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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.
More than two million cases of non-melanoma skin cancer occur each year in the United States (Siegal et al., 2012), and the number of these cancers has been increasing (Athas et al., 2003; Rogers et al., 2010). Indeed, the number of cases of non-melanoma skin cancer in the United States each year exceeds that for all of the other cancers combined. Accordingly, approaches for preventing these cancers, which are caused predominantly by overexposure to sunlight, are important. The inhibitory effect of caffeine on UVB-induced skin cancer in animal models described in this manuscript is paralleled by epidemiology studies indicating that drinking regular coffee, but not decaffeinated coffee, decreases the risk of non-melanoma skin cancer in humans (Jacobson et al., 1986; Abel et al., 2007; Song et al., 2012).
Several investigators have evaluated the effect of administration of caffeine post-UVC irradiation on the removal of thymine dimers and on the formation of UV-induced mutations in bacteria and cultured mammalian cells. Different results were obtained in different cell systems. In early studies, Sideropoulos and Shankel (1968) demonstrated that caffeine inhibited excision of thymine dimers from an excision repair proficient strain of E. coli that had been irradiated with UV, and this inhibitory effect of caffeine on excision repair was associated with increased UV-induced mutations. In E. coli strains (Hcr−) that lacked excision repair, post-UV administration of caffeine inhibited UV-induced mutations, and it was suggested that “caffeine could diminish mutagenesis in Hcr− strains by modifying the process of recombinational repair in a way that reduces its inaccuracy” (Witkin and Farquharson, 1969). In Chinese hamster cells lacking appreciable excision repair of thymine dimers, post-UV treatment with caffeine also inhibited UV-induced mutations (Trosko and Chu, 1973) possibly by inhibiting an error-prone repair system. In an additional study with Chinese hamster cells exposed to UV, caffeine inhibited the rescue of stalled replication forks by translesional DNA synthesis thereby causing a switch to bypass via homologous recombination (Johansson et al., 2006). The caffeine-induced decrease in translesional DNA synthesis was associated with decreased UV-induced mutations and with increased homologous recombination that resulted in increased chromosome aberrations (Johansson et al., 2006). Additional studies are needed to determine the effects of caffeine on error-free and error-prone repair of UV damage in bacteria, cultured mammalian cells, and in mice.
In the present manuscript, we describe animal data indicating that caffeine administration inhibits UVB-induced carcinogenesis by functioning as a sunscreen, as well as by enhancing UVB-induced apoptosis in the epidermis of UVB-treated mice by p53-dependent and p53-independent mechanisms. In long-term studies, caffeine administration enhances apoptosis in tumors of mice treated chronically with UVB.
Studies in our laboratory (AHC) demonstrated a strong inhibitory effect of ellagic acid, quercetin, myricetin, tannic acid, and many other plant phenols on the mutagenic action of the bay-region diol epoxide that is an ultimate carcinogenic metabolite of benzo[a]pyrene (Wood et al., 1982; Huang et al., 1983, 1985). Additional studies demonstrated that the mechanism of inhibition was by formation of a covalent adduct of the diol epoxide with the polyphenol (Sayer et al., 1982). In vivo studies indicated that ellagic acid strongly inhibited the tumorigenic activity of the bay-region diol epoxide of benzo[a]pyrene in mice (Chang et al., 1985), but ellagic acid and other polyphenols were only weak inhibitors of the tumorigenic action of benzo[a]pyrene (Chang et al., 1985). Because of our interest in plant phenols as inhibitors of carcinogenesis, we noted studies by Yoshizawa et al. (1987), who demonstrated that epigallocatechin gallate, a major polyphenol in green tea, inhibited tumor promotion on mouse skin. We also noted the studies by Mukhtar and his colleagues indicating an inhibitory effect of a green tea polyphenol fraction on UVB-induced carcinogenesis (Wang et al., 1991). These studies stimulated us to start studies with tea that led to our finding of caffeine as a strong inhibitor of UVB-induced carcinogenesis (Huang et al., 1997).
