| IntroductionOvarian cancer is the leading cause of death in gynecological cancers, the occurrence and development mechanism of ovarian cancer has been the focus of basic and clinical research in gynecologic cancer, understand the methods of controlling the biological behavior of ovarian cancer and the occurrence and development molecular mechanisms of ovarian cancer is very important for prevention and treatment of ovarian cancer.The occurrence and development of ovarian cancer is an extremely complicated multi-factor process, currently its pathogenesis is not clear. In recent years, the research on the relationship between glycans and tumors has attracted wide attention. Glycans are important components of cell membrane, which play essential roles in cell-cell interaction, cell-molecule recognition, as well as involve in signal transduction and molecule adhesion, therefore closely relate to many important life processes such as cell growth, apoptosis, mobility and differentiation etc. Upon cancerous transformation, cell membrane glycans, especially the carbohydrate chain of them, undergo structural and quantitative changes. The major presentation of ovarian cancer is alteration in type II carbohydrate chains, such as Lewis y antigen. Research shows that 75% of epithelial ovarian cancers have varying degree of Lewis y overexpression, and increased expression is associated with poor prognosis of patients. In our preliminary study, we introduced al,2-fucosyltransferase (al,2-FT) gene into human ovarian cancer cell line RMG-I through gene transfection and established cell model overexpressing Lewis y antigen. It was discovered that the RMG-I-H cells after transfection becomes highly tolerant to the anti-tumor drug,5-FU. It suggested that the Lewis y antigen possessed the function of boosting the survival ability of ovarian carcinoma cells.Most cell surface receptors are glycoproteins, studies showed that changes in glycosyltransferase expression might affect structure of carbohydrate chains on cell surface receptors and therefore impacted the expression and function of those glycoprotein receptors. In previous studies, we tested the differences in oncogene expression before and after al,2-FT gene transfection using gene chips technology. Results showed that:there were 88 differentially expressed genes after cell transfection. Altered genes mainly involved these genes regulating cell proliferation, signal transduction, protein amino acid phosphorylation, transcription, apoptosis and so on. Thus, it is possible that Lewis y may be an important component in signaling transduction pathway participating in signal transduction inside cell and further promoting proliferation of ovarian cancer cells.This study mainly on the basis of the preliminary work, and the use of a1,2-FT and Lewis y stable high expression cell model which has been already established, discuss the effect and molecular mechanism of Lewis y antigen on the proliferation of ovarian cancer cells. The results will lay the ground work for studying occurrence and development of ovarian cancer, provide a theoretical basis for ovarian cancer early diagnosis and treatment of target organ.MethodsThe first part:Reverse transcription-polymerase chain reaction (RT-PCR) was used to test the expression of a1,2-FT mRNA in the pre-and post-transfection cell lines; immunocytochemical staining was performed to detect the expression of Lewis y antigen in the cell lines; MTT assay was carried out to test the proliferation of the pre-and post-transfection cell lines; flow cytometry was used to analysis the cell cycle; the ovarian cancer cell lines were treated with a-L-fucosidase and anti-Lewis y antibody respectively, then MTT assay was carried out to test the proliferation.The second part:RT-PCR was used to test the expression of EGFR, HER2/neu mRNAs in the pre-and post-transfection cell lines; Western blot was carried out to detect the the expression of EGFR, HER2/neu proteins in the pre-and post-transfection cell lines; immunoprecipitaion using specific antibodies of EGFR, HER2/neu combined with Western blot using monoclonal antibody against Lewis y as primary antibody was used to test the expression level of Lewis y on the EGFR, HER2/neu; Western blot was performed to detect the the phosphorylation level of EGFR, HER2/neu proteins in cell lines.The third part:Western blot was carried out to detect the expression and phosphorylation level of PI3K/Akt and Raf/MEK/MAPK signaling pathways related-proteins in the pre-and post-transfection cell lines; the cells were treated with EGFR tyrosine kinase specific inhibitor ZD1839 and anti-Lewis y antibody respectively, then Western blot was used to detect the the expression and phosphorylation level of above mentioned signal proteins; MTT assay was carried out to test the survival of the cells treated with various PI3K inhibitor LY294002.ResultsThe first part:RT-PCR results showed the expression of a1,2-FT mRNA was significantly upregulated in post-transfection cell line RMG-I-H. Immunocytochemical staining results showed the expression of Lewis y antigen was significantly upregulated in RMG-I-H cells. MTT assay results showed the cell proliferation sped up as the Lewis y antigen was increased. Both of a-L-fucosidase and anti-Lewis y antibody inhibited the cell proliferation. Cell cycle test results showed Lewis y overexpression enhanced DNA synthesis and promoted cells in G0-G1 phase to enter S and G2-M phase, leading to shortened cell cycle.The second part:RT-PCR results showed the expression of HER2/neu mRNA was significantly upregulated in post-transfection cell line RMG-I-H, while the mRNA of EGFR was unchanged. Immunoprecipitation combined with Western blot showed the expression of EGFR protein in the transfected cells had no significant change, but the relative content of Lewis y on EGFR afterα1,2-FT gene transfection increased; in opposite to EGFR, although the expression of HER2/neu protein significantly increased afterα1,2-FT gene transfection, but the relative content of Lewis y on HER2/neu had no significant change. Western blot showed the phosphorylation level of EGFR, HER2/neu proteins were all significantly upregulated after transfection.The third part:Western blot showed the expression of Akt and ERK1/2 proteins were not obviously altered inα1,2-FT transfected cells, but the relative phosphorylation of Akt and ERK1/2 were all increased. When the cells were treated with EGFR tyrosine kinase specific inhibitor ZD1839 and anti-Lewis y antibody respectively, phosphorylation of both Akt and ERK1/2 were apparently decreased in non-andα1,2-FT transfected cells, especially theα1,2-FT transfected cells. By contrast, differences in phosphorylation intensity for Akt and ERK1/2 among non-andα1,2-FT transfected cell groups were attenuated in ZD1839 or anti-Lewis y antibody-treated cells. MTT assay results showed LY294002 inhibited the growth of Lewis y-overexpressing cells.Conclusions1. Lewis y antigen promotes the proliferation of the ovarian cancer cells, accelerates cell cycle progress.2. Lewis y antigen is not only an integral part of EGFR, but also a component of HER2/neu.3. Lewis y antigen as an integral part of EGFR family, its overexpression not only activates the tyrosine kinase of EGFR and HER2/neu, but also activates the downstream PI3K/Akt and Raf/MEK/MAPK signal transduction pathways of EGFR family, leads to accelerated gene transcription of HER2/neu in nucleus, enhances DNA synthesis and promotes cells in G0-G1 phase to enter S and G2-M phase, finally promotes the proliferation of the ovarian cancer cells.4. PI3K/Akt signaling pathway plays an important role in Lewis y antigen promoting cell proliferation. |
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