Effects Of Hypoxia On Neuroblastoma SK-N-SH Cells Induced By Cobalt Dichloride

Objective:The studies on hypoxia in solid tumors have attracted so much attention in recent years. This study is aimed at the optimal hypoxic condition induced by CoCl2which include the culture concentration and time of CoCl2.Methods:SK-N-SH cell line was incubated in cell culture mediums with different concentrations of CoCl2(the final concentrations of CoCl2was0,50,100,150,200,300,600μM) for different times(6,12,24,48,72,96hours,respectively). Then CCK-8was used to detect the proliferation of cells, and the morphology of cells was observed. The expression of HIFs at the levels of transcription and translation was measured by Quantitative PCR and Immunofluorescence.Results:1).CCK-8assay showed the inhibition of the proliferations of SK-N-SH cells treated with100μM CoCl2for72h were significant difference, and was in a dose-and time-dependent manner, along with the disappearance of cells which are small and have neurites.2). HIF-la mRNA, HIF-2a mRNA, and CXCR4mRNA all were significantly up-regulated at the transcriptional level.3). At normoxia, HIF-la was hardly detected in SK-N-SH cells, and HIF-2a was mainly presented in cytoplasm. After72h of hypoxia induced by CoCl2, both HIF-la and HIF-2a were found in nuclear fractions, and only HIF-2a could be detected both in the cytoplasm and in the nucleus.Conclusions:The optimal hypoxic condition was that the concentration of CoCl2in cell culture medium was100μM and the incubation time72h. The expressions of HIF-1αmRNA and HIF-2amRNA at transcription level significantly increased, in accordance with CXCR4mRNA, indicted that HIFs may be associated with mestastasis.