Objective: To investigate the effects of berberine in combination with bortezomib on proliferation and apoptosis of multiple myeloma (MM) cell line. Methods: MM cell line U266 cells were treated with berberine and/or bortezomib. The effects of berberine and/or bortezomib on proliferation of cells were measured by methylthiazolyl tetrazolium bromide (MTT). Flow cytometric Annexin Ⅴ/PI double staining method was used to detect effect of either drug alone or in combination on apoptosis of MM cell line U266. ELISA was used to measure the expression of casepase-3,-8,-9 affected by the two drugs. Western blot was used to detect the expression of the apoptosis-related protein TRADD and FADD. King formula was used to determine if there was a synergistic effect of berberine in combination with bortezomib. Results: ① Both berberine and bortezomib as single agent had dose- and time-dependent effects of proliferation inhibition on U266 cells. Berberine (20 μmol/L) and bortezomib (5 nmol/L) had a synergistic effect of proliferation inhibition (Q value: 1.31-1.65). ② The proportion of early stage apoptosis in both single agent groups and combination group significantly increased compared to control group (P< 0.05). Berberine and bortezomib had a synergistic effect on cell apoptosis (Q value after 6 h and 12 h were 0.896 and 1.197, respectively). ③ Berberine in combination with bortezomib significantly upregulated expressions of caspase-3, -8 and -9, which were statistically significant (P<0.05). ④Berberine in combination with bortezomib significantly upregulated expressions of TRADD (0.91±0.01, 0.70±0.01) and FADD (0.98±0.01, 0.98±0.01) compared with control group (both P<0.05). Conclusion: Berberine in combination with bortezomib had synergistic effects on proliferation inhibition and apoptosis, which were mediated by up-regulated levels of TRADD and FADD.
目的: 探讨小檗碱与硼替佐米联合对多发性骨髓瘤（MM）细胞增殖及诱导细胞凋亡的作用及其可能的机制。
方法: 以MM细胞株U266细胞为研究对象，以小檗碱和硼替佐米作用后，采用MTT法检测其对U266细胞增殖的影响，采用流式细胞术检测其对U266细胞凋亡的影响，采用ELISA法检测其对细胞caspase-3、-8、-9表达的影响，采用Western blot法检测其对细胞Fas相关死亡结构域蛋白（FADD）、TNF受体相关死亡域蛋白（TRADD）表达的影响。采用金（正均）氏公式计算协同系数Q值分析两药的协同作用。
结果: ①两药单独作用时对U266细胞增殖的抑制作用均呈时间、剂量依赖关系；小檗碱（20 µmol/L）与硼替佐米（5 nmol/L）联合作用对U266细胞增殖具有协同抑制作用（Q值为1.31~1.65）。②无论单独用药还是联合用药，与对照组比较，用药组早期凋亡细胞的比例均显著增加（P值均<0.05）；两药联合对U266细胞具有协同促进凋亡的作用（作用6、12 h, Q值分别为0.896和1.197）。③两药联合应用后U266细胞caspase-3、-8、-9、表达水平均较对照组、单药组明显增加，差异有统计学意义（P值均<0.05）。④两药联合应用（24、48 h）后U266细胞TRADD（0.91±0.01、0.70±0.01）、FADD（0.98±0.01、0.98±0.01）蛋白水平均较对照组（0.54±0.01、0.41±0.01）明显增加，差异有统计学意义（P值均<0.05）。
结论: 小檗碱联合硼替佐米可协同促进MM细胞凋亡，这种作用可能是通过上调TRADD及FADD蛋白表达水平而实现的。