依维莫司和MK-2206联合用药协同抑制肝癌细胞增殖  

Combination of everolimus and MK-2206 for synergistic inhibition of hepatocarcinoma cell proliferation

作　　者：乔振宇 王健[2] 吕晓业 黄芳[2] 王芃[2] 李山虎[2] 韩民 

机构地区：[1]贵州医科大学附属医院肝胆外科,贵州贵阳550001 [2]军事医学科学院生物工程研究所医学分子生物学实验室,北京100850 [3]解放军总医院普外科,北京100853

出　　处：《中国药理学与毒理学杂志》2017年第8期793-799,共7页Chinese Journal of Pharmacology and Toxicology

基　　金：国家自然科学基金(81372140);国家自然科学基金(81372770);国家自然科学基金(81470138)~~

摘　　要：目的研究哺乳动物西罗莫司(雷帕霉素)靶蛋白(mTOR)抑制剂与AKT激酶抑制剂联合用药对肝癌细胞增殖的抑制作用。方法 mTOR抑制剂西罗莫司及衍生物依维莫司单独作用HepG2和BEL-7402肝癌细胞0,1,3,6,12和24 h,或与胰岛素样生长因子1受体抑制剂NVP-AEW541或AKT激酶抑制剂MK-2206联合作用24 h,Western蛋白质印迹法检测不同时间点mTOR激酶下游分子p70S6K和AKT激酶活性;依维莫司与MK-2206单独或联合作用肝癌细胞72 h,分别采用平板克隆和CCK8法检测细胞生长增殖和存活力。结果单用西罗莫司和依维莫司可显著抑制肝癌细胞中p70S6K激酶活性(P<0.01),并在不同时间点反馈激活AKT激酶活性(P<0.05);NVP-AEW541或MK2206和依维莫司联合作用肝癌细胞后显著抑制AKT激酶的反馈激活(P<0.01);与使用依维莫司或MK-2206相比,联合使用依维莫司和MK-2206作用肝癌细胞后,显著抑制肿瘤细胞的克隆形成能力;肝癌细胞增殖率较依维莫司单用下降>45%(P<0.01)。结论肝癌细胞对西罗莫司及衍生物的耐药可能是通过反馈激活PI3K/AKT通路导致的,依维莫司联合使用MK-2206可协同抑制肝癌细胞的生长和增殖。OBJECTIVE To examine the synergistic inhibiory effect of combination of mammalian target of sirolimus （Rapamycin） （mTOR） inhibitor everolimus and AKT inhibitor MK-2206 on hepatocarcinoma cell proliferation. METHODS HepG2 and BEL-7402 cells were treated with sirolimus and everolimus alone for 0, 1, 3, 6, 12 and 24 h or in combination with insulin-like growth factor 1 receptor （IGF- 1R） inhibitor NVP-AEW541 or AKT inhibitor MK2206 for 24 h. p70S6K and AKT kinase activityies were detected by Western blotting. Plate clone formation assay and CCK8 assay were used to detect the growth and proliferation of hepatocarcinoma cells treated with everolimus and MK2206 alone or in combi- nation. RESULTS Sirolimus and everolimus inhibited p70S6K activity while causing feedback activation of AKT kinase activity at different time points （P〈0.01）. NVP-AEW541 and MK-2206 could inhibit AKT kinase feedback activation by everolimus （P〈0.05）. Colony formation of hepatocarcinoma cells treated with everolimus and MK-2206 in combination was significantly inhibited compared with everolimus or MK-2206 alone （P〈0.01）. Everolimus and MK-2206 in combination inhibited the proliferation rate of two types of hepatocarcinoma cancer cells by more than 45% compared with everolimus used alone （P〈0.01）. CONCLUSION The resistance of sirolimus and its derivatives in hepatocellular carcinoma cells may be achieved throngh the feedback-activated PI3K/AKT pathway, and the combination therapy can synergistically inhibit the growth and proliferation of hepatocarcinoma cells.

关 键 词：肝肿瘤 西罗莫司 依维莫司 MK2206 联合用药 抗药性 肿瘤 

分 类 号：R96[医药卫生—药理学]