Li Yan, Weng Dan-hui, Kong Fan-fei, Fan Liang-sheng, Hu Yi, Song Xiao-hong, Xing Hui, Wang Wei, Ma Ding, Wang Shi-xuan
Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Zhonghua Fu Chan Ke Za Zhi. 2010 Jun;45(6):445-8.
To explore the sensitivity and the molecular mechanism of cisplatin-resistance ovarian cancer cell line C13 to proteasome inhibitors and the combination with cisplatin.
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.
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 down-regulated 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].
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.
探讨蛋白酶体抑制剂及与顺铂联合应用对顺铂耐药卵巢癌细胞系C13的敏感性及其分子机制。
经不同处理后，采用甲基噻唑基四氮唑蓝（MTT）法检测细胞活力，用膜联蛋白V/碘化丙啶（PI）凋亡检测试剂盒测定不同组的凋亡率，采用蛋白质印迹法评估类Fas相关死亡结构域白介素-1β转化酶抑制蛋白（cFLIPs）的表达水平，并检测半胱天冬酶-8的活性。
MTT法显示，联合组在12、24、36、48、60、72小时各时间点的细胞活力分别为（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）%，均显著低于顺铂组（P<0.05）。处理24小时后，顺铂组、硼替佐米组和联合组的凋亡率分别为（16.7±1.7）%、（23.4±2.1）%和（26.9±1.6）%。联合组凋亡率显著高于未处理组、顺铂组或硼替佐米组（P<0.05）。蛋白质印迹法显示cFLIPs表达水平变化，顺铂、硼替佐米或联合处理后其表达均显著下调[（43.2±2.3）%对（75.7±3.0）%对（67.9±2.1）%，P<0.05]。联合组半胱天冬酶-8活性是未处理组的（5.6±1.6）倍，高于其他两组[（2.3±1.0）倍和（4.2±0.9）倍，P<0.05]。
蛋白酶体抑制剂硼替佐米联合应用可显著增强顺铂对肿瘤细胞的杀伤作用。cFLIPs/半胱天冬酶-8信号通路可能在联合治疗的分子机制中起重要作用。
