PDE2 is a novel target for attenuating tumor formation in a mouse model of UVB-induced skin carcinogenesis.

Our previous studies demonstrated that the topical application of caffeine is a potent inhibitor of UVB-induced carcinogenesis and selectively increases apoptosis in tumors but not in non-tumor areas of the epidermis in mice that are at a high risk for developing skin cancer. While this effect is mainly through a p53 independent pathway, the mechanism by which caffeine inhibits skin tumor formation has not been fully elucidated. Since caffeine is a non-specific phosphodiesterase inhibitor, we investigated the effects of several PDE inhibitors on the formation of sunburn cells in mouse skin after an acute exposure to ultraviolet light B (UVB). The topical application of a PDE2 inhibitor, erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (EHNA hydrochloride), stimulated epidermal apoptosis compared to control (P<0.01) and to a greater extent than caffeine whereas a PDE4 inhibitor attenuated the epidermal apoptosis compared to control (P<0.01). Since PDE2 hydrolyzes cyclic nucleotides, mainly cGMP, the effects of EHNA hydrochloride on epidermal apoptosis following UVB exposure may be mediated, in part, by increased cGMP signaling. Data demonstrated that the topical application of dibutyryl cGMP stimulated epidermal apoptosis (P<0.01) following an acute exposure to UVB. Treating UVB-pretreated mice topically with 3.1 µmole or 0.8 µmole of EHNA hydrochloride attenuated tumor formation to a greater extent than treating with 6.2 µmole caffeine when these compounds were applied once a day, five days a week for 18 weeks. These observations suggest a novel role for PDE2 in UVB-induced tumorigenesis and that PDE2 inhibitors that mediate cGMP signaling may be useful for the prevention and treatment of skin cancer.

Requirement and epigenetics reprogramming of Nrf2 in suppression of tumor promoter TPA-induced mouse skin cell transformation by sulforaphane.

Nrf2 is a transcription factor that plays critical roles in regulating the expression of cellular defensive antioxidants and detoxification enzymes. However, the role of Nrf2 and Nrf2's epigenetics reprogramming in skin tumor transformation is unknown. In this study, we investigated the inhibitory role and epigenetics of Nrf2 on tumor transformation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in mouse skin epidermal JB6 (JB6 P+) cells and the anticancer effect of sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables. After five days of treatment, SFN significantly inhibited TPA-induced JB6 cellular transformation and SFN enhanced the nuclear translocation of Nrf2 and increased the mRNA and protein levels of the Nrf2 target genes HO-1, NQO1 and UGT1A1. Knockdown of Nrf2 attenuated the induction of Nrf2, HO-1 and NQO1 by SFN, enhanced TPA-induced colony formation and dampened the inhibitory effect of SFN on TPA-induced JB6 transformation. Epigenetics investigation using bisulfite genomic sequencing showed that SFN decreased the methylation ratio of the first 15 CpGs of the Nrf2 gene promoter, which was corroborated by increased Nrf2 mRNA expression. Furthermore, SFN strongly reduced the protein expression of DNA methyltransferases (DNMT1, DNMT3a and DNMT3b). SFN also inhibited the total histone deacetylase (HDAC) activity and decreased the protein expression of HDAC1, HDAC2, HDAC3 and HDAC4. Collectively, these results suggest that the anti-cancer effect of SFN against TPA-induced neoplastic transformation of mouse skin could involve the epigenetic reprogramming of anti-cancer genes such as Nrf2, leading to the epigenetic reactivation of Nrf2 and the subsequent induction of downstream target genes involved in cellular protection.

Inverse relationship between p53 and phospho-Chk1 (Ser317) protein expression in UVB-induced skin tumors in SKH-1 mice.

