TRAIL增强低剂量阿霉素高效诱导人骨肉瘤细胞凋亡的实验研究

目的探讨可溶性肿瘤坏死因子相关凋亡诱导配体(TRAIL)与阿霉素(ADM)联用是否存在协同性杀伤人骨肉瘤细胞的作用以及分子机制。方法TRAIL、ADM或两药联用24h,MTT法测试细胞毒作用,吖啶橙染色荧光显微镜和透射电镜下观察细胞及亚细胞结构形态,流式细胞术检测细胞凋亡比例。RT-PCR和Westernblot分别检测药物作用前后cFLIPmRNA和cFLIP蛋白的表达。结果(1)人骨肉瘤U2OS细胞对TRAIL不敏感,IC50>1mg/L。U2OS细胞对ADM相对敏感,存在剂量依赖性细胞毒效应。(2)TRAIL与ADM联用呈现高效协同作用,表现为亚毒性浓度TRAIL(0.1mg/L)与亚毒性浓度ADM(1.0μmol/L)联用可杀伤49.54%±2.79%的U2OS细胞。(3)流式细胞术、透射电镜及荧光显微镜观察证实,协同性杀伤作用主要通过诱导细胞凋亡实现。(4)cFLIPmRNA和cFLIP蛋白的表达下调促进了药物协同诱导凋亡的发生。结论TRAIL与亚毒性浓度ADM联用表现出的高效杀灭肿瘤细胞作用主要由凋亡介导实现,cFLIPmRNA下调和cFLIP蛋白的表达减少可能参与这一过程。

Synergistic induction of apoptosis by the combination of TRAIL and low dose adriamycin in human osteosarcoma cell line U2OS

OBJECTIVE: To investigate whether TRAIL can synergize with adriamycin (ADM) to kill osteosarcoma cells (U2OS) in vitro, and its possible molecular mechanism. METHODS: MTT was used to evaluate the cytotoxic effect of TRAIL and ADM either used alone or in combination at 24 hours after treatment to U20S cells. Cell apoptosis and its proportion were detected by flow cytometry assay. Acridine orange fluorescence microscopy and transmission electron microscopy were used to examine cellular and ultrastructural changes of apoptosis. The changes of cFLIP in mRNA and protein level were semi-quantified by RT-PCR and Western blot analysis. RESULTS: (1) U2OS cells were not sensitive to TRAIL (IC(50) > 1 mg/L); the cells were relatively more responsive to ADM in an apparent dose-effect fashion. (2) The combination of TRAIL and ADM presented a synergistic effect on U2OS cells. Subtoxic concentration of TRAIL (0.1 mg/L) combined with subtoxic concentration of ADM (1.0 micromol/l) killed (49.54 +/- 2.79)% of U2OS cells. (3) The cytotoxicity was mainly attributed to cell apoptosis as demonstrated by flow cytometry assay, fluorescence microscopy and electron microscopy. CONCLUSION: Subtoxic dose of TRAIL can effectively kill osteosarcoma cells (U2OS) in combination with subtoxic dose of ADM, but not effective when used alone. Apoptosis is the main mechanism of this killing effect induced by combination of TRAIL and ADM. Down regulation of cFLIP at mRNA and protein level is involved in this apoptosis pathway.