Activation of a cysteine protease in MCF-7 and T47D breast cancer cells during beta-lapachone-mediated apoptosis.
beta-Lapachone (beta-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, beta-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 beta-lap, a novel non-caspase-mediated "atypical" cleavage of PARP (i.e., an approximately 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 beta-lap-mediated activation of calpain. beta-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 beta-lap kills cells through the cell-cycle-independent activation of a noncaspase proteolytic pathway.
Pink
J J
JJ
Laboratory of Molecular Stress Responses, Case Western Reserve University, Cleveland, Ohio, 44106, USA.
Wuerzberger-Davis
S
S
Tagliarino
C
C
Planchon
S M
SM
Yang
X
X
Froelich
C J
CJ
Boothman
D A
DA
D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001943:Breast Neoplasms / Q000201:enzymology* / Q000473:pathology*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D006801:Humans; D009285:Naphthoquinones / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D000903:Antibiotics, Antineoplastic; D009285:Naphthoquinones; C014638:beta-lapachone; D003546:Cysteine Endopeptidases