Resveratrol induces apoptosis involving mitochondrial pathways in mouse skin tumorigenesis

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Life Sciences 82 (20

Resveratrol induces apoptosis involving mitochondrial pathways in mouseskin tumorigenesis

Neetu Kalra 1, Preeti Roy 1, Sahdeo Prasad, Yogeshwer Shukla ⁎

Proteomics Laboratory, Industrial Toxicology Research Centre, P.O. Box 80, M.G. Marg, Lucknow-226001, India

Received 24 July 2007; accepted 3 November 2007

Abstract

Resveratrol, a plant constituent enriched in the skin of grapes, is one of the most promising agents for chemoprevention. In the present study,resveratrol-induced apoptosis in 7, 12-dimethylbenz[a]anthracene (DMBA)-initiated and 12-O-tetradecanoylphorbol-13-acetate (TPA) promoted,mouse skin tumors. The chemopreventive effects of resveratrol in terms of delayed onset of tumorigenesis, cumulative number of tumors andaverage number of tumors/mouse were recorded. Resveratrol treatment resulted in regression of tumors (28%) after withdrawal of the TPAtreatment. Induction of apoptosis by resveratrol in DMBA-TPA induced skin tumors was recorded by the appearance of a sub-G1 fraction (30%)using flow cytometry and an increase in the number of apoptotic cells by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling(TUNEL) assay. Western blot analysis combined with multivariable flow cytometry, showed that resveratrol application induces the expression ofthe p53 and pro-apoptotic Bax, with concomitant decrease in anti-apoptotic protein Bcl-2. Alteration in Bax/Bcl2 ratio by resveratrol treatmentresulted in apoptosis, which was associated with the release of cytochrome c and induction of apoptotic protease-activating factor-1(APAF-1).Further, this effect was found to result in cleaved fragments of caspase-9,-3, and poly (ADP-ribose) polymerase (PARP). These findingsdemonstrate for the first time that resveratrol induces apoptosis through activation of p53 activity in mouse skin tumors, thereupon suggesting itschemopreventive activity, through the modulation of proteins involved in mitochondrial pathway of apoptosis.© 2007 Elsevier Inc. All rights reserved.

Keywords: Chemoprevention; Resveratrol; Mouse skin tumors; Apoptosis; Mitochondrial pathway; APAF-1

Introduction

Resveratrol (trans-3,5,4'-trihydroxystilbene), a well knownanti-oxidant ingredient of red wine, is a phytoalexin present inabout 70 plant species including grape, peanut, mulberry.Resveratrol possesses anti-inflammatory, immunomodulatory,anti-oxidative, cardioprotective and cancer chemopreventiveproperties (Gusman et al., 2001; Dong, 2003). Multiple linesof evidence from preclinical studies suggest that resveratrolhas an ability to intervene multi-stage carcinogenesis (Aziz etal., 2005; Sengottuvelan et al., 2006; Li et al., 2002). A studyon the apoptosis inducing activity of resveratrol in humancancer cells, has brought attention to this molecule not only aspotential chemotherapeutic agent but also signifying its

⁎ Corresponding author. Tel.: +91 522 2620207; fax: +91 522 2628227.E-mail addresses: [email protected], [email protected]

(Y. Shukla).1 Authors contributed equally.

0024-3205/$ - see front matter © 2007 Elsevier Inc. All rights reserved.doi:10.1016/j.lfs.2007.11.006

putative role in the mechanism of action (Pervaiz, 2001).Resveratrol can potentially interfere with all three major stagesof carcinogenesis (i.e., initiation, promotion and progression)where its treatment significantly reduced the tumors in 7,12-dimethylbenz[a]anthracene (DMBA)-initiated and 12-O-tetra-decanoylphorbol-13-acetate (TPA)-promoted skin carcinogen-esis (Jang et al., 1997). Topical application of resveratrolresulted in about 60% reduction in TPA-promoted mouse skinpapilloma formation (Kapadia et al., 2002). The chemopre-ventive properties of resveratrol are not limited only tochemical carcinogens but has also been shown to protectagainst UVB-induced skin tumorigenesis in SKH-1 hairlessmice (Reagan-Shaw et al., 2004; Afaq et al., 2003). Althoughthe mechanistic aspect of chemoprevention with resveratrol hasbeen studied extensively in cultured cell lines (Dong, 2003)and in mouse skin in-vivo (Kundu et al., 2006), yet, details ofmechanisms underlying its anti-tumorigenic effect in DMBA-TPA stimulated tumorigenesis are not clearly elucidated. Thus,in the present study, for the first time we have shown apoptosis

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http://dx.doi.org/10.1016/j.lfs.2007.11.006

349N. Kalra et al. / Life Sciences 82 (2008) 348–358

as a mechanism of anti-tumorigenic effect of resveratrol in 2-stage mouse skin carcinogenesis.

