Exploiting an NQ01-Directed, Calpain-Medicated Apoptotic Pathway for Breast Cancer Therapy
Abstract
The purpose of this proposal is to further understand the molecular mechanisms of (beta-lap-induced apoptosis. We examined alterations in intracellular Ca2+ homeostasis using NQOl-expressing MCF-7 cells. Beta-Lap-exposed MCF-7 cells exhibited an early increase in intracellular Ca2+, from endoplasmic reticulum Ca2+ stores. BAPTA-AM, an intracellular Ca2+ chelator, blocked early increases in Ca2+ levels and inhibited (3-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 suggest a critical, but not sole, role for Ca2+ in the NQOl-dependent cell death pathway initiated by (3-lap. Further work was directed at elucidating the execution phase of the apoptotic pathway induced by (3-lap. We demonstrated that (3-lap mediated a unique proteolytic apoptotic pathway in NQOl-expressing cells via mu-calpain activation, and upon activation, mu calpain translocated to the nucleus. The apoptotic events in NQOl-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.
Document Details
- Document Type
- Technical Report
- Publication Date
- Oct 01, 2001
- Accession Number
- ADA398218
Entities
People
- Collen Tagliarino
- David A. Boothman
Organizations
- Case Western Reserve University