Immunohistochemical evaluation of serial stored paraffin sections from 42 keratoacanthomas and 11 squamous cell carcinomas demonstrated that skin tumors from UVB-exposed mice showed an inverse relationship (>95%) between p53 protein expression and phospho-Chk1 (Ser317), but not phospho-Chk1 (Ser345) protein expression. Tumors expressing high levels and large areas of p53 protein had no detectable phospho-Chk1 (Ser317), whereas tumors expressing high levels and large areas of phospho-Chk1 (Ser317) protein had no detectable p53. Squamous cell carcinomas that demonstrated heterogeneous p53 and phospho-Chk1 (Ser317) protein expression within the same tumor showed that areas expressing p53 were negative for phospho-Chk1 (Ser317) immunostaining while areas expressing phospho-Chk1 (Ser317) were negative for p53. Similar patterns were observed for keratoacanthomas. These findings were also observed in epidermal areas distant from tumors that demonstrated no detectable phospho-Chk1 (Ser317), but appreciable p53 protein in the basal layer. Tumors from congenic hairless p53 knockout mice had elevated levels of phospho-Chk1 (Ser317) compared to tumors from p53 wild-type SKH-1 controls. After a single exposure to UVB, normal epidermal cells from a p53 knockout mouse expressed a relatively high level of phospho-Chk1 (Ser317) whereas epidermal cells from a p53 wild-type littermate induced p53 protein and expressed a relatively low level of phospho-Chk1 (Ser317). These data illustrate the dynamic regulation of checkpoint function, suggesting that phosphorylation of Chk1 on Serine 317 is regulated by p53 status and that p53 may act as a molecular on/off switch for phosphorylation at this site.

Parametrial fat tissue from high fat diet-treated SKH-1 mice stimulates transformation of mouse epidermal JB6 cells.

Our previous studies indicated that decreasing visceral adipose tissue by surgical removal of the parametrial fat pads inhibited UVB-induced carcinogenesis in SKH-1 mice fed a high fat diet (HFD), but not a low fat diet (LFD) indicating that the parametrial fat tissue from mice fed a HFD played a role in skin carcinogenesis. OBJECTIVE: In the present study, we sought to investigate how a HFD may influence the intrinsic properties of the parametrial fat tissue to influence UVB-induced skin tumor formation. METHODS AND RESULTS: Immunohistochemical staining, adipokine array, and flow cytometry showed that parametrial fat tissue from mice fed a HFD had a higher density of macrophage-fused dead adipocytes (crown-like structures), more adipokines, and stimulated the production of more reactive oxygen species compared with parametrial fat tissue from mice fed a LFD. These differences between parametrial fat tissue from mice fed a HFD and LFD were associated with their effect on the in vitro transformation of mouse epidermal JB6 cells. Our results indicated that fat tissue filtrate (an aqueous filtrate made from the parametrial fat pad) from mice fed a HFD enhanced the conversion of JB6 cells from an epithelial-like morphology to cells with a fibroblast-like morphology to a greater extent than fat tissue filtrate from mice fed a LFD. Studies indicated that the fibroblast-like cells had decreased levels of E-cadherin, increased levels of Twist as assayed by western blot. Fat tissue filtrate made from the parametrial fat tissue of mice fed a HFD had 160% more transforming activity than that from mice fed a LFD and formed malignant mesenchymal tumors in vivo. CONCLUSION: These studies provide the first in vitro demonstration of a parametrial fat tissue-induced transformation of an epidermal cell.

Mechanisms of Caffeine-Induced Inhibition of UVB Carcinogenesis.

