摘要
目的研究自噬在拓扑替康诱导卵巢癌OVCAR3细胞死亡中的作用。方法采用不同浓度的拓扑替康处理卵巢癌OVCAR3细胞,MTT法测定拓扑替康对OVCAR3细胞增殖的抑制作用,单丹磺酰尸胺(MDC)荧光染色观察给药后OVCAR3细胞的自噬征象,LDH漏出率法检测拓扑替康联合自噬抑制剂3-甲基腺嘌呤(3-MA)对拓扑替康诱导OVCAR3细胞毒性的影响,MTT法测定拓扑替康联合自噬诱导剂雷帕霉素对OVCAR3细胞增殖抑制的影响,金氏公式分析联合作用效果。结果拓扑替康对OVCAR3细胞的生长有显著的抑制作用,呈明显的剂量依赖性,半数抑制浓度(IC50)为0.057μg/ml。MDC荧光染色显示拓扑替康可诱导OVCAR3细胞产生自噬囊泡,3-MA可以抑制拓扑替康诱导的自噬产生,雷帕霉素可以增强拓扑替康诱导的细胞自噬。细胞培养24h后,拓扑替康组LDH漏出率为22.5%,3-MA+拓扑替康组为16.8%;48h后拓扑替康组LDH漏出率为47.4%,3-MA+拓扑替康组为32.6%。雷帕霉素联合各浓度拓扑替康用药组的抑制率较拓扑替康单药组增加,按金氏公式计算,除最高浓度组(拓扑替康0.2μg/ml)外,其他各组q值均>1.15。结论拓扑替康对OVCAR3细胞有显著抑制作用并能够诱导其发生自噬,诱导自噬性细胞死亡可以提高拓扑替康对OVCAR3细胞的抗肿瘤作用。
Objective To study the effects of autophagy on topotecan-induced death of human ovarian cancer OVCAR3 cell line. Methods The growth inhibition of topotecan on OVCAR3 cells was detected by MTT assay.Monodansylcadaverine（MDC） staining was used to examine autophagosome.The autophagy specific inhibitor 3-methyladenine（3-MA） was added with topotecan and the cytotoxicity of topotecan was measured by lactose dehydrogenase（LDH） leakage.MTT assay was used to detect the growth inhibition effect of topotecan combined with classic autophagy inducer rapamycin and the Jin formular was used to analyze combined effect. Results MTT assay showed that topotecan evidently inhibited the proliferation of OVCAR3 cells in a dose-dependent manner.The half maximal inhibitory concentration（IC50） was 0.057μg/ml.MDC staining indicated that autophagy was induced in OVCAR3 cells when they were treated by topotecan and 3-MA could attenuate topotecan-induced autophagy of OVCAR3 cells.LDH leakage rate of topotecan single agent group was 22.5% and 47.4% after treatment for 24h and 48h,while that of 3-MA combined with topotecan group was 16.8% and 32.6%.MDC staining indicated that rapamycin could enhance topotecan-induced autophagy in OVCAR3 cells.Compared with topotecan single agent,the growth inhibition effect of the combined group was increased and the synergistic effect of topotecan and rapamycin was calculated according to the Jin formular.Q value of each group was higer than 1.15,except for the highest concentration group（topotecan 0.2μg/ml）. Conclusion Topotecan can inhibit proliferation of OVCAR3 significantly.It can also induce an autophagic cell death in OVCAR3 cells.Autophagy is one of the mechanisms that induces the death of ovarian cancer cells.Rapamycin combined with topotecan has synergistic inhibitory effect on the growth of OVCAR3 cells.
出处
《临床肿瘤学杂志》
CAS
2011年第11期974-978,共5页
Chinese Clinical Oncology
