Inhibition of photocarcinogenesis
Nonmelanoma skin cancers, including basal cell and squamous cell carcinomas, represent the most common malignant neoplasms in humans [65, 84, 88]. Epidemiological, clinical and biological studies have indicated that solar UV radiation is the major etiological agent in the development of skin cancers [7, 65, 81, 84]. Various animal models have been employed to examine the anti-photocarcinogenic effects of phytochemicals, like polyphenols. Following standard photocarcinogenesis protocols, it has been found that oral administration of GTPs (a mixture of green tea polyphenols or catechins) in drinking water of mice resulted in significant protection against skin tumorigenesis in terms of tumor incidence, tumor multiplicity and tumor size per group compared to non-GTPs-treated animals [reviewed in 36, 38, 44, 45, 99]. A water extract of green tea leaves, which primarily contained a mixture of polyphenolic ingredients, when provided as the sole source of drinking water to mice afforded protection against UVB radiation-induced tumorigenesis , and also promoted partial regression of established skin papillomas in mice . Topical treatment of SKH-1 hairless mouse skin with GTPs or (−)-epigallocatechin-3-gallate (EGCG) in a hydrophilic ointment significantly inhibited UVB-induced skin tumor development . Dietary grape seed proanthocyanidins (GSPs, 0.2 and 0.5%, w/w) supplementation of a control AIN76A diet inhibited photocarcinogenesis in SKH-1 hairless mice in terms of tumor incidence (% mice with tumors), tumor multiplicity and tumor size . Dietary GSPs also resulted in prevention of malignant progression of UVB-induced papillomas to carcinomas as compared to the malignant progression observed in non-GSPs-treated UVB-exposed control mice . Resveratrol is found in the skin of colored grapes, peanuts, red wine and mulberries. Topical application of resveratrol inhibits UVB-induced skin tumor initiation, promotion and progression [4, 29]. Silymarin, a flavonoid obtained from milk thistle, also has been shown to have anti-photocarcinogenic activity in laboratory animals. We  have shown that topical application of silymarin to SKH-1 hairless mice inhibited UVB-induced skin tumor development in terms of tumor incidence, tumor multiplicity and growth of the tumors. Silibinin, which is a major component of silymarin, has been shown to inhibit photocarcinogenesis in mice when applied topically or in the diet [21, 22]. As multiple in vivo animal studies suggest that plant polyphenols possess anti-photocarcinogenic activity, we will briefly summarize and discuss the molecular targets or mechanisms of action of these selected polyphenols against photocarcinogenesis.
Mechanism of Action and Molecular Targets of Polyphenols
Most of the natural polyphenols are pigments, typically yellow, red or purple, and can absorb UV radiation. Therefore, when applied topically, they can prevent penetration of the radiation into the skin. The radiation that polyphenols can absorb includes the entire UVB spectrum of wavelengths and part of the UVC and UVA spectra. Thus polyphenols may act as a sunscreen. This ability of natural polyphenols to act as sunscreens can reduce inflammation, oxidative stress and DNA damaging effects of UV radiation in the skin and, thus, on topical application the photoprotective effects of polyphenols are due in part to this sunscreen effect.
UV radiation-induced erythema, edema and hyperplastic epithelial responses are considered as inflammatory markers, and play crucial roles in skin tumor promotion [reviewed in 71]. UVB-induced cyclooxygenase-2 (COX-2) expression and a subsequent increase in the production of prostaglandin (PG) metabolites in the skin is a characteristic response of keratinocytes to acute or chronic exposure to UVB radiation. COX-2 is a rate-limiting enzyme for the generation of PG metabolites from arachidonic acid , and COX-2 expression has been linked to the pathophysiology of inflammation and cancer . A number of studies have demonstrated overexpression of COX-2 in chronically UVB-irradiated skin, as well as in UVB-induced premalignant lesions and squamous- and basal-cell carcinomas of the skin [8, 89]. Mechanistic studies of photocarcinogenesis have revealed that oral administration of GTPs (through addition to the drinking water) to SKH-1 hairless mice resulted in significant inhibition of UV radiation-induced cutaneous edema, erythema, and bi-fold skin thickness (a biomarker of inflammation). Treatment with GTPs also inhibits UVB-induced expression of COX-2 and its prostaglandin metabolites, which have been implicated in skin