Chemoprevention of skin cancer by grape constituent resveratrol: relevance to human disease?

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1 The FASEB Journal express article /fj fje. Published online April 18, Chemoprevention of skin cancer by grape constituent resveratrol: relevance to human disease? Moammir Hasan Aziz,* Shannon Reagan-Shaw,* Jianqiang Wu,* B. Jack Longley,*, and Nihal Ahmad*,, *Department of Dermatology; University of Wisconsin Comprehensive Cancer Center; Molecular and Environmental Toxicology Center, University of Wisconsin, Madison, Wisconsin 2005 FASEB Corresponding author: Nihal Ahmad, Department of Dermatology, University of Wisconsin, Medical Science Center, 1300 University Ave., Madison, WI, ABSTRACT According to the World Cancer Report, skin cancer constitutes ~30% of all newly diagnosed cancers in the world, and solar ultraviolet (UV) radiation (particularly, its UVB component; nm) is an established cause of ~90% of skin cancers. The available options have proven to be inadequate for the management of skin cancers. Therefore, there is an urgent need to develop mechanism-based novel approaches for prevention/therapy of skin cancer. In this study, we evaluated the chemopreventive effects of resveratrol against UVB radiation-mediated skin tumorigenesis in the SKH-1 hairless mouse model. For our studies, we used a UVB initiationpromotion protocol in which the control mice were subjected to chronic UVB exposure (180 mj/cm 2, twice weekly, for 28 weeks). The experimental animals received either a pretreatment (30 min before each UVB) or post-treatment (5 min after UVB) of resveratrol (25 or 50 micro mole/0.2 ml acetone/mouse). The mice were followed for skin tumorigenesis and were killed at 24 h after the last UVB exposure, for further studies. The topical application of skin with resveratrol (both pre- and post- treatment) resulted in a highly significant 1) inhibition in tumor incidence, and 2) delay in the onset of tumorigenesis. Interestingly, the post-treatment of resveratrol was found to impart equal protection than the pretreatment; suggesting that resveratrol-mediated responses may not be sunscreen effects. Because Survivin is a critical regulator of survival/death of cells, and its overexpression has been implicated in several cancers, we evaluated its involvement in chemoprevention of UVB-mediated skin carcinogenesis by resveratrol. Our data demonstrated a significant 1) up-regulation of Survivin (both at proteinand mrna- levels), 2) up-regulation of phospho-survivin protein, and 3) down-regulation of proapoptotic Smac/DIABLO protein in skin tumors; whereas treatment with resveratrol resulted in the attenuation of these responses. Our study also suggests that resveratrol enhanced apoptosis in UVB-exposure-mediated skin tumors. Our study, for the first time, demonstrated that 1) resveratrol imparts strong chemopreventive effects against UVB exposure-mediated skin carcinogenesis (relevant to human skin cancers), and 2) the chemopreventive effects of resveratrol may, at least in part, be mediated via modulations in Survivin and other associated events. On the basis of our work, it is conceivable to design resveratrol-containing emollient or Page 1 of 18

2 patch, as well as sunscreen and skin-care products for prevention of skin cancer and other conditions, which are believed to be caused by UV radiation. Key words: ultraviolet photocarcinogenesis Survivin A ccording to the World Cancer Report, skin cancer constitutes ~30% of all newly diagnosed cancers in the world, and solar ultraviolet (UV) radiation (particularly, its UVB component; nm) is an established cause of ~90% of skin cancers (1, 2). UVB functions as a complete carcinogen and plays a critical role in the initiation, promotion, and progression phases of the carcinogenesis process (3 9). The two most frequent types of skin cancer, collectively known as nonmelanoma skin cancer, are basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Exposure to UV radiation is also regarded as the major causative factor for the most invasive melanoma, as well as the precancerous conditions such as actinic keratosis (AK) (4, 8 12). It also elicits a variety of other adverse effects, including