Materials and methods

Materials

DMBA, TPA, purified resveratrol, and antibody specific forβ-actin (clone AC-74) were purchased from Sigma (St Louis,USA). The mouse monoclonal caspase 3, caspase 9, cyto-chrome c, Apaf1, PARP antibody were procured from CellSignaling Technology (Beverly, Massachusetts, USA). Anti-p53 mouse monoclonal antibody specific for wild type protein(clone PAb 1620, ab-5), Bcl-2 (ab-2) rabbit polyclonal IgG andBax (ab-1) rabbit polyclonal IgG antibody were procured fromOncogene Research Products (Cambridge, USA). Mutant p53(clone PAb 240) was obtained from Boerhinger (Mannheim,Germany). The rabbit anti-mouse horseradish peroxidase orgoats anti-rabbit horseradish peroxidase conjugate secondaryantibodies were obtained from Bangalore Genei (Bangalore,India) and the FITC conjugates were procured from BectonDickinson (Franklin Lakes, NJ, USA). The nitrocellulosemembrane was obtained from Millipore, (Bedford, Massachu-setts, USA). The rest of the chemicals were of analytical gradeof purity and were procured locally.

Animals and treatment

Male, Swiss albino mice (10–12 g body weight) were takenfrom Industrial Toxicology Research Centre animal-breedingcolony. The ethical approval for the experiment was obtainedfrom Institutional Animal Ethics Committee. Total number of90 animals were caged in polypropylene cages and housed 15animals per cage on wood chip bedding in an air-conditioned(temperature 23+2 °C, relative humidity 55+5%) animal room.Animals were quarantined for one week on a 12/12 h light–darkcycle and were fed synthetic solid pellet diet [Crude protein24%, ether extract 4%, crude fibre 4%, calcium 1%,phosphorous 0.6%, ash 8%, nitrogen source 50%] (Ashirwad,Chandigarh, India) and water ad libitum. Tumor inductionstudies were carried out as described earlier (Singh and Shukla,1998), in mouse skin model using DMBA as tumor initiator andTPA as promoter. Hair were clipped on the inter-scapular regionusing electric clipper and the mice with hair cycles in the restingphase (telogen) were used for the studies. The animals werekept under observation for food and water intake and tumorgrowth for the entire period of study. Animals from all thegroups were examined every week for gross morphologicalchanges including body weight and development of tumors onthe skin through out the study period.

The animals were equally divided into six groups. Animals ofthe Gr. I (control group) were given no treatment. Animals of theGr. II were topically applied with acetone (200 µl) which wasfollowed 1 h later by single dose of DMBA (52 µg/animal) inacetone (200 µl). A week later TPA (5 µg/animal), was giventhrice a week for a period of 24 weeks. In order to study the anti-initiating effect of resveratrol, animals of Gr. III were topically

applied with resveratrol (50 µM/mouse) in acetone (200 µl) forthree weeks, followed by single dose of DMBA (52 µg/animal)in acetone (200 µl). TPA 5 µg/animal was consecutively giventhrice a week for 24 weeks. Anti-promoting effect of resveratrolwas studied in animals of Gr. IV by giving a single dose ofDMBA (52 µg/mouse) in acetone (200 µl). After one week,resveratrol (50 µM/mouse) was given thrice a week 1 h prior toTPA (5 µg/animal) for 24 weeks. Animals of Gr. V (anti-initiating control) was topically applied with resveratrol (50 µM/mouse) in acetone (200 µl), for three weeks, followed by singledose of acetone (200 µl). A week later TPA (5 µg/mouse) in200 µl acetone was given thrice a week, for 24 weeks. Gr. VI(anti-promoting control) was topically given a single sub-carcinogenic dose of DMBA (52 µg/mouse) in acetone (200 µl),followed by resveratrol (50 µM/mouse) treatment for threeweeks, followed by 1 h acetone treatment (200 µl) for 24 weeks.

At the end of 24 weeks, all the surviving animals werescreened for the cumulative number of tumors and tumorvolume. Tumor volume per mouse was calculated in each groupusing formula V=D×d2 ×π/6 (where D=bigger dimension andd=smaller dimension). By the end of 24 weeks, treatment ofTPA was withdrawn and half of the animals from each groupwere sacrificed. Skin from the painted area (with or withouttumors) was excised, cleaned, snap frozen in liquid nitrogen,and stored at −80 °C until further use for performing flowcytometry, western blotting and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assays.At the end of 36 weeks the remaining animals with tumors werescreened for tumor regression and sacrificed thereafter.

