Limei Yan

Enhancement of the adhesive and spreading potentials of ovarian carcinoma RMG-1 cells due to increased expression of integrin α5β1 with the Lewis Y-structure on transfection of the α1,2-fucosyltransferase gene

Le(Y) antigen is known to be associated with malignant properties including metastasis and a poor prognosis of ovarian carcinomas. To clarify the mechanisms underling these properties, we established ovarian carcinoma-derived cells exhibiting enhanced expression of Le(Y) by transfection with alpha1,2-fucosyltransferase and compared their cellular properties with those of the original cells. So the human alpha1,2-fucosyltransferase gene was transfected into ovarian carcinoma-derived RMG-1 cells, which are known to contain Le(X), a precursor of Le(Y), and RMG-1-hFUT cells exhibiting enhanced expression of Le(Y) were established by selection with anti-Le(Y) antibodies, and their adhesive and spreading potentials on fibronectin-coated plates were compared with those of RMG-1 cells. Results showed that the relative expression of Le(Y) in RMG-1-hFUT cells was about 20-fold that in RMG-1 cells, and that of integrin alpha5beta1 and an integrin-mediated signal transduction molecule, focal adhesion kinase, was also increased in RMG-1-hFUT cells. Interestingly, anti-Le(Y) antibodies were revealed to immunoprecipitate integrin alpha5beta1, indicating that its oligosaccharides are composed of Le(Y), the amounts of which was substantially elevated in RMG-1-hFUT cells. The adhesion and spreading potentials on fibronectin-coated plates of RMG-1-hFUT cells were significantly enhanced in comparison to those of RMG-1 cells, and were greatly suppressed by anti-Le(Y) antibodies, indicating that Le(Y) is involved in the integrin-fibronectin interaction. These results suggested that transfection of the alpha1,2-fucosyltransferase gene into ovarian carcinoma-derived cells brought about elevated expression of integrin alpha5beta1 with Le(Y), resulting in enhancement of the adhesion and spreading potentials of cells through the integrin-fibronection interaction, which was inhibited by anti-Le(Y) antibodies. Thus, Le(Y) in integrin alpha5beta1 was thought to be involved in the enhanced cell adhesion properties of malignant ovarian carcinomas.

Enhancive effects of Lewis y antigen on CD44-mediated adhesion and spreading of human ovarian cancer cell line RMG-I

BackgroundThis study aimed to investigate the molecular structural relationship between cell adhesive molecule CD44 and Lewis y antigen, and determine the effects of Lewis y antigen on CD44-mediated adhesion and spreading of ovarian cancer cell line RMG-I and the Lewis y antigen-overexpressed cell line RMG-I-H.MethodsThe expression of CD44 in RMG-I and RMG-I-H cells before and after treatment of Lewis y monoclonal antibody was detected by immunocytochemistry; the expression of Lewis y antigen and CD44 was detected by Western Blot. The structural relationship between Lewis y antigen and CD44 was determined by immunoprecipitation and confocal laser scanning microscopy. The adhesion and spreading of RMG-I and RMG-I-H cells on hyaluronic acid (HA) were observed. The expression of CD44 mRNA in RMG-I and RMG-I-H cells was detected by real-time RT-PCR.ResultsImmunocytochemistry revealed that the expression of CD44 was significantly higher in RMG-I-H cells than in RMG-I cells (P < 0.01), and its expression in both cell lines was significantly decreased after treatment of Lewis y monoclonal antibody (both P < 0.01). Western Blot confirmed that the content of CD44 in RMG-I-H cells was 1.46 times of that in RMG-I cells. The co-location of Lewis y antigen and CD44 was confirmed by co-immunoprecipitation. The co-expression of CD44 and Lewis y antigen in RMG-I-H cells was 2.24 times of that in RMG-I cells. The adhesion and spreading of RMG-I-H cells on HA were significantly enhanced as compared to those of RMG-I cells (P < 0.01), and this enhancement was inhibited by Lewis y monoclonal antibody (P < 0.01). The mRNA level of CD44 in both cell lines was similar (P > 0.05).ConclusionLewis y antigen strengthens CD44-mediated adhesion and spreading of ovarian cancer cells.

Lewis (y) antigen overexpression increases the expression of MMP-2 and MMP-9 and invasion of human ovarian cancer cells.

