Yao-Ping Lu

Effects of oral administration of tea, decaffeinated tea, and caffeine on the formation and growth of tumors in high-risk SKH-1 mice previously treated with ultraviolet B light.

Treatment of SKH-1 mice with ultraviolet B light (UV-B, 30 mJ/cm2) twice a week for 22-23 weeks resulted in tumor-free animals with a high risk of developing malignant and nonmalignant tumors during the next several months in the absence of further UV-B treatment (high-risk mice). In three separate experiments, oral administration of green tea or black tea (4-6 mg tea solids/ml) as the sole source of drinking fluid for 18-23 weeks to these high-risk mice inhibited the formation and decreased the size of nonmalignant squamous cell papillomas and keratoacanthomas as well as the formation and size of malignant squamous cell carcinomas. In one experiment all these inhibitory effects of tea were statistically significant, whereas in the two other experiments many but not all of the inhibitory effects of tea were statistically significant. The decaffeinated teas were inactive or less effective inhibitors of tumor formation than the regular teas, and adding caffeine back to the decaffeinated teas restored biological activity. Oral administration of caffeine alone (0.44 mg/ml) as the sole source of drinking fluid for 18-23 weeks inhibited the formation of nonmalignant and malignant tumors, and this treatment also decreased tumor size in these high-risk mice.

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.

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 Nrf2s 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. Cancer Prev Res; 7(3); 319–29. ©2014 AACR.

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.

Inhibition by dietary dibenzoylmethane of mammary gland proliferation, formation of DMBA–DNA adducts in mammary glands, and mammary tumorigenesis in Sencar mice

Dibenzoylmethane (DBM) is a minor constituent of licorice and a beta-diketone analogue of curcumin. Feeding 1% DBM in the diet to Sencar mice during both the initiation and the post-initiation periods strongly inhibited 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumor multiplicity and mammary tumor incidence by 97%. In further in vivo studies to elucidate the possible mechanisms of the inhibitory action of DBM, feeding the 1% DBM in the AIN-76A diet to immature Sencar mice for 4-5 weeks decreased the uterine wet weight by 43%, inhibited the proliferation rate of mammary gland epithelial cells by 53%, uterine epithelium by 23%, and uterine stroma by 77%, when mice were killed during the first estrus phase of estrous cycle. In addition, feeding 1% DBM in the diet to Sencar mice at 2 weeks before, during and 1 week after DMBA treatment (intubation of 1 mg DMBA per mouse once a week for 5 weeks) inhibited formation of total DMBA-DNA adducts in mammary glands by 72% using a post-32P-labeling assay. Thus, feeding 1% DBM diet to Sencar mice inhibited formation of DMBA-DNA adducts in mammary glands and lowered the proliferation rate of the mammary gland in vivo. These results may explain the strong inhibitory actions of dietary DBM on mammary carcinogenesis in mice.

Nrf2 null enhances UVB-induced skin inflammation and extracellular matrix damages

Nrf2 plays a critical role in defending against oxidative stress and inflammation. We previously reported that Nrf2 confers protection against ultraviolet-B (UVB)-induced inflammation, sunburn reaction, and is involved in sulforaphane-mediated photo-protective effects in the skin. In this study, we aimed to demonstrate the protective role of Nrf2 against inflammation-mediated extracellular matrix (ECM) damage induced by UVB irradiation. Ear biopsy weights were significantly increased in both Nrf2 wild-type (Nrf2 WT) and knockout (Nrf2 KO) mice one week after UVB irradiation. However, these weights increased more significantly in KO mice compared to WT mice, suggesting a greater inflammatory response in KO mice. In addition, we analyzed the protein expression of numerous markers, including macrophage inflammatory protein-2 (MIP-2), pro-matrix metalloproteinase-9 (MMP-9), and p53. p53, a regulator of DNA repair, was overexpressed in Nrf2 KO mice, indicating that the absence of Nrf2 led to more sustained DNA damage. There was also more substantial ECM degradation and increased inflammation in UVB-irradiated Nrf2 KO mice compared to UVB-irradiated WT mice. Furthermore, the protective effects of Nrf2 in response to UVB irradiation were mediated by increased HO-1 protein expression. Collectively, our results show that Nrf2 plays a key role in protecting against UVB irradiation and that the photo-protective effect of Nrf2 is closely related to the inhibition of ECM degradation and inflammation.

