Effect of simvastatin on the proliferation, apoptosis and migration of hypoxic A375 melanoma cells

Abstract

Objective To evaluate the regulatory effect of simvastatin on the proliferation, apoptosis and migration of hypoxic A375 melanoma cells, and to explore their molecular mechanisms. Methods Under normoxic or hypoxic culture conditions, A375 cells were treated with 0.25, 0.5, 1, 2, 4, 8 μmol/L simvastatin (simvastatin groups) and dimethyl sulfoxide (DMSO, control group) separately for 48 hours. Cell counting kit-8 (CCK-8) assay, Transwell assay using immersion culture system and flow cytometry using annexin-V/propidium iodide (PI) staining were conducted to assess the proliferative and migratory activities and apoptosis of A375 cells, respectively. RT-PCR and Western blot analysis were performed to measure the mRNA and protein expression of hypoxia-inducible factor 1α (HIF-1α) , survivin and cyclin-dependent kinase inhibitor p27 in the simvastatin groups and control group, as well as in the HIF-1α-silenced hypoxic A375 cells. Results Under normoxic or hypoxic culture conditions, there were significant differences in the proliferative activities of A375 cells, proportion of apoptotic cells and number of migrating cells between the simvastatin groups and control groups (F= 95.84, 37.22 and 177.5 respectively, P < 0.001) . The hypoxic control group showed significantly higher cellular proliferative activity (1.391 ± 0.129) and higher number of migrating cells (322.550 ± 26.226) compared with the normoxic control group (0.807 ± 0.049, 125.583 ± 17.256 respectively, both P < 0.001) and hypoxia+ simvastatin group (0.685 ± 0.417, 115.167 ± 12.050 respectively, both P < 0.001) , but significantly lower proportion of apoptotic cells (6.167% ± 2.714%) compared with the normoxic control group (11.833% ±2.483%, P < 0.01) and hypoxia+ simvastatin group (17.833% ± 2.714%, P < 0.01) . Under the hypoxic condition, the simvastatin group showed significantly lower mRNA and protein expression of HIF-1α and survivin compared with the control group (all P < 0.05) , but significantly higher mRNA and protein expression of P27 compared with the control group (both P < 0.05) . Under the hypoxic condition, HIF-1α-silenced A375 cells showed significantly decreased mRNA and protein expression of survivin (t= 5.346 and 8.281 respectively, both P < 0.05) , but significantly increased mRNA and protein expression of p27 (t= 31.37 and 9.954 respectively, both P < 0.01) compared with the unsilenced cells. Conclusion Simvastatin can inhibit the proliferation and migration of hypoxic A375 cells, and promote their apoptosis, likely by activating the HIF signaling pathway. Key words: Melanoma; Cell line, tumor; Anoxia; Cell proliferation; Apoptosis; Cell migration assays; Survivin; Hypoxia inducible factor; Simvastatin