Duale Inhibition von B-Raf und VEGFR2 reduziert die Vaskularisierung und das Wachstum des hepatozellulären Karzinoms

Abstract

The Ras/Raf/MEK/ERK signaling pathway plays an important role in progression and angiogenesis of hepatocellular carcinoma (HCC). In addition, STAT3 is a central promoter of HCC growth. Both signaling pathways are also involved in motility and proliferation of endothelial cells. We hypothesized that dual inhibition of B-Raf and VEGFR2 with small molecule inhibitor (NVP-AAL881) will affect tumor cells, endothelial cells and pericytes thereby reducing tumor growth and angiogenesis of HCC in vivo. Human HCC cell lines (HepG2, Huh-7), endothelial cells (HUVEC) and pericytes (vascular smooth muscle cells, VSMC) were used for the experiments. A small molecule inhibitor (NVPAAL881, Novartis, Basel) was used for simultaneously blocking B-Raf kinase and VEGFR2. Cytotoxic effects in vitro were determined using MTT assays. Effects of NVP-AAL881 on activation of signaling intermediates (MEK, ERK) and STAT3 in tumor cells and EC were investigated by Western blotting. Changes in constitutive and growth factor induced migration of HCC cells, EC and VSMC were evaluated in Boyden chamber assays. Effect of combined B-Raf/VEGFR2-blockade on tumor growth were subsequently determined in a subcutaneous tumor model. Blockade of Raf in HCC cells blocked activation of MEK, ERK and STAT3. In addition, constitutive and growth factor induced tumor cell migration and invasion were significantly reduced (P < 0.05). In EC, NVP-AAL881 diminished activation of AKT, ERK as well as VEGFR2-independent STAT3 activation. Furthermore, migration and proliferation of EC and VSMC were significantly reduced (P < 0.05). In the subcutaneous model, tumor growth was significantly inhibited (P < 0.05). Immunohistological analyses revealed a marked reduction of CD31 vessel area in NVP-AAL881 treated tumors. In conclusion, combined inhibition of B-Raf and VEGFR2 efficiently reduces tumor growth and vascularization of HCC in vivo and, therefore, might be a promising approach for molecular-based therapy of HCC.