Exploiting and NQ01-Directed, Calpain-Medicated Apoptotic Pathway for Breast Cancer Therapy
Abstract
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.
Document Details
- Document Type
- Technical Report
- Publication Date
- Apr 01, 2003
- Accession Number
- ADA417425
Entities
People
- David A. Boothman
- Mark W. Wagner
Organizations
- Case Western Reserve University