Sunlight-induced non-melanoma skin cancer is the most prevalent cancer in the United States with more than two million cases per year. Several studies have shown an inhibitory effect of caffeine administration on UVB-induced skin cancer in mice, and these studies are paralleled by epidemiology studies that indicate an inhibitory effect of coffee drinking on non-melanoma skin cancer in humans. Strikingly, decaffeinated coffee consumption had no such inhibitory effect. Mechanism studies indicate that caffeine has a sunscreen effect that inhibits UVB-induced formation of thymine dimers and sunburn lesions in the epidermis of mice. In addition, caffeine administration has a biological effect that enhances UVB-induced apoptosis thereby enhancing the elimination of damaged precancerous cells, and caffeine administration also enhances apoptosis in tumors. Caffeine administration enhances UVB-induced apoptosis by p53-dependent and p53-independent mechanisms. Exploration of the p53-independent effect indicated that caffeine administration enhanced UVB-induced apoptosis by inhibiting the UVB-induced increase in ATR-mediated formation of phospho-Chk1 (Ser345) and abolishing the UVB-induced decrease in cyclin B1 which resulted in caffeine-induced premature and lethal mitosis in mouse skin. In studies with cultured primary human keratinocytes, inhibition of ATR with siRNA against ATR inhibited Chk1 phosphorylation and enhanced UVB-induced apoptosis. Transgenic mice with decreased epidermal ATR function that were irradiated chronically with UVB had 69% fewer tumors at the end of the study compared with irradiated littermate controls with normal ATR function. These results, which indicate that genetic inhibition of ATR (like pharmacologic inhibition of ATR via caffeine) inhibits UVB-induced carcinogenesis support the concept that ATR-mediated phosphorylation of Chk1 is an important target for caffeine's inhibitory effect on UVB-induced carcinogenesis.

Inhibition of UVB-induced nonmelanoma skin cancer: a path from tea to caffeine to exercise to decreased tissue fat.

Oral administration of green tea, black tea, or caffeine (but not the decaffeinated teas) inhibited ultraviolet B radiation (UVB)-induced skin carcinogenesis in SKH-1 mice. Studies with caffeine indicated that its inhibitory effect on the ATR/Chk1 pathway is an important mechanism for caffeine's inhibition of UVB-induced carcinogenesis. The regular teas or caffeine increased locomotor activity and decreased tissue fat. In these studies, decreased dermal fat thickness was associated with a decrease in the number of tumors per mouse. Administration of caffeine, voluntary exercise, and removal of the parametrial fat pads all stimulated UVB-induced apoptosis, inhibited UVB-induced carcinogenesis, and stimulated apoptosis in UVB-induced tumors. These results suggest that caffeine administration, voluntary exercise, and removal of the parametrial fat pads inhibit UVB-induced carcinogenesis by stimulating UVB-induced apoptosis and by enhancing apoptosis in DNA-damaged precancer cells and in cancer cells. We hypothesize that tissue fat secretes antiapoptotic adipokines that have a tumor promoting effect.

Surgical removal of the parametrial fat pads stimulates apoptosis and inhibits UVB-induced carcinogenesis in mice fed a high-fat diet.

Removal of the parametrial fat pads (partial lipectomy) from female SKH-1 mice fed a high-fat diet inhibited UVB-induced carcinogenesis, but this was not observed in mice fed a low-fat chow diet. Partial lipectomy in high-fat-fed mice decreased the number of keratoacanthomas and squamous cell carcinomas per mouse by 76 and 79%, respectively, compared with sham-operated control mice irradiated with UVB for 33 wk. Immunohistochemical analysis indicated that partial lipectomy increased caspase 3 (active form) positive cells by 48% in precancerous epidermis away from tumors, by 68% in keratoacanthomas, and by 224% in squamous cell carcinomas compared with sham-operated control mice. In addition, partial lipectomy decreased cell proliferation away from tumors and in tumors. RT-PCR analysis for adipokines revealed that mRNAs for TIMP1, MCP1, and SerpinE1 (proinflammatory/antiapoptotic cytokines) in the parametrial fat pads of sham-operated control mice were 54- to 83-fold higher than levels in compensatory fat that returned after surgery in partially lipectomized mice at the end of the tumor study. Feeding mice high-fat diets for 2 wk increased levels of TIMP1 and other adipokines in serum and epidermis, and these increases were inhibited by removal of the parametrial fat pads. Our results are a unique demonstration that surgical removal of a specific tissue fat results in inhibition of carcinogenesis in obese mice. This inhibition was associated with an increase in apoptosis and a decrease in proliferation in tumors and in precancerous areas away from tumors.

