Vector-based RNAi, a novel tool for isoform-specific knock-down of VEGF and anti-angiogenesis gene therapy of cancer☆

https://doi.org/10.1016/S0006-291X(03)00495-9Get rights and content

Abstract

Vascular endothelial growth factor (VEGF) carries out multifaceted functions in tumor development, and it exists as at least five isoforms with distinct biologic activities and clinical implications. Several strategies have been developed to block VEGF for cancer therapy; however, the approach to target-specific VEGF isoform(s) has not been explored to date. In the present study, we show that DNA vector-based RNA interference (RNAi), in which RNAi sequences targeting murine VEGF isoforms are inserted downstream of an RNA polymerase III promoter, has potential applications in isoform-specific “knock-down” of VEGF. Large molecular weight VEGF isoforms were specifically reduced in vitro in the presence of isoform-specific RNAi constructs. Additionally, H1 promoter may be superior to U6 promoter when used for vector-based RNAi of VEGF isoforms. This strategy provides a novel tool to study the function of various VEGF isoforms and may contribute to VEGF isoform-specific treatment in cancer.

Section snippets

Materials and methods

Cell culture. ID8, a murine ovarian cancer cell line, was generously provided by Dr. Terranova and co-workers [44]. ID8, HeLa (ATCC, Manassas, VA), and human osteosarcoma cell line 143B (Wistar Institute, Philadelphia, PA) were grown in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin (all from Invitrogen) in a humidified, 5% CO2 incubator at 37 °C.

Plasmid constructs. RNA

Vector-based RNAi knocks down the exogenous expression of GFP gene in epithelial ovarian cancer cell line

It has been reported that both chemically synthetic and vector-based siRNA can successfully knock down specific gene expression in mammalian cells, including malignant cells [29], [47], [48]. To explore the potential application of siRNA in ovarian cancer, we used ID8 cell line derived from spontaneous in vitro malignant transformation of mouse ovarian epithelial cells [44] in this study. Because epithelial ovarian carcinomas represent more than 90% of human ovarian cancer and they are believed

Discussion

In this study, we present a strategy to “knock down” VEGF in an isoform-specific manner using DNA-vector-based RNAi. This method may provide a novel tool to study the function of different VEGF isoforms in tumor angiogenesis, immune suppression, and autocrine effect of tumor cell survival. Most importantly, this strategy finds potential applications in VEGF isoform-specific therapy for cancer. Up to date, most VEGF-targeting therapies in place are incapable of specifically inhibiting a selected

References (55)

  • Y. Zeng et al.

    Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells

    Mol. Cell

    (2002)
  • L. Zhang et al.

    Different effects of glucose starvation on expression and stability of VEGF mRNA isoforms in murine ovarian cancer cells

    Biochem. Biophys. Res. Commun.

    (2002)
  • H. Ilves et al.

    Retroviral vectors designed for targeted expression of RNA polymerase III-driven transcripts: a comparative study

    Gene

    (1996)
  • H. Hasuwa et al.

    Small interfering RNA and gene silencing in transgenic mice and rats

    FEBS Lett.

    (2002)
  • P. Carmeliet et al.

    Angiogenesis in cancer and other diseases

    Nature

    (2000)
  • D.I. Gabrilovich et al.

    Production of vascular endothelial growth factor by human tumors inhibits the functional maturation of dendritic cells

    Nat. Med.

    (1996)
  • L. Zhang et al.

    Intratumoral T cells, recurrence, and survival in epithelial ovarian cancer

    N. Engl. J. Med.

    (2003)
  • R.E. Bachelder et al.

    Vascular endothelial growth factor is an autocrine survival factor for neuropilin-expressing breast carcinoma cells

    Cancer Res.

    (2001)
  • Y.S. Ng et al.

    Differential expression of VEGF isoforms in mouse during development and in the adult

    Dev. Dyn.

    (2001)
  • C.J. Robinson et al.

    The splice variants of vascular endothelial growth factor (VEGF) and their receptors

    J. Cell Sci.

    (2001)
  • J.E. Park et al.

