Pathways involved in sunburn cell formation: deregulation in skin cancer.

The incidence of squamous cell carcinoma of the skin is rising worldwide for decades. Chronic exposure to sunlight is the most important environmental risk factor for this type of skin cancer. This is predominantly due to the DNA damaging effect of ultraviolet-B (UVB) in sunlight. UVB induces also sunburn cells, i.e. apoptotic keratinocytes, which is a crucial protective mechanism against the carcinogenic effects of UVB irradiation. This process is regulated by a wide range of molecular determinants involved in the balance between pro- and anti-apoptotic pathways. Growing evidence suggests that the deregulation of this balance by chronic UVB irradiation, contributes to the development of skin cancer. This review gives a brief summary of major known pathways involved in the regulation of keratinocyte survival and cell death upon UVB damage and discusses the contribution of the deregulation of these cascades to photocarcinogenesis.     

Ultra-violet B (UVB)-induced skin cell death occurs through a cyclophilin D intrinsic signaling pathway

UVB-induced skin cell damage involves the opening of mitochondrial permeability transition pore (mPTP), which leads to both apoptotic and necrotic cell death. Cyclophilin D (Cyp-D) translocation to the inner membrane of mitochondrion acts as a key component to open the mPTP. Our Western-Blot results in primary cultured human skin keratinocytes and in HaCaT cell line demonstrated that UVB radiation and hydrogen peroxide (H(2)O(2)) induced Cyp-D expression, which was inhibited by anti-oxidant N-acetyl cysteine (NAC). We created a stable Cyp-D deficiency skin keratinocytes by expressing Cyp-D-shRNA through lentiviral infection. Cyp-D-deficient cells were significantly less susceptible than their counterparts to UVB- or H(2)O(2)-induced cell death. Further, cyclosporine A (Cs-A), a Cyp-D inhibitor, inhibited UVB- or H(2)O(2)-induced keratinocytes cell death. Reversely, over-expression of Cyp-D in primary keratinocytes caused spontaneous keratinocytes cell death. These results suggest Cyp-D's critical role in UVB/oxidative stress-induced skin cell death.     

Activation of T-cell Protein-tyrosine Phosphatase Suppresses Keratinocyte Survival and Proliferation following UVB Irradiation

Chronic exposure to UV radiation can contribute to the development of skin cancer by promoting protein-tyrosine kinase (PTK) signaling. Studies show that exposure to UV radiation increases the ligand-independent activation of PTKs and induces protein-tyrosine phosphatase (PTP) inactivation. In the present work, we report that T-cell PTP (TC-PTP) activity is stimulated during the initial response to UVB irradiation, which leads to suppression of keratinocyte cell survival and proliferation via the down-regulation of STAT3 signaling. Our results show that TC-PTP-deficient keratinocyte cell lines expressed a significantly increased level of phosphorylated STAT3 after exposure to low dose UVB. This increase corresponded with increased cell proliferation in TC-PTP-deficient keratinocytes following UVB irradiation. Loss of TC-PTP also reduced UVB-induced apoptosis. Corroborating with these results, overexpression of TC-PTP in keratinocyte cell lines yielded a decrease in phosphorylated STAT3 levels, which corresponded with a significant decrease in cell proliferation in response to low dose UVB. We demonstrate that TC-PTP activity was increased upon UVB exposure, and overexpression of TC-PTP in keratinocyte cell lines further increased its activity in the presence of UVB. Treatment of TC-PTP-deficient keratinocytes with the STAT3 inhibitor STA21 significantly reduced cell viability following UVB exposure in comparison with untreated TC-PTP-deficient keratinocytes, confirming that the effect of TC-PTP on cell viability is mediated by STAT3 dephosphorylation. Combined, our results indicate that UVB-mediated activation of TC-PTP plays an important role in the STAT3-dependent regulation of keratinocyte cell proliferation and survival. Furthermore, these results suggest that TC-PTP may be a novel potential target for the prevention of UVB-induced skin cancer.     

