resveratrol selectively induces dna damage,...

Cancer Therapy: Preclinical

Resveratrol Selectively Induces DNA Damage, Independentof Smad4 Expression, in Its Efficacy against Human Headand Neck Squamous Cell Carcinoma

Alpna Tyagi1, Mallikarjuna Gu1, Takenori Takahata2,5, Barbara Frederick3, Chapla Agarwal1,4,Sunitha Siriwardana2, Rajesh Agarwal1,4, and Robert A. Sclafani2,4

AbstractPurpose: Alterations in Smad4 signaling and its loss cause genomic instability and head and neck

squamous cell carcinoma (HNSCC), suggesting that agents that target both Smad4-dependent and

-independent pathways could control HNSCC.

Experimental Design: Resveratrol efficacy was evaluated against the HNSCC cells FaDu, Cal27,

Det562, and Cal27-Smad4 for viability, DNA damage, cell-cycle progression, and apoptosis, as well

as g-H2AX expression, and focus formation (g-H2AX and Brca1). Resveratrol efficacy was also

examined in nude mice for FaDu xenograft growth. Xenografts were analyzed for g-H2AX and cleaved

caspase-3.

Results: Resveratrol (5–50 mmol/L) suppressed viability and induced DNA damage in FaDu and Cal27

cells but not in normal human epidermal keratinocytes and human foreskin fibroblasts, showing its

selectivity toward HNSCC cells; however, Det562 cells were resistant to resveratrol even at 100 mmol/L.

Cal27 cells stably transfected with Smad4 showed similar resveratrol effects as parental Cal27, indicating

that a lack of resveratrol effect in Det562 cells was independent of Smad4 status in these cells. Furthermore,

resveratrol caused S-phase arrest and apoptotic death of FaDu and Cal27 cells together with induction of

Brca1 and g-H2AX foci. Resveratrol (50 mg/kg body weight) treatment also inhibited FaDu tumor growth

in nude mice, and g-H2AX and cleaved caspase-3 were strongly increased in xenografts from resveratrol-

treated mice compared with controls.

Conclusion: Our findings for the first time showed antiproliferative, DNA damaging, and apoptotic

effects of resveratrol in HNSCC cells independent of Smad4 status, both in vitro and in vivo, suggesting

that more studies are needed to establish its potential usefulness against HNSCC. Clin Cancer Res;

17(16); 5402–11. �2011 AACR.

Introduction

Head and neck squamous cell carcinoma (HNSCC) is thesixth most frequent cancer worldwide, and the 5-yearsurvival rates are among the lowest (<60%) of the majorcancers. HNSCCs are mainly caused by alcohol consump-tion, tobacco intake (smoking and smokeless products,such as betel quid), poor oral hygiene, and infection tohigh-risk types of human papilloma virus (1). According to

the statistical estimates given by the American CancerSociety, approximately 73,080 new cases of HNSCC wouldhave been diagnosed in the United States in the year 2010(2). Despite advancements in surgical procedures, che-motherapy, radiation therapy, as well as combination ofthese, the patient survival rates have not improved in thelast several decades. Moreover, it has been shown that thehigh rate of morbidity is due to both locoregional recur-rence and distant metastasis (1, 3).

TGF-b receptor (TGF-bR) II is the main pathway that isderegulated in HNSCC (4) in which aberrant signalingdownstream of this receptor contributes to tumorgrowth. TGF-b regulates its pleiotropic biological activ-ities through a complex formation between TGF-bRI andTGF-bRII. TGF-b receptors then transmit signal throughthe activation of the downstream Smad signaling path-ways, which consists of the Smad family of proteins(Smad1–5; 4). Receptor-activated Smads (Smad2 and 3or Smad1 and 5) associate with Smad4 and form aheterooligomeric complex, which translocates to the

Authors' Affiliations: Departments of 1Pharmaceutical Sciences, 2Bio-chemistry and Molecular Genetics, and 3Radiation Oncology, and 4Uni-versity of Colorado Cancer Center, University of Colorado Denver, Aurora,Colorado; and 5Department of Medical Oncology, Hirosaki UniversityGraduate School of Medicine, Aomori, Japan

Corresponding Author: Robert A. Sclafani, University of Colorado Schoolof Medicine, Campus Box 8101, Room 9100, Aurora, CO 80045. Phone:303-724-3271; Fax: 303-724-3215; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-11-1072

