硼替佐米联合二硫化二砷诱导慢性粒细胞白血病细胞凋亡机制研究

Effect of bortezomib combined with arsenic disulfide on induction of apoptosis in chronic myeloid leukemic cells

目的:探讨硼替佐米(bortezomib)与二硫化二砷(As2S2)对慢性粒细胞白血病(chronic myeloid leukemia,CML)细胞株K562和对伊马替尼耐药的同源细胞株K562R的增殖抑制、诱导凋亡及其作用机制。方法:应用四甲基偶氮唑盐比色法(MTT)增殖抑制实验、瑞氏染色细胞形态观察法、膜联蛋白Ⅴ(Annexin Ⅴ)流式细胞仪检测硼替佐米和As2S2单独或联合作用于CML细胞株的增殖抑制和凋亡情况;蛋白免疫印迹法(Western blot)检测药物对Bcr-Abl蛋白以及凋亡相关蛋白的影响;反转录PCR(RT-PCR)检测Bcr-Abl mRNA的表达。结果:硼替佐米联合As2S2能有效抑制CML细胞增殖,6nmol/L硼替佐米和3μmol/L As2S2联合作用于K562细胞48h,细胞生长抑制率达(76.4±3.9)%以上,坏死和凋亡细胞比例达54.0%,与单药作用(硼替佐米13.9%;As2S2 8.2%)相比,差异有统计学意义(P<0.05)。6nmol/L硼替佐米和3μmol/L As2S2联合作用于K562R细胞48h,细胞生长抑制率仅(55.7±5.5)%。12nmol/L硼替佐米和6μmol/L As2S2联合作用于K562R细胞48h,坏死和凋亡细胞比例为38.0%,显著高于单药(硼替佐米24.0%;As2S2 11.2%)(P<0.05)。药物联合作用通过线粒体途径协同诱导CML细胞凋亡,并使K562和K562R细胞的Bcr-Abl蛋白表达和蛋白磷酸化水平下降。结论:硼替佐米联合As2S2有效抑制K562细胞增殖和诱导凋亡,提高浓度后对K562R细胞也产生类似的作用,有增殖抑制和诱导凋亡作用,两者联合应用可能具有克服伊马替尼耐药的作用。

Objective To study the effect of bortezomib combined with arsenic disulfide （As2S2） on growth inhibition and apoptosis of two kinds of homologous chronic myeloid leukemic （CML） cell line K562 and K562R, sensitive or resistant to imatinib, respectively. Methods Tetrazolium-based colorimetric assay （MTF）, Giemsa stain cell morphology observation and Annexin V flow eytometry were used to assess the effects of bortezomib and AsaS2, in single use or in combination, on inhibition of proliferation and induction of apoptosis in CML cell line. The effects on Bcr-Abl and apoptosis-related proteins was evaluated by Western blot analysis and expression of Ber-Abl mRNA was determined by reverse transcriptase-polymerase chain reaction （RT-PCR）. Results Bortezomib and As2S2 exerted synergistic effects on growth inhibition in both the K562 and K562R cell lines. The growth inhibition rate was over 70% and the rate of dead and apoptosis cells reached 54% in K562 cell line after treated with 6 nmol/L bortezomib plus 3 p, mol/L As2S2 for 48 h. The difference was statistically significant （P〈0.05） when compared with these drugs in single use. In K562R cell line, the growth inhibition rate was about 55% and the rate of dead and apoptosis cells was 38% after treated with t2 nmol/L bortezomib plus 6 μmol/L As2S2 for 48 h, and the difference was also statistically significant when compared with these drugs in single use （P〈0.05）. Combined use of bortezomib and As2S2 induced apoptosis by mitoehondrial dependent apoptosis pathway, decreased Bcr-Abl protein expression and level of protein phosphorylation in both the K652 and K652R cell lines. Conclusions Combined use of bortezomib and As2S2 has significant synergistic growth inhibiting effect and apoptosis inducing effect both on imatinib-sensitive K562 and imatinib-resistant K562R cells. This combination regimen may be a potential therapeutic remedy for overcoming the imatinib resistance.