多西环素对骨髓瘤细胞株H929内源性凋亡的影响及其作用机制

Effect of Doxycycline on Intrinsic Apoptosis of Myeloma Cell Line H929 and Its Mechanism

目的:探索多西环素(DOX)诱导骨髓瘤H929细胞株凋亡的作用及其可能机制。方法:用DOX、MEK抑制剂U0126和RAS激动剂ML-098单独或联合处理H929细胞,Western blot检测p-MEK、caspase-3、caspase-9、c-Jun等蛋白的表达;CCK8法检测细胞增殖;Annexin-V-FITC/PI双染法检测细胞凋亡。结果:经DOX处理H929细胞后,p-MEK蛋白表达明显减少(P<0.05),伴有caspase-3、9蛋白剪切体的明显增加(P<0.05)。U0126处理H929细胞后,p-MEK蛋白水平明显下降(P<0.05),伴有caspase-3、9蛋白剪切体的明显增加(P<0.05)。DOX联合ML-098处理H929细胞后,H929细胞凋亡比例较DOX单独处理组减少;两药联合处理组p-MEK及p-Erk1/2蛋白表达明显高于DOX单独处理组(P<0.05), Caspase-3、9蛋白剪切体的水平较DOX单药处理组均明显下降(P<0.05)。DOX处理H929细胞后,c-Jun mRNA和蛋白表达水平均明显增加(P<0.05)。结论:DOX可诱导H929细胞内源性凋亡途径的激活,而MEK/ERK通路及c-Jun可能参与DOX诱导细胞凋亡。

Objective: To investigate the mechanism of the in vitro toxicity of doxycycline to myeloma cell line H929 and also the possible pathway involved its toxicity. Methods: Myeloma cell line H929 was treated with DOX, MEK inhibitor U0126 or RAS agonist ML-098, either alone or in combination. Then, the expression of p-MEK, caspase-3, caspase-9 and c-Jun in H929 were used to detected by Western blot;the cells proliferation and apoptosis were detected by CCK-8 assay and flow cytometry, respectively. Results: DOX significantly increased the levels of cleaved caspase-3 and caspase-9, and down-regulated the level of p-MEK in H929(P<0.05). MEK antagonist U0126 significantly increased the levels of cleaved caspase-3 and caspase-9, and down-regulated the level of p-MEK(P<0.05). After Dox combined with ML-098 treatment of H929 cells, the apoptosis rate of H929 cells was lower than that of DOX alone treatment group(P<0.05). Compared with DOX alone treatment group, the expressions of p-MEK and p-ERK1/2 in DOX+ML-098 combined treatment group were increased, and the levels of cleaved caspase-3,9 in H929 cells were decreased(P<0.05). The levels of c-Jun mRNA and protein increased in H929 when treated by DOX alone(P<0.05). Conclusion: DOX can induce apoptosis of H929 via intrinsic apoptosis pathway, and MEK/ERK pathway and c-Jun possibly play a role in this process.