�2011 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 17(16) August 15, 20115402

on May 7, 2018. © 2011 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst June 24, 2011; DOI: 10.1158/1078-0432.CCR-11-1072

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nucleus where it activates transcription of various targetgenes, namely, p53, cyclin-dependent kinase (cdk) inhi-bitors, c-Myc, and those regulating apoptosis (5–7).Mutation in TGF-bRII causes deregulation in growthinhibitory control and tumorigenesis (4). Mechanisticstudies have established that alteration in Smad4 signal-ing is due to frequent mutations in TGF-bRII, and thisalteration is common in HNSCC. Furthermore, impor-tance of Smad4 in HNSCC is recently established by thestudy in which Smad4 loss caused defects in Fanconianemia/Brca (Fanc/Brca) pathway, leading to genomicinstability in mice and spontaneous HNSCC (8). Theseobservations suggest that agents are needed that couldtarget TGF-b/Smad4 pathway for HNSCC treatment.Resveratrol, a nontoxic polyphenolic phytoalexin

found in grapes skin, red wine, peanuts, berries, etc.,has shown strong efficacy against various cancers in bothin vitro and in vivo studies (9, 10). Recently, a number ofinterventional clinical trials in humans have beeninitiated using resveratrol as a therapeutic, oral com-pound (11). The voluminous literature about anticancerefficacy of resveratrol is based on an initial study byPezzuto and colleagues showing that resveratrol inhibitspreneoplastic lesions in carcinogen-treated mouse mam-mary glands in culture and 7,12-dimethylbenz(a)anthra-cene–induced skin tumorigenesis in a mouse model (12).Despite extensive studies with resveratrol in various mod-els and organ sites of malignancies, to date, nothing isknown about the anticancer efficacy of resveratrol againstHNSCC. Because of these issues, we assessed whetherresveratrol is effective against HNSCC and if the efficacyof resveratrol is dependent on Smad4, a key signalingpathway deregulated in HNSCC.

Materials and Methods

Cell culture and reagentsThe human HNSCC cells FaDu, Cal27, and Det562 were

taken from American Type Culture Collection (ATCC) in2006 and were confirmed by DNA fingerprinting with theABI Identifiler kit using the following identifiler loci:D3S158, vWA, FGA, amelogenin, D8S1179, D21S11,D18S51, D13S317, D7S820, CSF1PO, D16S539, THO1,TPOX, D2S1338, and D19S433. All results matched thefingerprint profiles in the ATCC database. Normal humanepidermal keratinocytes (NHEK) and human foreskinfibroblasts (HFF) were from Lonza Walkersville, Inc.Dulbecco’s modified Eagle’s medium (DMEM) and othercell culture materials were from Invitrogen Corporation.Antibodies for g-H2AX and cleaved caspase-3, and anti-rabbit peroxidase-conjugated secondary antibody werefrom Cell Signaling. Smad4 and Brca1 antibodies werepurchased from Santa Cruz Biotechnology. Annexin V/Vybrant Apoptosis Assay Kit was from Molecular Probes.Resveratrol and b-actin were from Sigma-Aldrich. FaDu,Cal27, Det562, and HFF cells were cultured in DMEMcontaining 10% FBS and 1% penicillin/streptomycin.NHEK cells were cultured in KGM-Gold Bullet Kit (Lonza;catalogue # 00192060) under standard culture conditions.

Generation of Cal27 vector control (pcDNA3) andSmad4-overexpressing stable cell lines

Following similar procedures (8), Cal27 cells were trans-fected with pcDNA Flag-Smad4M (plasmid 14959) orvector control pcDNA3 (Addgene), using Lipofectamine2000. Stable transfectants were selected using G418 at 0.2mg/mL in DMEM (10% FBS) for 4 weeks, and cells werepooled and maintained in the same selective medium.

MTT assayCells were plated in 96-well plate under standard culture

conditions, next day treated with resveratrol, and at theend of desired treatment time, 20 mL MTT stock solution(5 mg/mL) was added to each well and incubated foranother 5 hours. Thereafter, media was aspirated, 200 mLof dimethyl sulfoxide (DMSO) was added to each well, andabsorbance was measured at 570 nm.

