptyvector, and 48 hours later, the cells were treated with orwithout dihydromyricetin. As shown in Fig. 4E and F,treatment with dihydromyricetin led to a downregulationof endogenous and exogenous Sox2 expression, and over-expressed Sox2 enhanced the ability of osteospheres inU2OS cells, which was partly recovered, when combinedwith dihydromyricetin. In addition, we performed IHCanalysis for Sox2 on tumor samples, and the resultsshowed that the tumor treated with dihydromyricetinhad a decrease in the expression of Sox2 (Fig. 4G). Theabove findings indicated that dihydromyricetin reducedthe ability of cells to form osteospheres due to thedepletion of Sox2, which may be responsible for main-

taining stem-cell characteristics in osteosarcoma stemcells.

Activationof AMPKa andp38MAPKmayplay a role in theantitumor potential of dihydromyricetin

To identify the upstream signaling kinases responsible fordihydromyricetin-induced cell-cycle arrest, apoptosis, andreduction of Sox2 expression in osteosarcoma cells, weperformed the proteome profiler antibody array. As shownin Fig. 5A and Supplementary Table S1, the phosphoryla-tion levels ofmultiple kinaseswere altered after treatment ofdihydromyricetin, including AMPKa, p38MAPK, GSK3b,JNK pan, and MSK whose expression have increased 10 to40 fold. Recently, many studies have indicated that activa-tion of AMPK is related to apoptosis in various cancer celllines (28, 29).Metformin andAICAR treatments activate theAMPKa signaling pathway in various cancer cell types,including osteosarcoma cells, and these drugs have beenshown to have antitumor properties (12, 30, 31). It has alsobeen reported that the activation of the MAPK signalingpathway, which includes p38MAPK, JNK pan, and the down-stream target MSK1/2, could induce cancer cell death

U2OS ZOSDHM (μmol/L)

DHM (μmol/L)

DHM (μmol/L)

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ATM

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CHK2

γH2AX

H2AX

GAPDH

0 15 30 60 0 15 30 60

0 15 30 60 0 15 30

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%)

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ld)

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00 030 3015 1560

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U2OS

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0 15 30 60

A B

C

D

E

Figure 2. Dihydromyricetin induces DNA damage and apoptosis in osteosarcoma cells. A,Western blot analysis of expression of P-ATM, P-CHK2, and gH2AXin U2OS and ZOS cells. B, inhibitory effect of dihydromyricetin on viability of U2OS and ZOS cells. C, Hoechst staining showed typical apoptotic morphologychanges after dihydromyricetin treatment in U2OS, U2OS/MTX, and ZOS cells. D, Western blot analysis for apoptosis-related proteins PARP and cleavedcaspase-3 in dihydromyricetin-treated U2OS and ZOS cells. E, caspase-3 activity assays were performed in U2OS and ZOS cells after treatment withdihydromyricetin.

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(15, 32). In osteosarcoma cells, the P38MAPK phosphoryla-tion subsequently activated caspase-3, leading to apoptosisor regulated Eag channel functions (33, 34). Therefore, wefurther investigated the expression of the AMPKa andp38MAPK signaling pathways in U2OS and ZOS cells treatedwith the indicated doses of dihydromyricetin. Our resultswere consistent with the proteome profiler antibody arrayanalysis (Fig. 5B). Because of the role of the AMPK andp38MAPK signaling pathway in the development of osteo-sarcomas, these results indicated that activation of AMPKand p38MAPK may play a role in the antitumor potential ofdihydromyricetin.Our recent data have demonstrated that GSK3b plays a

key oncogenic role in osteosarcoma growth by regulating

NF-kB signaling (23). In Fig. 5A and B, we also showed thatdihydromyricetin could suppress the activity of GSK3b.Therefore, we investigated whether the activation of AMPKwas correlated with the inactivation of GSK3b. Next, weusedmetformin andAICAR,which activate AMPKa, to treatU2OS and MG63 cells, and we detected an increasing levelof phosphorylated GSK3b, which suggested that dihydro-myricetin could inactivate GSK3b through the activation ofAMPKa (Fig. 5C). In addition, we demonstrated that dihy-dromyricetin treatment resulted in the downregulation ofSox2 and reduced the formation of osteospheres. We inves-tigated the levels of Sox2 and p21 expression after thedepletion of GSK3b or the inhibition of GSK3b by LiCltreatment in U2OS and MG63 cells. We found that

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Sox2 expression was downregulated and that p21 expres-sion increased; these results were similar to the resultsobtained from dihydromyricetin-treated cells (Fig. 5D).Taken together, these findings strongly suggest that theantitumor potential of dihydromyricetin in osteosarcomacells is correlated with the p38MAPK and AMPK– GSK3b–Sox2 signaling pathways.

