硼替佐米联合顺铂对卵巢上皮性癌顺铂耐药细胞化疗敏感性的影响

Proteasome inhibitors sensitize ovarian cancer cells to cisplatin

目的 研究蛋白酶体抑制剂--硼替佐米联合顺铂作用于卵巢上皮性癌（卵巢癌）顺铂耐药细胞的效果,并探讨其诱导细胞凋亡的分子学机制.方法 实验分组：硼替佐米（50 nmol/L）＋顺铂（40μmol/L）、硼替佐米（50 nmol/L）、顺铂（40 μmol/L）、对照组（未加药物）,以未加药物和细胞,仅加培养基为空白对照（空白组）.采用四甲基偶氮唑蓝比色法测定各组卵巢癌顺铂耐药细胞株C13细胞培养不同时间（12、24、36、48、60及72 h）后的增殖活性（以细胞存活率表示）,膜联蛋白-碘化丙啶双染色法流式细胞仪检测各组细胞的凋亡率;并采用蛋白印迹法检测各组细胞内凋亡抑制蛋白短亚型（cFLIPs）蛋白的表达水平,半胱氨酸天冬氨酸蛋白酶8（caspase-8）活性检测试剂盒检测各组细胞内caspase-8的活性.结果 硼替佐米＋顺铂组细胞显示较好的抑制效果,作用12、24、36、48、60及72 h时其细胞存活率分别为（56.0±8.4）%、（44.7±7.3）%、（33.7±11.2）%、（27.6±8.0）%、（27.6±7.6）%和（28.1±2.4）%,均明显低于顺铂组各时间点（P〈0.05）.作用24 h时,顺铂、硼替佐米、硼替佐米＋顺铂组细胞凋亡率分别为（16.7±l.7）%、（23.4±2.1）%和（26.9±1.6）%,硼替佐米＋顺铂组明显高于顺铂或硼替佐米组（P〈0.05）.各药物处理组细胞内cFLIPs蛋白表达水平均不同程度下降,以硼替佐米＋顺铂组[（43.2±2.3）%]降低最为明显,分别与顺铂、硼替佐米组[分别为（75.7±3.0）%、（67.9±2.1）%]比较,差异均有统计学意义（P〈0.05）.顺铂、硼替佐米和硼替佐米＋顺铂组细胞内caspase-8活性分别为2.3±1.0、4.2±0.9和5.6±1.6,两两比较,差异均有统计学意义（P〈0.05）.结论 蛋白酶体抑制剂--硼替佐米联合顺铂能增强卵巢癌顺铂耐药细胞的化疗敏感性;由药物作用后cFLIPs表达的降低和caspase-8活性的增强推测。

Objective To explore the sensitivity and the molecular mechanism of cisplatinresistance ovarian cancer cell line C13 to proteasome inhibitors and the combination with cisplatin. Methods After different treatments, methyl thiazolyl tetrazolium （MTT） assay was applied to examine the cell viability, annexin-V/propidium iodide（PI） apoptosis detection kit was used to determine the apoptosis rate of different groups, western blot assay was introduced to evaluate the expression levels of Fas-associated death domain-like interleukin-1 beta converting enzyme inhibitory protein （cFLIPs）, and the activity of caspase-8 was examined. Results MTT assay shown that the cell viability ratios of combination group at serial time points from 12, 24, 36, 48, 60, 72 hours were （ 56.0 ± 8.4 ） %, （44.7 ± 7.3 ） %, （ 33.7 ±11.2） %, （27.6 ± 8.0） %, （27. 6 ± 7.6） % and （28.1 ± 2.4） %, which were much lower than those of cisplatin group （P 〈0.05）. After treated for 24 hours, apoptosis rates of cisplatin group, bortezomib group and combination group were （ 16.7 ± 1.7） %, （23.4 ± 2.1 ） % and （26.9 ± 1.6） %, respectively. The rate of combination group was much higher than that of non-treated group and that of cisplatin group or bortezomib group （ P 〈 0.05 ）. Western blot assay showed the changes of expression levels of cFLIPs, which were downregulated seriously after cisplatin, bortezomib or combination treatment [ （43.2 ± 2.3 ）% vs（ 75.7 ± 3.0）%vs （67.9 ± 2.1 ） %, P 〈 0.05 ]. The caspase-8 activity of combination group was （5.6 ± 1.6） folds than that of non-treated group, which was higher than those of other two groups [ （ 2.3 ± 1.0） and （4.2 ± 0.9 ） folds,P 〈 0.05 ]. Conclusions The tumor cell lethal effect of cisplatin could be increase significantly by the combination application of proteasome inhibitors, bortezomib. And the cFLIPs/caspase-8 signaling pathway may be play an important role in the molecular mechanism of the combination treatment.