Open AccessAutophagy is involved in cytotoxic effects of crotoxin in human breast cancer cell line MCF‐7 cells
Author(s) -
YAN Cihui,
YANG Yaping,
QIN Zhenghong,
GU Zhenlun,
REID Paul,
LIANG Zhongqin
Publication year - 2007
Publication title -
acta pharmacologica sinica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.514
H-Index - 90
eISSN - 1745-7254
pISSN - 1671-4083
DOI - 10.1111/j.1745-7254.2007.00530.x
Subject(s) - autophagy , apoptosis , programmed cell death , mcf 7 , cytochrome c , viability assay , cathepsin d , biology , microbiology and biotechnology , cytotoxicity , cell culture , cytotoxic t cell , lactate dehydrogenase , cancer cell , biochemistry , enzyme , in vitro , cancer , genetics , human breast
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 cyto‐chrome 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. CrTX‐activated 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), NH 4 Cl, 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.