One hallmark of cancer is the ability to evade programmed cell death, or apoptosis; this allows uncontrolled proliferation and tumor growth. Our goal is to use a small molecule to activate to trigger apoptosis in cancer cells by targeting key proteins in the apoptotic pathway: executioner procaspases. We hope to develop a strategy for personalized cancer treatment in which cancers with elevated levels of procaspases will be treated with specialized small molecules. It is known that there are elevated levels of procaspase-7 in certain cancers, such as prostate cancer. We hypothesize that apoptosis can be selectively induced in cancer cells containing elevated levels of procaspase-7 via a direct small molecule activator of procaspase- 7 to caspase-7. This hypothesis is supported by recent research in our group in which we were able to induce apoptosis in cancer cells by small molecule activation of procaspase-3 to caspase-3. ? ? Specific Aim 1. Identification of small molecules which activate procaspase-7 to caspase-7. In this specific aim we intend to A) perform timecourse analyses to elucidate how procaspase-7 activates over time, B)identify a small molecule activator of procaspase-7 from a library screen, and C) validate procaspase-7-activating compounds found in the library screen. ? ? Specific Aim 2. Induction of apoptotic death using the identified small molecule activator of procaspase-7. During apoptosis, cells display a variety of characteristic traits. We will use various techniques to monitor the apoptotic hallmarks in HL60 and MCF-7 cells induced by the identified small molecule in Specific Aim 1. Techniques include Flow cytometry to detect phosphatidyl serine exposure on apoptotic cell membranes, Hoescht staining to detect chromatin condensation in apoptotic cells, Rescue from apoptotic death with a pan-caspase inhibitor ? ? Specific Aim 3. Identification of specific cancers in which executioner procaspase levels are elevated. A variety of paraffin-embedded primary cancer tissues are available to the Hergenrother laboratory. Using Western blotting and immunohistochemistry, we can determine executioner procaspase-3, -6, and -7 levels in prostate and breast cancer tissues. ? ? ?
