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Aim:To investigate the role of crotoxin (CrTX)-induced autophagy in the death ofMCF-7 cells,a caspase-3-deficient,human breast cancer cell line.Methods:Cul-tured MCF-7 cells were treated with various doses of CrTX,a phospholipase A2(PLA2) isolated from the venom of the South American rattlesnake,Crotalusdurissus terrificus.The cytotoxicity of CrTX in the presence and absence ofcaspase inhibitors was measured with methyl thiazolyl tetrazolium (MTT) andlactate dehydrogenase (LDH) leakage assays.The activation of autophagy wasdetermined with transmission electron microscope and monodansylcadaverin(MDC) labeling.The upregulation of lysosomal enzymes,the release of cyto-chrome c (cyto-c),and the nuclear translocation of the apoptosis inducing factor(AIF) were examined by immunoblotting and immunofluorescence.Results:CrTXinhibited the viability of MCF-7 cells in a dose-and time-dependent manner.CrTX-activated autophagy was revealed by punctuate MDC labeling,and an increase inthe formation of autophagosomes as well as apoptosis,as evidenced by nuclearcondensation and fragmentation.The activation of cathepsin B,D,and L,inaddition to the release of cytochrome c and the relocation of AIF into nuclei,wereobserved after CrTX treatment.Autophagy inhibitors 3-methyladenine (3-MA),NH_4Cl,and the pan-caspase inhibitor,Z-Val-Ala-Asp-fluoromethylketone (Z-Vad-fmk),attenuated CrTX-induced cell death.Conclusion:An autophagic mecha-nism contributes to the apoptosis of MCF-7 cells induced by CrTX.
Aim: To investigate the role of crotoxin (CrTX) -induced autophagy in the death ofMCF-7 cells, a caspase-3-deficient, human breast cancer cell line. Methods: Cul- tured 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. Cytotoxicity of CrTX in the presence and absence of caspase inhibitors was measured with methyl thiazolyl tetrazolium (MTT) and lactate dehydrogenase (LDH) leakage assays. activation of autophagy wasdetermined with transmission electron microscope and monodansylcadaverin (MDC) labeling. the upregulation of lysosomal enzymes, the release of cyto-chrome c (cyto-c), and the nuclear translocation of the apoptosis inducing factor (AIF) were examined by immunoblotting and immunofluorescence. Results: CrTXinhibited the viability of MCF-7 cells in a dose-and time-dependent manner. CrTX-activated autophagy was revealed by punctuate MDC labeling, and an increase inthe formation of autophagosomes as well as apoptosis, as evidenced by nuclear condensation and fragmentation. The activation of cathepsin B, D, and L, inaddition to the release of cytochrome c and the relocation of AIF into nuclei, wereobserved after CrTX treatment. Areophagy inhibitors 3-methyladenine ( 3-MA), NH_4Cl, and the pan-caspase inhibitor, Z-Val-Ala-Asp-fluoromethylketone (Z- Vad-fmk), attenuated CrTX- induced cell death. Conlusion: An autophagic mecha- nism contributes to the apoptosis of MCF-7 cells induced by CrTX.