Qing-Yun Peng

Stimulatory Effect of Topical Application of Caffeine on UVB-Induced Apoptosis in the Epidermis of p53 and Bax Knockout Mice

Shaved male or female p53(−/−) C57BL/6J mice and their wild-type littermates were irradiated once with UVB (60 mJ/cm2). The UVB-induced increase in apoptotic sunburn cells in p53(−/−) mice at 6–10 h after exposure to UVB was only 10–30% of that observed after treatment of p53(+/+) mice with UVB. Topical applications of caffeine immediately after UVB irradiation in female p53(+/+) or p53(−/−) mice enhanced the UVB-induced increase in apoptotic sunburn cells 6 h later by 127% and 563%, respectively. In another study, shaved female Bax(−/−) C57BL/6J mice and their wild-type littermates were irradiated once with UVB (60 mJ/cm2). The UVB-induced increase in apoptotic sunburn cells in Bax(−/−) mice at 6 h after exposure to UVB was only 14% of that observed after treatment of Bax(+/+) mice with UVB. Topical application of caffeine immediately after irradiation of Bax(+/+) or Bax(−/−) mice with UVB enhanced the UVB-induced increases in apoptotic sunburn cells at 6 h by 214% and 467%, respectively, and topical application of caffeine immediately after irradiation of Bax(+/+) or Bax(−/−) mice with UVB enhanced the UVB-induced increase in caspase 3 (active form) positive cells at 6 h by 253% and 750%, respectively. The results indicate that UVB-induced increases in apoptosis in the epidermis of wild-type mice are predominantly (but not entirely) by p53- and Bax-dependent pathways and that topical application of caffeine can enhance UVB-induced increases in apoptosis by p53- and Bax-independent pathways.

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.

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.

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.

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.

Oral caffeine during voluntary exercise markedly inhibits skin carcinogenesis and decreases inflammatory cytokines in UVB-treated mice

Ultraviolet B (UVB)-pretreated SKH-1 mice were treated with water, caffeine (0.1 mg/ml), voluntary running wheel exercise (RW) or caffeine together with RW for 14 wk. Treatment of the mice with caffeine, RW, or caffeine plus RW decreased skin tumors per mouse by 27%, 35%, and 62%, respectively, and the tumor volume per mouse was decreased by 61%, 70%, and 85%, respectively. In mechanistic studies, mice were treated with water, caffeine, RW, or caffeine plus RW for 2 wk prior to a single irradiation with UVB. Caffeine plus RW increased RW activity by 22% when compared with RW alone. Caffeine ingestion was not significantly different between groups. Treatment of mice with caffeine plus RW for 2 wk decreased the weight of the parametrial fat pads and stimulated the formation of UVB-induced apoptosis to a greater extent than treatment with caffeine or RW alone. An antibody array revealed that caffeine plus RW administered to mice fed a high-fat diet and irradiated with UVB decreased the epidermal levels of lipopolysaccharide-induced CXC chemokine, soluble TNF alpha receptor-1, and macrophage inflammatory protein-1γ. Overall, caffeine during RW exerts a stronger effect than either treatment alone for decreasing tissue fat, increasing UVB-induced apoptosis, lowering the levels of cytokines associated with inflammation and for inhibiting UVB-induced carcinogenesis.

Oral administration of caffeine during voluntary exercise markedly decreases tissue fat and stimulates apoptosis and cyclin B1 in UVB-treated skin of hairless p53-knockout mice

Treatment of p53(-/-) mice orally with caffeine, voluntary exercise or their combination for 2 weeks prior to a single irradiation with UVB (i) decreased the weight of the epididymal fat pads by 22, 40 and 56%, (ii) decreased the thickness of the dermal fat layer by 10, 26 and 42%, (iii) increased the number of apoptotic sunburn cells by 29, 100 and 489%, (iv) increased the number of caspase-3-positive cells by 33, 117 and 667% and (v) increased the number of mitotic cells with cyclin B1-positive staining by 40, 210 and 510%, respectively. Pearsons correlation coefficient indicated a statistically significant inverse relationship between the level of tissue fat and the number of mitotic cells with cyclin B1 in p53(-/-) mice but not in p53(+/+) littermates. Western blot analysis indicated that treatment of p53(-/-) mice with caffeine together with exercise increased the level of cyclin B1 significantly more than in p53(+/+) mice. p53(-/-) mice, but not p53(+/+) mice, treated with caffeine during exercise exhibited a dramatic decrease in the level of survivin. Our results suggest that voluntary exercise in combination with oral caffeine may exert a synergistic increase in UVB-induced apoptosis and that tissue fat may be a more important modulator of apoptosis and carcinogenesis in p53-deficient mice than in p53-normal mice. The stimulatory effects on apoptosis in p53(-/-) mice by the combination treatment might be associated with increased levels of cyclin B1 and decreased levels of survivin.

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.