erythema, sunburn cells, inflammation, hyperplasia, hyperpigmentation, immunosuppression, and premature skin aging (5, 8, 9, 12 14). Thus, a strategy aimed at preventing or protecting from UVB-mediated cellular damages could be a useful approach for the management of skin cancer and other skin disorders, including skin aging. This is particularly important because the available options have proven to be inadequate for the management of skin cancer and other skin conditions caused by UVB radiation. Thus, novel mechanism-based approaches for management of skin cancer and other adverse effects of UVB radiation should be pursued. Chemoprevention, a newer dimension in the management of neoplasia (or other diseases), could offer hope in this direction (6, 12 20). In recent years, chemoprevention is being increasingly appreciated as a plausible strategy for the management of a variety of neoplasm, including skin cancer (17, 19 33). Trans-resveratrol (trans-3,4',5-trihydroxystilbene), a relatively new chemopreventive agent amply presents in red wine and grapes, is believed to be responsible for the much-acclaimed phenomenon of the French Paradox (17, 34, 35). Resveratrol has shown remarkable cancer chemopreventive effects in a variety of tumor bioassay models (17, 19, 20, 28, 29, 32, 34 37). Jang et al. (19), for the first time, defined the cancer chemopreventive activity of resveratrol in assays representing the three major stages of carcinogenesis. This landmark study also demonstrated that resveratrol inhibited chemically induced skin tumorigenesis in a mouse model (19). However, this mouse model possesses only limited relevance to human skin cancers, as solar UV radiation (rather than chemicals) is believed to be the major contributor to the development of skin cancers in humans. On the basis of these facts, we designed this study to define the skin cancer chemopreventive effects of resveratrol in a photocarcinogenesis model of skin cancer that closely resembles human situations. Further, we investigated our hypothesis that the skin cancer-chemopreventive effects of resveratrol are mediated via modulations in the expression and function of Survivin, a member of the inhibitor of apoptosis (IAP) gene family. The rationale for this hypothesis is based on recent studies that have shown that apoptosis inhibition by Survivin might participate in the onset and progression of BCC and SCC, as well as melanoma, and suggested that therapeutic targeting of Survivin could be beneficial in patients with recurrent or advanced disease (38 41). Further, our recent study has suggested the involvement of Survivin (and the associated events) during short-term UVB exposure-mediated responses in skin in vitro and in vivo (42). Page 2 of 18

3 The present study demonstrated that resveratrol imparts significant protection against UVB exposure-mediated skin carcinogenesis in SKH-1 hairless mice. Our data also suggested that the chemopreventive effects of resveratrol may, at least in part, be mediated via modulations in Survivin and associated events. MATERIALS AND METHODS Reagents The antibodies against Survivin, phospho-survivin, and Smac/DIABLO were purchased from Novus Biologicals, Inc. (Littleton, CO), and the β-actin antibody was obtained from Sigma (St. Louis, MO). The M30 CytoDEATH kit (M30 monoclonal antibody) was purchased from Roche Molecular Biochemicals, (Indianapolis, IN). The BCA protein assay reagent kit was obtained from Pierce (Rockford, IL). Survivin probe and primers were purchased from Applied Biosystems (Foster City, CA). The TRIZOL reagent was purchased from GIBCOBRL Life Technologies (Grand Island, NY). The chemiluminescence detection kit was obtained from Pierce. All the other chemicals were obtained in the purest form commercially available. UVB source and animal exposure For UVB irradiation, we used a Daavlin Research Irradiator obtained from Daavlin Company (Bryan, OH). This equipment consists of a fixture mounted on fixed legs and contains four UVA and four UVB lamps. The exposure system is controlled using two Daavlin Flex Control Integrating Dosimeters. The dose units, in this equipment, could be entered as mj/cm 2 (for UVB) or Joules (for UVA). For accuracy, the machine is periodically