Arief Budiyanto

UV-induced skin damage

Solar radiation induces acute and chronic reactions in human and animal skin. Chronic repeated exposures are the primary cause of benign and malignant skin tumors, including malignant melanoma. Among types of solar radiation, ultraviolet B (290-320 nm) radiation is highly mutagenic and carcinogenic in animal experiments compared to ultraviolet A (320-400 nm) radiation. Epidemiological studies suggest that solar UV radiation is responsible for skin tumor development via gene mutations and immunosuppression, and possibly for photoaging. In this review, recent understanding of DNA damage caused by direct UV radiation and by indirect stress via reactive oxygen species (ROS) and DNA repair mechanisms, particularly nucleotide excision repair of human cells, are discussed. In addition, mutations induced by solar UV radiation in p53, ras and patched genes of non-melanoma skin cancer cells, and the role of ROS as both a promoter in UV-carcinogenesis and an inducer of UV-apoptosis, are described based primarily on the findings reported during the last decade. Furthermore, the effect of UV on immunological reaction in the skin is discussed. Finally, possible prevention of UV-induced skin cancer by feeding or topical use of antioxidants, such as polyphenols, vitamin C, and vitamin E, is discussed.

8-Oxoguanine Formation Induced by Chronic UVB Exposure Makes Ogg1 Knockout Mice Susceptible to Skin Carcinogenesis

8-Oxoguanine is one of the oxidative DNA damages that can result in stable mutations. The Ogg1 gene encodes the repair enzyme 8-oxoguanine-DNA glycosylase, which removes the oxidized base from DNA. In this study, we investigated the role of 8-oxoguanine in skin carcinogenesis induced by UVB irradiation using Ogg1 knockout mice (C57Bl/6J background). We examined the effect of UVB irradiation on the formation of 8-oxoguanine in epidermal cells using immunostaining and found that the level of 8-oxoguanine in Ogg1 knockout mice 24 hours after UVB irradiation remained high compared with that in wild-type and heterozygous mice. To verify the effect of chronic UVB irradiation on 8-oxoguanine formations in epidermal cells, we irradiated wild-type, heterozygous, and Ogg1 knockout mice with UVB at a dose of 2.5 kJ/m2 thrice a week for 40 weeks. We found that the mean number of tumors in Ogg1 knockout mice was 3.71, which was significantly more than in wild-type and heterozygous mice, being 1.71 and 2.28, respectively. The rate of developing malignant tumors in Ogg1 knockout mice was also significantly higher (88.5%; squamous cell carcinomas, 73.1%; sarcomas, 15.4%) than in wild-type mice (50.0%; squamous cell carcinomas, 41.7%; sarcomas, 8.3%). Moreover, the age of onset of developing skin tumors in Ogg1 knockout mice was earlier than in the other types of mice. These results clearly indicate that oxidative DNA damage induced by sunlight plays an important role in the development of skin cancers.

Preventive effect of antioxidant on ultraviolet-induced skin cancer in mice

Reactive oxygen species (ROS) have been shown to be responsible for inducing DNA damage after ultraviolet radiation (UV). Antioxidant, vitamin E and epigallocatechin gallate extracted from green tea, applied topically to the skin, delayed the onset of UV-induced skin cancer in mice. Since olive oil is reported to have a potent antioxidative effect in in vitro system, we asked whether, topical use of olive oil reduces the number and delays the onset of UV-induced skin cancer in mice. We found that super virgin olive oil painted immediately after UVB radiation significantly delayed the onset and reduced the number of skin cancer, but pretreatment of super virgin olive oil and pre- and/or post treatment by regular olive oil neither retarded nor reduced skin cancer formation in UV-irradiated mice. Further, 8-hydroxy-deoxyguanosine (8-OHdG) formation in mice epidermis was apparently reduced by super virgin olive oil painted immediately after UV radiation, although cyclobutane pyrimidine dimers and (6-4) photoproducts were not reduced by olive oil treatment. Our results suggest that daily topical use of super virgin olive oil after sun bathing may delay and reduce UV-induced skin cancer development in human skin, possibly by decreasing ROS-induced 8-OHdG which is responsible for gene mutation.

Involvement of EGF receptor activation in the induction of cyclooxygenase‐2 in HaCaT keratinocytes after UVB

Abstract:  Because selective inhibition of cyclooxygenase‐2 (COX‐2) suppressed the induction of skin tumors in mice by UV and as UV has been shown to induce expression of COX‐2 in skin and cells, COX‐2 may be crucial for photocarcinogenesis of the skin. We studied the mechanism of UVB‐induced expression of COX‐2 focusing on the signal transduction pathway involved. Hydrogen peroxide (H2O2) treatment of HaCaT cells induced expression of COX‐2 and pretreatment with the antioxidant N‐acetylcysteine (NAC) partly inhibited the UVB‐induced expression of COX‐2 protein in HaCaT cells, suggesting that oxidative stress contributes to COX‐2 induction. To examine the signaling pathways involved in the UVB‐induced expression of COX‐2 in HaCaT cells, we analysed the expression of COX‐2 protein after treatment with various inhibitors of signaling molecules. Inhibition of EGFR by a specific inhibitor and by a neutralizing antibody suppressed the induction of COX‐2 expression by UV. Although a neutralizing antibody to transforming growth factor‐α (TGF‐α) suppressed COX‐2 expression induced by TGF‐α, it did not suppress COX‐2 expression by UV, indicating that a direct activation of EGFR is involved. Treatment of cells at low temperature (4°C) inhibited UVB‐induced JNK activation, but it did not inhibit COX‐2 expression by UV. Inhibitors of MEK, p38 MAP kinase and PI3‐kinase, suppressed the induction of COX‐2 expression by UV. In contrast, an erbB‐2 inhibitor augmented the UVB‐induced increase of COX‐2 protein. These data indicate that oxidative stress in association with activation of EGFR, ERK, p38 MAP kinase, and PI3‐kinase plays crucial roles in the UVB induction of expression of COX‐2.