Our interest in caffeine was enhanced after finding that although oral administration of green and black tea inhibited UVB-induced complete carcinogenesis in SKH-1 mice, the decaffeinated teas were inactive, and high dose levels of the decaffeinated teas actually increased UVB-induced carcinogenesis (Huang et al., 1997). Adding back caffeine to the decaffeinated teas restored their inhibitory effects on UVB-induced carcinogenesis, and administration of caffeine alone had a strong inhibitory effect (Huang et al., 1997). The effects of oral administration of green tea, decaffeinated green tea, decaffeinated green tea plus caffeine, and caffeine alone on UVB-induced complete carcinogenesis are shown in Table Table1.1. The results indicated that oral caffeine during the course of twice a week exposure to UVB for 40weeks and for an additional 4weeks post-UVB strongly inhibited tumorigenesis. Shortly after we found an inhibitory effect of oral caffeine on UVB-induced carcinogenesis, we became aware of an earlier report indicating that topical application of caffeine just prior to each irradiation with UVB inhibited UVB-induced carcinogenesis in mice (Zajdela and Latarjet, 1978). Since UVA and solar radiation (UVA+UVB) are tumorigenic in mice (de Laat et al., 1997; Li et al., 2012), additional studies are needed to determine the effects of caffeine on tumorigenesis in animals exposed to UVA or solar radiation.
Since caffeine and caffeine sodium benzoate (a related, more potent inhibitor of UVB-induced skin cancer) have appreciable UV absorption between 260 and 300nm (with a peak at ~273nm), we studied the effect of topical application of these compounds prior to UVB irradiation on UVB-induced thymine dimers and sunburn lesions in the epidermis of SKH-1 mice. Topical application of caffeine or caffeine sodium benzoate 0.5h prior to UVB irradiation inhibited UVB-induced formation of thymine dimers and inhibited UVB-induced sunburn lesions, and caffeine sodium benzoate was more effective than caffeine (Figure (Figure1)1) (Lu et al., 2007).
Since caffeine and caffeine sodium benzoate have a sunscreen effect, this was avoided by evaluating the effect of the compounds on UVB-induced apoptosis by applying them immediately after UVB irradiation. Topical application of 3.1–24.8μmol of caffeine or caffeine sodium benzoate to SKH-1 mice immediately after irradiation with UVB (30mJ/cm2) caused a dose-dependent increase in UVB-induced apoptosis (Figure (Figure2)2) (Lu et al., 2007), and caffeine sodium benzoate was more effective than caffeine. Administration of caffeine or caffeine sodium benzoate in the absence of UVB was inactive (data not presented).
SKH-1 mice were treated orally with green tea (6mg tea solids/ml drinking fluid) or caffeine (0.44mg/ml in the drinking fluid) for 2weeks prior to irradiation with UVB (30mJ/cm2). Pretreatment with oral green tea or caffeine enhanced UVB-induced increases in p53 positive cells, p21 positive cells, and apoptotic sunburn cells (Figure (Figure3)3) (Lu et al., 2000). Oral administration of coffee (10mg coffee solids/ml) had a similar stimulatory effect on UVB-induced apoptosis (Conney et al., 2007). Oral administration of green tea or caffeine had no effect on p53, p21, or apoptosis in the absence of UVB irradiation indicating that these agents enhanced apoptosis only in DNA damaged epidermis but not in normal epidermis (Figure (Figure3).3). To our knowledge, these studies provided the first in vivo demonstration of the upregulation of a tumor suppressor by a cancer preventive agent.
In normal human and mouse keratinocytes with functional p53, UVB irradiation increased the levels of p53 and p21 (Lu et al., 2000; Lei et al., 2010). However, in studies with cultured human HaCaT keratinocytes with defective p53 by Lei et al. (2010) UVB irradiation markedly decreased the level of p21, which was associated with enhanced apoptosis. Inhibition of MDM2 and GSK3β (but not inhibition of ATR/ATM by siRNAs or caffeine) prevented the UVB-induced downregulation of p21 and prevented UVB-induced apoptosis (Lei et al., 2010). It will be important to determine the in vivo significance of these studies by doing studies in mice with UVB-induced p53 mutations in their epidermal cells. Studies of p21 expression and apoptosis in p53 knockout mice will also help define the relationship of UVB and caffeine to p21 in this particular situation.