Preparation of cytosolic fraction

The skin/tumor tissue was removed with sharp scalpelblades, and fat was scrapped off, on ice. The tissues were thenhomogenized in lysis buffer (50 mM Tris–HCl, 150 mM NaCl,1 mM EDTA, 20 mMNaF, 100 mM Na3VO4, 0.5% NP-40, 1%Triton X-100, 1 mM PMSF, pH 7.4) with freshly added proteaseinhibitor cocktail (Protease Inhibitor Cocktail Set III; Calbio-chem, La Jolla, CA, USA) in ice for 30 min for total cell lysatepreparation. After incubation, cells were centrifuged at12,000 ×g for 10 min at 4 °C. The supernatant was transferredto cryopreservation tubes and stored at −80 °C in ultra deepfreezer (Revco, USA) until used for further experimentation.

Western blotting

Western blotting was carried out as described previously(Arora et al., 2004). Protein contents were estimated by themethod of Lowry et al. (1951) using bovine serum albumin as astandard. Proteins (50 µg) were resolved on 10% SDS-PAGEand then electroblotted onto nitrocellulose membrane. The blotswere blocked for 1 h with 5% non-fat dry milk and probed withappropriate primary and secondary antibodies at dilutionsrecommended by the suppliers. Immunoblots were detected byhorseradish peroxidase conjugated anti-mouse or anti-rabbit IgGusing chemiluminescence kit of Millipore (Bedford, Massachu-setts, USA) and visualized by Versa Doc Imaging System

Table 1Chemopreventive effects of resveratrol on 2-stage mouse skin tumorigenesis after 24 weeks of treatment

Groups Treatment# Number of animalswith tumors

Ist induction oftumor (in days)

Protection in terms of percentageof tumor free survival

Cumulative numberof tumors

Average tumor/mouse(Mean+SE)

I Untreated 0/28 – – – –II Ace+DMBA+TPA 25/25 42 – 183 7.3+1.8III Res+DMBA+TPA 16/27 74 41%⁎ 76 4.8±0.5IV DMBA+Res+TPA 11/27 76 59%⁎ 50 4.5±0.4V Res+Ace+TPA 0/23 – – – –VI DMBA+Res+Ace 0/23 – – – –# The doses and treatment schedule are described in the Materials and methods section.⁎Shows significant protection by resveratrol in comparison to DMBA+TPA treated group, pb0.05.

Fig. 1. Effect of resveratrol on DMBA-TPA induced mouse skin tumors in termsof (A) reduction in total cumulative number of tumors, (B) induction of tumorfree survival, (C) reduction in tumor volume per mouse (mm3). For the statisticalanalysis of skin tumor appearance dynamics, the Kaplan Meir method of tumorfree survival estimation was applied. Student “t” test was done for multiplecomparisons. ⁎Indicates significant reduction over DMBA-TPA treated Gr. II.⁎pb0.05.

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(BioRad Model 4000). To quantify equal loading, membraneswere probed with β-actin antibody. The intensity was given interms of relative pixel density for each band normalized to bandof β-actin. The intensity of the bands were measured usingsoftware UNSCAN-IT automated digital system version 5.1.

Flow cytometric analysis for proteins

The single cell suspensions of skin/tumor tissue fromdifferent experimental and control groups were prepared usingMedimachine (Becton Dickinson, San Jose, CA, USA) asdescribed earlier (Arora et al., 2004). For the flow cytometricanalysis, cells (1×106) in suspension were taken and fixed inchilled 70% ethanol. After fixation, cells were permeabilizedwith 0.2% Triton X-100 and centrifuged. The pellets wereresuspended in PBS and incubated with respective antibodiesfor 1 h at room temperature. After incubation, the cells weretreated with FITC conjugated isotype-specific secondary anti-body. Cells were then analyzed on flow cell cytometer (BD-LSR II, Becton Dickinson, San Jose, CA, USA) equipped with488 nm argon laser light source. Total 15,000 events wereacquired for analysis using Cell Quest 3.3 Software. Cells wereproperly gated and histogram plot of FITC fluorescence (x axis)versus counts (y axis) was shown in logarithmic fluorescenceintensity.