Effects of green tea polyphenol (-)-epigallocatechin-3-gallate on newly developed high-fat/Western-style diet-induced obesity and metabolic syndrome in mice.

The aim of this study was to investigate the effects of (-)-epigallocatechin-3-gallate (EGCG) on newly developed high-fat/Western-style diet-induced obesity and symptoms of metabolic syndrome. Male C57BL/6J mice were fed a high fat/Western-style (HFW; 60% energy as fat and lower levels of calcium, vitamin D(3), folic acid, choline bitartrate, and fiber) or HFW with EGCG (HFWE; HFW with 0.32% EGCG) diet for 17 wks. As a comparison, two other groups of mice fed a low-fat diet (LF; 10% energy as fat) and high-fat diet (HF; 60% energy as fat) were also included. The HFW group developed more body weight gain and severe symptoms of metabolic syndrome than the HF group. The EGCG treatment significantly reduced body weight gain associated with increased fecal lipids and decreased blood glucose and alanine aminotransferase (ALT) levels compared to those of the HFW group. Fatty liver incidence, liver damage, and liver triglyceride levels were also decreased by the EGCG treatment. Moreover, the EGCG treatment attenuated insulin resistance and levels of plasma cholesterol, monocyte chemoattractant protein-1 (MCP-1), C-reactive protein (CRP), interlukin-6 (IL-6), and granulocyte colony-stimulating factor (G-CSF). Our results demonstrate that the HFW diet produces more severe symptoms of metabolic syndrome than the HF diet and that the EGCG treatment can alleviate these symptoms and body fat accumulation. The beneficial effects of EGCG are associated with decreased lipid absorption and reduced levels of inflammatory cytokines.

Protection from UV-induced skin carcinogenesis by genetic inhibition of the ataxia telangiectasia and Rad3-related (ATR) kinase.

Multiple human epidemiologic studies link caffeinated (but not decaffeinated) beverage intake with significant decreases in several types of cancer, including highly prevalent UV-associated skin carcinomas. The mechanism by which caffeine protects against skin cancer is unknown. Ataxia telangiectasia and Rad3-related (ATR) is a replication checkpoint kinase activated by DNA stresses and is one of several targets of caffeine. Suppression of ATR, or its downstream target checkpoint kinase 1 (Chk1), selectively sensitizes DNA-damaged and malignant cells to apoptosis. Agents that target this pathway are currently in clinical trials. Conversely, inhibition of other DNA damage response pathways, such as ataxia telangiectasia mutated (ATM) and BRCA1, promotes cancer. To determine the effect of replication checkpoint inhibition on carcinogenesis, we generated transgenic mice with diminished ATR function in skin and crossed them into a UV-sensitive background, Xpc(-/-). Unlike caffeine, this genetic approach was selective and had no effect on ATM activation. These transgenic mice were viable and showed no histological abnormalities in skin. Primary keratinocytes from these mice had diminished UV-induced Chk1 phosphorylation and twofold augmentation of apoptosis after UV exposure (P = 0.006). With chronic UV treatment, transgenic mice remained tumor-free for significantly longer (P = 0.003) and had 69% fewer tumors at the end of observation of the full cohort (P = 0.019), compared with littermate controls with the same genetic background. This study suggests that inhibition of replication checkpoint function can suppress skin carcinogenesis and supports ATR inhibition as the relevant mechanism for the protective effect of caffeinated beverage intake in human epidemiologic studies.

Effects of high-fat diets rich in either omega-3 or omega-6 fatty acids on UVB-induced skin carcinogenesis in SKH-1 mice.

