Zhu Jie, Zheng Ya, Zhang Haiyan, Zhu Jing, Sun Hong
Department of Gynecology, Obstetrics and Gynecology Hospital of Fudan UniversityShanghai 200011, China.
Shanghai Key Laboratory of Female Reproductive Endocrine Related DiseasesShanghai 200011, China.
Am J Transl Res. 2017 Sep 15;9(9):4046-4058. eCollection 2017.
Cisplatin is a common used anti-tumor drug in ovarian cancer therapy with potent effect. Studies have reported that autophagy works as a cell-survival process in cancer, chloroquine has been added to various chemotherapeutic drugs. In the current study, we aim to evaluate whether chloroquine can enhance the effects of cisplatin in treating ovarian cancer.
CCK-8 assay was used to detect cell viability. Transwell assay was used to examine cell migration and invasion. Flow cytometry assay was applied to evaluate cell apoptosis. Western-blot assay was used to detect proteins related to apoptosis, autophagy and the AKT/mTOR pathway.
In the current study, we showed that low concentration of chloroquine alone did not affect cell viability, migration or invasion, but it could enhance the efficacy of cisplatin in inhibiting cell viability, migration and invasion in both SKOV3 and hey cells. Afterwards, we observed that cisplatin triggered apoptosis and autophagy in both SKOV3 and hey cells in a dose-dependent manner. After treatment of cisplatin, SKOV3 and hey cells showed increased apoptotic rate in flow cytometry assay, increased protein levels of cleaved caspase 3, cleaved PARP and Bax, and decreased protein levels of Bcl-2 and Bcl-XL. Cisplatin also induced the formation of autophagosomes and increased autophagy-related proteins ATG 5, ATG 7, Beclin 1 and LC3B II/LC3B I. Meanwhile, cisplatin activated the AKT-mTOR pathway in both SKOV3 and hey cells. Next, chloroquine was added to ovarian cancer cells, flow cytometry assay revealed that chloroquine alone did not affect cell apoptosis and expressions of apoptosis-related proteins, while chloroquine plus cisplatin induced more apoptotic rate than cisplatin alone (p < 0.05). Meanwhile, apoptosis-related proteins had the same change trend. In vivo experiment demonstrated that chloroquine plus cisplatin was more effective than cisplatin alone in suppressing the growth of xenograft tumors, with lower ki-67 expression and higher cleaved caspase 3 expression.
Based on our study, we propose that cisplatin activates the AKT/mTOR signaling pathway, which subsequently induces cytoprotective autophagy in ovarian cancer cells. Meanwhile, inhibition of autophagy via chloroquine enhances the anti-tumor effect of cisplatin.
顺铂是卵巢癌治疗中常用的具有强效作用的抗肿瘤药物。研究报道自噬在癌症中作为一种细胞存活过程发挥作用，氯喹已被添加到各种化疗药物中。在本研究中，我们旨在评估氯喹是否能增强顺铂治疗卵巢癌的效果。
采用CCK-8法检测细胞活力。采用Transwell法检测细胞迁移和侵袭能力。应用流式细胞术评估细胞凋亡情况。采用蛋白质免疫印迹法检测与凋亡、自噬及AKT/mTOR通路相关的蛋白质。
在本研究中，我们发现低浓度氯喹单独使用不影响细胞活力、迁移或侵袭，但它能增强顺铂对SKOV3和hey细胞活力、迁移及侵袭的抑制作用。之后，我们观察到顺铂以剂量依赖性方式诱导SKOV3和hey细胞凋亡和自噬。顺铂处理后，流式细胞术检测显示SKOV3和hey细胞凋亡率增加，裂解的caspase 3、裂解的PARP和Bax蛋白水平升高，Bcl-2和Bcl-XL蛋白水平降低。顺铂还诱导自噬体形成并增加自噬相关蛋白ATG 5、ATG 7、Beclin 1和LC3B II/LC3B I。同时，顺铂激活SKOV3和hey细胞中的AKT-mTOR通路。接下来，将氯喹添加到卵巢癌细胞中，流式细胞术检测显示氯喹单独使用不影响细胞凋亡及凋亡相关蛋白的表达，而氯喹联合顺铂诱导的凋亡率高于顺铂单独使用（p<0.05）。同时，凋亡相关蛋白有相同的变化趋势。体内实验表明，氯喹联合顺铂在抑制异种移植瘤生长方面比顺铂单独使用更有效，其Ki-67表达更低，裂解的caspase 3表达更高。
基于我们的研究，我们提出顺铂激活AKT/mTOR信号通路，随后在卵巢癌细胞中诱导细胞保护性自噬。同时，通过氯喹抑制自噬可增强顺铂的抗肿瘤作用。
