Transforming Growth Factor β Pathway Mediated By Lewis Y Antigen Promotes Cell Proliferation In Ovarian Carcinoma-derived RMG-ⅠCells

IntroductionOvarian cancer is the most lethal gynecologic malignancy. The occurrence and development of ovarian cancer is an extremely complex multi-factor process, and the mechanism has always been the key that basis and clinical research of gynecologic oncology. With a detailed study, it was found that, in addition to DNA and proteins play a vital role in complex cell activity, carbohydrate also plays an important role. The outside of membrane lipid bilayer is almost entirely covered by the sugar chain of some glycoproteins or glycolipids, forming a layer of sugar. These glycoproteins embedded in or connected to the cell membrane in different ways. The exposed part of the sugar chains play an important part in recognition of cell-cell, cell-molecular, take participation in ligand binding, signal transduction, adhesion molecules and other molecules, and are closely related to cell growth, apoptosis, sports, differentiation and other important life processes. The sugar chains are known as the messengers. Reports show that the malignant behaviors of tumors (proliferation, invasion, metastasis, etc.) are closely related to the composition and structure of cell surface glycoconjugates. Structural abnormalities of sugar chains on cell surface in tumors may be recognised as non-self foreign matter by human immune system, thus was designated the tumor-associated carbohydrate antigens (TACA). TACA on cell surface are more abundant than any other protein antigens. Lewis y antigen is a TACA, extensive attention is received in recent years by researchers.Lewis y antigen is high-expressed in many malignant tumors and closely linked to the pathological staging and prognosis. However, the role of Lewis y antigen in ovarian cancer occurrence and development is not yet clear. Proliferate capacity can effectively reflect the malignant degree, which is an important tool to understand the biological behavior of tumors. We established previously a stably Lewis y high expression cell line RMG-I-H by transfection ofα1,2-FT (al,2-fucosyltransferase) gene, key enzyme in synthesis of Lewis y antigen, into Lewis y low-expression ovarian cancer cell line RMG-I. The establishment of Lewis y antigen high expression cell line RMG-I-H provides a good tool to investigate the effect of high expression of Lewis y on RMG-I cell proliferation and the mechanism.This thesis was divided into three parts:Part 1. The Correlation between Lewis y antigen and TGF-P and its receptors in ovarian cancer cells RMG-IObjectiveTo study the effects of high-expression of Lewis y antigen on ovarian cancer cells RMG-I in nude mice and the mechanisms of Lewis y antigen promoting ovarian cancer cell growth and proliferation. The Lewis y antigen closely related growth factor superfamily member transforming growth factor-P (TGF-P) was choosed to explore the effect of Lewis y on the expression of the growth factors and to further explore the impacts of TGF-β1 on the proliferation ability of cells before and afterα1,2-FT gene transfection and the expression of Lewis y antigen in TGF-βreceptors.MethodsA xenograft model was used to determine the effect of LeY on tumorigenecity in vivo. Using immunohistochemistry assay to detect the expression of TGF-β1 in nude mice transplanted tumor tissue. MTT assay was used to determine the effect of TGF-β1 on the proliferation capacity of cells before and after transfection. Fluorescence quantitative Real Time-PCR was used to to detect expression of TGF-β1, TPRI and TβRⅡin transplanted tumor tissue. Immunoprecipitation and western blot was used to detect the expression of Lewis y in TβRⅠand TβRⅡ. Using the laser confocal microscope to observe the co-localization of TPRⅠ(or TβRⅡ) and Lewis y.Results1. When subcutaneously inoculated in nude mice, RMG-I-H cells produced large tumors, while RMG-I-C and RMG-I cells produced small tumors. However, the tumor formation by RMG-I-H cells was significantly inhibited by LeY antibdoy. 2. Lewis y upregulated the expression of TGF-β1 in nude mice transplanted tumor tissue.3. TGF-β1 promoted the proliferation of RMG-I-H cells in a dose-dependent way, while the extent of TGF-β1 on proliferation was significantly lower than the corresponding concentration of RMG-I-H group in the two control groups.4. Lewis y did not change the TβRⅠexpression, while upregulate the TβRⅡexpression.5. Immunoprecipitation assay showed that TβRⅠand TβRⅡboth contain Lewis y structure, and expression of Lewis y in TβRⅠand TβRⅡon the cell surface increased significantly afteral,2-FT gene transfection.6. Using laser confocal microscope revealed the co-localization of TβRⅠ(Ⅱ) and Lewis y in RMG-I-H cell surface.ConclusionLeY enhances tumorigenicity of RMG-I cells. Lewis y upregulate the expression of TGF-β1, and TGF-β1 promote the growth and proliferation of ovarian cancer RMG-I-H cells.TGF-βreceptor, TβRⅠand TβRⅡ, do contain Lewis y structure, and compared to non-transfected group and empty vector transfected group, Lewis y antigen expression in TβRⅠand TPRⅡwas increased in RMG-I-H cells.Part 2. Effect of Lewis y antigen on the important signaling pathway mediated by TGF-βin ovarian cancer cellsObjectiveWe found in the second part thatα1,2-FT gene transfection changed the expression of Lewis y antigen in TβRⅠand TβRⅡon the surface of ovarian cancer RMG-I cells, then would the structural changes of TβRⅠand TβRⅡon cell surface can further lead to the expression of downstream signaling molecules or activation? This is the point of this study.Methods Western blot analysis was used to test the expression and phosphorylation level of downstream signal elements of TGF-P receptors, Smad2/3, Smad7, Akt1/2/3, MEK1/2 and ERK1/2, and the changes of the phosphorylation level of Smad2/3, Akt1/2/and ERK1/2 after handling different times (1min lOmin 30min) with Lewis y antibody.Results1. Lewis y did not change the expression of Smad2/3. However, the expression of p-Smad2/3 in RMG-I-H cells significantly down-regulated, the expression of Smad7 in RMG-I-H cells significantly up-regulated.2. TGF-β1 can activate ERK and PI3K signaling pathways in ovarian cancer RMG-I-H cells.3. Lewis y did not change the total expression of ERK1/2 and MEK1/2. However, p-ERKl/2 and p-MEK1/2 in RMG-I-H group was significantly higher in RMG-I-H cells than that in RMG-I and RMG-I-C cells.4. Lewis y did not change the expression of Akt1/2/3. However the p-Akt1/2/3 expression in RMG-I-H cells was significantly higher than RMG-I and RMG-I-C cells.5. Expression of p-ERK1/2, p-Akt1/2/3 and p-Smad2/3 reduced with the extension of the processing time of Lewis y monoclonal antibody treatment.ConclusionLewis y promotes cell proliferation probably due to involvement in regulating the Smad, ERK and PI3K pathways mediated through the TGF-βreceptors.