Our previous studies reported that caffeine or voluntary exercise decreased skin tumor multiplicity, in part, by decreasing fat levels in the dermis. These data suggest that tissue fat may play an important role in regulating ultraviolet light (UV) B-induced skin tumor development. In the present study, we explored the effects of high-fat diets rich in either omega-3 or omega-6 fatty acids on UVB-induced skin carcinogenesis. SKH-1 mice were irradiated with 30 mJ/cm(2) of UVB once a day, two times per week for 39 weeks. During UVB treatment, one group of mice was given a high-fat fish oil (HFFO) diet rich in omega-3 fatty acids and the other group of mice was given a high-fat mixed-lipids (HFMLs) diet rich in omega-6 fatty acids. The results showed that, compared with HFML diet, HFFO treatment (i) increased latency for the development of UVB-induced skin tumors; (ii) decreased the formation of papilloma, keratoacanthoma and carcinoma by 64, 52 and 46%, respectively and (iii) decreased the size of papilloma, keratoacanthoma and carcinoma by 98, 80 and 83%, respectively. Mechanistic studies with antibody array revealed that compared with HFML diet, administration of HFFO to the mice significantly decreased the UVB-induced increases in the levels of TIMP-1, LIX and sTNF R1 as well as other several proinflammatory cytokines and stimulated the UVB-induced apoptosis in the epidermis. Our results indicate that omega-3 fatty acids in HFFO diet have beneficial effects against UVB-induced skin carcinogenesis, and these effects may be associated with an inhibition on UVB-induced inflammatory response.

Caffeine decreases phospho-Chk1 (Ser317) and increases mitotic cells with cyclin B1 and caspase 3 in tumors from UVB-treated mice.

Oral administration of caffeine to mice inhibits UVB-induced carcinogenesis, and these results are paralleled by epidemiology studies indicating that caffeinated coffee and tea intake (but not decaffeinated beverage intake) is associated with decreased incidence of nonmelanoma skin cancer. Topical applications of caffeine to the skin of SKH-1 mice that had previously been treated with UVB inhibited subsequent skin tumor development and stimulated apoptosis in tumors but not in nontumor areas of the epidermis. This study sought to determine the basis of these differential effects on tumor versus nontumor sites that can be induced by caffeine, long after all UVB treatment has ceased. The activation status of the ATR/Chk1 pathway in UVB-induced tumors and uninvolved skin was determined by quantitating phospho-Chk1 (Ser317) and induction of lethal mitosis in vivo in the presence and absence of topical caffeine treatment. In the absence of caffeine, we found that UVB-induced tumors often had islands of phospho-Chk1 (Ser317) staining cells that were not present in nontumor areas of the epidermis. Treatment of mice with topical caffeine significantly diminished phospho-Chk1 (Ser317) staining and increased the number of mitotic cells that expressed cyclin B1 and caspase 3 in tumors, consistent with caffeine-induced lethal mitosis selectively in tumors. We hypothesize that compared with adjacent uninvolved skin, UVB-induced skin tumors have elevated activation of, and dependence on, the ATR/Chk1 pathway long after UVB exposure has ceased and that caffeine can induce apoptosis selectively in tumors by inhibiting this pathway and promoting lethal mitosis.

Tumorigenic effect of some commonly used moisturizing creams when applied topically to UVB-pretreated high-risk mice.

Irradiation of SKH-1 mice with UVB (30 mJ cm(-2)) twice a week for 20 weeks resulted in mice with a high risk of developing skin tumors over the next several months in the absence of further irradiation with UVB (high-risk mice). Topical applications of 100 mg of Dermabase, Dermovan, Eucerin Original Moisturizing Cream (Eucerin), or Vanicream once a day, 5 days a week for 17 weeks to these high-risk mice increased significantly the rate of formation of tumors and the rate of increase in tumor size per mouse. Additional studies indicated that treatment of high-risk mice with Dermabase, Dermovan, Eucerin, or Vanicream for 17 weeks increased the total number of histologically characterized tumors by 69% (average of two experiments; P<0.0001 in each experiment), 95% (P<0.0001), 24% (P<0.01), and 58% (P<0.0001), respectively. Topical applications of a specially designed Custom Blend cream to high-risk mice was not tumorigenic. The results indicate that several commercially available moisturizing creams increase the rate of formation and number of tumors when applied topically to UVB-pretreated high-risk mice. Further studies are needed to determine the effects of topical applications of moisturizing creams on sunlight-induced skin cancer in humans.