Flow cytometric analysis for cell cycle distribution

The pellet was resuspended in 50 µl cold PBS and fixed in2 ml of 70% ice-cold ethanol. Cells were centrifuged from thefixative and treated with 0.1% Triton X-100 for 5 min. Afterincubation, cells were again centrifuged and resuspended in1 ml of PBS, ribonuclease (100 µg/ml) was added, and the cellswere incubated at 37 °C for 30 min. After further centrifugation,cells were resuspended in 1 ml of PBS and 50 µg/ml propidiumiodide (PI) and incubated for 30 min at 4 °C. The samples wereacquired and analyzed on flow cell cytometer using Cell Quest3.3 software (Nicoletti et al., 1991).

Terminal deoxynucleotidyl transferase-mediated dUTP nickend labeling (TUNEL) assay

Paraffin embedded sections of skin/tumor tissues werestudied for their apoptotic status by utilizing the TUNEL

assay, which detects fluorescein-12-dUTP labeled 3'-OH endsof genomic DNA as described previously (Arora and Shukla,2002). The apoptotic cells were detected in-situ using Promega

Table 2Tumor regression pattern after deprivation of treatments (upto 36 weeks)

Groups Treatment Number ofanimals withtumors

Total numberof tumors at24 weeks

Total numberof tumors at36 weeks

II ACE+DMBA+TPA 14 92 85 (8%)III Res+DMBA+TPA 8 39 29 (25.6%)⁎

IV DMBA+Res+TPA 6 25 18 (28%)⁎

⁎Shows significant tumor regression in comparison to DMBA+TPA treatedgroup, pb0.05.


Detection System, as per manufacturer instructions. Tissuesections (10 μm thick), were fixed in 4% methanol andpermeabilized with 100 µl of the 20 µg/ml Proteinase K for20 min, and again fixed in 4% methanol free formaldehydesolution. After equilibration, 50 µl of TdT incubation buffercontaining 5 µl nucleotide mixture and 1 µl TdT enzymes/section was added and the slides were then incubated at 37 °C for60 min inside a humid chamber. The reaction was terminated byimmersing the slides in 2×Standard Saline Citrate in a coplin jarfor 15 min and the slides were washed thrice in PBS to removethe un-incorporated fluorescein-12-dUTP. Sections were stainedwith 1 mg/ml PI solution for 15 min at room temperature in darkand washed in deionized water thrice. Apoptotic cells werevisualized immediately under a fluorescence microscope (Leica,Wetzlar, Germany) attached with CCD camera (JVC, Tokyo,Japan) for the incorporation of fluorescein 12-dUTP at the 3'-OH ends of fragmented DNA, resulting in localized greenfluorescence within the nucleus of the apoptotic cells. Theapoptotic index is expressed as the amount of positive staining/lesion and quantitatively assessed by estimation of the numberand intensity of the stained cells/field using Leica image analysissystem in 25 longitudinal sections.

Statistical analysis

For the statistical analysis of skin tumor appearancedynamics, the Kaplan Meir method of tumor free survivalestimation was applied using S-plus software (Insightful,Seattle, WA). Differences between the survival in the groupswere compared by means of a log-rank test (Mantel, 1966).Student's “t” test was performed for comparison of other dataobtained. pb0.05 was considered to be statistically significant.

Results

Effect of resveratrol in 2-stage mouse skin carcinogenesis

The results showed the chemopreventive properties ofresveratrol in 2-stage mouse skin carcinogenesis. A significantdelay in the onset of tumorigenesis was recorded in theanimals supplemented with resveratrol along with DMBA-TPA (Table 1). Tumor development started in the Gr. II after42 days, while tumors started developing after 74 and 76 daysin Gr. III and Gr. IV respectively, which were supplementedwith resveratrol (Table 1). The chemopreventive effect ofresveratrol was also evident in terms of reduction in the

cumulative number of tumors (CNT) and average number oftumors per mouse. The CNT was 76±7.7 and 50±5.6 inresveratrol supplemented Gr. III and Gr. IV respectively, while183 in positive control Gr. II (Fig. 1A) by the end of the24 weeks of treatment. Thus, resveratrol treatment resulted in59% (Gr. III) and 73% (Gr. IV) prevention in CNT. Similarly,in terms of average number of tumors per mouse, comparedwith DMBA-TPA with 7.3±1.8 tumors, topical treatment ofresveratrol resulted in 4.8±0.5 and 4.5±0.4 tumors/mouserespectively in Gr. III and Gr. IV (Table 1). A significant(pb0.05) population of the animals of Gr. III (41%) and Gr.IV (59%) remained tumor free at the end of the 24 weeks ofthe experiment (Fig. 1B). The chemopreventive effects ofresveratrol were also evident in terms of tumor volume amongpositive control and experimental groups. The tumor volumewas 108±12.4 mm3 per mouse in DMBA-TPA group, whileonly 40±6 mm3 per mouse and 35±5 mm3 per mouse inresveratrol supplemented Gr. III and Gr. IV respectively (Fig.1C). Thus, resveratrol supplementation resulted in 63% (Gr.III) and 67.6% (Gr. IV) suppression in tumors volume overGr. II.