calibrated using the International Light IL 1400, digital light meter (Daavlin, Bryan, OH). For our experiments, female SKH-1 hairless mice (6 weeks old) were divided into seven groups of eight animals each (except in UVB alone group, where 12 animals were used). The mice in the first group received a topical application of 200 µl acetone alone, and those in the second group received 50 µm resveratrol (topically in 200 µl acetone per mouse). The third group of mice received a topical application of 200 µl acetone before UVB (180 mj/cm 2 ) exposure twice a week for 28 weeks. The mice in the fourth and fifth groups received a topical application of resveratrol (25 or 50 µm/200 µl acetone/mouse respectively), 30 min before UVB (180 mj/cm 2 ) exposure for 28 weeks (twice a week). The animals in the sixth and seventh groups were exposed to UVB (180 mj/cm 2 ), and immediately following UVB irradiation, they received 25 or 50 µm resveratrol applied topically (in 200 µl acetone per mouse) for 28 weeks (twice a week). The mice were closely followed for skin tumorigenesis and were killed at 24 h after the last UVB exposure, for further studies. The selection of the dose of resveratrol is based on our earlier studies where similar doses of this antioxidant were found to impart protection from short-term UVB exposure-mediated cutaneous damages in SKH-l hairless mice (30, 36). It is also important to mention here that the incidence of UVB-induced tumors in our study was much lower than observed by other authors (43, 44). The reason for this difference is that in our study, we subjected the animals to a twice weekly exposure of UVB (180 mj/cm 2 ); whereas other studies have used a 3 times per week exposure of UVB (180 mj/cm 2 ). Page 3 of 18

4 Histophathology For histopathological evaluation of the tumors and/or skin, the tissue was removed, fixed in 10% formalin, and embedded in paraffin. Tissue sections (5 µm) were cut, mounted on a glass slide, and stained with hematoxylin and eosin. Preparation of skin epidermal lysate Epidermis from the whole skin was separated as described earlier (30, 45). The epidermis (from control animals) or the tumors (pooled from single animals) were homogenized in ice-cold lysis buffer (in mm): 50 Tris-HCl, 150 NaCl, 1 EGTA, 1 EDTA, 20 NaF, 100 Na 3 VO 4, 1 PMSF (ph 7.4), as well as 0.5% NP-40, 1% Triton X-100, with freshly added protease inhibitor cocktail (Protease Inhibitor Cocktail Set III; Calbiochem, La Jolla, CA). The homogenate was then centrifuged at 13,000 g for 25 min at 4 C, and the supernatant (total cell lysate) was collected, aliquoted, and stored at 80 C. The protein content in the lysates was measured by the BCA assay (Pierce, Rockford, IL), as per the manufacturer's protocol. Immunoblot analysis For Western blot analysis, the tissue lysate (25 µg protein) was subjected to 12-15% Tris-glycine SDS-PAGE and transferred onto a nitrocellulose membrane. The nonspecific sites were blocked by incubating the membrane with 5% nonfat dry milk in buffer (10 mm Tris, 100 mm NaCl, 0.1% Tween-20, ph 7.6) for 1 h at room temperature, followed by incubation with the appropriate monoclonal primary antibody in the blocking buffer for overnight at 4 C. The antibody was used at dilution specified by the manufacturer. The blot was washed for 2 10 min and then incubated with the corresponding secondary antibody conjugated to horseradish peroxidase (Upstate, Lake Placid, NY) at 1:10,000 dilution for 1 h at room temperature. The blot was washed for 3 10 min, and the protein expression was detected by enhanced chemiluminescence (Pierce, Rockford, IL). Immunoblots were sequentially stripped of antibodies by incubation for 15 min at 37 C with Restore Western Blot Stripping Buffer (Pierce), blocked again, and reprobed with β-actin antibody to confirm the equal loading of proteins in each well. The quantification of protein was performed by densitometric analysis of the protein bands (as scanned TIFF images) using Scion Image Software (Scion Corp., Frederick, MD). Quantitative real-time PCR For determining the effect of treatments on gene expression analyses by real-time PCR, total RNA was isolated from the epidermis (from control animals) or the tumors (pooled from