Experimental Studies On The Growth Inhibition Of Pancreatic Carcinoma By Gene Transfer Of Antisense Vascular Endothelial Growth Factor

Angiogenesis, the sprouting of new capillaries from preexisting blood vessels, has been found to be a crucial components in tumor growth progression and metastasis. The mechanisms by which neovascularization stimulates tumor progression are the delivery of the nutrients and oxygen necessary for tumor growth. Intratumoral microvessel density (MVD) was a significant predictor of a poor prognosis of about all solid tumors. A number of naturally occurring growth factors can induce and/or promote angiogenesis by stimulating endothelial cell growth and migration. One of the key factors that enhance the sprouting of new blood vessels is vascular endothelial growth factor (VEGF). VEGF is a homodinieric glycoprotein consisting of 5 major isoforms hanving,respectively 121 145 165,189 or 206 amino acid residues in the mature monomer. These monomers are generated by differential splicing of mRNA derived from a single gene. All 5 isoforms are mitogenic toward vascular endothelial cells and induce vascular permeabilization. VEGF is over-expressed in numerous human malignancies, including colorectal,renal. liver, ovarian, pancreatic and gastric carcinomas, and this over-expression is associated with disease progression. All human pancreatic cancer cell lines could express and secrete VEGF protein and VEGF was to be believed the main angiogemc pathway in human pancreatic cancer. It is reported that high expression of VEGF is associated with liver metastasis and a poor prognosis for patients with ductal pancreatic adenocarcinoma. Recently,VEGF receptors were found expressed by some pancreatic cancer cells. This suggests that in some instances VEGF may directly promote pancreatic cancer proliferation. So,anti-angiogenic approaches targeting the VEGFIVEGF receptor cascade may prove useful in the therapy of this malignancy. In this study, we constructed a replication deficient recombinant adenoviral vector (Ad-aVEGF) containing the cDNA for VEGF 165 in an antisense orientation and studied the 5 effect on the biologicai behainr and angogenesis of panCreac cancer haferrd withAd-aVEGF .The effect and feasibility of a pancreatic cancer trethent based on antisenseVEGF will be bed. The main work is described as fol1ows:l.Analy8e of rsGF e1pression by hamunoki8tockemistw and RT-PCR assay.lihatumorai ndcrovessel densty (MVD) and exPression Of VEGF PrOtein wer StUdiedby innnonohistOCWcai stainin. We found that the over-exPression of VEGF protein waspositively correlated with high MVD and both were si~cantiy associated withhistopathOlogical Ng and lymPh node metaStaSis in patients with pancreatic carcinoma.VEGF W isofOnns were dwtined in l l patients with pancreatic cancer and 2 hUmanpancreatic cancer cell lines (SWl990 and PaTu8988) by reverse hacriPtaSe-polymeraSeM reaction or-PCR). VEGase was detected in aIl two hUInan pancreatic cancer ceIllines and VEGFl21 and VEGFl65 were identified as the PredoIninan SPecies PrOduced inpancreatic cancer cells.2.Con8thection of the replication-deficient recombinant adenovirus of antisen8e VEGF(Ad-MGFl and Lac-Z (Ad-LacZ).The VEGFl65cDNA was inserted into El and E3-substituted adenovirus vector pAxCAwtin an antisense orientation following the co-transfection of 293host cell line. The righStrUC'tUr and direction of the insert DNA was confinnd by restriction anaiysis. TherePlicationdeficient adenovirus in Which the CMV Promotes the tranScriPtion of theantisense VEGF gene or Las-Z gene was generated by the method of calcium phOsPhaeco-Precipitation from the homologous recombination of betWen the cosmid DNA of VEGFand Ad5-TPC. All recombinan adenviruses were ProP...