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The Stimulation of IGF-1R Expression by Lewis(y) Antigen Provides a Powerful Development Mechanism of Epithelial Ovarian Carcinoma
Dawo Liu,Juanjuan Liu,Changzhi Wang,Bei Lin,Qing Liu,Yingying Hao,Shulan Zhang,Masao Iwamori
International Journal of Molecular Sciences , 2011, DOI: 10.3390/ijms12106781
Abstract: Objective: This study aimed to measure and correlate the expression of insulin-like growth factor receptor-1 (IGF-1R) and the Lewis(y) antigen in ovarian cancer cell lines and tissue samples. Methods: Reverse transcriptase PCR (RT-PCR), Western blotting, immunoprecipitation, immunohistochemistry, and immunofluorescence double-labeling techniques were applied to detect and measure the expression of Lewis(y) and IGF-1R. Results: In α1,2-fucosyltransferase (α1,2-FT)-transfected cells, IGF-1R expression was significantly upregulated compared with cells that do not overexpress α1,2-FT ( P < 0.05). The amount of Lewis(y) expressed on IGF-1R increased 1.81-fold in α1,2-FT-overexpressing cells ( P < 0.05), but the ratio of Lewis(y) expressed on IGF-1R to total IGF-1R was unaltered between two cells ( P > 0.05). In malignant epithelial ovarian tumors, the positivity rates of Lewis(y) and IGF-1R detection were 88.3% and 93.33%, respectively, which is higher than the positivity rates in marginal (60.00% and 63.33%, all P < 0.05), benign (33.00% and 53.33%, all P < 0.01), and normal (0% and 40%, all P 0.05). Both IGF-1R and Lewis(y) were highly expressed in ovarian cancer tissues, and their expression levels were positively correlated ( P < 0.05). Conclusion: Overexpression of Lewis(y) results in overexpression of IGF-1R. Both IGF-1R and Lewis(y) are associated with the occurrence and development of ovarian cancers.
Lewis y Regulate Cell Cycle Related Factors in Ovarian Carcinoma Cell RMG-I in Vitro via ERK and Akt Signaling Pathways
Dawo Liu,Juanjuan Liu,Bei Lin,Shuice Liu,Rui Hou,Yingying Hao,Qing Liu,Shulan Zhang,Masao Iwamori
International Journal of Molecular Sciences , 2012, DOI: 10.3390/ijms13010828
Abstract: Objective: To investigate the effect of Lewis y overexpression on the expression of proliferation-related factors in ovarian cancer cells. Methods: mRNA levels of cyclins, CDKs, and CKIs were measured in cells before and after transfection with the α1,2-fucosyltransferase gene by real-time PCR, and protein levels of cyclins, CDKs and CKIs were determined in cells before and after gene transfection by Western blot. Results: Lewis y overexpression led to an increase in both mRNA and protein expression levels of cyclin A, cyclin D1 and cyclin E in ovarian cancer cells, decrease in both mRNA and protein expression levels of p16 and p21, and decrease of p27 at only the protein expression level without change in its mRNA level. There were no differences in proteins and the mRNA levels of CDK2, CDK4 and CDK6 before and after gene transfection. Anti-Lewis y antibody, ERK and PI3K pathway inhibitors PD98059 and LY294002 reduced the difference in cyclin and CKI expression caused by Lewis y overexpression. Conclusion: Lewis y regulates the expression of cell cycle-related factors through ERK/MAPK and PI3K/Akt signaling pathways to promote cell proliferation.
Increase in Docetaxel-Resistance of Ovarian Carcinoma-Derived RMG-1 Cells with Enhanced Expression of Lewis Y Antigen
Fan Zhang,Juanjuan Liu,Bei Lin,Qing Liu,Yue Zhao,Liancheng Zhu,Yingying Hao,Shulan Zhang,Masao Iwamori
International Journal of Molecular Sciences , 2011, DOI: 10.3390/ijms12117323
Abstract: Epithelial carcinomas of the ovary exhibit the highest mortality rate among gynecologic malignancies. Studies found that the metabolism of glycolipids or carbohydrates is associated with acquirement of anticancer drug-resistance by cancer cells. This study was to characterize possible involvement of Lewis Y (Le Y) antigen in the drug-resistance of cancer cells. We transfected the α1,2-fucosyltransferase gene into human ovarian carcinoma-derived RMG-1 cells and established RMG-1-hFUT cells with enhanced expression of Le Y. We determined the effects of docetaxel on the survival of cells by MTT assaying and observed the apoptosis of cells in the presence of docetaxel by flow cytometric analysis and by transmission electron microscopy. Plasma membranes and intracellular granules in RMG-1-hFUT cells were stained with anti-Le Y antibody, the intensity of the staining was higher than that in control cells. The RMG-1-hFUT cells exhibited higher resistance to docetaxel than the control cells with regard to the docetaxel concentration and time course. After treatment with 10 μg/mL docetaxel for 72 h, the control cells, but not RMG-1-hFUT, contained abundant positively stained cell debris due to disintegration of the cytoskeleton. On transmission electron microscopy, although the control cells treated with docetaxel as above showed the following morphology, i.e., absence of villi, cells shrunken in size, pyknosis, agglutinated chromatin and cell buds containing nuclei in the process of apoptosis, the RMG-1-hFUT cells showed only agglutinated chromatin and vacuoles in the cytoplasm. In summary, cells with enhanced expression of Le Y were shown to acquire docetaxel-resistance, indicating the possible involvement of glycoconjugates in the drug-resistance.