single animals) using the TRIZOL reagent (Invitrogen Corporation, Carlsbad, CA). The tissue was then homogenized with TRIzol (0.75 ml TRIzol per mg of tissue) in a glass-teflon homogeziner, and RNA was extracted and purified as per the manufacturer s protocol. The quality of RNA was checked by the ratio of optical densities of the RNA samples at 260 and 280 nm, which was consistently >1.8. From the RNA samples, the cdna was synthesized by reverse transcription of 1 µg of RNA with 200 U MMLV-reverse transcriptase (Promega, Madison, WI) in the presence of 1 µg random hexamer primers and 0.5 mm dntps (Promega). In the Survivin cdna-specific realtime quantitative PCR assay, the reaction mixture contained 100 µm of Survivin-forward and Page 4 of 18

5 100 µm of Survivin-reverse primers, 100 µm TaqMan probe, 12.5 µl TaqMan Universal PCR Master Mix (Applied Biosystems), and 1 µl of cdna, in a total volume of 25 µl. After enzyme activation for 10 min at 95 C, 50 two-step cycles were performed (20 s at 95 C and 60 s at 60 C) by ABI PRISM 7000 Sequence Detection System (Applied Biosystems). Mouse Survivin primers and TaqMan probe used were as follows: forward primer, 5 - GCGGAGGCTGGCTTCA-3 ; reverse primer, 5 -AGAAAAAACACTGGGCCAAATC-3 ; TaqMan Survivin probe, 5 -VIC-CCACTGCCCTACCGAGGACGAGCCMGBNFQ -3 (Applied Biosystems; Foster City, CA). Immunohistochemical analysis The induction of apoptosis in the skin was determined using M30 monoclonal antibody with reagents from the M30 CytoDEATH kit, as suggested by the manufacturer. Briefly, paraffinembedded tissue sections (5-µm thickness) were placed on silane-coated (Sigma Chemicals) glass slides, air dried overnight, and rehydrated with xylene and graded alcohol. The sections were then incubated with 3% H 2 O 2 in methanol for quenching the endogenous peroxidase. Antigen retrieval was performed by boiling the sections in citric acid buffer (ph 6.0) in a microwave oven (at 750 W) until boiling, followed by heating at low power (100 W) for 15 min. The samples were allowed to cool to room temperature and rinsed with PBS (2 min 3 times). The nonspecific binding sites were blocked by incubating with 1% BSA (in PBS, 0.1% Tween- 20) for 10 min, followed by incubation with appropriate primary antibodies for 1 h at 25 C in a humid chamber. The slides were rinsed in washing buffer (5 min 3 times) and incubated with streptavidin-pod for 30 min at C in a humid chamber. The sections were thoroughly washed with washing buffer followed by an incubation (at 25 C for 3 min) with a freshly prepared substrate solution (diaminobenzidine tetrahydrochloride with hydrogen peroxide, and copper sulfate), for the enhancement of color. The sections were then washed with water and counterstained with methyl green or eosin for 5 min, followed by microscopic analysis. Statistical analysis A Kaplan-Meier analysis was used to measure tumor incidence as a function of tumor-free survival using S-plus Software (Insightful; Seattle, WA). A log-rank analysis was used to determine whether the mean number of weeks tumor-free (as determined by the Kaplan-Meier analysis) was significant. A linear regression analysis was used to measure the rate of tumor multiplicity. A P value of >0.01 was considered to be statistically significant. S-plus Software (Insightful) was used to perform all the statistical tests. RESULTS AND DISCUSSION Chemoprevention by naturally occurring agents is gaining the attention of scientists and consumers as a plausible approach for the management of neoplasia (including skin cancer), as well as for certain precancerous conditions. Resveratrol, a naturally occurring antioxidant phytoalexin produced by some plants in response to injury or fungal infection, has shown cancer chemopreventive effects in a variety of tumor bioassay systems (17). Resveratrol has been shown to prevent chemical carcinogenesis in a mouse model (19). However, except for industrial occupational exposure, chemical skin carcinogenesis has little relevance, if any, to human situations as UV radiation is the established cause of most of the skin cancers diagnosed in humans (1, 2). Earlier studies from this laboratory have shown that resveratrol imparts protection Page 5 of 18

