摘要
目的探讨自噬激活在紫杉醇(paclitaxel,PTX)耐药卵巢癌(ovarian cancer)细胞中的作用及自噬抑制对耐药细胞化疗的影响。方法以卵巢癌细胞系SKOV3和紫杉醇耐药卵巢癌细胞(SKOV3/PTX)为研究对象;利用MTT实验检测细胞活力变化;Annexin V/PI双染法检测细胞凋亡;免疫印迹(Western blot,WB)法检测蛋白表达变化;利用脂质体瞬时转染法使细胞表达LC3-GFP-RFP荧光蛋白;利用激光共聚焦显微镜检测细胞荧光蛋白定位及表达。结果相比SKOV3细胞,相同剂量的PTX对SKOV3/PTX细胞的活力抑制能力明显降低,紫杉醇对SKOV3细胞和SKOV3/PTX细胞的半数抑制浓度((inhibitory concentration 50%,IC50)分别为(41.91±2.92)μM和(114.6±17.6)μM。此外,在凋亡水平上,PTX对SKOV3/PTX细胞的诱导凋亡作用显著降低。通过LC3-GFP-RFP过表达和LC3、p62蛋白检测,证明SKOV3/PTX细胞的自噬激活程度增高。采用自噬抑制剂氯喹(chloroquine,CQ)和3-MA可以阻断细胞自噬,并提高PTX对SKOV3/PTX细胞的活力抑制能力和诱导凋亡作用。结论自噬激活在紫杉醇耐药的卵巢癌细胞中发挥了保护作用,抑制自噬可以增强紫杉醇对耐药细胞的杀伤作用。
Objective To explored the role of autophagy activation in paclitaxel(PTX)-resistant ovarian cancer cells and the effects of autophagy inhibition on the treatment of resistant cancer cells.Methods The ovarian cancer cell lines SKOV3 and PTX-resistant ovarian cancer cells were used.MTT assay was used to determine the cell viability.Apoptosis rates were detected by Annexin V/PI staining assay.The protein expression was determined by western blot(WB).The cells were transfected by LC3-GFP-RFP using lipofectamine 2000 and the fluorescence was detected by laser scanning confocal microscope.Results The inhibitory effect of PTX on SKOV3/PTX cells was significantly reduced compared with SKOV3 cells and inhibitory concentration 50%(IC50)of PTX on SKOV3 cells and SKOV3/PTX cells were 41.91±2.92μM and 114.6±17.6μM,respectively.The apoptotic effect of PTX on SKOV3/PTX cells was significantly reduced.It was proved that SKOV3/PTX cells showed increased autophagy activation after LC3-GFP-RFP overexpression through detecting the expression of LC3 and P62.Autophagy of SKOV3/PTX cells was inhibited after treatment with autophagy inhibitors,chloroquine(CQ)and 3-MA and the inhibitory effect and apoptosis-inducing ability of PTX on SKOV3/PTX cells was improved.Conclusion Autophagy activation has a protective role in the PTX-resistant ovarian cancer cells.Inhibition of autophagy enhanced the cytotoxic effect of PTX-resistant ovarian cancer cells.
作者
杨璇
张玉洲
郭靖
YANG Xuan;ZHANG Yuzhou;GUO Jing(Kaifeng Obstetrics and Gynecology Hosipital,Kaifeng,475000)
出处
《实用癌症杂志》
2024年第2期181-185,共5页
The Practical Journal of Cancer