Study Of Vascular Endothelial Growth Factor (VEGF) Mediated Invasiveness And Metastasis In Gastric Carcinoma And Cholangiocarcinoma

Background and significationTumor metastasis is a complex and multi-stage process. A number of cellular and tissue changes occur as a cancer spreads, including cellular transformation and tumor growth, angiogenesis and lymphangiogenesis, and release of cancer cells into the circulation; then attachment, invasion, and proliferation within the new target organ. Several cytokines are now known to be involved in the process of tumor metastasis. Among these is vascular endothelial growth factor (VEGF), a factor that is associated not only with the angiogenesis of cancers but also with those involved in wound healing and other important pathologies. There are two mechanisms involved in VEGF-mediated angiogenesis; one is through its cognate receptor VEGFR2, the other is via up-regulation of the expression and/or activation of integrins.Integrins are heterodimeric transmembrane proteins that can induce cell adhesion to the extracellular matrix (ECM), coordinated by VEGF and its receptors. The avP6 integrin is restricted to epithelial cells and is highly expressed in developing fetal tissues as well as in a number of epithelial carcinomas and cancers. In our previous studies, we demonstrated that integrin avP6 can be used as a prognostic indicator of gastric carcinoma. In addition,αvβ6 expression in colon cancer cells leads to the increase in secretion of matrix metalloprotein-9 (MMP-9). In gastric cancer, degradation of ECM barriers by MMP-9 is important in facilitating tumor cellular dissemination and metastasis. Thus, integrinαvβ6 appears to have roles in promoting ECM degradation and in mediating tumor cell invasiveness via MMP-9.There are several confirmed collaborative relationships between integrins and VEGF in mediation of cell adhesion, migration, and proliferation. Theα1β1 andα2β2 integrins support VEGF-stimulated signal transduction and endothelial cell migration. Migration of multiple myeloma cells is induced by VEGF and associated withβ1 integrin. In colon cancer cells,α9β1-blocking antibody has been demonstrated to specifically inhibit the angiogenesis induced by VEGF-A. However, a role for integrinαvβ6 in VEGF-induced tumor progression has not yet been established.VEGF-C is a new member of the VEGF family, a tyrosine kinase receptor that is predominantly expressed in the endothelium of lymphatic vessels. The expression of VEGF-C is high in several types of human malignant tumors, including breast, lung, and gastric carcinomas and some investigators have shown that VEGF-C expression was closely associated with tumor invasion and lymph node metastasis. However, there have been very few reports on the correlation between VEGF-C gene expression and invasive phenotype in cholangiocarcinoma.The goal of the present study was therefore to investigate the relationship between VEGF and expression of integrinαvβ6 and their effects on invasiveness of gastric carcinoma cells in vitro. And the effect of VEGF-C in invasiveness and metastasis of cholangiocarcinoma.PARTⅠExpression of VEGF and integrinαvβ6 in gastric carcinoma tissues and cells Objective To study the expression of VEGF and integrinαvβ6 in gastric carcinoma tissues and cells.Methods Thirty gastric cancer tissues were collected from patients requiring clinically indicated surgical resection in QiLu Hospital of Shandong University (Jinan, China), with informed patient consent. Frozen gastric cancer tissue samples were stored at -80℃until processing. Immunohistochemical analysis was used to detect the expression of VEGF and integrinαvβ6 in gastric carcinoma tissues. The human gastric cancer cell line AGS was maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100U/ml penicillin, 10μg/ml streptomycin, and 2mM L-glutamine. Immuno-precipitation, FAC scanning and immunofluorescence staining were used to confirme the presence ofαvβ6 in gastric cancer AGS cells.Results Immunohistochemical analysis of tumor samples from 30 patients revealed that 26/30 samples stained positively for VEGF and 13/30 samples stained positively for integrinαvβ6. In 10/30 patients tumors were positive for both VEGF and integrinαvβ6. High surface expression ofαvβ6 integrin was detected by flow cytometry analysis, and the positive rate was higher than 90%. Expression ofαvβ6 was detected with immunofluorescent staining at the cell membrane in AGS cells. AGS cells were examined by immunoprecipitation analysis using antibody of 10D5 for expression ofαvβ6 integrin at protein levels.Conclusions VEGF and integrinαvβ6 are all expressed in gastric carcinoma tissues. And the high surface expression ofαvβ6 integrin is detected in gastric cancer AGS cells. PARTⅡVEGF enhances gastric carcinoma invasiveness via integrinαvβ6Objective To investigate the effect of