Simultaneous silencing of VEGF and KSP by siRNA cocktail inhibits proliferation and induces apoptosis of hepatocellular carcinoma Hep3B cells

Chung Doan,Dong Le,Si Do,Long Le,Son Hoang

doi:10.1186/0717-6287-47-70

Abstract

BackgroundVascular endothelial growth factor (VEGF) is involved in the growth of new blood vessels that feed tumors and kinesin spindle protein (KSP) plays a critical role in mitosis involving in cell proliferation. Simultaneous silencing of VEGF and KSP, an attractive and viable approach in cancer, leads on restricting cancer progression. The purpose of this study is to examine the therapeutic potential of dual gene targeted siRNA cocktail on human hepatocellular carcinoma Hep3B cells.ResultsThe predesigned siRNAs could inhibit VEGF and KSP at mRNA level. siRNA cocktail showed a further downregulation on KSP mRNA and protein levels compared to KSP-siRNA or VEGF-siRNA, but not on VEGF expression. It also exhibited greater suppression on cell proliferation as well as cell migration or invasion capabilities and induction of apoptosis in Hep3B cells than single siRNA simultaneously. This could be explained by the significant downregulation of Cyclin D1, Bcl-2 and Survivin. However, no sigificant difference in the mRNA and protein levels of ANG2, involving inhibition of angiogenesis was found in HUVECs cultured with supernatant of Hep3B cells treated with siRNA cocktail, compared to that of VEGF-siRNA.ConclusionSilencing of VEGF and KSP plays a key role in inhibiting cell proliferation, migration, invasion and inducing apoptosis of Hep3B cells. Simultaneous silencing of VEGF and KSP using siRNA cocktail yields promising results for eradicating hepatocellular carcinoma cells, a new direction for liver cancer treatment.

Highlights

Vascular endothelial growth factor (VEGF) is involved in the growth of new blood vessels that feed tumors and kinesin spindle protein (KSP) plays a critical role in mitosis involving in cell proliferation
Effects of pre-designed small-interfering RNA (siRNA) on KSP and VEGF Messenger RNA (mRNA) expression in Hep3B cells To address the functions of VEGF and KSP, Hep3B cells were transfected with VEGF-siRNAs and KSP-siRNAs
The results showed that VEGF mRNA was downregulated by 77.54 ± 3.22% (p < 0.01, Figure 2A) and VEGF protein level was downregulated by 59.42 ± 2.14% (p < 0.05, Figure 3B), which was confirmed by Enzyme linked immunosorbent assay (ELISA) analysis compared to untreated cells (Figure 3D)

Summary

Introduction

Vascular endothelial growth factor (VEGF) is involved in the growth of new blood vessels that feed tumors and kinesin spindle protein (KSP) plays a critical role in mitosis involving in cell proliferation. Hepatoblastoma (HB) and hepatocellular carcinoma (HCC), is one of the most common solid tumors, ranking the fifth in most common malignancy worldwide and the second cause of cancer-related deaths. KSP gene was frequently expressed in HCC tissues and there was a strong correlation between the level of KSP expression and HCC development [10]. These findings have indicated that the important role of KSP in mitotic progression makes it an significant candidate of anticancer therapy. Several KSP inhibitors have been studied in clinical trials and showed efficacy in preclinical models of human tumors [10,11]. More trials must be studied to test their efficacy in clinic due to the toxicological side effects of KSP inhibitors, such as the observed neutropenia and leukopenia [12]

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