Comet assayFollowing desired treatments, cells were harvested,

washed with PBS, 50 mL cell suspension containing5,000 cells mixed with 500 mL of 0.5% agarose at 37�C,and 75 mL of this mixture instantly added to comet slides(OxiSelect Comet Assay Kit; Cell Biolabs, Inc.). Embeddedcells were immersed in lysis buffer at 4�C for 1 hour andthen replaced with cold alkaline buffer and placed at 4�Cfor 30 minutes in dark followed by electrophoresis (35 Vfor 15 minutes at 4�C) in a horizontal electrophoresischamber filled with neutral Tris borate EDTA (TBE) elec-trophoresis buffer. Thereafter, slides were transferred to acontainer filled with cold distilled water for 2 to 4 minutesand then placed in 70% ethanol for 5minutes and air dried

Translational Relevance

DNA repair processes, which are defective in manycancer cells, offer avenues for therapeutic intervention.Thus, preclinical/clinical development of nontoxic che-mopreventive agents that can selectively induce DNAdamage in cancer cells is highly desirable. Supportingthis rationale, results of the present study are highlysignificant with major implications wherein we reportthat the anticancer effect of resveratrol is predominatelydue to its ability to induce DNA damage selectively inhead and neck squamous cell carcinoma (HNSCC) cellswithout affecting normal, repair-proficient cells. Thisstudy reports that resveratrol causes S-phase arrest andapoptotic death of FaDu, Cal27, and Cal27-Smad4 cellstogetherwith inductionof g-H2AXandBrca1 foci,whichare independent of Smad4 expression. The resveratrol-mediated upregulation of g-H2AX and cleaved caspase-3protein expression is also observed in FaDu tumorxenografts in athymic nude mice. These preclinicalresults hereby warrant further clinical investigation todetermine the efficacy of resveratrol against HNSCC.

DNA Damage by Resveratrol in HNSCC

www.aacrjournals.org Clin Cancer Res; 17(16) August 15, 2011 5403




overnight at room temperature. After staining with VistaGreen for 15 minutes at room temperature, comets (bothdouble- and single-strand DNA breaks) were observed byfluorescent microscopy (Olympus BH2) at 100� totalmagnification.

Cell-cycle distribution and apoptosis analysesCells were treated with resveratrol, trypsinized, and

washed twice with ice-cold PBS, and pellets were incubatedin 0.5 mL of saponin/propidium iodide (PI) solution at4�C for 24 hours in dark as reported earlier (13). Alter-natively, cells were subjected to Annexin V and PI stainingusing Vybrant Apoptosis Assay Kit 2 following the vendorprotocol. Stained cells were analyzed by flow cytometry atthe fluorescence-activated cell-sorting (FACS) AnalysisCore of the University of Colorado Cancer Center (UCCC)for cell-cycle distribution and apoptotic cell populations,respectively.

ImmunoblottingAt 60% to 65% confluency, cells were treated with

resveratrol, cell lysates were prepared, 30 to 60 mg proteinper sample was denatured with 2� sample buffer andsubjected to SDS-PAGE on 12% or 16% gel, and separatedproteins were transferred onto membrane by Westernblotting as described (13). Membranes were blockedwith blocking buffer for 1 hour at room temperatureand probed with primary antibody overnight at 4�C fol-lowed by incubation with peroxidase-conjugated appropri-ate secondary antibody and enhanced chemiluminescence(ECL) detection.

Immunofluorescence staining and confocalmicroscopy

Cells were plated on cover slips overnight and followingdesired treatment, washed gently with PBS, fixed in 4%formaldehyde for 15 minutes, incubated with ice-cold100% methanol for 10 minutes and rinsed twice withPBS, and blocked in 5% bovine serum albumin in PBSfor 60 minutes. Thereafter, cells were incubated withg-H2AX or Brca1 antibody overnight at 4�C, washed withPBS, incubated for 45 minutes with Texas Red goat anti-rabbit and Alexa Flour goat anti-mouse secondary antibo-dies, and counterstained with 40,6-diamidino-2-phenylin-dole (DAPI) for 5 minutes. Cell images were captured at1,000� magnification on a Nikon D Eclipse C1 confocalmicroscope and analyzed by EZ-C1 Free viewer software.