6 from short-term UVB exposure-mediated cutaneous damages in SKH-l hairless mice (30, 36). This study was designed to evaluate the skin cancer chemopreventative effects of resveratrol against chronic long-term UVB exposure in SKH-1 hairless mice and the mechanisms associated with the observed chemopreventive effects of resveratrol. It is important to mention here that the SKH-1 hairless mouse model of photocarcinogenesis is considered to mimic the development of skin carcinogenesis in humans (7). The control SKH-1 hairless mice were subjected to chronic UVB exposure (180 mj/cm 2, twice weekly) for 28 weeks; and this treatment regimen resulted in a variety of skin tumors. To study the skin cancer chemoprevention by resveratrol, we employed two different protocols. In the first set of animals, resveratrol (25 or 50 µm/0.2 ml acetone/mouse) was topically applied to the mouse skin 30 minutes before each UVB exposure. In the second set of animals, the mice were exposed to UVB radiation followed (5 min later) by topical application of similar amount of resveratrol. The later protocol (postapplication of resveratrol following UVB) was used to rule out the possibility that resveratrol might be acting solely as a sunscreen. Thus, in this protocol, since resveratrol was applied when UVB radiations have already penetrated the skin, the observed effect does not appear to be a sunscreen effect. Our data clearly demonstrated that the topical application of mouse skin with resveratrol (both pretreatment and post-treatment) resulted in a highly significant inhibition in the tumor incidence (% mice with tumors) as examined by Kaplan-Meier Analysis (Fig. 1). Interestingly, resveratrol treatment also resulted in a significant delay in the onset of tumorigenesis as shown by the Log- Rank analysis (Fig. 1). Resveratrol treatment significantly reduced tumor multiplicity (tumors per mouse), measured as a function of time, compared with UVB alone group (Fig. 1). Interestingly, the post-treatment of resveratrol was found to impart equal, if not better, protection than the pretreatment. This clearly suggests that the observed effects of resveratrol are not through a sunscreen mechanism; rather, it is absorbed in the skin and activates a protective signaling cascade. A close histological examination of the tumors and skin of the animals, by a board-certified dermatopathologist (BJL), revealed that UVB exposure resulted in a variety of tumors, including squamous cell carcinoma, Bowen s disease, invasive carcinomas in situ, and actinic keratoses (Table 1). However, in resveratrol-treated group (both pre- and post- treatment), a majority (in terms of percent) of the lesions were identified to be actinic keratoses with or without inflammation (Table 1). This is an important observation which suggested that resveratrol might be inhibiting the malignant conversion of premalignant conditions such as actinic keratoses. However, many detailed studies are needed to firmly establish this hypothesis. Sunlight-induced cancer develops as a result of a complex cascade of events initiated by chromosomal alterations and mutations via direct DNA damage and/or production of reactive oxygen species (4, 8, 9). Because Survivin is a critical regulator of survival/death of cells and its overexpression has been implicated in several cancers (46 50), we evaluated the involvement of Survivin during chemoprevention of UVB-mediated skin carcinogenesis by resveratrol. The Western blot and real-time-pcr analyses demonstrated an appreciable overexpression in the levels of Survivin in skin tumors (at protein- and mrna- levels), whereas the treatment of skin with resveratrol resulted in a downmodulation in protein and mrna levels of Survivin (Fig. 2). Survivin is related to the baculovirus iap gene that is selectively expressed in most of the common types of human cancers (47 51). Survivin has been shown to inhibit apoptosis and its expression may be cell-cycle regulated (associated with microtubules during mitosis) (47 51). Conversely, the down-regulation of Survivin by antisense oligonucleotides induces apoptosis in vitro (52). Page 6 of 18

