肿瘤坏死因子相关凋亡诱导配体(TRAIL)联合低剂量顺铂诱导多药耐药人卵巢癌细胞凋亡的实验研究

Induction of Apoptosis by the Combination of TRAIL and Lower Dosage DDP in Multidrug Resistant Human Ovarian Carcinoma Cell

为研究卵巢癌多药耐药机制和逆转耐药,探讨在低剂量顺铂存在下,人可溶性肿瘤坏死因子相关凋亡诱导配体(TRAIL)对多药耐药人卵巢癌细胞的杀伤增效作用及其可能存在的分子病理机制。用顺铂为诱导剂,逐步建立多药耐药人卵巢癌细胞株SKOV3/cDDP。(此细胞株经免疫组化检测P-gp高表达)。TRAIL、顺铂或两药联用12h,24h,48h,采用MTT法测试细胞毒作用,Hochest33258染色荧光显微镜下观察细胞形态学改变,透射电镜下观察亚细胞结构形态,通过流式细胞术检测细胞凋亡比例,Western blot分别检测在联用TRAIL作用前后cFLIP蛋白的表达情况。结果显示:1)单用100ng/mL TRAIL只能杀伤4.67%±0.26%的SKOV3/cDDP细胞,IC50>1000ng/mL。顺铂对SKOV3/cDDP细胞的作用存在剂量依赖性细胞毒效应,这种效应主要通过诱导细胞凋亡实现;2)亚毒性浓度TRAIL(100ng/mL)与亚毒性浓度DDP(1.0μg/mL)联用可杀死52.12%±2.84%的SKOV3/cDDP细胞。呈现出高效协同作用;3)流式细胞术分析、透射电镜观察及荧光显微镜证实其协同性杀伤作用主要通过促进诱导细胞凋亡实现;4)亚毒性浓度TRAIL(100ng/mL)与亚毒性浓度DDP(1.0μg/mL)联用,可使细胞cFLIP蛋白的表达下调,促进了细胞凋亡的发生。结论:TRAIL与亚毒性浓度顺铂联用表现出对多药耐药人卵巢癌细胞的高效杀伤作用,这种作用可能主要由促进细胞凋亡介导实现,cFLIP蛋白的表达减少可能参与这一过程。

In order to explore the multidmg resistant（MDR） mechanism in ovarian carcinoma and to understand the development of MDR and possible ways to overcome this phenomenon, we detected whether TRAIL can synergize with DDP to kill multidrug resistant human ovarian carcinoma cell （SKOV3/cDDP）, and its possible molecular pathology mechanism. SKOV3/cDDP, a MDR cell line, was established by SKOV3 parent cells exposed to progressively and intermittently increasing higher concentration of DDP and had been identified. MTT was used to evaluate the cytotoxic effects for 12,24,48 hours after TRAIL and DDP alone or combined were added to cells. Cell apoptosis and the apoptosis proportion were detected by Flow Cytometry Assay. Hochest33258 fluorescence microscopy and transmission electron microscopic were used to observe cellular and the ultrastructural changes of apoptosis. The changes of cFLIP in protein level were quantified by Western blot analysis. Results： 1 ） SKOV3/cDDP cells were not sensitive to TRAIL. The rate of killing was 4.67 % ± 0.26% with TRAIL concentration of 100ng/mL. And ID50 〉 1000ng/mL; the cells were comparatively more responsive to DDP with an apparent dose-effect relationship. This cytotoxicity was mainly due to cell apoptosis; 2 ） The combination of TRAIL and DDP presented synergistic effect on SKOV3/cDDP cells. Subtoxic concentration of TRAIL（100ng/mL） together with subtoxic concentration of DDP（1.0μg/mL） could kill 52.12% ± 2.84% of SKOV3/cDDP cells; 3）The cytotoxicity was mainly attributed to cell apoptosis as proved by flow cytometry assay, AO fluorescence microscopy and electron microscopy; 4） The combination of TRAIL and DDP, could downregulate the express of cFLIP in protein level. Conclusion： TRAIL can synergize with subtoxic DDP to effectively kill multidrug resistant human ovarian carcinoma cell （ SKOV3/cDDP）, which did not respond to TRAIL alone. Apoptosis is the main mechanism of this killing effect, the apoptosis pathway induced by TRAIL and DDP involved in the downregulate of the expression of cFLIP in protein level.