    The vascular endothelial growth factor (VEGF) isoforms: differential deposition into the subepithelial extracellular matrix and bioactivity of extracellular matrix-bound VEGF

    Mol. Biol. Cell

    (1993)
  • A. Yuan et al.

    Vascular endothelial growth factor 189 mRNA isoform expression specifically correlates with tumor angiogenesis, patient survival, and postoperative relapse in non-small-cell lung cancer

    J. Clin. Oncol.

    (2001)
  • Y. Oshika et al.

    Expression of cell-associated isoform of vascular endothelial growth factor 189 and its prognostic relevance in non-small cell lung cancer

    Int. J. Oncol.

    (1998)
  • T. Tokunaga et al.

    Vascular endothelial growth factor (VEGF) mRNA isoform expression pattern is correlated with liver metastasis and poor prognosis in colon cancer

    Br. J. Cancer

    (1998)
  • C. de Vries et al.

    The fms-like tyrosine kinase, a receptor for vascular endothelial growth factor

    Science

    (1992)
  • F. Shalaby et al.

    Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice

    Nature

    (1995)
  • G.H. Fong et al.

    Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium

    Nature

    (1995)

    • Cited by (120)

    • Low expression of vascular endothelial growth factor and high serum level of cyclic guanine monophosphate as the risk factors of femoral head osteonecrosis in alcohol-exposed Wistar rat

      2020, Chinese Journal of Traumatology - English Edition
      Citation Excerpt :

      Other studies have shown that in inducing bone resorption, NO follows a different pathway with the collaboration of high levels of prostaglandins. There is also the opinion that NO is supposed to increase perfusion through the effects of internal vasodilators which lead to femoral head preservation.18–21 However, Koo et al.19 also conveyed the need for further research to ensure this, given the lack of research that was well designed in his study.

    • Sprengerinin C exerts anti-tumorigenic effects in hepatocellular carcinoma via inhibition of proliferation and angiogenesis and induction of apoptosis

      2013, European Journal of Pharmacology
      Citation Excerpt :

      Tumor hypoxia can activate hypoxia-inducible factor (Hif)-1α, which targets the VEGF gene, further resulting in VEGF-dependent tumor angiogenesis. Therefore, compounds showing inhibitory effects on VEGF expression and VEGF-stimulated neovascularization can be used as promising drug candidates for anti-cancer therapy (Díaz-Rubio and Schmoll, 2005; Mendel et al., 2000; Zhang et al., 2003). Treatment with drugs inducing tumor cell cycle arrest and apoptosis may be another effective strategy for affecting tumor progression by suppressing the growth and expansion of the cells.

    • The effects of CD59 gene as a target gene on breast cancer cells

      2011, Cellular Immunology
      Citation Excerpt :

      Our studies focused on what role CD59 plays in the progression of breast cancer cells (MCF-7) under the silencing or overexpression of CD59. In mammalian cells, retroviral vector-mediated RNAi can be further applied to functional genomics studies, so that a group of related individual genes can be silenced simultaneously and their synergic functions can be systematically assessed [27–29]. In addition, viral vector-mediated RNAi holds promises in gene therapy for cancers and infectious diseases because it can result in loss-of-function phenotypes of disease-related genes [30,31].

    • Lipid-based vehicles for siRNA delivery in biomedical field

      2020, Current Pharmaceutical Biotechnology

    • Modeling and Analyzing the Flow of Molecular Machines in Gene Expression

      2018, RNA Technologies

    • Targeting VEGF pathway to normalize the vasculature: An emerging insight in cancer therapy

      2018, OncoTargets and Therapy
    View all citing articles on Scopus
    ☆

    Supported by a grant by the Sidney Kimmel Foundation and institutional funding from the Abramson Family Cancer Center and Cancer Research Institute, and the Department of Obstetrics and Gynecology at the University of Pennsylvania.

    View full text
    Copyright © 2003 Elsevier Science (USA). All rights reserved.