We further screened half of the animals for tumor regressionpattern among various groups. About 8% tumors were regressedin DMBA-TPA treated group after withdrawal of the treatment(Table 2) at the end of 36 weeks, while in resveratrolsupplemented groups a much higher magnitude tumor regres-sion was recorded in Gr. III (25%) and Gr. IV (28%) (Table 2).Overall our result shows a chemopreventive effect of resveratrolin mouse skin tumorigenesis.

Flow cytometric analysis for sub-G1 phase

Results of the flow cytometric analysis of skin/tumortissues showed marked increase in the ‘S’ phase population inDMBA-TPA treated mouse skin tumors (Gr. II) (Fig. 2A). Amarked increase in the sub-G1 cells population, characteristicof apoptosis, with concomitant decrease in S phase populationin resveratrol supplemented groups (Gr. III & IV) (Fig. 2A)was recorded. Sub-G1 cell population was found to be 25%and 30% of total cell population in Gr.III and Gr. IVrespectively. No sign of apoptosis could be seen either in theuntreated group (Gr. I) or control groups (Gr. V & Gr. VI)(Fig. 2A).

TUNEL assay

Apoptotic status of skin tumors following resveratroltreatment by TUNEL assay showed that apoptotic index inDMBA-TPA (Gr. II) treated tumors was 0.14±0.08 whichincreased to 0.26±0.2 and 0.27±0.2 in resveratrol treatedgroups (Grs. IV, and VI) (Fig. 2B and C) respectively. Thus,resveratrol causes a significant (pb0.05) increase in Grs. IV(86%) and VI (93%) respectively in the TUNEL positive cells(apoptotic bodies) in comparison to the DMBA-TPA exposedtumors. No significant change in apoptotic index was found inuntreated (Gr. I) and in anti-initiating, anti-promoting controlgroups (Gr. V, Gr. VI) (Fig. 2B and C).


Fig. 3. Effect of resveratrol on the expression of p53 protein. Western blot analysis of wild type-p53 (A), mutant p53 (B) and an overlay of flow cytometric analysis ofprotein expression wild type-p53 (C), mutant p53 (D) in mouse skin/tumor. For western blot analysis data is represented as the relative density of protein bandsnormalized to β-actin. ⁎⁎Shows significant increase over untreated Gr. I, ⁎shows significant decrease over Gr. II, pb0.05. For flow cytometric analysis a total of15,000 events were counted on argon 488 lasers. Result given is in terms of FITC fluorescence (x axis) versus counts (y axis). Data shown is the result of three differentexperiments with similar results. Gr. I: untreated, Gr. II: ACE+DMBA+TPA, Gr. III: R+DMBA+TPA, Gr. IV: DMBA+R+TPA, Gr. V: ACE+R+TPA, Gr. VI:DMBA+R+TPA.


Effect of resveratrol on p53 expression

Results of the western blot analysis showed no change in theexpression of tumor suppressor protein, p53, following topicaltreatment of DMBA-TPA (Fig. 3A). However, application ofresveratrol resulted in significant up-regulation (pb0.05) ofprotein expression in both Gr. III and Gr. IV (Fig. 3A). No effecton the expression of the p53 protein was seen in Gr. V and Gr.VI. However, an opposite trend could be observed in case ofmutant-p53 expression. Expression of mutant-p53 protein wasenhanced significantly in DMBA-TPA treated group (Gr. II)over untreated control (Gr. I). On the other hand resveratrol

Fig. 2. Effect of resveratrol on DMBA-TPA induced mouse skin tumors in terms of indin different phases of cell cycle, (B) Apoptotic cells are visualized under a fluorescTokyo, Japan) for the incorporation of fluorescein 12-dUTP at the 3'-OH ends of fragApoptotic index in different groups as measured by TUNEL assay. Data shown is the mapoptotic index over DMBA-TPA treated Gr. II. ⁎pb0.05. Gr. I: untreated, Gr. II: AACE+R+TPA, Gr. VI: DMBA+R+TPA.

