Abstract 2928: Withanolide E sensitizes renal carcinoma cells to TRAIL-induced apoptosis by increasing cFLIP degradation

Withanolide E (WE), a steroidal lactone from Physalis peruviana, was found to be highly active for sensitizing renal carcinoma cells and a number of other human cancer cells to TRAIL-mediated apoptosis. WE, the most potent and least toxic of 5 TRAIL sensitizing withanolides identified, enhanced death receptor-mediated apoptotic signaling via a rapid decline in the levels of both cFLIPL and cFLIPS proteins. Normal non-transformed human renal epithelial cells were completely resistant to the WE plus TRAIL combination. Other mechanisms by which TRAIL sensitizers have been reported to work: generation of reactive oxygen species (ROS), changes in various pro-and anti-apoptotic protein expression, death receptor upregulation, activation of intrinsic (mitochondrial) apoptotic pathways, ER stress, and proteasomal inhibition proved to be irrelevant to withanolide E activity. Loss of cFLIPL and cFLIPS was predominantly due to destabilization and/or aggregation of the proteins subsequently leading to their proteasomal degradation. Since cFLIPL and cFLIPS are reported to be HSP90 client proteins, and WE can inhibit HSP90 activity, the effect of WE on various other HSP90 client proteins was assessed. WE treatment of ACHN renal carcinoma cells altered the stability of a limited number of HSP90 client proteins when compared to the well-known HSP90 inhibitor geldanamycin. The destabilization of cFLIP proteins provides a potentially novel mechanism for sensitizing cancer cells to TRAIL mediated apoptosis. Sensitization of human renal carcinoma cells to TRAIL-induced apoptosis by WE and its lack of toxicity were confirmed in animal studies, in which a combination treatment of WE with an agonist antibody to TRAIL death receptor 5 (DR5) provided a significant therapeutic benefit compared to either agent alone in a renal cancer xenograft model. Due to its novel activity, WE is a promising reagent for analysis of mechanisms of TRAIL resistance, for understanding HSP90 function, and for further therapeutic development. Various structural analogs of WE are currently being tested for their TRAIL-sensitizing activity to provide more insight into Structure Activity Relationships (SAR) for these compounds. In marked contrast to bortezomib, among the best currently available TRAIL sensitizers, WE's more specific mechanism of action suggests minimal toxic side effects might accompany its combination with TRAIL death receptor agonists for cancer therapy. Citation Format: Brooks D. Alan,