Combined effect of dihydromyricetin withchemotherapeutic drugs on osteosarcoma cells

The combined treatment of cisplatin, doxorubicin, andmethotrexate was established as the standard treatment

regimen for osteosarcoma 30 years ago; however, improv-ing the survival of patients with osteosarcoma has proved tobe an enormously difficult challenge (35). Recent researchhas demonstrated that the activation of AMPK contributesto doxorubicin-induced cancer cell death and apoptosis,and that metformin could sensitize cancer cells to thechemotherapeutic drugs, which also is significantly associ-ated with increased survival among the patients (36, 37). Inaddition, the expression of p-AMPK in 25 pairs of biopsyand operation osteosarcoma samples was evaluated by IHCstaining. We compared the change of p-AMPK expressionbetween biopsy and operation samples from the same

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Zhao et al.





patient. In this cohort, 16 (64%) cases had a higher expres-sion after chemotherapy, 7 (28%) cases were lower, and 2(8%) cases almost had no change (Fig. 6A). Therefore, wehypothesize whether combining dihydromyricetin withthese chemotherapeutic drugs would have a synergisticeffect in killing osteosarcoma cells. For the experiment,ZOS cells were injected into nude mice, and 7 days later,themice were randomly divided into four groups and drugswere injected intraperitoneally. As single therapeuticmodalities, dihydromyricetin or adriamycin induced sim-ilar effects on tumor growth (meanvolumeof tumors, 1,381vs. 1,075 mm3; mean weight, 1.38 vs. 1.24 g), with a smalladvantage for adriamycin. Importantly, the combination ofdihydromyricetin with adriamycin worked additively toinhibit tumor growth fromosteosarcoma cells in nudemice(Fig. 6B, C, andD). Finally, we examined the bodyweight ofall the nude mice and found that dihydromyricetin had aslight potential of reducing adriamycin-induced weight lossside effect (Fig. 6E). These data suggest that the combinationof dihydromyricetin and adriamycin may be an attractivetherapeutic option for osteosarcoma.

DiscussionDifferent chemotherapy regimens have been compared

in patients with osteosarcoma, but the survival rates never

significantly improve (38). In addition, chemotherapyresistance is a common problem that can significantlydiminish clinical outcomes (39). Therefore, it is necessaryto develop several potentially useful therapeutic agents forovercoming the challenge, particularly those from naturalorigins (40, 41).

Recent studies have shown that dihydromyricetin hasmany biologic effects, including antialcohol intoxication,reducing blood pressure, antibacterial, antioxidant, andantitumor properties (4–6, 42, 43). This study demonstrat-ed that dihydromyricetin exhibits antitumor activity inosteosarcoma both in vitro and in vivo. We found thatdihydromyricetin treatment could inhibit the viability ofhuman osteosarcoma cells, including theMTX-resistant cellline U2OS/MTX. Cell-cycle arrest is an essential early eventin the inhibition of cell proliferation, and our result of cell-cycle assay showed that dihydromyricetin could induce aG2

–M cell-cycle arrest. The CDKI p21 regulates many cellularprocesses, such as cell-cycle arrest, DNA replication andrepair, cell proliferation and differentiation, senescence andapoptosis (44, 45). One potential consequence of dihydro-myricetin-induced cell-cycle arrest would be an increase inp21 levels in osteosarcoma cells. Indeed, p21 was increasedin osteosarcoma cells, includingU2OS,MG63,U2OS/MTX,and ZOS cells. Meanwhile, the tumor-suppressor P53 didnot increase in response to dihydromyricetin treatment,