Tumor xenograft studyExponentially growing FaDu cells were trypsinized,

washed, and resuspended in serum-free and antibiotic-freeDMEM. Athymic nu/nu male mice (6-week-old) weremaintained under standard conditions with free accessto water and AIN-76 diet, randomly divided into 5 groups(n ¼ 8/group), and injected s.c. with 3 � 106 cells mixedwith Matrigel (1:1) in the right flank to initiate tumorxenograft growth. Next day, mice in groups 2 and 3 weregavaged orally with 10 and 50 mg/kg body weight doses of

resveratrol in 0.2 mL of 0.5% carboxy methyl cellulose(CMC) for 5 d/wk for 30 days, respectively, and those ingroup 1 (control) with CMC alone. For groups 4 and 5,xenograft was allowed to grow for 15 days and then micewere gavaged orally with 0.5% CMC and 50 mg/kg bodyweight resveratrol dose in CMC for 5 d/wk for 15 days,respectively. Xenograft growth was measured twice weeklyin all groups of animals after seventh day of cells implanta-tion with a digital caliper, and tumor volume calculated asreported recently (14). Body weight and diet consumptionwere recorded twice weekly throughout study. At experi-ment terminations, tumors were excised, weighed, andstored at �80�C. Animal care and experiments were inaccordance with University of Colorado Denver–approvedInstitutional Animal Care and Use Committee protocol.

Immunohistochemical stainingTumor tissues, fixed in 10% phosphate-buffered forma-

lin for 12 hours at 4�C, were dehydrated in ascendingconcentrations of ethanol, cleared with xylene, embeddedin paraffin, and tissue blocks were cut with a rotarymicrotome into 4-mm sections and processed for immu-nohistochemical (IHC) staining. Briefly, sections afterdeparaffinization and rehydration were treated with0.01 mol/L citrate buffer (pH 6.0) in a microwave for 30minutes for antigen retrieval, followed by quenching ofendogenous peroxidase activity with 3%H2O2 inmethanol(v/v) for 5 minutes. Next, sections were incubated withspecific antibodies, g-H2AX and cleaved caspase-3, in PBSfor 2 hours at room temperature, followed by overnightincubation at 4�C in a humidified chamber. Negativecontrols were incubated only with universal negative con-trol antibodies under identical conditions. Sections werethen incubated with appropriate biotinylated secondaryantibody (1:200–400 dilutions), followed with conjugatedhorseradish peroxidase–streptavidin (Dako) and 3,30-dia-minobenzidine (Sigma Chemical Co.) working solutionand counterstained with hematoxylin (15). Microscopicimages were taken by AxioCam MrC5 camera at 400�magnification and processed using Axiovision 4.6 (CarlZeiss microimaging). IHC staining was quantified as thenumber of positive cells (brown cells) � 100/total numberof cells in 5 arbitrary selected 400� fields from each tumor.

Statistical analysisStatistical significance of differences between control and

treated samples was determined using Student’s t test(SigmaStat 3.5). The values of P < 0.05 were consideredsignificant. The data in all cases are representative of at least2 to 4 independent studies with reproducible results.

Results

Resveratrol inhibits cell viability and induces DNAdamage selectively in HNSCC cells

First, we evaluated resveratrol efficacy in a panel ofhuman HNSCC cell lines, namely, FaDu (homologousdeletion of Smad4), Cal27 (nonsense mutation in Smad4),

Tyagi et al.

Clin Cancer Res; 17(16) August 15, 2011 Clinical Cancer Research5404




and Det562 (wild-type Smad4) cells. Resveratrol at 50mmol/L dose caused 83% (P < 0.001) and 63% (P <0.001) decrease in viability of FaDu cells after 48 and 72hours, respectively (Fig. 1A). Cal27 cells at 50 mmol/Lresveratrol dose also showed up to 43% (P < 0.001)decrease in viability (Fig. 1A); however, such resveratroltreatments were not effective in Det562 cells, until a higherdose (100 mmol/L) and longer treatment of the agent(Fig. 1A). Several recent studies have shown that naturalchemopreventive agents cause DNA damage in tumori-genic cells leading to apoptotic cell death (9), and accord-ingly, next we assessed resveratrol effect on DNA damage inHNSCC cells employing the alkaline comet assay. Com-pared with controls wherein DNA remained within thenucleus, in resveratrol-treated cells, damaged DNA exitednucleus and stained as a tail (Fig. 1B). Overall, comet assayresults clearly showed DNA-damaged FaDu and Cal27 cellsfollowing resveratrol treatment; however, Det562 cells hadless DNA damage induced by resveratrol (Fig. 1B), which isconsistent with a lack resveratrol effect on Det562 cellviability (Fig. 1A), suggesting that the observed decreasein cell viability by resveratrol in FaDu and Cal27 cells ispossibly due to its DNA damage inducing effect. Impor-tantly, DNA damage was not observed in normal cells(NHEK and HFF) even at higher dose of resveratrol(Fig. 1C), showing its selectivity toward HNSCC cells. InFigure 1C, NHEK cells in the treatment groups are purpo-sely added less in number to see the comet tail. Thus, cellsare more in the control group than in resveratrol-treated

group. Furthermore, we have found that resveratrol had noeffect on the cell growth and cell number (data not shown)of NHEK cells.

