抗坏血酸增强三氧化二砷诱导多发性骨髓瘤细胞凋亡

Ascorbic acid enhances apoptosis of multiple myeloma RPMI 8226 cells induced by arsenic trioxide

目的:探讨抗坏血酸(AA)对三氧化二砷(As2O3)诱导人多发性骨髓瘤(MM)细胞株RPMI8226细胞凋亡的作用及其分子机制。方法:采用细胞计数、细胞形态学、透射电镜、吖啶橙溴化乙锭(AOEB)染色、AnnexinⅤ染色以及细胞周期测定,观察As2O3联合AA对RPMI8226细胞的增殖抑制及促凋亡作用;应用流式细胞仪检测RPMI8226细胞死亡受体DR4分子的表达。结果:单用AA对RPMI8226细胞的生长无影响;As2O3抑制RPMI8226细胞的生长,其作用呈时间和剂量依赖性;As2O3联合AA较单用As2O3对RPMI8226细胞的抑制作用强(P<0.01)。单用5μmol/L的As2O3对靶细胞作用后,无明显凋亡形态学改变;100μmol/L的AA联合5μmol/L的As2O3作用靶细胞后:光镜、电镜和AOEB染色显示出典型的凋亡形态学改变;AnnexinV染色检测到早期凋亡细胞;细胞周期显示:G1期细胞比例增高,S期减低,并有凋亡峰。As2O3能上调RPMI8226细胞表达DR4分子,AA联合As2O3可以增强上述效应(P<0.05)。结论:AA对As2O3诱导RPMI8226细胞凋亡有增敏效应,该效应过程可能依赖于死亡受体DR4的过度表达。

Objective： To investigate the effect of ascorbic acid （AA） on arsenic trioxide-induced apoptosis of multiple myeloma RPMI-8226 cells and the molecular mechanisms. Methods： RPMI 8226 cells were treated with 5μmol/L arsenic trioxide （As2O3）alone, or in combination with AA 100μmol/L. The growth inhibition was determined by Trypan blue exclusion assay. Apoptosis of cells was detected by Wrigh Giema, AO/EB, and Annexin V staining. Morphology of apoptotie cells was examined by transmission electron. Microscopy. Cell cycle and expression of death receptor 4 （DR4） on RPMI 8226 cells were tested by flow cytometry. Results： AA alone could not inhibit the growth and induce apoptosis of RPMI 8226 cells, while As2O3 inhibited the proliferation of RPMI 8226 cells in a time and dose-dependent manner. Compared with As2O3, treatment alone,growth inhibi tion was enhanced by combination therapy with As2O3 and AA（P〈0.01）. Wrigh Giema and AO/EB staining and transmission electron microscopy demonstrated the characteristic morphological changes of apoptosis of RPMI 8226 cells after treatment with As2O3 5 μmol/L in comhination with AA 100 μmol/L. Annexin V staining showed the earlier apoptotic cells. Flow cytometry showed that the proportion of G1 cells was increased and cell proportion in sub-S phase was decreased with appearance of apopto sis peak. As2O3 up-regulated DR4 expression on RPMI 8226 cells modestly. AA enhanced the up regulation of DR4 expression induced by As2O3, （P〈0.05）. Conclusions： AA enhances arsenic trioxide-induced apoptosis in RPMI 8226 cells. It may be dependent on the over-expression of death receptor DR4.