Release of cytokines/chemokines and cell death in UVB-irradiated human keratinocytes, HaCaT

Ultraviolet (UV) B can lead to inflammatory responses such as sunburn, which involves the production of various inflammatory cytokines and chemokines, and the induction of cell death. Keratinocytes in the skin has one of the highest risks of exposure to UV. However, the detailed mechanisms underlying UVB irradiation-induced inflammation and cell death are not well known. Thus, we investigated the effect of UVB irradiation on the production of various cytokines/chemokines and the induction of cell death in UVB-irradiated human keratinocytes (HaCaT cells). We evaluated 11 cytokines/chemokines in cell culture supernatants from HaCaT cells exposed to 0-400 mJ/cm(2) UVB irradiation. UVB at a dose 400 mJ/cm(2) induced the release of various cytokines; interleukin (IL)-1beta, IL-6, IL-8, interferon (IFN)-gamma, granulocyte-colony stimulating factor (G-CSF), macrophage inflammatory protein (MIP)-1beta, and tumor necrosis factor (TNF)-alpha. These results suggest that UVB irradiation-induced the release of several cytokines/chemokines and led to cell death in human keratinocytes. UV exposure may be associated with multiple physiological events in the human skin.     

Differential activity of UV-DDB in mouse keratinocytes and fibroblasts: impact on DNA repair and UV-induced skin cancer

UV-damaged DNA-binding protein (UV-DDB) is essential for global genome nucleotide excision repair of UV-induced cyclobutane pyrimidine dimers (CPD) and accelerates repair of 6-4 photoproducts (6-4PP). The high UV-induced skin cancer susceptibility of mice compared to man has been attributed to low expression of the UV-DDB subunit DDB2 in mouse skin cells. However, DDB2 knockout mice exhibit enhanced UVB skin carcinogenesis indicating that DDB2 protects mice against UV-induced skin cancer. To resolve these apparent contradictory findings, we systematically investigated the NER capacity of mouse fibroblasts and keratinocytes. Compared to fibroblasts, keratinocytes exhibited an increased level of UV-DDB activity, contained significantly higher levels of other NER proteins (i.e. XPC and XPB) and displayed efficient repair of CPD. At low UVB dosages, the difference in skin cancer susceptibility between DDB2 KO and wild type mice was even much more pronounced than previously reported with high dose UVB exposures. Hence, our observations show that mouse keratinocytes express sufficient levels of UV-DDB for efficient repair of photolesions and efficient protection against UV-induced skin cancer at physiological relevant UV exposure.     

Resveratrol Couples Apoptosis with Autophagy in UVB-Irradiated HaCaT Cells

UVB radiation causes about 90% of non-melanoma skin cancers by damaging DNA either directly or indirectly by increasing levels of reactive oxygen species (ROS). Skin, chronically exposed to both endogenous and environmental pro-oxidant agents, contains a well-organised system of chemical and enzymatic antioxidants. However, increased or prolonged free radical action can overwhelm ROS defence mechanisms, contributing to the development of cutaneous diseases. Thus, new strategies for skin protection comprise the use of food antioxidants to counteract oxidative stress. Resveratrol, a phytoalexin from grape, has gained a great interest for its ability to influence several biological mechanisms like redox balance, cell proliferation, signal transduction pathways, immune and inflammatory response. Therefore, the potential of resveratrol to modify skin cell response to UVB exposure could turn out to be a useful option to protect skin from sunlight-induced degenerative diseases. To investigate into this matter, HaCaT cells, a largely used model for human skin keratinocytes, were treated with 25 or 100 µM resveratrol for 2 and 24 hours prior to UVB irradiation (10 to 100 mJ/cm²). Cell viability and molecular markers of proliferation, oxidative stress, apoptosis, and autophagy were analyzed. In HaCaT cells resveratrol pretreatment: reduces UVB-induced ROS formation, enhances the detrimental effect of UVB on HaCaT cell vitality, increases UVB-induced caspase 8, PARP cleavage, and induces autophagy. These findings suggest that resveratrol could exert photochemopreventive effects by enhancing UVB-induced apoptosis and by inducing autophagy, thus reducing the odds that damaged cells could escape programmed cell death and initiate malignant transformation.     

