青蒿琥酯对硼替佐米耐药骨髓瘤细胞株的耐药逆转作用及机制

Drug Resistance Reversal Effect of Artesunate on Bortezomib-resistant Multiple Myeloma Cell Line and Its Molecular Mechanism

目的:探讨青蒿琥酯对硼替佐米耐药骨髓瘤(multiple myeloma,MM)细胞株的增殖抑制作用,并探讨其逆转骨髓瘤细胞耐药的作用机制。方法:采用硼替佐米浓度梯度递增法,以人MM细胞株NCI-H929为亲本,建立硼替佐米耐药的NCIH929细胞株NCI-H929BR。采用噻唑蓝(MTT)比色法检测青蒿琥酯对NCI-H929BR的增殖抑制及对硼替佐米的耐药逆转作用;流式细胞术检测细胞凋亡;蛋白免疫印迹法(Western blot)检测核转录因子-κB p65(nuclear factor-κB p65,NF-κB p65)蛋白,磷酸化核转录因子-κB p65(phospho-nuclear factor-κB p65,NF-κB p-p65)蛋白,P糖蛋白(P-glycoprotein,P-gp),B细胞淋巴瘤/白血病-2(B-cell lymphoma/leukemia-2,Bcl-2)蛋白以及Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)的表达变化。结果:采用浓度梯度递增法成功建立了硼替佐米耐药的NCI-H929细胞株NCI-H929BR,其耐药倍数为20.2倍。青蒿琥酯对NCI-H929BR有明显的增殖抑制作用,抑制效应呈浓度依赖性;硼替佐米(50 nmol·L-1)单药对NCI-H929BR无明显增殖抑制作用,青蒿琥酯(12.5 mg·L-1)单药的抑制率为(23.53±2.21)%(P<0.05),两药联合的抑制率为(60.71±3.43)%(P<0.01);青蒿琥酯处理后,NCI-H929BR的凋亡率上升,NF-κB p65,NF-κB p-p65,P-gp,Bcl-2表达下降,Bax表达上升,呈浓度依赖性。结论:青蒿琥酯可抑制NCI-H929BR的增殖,促进细胞凋亡,逆转其对硼替佐米的耐药性,下调NF-κB p65,p-p65,P-gp以及Bcl-2的表达,上调Bax的表达可能为其逆转肿瘤细胞耐药的作用机制。

Objective： To investigate the effects of Artesunate on suppressing the proliferation of Bortezomib-resistant multiple myeloma （MM） cell line and to explore its molecular mechanism on reversal of drug resistance. Method： Human MM cell line NCI-H929 was treated with Bortezomib in a dose-dependent manner to establish Bortezomib-resistant cell line NCI-H929BR. The inhibitory role of Artesunate on NCI-H929BR and its reversal effect against Bortezomib-resistance were determined by methylthiazolyldiphenyl-tetrazolium bromide（MTT） assay. Cell apoptosis was determined by flow cytometry, and the protein expression levels of nuclear factor-κB p65 （NF-κB p65）, phospho-nuclear factor-κB p65 （NF-κB p-p65）, P-glycoprotein （P-gp）, B-cell lymphoma/leukemia-2 （Bcl-2） protein and Bcl-2 associated X protein （Bax） were detected by Western blot assay. Result： Bortezomib-resistance index of NCI-H929BR was 20.2 times. Artesunate treatment had significant inhibitory effect on the proliferation of NCI-H929BR and the inhibitory effect was in a concentration-dependent manner. Bortezomib （50 nmol·L^-1）alone had less effect on NCI-H929BR proliferation, while the inhibition rate of artesunate （12.5 mg·L^-1） alone was （23.53±2.21）% （P〈0.05）; Bortezomib combined with artesunate treatment had greater inhibitory effect （60.71±3.43）% （P〈0.01）. Artesunate treatment increased NCI-H929BR apoptosis, down-regulated NF-κB p65, NF-κB p-p65, P-gp and Bcl-2 expression levels, and up-regulated Bax expression level in a concentration-dependent manner. Conclusion： Artesunate could inhibit NCI-H929BR proliferation, promote apoptosis and reverse Bortezomib-resistance, and its mechanism may be associated with down-regulating expression levels of NF-κB p65, NF-κB p-p65, P-gp, Bcl-2 and up-regulating expression level of Bax.