carcinogenesis and play a role in promoting tumors in the skin . Topical treatment with GTPs prior to UV exposure reduced the UV-induced hyperplastic response, myeloperoxidase activity and the numbers of infiltrating inflammatory leukocytes in the skin [37, 39, 43]. The relevance of the extensive in vitro and in vivo data that have been generated using animal models to the photoprotective effects of GTPs in human skin is not yet clearly understood. We have found, however, that topical application of GTPs prior to UV irradiation of the un-tanned backs of humans resulted in significantly less development of erythema as compared to the UV-irradiated skin that was not treated with GTPs [19, 41]. We also found that topical treatment of human skin with GTPs or EGCG (<1mg/cm2 skin area) prior to UVB exposure significantly reduced UVB-induced infiltration of inflammatory leukocytes and myeloperoxidase activity , which is used as a marker of tissue infiltration. Topical application of EGCG also resulted in inhibition of UVB-induced production of prostaglandin metabolites, including PGE2, PGF2α and PGD2, which play a critical role in inflammatory disorders and in proliferative skin diseases . Exposure of the skin to UV radiation is known to enhance the levels of proinflammatory cytokines. As the elevated levels of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6, contribute to the tumor promotion process, this effect would be expected to result in an earlier occurrence of tumors and more rapid progression [reviewed in 71]. The administration of GTPs in the drinking water of mice significantly reduced the levels of these proinflammatory cytokines in UVB-irradiated skin . The GTPs also reduced the levels of biomarkers of cellular proliferation in the UV-irradiated skin, including proliferating cell nuclear antigen (PCNA) and cyclin D1. The inhibitory effects of GTPs on these biomarkers of inflammation in UV-exposed skin provide further mechanistic evidence of the anti-carcinogenic effects of GTPs. Jeon et al. have examined the effects of dietary EGCG (1500 ppm in control diet) on UVB-induced inflammation in hairless mice . They observed that regular intake of EGCG strengthens the skin’s tolerance and appears to do so by increasing the minimal dose of radiation required to induce erythema thereby inhibiting the UV-induced perturbation of epidermal barrier function and skin damage. Zhao et al.  demonstrated that oral administration of green tea extract prior to and during multiple treatments with psoralen plus UVA reduced hyperplasia, hyperkeratosis, erythema and edema formation in murine skin. Treatment of EpiDerm, a reconstituted human skin equivalent, with a green tea extract also has been shown to inhibit the 8-methoxypsoralen-DNA adduct formation and p53 protein accumulation associated with exposure to psoralen plus UVA irradiation . Mnich et al.  found that topical treatment of human skin with green tea extract reduced UV-induced p53 expression and the number of apoptotic keratinocytes, suggesting that green tea extract ma y b e a suitable everyday photochemopreventive agent. These in vivo observations that have been generated using both animal and human systems provide insights into the possible protective mechanisms involved in the anti-inflammatory effects of green tea polyphenols.
The in vivo effects of other polyphenols like resveratrol, grape seed proanthocyanidins and silymarin also have been examined using animal models [reviewed in 6]. As was found for the GTPs and EGCG, topical treatment or dietary intake of the GSPs and/or silymarin inhibited UVB radiation-induced edema, erythema, infiltration of inflammatory leukocytes and myeloperoxidase activity in the mouse skin [21, 22, 40, 66]. Silymarin has been shown to inhibit UVB-induced COX-2 expression and subsequently the production of PG metabolites, which are considered to be tumor promoters in the skin. Silymarin also has been shown to inhibit the expression of ornithine decarboxylase, an enzyme required for polyamine biosynthesis, which has a role in tumor promotion in UVB-exposed skin . Topical application of resveratrol prior to UVB irradiation resulted in significant inhibition of UVB-induced increases in bi-fold skin thickness (a marker of edema development), hyperplastic response, leukocyte infiltration, and COX-2 and ornithine decarboxylase activity in SKH-1 hairless mouse skin [2, 3]. Collectively, the results concerning the inhibitory effects of these polyphenols on UVB-induced inflammatory responses revealed that anti-photocarcinogenic effects of polyphenols are mediated in part through their anti-inflammatory effects.