Arsenic Carcinogenesis in the Skin

Summary Chronic arsenic poisoning is a world public health issue. Long-term exposure to inorganic arsenic (As) from drinking water has been documented to induce cancers in lung, urinary bladder, kidney, liver and skin in a dose–response relationship. Oxidative stress, chromosomal abnormality and altered growth factors are possible modes of action in arsenic carcinogenesis. Arsenic tends to accumulate in the skin. Skin hyperpigmentation and hyperkeratosis have long been known to be the hallmark signs of chronic As exposure. There are significant associations between these dermatological lesions and risk of skin cancer. The most common arsenic-induced skin cancers are Bowen’s disease (carcinoma in situ), basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Arsenic-induced Bowen’s disease (As-BD) is able to transform into invasive BCC and SCC. Individuals with As-BD are considered for more aggressive cancer screening in the lung and urinary bladder. As-BD provides an excellent model for studying the early stages of chemical carcinogenesis in human beings. Arsenic exposure is associated with G2/M cell cycle arrest and DNA aneuploidy in both cultured keratinocytes and As-BD lesions. These cellular abnormalities relate to the p53 dysfunction induced by arsenic. The characteristic clinical figures of arsenic-induced skin cancer are: (i) occurrence on sun-protected areas of the body; (ii) multiple and recrudescent lesions. Both As and UVB are able to induce skin cancer. Arsenic treatment enhances the cytotoxicity, mutagenicity and clastogenicity of UV in mammalian cells. Both As and UVB induce apoptosis in keratinocytes by caspase-9 and caspase-8 signaling, respectively. Combined UVB and As treatments resulted in the antiproliferative and proapoptotic effects by stimulating both caspase pathways in the keratinocytes. UVB irradiation inhibited mutant p53 and ki-67 expression, as well as increased in the number of apoptotic cells in As-BD lesions which resulted in an inhibitory effect on proliferation. As-UVB interaction provides a reasonable explanation for the rare occurrences of arsenical cancer in the sun-exposed skin. The multiple and recurrent skin lesions are associated with cellular immune dysfunction in chronic arsenism. A decrease in peripheral CD4+ cells was noticed in the inhabitants of arsenic exposure areas. There was a decrease in the number of Langerhans cells in As-BD lesion which results in an impaired immune function on the lesional sites. Since CD4+ cells are the target cell affected by As, the interaction between CD4+ cells and epidermal keratinocytes under As affection might be closely linked to the pathogenesis of multiple occurrence of arsenic-induced skin cancer. In this review, we provide and discuss the pathomechanisms of arsenic skin cancer and the relationship to its characteristic figures. Such information is critical for understanding the molecular mechanism for arsenic carcinogenesis in other internal organs.     

Role for tumor necrosis factor-alpha in UVB-induced conversion of 7-dehydrocholesterol to 1alpha,25-dihydroxyvitamin D3 in cultured keratinocytes

UVB irradiation of cultured human keratinocytes induces both the conversion of 7-dehydrocholesterol (7-DHC) to calcitriol (1alpha,25(OH)(2)D(3)) and the release of tumor necrosis factor-alpha (TNF-alpha) in these cells. Calcitriol synthesis in human keratinocytes was reduced in the presence if a neutralizing polyclonal antibody directed against human TNF-alpha. On the other hand, we found a 1.7-fold higher stimulatory effect of UVB on liberation of TNF-alpha in cultured keratinocytes enriched with 7-DHC compared with irradiated cell cultures in absence of 7-DHC. These observations argue in favor of a synergetic relationship between generation of TNF-alpha and calcitriol in UVB irradiated keratinocytes. In addition, we found that TNF-alpha potently increases the conversion rate of Vitamin D(3) (cholecalciferol) to calcitriol in this cell system. The UVB-triggered formation of both TNF-alpha and calcitriol in cultured keratinocytes was wavelength-, time- and dose-dependent. Maximum formation of TNF-alpha and calcitriol was found at 300 nm and UVB doses of 30 mJ/cm2. The enhancement of both the formation of TNF-alpha and calcitriol in keratinocytes by UVB may be of relevance for regulation of growth and apoptosis in light-exposed epidermal cells and, in addition, may play a role in the UVB treatment of diseased skin including psoriasis.     

