Mu-calpain activation in beta-lapachone-mediated apoptosis.
Beta-lapachone (beta-Lap) triggers apoptosis in a number of human breast and prostate cancer cell lines through a unique apoptotic pathway that is dependent upon NQO1, a two-electron reductase. Recently, our laboratory showed that beta-lap-exposed MCF-7 cells exhibited an early increase in intracellular cytosolic Ca(2+) from endoplasmic reticulum stores, and that BAPTA-AM (an intracellular Ca(2+) chelator) blocked these early increases and partially inhibited all aspects of beta-lap-induced apoptosis. We now show that exposure of NQO1-expressing breast cancer cells to beta-lap stimulates a unique proteolytic apoptotic pathway involving mu-calpain activation. No apparent activation of m-calpain was noted. Upon activation, mu-calpain translocated to the nucleus concomitant with specific nuclear proteolytic events. Apoptotic responses in beta-lap-exposed NQO1-expressing cells were significantly delayed and survival enhanced by exogenous over-expression of calpastatin, a natural inhibitor of mu- and m-calpains. Furthermore, purified mu-calpain cleaved PARP to a unique fragment (approximately 60 kDa), not previously reported for calpains. We provide evidence that beta-lap-induced, mu-calpain-stimulated apoptosis does not involve any known apoptotic caspases; the activated fragments of caspases were not observed after beta-lap exposures, nor were there any changes in the pro-enzyme forms as measured by Western blot analyses. The ability of beta-lap to trigger an apparently novel, p53-independent, calpain-mediated apoptotic cell death further support the development of this drug for improved breast cancer therapy.
Departments of Radiation Oncology and Pharmacology,Case Western Reserve University, Cleveland, Ohio 44106-4942, USA.
Colleen
C
Tagliarino
John J
JJ
Pink
Kathryn E
KE
Reinicke
Sara M
SM
Simmers
Shelly M
SM
Wuerzberger-Davis
David A
DA
Boothman
D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology*; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000494:pharmacology; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020169:Caspases / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D003114:Colony-Forming Units Assay; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation; D005260:Female; D006801:Humans; D020287:In Situ Nick-End Labeling; D018613:Microscopy, Confocal; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D009285:Naphthoquinones / Q000494:pharmacology*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D021381:Protein Transport; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D000903:Antibiotics, Antineoplastic; D002135:Calcium-Binding Proteins; D015853:Cysteine Proteinase Inhibitors; D009285:Naphthoquinones; D016159:Tumor Suppressor Protein p53; C014638:beta-lapachone; C033668:calpastatin; D016660:NAD(P)H Dehydrogenase (Quinone); C447479:NQO1 protein, human; D011065:Poly(ADP-ribose) Polymerases; D002154:Calpain; D020169:Caspases; C449000:m-calpain; C449002:mu-calpain; D002118:Calcium