A NOVEL ORALLY BIOAVAILABLE SMALL MOLECULE COMPOUND HJC0416 INHIBITS LIVER FIBROGENESIS THROUGH THE HSP90/NF-κB PATHWAY
Jana E. DeJesus*, Xiaofu Wang, Yanping Gu, Rui-Min D. Mao, William S. Fagg, Jia Zhou, Ravi Radhakrishnan
Surgery, The University of Texas Medical Branch at Galveston, Galveston, TX
Introduction. Chronic inflammation leads to liver fibrosis and, potentially, cirrhosis. Hepatic stellate cell (HSC) activation represents the initial step of liver fibrogenesis since the HSC is the major producer of extracellular matrix (ECM). Understanding the mechanism of inflammation and fibrogenesis is critically important to developing treatments for liver fibrosis. NF-ΚB is a key inflammatory signaling pathway, and the survival of activated HSC was found to be NF-KB dependent. Using a fragment-based drug design approach, our team previously designed and synthesized a group of small molecule compounds for cancer therapy. After screening these molecules in cancer cells and activated HSC In vivo and In vitro, HJC0416 was identified to be a novel, orally bioavailable molecules with potent anti-cancer, anti-inflammatory and anti-fibrotic effects. For example, HJC0416 induced activated HSC cell cycle arrest and apoptosis, inhibited endogenous and TGFβ-stimulated ECM expression. However, the molecular mechanisms of HJC0416's anti-fibrogenetic effects in HSC remain largely unknown. In this study, we examined the effects of HJC0416 on NF-KB and its associate factor HSP90 in HSC in vitro.
Methods. Activated human HSC line LX-2 was treated with either JHC0416 or 17-AAG, then exposed to TNFα or TGFβ as indicated. Nuclear and cytosolic proteins were isolated for Western blots or immunofluorescence assay.
Results. HJC0416 treatment significantly attenuated TNFα–induced IκBα phosphorylation, NF-KBp65 nuclear translocation and DNA binding activity. Endogenous and TNFα–induced p65 phosphorylation of Ser536 was suppressed by HJC0416. Notably, HJC0416 treatment dose-dependently down-regulated the expression of IKKβ, RIP1 and AKT, FAK, CDK9, all of which are HSP90 interacting proteins, suggesting that HSP90 may be involved in HJC0416 regulated-NF-KB signaling and fibrogenesis in HSC. Our results confirmed that HSP90 specific inhibitor 17-AAG prevented TNFα-induced IκBα phosphorylation and degradation, p65 nuclear translocation and DNA binding. Similar with previous HJC0416 data, 17-AAG inhibited endogenous and TGFβ-stimulated fibrosis markers collagen type I and fibronectin. Conclusion. The anti-fibrogenetic effect of orally bioavailable compound HJC0416 is through the HSP90/NF-KB pathway. HJC0416 may be a promising drug candidate for liver fibrosis treatment.
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