| Hepatocellular carcinoma (HCC) is characteristic of diagnosis delay, early metastasis, poor prognosis and resistance to chemotherapy and radiotherapy. So it is important for longer survival period to find available therapeutic approaches. Anti-angiogenesis as a therapeutic approach has a number of theoretical advances over conventional cytotoxic therapy whose principal mechanism of action is directed principally at the tumor cell. This approach has a broad spectrum of antitumor activity with low toxicities and drug resistance and as such is applicable to solid tumors generally. Moreover, with the development of modern molecular biology techniques, more and more attention is given to antisense techniques. The most promising agent for clinical gene therapy appears to be antisene oligodeoxynucleotides (ASODN) and it will bring us an epoch for tumor therapy.Angiogenesis plays a key role hi the growth of solid tumors. Furthermore, angiogenesis is required for the progression of tumors from a benign to amalignant phenotype and for metastasis. Malignant tumor cells secrete factors to their cognate receptors on endothelial cells to induce angiogenesis. The most potent angiogenic factor is vascular endothelial growth factor (VEGF) that mediates endothelial proliferation and vascular permeability. Preclinical models have demonstrated the essential nature of VEGF in the angiogenesis of solid tumor growth and metastasis, whereas pathologic investigations have revealed strong correlations between VEGF production, microvessel density, and overall aggressiveness of many human solid tumors. Recent advances in the understanding of the molecular mechanisms of VEGF action have led to successful models for intervention in VEGF-mediated pathways in therapy for solid tumors. VEGF induces angiogenesis through the activation of its high-affinity transmembrane receptors, fins-like tyrosine kinase-1 (Flt-1) and kinase insert domain-containing receptor (KDR). That is, VEGF regulates angiogenesis of tumor in a paracrine manner. However, the expression of Flt-1 and KDR on tumor cells themselves suggests a potential autocrine function for VEGF (such as regulating tumor cell proliferation). Inhibition of VEGF or VEGF receptors inhibited the proliferation of these cell lines in vitro. Furthermore, this effect was abrogated by exogenous VEGF. Thus, VEGF is an autocrine growth factor for tumor cell lines that express VEGF receptors. These findings imply that a novel dual role may exist for VEGF, such that it is involved in tumor cell activation (autocrine), hi addition to paracrine actions whereby it regulates endothelial cell functions and subsequent neovascular development, hi KDR-expressing tumors, VEGF inhibition may have dual functions: direct inhibition of tumor cell growth and inhibition of angiogenesis. A high expression level of VEGF has been shown to be associated with tumor progression and metastasis in human HCC, but its autocrine mechanism is unclear.In order to investigate the effects of ASODN against VEGF on several human HCC cell lines, antisense 21-mer phosphorothioate oligodeoxynucleotide was synthesized complementary to the 5' translated initial region of human VEGF mRNA. Intracellular distribution of fluorescein-labeled ASODN in SMMC-7721 cell was observed by laser scanning confocal microscopy. And the principle of ASODN following its concentration and process time was analyzed quantitatively. The inhibition of expression for mRNA and protein of VEGF was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemical stain. For autocrine mechanism of VEGF in HCC cell lines, RT-PCR and immunocytochemical stain was performed to characterize the expression of VEGF receptors. Phenotypic alterations in HCC cells by antisense reduction of VEGF expression were observed. And the alterations for expression of VEGF receptors in HCC cell membranes were analyzed by flow cytometry. Following results were obtained:1. Intracellular density of ASODN against VEGF in SMMC-7721 cell was correlated w...
|
PDF Full Text Request