Resveratrol selectively causes cell-cycle arrest andapoptosis in HNSCC cells independent of Smad4

On the basis of results in Figure 1 showing resveratrolcaused decreased cell viability and DNA damage selec-tively in HNSCC cells carrying nonfunctional Smad4(FaDu and Cal27), next we assessed its effect on cell-cycleprogression and apoptosis in FaDu cells (deletion Smad4)and Cal27-Smad4 cells overexpressing Smad4 (Fig. 2A) toaddress whether a lack of resveratrol effect in Det562 cellsis due to their wild-type Smad4 status. Resveratrol (10mmol/L) arrested FaDu cells in S-phase (66%, P < 0.001)compared to controls with 34% S-phase population(Fig. 2B); however, higher resveratrol concentrations (25–50 mmol/L) for 24 hours caused G1 arrest (63-78%) com-pared to control (41%; Fig. 2B). For Cal27, compared tocontrols with 38% to 39% cells in S-phase, resveratrol(25 mmol/L) significantly induced S-phase arrest (53%and 56%; P < 0.001) in both vector control Cal27-pcDNA3and Smad4-overexpressing (based on overexpressedSmad4 protein; Fig. 2A) Cal27-Smad4 cells, respectively(Fig. 2C). These findings revealed that resveratrol efficacy inCal27 and a lack of efficacy in Det562 cells are independentof Smad4 expression. In other studies, resveratrol (50–100mmol/L) treatments did not alter cell-cycle progression inNHEK cells (Fig. 2D), further supporting its selectivity

Figure 1. Resveratrol inhibits cellviability and induces DNA damageselectively in HNSCC cells. A,resveratrol effect on cell viability ofFaDu, Cal27, and Det562 cellsemploying the MTT assay asdetailed in Materials and Methods.*, P < 0.001 between control andresveratrol; each bar representsmean � SE of 3 samples fromeach group. B and C, cells weretreated for 24 hours with indicatedconcentrations of resveratrol andthen comet assay was conductedas detailed in Materials andMethods. Representativephotographs of damaged andundamaged nuclei in HNSCCFaDu, Cal27, and Det562 cells (B)and NHEK and HFF cells (C) werecaptured as detailed in Materialsand Methods. Res, resveratrol.

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toward HNSCC cells. Resveratrol (25–50 mmol/L) treat-ment also induced apoptosis in FaDu, Cal27-pcDNA3, andCal27-Smad4 cells by 20- to 27-fold, 2.5- to 4-fold, and 3-to 5-fold (P < 0.001), respectively (Fig. 3). Similar to S-phase arrest results, Cal27 cells with overexpression ofSmad4 retained sensitivity to resveratrol-induced apoptosis(Fig. 3C), suggesting that the effect on apoptosis is alsoindependent of Smad4.

Resveratrol induces g-H2AX and Brca1 focusformation in HNSCC cells

H2AX serine-139 phosphorylation (the so-calledg-H2AX) is essential for recruiting and localizing DNArepair proteins (Brca1 and Rad51) at the sites containingdamaged chromatin and for checkpoint activation, whicharrest cell-cycle progression (16). Brca1 is a tumor suppres-sor protein involved in maintaining genome integrity, and

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Figure 2. Resveratrol selectively causes cell-cycle arrest in HNSCC cells independent of Smad4. A, Cal27 cells were transfected with pcDNA Flag-Smad4M(plasmid 14959) or vector control pcDNA3 (Addgene), using Lipofectamine 2000. Stable transfectants were selected using G418 at 0.2 mg/mL inDMEM (10% FBS) for 4 weeks, and cells were pooled and maintained in the same selective medium. Cells lysates were prepared and subjected to SDS-PAGEfollowed by immunoblotting, and membranes were probed with Smad4 antibody as detailed in Materials and Methods. Percentage cell-cycledistribution in FaDu (B), Cal27-pcDNA3 and Cal27-Smad4 (C), and NHEK (D) cells following resveratrol treatment for 24 hours at indicated concentrations andFACS analysis as detailed in Materials and Methods. Results shown are representative of triplicate samples from each treatment and were reproduciblein 2 independent experiments. NS, nonspecific; Res, resveratrol.

