The importance of calcium ions and calpains in beta-lapachone-mediated apoptosis

Beta-Lapachone (Beta-Lap) is a naturally occurring compound present in the bark of the Lapacho tree native to South America. Beta-Lap has anti-tumor activity against a variety of human cancers, including colon, prostate, promyelocytic leukemia and breast. It killed cells exclusively by apoptosis and independently of p53 status, which is critical since many cancers exhibit loss of p53 tumor suppressor function. Beta-Lap triggered apoptosis in a number of human breast and prostate cancer cell lines through a unique apoptotic pathway dependent upon NQO1, a two-electron reductases (Chapter 1).; This thesis further characterized the molecular mechanisms of Beta-lap-induced apoptosis. We examined alterations in intracellular Ca2+ homeostasis using NQO1expressing MCF-7 cells (Chapter 2). Beta-Lap-exposed MCF-7 cells exhibited an early increase in intracellular Ca2+, from endoplasmic reticulum Ca2+ stores. BAPTA-AM, an intracellular Ca 2+ chelator, blocked early increases in Ca2+ levels and inhibited Beta-lap-mediated mitochondrial membrane depolarization, intracellular ATP depletion, specific and unique substrate proteolysis, and apoptosis. The extracellular Ca2+ chelator, EGTA, inhibited later apoptotic endpoints (observed >8 h, e.g., substrate proteolysis and DNA fragmentation), suggesting that later execution events were triggered by Ca2+ influxes from the extracellular milieu. Collectively, these data suggested a critical, but not sole, role for Ca2+ in the NQO1-dependent cell death pathway initiated by Beta-lap.; Further work was directed at elucidating the execution phase of apoptosis induced by Beta-lap (Chapter 3). We demonstrated that Beta-lap mediated a unique proteolytic apoptotic pathway in NQO1-expressing cells via μ-calpain activation, and upon activation, mu-calpain translocated to the nucleus. The apoptotic events in NQO1-expressing cells in response to Beta-lap were significantly delayed and survival enhanced via exogenous expression of calpastatin. Furthermore, we showed that mu-calpain cleaved PARP to a unique fragment (∼60 kDa) different from that previously reported for calpains. We also provide evidence that Beta-lap-induced, mu-calpain stimulated, apoptosis does not involve any of the known caspases.; Understanding the molecular mechanisms of Beta-lap-induced apoptosis further explained the pleiotropic effects of this drug. Elucidating the molecular targets of Beta-lap will help better target this agent for breast cancer therapy, as well as provide a rationale for future drug development and/or modulation of existing agents.