Formation of Cyclobutane Pyrimidine Dimers and 8-Oxo-7,8-dihydro-2′-deoxyguanosine in Mouse and Organ-cultured Human Skin by Irradiation with Broadband or with Narrowband UVB¶

Abstract Narrowband UVB (NB-UVB) is a newly developed UVB source that, in addition to the previously used broadband UVB (BB-UVB), has been effectively used in phototherapy of various skin diseases. Besides its therapeutic effectiveness, NB-UVB also has some adverse effects that should be evaluated. As with all phototherapies, the photocarcinogenic potential of NB-UVB is the major concern. To assess the carcinogenic potential we measured the DNA damage induced by the two UVB sources because exposure of cells to UVB directly or indirectly induces DNA damage such as cyclobutane pyrimidine dimers (CPD) or 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo), respectively. These types of DNA damage cause mutations of oncogenes and tumor suppressor genes, which can lead to photocarcinogenesis. In the present study we measured the yield of CPD and the oxidative DNA damage marker, 8-oxodGuo, in organ-cultured human skin and in mouse skin after exposure to NB-UVB or BB-UVB at therapeutically equivalent doses. We show that a 10-fold higher dose of NB-UVB yields a similar amount of CPD compared with BB-UVB in two types of samples examined. In contrast to CPD, the formation of 8-oxodGuo after irradiation with NB-UVB at a 10-fold higher dose is 1.5–3 times higher than that caused by BB-UVB. These results suggest that although NB-UVB at equivalent erythema–edema doses is not more potent in inducing CPD formation than is BB-UVB, NB-UVB may generate a higher yield of oxidized DNA damage.

Inhibition of Epidermal Growth Factor Receptor and PI3K/Akt Signaling Suppresses Cell Proliferation and Survival through Regulation of Stat3 Activation in Human Cutaneous Squamous Cell Carcinoma

Recent studies have emphasized the important role of Stat3 activation in a number of human tumors from the viewpoint of its oncogenic and antiapoptotic activity. In this study, we examined the role and related signaling molecules of Stat3 in the carcinogenesis of human cutaneous squamous cell carcinoma (SCC). In 35 human cutaneous SCC samples, 86% showed overexpression of phosphorylated (p)-Stat3, and most of those simultaneously overexpressed p-EGFR or p-Akt. Constitutive activation of EGFR and Stat3 was observed in three SCC cell lines and four of five SCC tissues. AG1478, an inhibitor of the EGFR, downregulated Stat3 activation in HSC-1 human SCC cells. AG1478 inhibited cell proliferation and induced apoptosis of HSC-1 cells but did not inhibit the growth of normal human epidermal keratinocytes that did not show Stat3 activation. Furthermore, a PI3K inhibitor also suppressed Stat3 activation in HSC-1 cells to some degree. Combined treatment with the PI3K inhibitor and AG1478 strongly suppressed Stat3 activity and dramatically induced apoptosis of HSC-1 cells. These data suggest that Stat3 activation through EGFR and/or PI3K/Akt activation plays a critical role in the proliferation and survival of human cutaneous SCC.

Retinoic acid suppresses telomerase activity in HSC-1 human cutaneous squamous cell carcinoma

Background  Activation of telomerase is crucial for the continued growth and progression of cancer cells. In a previous study, we showed that telomerase is frequently activated in skin tumours.

Low-dose ultraviolet B radiation synergizes with TNF-α to induce apoptosis of keratinocytes

High-dose ultraviolet B (UVB) irradiation is known to induce apoptosis of keratinocytes, but low-dose UVB dose not. In this paper we present evidence that low-dose UVB can induce TNF-alpha-dependent apoptosis of keratinocytes. In our study, 5 mJ/cm(2) doses of UVB were not sufficient by themselves to induce apoptosis of cultured human keratinocytes, but 20 mJ/cm(2) doses of UVB were. The combination of 5 mJ/cm(2) doses of UVB and exogenous TNF-alpha (15 ng/ml) induced significant apoptosis of keratinocytes, although exogenous TNF-alpha without UVB did not. This phenomenon was accompanied by enhanced clustering of tumor necrosis factor receptor 1 (TNFR1). TNF-alphas promotion of the induction of apoptosis by low-dose UVB was seen until 30 min after irradiation but not at 1 h. We confirmed this finding using a skin organ culture system. UVB (20 mJ/cm(2)), which did not induce transformation of epidermal keratinocytes into sunburn cells, induced apoptosis when TNF-alpha was added to the culture medium. These results suggest that one of the possible mechanisms of inducing keratinocyte apoptosis by low-dose UVB and TNF-alpha is that low-dose UVB augments ligand-binding-induced TNFR1 clustering, resulting in increased apoptotic cell death.