腺苷酸活化蛋白激酶增强乳腺癌对多柔比星化疗敏感性的机制

Mechanism of AMPK-enhanced chemosensitivity of breast cancer MCF-7/adr cells to adriamycin

背景与目的:腺苷酸活化蛋白激酶(AMP-activated protein kinase,AMPK)在调控细胞代谢和能量平衡方面起着重要作用,并与细胞增殖、生存和多种信号通路密切相关。近年来发现AMPK参与肿瘤的抑制和耐药。该研究旨在探讨AMPK对多柔比星抑制乳腺癌作用的影响及其机制。方法:采用四甲基偶氮唑蓝(methylthiazolyl tetrazolium,MTT)法检测多柔比星作用后对MCF-7/adr、MCF-7/adr-vector及MCF-7/adr-AMPKα细胞增殖的影响;Hoechst染色法观察各组细胞凋亡形态;流式细胞术(flow cytometry,FCM)检测各组细胞凋亡率;荧光酶标仪检测3组细胞多柔比星累积量;蛋白[质]印迹法(Western blot)检测各组细胞中耐药蛋白及凋亡相关蛋白的表达。结果:多柔比星对MCF-7/adr细胞增殖抑制作用呈剂量和时间依赖性,其作用24、48 h的IC_(50)值分别为(36.8±2.1)和(28.8±1.3)μg/m L。过表达AMPKα可增加多柔比星对MCF-7/adr细胞的生长抑制作用,呈剂量和时间依赖性,其作用24、48 h的IC_(50)值分别为(16.0±0.7)和(4.2±0.2)μg/m L。荧光形态分析发现多柔比星联合AMPKα能诱导MCF-7/adr细胞凋亡。1.0μg/m L多柔比星作用48 h后,MCF-7/adr、MCF-7/adr-vector及MCF-7/adr-AMPKα细胞的凋亡率分别为(12.0±1.4)%、(12.7±1.6)%和(32.0±4.2)%,MCF-7/adr细胞中AMPKα过表达明显提高MCF-7/adr细胞对多柔比星的敏感性。荧光酶标仪检测显示,过表达AMPKα能明显提高细胞内多柔比星的累积量,具有浓度依赖性。Western blot实验结果显示,与MCF-7/adr和MCF-7/adr-vector细胞比较,MCF-7/adr-AMPKα细胞中Bax、细胞色素c(Cyto c)的释放、caspase-3和多聚腺苷二磷酸-核糖聚合酶降解产物(cleaved PARP)蛋白表达明显增加,而细胞外排泵P-糖蛋白(P-gp)和Bcl-2蛋白表达降低。结论:AMPKα可通过抑制耐药细胞外排泵以及调控凋亡相关蛋白的表达,从而增强乳腺癌耐药细胞对多柔比星的化疗敏感性。

Background and purpose： AMP-activated protein kinase （AMPK） plays an important role in the regulation of cell metabolism and energy balance and is associated with cell proliferation, survival and multiple signaling pathways. Recent reports found that AMPK is involved in tumor suppression and drug resistance. The aim of this study was to explore the effect of AMPK on the anti-tumor effect of adriamycin and underlying mechanism in breast cancer MCF-7/adr cells. Methods： The anti-proliferative effects of adriamycin was detected by methyl thiazolyl tetrazolium （MTT） assay in MCF-7/adr, MCF-7/adr-vector and MCF-7/adr-AMPKa cells. The cell morphology in each group was stained with the fluorescent dye Hoechst 33528, and the effects on apoptosis induction were examined by flow cytometry （FCM）. The intracellular concentration of adriamycin was detected by fluorescence assay. The resis- tance- and apoptosis-related proteins were analyzed by Western blot. Results： The growth of breast cancer MCF-7/adr cells was inhibited by adriamycin in a dose- and time-dependent manner. The IC50 values at 24 and 48 h were （36.8±2.1） and （28.8±1.3） μg/mL, respectively. AMPKa over-expression enhanced the cytotoxic effect of adriamycin in MCF-7/adr-AMPKa cells in a dose- and time-dependent manner. Its IC50 values at 24 and 48 h were （16.0±0.7） and （4.2±0.2） μg/mL, respectively. Fluorescent morphological assay showed that AMPKa overexpression contributed to adriamycin induced apoptosis in MCF-7/adr-AMPKa cells. After treatment with 1.0 μg/mL adriamycin for 48 h, the apoptosis rates of MCF-7/adr, MCF-7/adr-vector and MCF-7/adr-AMPKa cells were （12.0±1.4）%, （12.7±1.6）% and （32.0±4.2）%, respectively, indicating that overexpression of AMPKtt enhanced the adriamycin-induced apoptosis in MCF-7/adr cells. Fluorescence microplate assay showed that over expression of AMPKa significantly increased the intracellular accumulation of adriamycin, in a concentration dependent manner. Western blot analysis showed that, compared with MCF-7/adr and MCF-7/adr-vector cells, the expressions of Bax, caspase-3 and cleaved PARP proteins were increased. Meanwhile, Bcl-2 and P-gp protein expressions were decreased in MCF-7/adr-AMPKa cells. Furthermore, the release of eytochrome c from mitochondria into the cytosol was also observed in MCF-7/adr-AMPKct cells. Conclusion： AMP- Ka overexpression can enhance the chemosensitivity of breast cancer MCF-7/adr cells to adriamycin through inhibiting the drug efflux transporter and regulating the expression of apoptosis-related proteins.