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目的探讨亚毒性剂量的阿霉素影响抗DR4、DR5单克隆抗体(mAb)FMU1.4和FMU1.5诱导3株神经胶质瘤细胞株U343(TRAIL敏感株)、U138(TRAIL部分敏感株)及U373(TRAIL耐受株)凋亡的作用及可能的机制。方法采用流式细胞术检测DR4、DR5的表达及神经胶质瘤细胞中DNA倍增。用MTT比色法检测mAbFMU1.4和FMU1.5对3株神经胶质瘤细胞增殖的抑制作用。用共聚焦显微镜观察3株细胞中Ca2+的浓度。以Westernblot检测细胞内色素C、FLIP[FLICE-inhibitoryprotein,为一组含有死亡效应结构域(DED)的胞浆蛋白]的表达。结果亚毒性剂量的阿霉素作用后,DR5、DR4在3株细胞中的表达提高;而mAbFMU1.4、FMU1.5诱导U138和U373细胞凋亡的作用增强,细胞内细胞色素C的表达提高,FLIP的表达降低,Ca2+浓度增加。结论亚毒性剂量的阿霉素与抗DR4、DR5mAb联合应用后,可提高mAb诱导靶细胞凋亡的效应,其作用机制与DR4、DR5、细胞色素C、FLIP的表达及Ca2+的含量有关。
Objective To investigate the effects of sub-toxic doses of doxorubicin on the proliferation of three glioma cell lines, U343 (TRAIL-sensitive), U138 (TRAIL-sensitive), induced by anti-DR4 and DR5 monoclonal antibodies (mAb) FMU1.4 and FMU1.5, And U373 (TRAIL-resistant) apoptosis and its possible mechanism. Methods Flow cytometry was used to detect the expression of DR4 and DR5 and DNA doubler in glioma cells. The inhibitory effects of mAbFMU1.4 and FMU1.5 on the proliferation of three glioma cells were detected by MTT assay. Confocal microscope was used to observe the concentration of Ca2 + in the three cells. The expression of FLIP [FLICE-inhibitory protein, a group of cytoplasmic proteins containing death effect domain (DED)] was detected by Western blot. Results The sub-toxic dose of doxorubicin increased the expression of DR5 and DR4 in three cell lines. The effect of mAbFMU1.4 and FMU1.5 on the apoptosis of U138 and U373 cells was enhanced and the expression of intracellular cytochrome C was increased , FLIP expression decreased, Ca2 + concentration increased. Conclusion The combination of sub-toxic dose of doxorubicin with anti-DR4 and DR5 mAb can enhance the effect of mAb on target cell apoptosis. Its mechanism is related to the expression of DR4, DR5, cytochrome C and FLIP and the content of Ca2 +.