Exploiting and NQ01-Directed, Calpain-Medicated Apoptotic Pathway for Breast Cancer Therapy

摘要

The purpose of this proposal was to further understand the molecular mechanisms of beta-lap-induced apoptosis, and its ability to target cancer over normal cells. We believe that beta-lap induces apoptosis through changes in intracellular calcium homeostasis and micron-calpain activation. This will be tested via two specific aims using NQ01-expressing and non-expressing (beta-lap sensitive and resistant, respectively) MDA-MB-468 breast cancer cells as a model system. The first aim was to determine changes in intracellular calcium homeostasis before and after beta-lap exposure. Fluorescence calcium dye indicators will be used to determine changes in intracellular calcium levels as well as GFP-calmodulin calcium indicators (cameleons, that are targeted to intracellular organelles), for a more accurate determination of where calcium changes are occurring. Analysis of apoptosis via flow cytometric analyses will be performed in breast cancer cells in the presence of extracellular calcium chelators, to determine if changes in intracellular calcium concentrations are critical for DNA fragmentation and cell death. The second aim will be to determine the role of calpain and its downstream targets in beta-lap-induced apoptosis. Calpain activation will be assessed using fluorogenic substrates. Substrate cleavage analyses, in vitro, will be performed using specific downstream targets, as determined from western blot timecourse analyses (PARP, lamin B, and p53). Confocal microscopy with indirect immunofluorescence and Green Fluorescent Protein (OFP)-tagged micron-calpain will be used to examine calpain translocation and co-localization studies with downstream targets.