Brivanib attenuates hepatic fibrosis in vivo and stellate cell activation in vitro by inhibition of FGF, VEGF and PDGF signaling.

Brivanib is a selective inhibitor of vascular endothelial growth factor receptor (VEGFR) and fibroblast growth factor receptor (FGFR) tyrosine kinases, which are both involved in mechanisms of liver fibrosis. We hypothesized that inhibition of VEGFR and FGFR by brivanib would inhibit liver fibrosis. We therefore examined the effect of brivanib on liver fibrosis in three mouse models of fibrosis.In vivo, we induced liver fibrosis by bile duct ligation (BDL), chronic carbon tetrachloride (CCl4), and chronic thioacetamide (TAA) administration. Liver fibrosis was examined by immunohistochemistry and Western immunoblotting. In vitro, we used LX-2 human hepatic stellate cells (HSCs) to assess the effect of brivanib on stellate cell proliferation and activation.After in vivo induction with BDL, CCl4, and TAA, mice treated with brivanib showed reduced liver fibrosis and decreased expression of collagen Iα1 and α-smooth muscle actin in the liver. In vitro, brivanib decreased proliferation of HSCs induced by platelet-derived growth factor (PDGF), VEGF, and FGF. Brivanib also decreased stellate cell viability and inhibited PDGFBB-induced phosphorylation of its cognate receptor.Brivanib reduces liver fibrosis in three different animal models and decreases human hepatic stellate cell activation. Brivanib may represent a novel therapeutic approach to treatment of liver fibrosis and prevention of liver cancer

Establishment and characterization of a new human intrahepatic cholangiocarcinoma cell line LIV27

Cholangiocarcinoma (CCA) is a highly lethal cancer arising from the biliary tract epithelium. The cancer biology of this neoplasm is not well understood. To date, only a few CCA cell lines have been reported, which were mostly developed from Asian patients. In this study, we report and characterize a new intrahepatic CCA cell line, LIV27, derived from a surgically resected tumor in a 67-year-old Caucasian woman with primary sclerosing cholangitis (PSC). LIV27 cells grow well in collagen-coated flasks or plates with a doubling time of 57.8 h at passage 14. LIV27 cells have high tumorigenicity in nude mice and stain positive for CK7 and CK19, markers that differentiate CCA from hepatocellular carcinoma. Karyotype analysis showed that LIV27 is aneuploid. We established a single-locus short tandem repeat profile for the LIV27 cell line. This newly established cell line will be a useful model for studying the molecular pathogenesis of, and developing novel therapies for, cholangiocarcinoma

Brivanib decreases viability of PDGF-BB treated LX-2 cells.

<p>(A) The effect of increasing concentrations of brivanib on the viability of LX-2 HSCs cultured in DMEM/10% FBS was assessed using Cell Counting Kit-8 (CCK-8). Cells showed a brivanib dose-dependent decrease in viability, with half maximal inhibitory concentration (IC₅₀) of 16.98 µM (95% CI 13.95 µM–20.67 µM). (B) Cells stimulated with 5 ng/ml PDGF-BB in serum-free medium showed a similar trend albeit more pronounced decrease in viability compared to cells cultured in 10% FBS. The IC₅₀ of LX-2 cells cultured in serum-free media supplemented with PDGF-BB was 8.79 µM (95% CI 7.80 µM–9.99 µM).</p

Brivanib does not inhibit TGF-β1-induced α-SMA expression in human LX-2 hepatic stellate cells.

<p>(A) The expression of α-SMA in LX-2 HSCs after TGF-β1 treatment was assessed by Western immunoblotting. HSCs were incubated with 10% or 1% FBS for 24 hours, and TGF-β1 was added at different concentrations. The lysate was extracted 24 hours after addition of TGF-β1. Peak α-SMA expression was seen after treatment with 2 ng/ml of TGF-β1. (B) To determine the effect of brivanib on TGF-β1-induced activation of HSCs as assessed by α-SMA expression, LX-2 HSCs were partially serum-starved by incubation with 1% FBS for 24 hours. Brivanib was then added at increasing concentrations 2 hours before addition of 2 ng/ml TGF-β1. Cell lysates were prepared 24 hours after adding TGF-β1 and analyzed by Western immunoblotting.</p

PDGF, VEGF and FGF2 all induce proliferation of human LX-2 hepatic stellate cells.

<p>Relative proliferation of LX-2 cells as assessed by BrdU incorporation after addition of TGF-β1 (A); PDGF (B); VEGF (C); or FGF (D). The LX-2 HSCs were starved for 24 hours and then treated with the respective cytokine or growth factor. BrdU incorporation was measured 72 hours later. Data shown are representative of four samples per group and are presented as mean ± SEM. *, P<0.05 (normalized to BrdU incorporation in the absence of the growth factor).</p