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Abstract：
Currently, platinum agents remain the mainstay of chemotherapy for ovarian cancer (OC). However, cisplatin (DDP) resistance is a major reason for chemotherapy failure. Thus, it is extremely important to elucidate the mechanism of resistance to DDP. Here, we establish two DDP-resistant ovarian cancer cell lines and find that caseinolytic protease P (CLPP) level is significantly downregulated in DDP-resistant cell lines compared to wild-type ovarian cancer cell lines (SK-OV-3 and OVcar3). Next, we investigate the functions of CLPP in DDP-resistant and wild-type ovarian cancer cells using various assays, including cell counting kit-8 assay, western blot analysis, immunofluorescence staining, and detection of reactive oxygen species (ROS) and apoptosis. Our results show that CLPP knockdown significantly increases the half maximal inhibitory concentration (IC 50) and mitophagy of wild-type SK-OV-3 and OVcar3 cells, while CLPP overexpression reduces the IC 50 values and mitophagy of DDP-resistant SK-OV-3 and OVcar3 cells. Next, we perform database predictions and confirmation experiments, which show that heat shock protein family A member 8 (HSPA8) regulates CLPP protein stability. The dynamic effects of the HSPA8/CLPP axis in ovarian cancer cells are also examined. HSPA8 increases mitophagy and the IC 50 values of SK-OV-3 and OVcar3 cells but inhibits their ROS production and apoptosis. In addition, CLPP partly reverses the effects induced by HSPA8 in SK-OV-3 and OVcar3 cells. In conclusion, CLPP increases DDP resistance in ovarian cancer by inhibiting mitophagy and promoting cellular stress. Meanwhile, HSPA8 promotes the degradation of CLPP protein by regulating its stability.
摘要：
目前,铂类药物仍然是卵巢癌（OC）化疗的主要药物。然而,顺铂（DDP）耐药是化疗失败的主要原因。因此，阐明对DDP的耐药机制极为重要。这里,我们建立了两种耐DDP的卵巢癌细胞系，发现与野生型卵巢癌细胞系（SK-OV-3和OVcar3）相比，耐DDP的细胞系中酪蛋白分解蛋白酶P（CLPP）水平显着下调。接下来,我们使用各种方法研究了CLPP在DDP抗性和野生型卵巢癌细胞中的功能，包括细胞计数试剂盒-8测定，蛋白质印迹分析,免疫荧光染色，并检测活性氧（ROS）和细胞凋亡。我们的结果表明，CLPP敲低显着增加野生型SK-OV-3和OVcar3细胞的半最大抑制浓度（IC50）和线粒体自噬，而CLPP过表达降低了DDP抗性SK-OV-3和OVcar3细胞的IC50值和线粒体自噬。接下来,我们进行数据库预测和确认实验，这表明热休克蛋白家族A成员8(HSPA8)调节CLPP蛋白的稳定性。还检查了HSPA8/CLPP轴在卵巢癌细胞中的动态作用。HSPA8增加SK-OV-3和OVcar3细胞的线粒体自噬和IC50值，但抑制其ROS产生和凋亡。此外,CLPP部分逆转了HSPA8在SK-OV-3和OVcar3细胞中诱导的作用。总之,CLPP通过抑制线粒体自噬和促进细胞应激增加卵巢癌的DDP抗性。同时,HSPA8通过调节CLPP蛋白的稳定性促进其降解。
