Role Of Adenosine Phosphate-activated Protein Kinase (AMPK)in Doxorubicin Induced Apoptosis Of MCF-7 Human Breast Cancer Cell

Background and Objectives Breast cancer is one of the main health risks among worldwide women,which had an extremely highprevalence.Breast cancer,seriously affecting the lives of women and families quality,increasing the burden on families and countries,has increased dramatically in the incidence of morbidity and mortality rates in our country in recent years.Chemotherapy has currently been one of the main treatments.Doxorubicin,a glycoside antitumor antibiotic extracted from the fermentation broth Streptomyces peucetius var.caesius in 1970 s,inhibiting synthesis of RNA and DNA,especially for RNA,is a cycle non-specific drugs.DOX has been widely used in breast cancer,lung cancer,bladder cancer,stomach cancer,ovarian cancer,Hodgkin’s lymphoma,non-Hodgkin’s lymphoma,multiple myeloma,sarcoma and pediatric cancer treatment,however,due to the toxic effects of cardiac muscle cells make its use very limited,and mostly combined with other anticancer drugs.The mechanism of DOX anti-tumor action has not been fully elucidated,further studies is needed to clarify its molecular mechanism.Aiming to investigate the mechanism of anti-breast cancer cell proliferation induced by doxorubicin(DOX),we perform the study to expound the role of AMP-activated protein kinase(AMPK)in doxorubicin induced breast cancer MCF-7 cell apoptosis.Methods and materials MCF-7 Cells were cultured in DMEM medium supplemented with 10% fetal bovine serum(FBS)with antibiotics,and incubated at 37 °C in a humidified air atmosphere containing 5% CO2,logarithmic growth phase of the cancer cells were used in the experiment.AMP-Activated Protein Kinase(AMPK)activator AICAR,AMPK si RNA,AMPK inhibitor compound C and doxorubicin were used to treated MCF-7 cells at different time;then AMPK,acetyl Co A carboxylase(ACC),P38 activationby were detected by Western Blot,MTT was used as cell viability assay.Detect the role of AMPK si RNA in Doxorubicin-induced inhibition of cancer cell clone formation,Doxorubicin-induced cell apoptosis,in Doxorubicin-induced ROS generation.Statistical analysis The results were presented as mean ± SD.Comparisons between more than 2 groups were performed by analysis of variance(one-way ANOVA,SPSS 16.0),followed by Students-test.p < 0.05 was considered statistically significant.Results1.Doxorubicin-induced activation of AMPK,AMPK agonist(AICAR)or in combination with doxorubicin activated AMPK and Increased MCF-7 cell proliferation rate [the difference of cell viability between group AICAR+DOX(17.7±1.6%)and group DOX(58.4±1.8%)was significant(P <0.001)].After AMPK inhibitor(compound C)or AMPK si RNA and doxorubicin combined administration,P-AMPK and P-ACC expression was significantly decreased,the level of p-p38 was not affected,and MCF-7cell proliferation inhibition rate decreased[the cell viability of group AMPKi+DOX(72.7±1.8%)VS.group DOX(48.3±1.7%),P<0.001;group AMPK si RNA +DOX(76.9±2.2%)VS.group scramble si RNA+DOX(96.9±1.8%),P<0.001];AMPK si RNA inhibit Doxorubicin-induced inhibition of cancer cell clone formation,Doxorubicin-induced cell apoptosis and Doxorubicin-induced ROS generation.2.AMPKα and ACC phosphorylation level rised with time after added doxorubicin in MCF-7 cells,DOX activated AMPK signaling pathway;P-AMPK and P-ACC were significantly increased compared with single-use AICAR when DOX and AMPK activator AICAR treated;We took AMPKα si RNA stably transfected Eca-109 cell lines to gene silencing AMPK or inhibited the expression of AMPK by AMPK inhibitor compound C,then treated with DOX drugs,AMPK activation degree weakened,AMPK-RNAi and AMPK inhibitor compound C can inhibit DOX-induced activation of AMPK.Conclusions In cultured MCF-7 breast cancer cells,AMPK mediated DOX-induced apoptosis,possibly by activating caspase-3,induced ROS generation,eventually causing cancer cell death.