Abstract 4095: Decreased caspase 8 protein expression and incomplete caspase 3 activation in TRAIL-resistant normal human fibroblasts and epithelial cells

Abstract

Abstract TNF-related apoptosis-inducing ligand (TRAIL)-mediated cell death is currently being utilized in newly developed cancer therapies. The molecular basis of normal cell resistance to TRAIL-mediated apoptosis, and thus therapeutic index, remains largely unexplored. We investigated TRAIL sensitivity in normal fibroblasts (HFF, WI-38, MRC-5) and epithelial cells (FHs 74 Int). We found that MRC-5 cells were moderately sensitive to TRAIL after 18 hours of treatment while WI-38, HFF, and FHs 74 Int cells were not. Normal fibroblasts and epithelial cells expressed both pro-apoptotic TRAIL receptors DR4 and DR5, although normal cell DR4 and DR5 expression was slightly lower when compared to human lung and colon cancer cells. While normal and cancer cells showed comparable DcR2 expression, normal fibroblasts and epithelial cells expressed little DcR1. c-Myc, a major determinant of TRAIL sensitivity in cancer cells, was found to be decreased in all normal cells compared to cancer cells. Expression of initiator caspase 8, required for complete TRAIL signaling, was assessed. Markedly reduced caspase 8 protein expression was detected in WI-38, HFF and FHs 74 Int cells compared to tumor cells that generally show robust caspase 8 expression. MRC-5 caspase 8 protein expression was found to be noticeably increased compared to TRAIL-resistant normal fibroblasts and epithelial cells and may be a contributing factor in MRC-5 TRAIL sensitivity. Expression and activation of effector caspase 3 was also examined. After 4 hours of TRAIL exposure, expression of full length caspase 3 declined in normal fibroblasts, epithelial cells and tumor cells. Yet, appearance of the mature active caspase 3 subunit p17 was only seen in MRC-5 fibroblasts and cancer cells. Interestingly, cleavage of caspase 3 substrate, PARP, was only seen in cancer cells after 4 hours of TRAIL exposure suggesting that MRC-5 fibroblasts have a delayed cytotoxic response to TRAIL. TRAIL-insensitive normal cells were treated with TRAIL and a proteasome inhibitor to increase susceptibility to TRAIL-mediated cell death. TRAIL-induced cell death was observed in WI-38, HFF and FHs 74 Int cells after combined treatment of TRAIL and MG132. Our current studies are focused on further analysis of caspase 3 activation and stabilization after TRAIL treatment in TRAIL-resistant normal cells. Additionally, we are investigating whether treatment of normal fibroblasts and epithelial cells with histone deacetylase inhibitors, and/or DNA methylation inhibitors might result in increased caspase 8 protein expression and susceptibility to TRAIL-mediated apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4095. doi:10.1158/1538-7445.AM2011-4095