Figure 5. Dihydromyricetin regulates activity of AMPKa and p38MAPK in osteosarcoma cells. A, cell lysates of control and dihydromyricetin-treated U2OScells were applied to the proteome profiler antibody array analysis showing the phosphorylation of 45 kinases. The antibody arraywas composed of duplicatespots for each kinase on the singlemembrane. B, cell lysates of control and dihydromyricetin-treatedU2OS andZOS cellswere analyzed byWestern blotwithvarious antibodies, as indicated. C, effects of metformin and AICAR on the phosphorylation of GSK3b in U2OS and MG63 cells were confirmed byWestern blotting. D, the levels of Sox2 and p21 expression after the depletion of GSK3b or the inhibition of GSK3b by LiCl treatment in U2OS andMG63 cellswere assayed by Western blotting.






suggesting that increased P21 was p53 independent. TheDNA damage checkpoints are biochemical pathways thatdelay or arrest cell cycle in response to genomic DNAdamage, and cell would activate ATM, one of the sensorkinases. In turn, ATMphosphorylatesmultiple downstreamsubstrates, including the effector kinase CHK2 and histoneH2AX,which is the gold standard for early detectionofDNAdamage, resulting in cell-cycle arrest and/or apoptosis (46).In this study, we found that dihydromyricetin treatment notonly caused significant DNA damage but also inducedapoptosis of osteosarcoma cells in dose dependent, asdetected by PARP and caspase-3 activity. Further, we alsoreported that dihydromyricetin had antitumor potential innude mice, including orthotopic model, and the toxicityof combination was reduced compared with adriamycintreatment alone. This study is the first to show that dihy-dromyricetin exhibits strong antitumor effects againsthuman osteosarcoma cells both in vitro and in vivo withoutnoticeable side effects.

Sox2, as a transcription factor, marked and maintained adistinct cell population in osteosarcomas that had stem

cell–like properties and was responsible for their tumor-igenic potential. A recent study reported overexpressionof Sox2 in sphere-forming cells from human sarcomas,and Sox2 downregulation decreased the stem-cell popu-lation in murine and human osteosarcomas (27, 47). Ourprevious work also demonstrated that overexpressed Sox2could increase the capability of sphere formationin osteosarcoma cells (20). In the present study, wedemonstrated that dihydromyricetin could decrease thecapability of sphere formation in osteosarcoma cells.Furthermore, we investigated the expression of Sox2 inosteosarcoma cells treated with dihydromyricetin, andfound that Sox2 was downregulated through transcrip-tional level in dose dependent. Exogenous transfection ofSox2 enhanced the ability of osteospheres in U2OScells, and when combined with dihydromyricetin, theenhanced osteosphere was partly recovered. Overall,these results suggest that Sox2 is a key player in osteo-sphere and dihydromyricetin could suppress osteospherethrough downregulation of Sox2 and decrease the stemcell-population in human osteosarcomas.

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Figure 6. Combined effect ofdihydromyricetin withchemotherapeutic drugs onosteosarcoma cells. A, p-AMPKstaining was observed inosteosarcoma specimensbetween biopsy and operationsamples B, effect ofdihydromyricetin and/oradriamycin combinationtreatments on osteosarcomaxenografts in nudemice. ZOScellswere injected subcutaneouslynear the scapula of the nude mice.The mice bearing ZOS cells weretreated as described in "MaterialsandMethods." The tumor volumeswere monitored every 2 days, asindicated. C, xenografts excisedfrom the tumor-bearing micein A at day 27. D, weights of thexenografts from B at day 27. E, thebody weight of tumor-bearingmice in B at day 27. ��, P < 0.01;��, P < 0.001.)

