The Mechanism Of Vascular Endothelial Growth Factor (VEGF) In Micrometastasis Of Lewis Lung Carcinoma

The mechanism of micrometastasis of lung cancer is being paid more attention.Asgreen fluorescent protein(GFP) has been used to localize functional protein and trace targetcell, the micrometastasis process of lung cancer can be continually observed by a successfulexperimental model with GFP. It is well known that angiogenesis and invasion are twoessential events for metastasis of tumor. Vascular endothelial growth factor(VEGF) isclosely related to angiogenesis, growth and metastastasis of tumor, mediating by its tworeceptors (FLT and KDR). But the role of VEGF in micrometastasis has not been known.Some researches showed that VEGF could enhance the expression of Matrixmetalloproteinase-2(MMP-2) and matrix metalloproteinase-9(MMP-9) in leukemia, ovariancancer and other tissues. However, little is know about the similar effects of VEGF in lungcancer.Therefore, this research was designed to study the effects of VEGF in micrometastasisof lung cancer by three steps: first, Lewis lung carcinoma cell (LLC) were transfected byVEGF and sFLT recombinant plasmid or mock plasmid, named as LLC-VEGF, LLC-sFLTand LLC-mock, respectively. Before and after the transfection, cell growth, invasion,endothelial cell migration and expression levels of MMP-2 and MMP-9 were observed.Second, the transfected cell lines were inoculated into C57BL/6J mice, and themicrometastases foci, MVD and the expression levels of related factors such asMMP-2,MMP-9 in the xenografts were compared among the groups. At last, the serumVEGF level of non small cell lung cancer(NSCLC)patients and circulating CK19mRNAlevel that was usually severed as a micrometastasis marker of epithelial tumors, wereobserved in clinical stage of NSCLC.The main results are listed follows:1. Recombinant plasmid pEGFPS2-VEGF and pEGFPS2-sFLT were constructedsuccessfully with sequencing identification. Transfected cell lines stably expressed greenfluorescence. The growth of LLC in vitro was not interfered by transfection,suggestingVEGF might act its paracrine effects on the cell. In invasion assay, the highest migration of 3第三军医大学博士学位论文tumor cells was found in LLC-VEGF ( 75±15.11), comparison to LLC-mock(34±2.45) andLLC-sFLT(15±1.41) (P<0.05). Similar results were found when these tumor cells werecocultured with ECV304 cell. VEGF was found to increase activity of MMP-2 and MMP-9by RT-PCR,Western blot and zymogram analysis. The above mentioned results suggest thatoverexpression in LLC-VEGF might be promoted tumor cell invasive through upregulatingMMP-2 and MMP-9 ,while the activity was inhibited in LLC-sFLT cells.2. Experimental and spontaneous pulmonary micrometastases tagged with GFP weredetected on the 2nd and the 7th day after injection via vein and subcutaneous, respectively.The GFP positive foci on the 2nd, the 4th and the 7th day postinjection were about 20, 40and 100μm in diameter, up to 500 μm on the 10th day. VEGF was found to increase thenumber and size of micrometastasis foci in both models, sFLT inhibited micrometastsesformation. The MVD, MMP-2 and MMP-9 levels in xenografts were much higher inLLC-VEGF than in LLC-sFLT (P<0.01) .A positive correlation was found between serumVEGF levels and size of micrometastases foci in lungs of experimental metastases mice.3.Clinically, significant increased of serum VEGF levels was found in advaneedNSCLC patients.there was a positive correlation (r=0.9974, P<0.01)between serum VEGFlevel and circulating CK19mRNAlevel .Conclusion:1. An animal model of lung micrometastasis was established in mice via intravenousand subcutaneous injection of Lewis lung carcinoma cells(LLC) transfected with plassmidcontaining green fluorescent protein(GFP) gene.2. The cell invasive and angiogenic ability ,and pulmonary micrometastasis of LLCwere promoted by overexpression Vascular endothelial growth factor (VEGF) .while themetastasic effects of VEGF on LLC could be blocked by its soluble receptor sFLT.3. Serum VEGF levels as well a...