SKH-1 hairless mice were irradiated with ultraviolet B (UVB) twice weekly for 20 weeks. These tumor-free mice, which had a high risk of developing skin tumors during the next several months, were then treated topically with caffeine (6.2 mol) or (؊)-epigallocatechin gallate (EGCG; 6.5 mol) once a day 5 days a week for 18 weeks in the absence of further treatment with UVB. Topical applications of caffeine to these mice decreased the number of nonmalignant and malignant skin tumors per mouse by 44% and 72%, respectively. Topical applications of EGCG decreased the number of nonmalignant and malignant tumors per mouse by 55% and 66%, respectively. Immunohistochemical analysis showed that topical applications of caffeine or EGCG increased apoptosis as measured by the number of caspase 3-positive cells in nonmalignant skin tumors by 87% or 72%, respectively, and in squamous cell carcinomas by 92% or 56%, respectively, but there was no effect on apoptosis in nontumor areas of the epidermis. Topical applications of caffeine or EGCG had a small inhibitory effect on proliferation in nonmalignant tumors as measured by BrdUrd labeling (16 -22%), and there was also a similar, but nonsignificant, inhibitory effect on proliferation in malignant tumors. The results suggest a need for further studies to determine whether topical applications of caffeine or EGCG can inhibit sunlight-induced skin cancer in humans.tea constituents ͉ programmed cell death ͉ caspase 3 S kin cancer is a major cancer in the United States, and its incidence is expected to increase substantially because of increased recreational exposure to sunlight and depletion of the ozone layer (1, 2). The identification and use of protective agents should have an important impact on the formation of these cancers. Although the use of sunscreens is an approach that has decreased the risk of skin cancers (3, 4), there is also a need to identify additional approaches for skin cancer prevention in individuals previously exposed to high-dose levels of sunlight (high-risk individuals). Treatment of SKH-1 hairless mice with ultraviolet B (UVB) (30 mJ͞cm 2 ) twice a week for 20 weeks resulted in mice without tumors but with epidermal hyperplasia and a high risk of developing skin tumors during the next several months in the absence of further UVB treatment (initiated high-risk mice) (5). This animal model resembles humans who are heavily exposed to sunlight early in life and then have reduced exposure later in life. We have used UVB-pretreated high-risk mice for evaluating the effects of potential chemopreventive agents on skin tumor formation in the absence of further exposure to UVB. Oral administration of green tea, black tea, or caffeine to UVBpretreated high-risk mice inhibited tumorigenesis, but the decaffeinated teas had little or no activity, and reconstitution of the decaffeinated teas with caffeine restored biological activity (5). These observations indicate that caffeine is a major cancer chemopreventive constituent in tea. Potential antitumor mechanisms include...

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 ataxiatelangiectasia mutated-and Rad3-related (ATR) pathway, causing a severalfold increase in keratinocytes with phosphoChk1 (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 À/À 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 ATRmediated phosphorylation of Chk1 and prematurely increasing the number of cyclin B1-containing cells that undergo lethal mitosis. [Cancer Res 2008;68(7):2523-9]

Irradiation of female SKH-1 hairless mice with UVB (30 mJ/cm2) twice a week for 10-20 weeks resulted in the formation of a large number of cellular patches (>8 adjacent cells/patch) that are recognized with an antibody (Pab240) which recognizes mutated but not wild-type p53 protein. These patches are not recognized by an antibody (Pab1620) to wild-type p53 protein. The patches, which are considered putative early cellular markers of the beginning of tumor formation, started appearing after 4-6 weeks of UVB treatment, and multiple patches were observed after treatment for 10 weeks. The number and size of the patches increased progressively with continued UVB treatment. Discontinuation of UVB for 4 weeks resulted in an 80-90% decrease in the number of these patches. The number of the remaining patches did not decrease any further but remained relatively constant for at least 4-9 weeks. Oral administration of green tea (6 mg tea solids/ml) or caffeine (0.4 mg/ml) as the sole source of drinking fluid during irradiation with UVB, twice a week for 20 weeks, inhibited UVB-induced formation of mutant p53 positive patches by approximately 40%. Oral administration of green tea (6 mg tea solids/ml) as the sole source of drinking fluid or topical applications of caffeine (6.2 micromol) once a day 5 days a week starting immediately after discontinuation of UVB treatment enhanced the rate and extent of disappearance of the mutant p53-positive patches. Topical applications of caffeine to the dorsal skin of mice pretreated with UVB for 20 weeks resulted in enhanced apoptosis selectively in focal basal cell hyperplastic areas of the epidermis (putative precancerous lesions), but not in areas of the epidermis that only had diffuse hyperplasia. Our studies indicate that the chemopreventive effect of caffeine or green tea may occur by a proapoptotic effect preferentially in early precancerous lesions.

