Autophagy is involved in cytotoxic effects of crotoxin in human breast cancer cell line MCF-7 cells  

作　　者：Ci-hui YAN Ya-ping YANG Zheng-hong QIN Zhen-lun GU Paul REID Zhong-qin LIANG 

机构地区：[1]Department of Pharmacology, Soochow University School of Medicine, Suzhou 215123 China [2]Celtic Biotech, Ltd, Dublin, Ireland

出　　处：《Acta Pharmacologica Sinica》2007年第4期540-548,共9页中国药理学报（英文版）

摘　　要：Aim： To investigate the role of crotoxin （CrTX）-induced autophagy in the death of MCF-7 cells, a caspase-3-deficient, human breast cancer cell line. Methods： Cultured MCF-7 cells were treated with various doses of CrTX, a phospholipase A2 （PLA2） isolated from the venom of the South American rattlesnake, Crotalus durissus terrificus. The cytotoxicity of CrTX in the presence and absence of caspase inhibitors was measured with methyl thiazolyl tetrazolium （MTT） and lactate dehydrogenase （LDH） leakage assays. The activation of autophagy was determined with transmission electron microscope and monodansylcadaverin （MDC） labeling. The upregulation of lysosomal enzymes, the release of cytochrome c （cyto-c）, and the nuclear translocation of the apoptosis inducing factor （AIF） were examined by immunoblotting and immunofluorescence. Results： CrTX inhibited the viability of MCF-7 cells in a dose- and time-dependent manner. CrTXactivated autophagy was revealed by punctuate MDC labeling, and an increase in the formation of autophagosomes as well as apoptosis, as evidenced by nuclear condensation and fragmentation. The activation of cathepsin B, D, and L, in addition to the release of cytochrome c and the relocation of AIF into nuclei, were observed after CrTX treatment. Autophagy inhibitors 3-methyladenine （3-MA）, NH4Cl, and the pan-caspase inhibitor, Z-Val-Ala-Asp-fluoromethylketone （Z-Vad- fmk）, attenuated CrTX-induced cell death. Conclusion： An autophagic mechanism contributes to the apoptosis of MCF-7 cells induced by CrTX.Aim： To investigate the role of crotoxin （CrTX）-induced autophagy in the death of MCF-7 cells, a caspase-3-deficient, human breast cancer cell line. Methods： Cultured MCF-7 cells were treated with various doses of CrTX, a phospholipase A2 （PLA2） isolated from the venom of the South American rattlesnake, Crotalus durissus terrificus. The cytotoxicity of CrTX in the presence and absence of caspase inhibitors was measured with methyl thiazolyl tetrazolium （MTT） and lactate dehydrogenase （LDH） leakage assays. The activation of autophagy was determined with transmission electron microscope and monodansylcadaverin （MDC） labeling. The upregulation of lysosomal enzymes, the release of cytochrome c （cyto-c）, and the nuclear translocation of the apoptosis inducing factor （AIF） were examined by immunoblotting and immunofluorescence. Results： CrTX inhibited the viability of MCF-7 cells in a dose- and time-dependent manner. CrTXactivated autophagy was revealed by punctuate MDC labeling, and an increase in the formation of autophagosomes as well as apoptosis, as evidenced by nuclear condensation and fragmentation. The activation of cathepsin B, D, and L, in addition to the release of cytochrome c and the relocation of AIF into nuclei, were observed after CrTX treatment. Autophagy inhibitors 3-methyladenine （3-MA）, NH4Cl, and the pan-caspase inhibitor, Z-Val-Ala-Asp-fluoromethylketone （Z-Vad- fmk）, attenuated CrTX-induced cell death. Conclusion： An autophagic mechanism contributes to the apoptosis of MCF-7 cells induced by CrTX.

关 键 词：CROTOXIN AUTOPHAGY APOPTOSIS CYTOCHROMEC apoptosis inducing factor 

分 类 号：R737.9[医药卫生—肿瘤] R979.1[医药卫生—临床医学]