Tyagi et al.





following DNA damage, it accumulates in nucleus, where itconcentrates in nuclear foci with other DNA double-strandbreaks repair factors, including homologous recombina-tion protein, RAD51, and end-joining RAD50–MRE11–NBS1 protein complex (17). Accordingly, to further dissectresveratrol mechanism of action, we assessed g-H2AX and

Brca1 focus formation. Resveratrol enhanced g-H2AX levelsin both FaDu and Cal27 cells but had no effect in Det562cells. A higher resveratrol dose (100 mmol/L) also inducedg-H2AX in NHEK but to a lesser extent than in FaDu andCal27 cells (Fig. 4A). We also compared g-H2AX levels inparental Cal27 versus 2 independently derived Cal27-Smad4 cells (generated by us and the Wang laboratory;ref. 8) and found them to be comparable among theselines. Together, these results clearly indicated that resver-atrol triggered DNA damage in HNSCC cells independentof Smad4 expression. Consistently, compared with con-trols, resveratrol also induced discrete nuclear g-H2AX andBrca1 focus formation in FaDu cells where Brca1 coloca-lized with g-H2AX at the sites of DNA damage (Fig. 4B).

Another important observation related to DNA dama-ging effect of resveratrol in FaDu cells is an S-phase arrest at10 mmol/L, but a G1 phase arrest at higher concentration(25–50 mmol/L; Fig. 2A), and a higher g-H2AX levels withincreasing resveratrol concentration indicative of increas-ing levels of DNA damage (Fig. 4A), which is consistentwith G1 or G1–S arrest at higher concentrations. Accord-ingly, we also determined cyclin levels in FaDu cells afterresveratrol treatment and found that whereas cyclin D1levels decreased, cyclin E levels increased (data not shown)simultaneously, a situation representing inhibition of DNAreplication and a concomitant G1–S arrest and blockade toS-phase progression by resveratrol, as we had seen inovarian cancer cells (9).

Resveratrol-induced g-H2AX and Brca1 focusformation is independent of Smad4 in Cal27 cells

Recently, Smad4 loss was reported to cause genomicinstability in mice, which correlated with less expressionand function of genes encoding proteins in the Fanc/Brcain DNA repair pathway together with HNSCC development(8). Thus, we measured g-H2AX and Brca1 focus formationin Cal27-pcDNA3 and Cal27-Smad4 cells. Cal27-pcDNA3andCal27-Smad4 cells showedalmost parallel g-H2AX focusformation after resveratrol (50 mmol/L) treatment for 24hours (Fig. 5A). Mitomycin C (MMC; 20 ng/mL) treatmentalso induced the g-H2AX focus formation in both the celllines (Fig. 5A). However, MMC-treated cells displayed lessintense staining than resveratrol-treated cells (Fig. 5A). Brca1staining was also examined in both cell lines after resveratroltreatment. Resveratrol (50 mmol/L) treatment for 24 hoursinduced strong nuclear localization of Brca1 as comparedwith untreated control (Fig. 5B). However, Cal27-Smad4cells showed Brca1 focus formation similar to Cal27-pcDNA3 control cells (Fig. 5B). As shown previously (8),Smad4was required for the formation of Brca1 foci byMMC(Fig. 5B). Thus, unlike MMC, resveratrol can produce Brca1foci independently of Smad4.

Resveratrol inhibits FaDu tumor xenograft growthtogether with in vivo DNA damage and apoptoticeffects

We also extended studies to an in vivo xenograft modelto establish biological significance of all our in vitro

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Figure 4. Resveratrol inducesg-H2AX and Brca1 focusformation in HNSCC cells. A, cellswere treated with DMSO alone orresveratrol for 24 hours, total celllysates were prepared andsubjected to SDS-PAGE followedby immunoblotting, andmembranes were probed withg-H2AX and b-actin antibodies asdetailed in Materials andMethods.B, resveratrol induces the g-H2AXand Brca1 focus formation. FaDucells were treated with DMSO(control) or resveratrol (25–50mmol/L) for 24 hours followed byimmunocytochemical staining forg-H2AX and Brca1 by usingspecific antibody, cell imageswere captured at 1,000�magnification on a Nikon DEclipse C1 confocal microscope,and images were analyzed by EZ-C1 Free viewer software. Red andgreen fluorescence representsstaining for g-H2AX and Brca1,respectively. Res, resveratrol;C, control.