The skin possesses an elaborate antioxidant defense system to deal with UV-induced oxidative stress; however, excessive and chronic exposure to UV radiation can overwhelm the cutaneous antioxidant capacity, leading to oxidative stress and oxidative damage which may result in skin disorders, immunosuppression, premature aging of the skin and development of melanoma and non-melanoma skin cancers. GTPs have been shown to inhibit photo-enhanced lipid peroxidation . Topical treatment of the mouse and human skin with EGCG prior to UV exposure significantly reduced UVB-induced nitric oxide and hydrogen peroxide production, as well as leukocyte infiltration [19, 41, 43]. It is well established that the infiltrating leukocytes are the major source of nitric oxide and hydrogen peroxide production, which create the state of oxidative stress. EGCG has been shown to have the ability to block UVB-induced leukocyte infiltration in mouse as well as in human skin, and thus may be able to inhibit UVB-induced production of reactive oxygen species by these infiltrating leukocytes [34, 37, 41, 43]. Although reactive oxygen species help the host to destroy invading microorganisms , excessive and uncontrolled production can also damage host tissues and predispose it to various disease states [23, 49]. Thus, the application of EGCG may prove beneficial in ameliorating the harmful effects caused by UVB radiation through its ability to reduce the generation of reactive oxygen species. Treatment with EGCG also has been shown to result in a reduction in the numbers of hydrogen peroxide producing and inducible nitric oxide synthase expressing cells, as well as a reduction in the production of hydrogen peroxide and nitric oxide both in the epidermis and dermis of UVB-irradiated skin sites . Similar effects also have been observed in human skin when EGCG was applied topically before exposure to UVB (4x minimal erythema dose) . This EGCG treatment also inhibited UV-induced epidermal lipid peroxidation and protected the antioxidant defense enzymes in the UVB-exposed human skin . Based on the evidence of the photoprotective effects of GTPs/EGCG in animal and human systems, it appears that both GTPs and EGCG can induce preventive effects by acting at different active sites within the cascade of events that generates reactive oxygen species. Kim et al.  observed that EGCG treatment of the skin of guinea pigs inhibits UVB-induced lipid peroxidation and the erythema response. They also found that EGCG treatment of human fibroblasts in culture blocked the UV-induced increase in collagen secretion and collagenase mRNA levels, and also inhibited the binding activities of the UV-induced nuclear transcription factors nuclear factor-kappaB (NF-κB) and activated protein (AP)-1 . Wei et al.  have demonstrated that aqueous extracts of green tea have potent scavenging effects on oxygen species and block UV-induced oxidative DNA damage in the calf thymus, which may, at least in part, explain the mechanisms by which green tea inhibits photocarcinogenesis. Collectively, these data suggest that green tea may have the potential to reduce the risk of UV-induced oxidative stress-mediated skin diseases or disorders in humans, including premature aging of the skin and development of cutaneous malignancies.
Oxidation of some amino acid residues, such as lysine, arginine and proline, leads to the formation of carbonyl derivatives that affect the nature and function of the proteins . The presence of carbonyl groups in proteins has become a widely accepted measure of oxidative damage of proteins under conditions of oxidative stress. Multiple exposures of the skin to UV radiation results in a several-fold increase in the levels of protein carbonyls in comparison to non-UV exposed skin. In separate experiments, it has been shown that topical treatment with EGCG, GTPs or GSPs significantly inhibits acute or chronic UV irradiation-induced protein oxidation in the skin of mice [82, 91]. The inhibition of UVB-induced protein oxidation by green tea polyphenols or proanthocyanidins could result in a reduction in skin photodamage and, more specifically, may prevent premature aging of the skin.
Treatment of normal human epidermal keratinocytes with EGCG in vitro was found to inhibit UVB-induced intracellular release of hydrogen peroxide concomitantly with the inhibition of UVB-induced oxidative stress-mediated phosphorylation of epidermal growth factor receptor and mitogen-activated protein kinases signaling pathways . Similar effects also were observed when HaCaT cells were treated with (−)-epicatechin-3-gallate (ECG) and exposed to UVB radiation. These in vitro studies suggest that ECG can act as a free radical scavenger when keratinocytes are photodamaged [26, 27]. The treatment of HaCaT cells with ECG also demonstrated its free radical scavenging effects when cells were irradiated with UVA radiation. These observations indicate that EGCG could play an important role in the attenuation of oxidative stress-mediated cellular signaling responses, which are essential factors in various skin diseases in humans. Topical cream-based formulations of EGCG or GTPs for human use have been developed, and their photoprotective effects evaluated in vivo using an animal model. An exceptionally high photoprotective effect of EGCG or GTPs was observed against UV radiation-induced oxidative stress in the mouse skin when evaluated in terms of lipid peroxidation, hydrogen peroxide production and analysis of anti-oxidant defense enzymes [67, 90]. Topical treatment of EGCG or oral administration of GTPs in the drinking water of mice also has been shown to inhibit UVB radiation-induced depletion of antioxidant