The percentage of animals with some tumor regression after 21 days of treatment was 0% for the control group, 31% for the ATRA group, 62% for the TPA group, and 100% for the TPA؉ATRA group (13 mice/ group). Although treatment of the mice with TPA or TPA؉ATRA continued to inhibit tumor growth for the duration of the 46-day study, treatment of the mice with ATRA alone did not inhibit tumor growth beyond 28 days of daily injections (6 mice/group). Mechanistic studies indicated that treatment of the mice with TPA or TPA؉ATRA for 46 days increased apoptosis in the tumors, and treatment with TPA؉ATRA also decreased the mitotic index. Because the dose of TPA used in this study was effective and resulted in clinically achievable blood levels, clinical trials with TPA alone or in combination with ATRA in patients with prostate cancer may be warranted.

Female CD-1 mice were initiated with a single topical application of 7,12-dimethylbenz[a]anthracene and promoted with 12-O-tetradecanoylphorbol-13-acetate. Mice with established papillomas were then treated with black tea or decaffeinated black tea (approximately 4 mg tea solids/ml) as the sole source of drinking fluid for 11-15 weeks. In four separate experiments, oral administration of black tea inhibited the growth of papillomas (increase in tumor volume/mouse) by an average of 35%, 37%, 41% and 48%, respectively. Studies with decaffeinated black tea gave inconsistent results. In one experiment, administration of decaffeinated black tea inhibited papilloma growth (increase in tumor volume/mouse) by 27%, but in two additional experiments papilloma growth was stimulated by 14% and 193%, respectively. In a separate experiment, skin tumors were generated by treating SKH-1 female mice with ultraviolet B light (UVB; 30 mJ/cm2) twice weekly for 22 weeks, after which UVB administration was stopped. Tumors were allowed to develop during the following 13 weeks, and tumor-bearing mice were then treated with black tea (6 mg/ml tea solids) as the drinking fluid for 11 weeks. In this experiment, tumor growth (increase in tumor volume/mouse) was inhibited by 70%. Histological examination revealed that tea-treated mice had a 58% decrease in the number of nonmalignant tumors (primarily keratoacanthomas)/mouse and a 54% decrease in the number of squamous cell carcinomas/mouse. In addition, administration of black tea decreased the volume per tumor by 60% for nonmalignant tumors and by 84% for carcinomas. Mechanistic studies with tumors from these mice revealed that administration of black tea decreased the bromodeoxyuridine labeling index in squamous cell papillomas, keratoacanthomas and squamous cell carcinomas by 56%, 45% and 35%, respectively, and the apoptosis index was increased by 44%, 100% and 95%, respectively. Administration of black tea decreased the mitotic index in keratoacanthomas and squamous cell carcinomas by 42% and 16%, respectively. The results indicate that oral administration of black tea to tumor-bearing mice inhibited proliferation and enhanced apoptosis in nonmalignant and malignant skin tumors.