Tyagi et al.





findings. Resveratrol feeding by oral gavage resulted in adose- and time-dependent inhibition in tumor growth,and at the end of the study (30 days), tumor volume/mouse decreased from 2.00 � 0.25 in controls to 1.35 �0.12 and 1.01 � 0.17 cm3 in 10 and 50 mg/kg bodyweight resveratrol-fed groups, respectively, accounting for32% and 50% (P < 0.01) inhibition in tumor growth(Fig. 6A). A reduction in tumor weight/mouse furthersupported these results wherein resveratrol-treated groupsshowed 0.95 � 0.15 and 0.76 � 0.13 g/mouse tumorweight, respectively, as compared to controls with 2.00 �0.35 g/mouse tumor weight (Fig. 6A). In contrast, resver-atrol (50 mg/kg body weight) feeding for 15 days had lesseffect that was not statistically significant (Fig. 6A). Inboth efficacy studies, compared with controls, we did notobserve any significant changes in body weight and dietconsumption in resveratrol-fed groups of mice through-out the experiments (data not shown), suggesting that theresveratrol doses and treatment regimens employed inthis study are nontoxic.

The qualitative g-H2AX and cleaved caspase-3 IHC ana-lyses of tumor xenografts for DNA damage and apoptosismarkers, respectively, clearly showed a strong increase instaining in both resveratrol-treated tumors and controls(Fig. 6B). Quantitative nuclear g-H2AX immunostainingshowed 25% (P < 0.001) and 32% (P < 0.001) induction inFaDu tumor xenografts from 10 and 50mg/kg body weightresveratrol-treated mice, respectively, compared with 13%in controls (Fig. 6B, top). Similarly, cleaved caspase-3staining also increased 11% to 16% (P < 0.001) in FaDutumor xenografts from resveratrol-treated mice comparedwith 5% cells in controls (Fig. 6B, bottom).

Discussion

Following DNA damage, normal cells either repair thedamage or undergo apoptotic death mostly in p53-depen-dent manner. However, DNA repair processes are defec-tive in many cancer cells (18) that offer avenues fortherapeutic intervention. Consequently, the present study

Figure 5. Resveratrol-inducedg-H2AX and Brca1 focusformation is independent ofSmad4 in Cal27 cells. Resveratrolinduces g-H2AX (A) and Brca1 (B)focus formation in Cal27-pcDNA3and Cal27-Smad4 cells. Followingindicated treatments of the cells,immunocytochemical staining forg-H2AX and Brca1 was carried outusing specific antibody as detailedin Materials and Methods. Cellimages were captured at 1,000�magnification on a Nikon DEclipse C1 confocal microscopeand analyzed by EZ-C1 Freeviewer software. Red and greenfluorescence represents stainingfor g-H2AX and Brca1,respectively. Res, resveratrol.

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is highly significant, with major therapeutic implicationsin that the anticancer effect of resveratrol is predomi-nately due to its ability to induce DNA damage selectivelyin HNSCC cells without affecting normal, repair-profi-cient cells.

Our detailed mechanistic studies revealed that resvera-trol-induced DNA damage in HNSCC cells results in DNAstrand breaks, as evident by the production of comet tailsand "defective" g-H2AX and Brca1 repair foci resulting inirreversible arrest of HNSCC cells either at the G1 to S-phaseboundary or in S-phase, eventually leading to apoptoticdeath. Similar results have been seen in breast cancer cellslacking Brca2 (19) and in colon cancer cells lacking Cdc14(20) where foci form after DNA damage but are repairedinefficiently. Because p53 is important for DNA repairmechanisms, the novelty of our study is that resveratroleffects were independent of p53 because all 3 HNSCC celllines harbor mutant p53 mutations (21). Furthermore,sensitivity to resveratrol was directly proportional to extentof basal DNA damage. For example, FaDu cells havinghigher level of basal DNA damage (Fig. 1B) were moresensitive to resveratrol than Cal27 cells (Fig. 1A). Similarly,Det562 cells with no detectable g-H2AX even in 50 mmol/Lresveratrol were comparatively more resistant to treatmentthan the other 2 HNSCC cell lines. Because conventionalcancer therapy damages DNA in normal and tumor cells(22), it is of considerable importance that resveratrol doesnot cause DNA damage and checkpoint activation in nor-mal cells with efficient DNA repair pathways, therebyestablishing the selectivity of resveratrol in affectingHNSCC cells.