7 Survivin expression in tumors has been associated with increased aggressiveness and decreased patient survival (47 51). Studies have shown that inhibition of melonoma tumor growth in vivo by deregulation of Survivin or with chemotherapy may be useful in the treatment of melanoma (53). Studies have also demonstrated that apoptosis inhibition by Survivin may participate in the onset and progression of both BCC and SCC, and suggest that therapeutic targeting of Survivin may be beneficial in patients with recurrent or advanced disease (39, 41). Deregulation of Survivin was suggested to influence both epidermal homeostasis and the development of melanoma and nonmelanoma skin cancer (54). Importantly, Survivin expression was found to prevent papilloma regression and promotes conversion to SCC (46). Thus, our results represent a novel finding that resveratrol may protect the skin from UVB-mediated damages, including skin cancer development via modulating the expression and function of Survivin. To examine the mechanism of the observed modulation in Survivin by UVB and/or resveratrol treatments, we determined the effect of these treatments on Survivin-phosphorylation at Thr 34. As shown by immunoblot analysis (Fig. 3), our data demonstrated that the levels of Survivin phosphorylation at Thr 34 is up-regulated in UVB-induced tumors. Interestingly, the pre- and post-treatment of skin with resveratrol resulted in an appreciable down-regulation of Survivin phosphorylation at Thr 34 (Fig. 3). This is an important observation because one of the critical requirements of Survivin function has been identified in the phosphorylation on Thr34 by the mitotic kinase cdc2-cyclin B1 (55, 56). Recently, Wall and colleagues have demonstrated that Thr34 phosphorylation of Survivin critically regulates Survivin levels in tumor cells and that sequentially ablation of p34cdc2 kinase activity may remove the Survivin viability checkpoint and enhance apoptosis in tumor cells (56). Thus, we believe that the observed resveratrol-caused decrease in Survivin could be mediated via a decreased phosphorylation of this IAP protein at Thr 34 (55, 56). Studies have suggested that a direct interaction between Survivin and Smac/DIABLO is essential for the antiapoptotic activity of Survivin (57). It was shown that a point mutation (D71R) in the baculovirus IAP repeat motif and a C-terminal deletion mutant (Surv-BIR) of Survivin failed to bind to Smac/DIABLO and abrogate its ability to inhibit apoptosis (57). Thus, the N-terminal of mature Smac/DIABLO was suggested to be required for the function of Survivin (57). Because of these facts, we next determined the effect of UVB and/or resveratrol treatments on modulations in Smac/DIABLO protein in the epidermal tissues and tumors in SKH-1 hairless mice. As shown by immunoblot analysis (Fig. 3), our data demonstrated that the levels of Smac/DIABLO are significantly down-regulated in UVB-induced tumors, whereas the treatment of skin with resveratrol reversed this response. Our present observation suggesting an involvement of Survivin and associated events during chemoprevention of UVB exposuremediated skin carcinogenesis are supported by our very recent studies showing that short-term UVB exposure resulted in a significant up-regulation in protein and/or mrna levels of Survivin, phospho-survivin and p53 with a concomitant decrease in Smac/DIABLO in mouse skin, both in vitro (in normal human epidermal keratinocytes) and in vivo (in SKH-1 hairless mouse skin) (42). It has been shown that during tumorigenesis, Survivin expression is inversely correlated with apoptosis inhibition and positively correlated with proliferation and angiogenesis (52). Therefore, finally we determined the involvement of apoptosis during resveratrol-mediated chemoprevention of skin tumorigenesis. For this purpose, we employed M30 CytoDEATH detection kit that recognizes a specific caspase cleavage site within cytokeratin 18. This method Page 7 of 18