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.

Patches of Mutant p53-Immunoreactive Epidermal Cells Induced by Chronic UVB Irradiation Harbor the Same p53 Mutations as Squamous Cell Carcinomas in the Skin of Hairless SKH-1 Mice

Treatment of SKH-1 hairless mice with UVB (30 mJ/cm(2)) twice a week for 20 weeks results in the formation of cellular patches, long before the appearance of tumors, that are visualized in epidermal sheets with an antibody (PAb240) recognizing mutated p53 protein. Direct sequencing analysis of the whole coding region of the p53 gene (exons 2-11) detected one or two mutations in 64.4% of 104 analyzed patches and no mutations in nonstained adjacent normal controls. Homozygous mutation was detected in 22.4% of the mutant patches. Except for two nonsense mutations, all others were missense (exons 4-9) and mostly (95.5%) at the DNA-binding domain. Primer extension analysis of cloned PCR fragments found three of four double-mutated patches harboring different mutations in separate alleles. All mutation hotspots reported earlier in UVB-induced mouse squamous cell carcinomas (SCC) at codons 270 (Arg --> Cys), 149 (Pro --> Ser), 275 (Pro --> Leu and Pro --> Ser), and 176 (His --> Tyr) with a frequency of 32.1%, 7.1%, 14.7%, and 3.2% were detected in epidermal patches at a frequency 47.7%, 9.1%, 4.5%, and 2.3%, respectively. Mutations at codons 210 and 191 found in patches at respective frequencies of 8.0% and 4.5% were not previously detected in UVB-induced mouse SCC. In summary, (a) the p53 mutation profile of UVB-induced skin patches and SCC was very similar suggesting that patches are precursor lesions for SCC, (b) a small number of patches harbored mutations that were not before observed in SCC from UVB-treated mice, and (c) about 36% of the patches did not harbor a p53 mutation.

Stimulatory effect of oral administration of green tea and caffeine on locomotor activity in SKH-1 mice

Administration of green tea or caffeine was shown previously to inhibit ultraviolet B light-induced carcinogenesis in SKH-1 mice, and this effect was associated with a reduction in dermal fat. In the present study, oral administration of 0.6% green tea (6 mg tea solids/ml) or 0.04% caffeine (0.4 mg/ml; equivalent to the amount of caffeine in 0.6% green tea) as the sole source of drinking fluid to SKH-1 mice for 15 weeks increased total 24 hr locomotor activity by 47 and 24%, respectively (p<0.0001). Oral administration of 0.6% decaffeinated green tea (6 mg tea solids/ml) for 15 weeks increased locomotor activity by 9% (p<0.05). The small increase in locomotor activity observed in mice treated with decaffeinated green tea may have resulted from the small amounts of caffeine still remaining in decaffeinated green tea solutions (0.047 mg/ml). The stimulatory effects of orally administered green tea and caffeine on locomotor activity were paralleled by a 38 and 23% increase, respectively, in the dermal muscle layer thickness. In addition, treatment of the mice with 0.6% green tea or 0.04% caffeine for 15 weeks decreased the weight of the parametrial fat pad by 29 and 43%, respectively, and the thickness of the dermal fat layer was decreased by 51 and 47%, respectively. These results indicate that oral administration of green tea or caffeine to SKH-1 mice increases locomotor activity and muscle mass and decreases fat stores. The stimulatory effect of green tea and caffeine administration on locomotor activity described here may contribute to the effects of green tea and caffeine to decrease fat stores and to inhibit carcinogenesis induced by UVB in SKH-1 mice.

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.