Inhibitory effect of voluntary running wheel exercise on the growth of human pancreatic Panc-1 and prostate PC-3 xenograft tumors in immunodeficient mice.

In the present study, we investigated the effect of voluntary exercise on the formation and growth of the human pancreas Panc-1 and prostate PC-3 tumors in immunodeficient mice. Female severe combined immunodeficient (SCID) mice were injected subcutaneously with human pancreatic cancer Panc-1 cells, and male SCID mice were injected subcutaneously with human prostate cancer PC-3 cells. Voluntary running wheel exercise for 63 days, starting one week before the subcutaneous injection of Panc-1 or PC-3 tumor cells into SCID mice, suppressed the growth of Panc-1 and PC-3 tumors. The exercise regimen increased the food and fluid consumption in the female and male mice. Exercise also decreased the size of the parametrial fat pads in the female mice and the paradidymis fat pads in the male mice, but there was no effect on the body weight. Mechanistic studies showed that voluntary running wheel exercise inhibited proliferation as reflected by a decreased mitosis, and the exercise regimen also stimulated apoptosis as reflected by the increased caspase-3 (active form) expression in the Panc-1 and PC-3 tumors. Voluntary running wheel exercise decreased the ratio of the percent mitotic cells/apoptotic cells in Panc-1 and PC-3 tumors by 38 and 32%, respectively. The present study demonstrated an inhibitory effect of voluntary exercise on the growth of pancreas and prostate tumors in a SCID mouse xenograft model.

Effect of caffeine on the ATR/Chk1 pathway in the epidermis of UVB-irradiated mice.

Administration of caffeine was shown in earlier studies to enhance UVB-induced apoptosis and inhibit UVB-induced carcinogenesis in hairless SKH-1 mice. Here, we describe a potential mechanism for these in vivo effects. A single irradiation of mouse skin with UVB activated the ataxia-telangiectasia mutated- and Rad3-related (ATR) pathway, causing a severalfold increase in keratinocytes with phospho-Chk1 (Ser(345)) and a marked decrease in mitotic keratinocytes with cyclin B1 compared with baseline. When given in the drinking water for 1 to 2 weeks before UVB, caffeine (0.4 mg/mL) markedly inhibited the UVB-induced phosphorylation of Chk1 on Ser(345) and caused premature expression of cyclin B1 in the epidermis. Normal keratinocytes had delayed mitotic entry for >10 h following UVB. Caffeine administration reduced this mitotic delay to only 4 h and caused markedly increased apoptosis by 6 to 10 h after UVB. p53 knockout mice were used to determine the role of p53 in these processes. Irradiation with UVB markedly decreased the number of mitotic keratinocytes with cyclin B1 in p53 knockout mice, and topical caffeine immediately after UVB abrogated this response and increased UVB-induced apoptosis severalfold. These effects of caffeine in knockout mice were substantially greater than in wild-type mice. The ability of caffeine to promote the deletion of p53(-/-) keratinocytes may be relevant to its inhibitory effect on UVB-induced skin cancer. Our studies indicate that administration of caffeine enhances the removal of DNA-damaged cells by inhibiting the ATR-mediated phosphorylation of Chk1 and prematurely increasing the number of cyclin B1-containing cells that undergo lethal mitosis.

Effect of caffeine on UVB-induced carcinogenesis, apoptosis, and the elimination of UVB-induced patches of p53 mutant epidermal cells in SKH-1 mice.