8 is an excellent means to determine early apoptosis under in vivo situations (58, 59). As indicated by immunohistochemical analysis (Fig. 4), we found a modest induction of apoptosis in UVBinduced mouse skin tumors. However, resveratrol (both pre- and post-treatment) resulted in an enhancement in the induction of apoptosis (Fig. 4), suggesting that the chemopreventive effects of resveratrol against UVB-induced skin tumorigenesis are mediated by an apoptotic elimination of UVB-initiated (damaged precancerous cells) via modulations in Survivin and other associated events. Our study, for the first time, demonstrated that resveratrol imparts strong chemopreventive effects against UVB exposure-mediated skin carcinogenesis (relevant to human skin cancers). On the basis of our data, we suggest that resveratrol protects against UVB exposure-mediated damages via an inhibition of Survivin-phosphorylation at Thr 34, which results in a decrease in Survivin that leads to an up-regulation of Smac/DIABLO. This series of events finally leads to an apoptotic death of damages/premalignant or malignant cells. A simplified depiction of our proposed mechanism of resveratrol action is shown in Fig. 5. Thus, we believe that the chemopreventive effects of resveratrol against UVB-mediated damages including skin carcinogenesis are, at least in part, mediated via modulations in the Survivin pathway. However, the possibility of involvement of other signaling events in the observed chemopreventive effects of resveratrol is an intriguing possibility. On the basis of our work, it is conceivable to design resveratrol containing emollient or patch, as well as sunscreen and skin-care products for prevention of skin cancer and other conditions that are believed to be caused by UV radiation. ACKNOWLEDGMENTS This work was supported by funding from National Cancer Institute (CA ) and Elsa Pardee Foundation. REFERENCES 1. Miller, D. L., and Weinstock, M. A. (1994) Nonmelanoma skin cancer in the United States: incidence. J. Am. Acad. Dermatol. 30, Steward, B. W., and Kleihues (2004) P. Reduction of exposure to ultraviolet radiation. World Cancer Report; Lyon, France. IARC Press, Ahmad, N., Gilliam, A. C., Katiyar, S. K., O'Brien, T. G., and Mukhtar, H. (2001) A definitive role of ornithine decarboxylase in photocarcinogenesis. Am. J. Pathol. 159, Black, H. S., degruijl, F. R., Forbes, P. D., Cleaver, J. E., Ananthaswamy, H. N., defabo, E. C., Ullrich, S. E., and Tyrrell, R. M. (1997) Photocarcinogenesis: an overview. J. Photochem. Photobiol. B 40, Brash, D. E., Ziegler, A., Jonason, A. S., Simon, J. A., Kunala, S., and Leffell, D. J. (1996) Sunlight and sunburn in human skin cancer: p53, apoptosis, and tumor promotion. J. Investig. Dermatol. Symp. Proc. 1, Page 8 of 18

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13 Table 1 Histological characterization of tumors Groups AK and AK-H Bowen s disease SCP SCC UVB alone, mj/cm 2 25 µm Resveratrol UVB, 180 mj/cm 2 50 µm Resveratrol UVB, 180 mj/cm 2 UVB, 180 mj/cm µm Resveratrol UVB, 180 mj/cm µm Resveratrol + + The SKH-1 hairless mice were treated with resveratrol and/or exposed to UVB as detailed in the Materials and Methods section. The animals were followed for the development of skin tumors, and the experiment was terminated at 28 weeks. For histopathological evaluation of the tumors, epidermal tissues were removed, fixed in 10% formalin, and embedded in paraffin. Tissue sections (5 µm) were cut, mounted on a glass slide, stained with hematoxylin and eosin, and microscopically examined by a board-certified dermatopathologist. The representative data show at least two biopsies from each mouse. AK, actinic keratosis; AK-H, actinic keratosis hyperplasia; SCP, squamous-cell papilloma; SCC, squamous-cell carcinoma. Grading: +++ = very high incidence (>50%); ++ = high incidence (30 50%); +, moderate incidence (0 30%) and, = negative. Page 13 of 18

14 Fig. 1 Figure 1. Effect of resveratrol treatments on UVB-exposure mediated skin tumorigenesis in SKH-1 hairless mice. The SKH-1 hairless mice were treated with resveratrol and/or exposed to UVB as described in the Materials and Methods section, and the animals were followed for the development of skin tumors with the experiment terminated at 28 wk. Photographs were taken using a Kodak Digital camera before euthanizing the animals at the end of experiment. Representative pictures of mice are shown (A). The tumor incidence was analyzed by Kaplan-Meier analysis and Log rank analysis (B) to determine the number of weeks the animals were free from tumor (appearance of a papule). The tumor multiplicity was analyzed by linear regression analysis of the data (C). The data represent the means ± SE of 8 mice (12 mice in the UVB alone group). P < 0.01 was considered to be statistically significant. Page 14 of 18