Lewis (y) Antigen Overexpression Increases the Expression of MMP-2 and MMP-9 and Invasion of Human Ovarian Cancer Cells
Limei Yan,Bei Lin,Lili Gao,Song Gao,Chuan Liu,Changzhi Wang,Yifei Wang,Shulan Zhang,Masao Iwamori
International Journal of Molecular Sciences , 2010, DOI: 10.3390/ijms11114441
Abstract: 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.
Study on the Expression and Clinical Significances of Lewis y Antigen and Integrin αv, β3 in Epithelial Ovarian Tumors
Yifei Wang,Juanjuan Liu,Bei Lin,Changzhi Wang,Quanrong Li,Shuice Liu,Limei Yan,Shulan Zhang,Masao Iwamori
International Journal of Molecular Sciences , 2011, DOI: 10.3390/ijms12063409
Abstract: 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.
Enhancive effects of Lewis y antigen on CD44-mediated adhesion and spreading of human ovarian cancer cell line RMG-I
Lili Gao, Limei Yan, Bei Lin, Jian Gao, Xiuyun Liang, Yanyan Wang, Juanjuan Liu, Shulan Zhang, Iwamori Masao
Journal of Experimental & Clinical Cancer Research , 2011, DOI: 10.1186/1756-9966-30-15
Abstract: The 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.Immunocytochemistry 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).Lewis y antigen strengthens CD44-mediated adhesion and spreading of ovarian cancer cells.Glycosylated antigens, important components of glycolipids and glycoproteins, are widely expressed on cell membrane and are involved in cell adhesion, recognition, and signal transduction [1]. The alterations of type II sugar chains, such as Lewis × and Lewis y, are common in ovarian cancer: 75% of epithelial ovarian cancers have overexpression of Lewis y antigen which shows obvious relationship with prognosis; tumor marker CA125 in epithelial ovarian cancer also contains Lewis y structure [2,3]. Alpha1, 2-fucosyltransferase (α1, 2-FT) is a key e
Lewis y antigen promotes the proliferation of ovarian carcinoma-derived RMG-I cells through the PI3K/Akt signaling pathway
Juanjuan Liu, Bei Lin, Yingying Hao, Yue Qi, Liancheng Zhu, Feifei Li, Dawo Liu, Jianping Cong, Shulan Zhang, Masao Iwamori
Journal of Experimental & Clinical Cancer Research , 2009, DOI: 10.1186/1756-9966-28-154
Abstract: We constructed a plasmid encoding α1,2-fucosyltransferase (α1,2-FT) gene and then transfected it into ovarian carcinoma-derived RMG-I cells with lowest Lewis y antigen expression level. Effect of Lewis y on cell proliferation was assessed after transfection. Changes in cell survival and signal transduction were evaluated after α-L-fucosidase, anti-Lewis y antibody and phosphatidylinositol 3-kinase (PI3K) inhibitor treatment.Our results showed that the levels of α1,2-FT gene and Lewis y increased significantly after transfection. The cell proliferation of ovarian carcinoma-derived RMG-I cells sped up as the Lewis y antigen was increased. Both of α-L-fucosidase and anti-Lewis y antibody inhibited the cell proliferation. The phosphorylation level of Akt was apparently elevated in Lewis y-overexpressing cells and the inhibitor of PI3K, LY294002, dramatically inhibited the growth of Lewis y-overexpressing cells. In addition, the phosphorylation intensity and difference in phosphorylation intensity between cells with different expression of α1,2-FT were attenuated significantly by the monoantibody to Lewis y and by the PI3K inhibitor LY294002.Increased expression of Lewis y antigen plays an important role in promoting cell proliferation through activating PI3K/Akt signaling pathway in ovarian carcinoma-derived RMG-I cells. Inhibition of Lewis y expression may provide a new therapeutic approach for Lewis y positive ovarian cancer.Lewis y antigen is carried by glycoconjugates (glycoproteins and glycolipids) at cell surface. It is an oligosaccharide with two fucoses, and its chemical structure is Fucα1 → 2Galβ1 → 4 [Fucα1 → 3]GlcNAcβ1 → R, belonging to the A, B, H, Lewis blood group antigens family with specific fucosylation of the terminal end of carbohydrate structure catalyzed by the α1,2-fucosyltransferase [1,2]. The expression of Lewis y antigen primarily occurs during the embryogenesis period. Under physiologic conditions, its expression in adults is limited on the sur
A Piezoelectric Sensor-Based System for Objective Analyzing of the Preparation of Fluid Foods  [PDF]
Hajime Iwamori, Ichiro Ashida, Yozo Miyaoka
Journal of Sensor Technology (JST) , 2014, DOI: 10.4236/jst.2014.43014
