The Inhibitory Effects of HJC0416 on Liver Fibrogenesis in Activated Hepatic Stellate Cells

Abstract

IntroductionThe STAT3 signaling pathway regulates activation of hepatic stellate cells (HSCs) and promotes its fibrogenic behavior. Using a fragment‐based drug design approach, our group has developed HJC0416, a novel orally bioavailable small‐molecule inhibitor of STAT3. HJC0416 was found to induce apoptosis and decreased tumor growth in breast cancer models. In the present study, we studied the antifibrogenic properties of HJC0416 in activated hepatic stellate cells (HSC), which are the major effector cells of liver fibrosis.MethodsHuman (LX‐2) and rat (HSC‐T6) hepatic stellate cell lines were used. Cell proliferation was measured by Alamar Blue assay. Cellular protein expression levels were determined by Western blotting and immunofluorescence. Cell cycle was assessed by flow cytometry. Yo‐pro‐1 staining was used to measure apoptosis.ResultsHJC0416 treatment significantly inhibited LX‐2 and HSC‐T6 cell proliferation in a dose‐dependent fashion, and induced apoptosis and S‐phase cell cycle arrest. HSC activation marker α‐smooth muscle actin was also attenuated. HJC0416 suppressed Tyr705‐phosphorylated STAT3 (pSTAT3) and impaired pSTAT3 nuclear translocation and transcriptional activity. STAT3 target genes cyclin D1 and c‐myc were down‐regulated by HJC0416. Static, a well‐known STAT3 inhibitor, confirmed that HSC proliferation was suppressed by STAT3 inhibition. Extracellular matrix (ECM) proteins collagen type I (Coll.I) and fibronectin (FN) are major components of hepatic scar, with TGFβ being the most potent stimulator for ECM production. Our data revealed that endogenous Coll.I and FN expression were suppressed by HJC0416 in a dose‐dependent manner; TGFβ‐stimulated Coll.I and FN production were prevented by pretreatment with HJC0416.ConclusionHJC0416 inhibits HSC activation via the STAT3 signaling pathway. HJC0416 represents a promising anti‐hepatic fibrogenic agent.Support or Funding InformationThis work was supported by grants P50 CA097007, P30 DA028821, R21 MH098344 (JZ) from the National Institutes of Health, Cancer Prevention Research Institute of Texas award, R. A. Welch Foundation Chemistry and Biology Collaborative Grant (JZ) from the Gulf Coast Consortia, and John Sealy Memorial Endowment Fund and the Center for Addiction Research (JZ) from the University of Texas Medical Branch.