Enhancive effects of Lewis y antigen on CD44-mediated adhesion and spreading of human ovarian cancer cell line RMG-I
Journal of Experimental & Clinical Cancer Research
Enhancive effects of Lewis y antigen on CD44-mediated adhesion and spreading of human ovarian cancer cell line RMG-I
Lili Gao 0
Limei Yan 0
Bei Lin 0
Jian Gao 0
Xiuyun Liang 0
Yanyan Wang 0
Juanjuan Liu 0
Shulan Zhang 0
Iwamori Masao 1
0 Department of Obstetrics and Gynecology, Shengjing Hospital Affiliated to China Medical University , Shenyang, 110004, P R of China
1 Departments of Biochemistry, Faculty of Science and Technology, Kinki University , Osaka, 577-8502 , Japan
Background: This 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. Methods: 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. Results: 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 coimmunoprecipitation. 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). Conclusion: Lewis y antigen strengthens CD44-mediated adhesion and spreading of ovarian cancer cells.
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Background
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 (a1, 2-FT)
is a key enzyme for synthesizing Lewis y antigen. In our
previous study, we successfully transferred a1, 2-FT
gene into ovarian cancer cell line RMG-I and established
a cell line RMG-I-H with stable high expression of
Lewis y antigen, which showed obviously enhanced
malignant behaviors [4-6].
CD44, one of important adhesive molecules on cells, is
involved in the adhesion and metastasis of tumor cells
and plays an important role in tumor development
[7-10], but the regulatory mechanism is unclear yet. The
molecule CD44 is abundant of a-L-fucose, and is an
important a1, 2-fucose antigen-containing protein on
the surface of cells [11]. CD44 is expressed on several
tissue cells, binds to receptors in extracellular matrix
such as hyaluronic acid (HA) and laminin, and mediates
cell-cell and cell-matrix adhesion [12,13]. The present
study aimed to determine the impact of a1, 2-FT gene
transfection on the expression of CD44 on cells and the
effects of Lewis y antigen on CD44-mediated cell
adhesion and spreading.
Methods
Materials
Lewis y monoclonal antibody was purchased from
Abcam Co.; CD44 monoclonal antibody from Santa
Cruz Co. and Wuhan Boster Co.; Protein A-agarose,
ECL chromogenic agent, and 5 SDS-PAGE loading
buffer from Shanghai Beyotime Institute of
Biotechnology; SABC kit from Beijing Zhongshan Golden Bridge
Biotechnology Co., Ltd; HA from Hefei Bomei
Biotechnology Co., Ltd; DMEM culture medium from Gibco
Co.; fetal bovine serum (FBS) from Shenyang Boermei
Reagent Co.; Coomassie brilliant blue from Beijing
Solarbio Science & Technology Co., Ltd; Trizol reagent,
PrimeScriptRT reagent kit, and SYBR Premix Ex
Taqfrom Dalian TaKaRa Biotechnology Co. The
sequences of primers were synthesized by Shanghai
Invitrogen Co.
Cell line and cell culture
The cell line RMG-I was originated from ovarian clear
cell cancer tissues. The cell line RMG-I-H with high
expression of a1, 2-FT and Lewis y antigen was
established in our lab [14]. RMG-I and RMG-I-H cells
were cultured in DMEM medium containing 10% FBS
at 37C in 5% CO2 and saturated humidity. Cells are
grouped in immunocytochemistry, c (...truncated)