Although UVB-induced apoptosis in the epidermis was markedly decreased in p53 knockout mice, topical application of caffeine immediately after UVB irradiation in these knockout mice markedly stimulated UVB-induced apoptosis by a p53-independent effect (Figure (Figure4)4) (Lu et al., 2004).
Treatment of primary human keratinocytes with caffeine inhibited UVB-induced increase in ATR-mediated formation of p-Chk1 (Ser 345), and apoptosis was increased (Figure (Figure6)6) (Heffernan et al., 2009). Genetic inhibition of ATR by siRNA for ATR also enhanced UVB-induced apoptosis, which was not increased further by the addition of caffeine (Figure (Figure6)6) (Heffernan et al., 2009). These results suggest that caffeine enhances UVB-induced apoptosis in primary human keratinocytes predominantly by inhibiting ATR-mediated phosphorylation of Chk1. Our cell culture studies are in agreement with in vivo studies presented later in this review indicating that transgenic mice with diminished ATR function have decreased UVB-induced tumorigenesis when compared with littermate controls with normal ATR.
In studies with cultured human HaCaT keratinocytes with defective p53 by Han et al. (2011), treatment with caffeine enhanced UVB-induced apoptosis, inhibited UVB-induced phosphorylation of Chk1 as well as the phosphorylation of AKT and the upregulation of COX-2. In these cells, siRNA inhibition of ATR did not have a significant effect on UVB-induced apoptosis (Han et al., 2011), which differed from our data obtained from studies with primary human keratinocytes (Figure (Figure6)6) (Heffernan et al., 2009). The reasons for differences in the effects of siRNA for ATR on UVB-induced apoptosis between the two cell lines may be related to different properties of the various cells lines or to the use of different incubation conditions for the two studies. The lack of effect of siRNA for ATR on UVB-induced apoptosis observed by Han and his colleagues may have resulted from incubation conditions whereby maximum apoptosis already occurred prior to the addition of siRNA for ATR. The in vivo importance of decreasing ATR function for inhibition of UVB carcinogenesis was demonstrated by showing that decreasing ATR function in transgenic mice inhibited UVB-induced carcinogenesis (see Figure Figure11).11). Additional studies are needed to determine the in vivo effects of caffeine administration on UVB-induced phosphorylation of AKT and UVB-induced upregulation of COX-2 in the epidermis of p53 proficient and p53 deficient mice.
Treatment of SKH-1 mice with caffeine or caffeine sodium benzoate (2.1mmol/l as the drinking fluid) for 1week inhibited the UVB-induced increase in p-Chk1 (Ser 345) by 82 and 99%, respectively at 6h post-UVB, and cyclin B1 was increased 153 and 201%, respectively at 6h post-UVB (Figure (Figure7)7) (Lu et al., 2008). The time course for the UVB-induced increase in p-Chk1 (Ser 345), and the effect of caffeine to abrogate the UVB-induced increase in p-Chk1 (Ser 345) and the decrease in cyclin B1 is shown in Figure Figure88 (Lu et al., 2008). Caffeine-induced abrogation of the UVB-induced decrease in cyclin B1 is associated with premature mitosis and cell death (Figure (Figure88).