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To determine the potential molecular mechanism ofaction of dihydromyricetin in osteosarcoma, we used aproteomeprofiler array and found that thephosphorylationlevels ofmultiple kinaseswere affected by dihydromyricetintreatment. Specifically, we observed the dihydromyricetin-mediated activation of AMPKandp38MAPK in osteosarcomacells. Activation of AMPK usually occurs under conditionsof metabolic stress or when the ATP:AMP ratio decreases(48). Activation of AMPK in response to metabolic stressresults in the silencing of intracellular energy-consuminganabolic processes and activates energy-producing catabol-ic processes (10). The energy status of the cell is a crucialfactor in all aspects of cell function; it is possible that AMPKhas many downstream targets whose phosphorylationmediates dramatic changes in cell metabolism, cell growth,and other functions. The potent AMPK activator, metfor-min, has been demonstrated as an effective antitumor agentthrough induction DNA damage and apoptosis in osteo-sarcoma (14). GSK3b, a serine/threonine protein kinase,also plays key roles in multiple pathways. AlthoughGSK3bis generally recognized as a tumor suppressor thatis frequently inactivated in a variety of tumors, we demon-strated that GSK3b activity may promote osteosarcomatumor growth and induce apoptosis in osteosarcoma cells(23). In this study, we demonstrated that cells treated withAMPKa activators resulted in the increased phosphoryla-tion of GSK3b, suggesting that dihydromyricetin treatmentcould inactive GSK3b through the activation of AMPKa. Wefurther investigated the expression of Sox2 and p21 afterGSK3b depletion or GSK3b inhibition by LiCl treatment inosteosarcoma cells and found that Sox2 expression wasdownregulated and that p21 was upregulated; these resultswere consistent with the results of the dihydromyricetintreatment. On the basis of our results, we suggest thatdihydromyricetin possesses antitumor activity due to itsability to affect the AMPK–GSK3b–Sox2 signaling pathway.Another important observation was that dihydromyricetininduces the activation of p38MAPK and the JNK proteinkinases. TheMAPK signaling pathway has been exploited incancer treatment because of its key roles in inflammation,cell-cycle regulation, cell death, development, differentia-tion, senescence, and tumorigenesis (15, 16). A recent studyalso demonstrated that anandamide activated caspase-3through an increase in p38MAPK phosphorylation in oste-osarcoma cells (18). However, further studies are needed toclearly understand how dihydromyricetin induces AMPK

activation, which can be caused by generatingmore reactiveoxygen species (ROS) or other metabolic stress.

In conclusion, we have demonstrated that dihydromyr-icetin possesses strong antitumor effects against humanosteosarcoma cells without noticeable side effects, and thatdihydromyricetin can reduce the toxicity of the chemother-apeutic adriamycin when the two agents are used in com-bination. Molecular study revealed that dihydromyricetinactivated AMPK and p38MAPK in osteosarcoma cells andinduced strong apoptotic response. Moreover, dihydromyr-icetin also decreases the population of stem cells and sphereformation capability in osteosarcoma through downregu-lation of Sox2. Considering the current clinical treatmentoutcome, these results suggest that dihydromyricetin maybe a promising agent for the treatment of osteosarcoma.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: Z. Zhao, J. Yin, M. Wu, X. Xie, Q. Jia, J. ShenDevelopment of methodology: Z. Zhao, J. Yin, M. Wu, C. Zou, J. Wang,Q. Jia, J. ShenAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): Z. Zhao, J. Yin, M. Wu, C. Zou, J. Lu, Y. Wang,Q. JiaAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): Z. Zhao, J. Yin, M.Wu, Y.Wu, J. Lu, Q. JiaWriting, review, and/or revision of the manuscript: Z. Zhao, J. Yin,J. Wang, Q. Jia, J. ShenAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): Z. Zhao, J. Yin, G. Song, X. Xie,C. Zou, Q. Tang, Y. Wang, J. Wang, Q. JiaStudy supervision: Z. Zhao, J. Yin, J. Wang, T. Kang, Q. Jia, J. Shen

AcknowledgmentsThe authors thank the graduate students in Prof. Tiebang Kang’s labora-

tory in Sun Yat-Sen University Cancer Center, Guangzhou, China.

Grant SupportThis work was supported by the Natural Science Foundation of China

(No. 81272939, to J.N. Shen and No. 8110 2040, to J.Q. Yin); Sun Yat-SenUniversity Clinical Research 5010 Program (No. 200709, to J.N. Shen), andtheMinistry of Science and Technology of Guizhou Province, China [(2012)7006, to Q. Jia].

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received February 24, 2014; revisedMay 12, 2014; acceptedMay 20, 2014;published OnlineFirst June 3, 2014.

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2014;7:927-938. Published OnlineFirst June 3, 2014.Cancer Prev Res Zhiqiang Zhao, Jun-qiang Yin, Man-si Wu, et al.

Exerting Antitumor Potential in OsteosarcomaMAPKDihydromyricetin Activates AMP-Activated Protein Kinase and P38

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