A specific 17-BHW (LG-02) sensitizes cancer cells to apoptosis in response to TRAIL and toll-like receptor (TLR) ligands Poonam Tewary1,2, Alan D. Brooks1,2, Ya-ming Xu3, E. M. Kithsiri Wijeratne3, A. A. Leslie Gunatilaka3, Thomas J. Sayers1,2 1Leidos Biomedical Research, Inc.; Frederick National Laboratory for Cancer Research Frederick, MD; 2CIP, 3Natural Products Center, School of Natural Resources and the Environment, University of Arizona, Tucson, AZ
Despite many therapeutic successes, cancer is the second-most frequent cause of mortality in the US. Strategies for cancer therapy aim to overcome excessive proliferation and avoidance of apoptosis. Recent studies have implicated the role of TLR3 signaling to initiate apoptosis in malignant cells and promote anticancer immune responses. We have previously shown that 17β-hydroxywithanolides (17-BHWs), withanolide E and LG-02 derived from medicinal plants of the genus, Physalis were capable of sensitizing tumor cells to TRAIL-mediated apoptosis by reducing cellular levels of the anti-apoptotic protein cFLIP. 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 those tested, LG-02 was found to be 4-5-fold more potent than WE in sensitizing tumor cells to apoptosis in response to TRAIL as well as to the synthetic polynucleotide poly (I:C), a viral mimic which activates TLR signaling. This is the first report of withanolides having this dual activity. LG-02 and poly (I:C) mediated apoptosis was blocked by the pan caspase inhibitor zVAD and was dependent on endosomal acidification but independent of IRF3 and Interferon α/β signaling. Loss of cIAPs promote spontaneous formation of ripoptosome that activates either apoptosis or necroptosis. Immunoprecipitation of this complex showed enhanced levels of FADD and RIP1. Surprisingly, LG-02 mediated sensitization of melanoma cells to poly (I:C) mediated cell death was dependent on both intrinsic and extrinsic apoptotic pathways as indicated by cFLIP degradation and Caspase 9 and Bid cleavage. Intra tumor 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 17-BHWs could lead to identification of novel and common therapeutic targets involved in apoptosis in response to both TNF death receptor family members as well as TLR ligands.
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