The Significance Of Expression Of Vascular Endothelial Cell Growth Factor Families And Their Receptors In Non-small Cell Lung Carcinoma And Being A Target For Gene Therapy

INTRODUCTIONLung carcinoma is the leading cause of cancer death amongst men and has become second amongst women in industrialized countries. Despite therapeuticefforts, fewer than 10 per cent of patients can be cured and enjoy long-term survival.Angiogenesis is an essential step in tumor growth and metastasis. During this step, endothelial cells, which form the inner lining of blood vessels, migrate, proliferate and form tubes, and participate in the proteolytic degradation of the basement membrane and extracellular matrix. The ability of a primary tumor to induce angiogenesis upsets the balance between positive and negative regulatory factors . Vascular endothelial growth factor (VEGF)is unique among angiogenic factors by virtue of its paracrine/autocrine effect on the proliferation and motogenesis of endothelial cells. Although expression of VEGF-A in cancer has been studied extensively, the roles of other VEGF family members, i. e., VEGF-(?), vEGF-C, and VEGF-D, in tumor angiogenesis and lymphoid metastasis are poorly understood . The biological effects of VEGF are mediated through the activation of specific tyrosine kinase receptors, i.e. , Flt-1, Flk-1/KDR, Flt-4, expressed mainly on angioblasts and endothelial cells.Inhibition of VEGF to interfere with tumor neovascularization is one of the most important strategies in anti-angiogenesis therapy. Various VEGF-targeting approaches have been developed and some of them are in clinical trials, including small molecule inhibitors of VEGF-receptors , dominant negative VEGF .humanized antibodies to VEGF , soluble VEGF receptors , as well as antisense constructs against VEGF . However, except for antisense approach, the above strategies are incapable of specific targeting of VEGF isoforms; and even the effciency of antisense is usually very low . RNA interference (RNAi) is a sequence-specific posttranscriptional gene silencing mechanism, which is triggered by double-stranded RNA (dsRNA) and causes degradation of mRNA homologous in sequence to the dsRNA. This new approach has been successfully applied to inhibit virus replication and tumorigenicity.In the present study according to mRNA sequence of VEGF165 (ABO21221, GenBank), shRNAs was synthesized and transfected into A549 cells in vitro using shRNAs-liposome complex in order to find a new effective method to the gene therapy for NSCLC.Part 1Expression of Vascular Endothelial Growth Factor Family and their receptors Messenger RNA in non-samll lung caner tissues and paratumor tissues Purpose. Because the crucial role of angiogenesis and lymphangiogenesis has been played in the tumor growth and metastasis, the present study was undertaken to characterize the relative expression of vascular endothelial growth factors VEGF-A(vascular endothelial growth factor-A), VEGF-C, VEGF-D, and their receptors; KDR (kinase insert domain-containing receptor), Flt-1 (fms-like-tyrosine kinase), and Flt-4 in non-small lung cancer. The relationships between related mRNAs expression and the pathological classification, prognosis were analyzed.Methods. 36 surgical specimens of lung cancer tissue and the paratumour tissue responded except 10 surgical specimens of normal lung tissue were examined by semi-quantitative multiplex reverse transcription-PCR (RT-PCR) to detected the expression of different VEGF and their receptors mRNA.Results. The expressions of VEGF-A 121? 165 and KDR mRNA were significantly increased in lung Cv_.-.~cr tissue. There was no difference between VEGF-C mRNA expression and its receptor Flt-4 mRNA in all of specimens, in contrast, theexpression of VEGF-D and Flt-lmRNA was significantly decreased in tumor tissues.The significant correlations among the expression of VEGF-A121, 165 mRNA and the expression of Fit—1 and KDR mRNA in lung cancer tissues were found, and the significant correlation between VEGF-C mRNA and Flt-4 mRNA was shown in paratumor tissues as well. There was not any correlation between VEGF-D mRNA expression and all of receptors. Through analysis we found that expression of KDR mRNA was closely correlated with the tumor size, pathology grade and P-TNM stage, etc, while VEGF-A121 mRNA correlated with tumor size only. None of the VEGF family members and