蛋白酶体抑制剂Oprozomib对顺铂耐药卵巢癌细胞的作用及机制研究

Effect of proteasome inhibitor Oprozomib on ovarian cancer cells to cis-platinum

目的探讨蛋白酶体抑制剂Oprozomib(OZ)对顺铂(DDP)耐药卵巢癌细胞的作用,并初步揭示其机制。方法体外培养DDP耐药卵巢癌细胞SKOV3/DDP、A2780/DDP,传代后选取对数生长期细胞进行实验。研究OZ对耐药卵巢癌细胞活力的影响时,OZ浓度设为0、3、9、27、81、243、729、2181、6561 nmol/L,与细胞共培养;研究DDP对耐药卵巢癌细胞活力的影响时,DDP浓度设为0、3、9、27、81、243、729、2181、6561 nmol/L,另加入125 nmol/L OZ,采用CCK-8法检测细胞活力;采用流式细胞术检测DDP+OZ联用时的细胞凋亡情况。将实验分为对照组和OZ组,检测OZ对蛋白酶体糜蛋白酶(CT-L)活性和谷胱甘肽(GSH)合成的影响;采用Western blotting检测胞内谷胱甘肽合成酶(GSS)蛋白的表达。将实验分为对照组、DDP组、OZ组、DDP+OZ、DDP+OZ+GSH组和DDP+OZ+Tempol组,检测胞内活性氧簇(ROS)水平;采用流式细胞术检测细胞凋亡情况。结果OZ对SKOV3/DDP、A2780/DDP细胞的IC50分别为140、350 nmol/L;与OZ联用时,DDP在两株细胞中的IC50分别为154、232 nmol/L。DDP+OZ联用时,两株细胞凋亡率均明显升高(P<0.01)。OZ可抑制蛋白酶体CT-L活性,并呈剂量依赖性。OZ可抑制胞内GSH合成及GSS蛋白表达(P<0.01),GSH水平降低导致ROS清除受阻,ROS清除剂Tempol可明显降低胞内ROS水平(P<0.05,P<0.01)。Tempol对DDP+OZ诱导的细胞凋亡具有明显抑制作用(P<0.01)。结论OZ通过抑制GSH生成阻碍胞内ROS清除,进而增强SKOV3/DDP和A2780/DDP细胞对DDP的敏感性。

Objective To study the enhancement of sensitivity to cisplatin(DDP)mediated by the addition of proteasome inhibitor Oprozomib(OZ)in ovarian cancer cells.Methods SKOV3/DDP and A2780/DDP cells were cultured in vitro,and the logarithmic growth phase cells were used in this study.To study the effect of OZ on drug-resistant cell viability,a series dilutions of OZ(0,3,9,27,81,243,729,2181,and 6561 nmol/L)was used.The effect of DDP was also investigated with a series titrations(0,3,9,27,81,243,729,2181 and 6561 nmol/L)in the presence of 50μl of 500 nmol/L OZ.CCK-8 method was used to detect cell viability;flow cytometry was used to detect the apoptosis rate after treated with DDP+OZ.The experiments were divided into the control group and OZ group to detect the effects of OZ on the activity of proteasome chymotrypsin(CT-L),the synthesis of intracellular glutathione(GSH);the expression of intracellular glutathione synthetase(GSS)was examined using western blotting.The experiments were divided into the control group,DDP group,OZ group,DDP+OZ group,DDP+OZ+GSH group and DDP+OZ+Tempol group to detect the ROS level and apoptosis rate.Results The IC50 of OZ to SKOV3/DDP and A2780/DDP cells were 140 and 350 nmol/L,respectively;In the presence of OZ,the IC50 of DDP to SKOV3/DDP and A2780/DDP cells were 154 and 232 nmol/L,respectively.The apoptosis rate of ovarian cancer cells increased significantly(P<0.01)after treated with DDP+OZ together.OZ can inhibit the CT-L activity of the proteasome in a dose-dependent manner and can inhibit GSH synthesis and GSS protein expression(P<0.01).The decrease of GSH level leads to the obstruction of ROS clearance,and the ROS level was significantly reduced by the ROS scavenger Tempol(P<0.05,P<0.01).Tempol significantly inhibits the apoptosis induced by DDP+OZ(P<0.01).Conclusion OZ enhanced sensitivity of SKOV3/DDP and A2780/DDP to cis-platinum by inhibiting the generation of GHS which resulted in the accumulation of ROS.