supplementation resulted in decreased of mutant-p53 expres-sion in both Gr. III and Gr. IV in comparison to DMBA-TPAtreated group (Gr. II) (Fig. 3B). Analysis of wild type-p53 levelsamong various groups by flow cytometry further lend support toresults obtained by western blotting. The flow cytometricanalysis of wild type-p53 in skin/tumors from various groupsrevealed shifting of the peak towards higher fluorescence asmean fluorescence intensity (MFI) value of 16.4±3.1 and13.2±2.4 in resveratrol-supplemented groups (Gr. III & IV,respectively) over 5.31±1.0 for Gr. II (DMBA+TPA) (Fig.3C). An increase in the MFI for mutant p53 was apparentfrom 1.4±0.6 for untreated group (Gr. I) to 17.4±2.4 for

uction of apoptosis (A) Flow cytometric analysis to show the distribution of cellsence microscope (Leica, Wetzlar, Germany) attached with CCD camera (JVC,mented DNA, resulting in localized green fluorescence of the apoptotic cells. (C)ean of three different experiments with similar results. ⁎Significant induction in

CE+DMBA+TPA, Gr. III: R+DMBA+TPA, Gr. IV: DMBA+R+TPA, Gr. V:


DMBA-TPA induced tumor cells (Gr. II) (Fig. 3D). Thisincrease in the MFI by DMBA-TPA recorded an inhibitorytrend in resveratrol-supplemented groups as observed to be6.1±1.0 in Gr. III and 4.7±0.9 in Gr. IV (Fig. 3D).

Resveratrol administration downregulates the expression ofBcl-2 and upregulates the expression of Bax protein

Western blot analysis revealed that topical administrationof DMBA-TPA treatment resulted in down-regulation of Bax,(Fig. 4A) with concomitant increase in the Bcl-2 expression(pb0.05) (Fig. 4B) over untreated control (Gr. I). However,resveratrol supplementation up-regulated the expression ofBax, (Fig. 4A) and down-regulated the expression of Bcl-2,(Fig. 4B) in both Gr. III and Gr. IV. The increase in the ratioof Bax/Bcl-2 (Fig. 4C) in skin tumors suggested thesusceptibility of tumor cells to undergo apoptosis, and thismay be the reason for tumor suppression in resveratrol-treatedmice. Flow cytometric analysis of the cells from the skin ofvarious groups, further confirmed the observations, as shownby the shift in MFI for Bax from 8.3±1.2 for Gr. I (untreated)to 4.3±1.0 for Gr. II (DMBA+TPA). This shift in MFI was,however, magnified in resveratrol supplemented groups to46.5±4.7 and 53.4±5.6 for Gr. III and Gr. IV, respectively(Fig. 4D). In case of Bcl-2, MFI was 3.6±0.9 for untreatedGr. I. Clear shift towards right showed an increased expres-sion in DMBA+TPA treated Gr. II with MFI of 53.1±5.4.This increase in the MFI by DMBA+TPA was restored in

Fig. 4. Effect of resveratrol on the expression of Bcl-2 family proteins. Western blcytometric analysis of protein expression Bax (D), Bcl-2 (E) in mouse skin/tumor. Fonormalized to β-actin. ⁎⁎Shows significant decrease over untreated Gr. I, ⁎shows s15,000 events were counted on argon 488 laser. Result given is in terms of FITC fluorexperiments with similar results. Gr. I: untreated, Gr. II: ACE+DMBA+TPA, Gr. IDMBA+R+TPA.

resveratrol-supplemented groups to 8.5±1.3 in Gr. III and 7.6±0.8 in Gr. IV (Fig. 4E).

Resveratrol treatment induces mitochondrial disruption andthus release of cytochrome c, induction of Apaf-1, and cleavageof caspase-9, caspase-3 and PARP

In mitochondrial pathway of apoptosis, a number of signalscan cause changes in mitochondrial membrane potential andmitochondrial permeability transition resulting in the release ofcytochrome c. The release of cytochrome c from themitochondria is facilitated by Bax and blocked by Bcl-2(Kluck et al., 1997; Budihardjo et al., 1999). Therefore, westudied the role of resveratrol on the expression of proteinsinvolved in the mitochondrial pathway. The results showed thatDMBA-TPA treatment resulted in down-regulation of the levelof Apaf-1 (Fig. 5A), release of cytochrome c (Fig. 5B) and hadno effect on caspases cleavage. However, resveratrol treatmentresulted in marked increase in cytochrome c release (Fig. 5A),induction of Apaf-1 (Fig. 5B), and cleavage of caspase 9 (Fig.5C), caspase 3 (Fig. 5D) and PARP (Fig. 5E) in both Gr. III andGr. IV. The cleaved caspase 3 (19 and 17 kDa) and PARP (116,85, and 62 kDa) are the characteristic hallmarks of apoptosis(Decker et al., 2000) which were observed in this system. Flowcytometric analysis of the cells from various groups furtherconfirmed the observations, as shown by these shift in MFI forcytochrome c from 10.3±2.0 for Gr. I (untreated) to 3.3±0.8 forGr. II (DMBA+TPA). This decrease in MFI was however,