Oral administration of green tea or caffeine to SKH-1 mice during UVB irradiation for several months inhibited the formation of skin cancer. Similar effects were observed when green tea or caffeine was given to tumor-free UVB-initiated mice with a high risk of developing skin tumors in the absence of further UVB irradiation (high risk mice). Mechanistic studies indicated that topical application of caffeine stimulated UVB-induced apoptosis as well as apoptosis in UVB-induced focal hyperplasia and tumors in tumor-bearing mice. Oral or topical administration of caffeine enhanced the removal of patches of epidermal cells with a mutant form of p53 protein that appeared early during the course of UVB-induced carcinogenesis, and oral administration of caffeine altered the profile of p53 mutations in the patches. In additional studies, topical application of caffeine was shown to have a sunscreen effect, and topical application of caffeine sodium benzoate was more active than caffeine as a sunscreen and for stimulating UVB-induced apoptosis. Caffeine sodium benzoate was also highly active in inhibiting carcinogenesis in UVB-pretreated high risk mice. Our studies indicate that caffeine and caffeine sodium benzoate may be useful as novel inhibitors of sunlight-induced skin cancer.

Voluntary exercise together with oral caffeine markedly stimulates UVB light-induced apoptosis and decreases tissue fat in SKH-1 mice.

Treatment of SKH-1 mice orally with caffeine (0.1 mg/ml in the drinking water), voluntary running wheel exercise, or a combination of caffeine and exercise for 2 weeks (i) decreased the weight of the parametrial fat pads by 35, 62, and 77%, respectively; (ii) decreased the thickness of the dermal fat layer by 38, 42, and 68%, respectively; (iii) stimulated the formation of UVB-induced apoptotic sunburn cells in the epidermis by 96, 120, and 376%, respectively; and (iv) stimulated the formation of UVB-induced caspase 3 (active form)-positive cells in the epidermis by 92, 120, and 389%, respectively (average of two experiments). Oral administration of caffeine (0.4 mg/ml in the drinking water) in combination with voluntary exercise was less effective than administration of the low dose of caffeine in combination with exercise in stimulating UVB-induced apoptosis. Although orally administrated caffeine (0.1 mg/ml in the drinking water) or voluntary exercise for 2 weeks caused only a small nonsignificant stimulation of UVB-induced increase in the percentage of phospho-p53 (Ser-15)-positive cells in the epidermis (27 or 18%, respectively), the combination of the two treatments enhanced the UVB-induced increase in phospho-p53 (Ser-15)-positive cells by 99%. The plasma concentration of caffeine in mice ingesting caffeine (0.1-0.4 mg/ml drinking water) is similar to that in the plasma of most coffee drinkers (one to four cups per day). Our studies indicate a greater than additive stimulatory effect of combined voluntary exercise and oral administration of a low dose of caffeine on UVB-induced apoptosis.

Caffeine and caffeine sodium benzoate have a sunscreen effect, enhance UVB-induced apoptosis, and inhibit UVB-induced skin carcinogenesis in SKH-1 mice.

Topical application of caffeine sodium benzoate (caffeine-SB) immediately after UVB irradiation of SKH-1 mice enhanced UVB-induced apoptosis by a 2- to 3-fold greater extent than occurred after the topical application of an equimolar amount of caffeine. Although topical application of caffeine-SB or caffeine enhanced UVB-induced apoptosis, both substances were inactive on non-UVB-treated normal skin. Topical application of caffeine-SB or caffeine (each has UVB absorption properties) 0.5 h before irradiation with a high dose of UVB decreased UVB-induced thymine dimer formation and sunburn lesions (sunscreen effect). Caffeine-SB was more active than an equimolar amount of caffeine in exerting a sunscreen effect. In additional studies, caffeine-SB strongly inhibited the formation of tumors in UVB-pretreated 'high-risk mice' and in tumor-bearing mice, and the growth of UVB-induced tumors was also inhibited. Caffeine-SB and caffeine are the first examples of compounds that have both a sunscreen effect and enhance UVB-induced apoptosis. Our studies suggest that caffeine-SB and caffeine may be good agents for inhibiting the formation of sunlight-induced skin cancer.

Stimulatory effect of voluntary exercise or fat removal (partial lipectomy) on apoptosis in the skin of UVB light-irradiated mice.