15 Fig. 2 Figure 2. Effect of resveratrol treatments on UVB exposure-mediated modulations on mrna and protein levels of Survivin in SKH-1 hairless mice. The SKH-1 hairless mice were treated with resveratrol and/or exposed to UVB as described in the Materials and Methods section, and the animals were killed at the end of experiment. For immunoblot analysis, the protein lysates were prepared from the epidermis (control animals) or the tumors (pooled from single animals). The Survivin levels were determined using anti-survivin antibody. Equal loading was confirmed by stripping the blot and reprobing it with a β-actin antibody. The protein bands shown here are representative of three independent experiments with similar results. The quantification of protein was performed by densitometric analysis using Scion Image Software, and the data (relative density normalized to β-actin) are expressed as the means ± SE of three experiments. Lane 1 = acetone alone; lane 2 = resveratrol (50 µm) alone; lane 3 = UVB alone; lane 4 = resveratrol (25 µm) + UVB; lane 5 = resveratrol (50 µm) + UVB; lane 6 = UVB + resveratrol (25 µm); and lane 7 = UVB + resveratrol (50 µm). For real-time PCR, total RNA was isolated from the epidermis (control animals) or the tumors (pooled from single animals) using the TRIZOL reagent. Real-time PCR analysis was performed with ABI PRISM 7000 Sequence Detection System. Mouse Survivin primers and TaqMan probe used were as follows: forward primer, 5 -GCGGAGGCTGGCTTCA-3 ; reverse primer, 5 -AGAAAAAACACTGGGCCAAATC-3 ; TaqMan Survivin probe, 5 -VIC- CCACTGCCCTACCGAGGACGAGCCMGBNFQ -3. The relative fluorescent intensity shown represents two independent experiments with similar results. Page 15 of 18

16 Fig. 3 Figure 3. Effect of resveratrol treatments on UVB exposure-mediated modulations on protein levels of Thr 34 - phosphorylated Survivin and Smac/DIABLO, in SKH-1 hairless mice. The SKH-1 hairless mice were treated with resveratrol and/or exposed to UVB as described in the Materials and Methods section, and the animals were killed at the end of experiment. For immunoblot analysis, the protein lysates were prepared from epidermis (control animals) or the tumors (pooled from single animals). The effect on Survivin-phosphorylation was determined using anti-phospho Thr 34 - Survivin antibody. Equal loading was confirmed by stripping the blot and reprobing it with a β-actin antibody. The protein bands shown here are representative of three independent experiments with similar results. The quantification of protein was performed by densitometric analysis using Scion Image Software, and the data (relative density normalized to β-actin) are expressed as the mean + SE of three experiments. Lane 1 = acetone alone; lane 2 = resveratrol (50 µm) alone; lane 3 = UVB alone; lane 4 = resveratrol (25 µm) + UVB; lane 5 = resveratrol (50 µm) + UVB; lane 6 = UVB + resveratrol (25 µm); lane 7 = UVB + resveratrol (50 µm). Page 16 of 18

17 Fig. 4 Figure 4. Effect of resveratrol on multiple UVB-exposure-mediated modulations in M30 CytoDEATH immunostaining in SKH-1 hairless mouse skin. The SKH-1 hairless mice were treated with resveratrol and/or exposed to UVB as detailed in the Materials and Methods section. The animals were killed at the end of experiment, and the tumors or epidermal tissue were surgically removed and frozen for further study. From the skin biopsies, 5-µm-thick paraffin-embedded sections were cut, and the effect of treatments on M30 CytoDEATH immunostaining was determined by immunohistochemical analysis using the mouse monoclonal M30 CytoDEATH antibody. The images were captured a Spot Insight QE Camera attached to a Nikon microscope. The representative pictures are from 5 or 6 tissue samples. Apoptosis is evident by brown color staining. Details of experiments are provided in Materials and Methods. Page 17 of 18

18 Fig. 5 Figure 5. Proposed mechanism of the chemopreventive effects of resveratrol against UVB-mediated damages, including skin carcinogenesis. Page 18 of 18