17-丙烯胺基-17-去甲氧基格尔德霉素增强慢性粒细胞白血病细胞对伊马替尼敏感性的机制研究

Mechanisms of Imatinib Resistance on K562 and K562A02 Reversing by 17-AAG

目的探索伊马替尼(IM)联合使用17-丙烯胺基-17-去甲氧基格尔德霉素(17-AAG)增加慢性粒细胞白血病(CML)细胞株K562和K562A02凋亡率的机制。方法采用四甲基偶氮唑蓝法观察细胞增殖抑制率,Annexin V/PI双染色法检测细胞凋亡率,逆转录-聚合酶链反应法检测Bcr-abl、MDR1、Bcl-2 mRNA的表达及Western blot法检测Bcr-abl、p-gp、Bcl-2蛋白的表达。结果0.31μg·mL-1伊马替尼联合1.25μg·mL-117-丙烯胺基-17-去甲氧基格尔德霉素分别作用于K562和K562A02细胞48 h,增殖抑制率分别为(70.78±1.01)%和(39.27±1.52)%,凋亡率为(48.74±2.51)%和(45.0±0.4)%。2.5μg·mL-1伊马替尼联合10μg·mL-117-丙烯胺基-17-去甲氧基格尔德霉素作用于K562A02细胞48 h,增殖抑制率达(65.63±0.93)%。逆转录-多聚酶链反应和Western blot结果显示,联合用药后K562细胞的Bcr-abl和Bcl-2基因显著降低,MDR1呈弱表达,无明显变化。而K562A02细胞中,Bcr-abl、Bcl-2和MDR1基因的表达均有明显下调(P<0.05)。结论伊马替尼联合17-丙烯胺基-17-去甲氧基格尔德霉素可有效抑制K562和K562A02细胞增殖和诱导凋亡,两者联合应用具有克服伊马替尼耐药的作用。

OBJECTIVE To observe the effect of imatinib （IM） combined with 17-allylamino-17-demethoxygeldanamycin （ 17- AAG） on the growth inhibition and apoptosis of two homologous chronic myeloid leukemic （CML） cell lines K562 and K562A02, and to evaluate their sensitivity or resistance to imatinib. METHODS Proliferation inhibition was detected by tetrazolium-based colorimetric assay （MTF） , and drug-induced apoptosis was evaluated by Annexin V/PI assay. The mRNA expressions of Bcr-abl, MDR1 and Bcl-2 were determined by reverse transcriptase-polymerase chain reaction （ RT-PCR）, and the protein expressions of Bcr-abl, p-gp and Bcl-2 were evaluated by Western blot. RESULTS The proliferation inhibition rate were （70. 78 ±1.01 ） % and （39. 27 ±1.52） % , respectively, as the apoptotic rate reached （48.74 ±2. 51 ） % and （45.0± 0. 4） % in K562 and K562A02 cell lines after being treated with 0. 31 μg·mL^-1 IM plus 1.25 μg·mL^-1 17-AAG for 48 h. In K562A02 cell line, however, the rate was about （65.63 ± 0.93 ） % after being treated with 2.50 μg·mL^-1 IM plus 10 μg·mL^-1 17-AAG for 48 h （P 〈 0. 05 ）. Combined use of IM and 17- AAG significantly affected the expressions of Bcr-abl, Bcl-2 in mRNA and proteins level, while MDR1 mRNA was not expressed in K562 cell. However, the Bcr-abl, MDR1 mRNA and Bcr-abl, p-gp protein expressions in K562A02 cells decrease significantly after being treated with IM plus 17-AAG for 48 h. CONCLUSION Combined use of IM and 17-AAG exerted significant synergistic effects on the growth inhibition and apoptosis of imatinib-sensitive K562 and imatinib-resistant K562A02 cells. This combination regimen may be a potential therapeutic remedy tor overcoming the resistance to imatinib.