defense enzymes, such as catalase, glutathione peroxidase, superoxide dismutase and the levels of glutathione . A study has been conducted in an attempt to determine whether the sunscreen-containing green tea extracts protect human subjects from UV irradiation-induced photoaging and photoimmunosuppression . The investigators reported that a sunscreen containing different concentrations of green tea extracts conferred significant protection against biological events associated with photoaging (MMP-2, MMP-9) and photoimmunology (CD1a+ Langerhans cells). Similar to green tea, chemopreventive effects also were noted when mice were given a GSPs-supplemented AIN76A diet. The provision of dietary GSPs (0.2 and 0.5%, w/w) to mice exposed to either acute or chronic UVB irradiation was found to inhibit depletion of glutathione peroxidase, catalase, and glutathione, and to inhibit UVB-induced hydrogen peroxide, lipid peroxidation, protein oxidation and nitric oxide, in mouse skin . As UVB-induced oxidative stress mediates activation of mitogen-activated protein kinases (MAPK) and NF-κB signaling pathways, the effects of GSPs in vivo in the same animal model on these pathways also were examined. It was observed that the treatment with GSPs inhibited UVB-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun-N-terminal-kinase and p38 proteins of the MAPK family, which seemed to be mediated through reactivation of MAPK phosphatases . It has been shown that the GSPs can inhibit the UVB-induced activation of NF-κB through inhibition of degradation of IκBα and activation of IκB kinase α. Using an identical mouse model, it has been further demonstrated that dietary administration of GSPs resulted in inhibition of the expression of PCNA, cyclin D1, inducible nitric oxide synthase (iNOS) and COX-2 in the skin, which are NF-κB-targeted proteins. Similar studies were conducted in vitro using normal human epidermal keratinocytes with and without treatment with GSPs and UVB irradiation. The results were identical to those obtained in the mouse model, which suggests that results generated in this animal model of the photoprotective effects of GSPs can be extrapolated to the human system . Study also has been conducted to determine the effects of oligomeric proanthocyanidins on UV-induced melanogenesis of human melanocytes in vitro. The results of this study suggested that oligomeric proanthocyanidins have potential photoprotective effects on human melanocytes including scavenging of intracellular reactive oxygen species and adjustment of cell cycle check points . In an in vitro cell culture model, treatment of human epidermoid carcinoma A431 cells with GSPs resulted in inhibition of cell proliferation and induction of apoptotic cell death. This effect of GSPs was associated with the inhibition of constitutive expression of NF-κB/p65 and its targeted genes, such as COX-2, iNOS, PCNA, cyclin D1 and matrix metalloproteinase (MMP)-9 . These observations provide a molecular basis for the photoprotective effects of GSPs and GTPs in an in vivo model. Studies conducted by the authors have shown that topical treatment of SKH-1 hairless mouse skin with silymarin resulted in inhibition of UVB-induced intracellular production of H2O2 in both the epidermis and dermis when analyzed by immunohistochemistry and biochemical analytical procedures and compared with the results obtained using non-silymarin-treated control mice . In these experiments, it was found that the significant inhibition of the UVB-induced oxidative stress was associated with significant inhibition of UV-induced infiltration of activated macrophages and neutrophils. Treatment with silymarin also inhibits UVB-induced expression of inducible nitric oxide synthase and subsequently nitric oxide production [31, and reviewed in 32]. Resveratrol is also a potential polyphenolic antioxidant. Pretreatment of human epidermal keratinocytes with resveratrol inhibited UVB-mediated activation of the NF-κB pathway [1, 2]. In SKH-1 hairless mice, topical application of resveratrol inhibited UVB-induced inflammatory responses and hydrogen peroxide production, which is a stable source of oxidative stress, in the skin . Inhibition of these critical events by resveratrol may have contributed to the prevention of UV radiation-induced skin cancer in these mice. Park and Lee  have demonstrated that treatment of HaCaT cells with resveratrol before UVB irradiation resulted in an increase in cell survival of UVB-irradiated cells which was associated with the reduction of reactive oxygen species production. Additionally, the activation of caspase-3 and -8 was partially reduced in the resveratrol-pretreated HaCaT cells, implying that the attenuation of caspase-3 and -8 activation is involved in cell survival after UVB irradiation. Soybeans are a rich source of the isoflavones, genistein and daidzein, and are photoprotective [reviewed in 97]. Studies using SENCAR mice have shown that topical genistein treatment reduced UV radiation-induced activation of c-fos and c-jun in a dose-dependent manner . Genistein also has been shown to reduce UV radiation-induced oxidative and photodynamic DNA damage . Treatment of the human keratinocyte cell line NCTC 2544 with genistein prevented UV-induced enhancement of the DNA-binding activity of the signal transducer and activator of transcription-1 by acting as a tyrosine kinase inhibitor, thus limiting lipid peroxidation and increases in reactive oxygen species generation .
To further illustrate the role of polyphenols in dermatologic conditions as well as in skin photoprotection, we are providing an in-depth review of the studies on the effects of polyphenols from green tea.