Abstract 4286: Oral caffeine during voluntary exercise markedly inhibits skin carcinogenesis and decreases cytokines associated with inflammation in UVB-treated mice

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL In previous studies, we found that treatment of female SKH-1 mice with either oral administration of caffeine or voluntary running wheel exercise (RW) inhibited the formation of UVB-induced skin tumors. In the present study, we determined whether RW in combination with oral caffeine has a synergistic inhibitory effect on UVB-induced skin carcinogenesis. UVB-pretreated female high-risk SKH-1 mice (7-8 weeks of age, total 160 mice) have no tumors but they develop tumors in the absence of further UVB irradiation over the next several months. These high-risk mice (40 mice per group) were then treated with water (control), caffeine (0.1 mg/ml in drinking fluid), RW or caffeine together with RW for 14 weeks. Although there were no differences in body weight between the four groups during the course of the study, treatment with caffeine increased running wheel activity by 40% when compared with RW alone. Treatment of the mice with caffeine, RW or caffeine together with RW decreased skin tumors per mouse by 27, 35 and 62%, respectively, and the tumor volume per mouse was decreased by 61, 70 and 85%, respectively. In mechanistic studies, female SKH-1 mice were treated with water (control), caffeine (0.1 mg/ml), RW, or a combination of caffeine plus RW for 2 weeks. All mice were then exposed to a single dose of UVB (30 mJ/cm2) and sacrificed before (control) and at 0.1, 0.5, 1, 2, 4, 6, 10, 16, 24, and 48 h post UVB. The results showed that mice treated with oral caffeine for 2 weeks increased RW activity by 22% when compared with the mice treated with RW alone. Treatment of mice orally with caffeine, RW, or a combination of caffeine and RW for 2 weeks (a) decreased the weight of the parametrial fat pads by 30, 56 and 63%, respectively, (b) stimulated the formation of UVB-induced apoptotic sunburn cells in the epidermis by 21, 124 and 298% at 6 hours post-UVB, and stimulated the formation of UVB-induced caspase 3 (active form) positive cells in the epidermis by 30, 164 and 333%, respectively at 6 hours post-UVB. Combination treatment of the mice had a small but significant inhibitory effect on cell proliferation as measured by bromodeoxyuridine incorporation into DNA in the epidermis. Antibody array with 40 cytokines associated with inflammation revealed that oral caffeine together with RW administered to mice fed a high fat diet rich in omega-6 fatty acids for 2 weeks significantly decreased the UVB-induced increases in the levels of LIX, sTNF R1 and MIP-1α as well as several other cytokines associated with inflammation. Our results indicate that voluntary exercise in combination with oral caffeine exerts a stronger effect than either treatment alone for decreasing tissue fat, increasing UVB-induced apoptosis, lowering the levels of cytokines associated with inflammation and for inhibiting UVB-induced carcinogenesis in UVB-pretreated high-risk mice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4286. doi:1538-7445.AM2012-4286

Abstract 1815: Stimulation of apoptosis by caffeine or exercise is associated with an increase in thymine dimer formation in the epidermis of UVB-treated SKH-1 mice

In early studies, we found that oral administration of caffeine or voluntary running wheel exercise (RW) for 1 or 2 weeks prior to UVB irradiation enhanced UVB-induced apoptosis in the epidermis of SKH-1 hairless mice. In additional studies, we found that oral administration of caffeine or RW during the course of irradiation with UVB inhibited UVB-induced skin carcinogenesis. In the present study, we explored the possible mechanism for the effect of caffeine administration or RW to stimulate UVB-induced apoptosis in the epidermis of SKH-1 mice. Female SKH-1 mice (5 mice per time point) were treated with: (1) oral water (control), (2) oral caffeine (0.1 mg/ml in the drinking water) (3) RW, or (4) 0.1 mg/ml oral caffeine plus RW for 2 weeks. The pretreated mice were then exposed to a single irradiation with UVB (30 mJ/cm2) and sacrificed at 0 (no UVB treatment), or at 5 min, 0.5h, 1h, 2h, 4h, 6h, 10h, 16h, 24h and 48h after UVB. The results showed that oral caffeine, RW, or caffeine plus RW for 2 weeks stimulated UVB-induced caspase 3 positive cells by 30%, 164% or 333%, respectively, at 6 hr after UVB (peak timepoint for UVB-induced apoptosis). These treatments had little inhibitory effect on cell proliferation. Unexpectedly, all of these treatments enhanced UVB-induced thymine dimer formation as measured by immunohistochemistry. Oral caffeine, RW, or the combination of caffeine and RW increased UVB-induced thymine dimer positive cells by 52%, 56% or 75%, respectively at 0.5 hr after UVB (peak for UVB-induced thymine dimer formation), when compared to the water control group. Thymine dimer positive cells were gone by 48 h post-UVB. The effect of caffeine and RW to increase the number of thymine dimer positive cells preceded the increase in apoptosis in the epidermis of UVB irradiated mice. Slot-blot analysis of isolated epidermal DNA from the mice treated with oral caffeine, RW or their combination for 2 weeks for thymine dimers confirmed the immunohistochemistry data. In a separate experiment, mice treated with instant coffee (10 mg coffee solids/ml) or caffeine (0.4 mg/ml) in the drinking fluid for one week prior to UVB irradiation showed enhanced UVB-induced increase in thymine dimer positive cells by 75-80% at 15-30 min post-UVB compared with water control mice irradiated with UVB. The coffee-induced increase in thymine dimer positive cells was followed by an increase in apoptosis (about 2-fold) at 6-10 hr post-UVB. Our results indicate that RW, oral administration of caffeine or coffee increase UVB-induced thymine dimers in DNA followed by an increase in apoptosis in the epidermis. We hypothesize that caffeine- or voluntary running wheel exercise-induced increase in thymine dimers may play a role in the ability of caffeine administration and RW to increase UVB-induced apoptosis and to inhibit UVB-induced carcinogenesis (Supported by NIH Grants RO1 CA128997 and RO1CA114442). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1815. doi:10.1158/1538-7445.AM2011-1815