Earlier studies indicated that high dietary fat and obesity are associated with an increased risk of cancer at several organ sites in experimental animals and in humans. In a recent study we found that voluntary running wheel exercise decreased body fat and inhibited ultraviolet B light (UVB)-induced carcinogenesis in the epidermis of SKH-1 mice. In the present study we demonstrate that voluntary running wheel exercise stimulated UVB-induced apoptosis in the epidermis by a p53-independent mechanism, and voluntary exercise also stimulated apoptosis in UVB-induced tumors in tumor-bearing mice. Exercise had no effect in non-UVB-treated epidermis or in areas of the epidermis away from tumors in tumor-bearing mice. In addition, we found that removal of the parametrial fat pads (partial lipectomy) 2 weeks before UVB irradiation enhanced UVB-induced apoptosis. The results of our studies suggest that fat cells secrete substances that inhibit apoptosis in cells with DNA damage and possibly also in tumors. Our results help explain why exercise or various dietary regimens that decrease tissue fat inhibit carcinogenesis.

Inhibitory effects of voluntary running wheel exercise on UVB-induced skin carcinogenesis in SKH-1 mice.

Earlier studies showed that oral administration of green tea or caffeine to SKH-1 mice inhibited ultraviolet B light (UVB)-induced skin carcinogenesis, decreased dermal fat thickness and increased locomotor activity. In the present study, the effects of voluntary running wheel exercise on thickness of dermal fat as well as on UVB-induced tumorigenesis in SKH-1 mice were studied in UVB-initiated high-risk and UVB-induced complete carcinogenesis models. In the high-risk model, animals were exposed to UVB (30 mJ/cm(2)) 3 times/week for 16 weeks. For 14 weeks subsequent to UVB exposure, half of the animals had access to running wheels in their cages whereas the other half did not. In the complete carcinogenesis model, animals were exposed to UVB (30 mJ/cm(2)) 2 times/week for 33 weeks. From the beginning, half of the animals had access to running wheels whereas the other half did not. At the conclusion of each study, body weights were not different between groups, although animals with running wheels consumed significantly more food and water than animals without running wheels. In addition, animals with running wheels had decreases in parametrial fat pad weight and thickness of the dermal fat layer. In both UVB-initiated high-risk and complete carcinogenesis models, voluntary running wheel exercise delayed the appearance of tumors, decreased the number of tumors per mouse and decreased tumor volume per mouse. Histopathology studies revealed that running wheel exercise decreased the number of non-malignant tumors (primarily keratoacanthomas) by 34% and total tumors per mouse by 32% in both models, and running wheel exercise decreased the formation of squamous cell carcinomas in the UVB-induced complete carcinogenesis model by 27%. In addition, the size of keratoacanthomas and squamous cell carcinomas were decreased substantially in both models. The effects described here indicate that voluntary running wheel exercise inhibits UVB-induced skin tumorigenesis and may also inhibit tumor growth.

Inhibition of estradiol-induced mammary proliferation by dibenzoylmethane through the E2-ER-ERE-dependent pathway.

The phytochemical dibenzoylmethane (DBM) has been shown to inhibit 7,12-dimethylbenz[a]anthracene induced mammary tumorigenesis in Sencar mice. However, the molecular basis of this activity is still elusive. In the present study, we demonstrated that DBM inhibits estradiol (E2)-induced incorporation of bromodeoxyuridine into mammary DNA in immature female Sencar mice by 52%, when 10 micromol of DBM was intraperitoneally injected into mice prior to the injection of E2. Examination of the influence of DBM on the expressions of E2-ERE-dependent oncogenes in MCF-7 cells indicated that DBM inhibits the E2-induced cell growth as well as the expressions of four oncogenes, telomerase, c-myc, Ha-ras and bcl-2. Further mechanistic study using chromatin immunoprecipitation assay demonstrated that DBM acts as a pure antagonist by attenuating the binding of estrogen receptor to the estrogen response elements in the regulatory regions of c-myc, hTERT and bcl-2 genes in vivo. Taken together, our results strongly suggest that DBM plays an inhibitory role in E2-induced proliferations, which establishes DBM as a model molecule for studying the antiestrogenic drugs.