mTORC1/2抑制剂与伊马替尼联用抑制Ph^+ ALL细胞株生长的作用机制

The Mechanism of Combination using mTORC1/2 Inhibitor and Imatinib to Suppress Cell Proliferation of Ph^+ ALL Cell Line

目的探讨哺乳动物雷帕霉素靶蛋白C1/2(mTORC1/2)抑制剂PP242与伊马替尼(IM)联用对费城染色体阳性(Ph+)急性淋巴细胞白血病(ALL)细胞株SUP-B15的抗白血病作用及机理。方法以SUP-B15细胞为研究模型,MTT法检测IM与PP242联合处理SUP-B15细胞72h后的半数抑制浓度(IC50)及联合作用指数(CI);Western blot法检测PP242处理SUP-B15细胞后对磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/mTOR通路的影响,以及IM与PP242联合处理SUP-B15细胞后对PI3K/Akt/mTOR通路及凋亡相关蛋白的影响。结果IM单用于SUP-B15细胞的IC50值为(1.50±0.09)μmol/L。而IM与20、30、50nmol/L PP242联用于SUP-B15细胞,其IC50分别下降为(0.81±0.030)μmol/L、(0.36±0.140)μmol/L、(0.02±0.002)μmol/L,CI值分别为0.764、0.545、0.507,提示两药有较高的协同作用。PP242单独作用于SUP-B15细胞后,磷酸化Akt(p-Akt)、p-4EBP1、p-elF4E、p-ABL、p-mTOR、p-P70等PI3K/Akt/mTOR通路上的关键磷酸化蛋白表达下调,且这种变化呈现浓度和时间依赖性。PP242与IM联合用于SUP-B15细胞时,SUP-B15细胞PI3K/Akt/mTOR通路的各关键蛋白下调较PP242或IM单用时更明显,凋亡相关蛋白Bax、cleaved Caspase-3上调也较两药单用时更明显。结论PP242与IM联合使用时可增强对于PI3K/Akt/mTOR通路的抑制,增加由Bax、Caspase-3所介导的凋亡作用。

Objective To investigate the anti-leukemia effect and mechanism of mTORC1/2 inhibitor PP242 combined with imatinib （IM） on the proliferation of Ph＋ acute lymphoblastic leukemia （ALL） cell line SUP-B15. Methods SUP-B15 cell line was treated with PP242, imatinib （IM）, or PP242 plus IM for 72 h, IC50 values （the concentration of drug required to kill 50% of the cells） and the combination index （CI ） of synergistic cytotoxicity was determined using MTT methods. The expressions of PI3K/Akt/mTOR and apoptosis associated proteins were examined by Western blot test. Results The IC50 value of IM alone was （1.50±0.09） μmol/L, however, the IC50 values were （0.81±0.030） μmol/L, （0.36±0.140） μmol/L and （0.02±0.002） μmol/L combined with 20 nmol/L, 30 nmol/L and 50 nmol/L of PP242, and the CI values were 0.764, 0.545 and 0.507, indicating two drugs had highly synergistic effect on anti-proliferation in the SUP-B15 cell line. The expressions of p-Akt, p-4EBP1, p-elF4E, p-cAbl, p-mTOR and p-P70 were down-regulated significantly in a dose-dependent and time-dependent manner after PP242 treatment．Compared with PP242 or IM alone, the down-regulation of PI3K/Akt/mTOR signaling pathway and the up-regulation of the apoptosis associated proteins （bax and cleaved caspase-3） were more significant in the combination of two drugs. Conclusion The combination of IM and PP242 could increase the inhibition of PI3K/Akt/mTOR signaling pathway and apoptosis mediated by bax and caspase-3 in SUP-B15 cell line.