Lewis (y) antigen is a difucosylated oligosaccharide present on the plasma membrane, and its overexpression is frequently found in human cancers and has been shown to be associated with poor prognosis. Our previous studies have shown that Lewis (y) antigen plays a positive role in the process of invasion and metastasis of ovarian cancer cells. However, the mechanisms by which Lewis (y) antigen enhances the invasion and tumor metastasis are still unknown. In this study, we established a stable cell line constitutively expressing Lewis (y) antigen (RMG-1-hFUT) by transfecting the cDNA encoding part of the human α1,2-fucosyltransferase (α1,2-FUT) gene into the ovarian cancer cell line RMG-1, and investigated whether Lewis (y) antigen regulates the expression of matrix metalloproteinase-2 (MMP-2) and MMP-9, and tissue inhibitors of metalloproteinases (TIMP-1) and TIMP-2. We found that RMG-1-hFUT cells exhibited higher invasive capacities than their control cells. In addition, expression of TIMP-1 and TIMP-2 was down-regulated and expression of MMP-2 and MMP-9 was up-regulated. Anti-Lewis (y) antigen antibody treatment significantly reversed the expression of TIMP-1, TIMP-2, MMP-2 and MMP-9. Taken together, we provide the first evidence that down-regulation of TIMP-1 and TIMP-2 and up-regulation of MMP-2 and MMP-9 represents one of the mechanisms by which Lewis (y) antigen promotes cell invasion.

High Expression of Lewis y Antigen and CD44 Is Correlated with Resistance to Chemotherapy in Epithelial Ovarian Cancers

Objectives To measure Lewis y antigen and CD44 antigen expression in epithelial ovarian carcinoma and to correlate the levels of these antigens with clinical response to chemotherapy. Methods The study cases included 34 cases of ovarian carcinoma with resistance to chemotherapeutic drugs, 6 partially drug-sensitive cases, and 52 drug-sensitive cases (92 total). Results The rates of expression of Lewis y antigen and CD44 antigen were significantly greater in the drug-resistant group than that in the partially-sensitive or sensitive groups. Surgical stage, residual tumor size and expression of CD44 and Lewis y antigen in ovarian carcinoma tissues were independent risk factors for chemotherapeutic drug resistance. Conclusions Over-expression of Lewis y and CD44 antigen are strong risk factors for chemotherapeutic drug resistance in ovarian carcinoma patients.

Study on the expression and clinical significances of lewis y antigen and integrin αv, β3 in epithelial ovarian tumors.

Objective To detect the expression and clinical significances of Lewis y antigen and integrin αv, β3 in epithelial ovarian tumors, and to explore the expression correlation between Lewis y antigen and integrin αv, β3. Methods Immunohistochemical staining was performed in 95 cases of epithelial ovarian cancer, 37 cases of borderline tumors, 20 cases of benign tumors, and 20 cases of normal ovarian tissue, for the detection of Lewis y antigen and integrin αv, β3 expressions, and to analyze the relationship between Lewis y antigen and integrin, and the relationship between clinical and pathological parameters of ovarian cancer. In addition, immunofluorescence double labeling was utilized to detect the expression correlation between Lewis y antigen and integrin αv, β3 in ovarian cancer. Results In epithelial ovarian tumors, the expression rate of Lewis y antigen was 81.05%, significantly higher than that of borderline (51.53%) (P < 0.05) and benign (25%) (P < 0.01) tumors, and normal ovarian tissues (0) (P < 0.01). The expression rate of integrin αv, β3 in malignant epithelial ovarian tumors was 78.95% and 82.11%, respectively, significantly higher than that of the borderline (45.94%, 40.54%) (both P < 0.05), benign group (10.00%, 15.00%) (both P < 0.01) and normal ovary group (5%, 15%) (both P < 0.01). Conclusions Lewis y and integrins αv, β3 are relevant to pelvic and abdominal diffusion and metastasis of ovarian cancer cells, suggesting that these two molecules mediate a boosting function for tumor metastasis.

Overexpression of Lewis (y) Antigen Protects Ovarian Cancer RMG‐1 Cells from Carboplatin‐Induced Apoptosis by the Upregulation of Topo‐I and Topo‐II β

Lewis (y) antigen, a difucosylated oligosaccharide, has been shown to be associated with malignant properties of ovarian carcinomas. In this study, we have investigated the potential role of Lewis (y) antigen, which was stably transfected into ovarian cancer RMG‐1 cells, on carboplatin‐induced apoptosis. Overexpression of Lewis (y) antigen effectively protected vitronectin‐adherent RMG‐1 cells from carboplatin‐induced apoptosis as assessed by Hoechst 33258 staining and flow cytometry. Treatment with anti‐Lewis (y) antigen, anti‐integrin αv, or anti‐integrin β3 antibody partially abolished the protective effect on apoptosis and markedly inhibited the expression of Topo‐II β in cells overexpressing Lewis (y) antigen (all P < 0.01). Moreover, elevated expression of Topo‐I and Topo‐II β was found in Lewis (y) antigen‐overexpressing cells (P < 0.01). However, no obvious changes in Topo‐II α were observed throughout the study (P > 0.05). Taken together, these data suggest that the overexpression of Lewis (y) antigen confers cell adhesion‐mediated drug resistance to apoptosis in ovarian cancer cells by the upregulation of Topo‐I and Topo‐II β. Therefore, the inhibition of Lewis (y) antigen may be a novel strategy of cancer chemotherapy. Anat Rec, 2011.

