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

Abstract

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 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).

Conclusion: Lewis y antigen strengthens CD44-mediated adhesion and spreading of ovarian cancer cells.

Figure 1

The expression of CD44 in RMG-I and RMG-I-H cells detected by immunocytochemistry (×400). Panels 1 and 5 are negative controls; panels 2 and 6 are Lewis y antibody-untreated cells; panels 3 and 7 are Lewis y antibody-treated cells; panels 4 and 8 are cells treated by irrelevant isotype-matched control. The expression of CD44 was detected by SABC methods in RMG-I and RMG-I-H cells, and brown color degree by DAB staining indicated the expression level of CD44. It can be seen from the figure that the expression of CD44 in the RMG-I-H cells was stronger than that in RMG-I cells, which was decreased after Lewis y antibody blocking.

Figure 2

Co-location of CD44 and Lewis y antigen on RMG-I-H cells observed under confocal laser scanning microscope. Red fluoscence on the upper left panel indicates CD44 expression; green fluoscence on the upper right panel indicates Lewis y antigen expression; blue fluoscence on the upper right panel indicates cell nuclear location; the lower right panel is a merged image of the other three panels. Lewis y antigen CD44 mainly expressed in the cell membrane observed under the confocal laser scanning microscope, and it were seen as yellow fluorescence after the two overlap, suggesting that Lewis y antigen and CD44 co-localizated in the cell membrane.

Figure 3

The expression of CD44 and Lewis y antigen in RMG-I and RMG-I-H cells. Panel A shows the expression of Lewis y antigen in RMG-I-H cells was higher than that in RMG-I; panel B shows the expression of CD44 in RMG-I-H cells was higher than that in RMG-I; panel C shows that Lewis y antigen, which in RMG-I-H cells was higher than that in RMG-I, was expressed both in RMG-I and RMG-I-H cells after CD44 immunoprecipitation; panel D Quantitative data were expressed as the intensity ratio target genes to beta-actin. (P < 0.01)

Figure 4

The mRNA expression of CD44 and α1, 2-FT in RMG-I and RMG-I-H cells were tested by quantitative Real-Time RT-PCR. The mRNA level of α1, 2-FT was significantly increased, but the mRNA level of CD44 was almost the same in RMG-1-hFUT cells and RMG-1 cells. (**P < 0.01, * P > 0.05).