The Study Of Breast Cancer Treatment Using Antisense VEGF₁₆₅ CDNA

It is now widely recognized that angiogenesis plays a pivotal role in the development of solid tumors. Any tumor mass in excess of 2 mm in diameter depends on the formation of a vascular network that provides the growing tumor with oxygen and essential nutrients. Formation of new vessels is needed for tumor development as well as invasion and metastasis. Therapies aimed at destroying tumor vasculature can achieve rapid regression of experimental tumors and it has been shown that tumor cell apoptosis is significantly increased by treatment with antiangiogenic method. It has been shown that antiangiogenic therapy showed less drug resistance and cell toxicity than conventional chemotherapy. Antiangiogenic therapies represent a potential new approach to treating cancer. There are several methods of inhibiting angiogenesis currently under study. To date, many angiogenic factors have been identified. Vascular endothelial growth factor (VEGF) is one of the most potent of these and is known to play a pivotal role in angiogenesis. Tumor neovascularization is regulated by cell-surface receptors and extracellular factors. The factors regulating neovascularization, e.g., vascular endothelial growth factor (VEGF),is highly specific for endothelial cells. There is a balance between proangiogenic and antiangiogenic factors in normal. When the balance is disturbed, angiogenesis is induced. In tumors, this imbalance may be caused by either hypoxia (low oxygen tension) or genetic alterations that activate oncogenes or inactivate tumor suppressor genes . Angiogenesis consists of several steps.. Endothelial cells that form the wall of existing small blood vessels are activated, degrade the extracellular matrix, migrate through the matrix, and proliferate. The new endothelial cells organize into hollow tubes, which ultimately anastomose to form new capillaries. Vascular endothelial growth factor (VEGF) is essential for angiogenesis in health and pathophysiology, VEGF is also thought to play a key role in postnatal angiogenesis in human pathophysiology including cancer, rheumatoid arthritis, ocular neovascularizing disorders, and cardiovascular disease. The VEGF in our study is VEGF165 of VEGF-A. VEGF (VEGF-A) is a member of a family of related growth factors that now includes VEGF-B, -C, -D, and -E and placenta growth factor (PlGF). Alternative splicing of human VEGF mRNA from a single gene containing eight exons gives rise to at least five different isoforms of 121, 145, 165, 189 and 206 amino acid residues. VEGF121, VEGF145, and VEGF165 are secreted protein. Human VEGF165 is the most abundant and, in in vitro studies, the most biologicallyactive form. VEGF expression is induced by a number of stimuli including hypoxia, activated oncogenes, and inflammatory cytokines. Negative regulators of VEGF expression include wild type von Hippel-Lindau (VHL) and p53 tumor suppressor genes. Two distinct receptor tyrosine kinases (RTKs) have been identified for VEGF, VEGFR1 and VEGFR2, which bind VEGF-A. Vascular endothelial growth factor (VEGF) is a potent angiogenic peptide with diverse biologic effects. VEGF plays a central role in tumor growth, affecting not only angiogenesis but also serving to directly stimulate the growth and survival in tumors expressing its receptors. Since angiogenesis is essential for the growth of any solid tumor, emerging efforts are being made to develop antiangiogenic therapy. Objective:To construct eukaryotic expression vector carrying antisense VEGF165 and investigate whether the recombined plasmids could regulate the secretion level of VEGF protein and affect the growth cycle of the transfected human breast cancer cell line MCF-7. Methods: VEGF165cDNA was amplified by reversetranscription PCR(RT-PCR)from MCF-7 cell,and was then inserted into the expression plasmid pcDNA3 to construct the recombined plasmids that encoding VEGF165cDNA in an antisense orientation.The MCF-7 were transfected with these plasmids.The VEGF expression of MCF-7 cells before or after transfection was detected by immunochemistry.The growth cycle was also detected. Result: The eukaryotic expression vector pcDNA3/anti-VEGF165...