ot analysis of Bax (A), Bcl-2 (B), Bax/Bcl-2 ratio (C) and an overlay of flowr western blot analysis data is represented as the relative density of protein bandsignificant increase over Gr. II, pb0.05. For flow cytometric analysis a total ofescence (x axis) versus counts (y axis). Data shown is the result of three differentII: R+DMBA+TPA, Gr. IV: DMBA+R+TPA, Gr. V: ACE+R+TPA, Gr. VI:

Fig. 5. Effect of resveratrol on the expression proteins involved in mitochondrial pathway. Western blot analysis of cytochrome c (A), Apaf 1(B), caspase 9(C), caspase3(D) and PARP (E) and an overlay of flow cytometric analysis of protein expression cytochrome c (F), Apaf 1 (G) in mouse skin/tumor. For western blot analysis datais represented as the relative density of protein bands normalized to β-actin. ⁎⁎Shows significant decrease over untreated Gr. I, ⁎shows significant increase over Gr. II,pb0.05. For flow cytometric analysis a total of 15,000 events were counted on argon 488 laser. Result given is in terms of FITC fluorescence (x axis) versus counts (yaxis). Data shown is the result of three different experiments with similar results. Gr. I: untreated, Gr. II: ACE+DMBA+TPA, Gr. III: R+DMBA+TPA, Gr. IV:DMBA+R+TPA, Gr. V: ACE+R+TPA, Gr. VI: DMBA+R+TPA.


intensified in resveratrol supplemented groups to 50.5±6.1 and55.4±6.4 for Gr. III and Gr. IV, respectively (Fig. 5F). In case ofApaf1, MFI was 8.6±2.4 for untreated Gr. I. It was decreased to4.2±1.4 in Gr. II (DMBA+TPA). Conversely, resveratrolsupplementation resulted in increased level of Apaf1 as

indicated by shift in the peak towards right (Fig. 5G). It was60.1±7.6 and 65.2±8.1 in Gr. III and Gr. IV respectively (Fig.5G). These observations further show the involvement ofmitochondrial pathway in resveratrol-induced apoptosis intumor cells.

Fig. 6. Possible mechanism of protection by resveratrol in 2-stage mouse skintumorigenesis model. Indicate increase in expression of protein. Indicatedecrease in expression of protein.


Discussion

Functional abnormalities of intracellular signaling networkcauses the disruption in homeostasis maintained by criticalcellular components, thereby accelerating premalignant andmalignant transformation (Kundu et al., 2006). Chemopreven-tive agents plays a decisive role in induction of apoptosis bydisrupting mitochondrial functions which lead to apoptoticdeath of tumor cells (Baliga et al., 2005). Our results showedthat resveratrol effectively prevents development of DMBA-TPA induced mouse skin tumors, through induction ofapoptosis, characterized by induction of cytochrome c release,expression of Bax and inhibition of Bcl-2.

Growing evidence from both in-vitro and in-vivo studiesdemonstrate that suppression of apoptosis is involved in tumorpromotion by chemical agents (Huang et al., 1999). The lack ofa p53-dependent apoptotic response in papillomas suggestsother functions of p53 besides apoptosis, such as their role in G1cell cycle arrest or differentiation, which are more critical for thesuppression of malignant progression by p53 (Kemp et al.,2001). It has been shown that DNA damage caused by apolycyclic aromatic hydrocarbon, benzo[a]pyrene (BaP)induces p53 protein as a protective measure to eliminate thepossibility of fixation of the DNA damage. However, TPAinhibits p53 response induced by B(a)P and causes tumorpromotion (Mukherjee and Sikka, 2006). We also demonstratedthat DMBA-TPA treatment resulted in down-regulation of theexpression of p53 (Fig. 3A), whereas resveratrol treatment leadsto bring back the near normal levels of p53. The expression ofp53 was up-regulated due to DNA damage by mutagen in in-vitro and in-vivo. p53 is reported to be up-regulated in the cellsby its increased half-life through inhibition of its degradationtime (Harris, 1996; Colman et al., 2000), as well as modulationof its stability by post-translational events such as phosphoryla-tion and acetylation (Kapoor and Lozano, 1998; Oshiro et al.,2003; Fraser et al., 2006). Therefore, it is likely that up-regulation of p53 by resveratrol can occur via a similar pathway.Since, the balance between wild type-p53 and mutant-p53determines the fate of the cell; many chemopreventive agentsare known to exert their anti-cancer effects by modulating theirexpression levels (Arora et al., 2004). The results of the presentstudy showed up-regulation of wild type-p53, and down-regulation of the mutant-p53 expression (Fig. 3B) by theresveratrol treatment which is in accordance with the previousreports where natural and dietary compounds have been shownto exert their preventive property through modulating thebalance between wild type- and mutant-p53 protein expression(Arora et al., 2004).