Treatment of SKH-1 mice with UVB (30mJ/cm2) twice a week for 20weeks resulted in mice without tumors but with epidermal hyperplasia and a high risk of developing tumors over the next several months in the absence of further treatment with UVB (“high-risk mice”) (Lou et al., 1999). Treatment of these UVB-pretreated “high-risk mice” with oral caffeine (Lou et al., 1999) or with topical applications of caffeine (Lu et al., 2002) inhibited tumor formation. The inhibitory effect of topical applications of caffeine on tumor formation in high-risk mice is shown in Table Table2.2. Treatment of high-risk mice with topical applications of caffeine (6.2μmol) once a day, five times per week for 18weeks inhibited the percent of mice with squamous cell carcinomas by 64% and the number of carcinomas/mouse by 72%. Administration of caffeine increased apoptosis in the tumors but not in areas of the epidermis away from tumors indicating selectivity for caffeine action on tumors but not in areas away from tumors (Table (Table3)3) (Lu et al., 2002).
Topical applications of caffeine to UVB-pretreated high-risk mice inhibited carcinogenesis as described in Table Table22 and enhanced apoptosis in the tumors (but not in non-tumor areas) as described in Table Table3.3. Immunohistochemistry revealed islands of phospho-Chk1 (Ser 317) in the tumors but not in areas away from the tumors, and caffeine administration decreased the number of positively stained islands and also decreased the intensity of staining (Lu et al., 2011). We also observed that some mitotic cells in the tumors were positive for cyclin B1 staining and for caspase 3 (active form) staining. Representative mitotic tumor cells with cyclin B1 or apoptosis (caspase 3, active form) staining are shown in Figure Figure99 (Lu et al., 2011).
Topical caffeine increased the percentage of mitotic tumor cells with cyclin B1 by 70% (Lu et al., 2011), and the percentage of mitotic keratoacanthoma and squamous cell carcinoma cells with caspase 3 (active form) was increased by 214 and 317%, respectively (Figure (Figure10)10) (Lu et al., 2011). Interestingly, caffeine administration did not increase the percentage of mitotic cells with caspase 3 in non-tumor areas of the epidermis in tumor-bearing mice.
To determine the effect of genetic inhibition of ATR on UVB-induced carcinogenesis, we generated transgenic mice with diminished ATR function in skin and crossed them into a UV-sensitive Xpc−/− background. Unlike caffeine, this genetic approach was selective for ATR function and had no effect on ATM activation by DNA damage. Primary keratinocytes from these mice had diminished UV-induced Chk1 phosphorylation and a twofold increase in apoptosis after UV exposure. With chronic UV treatment, transgenic mice remained tumor-free for significantly longer and had 69% fewer tumors at the end of observation of the full cohort than in littermate controls (Figure (Figure11)11) (Kawasumi et al., 2011).
Since oral administration of caffeine increased locomotor activity and decreased tissue fat in SKH-1 mice (Lu et al., 2001; Michna et al., 2003), we evaluated the effect of voluntary running wheel exercise and removal of the parametrial fat pads on UVB-induced apoptosis and UVB-induced skin cancer (Lu et al., 2006, 2012; Michna et al., 2006). Both running wheel exercise and removal of the parametrial fat pads had the same effects as oral caffeine administration – stimulation of UVB-induced apoptosis, inhibition of UVB-induced carcinogenesis, and enhanced apoptosis in the tumors of SKH-1 mice treated chronically with UVB (Lu et al., 2006, 2012; Michna et al., 2006). The results suggest that oral administration of caffeine may inhibit UVB-induced carcinogenesis in part by increasing locomotor activity and by decreasing tissue fat.
Animal data indicate that caffeine administration inhibits UVB-induced carcinogenesis by functioning as a sunscreen as well as by enhancing UVB-induced apoptosis and apoptosis in UVB-induced tumors. The stimulatory effect of caffeine on UVB-induced apoptosis occurs by p53-dependent and p53-independent mechanisms. Inhibition of the ATR/Chk1 pathway by caffeine is a major contributor to caffeine inhibition of UVB-induced carcinogenesis. The inhibitory effects of caffeine on UVB-induced carcinogenesis in animal studies described here are paralleled by an inhibitory effect of regular but not decaffeinated coffee on non-melanoma skin cancer in humans (Jacobson et al., 1986; Abel et al., 2007; Song et al., 2012).
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.