Abstract 1334: Withaferin A induces cytoprotective autophagy in hepatocellular cancer cells and concomitant autophagic inhibition augments the efficacy of Withaferin A in hepatocellular carcinoma
Authors: Siddharth Sumit , Muniraj Nethaji , Saxena Neeraj K. , Sharma Dipali 
Published: August 2018

Background and Aim: Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world and the third most common cause of cancer-related deaths. While multiple chemotherapeutic and targeted agents have been developed for other cancers, Sorafenib, an oral multikinase inhibitor, is the only approved agent for the treatment of advanced HCC. Clearly, it is imperative to develop novel effective therapeutic strategies for HCC to improve long-term survival of HCC patients. We found Withaferin A (WFA), a bioactive alkaloid derived from Withania somnifera, as an effective agent for HCC inhibition. Interestingly, we observed that WFA also induces autophagy in HCC cells. Autophagy can be cancer inducing or cancer inhibiting at the functional level. We aim to systemically investigate the anti-HCC efficacy of WFA, investigate the functional impact of autophagy and explore the efficacy of combined regimens of WFA and autophagic inhibitors.

Methods: Using in vitro and in vivo models, we established that WFA inhibits liver tumorigenesis. tfLC3B, LC3B-GFP/Lysotracker and LC3B/Rab7 were used to examine the fusion of autophagosomes with lysosomes. Cathepsin-D activation and DQ-BSA assays were conducted to analyze the protein degradation activity in lysosomes. Multiple cell based assays and Chou-Talalay synergy quantification assays were conducted to analyze the synergistic effect of WFA with autophagic inhibitors. IHC staining was used to study autophagic markers in vivo.

Results: WFA inhibited anchorage dependent cell growth in vitro and reduced the HCC tumor burden in vivo. In addition to inducing apoptotic cell death, WFA induced cleavage of Light Chain 3B (LC3B) and increased the expression of autophagic proteins (ATG5, ATG7 and Beclin1) both in vitro and in vivo. The redistribution of LC3B-GFP from cytosol to autophagosome demonstrated enhanced autophagosome formation in WFA treated cells. The fusion of autophagosome with lysosome was observed in WFA-treated HCC cells indicating autolysosome formation. WFA treated HCC cells exhibited elevated lysosomal degradation activity. Inhibiting autophagy either by blocking autophagosome formation or elevating lysosomal pH (CQ and BAF) enhanced WFA-induced growth inhibition and apoptosis indicating the presence of cytoprotective autophagy. Combination index analysis of WFA and CQ showed synergism and increased efficacy as compared to either monotherapy. These in vitro findings were corroborated with in vivo experiments.

Conclusion: Our results reveal that WFA inhibits hepatocellular carcinoma by apoptotic induction but the efficacy of WFA is somewhat abrogated by the concomitant induction of cytoprotective autophagy. Our preclinical findings present evidence to support WFA and chloroquine as an effective combination treatment regimen for HCC.

Citation Format: Sumit Siddharth, Nethaji Muniraj, Neeraj K. Saxena, Dipali Sharma. Withaferin A induces cytoprotective autophagy in hepatocellular cancer cells and concomitant autophagic inhibition augments the efficacy of Withaferin A in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1334.