Activation of a Cysteine Protease in MCF-7 and T47D Breast Cancer Cells during β-Lapachone-Mediated Apoptosis |
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Authors: | John J Pink Shelly Wuerzberger-Davis Colleen Tagliarino Sarah M Planchon XiaoHe Yang Christopher J Froelich David A Boothman |
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Institution: | a Laboratory of Molecular Stress Responses, Department of Radiation Oncology, Case Western Reserve University, Cleveland, Ohio, 44106;b Evanston Northwestern Healthcare Research Institute, Evanston, Illinois, 60201 |
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Abstract: | β-Lapachone (β-lap) effectively killed MCF-7 and T47D cell lines via apoptosis in a cell-cycle-independent manner. However, the mechanism by which this compound activated downstream proteolytic execution processes were studied. At low concentrations, β-lap activated the caspase-mediated pathway, similar to the topoisomerase I poison, topotecan; apoptotic reactions caused by both agents at these doses were inhibited by zVAD-fmk. However at higher doses of β-lap, a novel non-caspase-mediated “atypical” cleavage of PARP (i.e., an 60-kDa cleavage fragment) was observed. Atypical PARP cleavage directly correlated with apoptosis in MCF-7 cells and was inhibited by the global cysteine protease inhibitors iodoacetamide and N-ethylmaleimide. This cleavage was insensitive to inhibitors of caspases, granzyme B, cathepsins B and L, trypsin, and chymotrypsin-like proteases. The protease responsible appears to be calcium-dependent and the concomitant cleavage of PARP and p53 was consistent with a β-lap-mediated activation of calpain. β-Lap exposure also stimulated the cleavage of lamin B, a putative caspase 6 substrate. Reexpression of procaspase-3 into caspase-3-null MCF-7 cells did not affect this atypical PARP proteolytic pathway. These findings demonstrate that β-lap kills cells through the cell-cycle-independent activation of a noncaspase proteolytic pathway. |
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Keywords: | apoptosis β -lapachone caspase breast cancer poly(ADP)-ribose polymerase PARP calpain topotecan |
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