| Background:As well known potential carcinogens, reactive oxygen species (ROS) are involved in both carcinogenesis and tumor progression via ROS-induced DNA damage. During normal metabolism, cells avoid oxidative damage by means of antioxidant defense mechanisms that control the balance between the generation and removal of oxygen radicals. The antioxidant system includes various enzymes, such as superoxide dismutase and catalase, as well as glutathione peroxidase (GPX). GPX3, a member of the GPX family, is an extracellular glycosylated enzyme that reduces both phospholipid hydroperoxides and fatty acid hydroperoxides using various electron donors, such as glutathione, thioredoxin, and glutaredoxin. It is generally known that hydroperoxides are involved in various biological behaviors of cancer cells such as proliferation, motility, and invasion. Therefore, some investigators expected that GPX3 might play a role in cancer carcinogenesis by controlling the hydroperoxide levels inside cells.Malignant melanoma is a common leading cause of cancer-related death among patients with skin cancer. The incidence of cutaneous melanoma has rapidly increased over the last fifty years. However, the molecular basis of melanoma is largely unknown.Objective:In the present study, we aimed to investigate whether GPX3 is also downregulated in melanoma. If so, whether promoter hypermethylation is associated with its repression. Implication of GPX3 expression on melanoma pathogenesis was also elucidated.Materials and Methods:1) GPX3 mRNA expression was detected by reverse transcription PCR (RT-PCR) analysis and quantitative real time PCR analysis in human epidermal melanocytes (HEMs), melanoma cell lines, normal skin tissues and melanoma tissue samples. GPX3 protein expression was detected by immunocytochemistry in HEMs and melanoma cell lines. 2) Promoter methylation of GPX3 was investigated in HEMs, melanoma cell lines, normal skin samples and melanoma tissue samples using methylation specific PCR (MSP) analysis.3) To determine whether methylation of GPX3 directly repressed GPX3 expresion, melanoma cell lines with GPX3 methylation and repression were treated with 5-Aza-2’-Deoxycitidine (5-Aza). Restoration of GPX3 expression was determined by RT-PCR analysis.4) GPX3 depleted stable cell line (Mock-SK-MEL-24 and GPX3a-SK-MEL-24) was constructed to evaluate the influence of GPX3 expression on biological behavior of melanoma cells in vitro and in vivo. Proliferation assay, wound healing assay and transwell assay were performed in the constructed stable cells, to investigate the influence of GPX3 expression on proliferation ability, motility and invasiveness of melanoma cells in vitro.5) Human melanoma xenograft mice models were constructed using Mock-SK-MEL-24 and GPX3A-SK-MEL-24 and the ability of tumorigenesis was comparatively investigated between the two cells.6) We further determined the GPX3 protein expression in 105 melanoma tissue samples using immunofluorescence and investigated the clinicopathological significance of GPX3 on melanoma.Results:1) Compared to HEMs, GPX3 mRNA’expression was downregulated in melanoma cell lines. Moreover, GPX3mRNA expression increased in normal skin tissue samples than melanoma tissue samples.2) GPX3 was unmethylated in HEMs and partial or full methylated in SK-MEL-24 and SK-MEL-2, respectively. In normal skin samples were showed unmethylation of GPX3, by contrast, all of melanoma tissue samples showed full or partial methylation of GPX3.3) GPX3 mRNA expression was restored by 5-Aza treatment in melanoma cell lines.4) GPX3 mRNA expression was decreased in GPX3△-SK-MEL-24 than Mock-SK-MEL-24. Compared to Mock-SK-MEL-24cells, GPX3△-SK-MEL-24 cells demonstrated 1.43-fold and 2.07-fold higher proliferative ability in each time point, respectively (P<0.001, and P<0.001, respectively). Moreover, GPX3△-SK-MEL-24 cells showed 3.06-fold (P<0.001), and 6.47-fold (P<0.001) higher migration and invasive ability, respectively.5) Based on the human melanoma xenograft analysis, we found that GPX3 downregulation can promotes tumorigenesis of melanoma cells.6) In the melanoma tissue samples, GPX3 expression was detected in the cytoplasm of cancer cells. GPX3 expression was more frequently detected in primary site than metastatic site of melanoma. (P=0.036) Moreover, GPX3 expression showed increased survival rates (median survival duration 71 months) compared to patients without GPX3 expression (median survival duration 18 months) (P=0.013)Conclusion:1) GPX3 expression was downregulated in melanoma.2) Downregulation of GPX3 was largely depended on the promoter hypermethylation in melanoma.3) GPX3 expression inhibits proliferation, motility, invasion as well as tumorigenesis of melanoma cells.4) GPX3 is a possible prognostic marker of melanoma patients. All of those indicated that GPX3 may have strong influence on melanoma pathogenesis. |
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