β-Lapachone (β-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 β-lap-exposed MCF-7 cells exhibited an early increase in intracellular cytosolic Ca2+ from endoplasmic reticulum stores, and that BAPTA-AM (an intracellular Ca2+ chelator) blocked these early increases and partially inhibited all aspects of b-lap-induced apoptosis. We now show that exposure of NQO1-expressing breast cancer cells to β-lap stimulates a unique proteolytic apoptotic pathway involving μ-calpain activation. No apparent activation of μ-calpain was noted. Upon activation, m-calpain translocated to the nucleus concomitant with specific nuclear proteolytic events. Apoptotic responses in β-lap-exposed NQO1-expressing cells were significantly delayed and survival enhanced by exogenous over-expression of calpastatin, a natural inhibitor of m- and μ-calpains. Furthermore, purified ...