IU1抑制USP14对人卵巢癌细胞缝隙连接及顺铂毒性影响的研究

Effect of USP14 inhibition by IU1 on the gap junction and cisplatin toxicity in ovarian cancer cells

目的探究1-[1-(4-氟苯基)-2,5-二甲基-1H-吡咯-3-基]-2-(1-吡咯烷基)乙酮(IU1)抑制泛素特异性蛋白酶14(USP14)对人卵巢癌细胞HO9810、OVCRA3的顺铂毒性的影响及其机制。方法采用CCK-8法测定不同浓度IU1对HO9810、OVCRA3细胞的增殖抑制率及IU1对顺铂细胞毒性的影响;采用去泛素化酶活性实验对IU1作用于HO9810、OVCRA3细胞后USP14活性的变化进行研究;采用Western blot检测IU1作用于HO9810、OVCRA3细胞后cleaved-caspase 3和连接蛋白32(Cx32)表达水平的变化;采用细胞接种荧光示踪法测定IU1对人卵巢癌细胞HO9810、OVCRA3细胞缝隙连接(GJ)的影响。结果 IU1能降低USP14的活性;IU1能降低顺铂的细胞毒性,抑制细胞凋亡;IU1对Cx32的蛋白表达水平无影响,但能抑制细胞间GJ通信功能。结论 IU1可以降低顺铂的细胞毒性,该作用可能与IU1抑制卵巢癌细胞HO9810、OVCRA3的GJ通讯功能有关,与Cx32蛋白表达水平变化无关。

Objective To determine the effect of ubiquitin specific protease 14(USP14)inhibition by 1-[1-(4-fluorophenyl)-2,5-dimethylpyrrol-3-yl]-2-pyrrolidin-1-ylethanone(IU1)on cisplatin toxicity in ovarian cancer HO9810 and OVCRA3 cells and its mechanism in vitro.Methods CCK-8 assay was used to explore the effect of different concentrations of IU1 on the cell proliferation and cisplatin toxicity in human ovarian cancer cells.We chose deubiquitinase trap assay to test USP14 activity.Western blot was used to determine the effect of IU1 on the expression of cleaved-caspase 3 and connexin 32(Cx32).The effect of IU1 on the gap junction(GJ)of human ovarian cancer cells HO9810 and OVCRA3 was determined by parachute assay.Results IU1 decreased the cisplatin cytotoxicity and suppressed the cell apoptosis.Moreover,IU1 had no effect on the protein expression of Cx32,but impaired the GJ function.Conclusion IU1 can reduce the cytotoxicity of cisplatin,which might relate to the inhibition of GJ function,but not to the Cx32 protein expression of HO9810 and OVCRA3 cells.