siRNA targeting VEGF inhibits hepatocellular carcinoma growth and tumor angiogenesis in vivo

Background/Aims

We have investigated whether siRNA targeted against VEGF inhibits functional properties of endothelial cells in vitro and HCC tumor growth and blood vessel formation in vivo.

Methods

The influence of siRNA-VEGF on endothelial cell proliferation, apoptosis and tube formation were analyzed in vitro. Antitumoral effects were examined in an orthotopic tumor model after ex vivo transfer or intraperitoneal treatment of siRNA, respectively. Intratumoral microvessel density was assessed by CD31 staining.

Results

VEGF expression was inhibited in Hepa129 by 70% and in SVEC4-10 by 48% within two days after transfection. In vitro, endothelial cell proliferation and tube formation was reduced by 23% and 38%, respectively. Interference with VEGF signaling was demonstrated by reduced pAKT in hepatoma cells. Tumor growth was inhibited by ex vivo transfer or intraperitoneal application of siRNA-VEGF by 83% or 63% in orthotopic tumors within 14 days. VEGF protein was reduced in both models by 29% and 44%. Microvessel density dropped to 34% for tumors from ex vivo transfected cells and 39% for systemic treated tumors.

Conclusions

The results show that VEGF knockdown can be associated with reduced endothelial cell proliferation and tube formation in vitro and decreased tumor growth and microvessel density in vivo.

Introduction

It has been acknowledged that hepatocellular carcinoma (HCC) progression is associated with tumor angiogenesis and upregulation of VEGF (vascular endothelial growth factor) [1], [2]. In HCC, increased VEGF levels correspond to increased tumor sizes [3]. Due to limited therapeutic options for HCC patients [4], [5], new strategies are needed to control HCC disease. VEGF, mainly VEGF-A, is known to be secreted by tumor cells, thereby activating quiescent endothelial cells (EC) in a paracrine manner [6], [7]. VEGF-A (referred here as VEGF) is bound by VEGF receptor 1 (VEGFR1), VEGFR2 and the novel VEGF receptor neuropilin-1 (Nrp1) mainly on the cell surface of endothelial cells.

Binding of VEGF to its receptor and subsequent receptor dimerization activates transduction pathways which promote migration, proliferation and prolongs cell survival [8], [9]. These effects of VEGF on EC proliferation, differentiation, migration and cell survival predispose VEGF for potential therapeutic antitumor strategies [8], [10].

Inhibition of tumor growth on the molecular level can be achieved by affecting pro-tumoral and pro-angiogenic factors, like VEGF or VEGF receptors. There are several options of inhibiting VEGF binding to its receptors, such as soluble VEGF receptors [11], or anti VEGF antibodies [12]. The disadvantage of these approaches is that already circulating VEGF has to be captured and neutralised to block VEGF mediated downstream effects. In our opinion a more appropriate approach is to knock down VEGF protein synthesis and secretion at the generation site by small interfering RNA (siRNA). A variety of studies has checked antitumor effects of siRNA-VEGF, but surprisingly, there is little data about VEGF knockdown and paracrine functional effects on EC [13], [14]. In vitro effects were mainly analyzed in the corresponding tumor cells used for the in vivo models[15], [16], [17], [18], [19], [20].

In this study, siRNA targeted against VEGF was used to reduce VEGF expression in hepatoma and endothelial cells to alter AKT signaling and to investigate functional endothelial cell specific effects in vitro. These results were transferred to an in vivo orthotopic hepatoma model to analyze antitumoral and angiostatic effects.