Effects of tumor suppressor gene Dickkopf-3 on proliferation and apoptosis of cutaneous malignant melanoma cells

Abstract

Objective To investigate the expression of Dickkopf-3 (DKK3) in human malignant melanoma cell lines and tissues, and to evaluate effects of DKK3 on the proliferation and apoptosis of malignant melanoma cell line A375. Methods Reverse transcription PCR (RT-PCR) and real-time fluores-cence-based quantitative PCR (qRT-PCR) were performed to measure the mRNA expression of DKK3 in human malignant melanoma cell lines HM, A375, WM451, WM35, SK-MEL-1, Hs-695T and MDA-MB-435s, as well as in 38 primary melanoma tissues, 4 metastatic melanoma tissues and 20 pigmented nevus tissues. Cultured malignant melanoma A375 cells were divided into 2 groups to be transfected with pcDNA3.1 (+) -Flag-DKK3 (experiment group) and pcDNA3.1 (+) -Flag-Vector (control group) respectively. The overexpression of DKK3 was verified by RT-PCR. Cell counting kit-8 (CCK8) assay and plate colony formation assay were performed to evaluate the proliferative activity of A375 cells, flow cytometry was conducted to detect apoptosis of A375 cells, and Western blot analysis was performed to determine the expression of cell cycle- and cell apoptosis-related proteins. Results The mRNA expression of DKK3 was downregulated in WM35 cells, absent in HM cells, A375 cells, WM451 cells, SK-MEL-1 cells and Hs-695T cells, but upregulated in MDA-MB-435s cells. Compared with pigmented nevus tissues, the mRNA expression of DKK3 was significantly decreased in malignant melanoma tissues (P < 0.001) . Compared with the control group (100%) , cell colony formation was markedly suppressed in the experiment group (23.22% ± 3.55%) , and the proliferative activity of A375 cells was also significantly inhibited in the experiment group 24, 48, 72 hours after the transfection (all P < 0.05) . Flow cytometry showed that compared with the control group, A375 cells were significantly arrested in G1 phase (48.68% ± 3.92% vs. 25.38% ± 2.92%, P < 0.001) , and the apoptosis rate of A375 cells was significantly increased in the experiment group (P < 0.001) . Compared with the control group, the experiment group showed significantly higher expression of p21, Bax, cleaved-parp and cleaved-casp3, but significantly lower expression of cyclin D1 and Bcl2 (all P < 0.001) . Conclusion DKK3 expression is downregulated in human malignant melanoma tissues, so it may serve as a potential tumor suppressor gene involved in the development of cutaneous malignant melanoma. Key words: Melanoma; Cell proliferation; Apoptosis; DKK3; Tumor suppressor genes