Concomitant Inhibition of Cytoprotective Autophagy Augments the Efficacy of Withaferin A in Hepatocellular Carcinoma

Abstract

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related mortality, and despite recent advances in early diagnosis and therapeutics, HCC related morbidity and mortality rate continue to rise. Clearly, it is imperative to develop novel effective therapies for HCC to improve long-term survival of HCC patients. We found that Withaferin A (WFA), a bioactive compound derived from Withania somnifera, is an effective agent for HCC inhibition. Interestingly, we observed that in addition to inducing apoptotic cell death, WFA also induces autophagy in HCC cells. Utilizing mRFP-EGFP-LC3B, LC3B-GFP/Lysotracker and LC3B-GFP/Rab7-RFP, we show that WFA induces autophagosomes-lysosomes fusion. WFA-induced autolysosomes exhibit intact protein degradation activity as evident with cathepsin-D activation and DQ-BSA assays. Importantly, we present that inhibiting WFA-induced autophagy either by blocking autophagosome-formation or by elevating lysosomal pH (Chloroquine and Bafilomycin) enhances WFA-induced growth-inhibition and apoptosis, indicating the presence of cytoprotective autophagy. Indeed, WFA and CQ combination shows synergism and higher efficacy in comparison to either monotherapy. Collectively, we reveal that the efficacy of WFA is somewhat diminished by the concomitant induction of cytoprotective autophagy which can be successfully conquered by cotreatment with CQ, and we pave the way for development of a novel combination therapeutic strategy for HCC.

The Preparation of Ginsenoside Rg5, Its Antitumor Activity against Breast Cancer Cells and Its Targeting of PI3K

“…In this work, Rg5 induced autophagy in breast cancer cells, which was demonstrated by the formation of autophagosomes observed by TEM, the upregulation of LC3-II, Beclin-1, Atg5, and Atg12 expression and the downregulation of p62 expression. Characterized by two main features, namely, the involvement of cytoplasmic material and culmination with lysosomal degradation, autophagy is a multistep process [35]. Rg5 treatment increased the level of LC3B puncta, and microscopic evaluation of tandem GFP-RFP-tagged LC3B in MCF-7 cells demonstrated that Rg5 induced the occurrence of autophagic flux.…”

Induction of STK11-dependent cytoprotective autophagy in breast cancer cells upon honokiol treatment

“…Multiple view-fields were examined and representative cells from 3 view-fields were photographed. For puncta, at least 50–100 cells/sample were counted in triplicates 45 , 77 , 78 .…”

Role of autophagic response induced by major phytochemicals in cancer prevention and treatment

“…Upon exposure to WA, MDA-MB-231 and MCF-7 breast cancer cells underwent autophagy, which was confirmed by analysis of acidic vesicular organelles and cleavage and recruitment to autophagosomes of LC3-II [56] . WA-induced autophagy was shown to be cytoprotective in hepatic cells, and by inhibiting its cytoprotective effects with chloroquine, tumor cells became more responsive to therapy [57] . Table 3 summarizes the WA-induced autophagic response in different cancer models.…”

“…Lung WA and pemetrexed, cisplatin, or gemcitabine Synergistic effect of WA and pemetrexed, cisplatin, or gemcitabine. WA downregulated mTOR/STAT3 signaling Siddharth et al [57] 2019 Cell culture, Huh7, HepG2, MHCC97H, MHCC97L…”