关键词: 乳腺癌, 耐药, 腺苷酸活化蛋白激酶, 凋亡, 多柔比星

Abstract: 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-AMPKα 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 resistance- 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. AMPKα over-expression enhanced the cytotoxic effect of adriamycin in MCF-7/adr-AMPKα 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 AMPKα overexpression contributed to adriamycin induced apoptosis in MCF-7/adr-AMPKα 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-AMPKα cells were (12.0±1.4)%, (12.7±1.6)% and (32.0±4.2)%, respectively, indicating that overexpression of AMPKα enhanced the adriamycin-induced apoptosis in MCF-7/adr cells. Fluorescence microplate assay showed that over expression of AMPKα 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-AMPKα cells. Furthermore, the release of cytochrome c from mitochondria into the cytosol was also observed in MCF-7/adr-AMPKα cells. Conclusion: AMPKα 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.

Key words: Breast cancer, Drug resistance, AMP-activated protein kinase, Apoptosis, Adriamycin