Autophagy is involved in cytotoxic effects of crotoxin in human breast cancer cell line MCF-7 cells.
OBJECTIVE
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. 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), NH4Cl, and the pan-caspase inhibitor, Z-Val-Ala-Asp-fluoromethylketone (Z-Vad-fmk), attenuated CrTX-induced cell death.


CONCLUSIONS
An autophagic mechanism contributes to the apoptosis of MCF-7 cells induced by CrTX.
Yan
Ci-hui
CH
Department of Pharmacology, Soochow University School of Medicine, Suzhou 215123, China.
Yang
Ya-ping
YP
Qin
Zheng-hong
ZH
Gu
Zhen-lun
ZL
Reid
Paul
P
Liang
Zhong-qin
ZQ
D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001343:Autophagy / Q000502:physiology*; D001943:Breast Neoplasms / Q000188:drug therapy*; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D003439:Crotoxin / Q000494:pharmacology*; D005260:Female; D006801:Humans
D000970:Antineoplastic Agents; D003439:Crotoxin; D053148:Caspase 3