A monitoring system using a piezoelectric sensor was developed for the quantitative analysis of the stirring process in which a mixture of a thickener with water was prepared. A thin piezoelectric sensor film was attached to the inside of a glass container in which the mixture was stirred and connected to a recorder. Seven healthy young participants were asked to conduct stirring for 90 s at three cycles per second (3 Hz). A triphasic wave with a large upward peak was recorded in each cycle of stirring. The average period across the seven participants was 0.337 s (n = 613 cycles), and statistical analysis revealed no significant difference between the average value and the expected value of 0.333 s. Video images captured during stirring were analyzed, and the analysis showed that the peak of the large upward wave appeared approximately 0.12 s (approximately 36% of the average period) after the time when the spoon head for stirring passed near the piezoelectric sensor. The monitoring system developed in this study allows the precise temporal analysis of stirring for the preparation of a mixture.
Characterization of Spermatogonial Stem Cells Lacking Intercellular Bridges and Genetic Replacement of a Mutation in Spermatogonial Stem Cells
Naoki Iwamori, Tokuko Iwamori, Martin M. Matzuk
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0038914
Abstract: Stem cells have a potential of gene therapy for regenerative medicine. Among various stem cells, spermatogonial stem cells have a unique characteristic in which neighboring cells can be connected by intercellular bridges. However, the roles of intercellular bridges for stem cell self-renewal, differentiation, and proliferation remain to be elucidated. Here, we show not only the characteristics of testis-expressed gene 14 (TEX14) null spermatogonial stem cells lacking intercellular bridges but also a trial application of genetic correction of a mutation in spermatogonial stem cells as a model for future gene therapy. In TEX14 null testes, some genes important for undifferentiated spermatogonia as well as some differentiation-related genes were activated. TEX14 null spermatogonial stem cells, surprisingly, could form chain-like structures even though they do not form stable intercellular bridges. TEX14 null spermatogonial stem cells in culture possessed both characteristics of undifferentiated and differentiated spermatogonia. Long-term culture of TEX14 null spermatogonial stem cells could not be established likely secondary to up-regulation of CDK4 inhibitors and down-regulation of cyclin E. These results suggest that intercellular bridges are essential for both maintenance of spermatogonial stem cells and their proliferation. Lastly, a mutation in Tex14+/? spermatogonial stem cells was successfully replaced by homologous recombination in vitro. Our study provides a therapeutic potential of spermatogonial stem cells for reproductive medicine if they can be cultured long-term.
H3K27 Demethylase, JMJD3, Regulates Fragmentation of Spermatogonial Cysts
Naoki Iwamori, Tokuko Iwamori, Martin M. Matzuk
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0072689
Abstract: The spermatogonial stem cell (SSC) compartment is maintained by self-renewal of stem cells as well as fragmentation of differentiating spermatogonia through abscission of intercellular bridges in a random and stochastic manner. The molecular mechanisms that regulate this reversible developmental lineage remain to be elucidated. Here, we show that histone H3K27 demethylase, JMJD3 (KDM6B), regulates the fragmentation of spermatogonial cysts. Down-regulation of Jmjd3 in SSCs promotes an increase in undifferentiated spermatogonia but does not affect their differentiation. Germ cell-specific Jmjd3 null male mice have larger testes and sire offspring for a longer period compared to controls, likely secondary to increased and prolonged maintenance of the spermatogonial compartment. Moreover, JMJD3 deficiency induces frequent fragmentation of spermatogonial cysts by abscission of intercellular bridges. These results suggest that JMJD3 controls the spermatogonial compartment through the regulation of fragmentation of spermatogonial cysts and this mechanism may be involved in maintenance of diverse stem cell niches.
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