Apoptosis is regulated by anti-apoptotic and pro-apoptoticeffectors, which involve a large number of biomolecules.Therefore, to have an insight of mechanisms involved inapoptosis, we looked at the role of resveratrol on pro-apoptoticand anti-apoptotic proteins of the Bcl-2 family. The members ofBcl-2 family play an important role in induction of apoptosisand are considered as a target for anti-cancer therapy (Baell andHuang, 2002; Goodsell, 2002). Bcl-2, an oncoprotein, functionsas a suppressor of apoptosis, and its down regulation causes

tumor regression (Kluck et al., 1997, Sedlak et al., 1995). Onthe other hand, predominance of Bax, a proapoptotic proteinover Bcl-2 promotes apoptosis (Wolter et al., 1997). Studieshave also shown that the ratio of Bax to Bcl-2 proteins increasesduring apoptosis (Green and Reed, 1998; Thornberry andLazebnik, 1998). Resveratrol treatment increased the ratio ofBax/Bcl-2, by enhancing the expression of Bax (Fig. 4A), andsuppressing the expression of Bcl-2 (Fig. 4B). Thus, our studyconfirms the involvement of these proteins in the induction ofapoptosis by resveratrol in mouse skin tumorigenesis.

The mitochondrion is a prominent participant in apoptosisand Bax plays an essential role for onset of mitochondrialdysfunction (Green and Reed, 1998). The intracellular move-ment of Bax induces release of cytochrome c through openingsin the outer membrane, formed as a consequence of transition ofpermeability and loss of mitochondrial membrane potential(Thornberry and Lazebnik, 1998). The released cytochrome cforms an “apoptosome” of Apaf-1, cytochrome c, and caspase-9, which subsequently cleaves the effector caspase 3 (Thorn-berry and Lazebnik, 1998). In our in-vivo system, resveratrolincreased the levels of cytochrome c, the adaptor Apaf-1,cleaved caspase 9, and caspase 3. The activated caspase 3 is thekey executioner of cell apoptosis. Activated caspase 3 cleavesintracellular proteins vital to cell survival and growth, such asPARP, a marker of apoptosis (Darmon et al., 1995). The releaseof cytochrome c also induces fragmentation of DNA (Karmakaret al., 2007). From these results, it can be inferred that PARPcleavage (Fig. 5E) is very prominent for causing apoptosis in


mouse skin tumors. Our findings also show the involvement ofDNA damage and PARP cleavage as a mechanism of inductionof apoptosis in resveratrol supplemented groups. Earlier, it wasshown that resveratrol induces apoptosis in JB6 P+ mouseepidermal cell line C1 41 through activation of p53 activity,suggesting that its anti-tumor activity might have occurredthrough the induction of apoptosis (Kemp et al., 2001). Further,it was shown that both ERKs and p38 kinase mediateresveratrol-induced activation of p53 and apoptosis throughphosphorylation of p53 at serine 15 in JB6 epidermal cell line(She et al., 2001).

Thus, the results of the present study validate our convictionthat resveratrol inhibits tumor development through inductionof apoptosis via mitochondrial cell death pathway (summarizedin Fig. 6). These observations hold promise for further in-vitroand molecular target oriented studies to examine the chemo-preventive efficacy of resveratrol as a potential chemother-apeutic agent.

Acknowledgments

Authors are thankful to Dr. Ashwani Kumar, DirectorIndustrial Toxicology Research Centre, Lucknow for his keeninterest in the study. Authors are also thankful to Indian Councilof Medical Research (India) for providing senior fellowship toMs. Neetu Kalra and to Mr. Sahdeo Prasad.

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Resveratrol induces apoptosis involving mitochondrial pathways in mouse skin tumorigenesis