Lewis Y Antigen Promotes The Expression Of Vascular Endothelial Growth Factor Receptor In Ovarian Carcinoma-derived RMG-I Cells

ObjectionOvarian carcinoma is the leading cause of death from gynecologic cancer,its development mechanism is always the emphasis of gynecology tumor fundamental and clinical research.The initiation and development of ovarian carcinoma is an extremely complicate and multifactorial process.At present,its molecular mechanism is still poorly understood.Just outside the plasma membrane is a coat of the sugar chains which linked to surface glycoproteins and glycolipids.The glycoproteins begin on the cytoplasmic face of the lipid bilayer,within it,or on its periphery.Sugar chains are important to cell-cell recognition and cell-molecule recognition,can serve as ligand binding,signal transduction and Intercellular Adhesion,was closely correlated with cell growth, apoptosis and cell differentiation,and was called intercellular messenger.At present, research of sugar chains isn't satisfied with the whole chains,deep into the function of glycon.The aspect has reported but don't elucidate clearly.Sugar chains has become a hot subject in Glycobiology and Cytobiology.Lewis y antigen is a member of a family of blood group-related difucosylated oligosaccharide which linked to surface glucolipid and glycoprotein,The expression of Lewis y antigen is mainly regulated by 1,2-fucosyltransferase.We have previously transfected 1,2-fucosyltransferase into human ovarian cancer cells RMG-I,and established RMG-I-H cell lines,which was overexpressed in 1,2-fucosyltransferase and Lewis y antigen.Through comparing biological behaviour of the two cells,our study has clearly shown that the tumor cell generation, invasion and metastasis were upregulated after transfection.The viability of the transfections in the medium containing the anticancer drug 5-fluorouracil(5-FU) and carboplatin was signignificantly elevated.Based on our previous work,we have found that VEGF overexpresses in RMG-I-H which is rich in Lewis y antigen,in order to investigate Lewis y antigen influence on VEGFR,to compare the VEGFR expression in RMG-I-H and RMG-I,to further elucidate the role of Lewis y in ovarian cancer development,and to provide a new approach for ovarian carcinogenesis and development research.Methodsovarian cancer cell lines and human ovarian carcinoma xenograft modelsRMG-I was clear cell carcinoma of the ovary cell lines.Cell lines RMG-I-H was obtained from RMG-I which was transfected with 1,2-fucosyltransferase gene and overexpressed by Lewis y antigen.Ten female athymic nude mice(20.420.4±0.6 g) were 4 to 6 weeks old.Animals were randomly devided into two groups,the experimental group were treated by subcutaneous injection with RMG-I-H cell(0.3 ml),the control group were treated by subcutaneous injection with RMG-I cell(0.3 ml).The animals were allowed a period of adaptation in a sterile and pathogen-free environment.Five weeks later,animals were sacrificed by decapitation.Tumor mass were removed,weighed and fixed in 10% formalin.Tumor mass were confimed high malignant ovarian clear carcinoma by pathologic diagnosis.SP immuocytochemical method was used to detect the expression of Flt-1 and KDR in RMG-I and RMG-I-HMonolayers of RMG-I-H and RMG-I cells were made and fixed in 4% paraformaldehyde.Flt-1 and KDR was detected using SP immuocytochemical method according to the kit direction.Assessment of the resuilts:yellow brown particles in the cytoplasm and cytomembrane represented positive reaction.With Meta Morph microscopic image analysis system for quantitative analysis of morphology,we detect integral optical density(IOD) and record average data.SP immunohistochemistry method was used to detect the expression of Flt-1 and KDR in nude mice tissue Paraffin-embedded tissues were cut up into section which are 4-mm-thick,then were converntional dewaxed,hydrated,and experiencing antigentic revival.The following is the same with the above.RT-PCR was used to determine the expression of Flt-1mRNA and KDR mRNA in RMG-I and RMG-I-HTotal RNAs were extracted from exponentially growing monolayers of RMG-I and RMG-I-H.RT-PCR was according to the TAKARA kit direction.The PCR products were electrophoresed on a 3%agarose gel.β-actin expression was used as a control for the amount of RNA.The relative intensities of the bands were determined, and the ratios toβ-actin were calculated.Each experiment was performed in triplicate.Statistical analysisThe data shown were mean values of at least three different experiments and expressed as mean±SD.Student's t test was used for comparison.P＜0.05 is considered statistically significant.All data were analyzed with SPSS13.0 for Windows.ResultsThe expression of Flt-1 and KDR in ovarian cancer cells RMG-I-H and RMG-IFlt-1 positive granules in RMG-I-H were brown or yellow brown,with integral optical density 21.000±1.255,while Flt-1 positive granules in RMG-I were pale brown granules with integral optical density:17.868±1.818.Staining intensity in RMG-I-H did not differ significantly from that in RMG-I(P=0.411).KDR positive granules in RMG-I-H were brown or yellow brown,with integral optical density: 24.245±2.168,while KDR positive granules in RMG-I were pale brown granules with integral optical density:14.469±1.0443.Staining intensity in RMG-I-H was signifancently higher than that in RMG-I(P=0.002).The expression of Flt-1 and KDR in in in nude mice tissueIn the tissue of the subcutaneously transplanted tumor of RMG-I-H,Flt-1 positive granules were brown or yellow,with integral optical density:45.540±2.373,while Flt-1 positive granules in the non-tranfection group were pale yellow with integral optical density:42.009±2.905.Staining intensity in transfection group did not differ significantly from that in non-tranfection group(P=0.270).In the tissue of the subcutaneously transplanted tumor of RMG-I-H,KDR positive granules were brown or yellow,with integral optical density:71.519±2.558,while KDR positive granules in the non-tranfection group were pale yellow with integral optical density:45.977±1.879.Staining intensity in transfection group was signifancently higher than that in non-tranfection group(P=0.001).The gene expression of Flt-1 and KDR in RMG-I and RMG-I-HThe relative intensity of mRNA expression of Flt-1 in RMG-I-Hand RMG-I were0.896±0.042 and 0.788±0.100,respectively.The Flt-1 mRNA in RMG-I-H did not differ significantly from that in RMG-I(P=0.120).The relative intensity of mRNA expression of KDR in RMG-I-Hand RMG-I were 0.8964±0.042 and 0.788±0.100,respectively.The expression of KDRmRNA in RMG-I-H was higher than that in RMG-I-H(P=0.003).ConclutionThe results suggested that Lewis y antigen promotes the expression of KDR, which is regulated in gene level,it maybe promote tumor angiogenesis through VEGF autocrine and paracrine loops,mediate ovarian cancer growth invasion,metaptosis and chemoresistance.