The TGF-b/Smad signaling pathway is altered bygenetic mutation that contributes to the carcinogenesisof HNSCC (23). Downregulation of the Smad4 tumorsuppressor, the central mediator of TGF-b/Smad signalingpathway is common in malignant HNSCC and correlateswith downregulated expression and an inefficient Fanc/Brca DNA repair pathway (8). Thus, DNA repair defectsoccur in HNSCC in which Smad4 is frequently mutated orpoorly expressed. In this regard, our results are highlysignificant and show resveratrol efficacy in FaDu andCal27 HNSCC cell lines, which both contain null muta-tions in Smad4. To determine whether the anticancereffects of resveratrol by the induction of DNA damageis dependent on Smad4-regulated DNA repair mechan-isms, matching Cal27 and Cal27-Smad4 stable transfec-tants were used. Resveratrol treatment caused comparableDNA damage, "defective" repair foci, cell-cycle arrest, andapoptotic death in both cell lines irrespective of theirSmad4 status, establishing the fact that Smad4 is dispen-sable for the anticancer effects of resveratrol in HNSCC.Perhaps, Smad4 is required to process MMC-induceddamage and form foci (8), whereas it is not required toform the unrepairable foci induced by resveratrol.Together, these results indicate that most of the humanHNSCC tumors will be sensitive to resveratrol, eventhough more than 80% of them are defective in Smad4(24), signifying strong translational potential of ourfindings.

An important aspect of our study is that the in vitroeffects of resveratrol were reproducible in xenograftstudies in both preventive and therapeutic treatment

AControl Control Control10 mg/kg Res

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B

Figure 6. Resveratrol inhibitsFaDu tumor xenograft growthtogether with in vivo DNA damageand apoptotic effects. A, growthinhibitory effects of resveratrol interms of tumor volume as afunction of treatment and tumorweight at study end following theexperiment detailed in Materialsand Methods. In each case, datashown are mean � SE of 8 miceper group. B, IHC analyses forg-H2AX and cleaved caspase-3 inFaDu tumor xenografts from the30 days of resveratrol treatmentstudy as detailed in Materials andMethods. Representative imagesare shown for both staining in allsamples of 3 groups. In each case,quantitative data are mean � SEof 5 tumor samples from 5individual mouse in each groupfrom 5 randomly selectedmicroscopic (magnification: 400�)fields from each tumor. NS, notstatistically significant; Res,resveratrol.

Tyagi et al.





regimens showing an inhibition in FaDu tumor xenograftgrowth in nude mice, which was associated with highlevels DNA damage and a marked induction in apoptosis.Overall, our results clearly show strong resveratrol efficacyagainst the growth and proliferation of HNSCC in vitroand in vivo. Specifically, resveratrol produces these effectson HNSCC via its selective ability to induce DNA damageand apoptotic death. Importantly, the anticancer effectsof resveratrol and all associated mechanisms are inde-pendent of Smad4 status, the mutation/absence of whichis one of the primary causes of failed cellular DNA repairmachinery in HNSCC. On the basis of these findings,more studies are needed in future to evaluate both antic-ancer and chemopreventive efficacy of resveratrol in rele-vant preclinical models to establish its potentialusefulness against human HNSCC.

Disclosure of Potential Conflicts of interest

No potential conflicts of interest were disclosed.

Grant Support

CCTSI (Colorado Clinical and Translational Sciences Institute NIH/NCRR #UL1-RR-025780) co-pilot grant and UCCC (University of ColoradoCancer Center) Aging and Cancer Seed grants (NIH/NCI P20-CA103680)were awarded to R.A. Sclafani. NIH R01 grant AT-003623 was awarded to C.Agarwal. UCCC Core Grant (NIH/NCI P30-CA046934) supported the Corefacilities for FACS analysis.

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 April 21, 2011; revised June 10, 2011; accepted June 16, 2011;published OnlineFirst June 24, 2011.

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2011;17:5402-5411. Published OnlineFirst June 24, 2011.Clin Cancer Res Alpna Tyagi, Mallikarjuna Gu, Takenori Takahata, et al. Squamous Cell Carcinoma

Expression, in Its Efficacy against Human Head and NeckSmad4Resveratrol Selectively Induces DNA Damage, Independent of

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