Lewis y enhances CAM-DR in ovarian cancer cells by activating the FAK signaling pathway and upregulating Bcl-2/Bcl-XL expression

Oligosaccharides on the surface of adhesion molecules may contribute to the process of CAM-DR. To investigate the role of the Lewis y antigen in this process, we established a cell adhesion model mediated by the integrin α5β1-FN interaction in the ovarian cancer cell line, RMG-1-hFUT, which highly expresses Lewis y by transfection with α1,2-fucosyltransferase into RMG-1 cells. Our results indicate that the rates of carboplatin-induced apoptosis and necrosis are reduced in FN-adhered tumor cells, and carboplatin resistance is significantly decreased in the presence of anti-Lewis y antibody. CAM-DR in tumor cells has been correlated with elevated expression of the nuclear anti-apoptotic proteins Bcl-2 and Bcl-XL. Lewis y promotes the expression of the Bcl-2 and Bcl-XL genes by activating the focal adhesion kinase signaling pathway and accelerating their transcription. Thus, Lewis y leads to inhibition of apoptosis and enhancement of CAM-DR by activation of the FAK signaling pathway and upregulation of Bcl-2/Bcl-XL expression in ovarian cancer cell lines.

c-Jun transcriptionally regulates alpha 1, 2-fucosyltransferase 1 (FUT1) in ovarian cancer

Alpha 1, 2-fucosyltransferase (FUT 1/2) is a rate-limiting enzyme that catalyzes the synthesis of Lewis y, a cell membrane-associated carbohydrate antigen. In human ovarian cancer, the upregulated expression of FUT1 and Lewis y is associated with advanced pathological stages and involved in cell proliferation, migration and invasion. However, the mechanism underlying the upregulation of FUT1 is largely unknown. Here, we identify an AP-1 binding site in FUT1 promoter in ovarian cancer cells. c-Jun promotes FUT1 expression, thereby enhancing Lewis y biosynthesis in various ovarian cancer cell lines. Moreover, EMSA, luciferase activity and ChIP assays demonstrate c-Jun directly interacts with FUT1 promoter. Furthermore, FUT1 mediates c-Jun-induced cell proliferation in ovarian cancer cells. In human ovarian cancer samples, c-Jun overexpression is linked to malignant degree and positively correlated to FUT1 and Lewis y expression. Taken together, c-Jun could transcriptionally modulate FUT1 expression in ovarian cancer, implicating the potential application of c-Jun inhibitors for human ovarian cancer therapy.

c-Fos mediates α1, 2-fucosyltransferase 1 and Lewis y expression in response to TGF-β1 in ovarian cancer

FUT1 is a key rate-limiting enzyme in the synthesis of Lewis y, a membrane-associated carbohydrate antigen. The aberrant upregulation of FUT1 and Lewis y antigen is related to proliferation, invasion and prognosis in malignant epithelial tumors. A c-Fos/activator protein-1 (AP-1) binding site was found in the FUT1 promoter. However, the mechanisms of transcriptional regulation of FUT1 remain poorly understood. TGF-β1 is positively correlated to Lewis y. In the present study, we investigated the molecular mechanism of FUT1 gene expression in response to TGF-β1. We demonstrated that c-Fos was highly expressed in 77.50% of ovarian epithelial carcinoma cases and was significantly correlated with Lewis y. Using luciferase activity and chromatin immunoprecipitation (ChIP) assay, we further revealed that c-Fos interacted with the FUT1 promoter in ovarian cancer cells and transcriptional capacity of the heterodimer formed by c-Fos and c-Jun was stronger than that of the c-Fos or c-Jun homodimers. Then, we demonstrated that TGF-β1 induced dose-dependent c-Fos expression, which was involved in TGF-β1-induced ovarian cancer cell proliferation. In addition, inhibition of MAPK activation or TGF-β1 receptor by pharmacological agents prevented TGF-β1-induced c-Fos and Lewis y expression. Silencing of c-Fos prevented TGF-β1-induced Lewis y expression. Collectively, the results of these studies demonstrated that TGF-β1 regulated FUT1 and Lewis y expression by activating the MAPK/c-Fos pathway.