BackgroundSleep disturbance is very common following traumatic brain injury (TBI), which may initiate or exacerbate a variety of co-morbidities and negatively impact rehabilitative treatments. To date, there are paradoxical reports regarding the associations between inherent characteristics of TBI and sleep disturbance in TBI population. The current study was designed to explore the relationship between the presence of sleep disturbance and characteristics of TBI and identify the factors which are closely related to the presence of sleep disturbance in TBI population.Methods98 TBI patients (72 males, mean age ± SD, 47 ± 13 years, range 18-70) were recruited. Severity of TBI was evaluated based on Glasgow Coma Scale (GCS). All participants performed cranial computed tomography and were examined on self-reported sleep quality, anxiety, and depression.ResultsTBI was mild in 69 (70%), moderate in 15 (15%) and severe in 14 (15%) patients. 37 of 98 patients (38%) reported sleep disturbance following TBI. Insomnia was diagnosed in 28 patients (29%) and post-traumatic hypersomnia in 9 patients (9%). In TBI with insomnia group, 5 patients (18%) complained of difficulty falling asleep only, 8 patients (29%) had difficulty maintaining sleep without difficulty in initial sleep and 15 patients (53%) presented both difficulty falling asleep and difficulty maintaining sleep. Risk factors associated with insomnia were headache and/or dizziness and more symptoms of anxiety and depression rather than GCS. In contrast, GCS was independently associated with the presence of hypersomnia following TBI. Furthermore, there was no evidence of an association between locations of brain injury and the presence of sleep disturbance after TBI.ConclusionOur data support and contribute to a growing body of evidence which indicates that TBI patients with insomnia are prone to suffer from concomitant headache and/or dizziness, report more symptoms of anxiety and depression and severe TBI patients are likely to experience hypersomnia.

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.skin cancer prevention | squamous cell carcinoma | xeroderma pigmentosum M ultiple human epidemiologic studies have revealed that coffee or tea intake decreases the risk of UV-associated nonmelanoma skin cancers (1-4) and non-UV-associated cancers, most prominently hepatocellular and endometrial cancers (5). In the largest study, among 93,676 women, each daily cup of caffeinated coffee consumption was dose-dependently associated with a 5% reduction in the prevalence of nonmelanoma skin cancers, whereas decaffeinated coffee had no effect, and tea had an intermediate effect, consistent with its caffeine content (3). These observations in humans hold true in multiple related studies in mice. Oral intake of caffeine in the drinking water of chronically irradiated SKH-1 hairless mice suppressed UVinduced skin cancer development (6). Topical application of caffeine in UV-pretreated "high-risk" mice also inhibited UVinduced squamous cell carcinomas (SCCs) in hairless mice by 72% (7).UV-induced skin tumor development is a complex, long-term pathological process in vivo, and it is unclear how caffeine sup-

Lactic acid bacteria have been categorized as probiotics and play a crucial role in human health by stimulating the supply of nutrients, shaping the immune system, and preventing the colonization of pathogenic microbes. This study investigated the mechanisms for the action of three potential probiotic Lactobacillus strains: Lactobacillus casei SR1, Lactobacillus casei SR2, and Lactobacillus paracasei SR4 isolated from human breast milk. These Lactobacillus strains were identified via 16S DNA sequencing and characterized via biochemical assays including acid resistance, bile resistance, antioxidant activity, and antibiotic susceptibility. The bioactivity of the cell-free culture supernatant (CFCS) secreted by these strains on the cervix cancer (HeLa) cell line was also evaluated via cytotoxicity assay and apoptosis analysis. The mechanism of anticancer activity was also investigated via RT-qPCR and western blotting. The results demonstrated that these newly isolated Lactobacillus strains from human milk displayed noticeable probiotic characteristics such as excellent antibiotic susceptibility, outstanding antioxidant activity, and promising resistance to low pH and high concentration of bile salts. The results of the conducted bioactivity assays verified that the CFCSs had acceptable anticancer effects on cervix cancer (HeLa) cells by upregulating the expression of apoptotic genes BAX, BAD, caspase3, caspase8, and caspase9 and by downregulating the expression of the BCl-2 gene. Overall, these results indicate that the Lactobacillus strains isolated from human breast milk could be considered as a topical medication with a potential therapeutic index due to their efficacy against cervix cancer cells.