Ultraviolet radiation-induced apoptosis in keratinocytes: on the role of cytosolic factors

Epidemiological and experimental evidences have established solar ultraviolet (UV) radiation as the leading cause of skin cancers. Specifically, the frequency of non-melanoma skin cancer, one of the malignancies with the most rapidly increasing incidence, is directly related to the total exposure to solar UV light. As part of a general effort to elucidate the components of cellular signal transduction pathways, the mechanisms of cellular responses to UV radiation have received considerable attention over the last few years. These efforts were driven mainly by the conviction that understanding how normal cells respond to extracellular stimuli such as exposure to UV radiation will undoubtedly help in deciphering what goes wrong in a variety of clinical disorders including skin cancers and will assist in the development of novel therapeutic strategies. Studies over the last decade have established that UV radiation induces a bewildering array of signal transduction pathways, some of which could lead to apoptotic cell death. UV-induced cell death by apoptosis is considered to be a natural protective mechanism that removes damaged keratinocytes and circumvents the risk of malignant transformation. In this review, we summarize some of the most important findings regarding the response and role of mitogen-activated protein kinases in UVA and UVB radiation-induced signaling to apoptosis in keratinocytes. We will also briefly discuss what is known about the role of the BCL-2 family of proteins, the emerging role of lysosomal proteases and other important cytosolic signaling proteins in UV-induced apoptosis.     

Granulocyte‐Macrophage Colony‐Stimulating Factor (GM‐CSF) Controls the Proliferation and Differentiation of Mouse Epidermal Melanocytes from Pigmented Spots Induced by Ultraviolet Radiation B

Repeated exposure of ultraviolet radiation B (UVB) on the dorsal skin of hairless mice induces the development of pigmented spots long after its cessation. The proliferation and differentiation of epidermal melanocytes in UVB‐induced pigmented spots are greatly increased, and those effects are regulated by keratinocytes rather than by melanocytes. However, it remains to be resolved what factor(s) derived from keratinocytes are involved in regulating the proliferation and differentiation of epidermal melanocytes. In this study, primary melanoblasts (c. 80%) and melanocytes (c. 20%) derived from epidermal cell suspensions of mouse skin were cultured in a basic fibroblast growth factor‐free medium supplemented with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). GM‐CSF induced the proliferation and differentiation of melanocytes in those keratinocyte‐depleted cultures. Moreover, an antibody to GM‐CSF inhibited the proliferation of melanoblasts and melanocytes from epidermal cell suspensions derived from the pigmented spots of UV‐irradiated mice, but not from control mice. Further, the GM‐CSF antibody inhibited the proliferation and differentiation of melanocytes co‐cultured with keratinocytes derived from UV‐irradiated mice, but not from control mice. The quantity of GM‐CSF secreted from keratinocytes derived from the pigmented spots of UV‐irradiated mice was much greater than that secreted from keratinocytes derived from control mice. Moreover, immunohistochemistry revealed the expression of GM‐CSF in keratinocytes derived from the pigmented spots of skin in UV‐irradiated mice, but not from normal skin in control mice. These results suggest that GM‐CSF is one of the keratinocyte‐derived factors involved in regulating the proliferation and differentiation of mouse epidermal melanocytes from UVB‐induced pigmented spots.     

Selective induction of cell death in melanoma cell lines through targeting of Mcl-1 and A1

Melanoma is an often fatal form of skin cancer which is remarkably resistant against radio- and chemotherapy. Even new strategies that target RAS/RAF signaling and display unprecedented efficacy are characterized by resistance mechanisms. The targeting of survival pathways would be an attractive alternative strategy, if tumor-specific cell death can be achieved. Bcl-2 proteins play a central role in regulating survival of tumor cells. In this study, we systematically investigated the relevance of antiapoptotic Bcl-2 proteins, i.e., Bcl-2, Bcl-xL, Bcl-w, Mcl-1, and A1, in melanoma cell lines and non-malignant cells using RNAi. We found that melanoma cells required the presence of specific antiapoptotic Bcl-2 proteins: Inhibition of Mcl-1 and A1 strongly induced cell death in some melanoma cell lines, whereas non-malignant cells, i.e., primary human fibroblasts or keratinocytes were not affected. This specific sensitivity of melanoma cells was further enhanced by the combined inhibition of Mcl-1 and A1 and resulted in 60% to 80% cell death in all melanoma cell lines tested. This treatment was successfully combined with chemotherapy, which killed a substantial proportion of cells that survived Mcl-1 and A1 inhibition. Together, these results identify antiapoptotic proteins on which specifically melanoma cells rely on and, thus, provide a basis for the development of new Bcl-2 protein-targeting therapies.     

