VEGF Regulation durch Activin A im humanen hepatozellulären Karzinom

Abstract

Introduction: Upregulation of vascular endothelial growth factor (VEGF) is known to play a critical role in hepatocellular tumour biology. In an attempt to identify factors responsible for VEGF induction in human hepatocellular carcinoma (HCC), we evaluated the effects of activin A on VEGF gene expression. Methods: Expression of VEGF, activin A and its receptors was analyzed in vivo by immunohistochemistry and in vitro by polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA). Activin A effects on VEGF gene regulation were analysed by ELISA and proliferation assays. VEGF promoter analysis and gel shift assays were performed to define minimal promoter requirements and potential transcription factors. Results: Expression of activin A., the four receptors (ActR-I, ActR-IB, ActR-II and ActR-IIB) and VEGF was demonstrated by immunohistochemistry in 9/9 HCC’s. In vitro the expression of VEGF and the activin A/receptor system could be confirmed by ELISA and RT-PCR in three HCC cell lines. Incubation of HCC cells with activin A lead to a significant time and dose dependent growth inhibition after 72 hours. The VEGF expression was significantly stimulated with a peak after 96 hours. Transient transfections, deletional and gel shift assays demonstrated activin A dependent stimulation of the VEGF promoter through Sp1 dependent activation of the −85 to −50 promoter region of the VEGF promoter. Conclusion: For the first time it could be demonstrated on a molecular basis, that activin A indirectly stimulates angiogenesis in human HCC through induction of VEGF. This autocrine/paracrine loop serves as a potential target for a therapeutical antiangiogenic approach.