Abstract
Apoptosis is a form of programmed cell death that occurs in multicellular organisms. Fibroblasts are the main cellular ingredients in keloid tissue, which has a relatively low apoptosis level. A natural metabolite of estradiol, 2-Methoxyestradiol (2ME2) exerts a pro-apoptotic effect on tumor cells. In this study, the expression levels of key factors in the apoptosis pathway and the expression level of the proliferating cell nuclear antigen (PCNA) were measured to assess the levels of apoptosis and proliferation in both normal skin fibroblasts and keloid fibroblasts. Twelve samples were obtained from 12 patients: 6 keloid patients and 6 non-keloid patients. All 12 of the patients were randomly selected from the Department of Plastic Surgery at Peking Union Medical College Hospital from June 2016 to December 2016. After cell culture, fibroblasts were divided into the following 6 groups: normal skin fibroblasts (S); keloid fibroblasts (K); keloid fibroblasts treated with 2ME2 (2ME2); keloid fibroblasts treated with DMSO (DMSO); keloid fibroblasts treated with the caspase inhibitor Ac-DEVD-CHO (IN); and keloid fibroblasts treated with both Ac-DEVD-CHO and 2ME2 (IN+2ME2). Fibroblasts at up to passage 3 were used for analysis. Cell activity was measured by the cell counting kit-8. TUNEL staining was used to observe the cell apoptotic morphology. The key apoptosis factors (caspase-3, caspase-8, caspase-9, Bcl-2, Bax, and cytochrome-c) and PCNA expression levels were detected by immunofluorescence analysis and Western blotting. A certain concentration of 2ME2 was also used in group S to evaluate the toxicity. Compared with that in the other groups, 2ME2 significantly inhibited cell activity and led to apoptotic appearance of fibroblasts. In protein analysis, 2ME2 remarkably increased the expression of apoptosis factors and decreased the PCNA expression. Apoptosis levels were reduced by both the caspase inhibitor and 2ME2; thus indicating that the pro-apoptosis effect of 2ME2 was achieved through a caspase-dependent mechanism in keloid fibroblasts. Toxicity assessment showed that 2ME2 had a very low influence on normal skin fibroblasts. 2ME2, considered to be a new promising type of chemotherapy drug, exerts a pro-apoptosis effect by regulating the caspase family and an anti-proliferation effect towards keloid fibroblasts, and it presents low toxicity towards normal fibroblasts in vitro.