Protective effect of inositol hexaphosphate against UVB damage in HaCaT cells and skin carcinogenesis in SKH1 hairless mice

UVB radiation damages keratinocytes, potentially inducing chronic skin damage, cutaneous malignancy, and suppression of the immune system. Naturally occurring agents have been considered for prevention and treatment of various kinds of cancer, including skin cancer. Inositol hexaphosphate (IP6), an antioxidant, is a naturally occurring polyphosphorylated carbohydrate that has shown a strong anticancer activity in several experimental models. We assessed the protective effects of IP6 against UVB irradiationinduced injury and photocarcinogenesis by using HaCaT cells (human immortalized keratinocytes) and SKH1 hairless mice. We found that IP6 counteracts the harmful effects of UVB irradiation and increases the viability and survival of UVB-exposed cells. Treatment with IP6 after UVB irradiation (30 mJ/cm(2)) arrested cells in the G(1) and G(2) M phases while decreasing the S phase of the cell cycle. Treatment with IP6 also decreased UVB-induced apoptosis and caspase 3 activation. Topical application of IP6 followed by exposure to UVB irradiation in SKH1 hairless mice decreased tumor incidence and multiplicity as compared with control mice. Our results suggest that IP6 protects HaCaT cells from UVB-induced apoptosis and mice from UVB-induced tumors.     

UV-radiation, apoptosis and skin

Apoptosis or programmed cell death is a key function in regulating skin development, homeostasis and tumorigenesis. The epidermis is exposed to various external stimuli and one of the most important is UV radiation. The UVA and UVB spectra differ in their biological effects and in their depth of penetration through the skin layers. UVB rays are absorbed directly by DNA which results in its damage. UVA can also cause DNA damage but primarily by the generation of reactive oxygen species. By eliminating photodamaged cells, apoptosis has an important function in the prevention of epidermal carcinogenesis. UV-induced apoptosis is a complex event involving different pathways. These include: 1. activation of the tumour suppressor gene p53; 2. triggering of cell death receptors directly by UV or by autocrine release of death ligands; 3. mitochondrial damage and cytochrome C release. The extrinsic pathway through death receptors such as fibroblast-associated, tumour necrosis factor receptor and TNF related apoptosis inducing ligand receptor activate caspase cascade. The intrinsic or mitochondrial pathway of apoptosis is regulated by the Bcl-2 family of proteins, anti-apoptotic (Bcl-2, Bcl-xl, Bcl-w) and the pro-apoptotic (Bax, Bak, Bid). The balance between the pro-apoptotic and anti-apoptotic proteins determines cell survival or death. We discuss recent findings in the molecular mechanisms of UV induced apoptosis.     

Death penalty for keratinocytes: apoptosis versus cornification

Homeostasis implies a balance between cell growth and cell death. This balance is essential for the development and maintenance of multicellular organisms. Homeostasis is controlled by several mechanisms including apoptosis, a process by which cells condemned to death are completely eliminated. However, in some cases, total destruction and removal of dead cells is not desirable, as when they fulfil a specific function such as formation of the skin barrier provided by corneocytes, also known as terminally differentiated keratinocytes. In this case, programmed cell death results in accumulation of functional cell corpses. Previously, this process has been associated with apoptotic cell death. In this overview, we discuss differences and similarities in the molecular regulation of epidermal programmed cell death and apoptosis. We conclude that despite earlier confusion, apoptosis and cornification occur through distinct molecular pathways, and that possibly antiapoptotic mechanisms are implicated in the terminal differentiation of keratinocytes.