摘要
目的探讨葡萄糖‑6‑磷酸脱氢酶(glucose‑6‑phosphate dehydrogenase,G6PD)在卵巢癌细胞顺铂耐药中的作用及其潜在的机制。方法通过低浓度加量持续诱导法构建卵巢癌顺铂耐药细胞株A2780/CDDP,采用Label‑free定量蛋白质组学法分析A2780和A2780/CDDP细胞差异蛋白G6PD,Western blot和RT‑qPCR检测G6PD表达情况。通过siRNA技术将siRNA‑G6PD及其阴性对照(siRNA‑NC)转染至A2780/CDDP细胞,然后采用Western blot验证G6PD基因沉默效果,MTT法检测细胞增殖情况,流式细胞术检测细胞凋亡情况,流式细胞仪和酶标仪检测铁死亡相关物质活性氧(ROS)、丙二醛(MDA)、还原型谷胱甘肽(GSH)和脂质过氧化物的表达水平。结果与A2780细胞相比,A2780/CDDP细胞中有95个上调蛋白和102个下调蛋白,其中上调蛋白中以G6PD表达差异最为显著;这些蛋白大多富集在代谢通路,且其功能主要与细胞氧化应激反应、核糖磷酸代谢途径相关。与A2780细胞相比,A2780/CDDP细胞中G6PD mRNA和蛋白表达水平均显著升高(均P<0.05),铁死亡相关物质ROS、MDA和脂质过氧化物水平均降低(均P<0.05),而GSH水平升高(P=0.001)。沉默G6PD后的A2780/CDDP细胞中顺铂的IC_(50)值降低(P=0.012),细胞凋亡率升高(P=0.006),铁死亡相关物质ROS、MDA和脂质过氧化水平均升高(均P<0.05),而GSH水平降低(P=0.007)。结论沉默G6PD可能通过诱导卵巢癌顺铂耐药细胞A2780/CDDP中的铁死亡水平而增强顺铂敏感性。
Objective To investigate the role and underlying mechanism of glucose‑6‑phosphate dehydrogenase(G6PD)in cisplatin resistance of ovarian cancer cells.Methods The cisplatin‑resistant ovarian cancer cell line A2780/CDDP was established by continuous induction with low concentration.The differential expressed protein G6PD in A2780 and A2780/CDDP cells were analyzed by a Label‑free protein quantification method.The expression levels of G6PD was detected by Western blot and RT‑qPCR.siRNA‑G6PD and its negative control(siRNA‑NC)were transfected into A2780/CDDP cells by siRNA technique and then the effect of G6PD gene silencing was verified by Western blot.MTT experiment was used to detect cell proliferation ability and flow cytometry was used to detect the apoptosis rate.The expression levels of reactive oxygen species(ROS),malondialdehyde(MDA),reduced glutathione(GSH)and lipid peroxides related to ferroptosis were detected by flow cytometry and microplate reader.Results Compared with A2780 cells,there were 95 up‑regulated proteins and 102 down‑regulated proteins in A2780/CDDP cells,among which G6PD had the most significant difference in expression.Most of these proteins were enriched in metabolic pathways,and their functions were mainly related to cellular oxidative stress response and ribose phosphoric acid metabolism pathways.Compared with A2780 cells,the expression levels of G6PD mRNA and protein in A2780/CDDP cells were significantly increased(all P<0.05),the levels of ROS,MDA and the lipid peroxides related to ferroptosis were decreased(all P<0.05),while the GSH level was increased(P=0.001).After silencing G6PD,the IC_(50) value of cisplatin in A2780/CDDP cells was decreased(P=0.012),the apoptosis rate was increased(P=0.006),and the levels of ROS,MDA and the lipid peroxides related to ferroptosis were increased(all P<0.05),while the GSH level was decreased(P=0.007).Conclusions Silencing G6PD may enhance cisplatin sensitivity by inducing the ferroptosis levels in cisplatin⁃resistant ovarian cance
作者
陈朝梅
奚敏
施秀
王芳
周金华
CHEN Chaomei;XI Min;SHI Xiu;WANG Fang;ZHOU Jinhua(Department of Gynaecology and Obstetrics,the First Affiliated Hospital of Soochow University,Suzhou 215024,China)
出处
《中国癌症防治杂志》
CAS
2022年第3期274-280,共7页
CHINESE JOURNAL OF ONCOLOGY PREVENTION AND TREATMENT
基金
国家自然科学基金项目(82172609)
苏州市民生科技项目(SYS2020106)
苏州大学附属第一医院自然科学基金博习培育计划项目(BXQN202108)。