integrinαvβ6 on VEGF regulated invasiveness and metastasis in gastric carcinoma cells, to probe into the key point of regulating metastasis of gastric carcinoma.Methods The AGS cells were starved for 4h in medium without fetal bovine serum and then stimulated with VEGF (30ng/ml) for 24h at 37℃. The expression at both mRNA and protein levels was also observed for integrinαvβ6, P-ERK and MMP-9 by using the real-time PCR, western blot analysis and immunoprecipitation.The roles ofαvβ6, P-ERK and MMP-9 at both mRNA and protein levels were detected by using monoclonal antibody for the humanαvβ6 integrin (10D5) and an inhibitor of the ERK signaling pathway (U0126).Specific P6 siRNA was designed according to the cDNA sequence of integrin subunit beta6 and the BLAST search was used to avoid unintentional silencing and ensure the uniqueness of target. Cells were grown subconfluently onto 24-well plates and transfected with 1μg ofβ6 siRNA, with nonspecific siRNA as control. After 48h of transfection, cells were washed and analyzed further as specifically indicated. The VEGF-stimulated transfected AGS cells were detected to calculate the change of integrinαvβ6, P-ERK and MMP-9 at both mRNA and protein levels.Migration of AGS cells through Matrigel-coated filters was examined in 24-well transwell inserts. After trypsinization, 1×105 cells in 100μl were incubated with 10D5 or U0126 for 1h and then were placed in the upper wells. Alternatively,β6 siRNA-treated cells were directly added to the transwell inserts. VEGF was added to the lower wells in a final volume of 1 ml. After 4 hours of incubation, while non-migrating cells were scraped away gently and cells on the lower surface were fixed and stained with Crystal Violet. The migrating cells were counted using a microscope.Results By using immunoprecipitation analysis, flow cytometry analysis and immunofluorescent staining, integrinαvβ6 was detected at the cell membrane in AGS cells and the positive rate was higher than 90%. AGS cells were treated with VEGF (30ng/ml) after separately pretreatment withβ6 siRNA, with an inhibitor of the ERK signaling pathway (U0126), or with a neutralizing antibody toβ6 (10D5). Expression of integrinαvβ6, P-ERK and MMP-9 at both mRNA and protein levels was measured by means of a real time PCR assay and immunoprecipitation analysis. A significant difference were observed (P<0.05) between VEGF-stimulated and un-stimulated cells. VEGF-induced expression of integrinαvβ6, P-ERK and MMP-9 at both mRNA and protein levels was significantly inhibited byβ6 siRNA, U0126, and 10D5 (P<0.05). Inhibition of the VEGF-induced migration of AGS. AGS cells were separately pretreated withβ6 siRNA, U0126, or 10D5 and were examined for their ability to migrate through a Matrigel filter in the absence/presence of VEGF (30ng/ml). Migration of AGS cells without VEGF or with VEGF stimulation was significantly inhibited byβ6 siRNA, U0126, and 10D5 (P<0.05).Conclusions Expression ofαvβ6 is detected at the cell membrane in AGS cells. The expression of integrinαvβ6, P-ERK and MMP-9 at both mRNA and protein levels and the invasiveness of AGS cells are stimulated by VEGF, but these effects are significantly inhibited by P6 siRNA, U0126, and 10D5 (P<0.05). These data suggest that VEGF is critical to the invasive process in human gastric cancer and that this occurs via up-regulation of integrin avP6 expression and activation of ERK.PARTⅢVEGF-C Expression and invasiveness in cholangiocarcinomaObjective To investigate the expression of VEGF-C in cholangiocarcinoma tissues and the VEGF-C induced invasiveness and metastasis in cholangiocarcinoma cells.Methods The cholangiocarcinoma tissues were collected from patients requiring clinically indicated surgical resection in QiLu Hospital of Shandong University (Jinan, China), with informed patient consent. Frozen cholangiocarcinoma tissue samples were stored at -80℃until processing. Immunohistochemical analysis was used to detect the expression of VEGF-C in cholangiocarcinoma tissues. And the relationships between the expression of VEGF-C and the clinical-pathological features of cholangiocarcinoma tissues were investigated. The human cholangiocarcinoma cell line FRH-0201 was maintained in RPMI 1640 medium. And the proliferation and invasiveness of FRH-0201 cells were detected by the MTT and Transwell experiment in the absence/presence of VEGF-C.Results The expression of VEGF-C was detected in 78.2% of cholangio-carcinoma tissues, and the expression was not associated with age, sex and the size of tumor, but associated with lymphatic invasion of the tumors. The proliferation and invasiveness of tumor cells were significantly promoted by VEGF-C (P<0.05).Conclusions Positive VEGF-C expression in cholangiocarcinoma is linked to significantly enhanced invasiveness, and its value as a prognostic marker is significant for early stage tumors.