内质网功能状态对卵巢癌细胞顺铂敏感度的影响

Effect of Endoplasmic Reticulum Status on Sensitivity of Ovarian Cancer Cells to Cisplatin

目的探讨内质网应激(endoplasmic reticulum stress,ERS)功能状态对卵巢癌细胞顺铂(DDP)敏感度的影响。方法卵巢癌细胞系SKOV3接受40μmol/L DDP处理24 h后,q RT-PCR检测ERS相关基因改变;同时内质网示踪染料(ER-tracker)观察内质网形态变化;分别用ERS稳定剂牛磺熊去氧胆酸钠(tauroursodeoxycholate,TUDCA)或ERS诱导剂毒胡萝卜素(thapsigargin,TG)预处理SKOV3,再接受DDP处理,应用流式细胞术检测细胞凋亡率;应用Western blot法检测内质网应激相关蛋白GRP78和CHOP是否表达;从临床卵巢癌患者腹水中分离与培养原代卵巢癌细胞后,采用流式细胞术比较TUDCA及TG预处理对原代卵巢癌细胞DDP敏感度影响,Western blot法检测凋亡相关蛋白PARP和Caspase-3的变化。结果顺铂处理卵巢癌细胞系SKOV3后能够引起明显的ERS,ERS相关蛋白明显上调,内质网形态呈现颗粒化;TUDCA可以显著下调DDP引起的杀伤作用(P<0.05),显著降低GRP78和CHOP蛋白水平;TG可以显著上调DDP引起的杀伤作用(P<0.05)、GRP78和CHOP蛋白水平;在卵巢癌腹水原代细胞中TUDCA亦可显著抑制DDP引起的杀伤作用(P<0.05),TG亦可显著上调DDP引起的杀伤作用(P<0.05)。结论改变ERS的功能状态能够影响卵巢癌细胞对顺铂的敏感度。

Objective To explore the effect of endoplasmic reticulum stress(ERS) on the sensitivity of ovarian cancer cells to cisplatin. Methods After 24 h treatment of 40μmol/L DDP on SKOV3 cells, q RTPCR was performed to examine the alteration of ERS related genes; ER-tracker was adopted to observe the morphological change; ERS stabilizer tauroursodeoxycholate(TUDCA) and inducer thapsigargin(TG) were applied respectively as pretreating agent before cisplatin. Furthermore, flow cytometry was employed to compare the relative apoptosis rate; Western blot was operated to show the relative level of ERS-related protein GRP78 and CHOP. After isolating and culturing primary ovary cancer cells from clinical patients’ ascites, we also used flow cytometry to check the sensitivity of primary ovarian cancer cells treated with TUDCA or TG to cisplatin. Meanwhile, Western blot was performed to check the apoptosis-related protein PARP and Caspase-3. Results Cisplatin caused evident ERS in SKOV3 cells, with obvious increase of ERS-related protein and granular distribution of ERS; TUDCA significantly alleviated the DDP cytotoxicity in SKOV3(P<0.05), and reduced the level of GRP78 and CHOP; while TG exerted the opposite effect compared with TUDCA, TG obviously enhanced the DDP cytotoxicity(P<0.05), and augmented the level of GRP78 and CHOP in SKOV3; As to the primary ovarian cancer cells, TUDCA dramatically inhibited while TG augmented the killing effect induced by DDP(P<0.05). Conclusion Altering ERS could modulate the sensitivity of ovarian cancer cells to cisplatin.