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Withaferin A suppresses and inhibits hepatocellular carcinoma‐cell proliferation and tumor burden via activation of ERK1/2‐ELK1‐RSK‐DR5 axis
Author(s) -
Kuppusamy Panjamurthy,
Nagalingam Arumugam,
Drachenberg Cinthia,
Sharma Dipali,
Saxeeeraj K
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.lb179
Subject(s) - withaferin a , cancer research , hepatocellular carcinoma , apoptosis , viability assay , medicine , cancer , liver cancer , withania somnifera , matrigel , cell growth , pharmacology , chemistry , pathology , angiogenesis , biochemistry , alternative medicine
Hepatocellular carcinoma (HCC) is the fifth most common highly lethal cancer and the third most common cause of cancer‐related mortality in the world. Available therapies are often limited by high toxicity, lower efficacy, therapeutic resistance and therapy‐related morbidity. Liver resection, ethanol injection, radiofrequency ablation, and chemoembolization have been used successfully for the local management of HCC; however, no single cytotoxic‐chemotherapeutic‐agent has been proven effective for systemic treatment. Therefore, more effective therapeutic strategies are needed to combat liver cancer related mortality. Withaferin A ( WA ) is a bioactive compound derived from Withania somnifera has been safely used for centuries in Indian‐Ayurvedic‐medicine against various ailments. For the first time, we demonstrated that WA displays substantial inhibitory activity against liver cancer cells in vitro and tumor burden in vivo in HCC‐cell‐xenograft‐nude mice and DEN induced HCC in C57BL/6. Methods Therapeutic efficacy of WA was assessed by determining cell viability of HCC cell lines, HepG2 and Huh7, using XTT reduction while apoptosis was quantified by TUNEL assay. Effect of WA on cell migration (spheroid assay), invasion (matrigel invasion), clonogenicity, anchorage independent growth (colony formation on soft agar) were determined. Additionally, in a quantitative real‐time, ECIS (Electric cell‐substrate impedance sensing)‐based assay was performed to evaluate efficacy of WA on migration and invasion of HCC cell lines. The mechanism for the inhibitory role of WA on HCC was determined by immunoblot assay for cleaved caspase 3, Bax, cleaved PARP, phosphorylated ERK, Elk1, RSK1 and DR5 in HCC cells, tumors derived from xenografts in nude mice and tumorous‐livers from DEN treated mice. Results WA treatment significantly inhibited HCC cell‐proliferation, migration, invasion, clonogenicity and anchorage‐independent growth. WA treatment significantly induced apoptosis to suppress cell and tumor growth as indicated by increased cleaved caspase 3 activity, cleaved PARP and Bax. Mechanistically, WA induced expression of death receptor 5 (DR5) that triggers apoptosis when overexpressed. Our study revealed that WA inhibits phosphorylation of Stat3 which in turn up‐regulates phosphorylation of Elk1. Inhibition of ERK or Elk1 phosphorylation inhibits WA‐induced DR5 induction, indicating that ERK and Elk1 cooperatively regulate DR5 expression. Conclusions Collectively, our studies show the efficacy of WA as a novel agent for HCC inhibition and provide mechanistic insight into the important role of ERK‐Elk1 axis in WA‐mediated HCC inhibition.