their receptors closely correlated with the lymphoid metastasis. However, Kendall stage analysis revealed that tumors with lymphoid metastasis and clinical pathology grade were closely correlated with COMVEGF-C-3 expression while C0MVEGF-O2 expression correlated with the tumor size.Conclusions. Compared with clinical features, members of VEGFs family and their receptors mRNA expression in non-small lung cancer tissues and paratumor tissues were investigated firstly. Although VEGF-C mRNA expression was required for nodal involvement, we assume that other factors, such as Flt-4, are involved, too. Members of VEGFs family and their receptors may play an important role in tumor growth and the hematogenous and lymphoid metastasis. There would be two pathways of antocrine and paracine participated in its mechanisms.Part 2Expression of Vascular Endothelial Growth Factor A> C and theirreceptors protein in non-samll lung caner tissuesPurpose. Because angiogenesis and lymphangiogenesis take crucial roles in tumor growth and metastasis, the present study focused to characterize the relative expressions among vascular endothelial growth factors-A(VEGF-A), VEGF-C, and their receptors KDR (kinase insert domain-containing receptor), FLT-1 (fmsliketyrosine kinase), and MVD, MLD in non-small lung cancer, in which the pathological classification, and prognosis involved.Methods. Imraunohistological features of VEGF-A > VEGF-C and their receptors protein were investigated in both 60 archival surgical specimens of non-smalltlung cancer and 10 specimens from benign lung neoplasms. Either microvessels or microlymphvessels which recognized by Anti~CD34 McAb(endothelial cell specific) and anti-Flt-4 McAb were quantified by vessels counting under microscope (200X) in the related area of the tumors specimens. Results. Out of the 60 surgical specimens of non-small lung cancer, exhibited intense positive staining in VEGF-A; 36 cases(60%), VEGF-C; 37cases(61. 7%), Flt-1; 38 cases (63. 3%) andKDR; 38 cases (63. 3%) in the cytoplasm of cancer cells, respectively. All of the expression of VEGFA, C and their receptors were higher than that of expression in benign lung neoplasms. In contrast, Flt-4 was mainly expressed on the lymphatic endothelial cells. There was a significant relationship between the expression of VEGF-A protein and the expression of KDR protein in lung T^cer tissues. In the VEGF-A positive tumors vessel count was significantly increased compared with in the negative tumors. VEGF-C expression also correlated with the expression of KDR protein and microlymphvessels count was significantly higher in the VEGF-C positive tumors than in the negative tumors. No correlation was observed between the expression of VEGF-A, VEGF-C, Flt-lprotein and any clinical pathologic features. While the KDR correlated with tumor size. The survival time of patients with higher MVD and MLD was significantly shorter than that of patients with lower MVD and MLD . Conclusions. These results overall suggest that VEGF-A , C and their receptors may play an important role in tumor progression via lymphangiogenesis and angiogenesis in non-small lung cancer. The mechanisms of them may result in two pathways; antocrine and paracine. MVD and MLD would be useful predictor of prognosis in NSCLC.Part 3Silencing effects of RNA interference (RNAi) on VEGF165 expressionin A549 cellsPurpose :To investigate whether RNA interference (RNAi ) induced by smallinterference RNA (siRNA) transfection in vitro could suppress vascularendothelial growth factor 165 mRNA expression in non-small lung carcinoma(NSCLC) cells.Methods: According to mRNA sequence of VEGF165 (AB021221, GenBank), shRNAs wassynthesized and transfected into A549 cells in vitro using shRNAs-liposomecomplex. The VEGF165 protein was analyzed by immunohistochemistry and westernblot between transfection and controls. The bioactive behalves of cells weredetermined by MTT and cell count.Results: Sequence specific shRNAs targeting VEGF165 showed targeting effectof down-regulated VEGF165 expression significantly after transfected into A549cells 24 hours. No influence was found on the growth and differentiation ofcells during shRNAs transfection.Conclusions: Sequence specific siRNAs targeting VEGF165 was capable ofsuppressing VEGF165 expression. The successful application of RNAi offers anew effective method to the gene therapy for NSCLC.