Abstract 2159: A specific 17-beta-hydroxywithanolide (LG-02) sensitizes cancer cells to apoptosis in response to TRAIL and TLR3 ligands
Authors: Tewary Poonam , Brooks Alan D. , Xu Ya-ming , Wijeratne Kithsiri E.M , Gunatilaka Leslie A. , Sayers Thomas J. 
Published: July 2017

Recent studies have demonstrated a role of toll-like receptor 3 (TLR3) signaling for the initiation of apoptosis in some malignant cells. We have previously shown that, withanolide E (WE), a 17β-hydroxywithanolide (17-BHW) natural product derived from the medicinal plant Physalis peruviana was capable of sensitizing tumor cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis by reducing cellular levels of the anti-apoptotic protein cFLIP. Animal studies also revealed that WE sensitized human renal carcinoma cells to apoptosis at concentrations that did not promote apoptosis in normal cells. Thus we further screened a library of 30 natural and semi-synthetic 17-BHWs for their ability to promote death ligand-mediated cancer cell death. Among the 30 compounds tested, LG-02 (physachenolide C) was found to be 4-5 fold more potent than WE in sensitizing some human renal carcinoma and melanoma cells to apoptotic cell death in response not only to TRAIL but also to the synthetic polynucleotide poly (I:C), which is known to mimic anti-viral responses by activating TLR3 signaling. To date there are no withanolides known to have this dual apoptosis sensitizing activity. LG-02 and Poly (I:C) treatment resulted in increased activation of caspase-8, and apoptosis was blocked by the pan caspase inhibitor zVAD-FMK. Poly (I:C)-driven apoptosis signaling was dependent on endosomal acidification, but independent of IRF 3 and Interferon α/β signaling. Molecular studies suggested a role for changes in the anti-apoptotic proteins cFLIP, IAPs, and Livin on apoptosis signaling in LG-02 treated cells. Loss of cIAP activity is reported to promote spontaneous formation of an intracellular death-inducing protein platform the ripoptosome, that can activate either apoptosis or necroptosis. Immunoprecipitation of either the TRAIL death-inducing signaling complex (DISC) or the Poly (I:C) ripoptosome, demonstrated enhanced levels of FADD and RIP1 and decreased levels of cFLIP in these macromolecular apoptosis signaling complexes in LG-02 treated cells. Intratumor administration of LG-02 and Poly (I:C) in a xenograft M14 melanoma model provided therapeutic benefit leading to complete tumor regression in 90 % of the mice as compared to control mice. Further studies with active 17-BHWs could lead to the identification of more potent analogues, and novel and common therapeutic targets involved in apoptosis signaling in response to both TNF death receptor family members as well as TLR3 ligands.Funded by FNLCR Contract HHSN261200800001E

Citation Format: Poonam Tewary, Alan D. Brooks, Ya-ming Xu, Kithsiri E.M Wijeratne, Leslie A. Gunatilaka, Thomas J. Sayers. A specific 17-beta-hydroxywithanolide (LG-02) sensitizes cancer cells to apoptosis in response to TRAIL and TLR3 ligands [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2159. doi:10.1158/1538-7445.AM2017-2159