Lewis y antigen promotes the proliferation of ovarian carcinoma-derived RMG-I cells through the PI3K/Akt signaling pathway

Abstract

Background: Lewis y antigen is difucosylated oligosaccharide and is carried by glycoconjugates at cell surface. Elevated expression of Lewis y has been found in 75% of ovarian tumor, and the high expression level is correlated to the tumor's pathological staging and prognosis. This study was to investigate the effect and the possible mechanism of Lewis y on the proliferation of human ovarian cancer cells.

Methods: We constructed a plasmid encoding alpha1,2-fucosyltransferase (alpha1,2-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 alpha-L-fucosidase, anti-Lewis y antibody and phosphatidylinositol 3-kinase (PI3K) inhibitor treatment.

Results: Our results showed that the levels of alpha1,2-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 alpha-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 alpha1,2-FT were attenuated significantly by the monoantibody to Lewis y and by the PI3K inhibitor LY294002.

Conclusions: 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.

Figure 1

Characterization of α1,2-FT-transfected cell lines. (A) RT-PCR profiles of α1,2-FT mRNA in non- and α1,2-FT-transfected cells. M: DNA ladder marker (100-2000 bp). (B) Relative expression of α1,2-FT mRNA in non- and α1,2-FT-transfected cells (n = 3). The data was expressed as the intensity ratio of α1,2-FT to β-actin (Mean ± SD). * p < 0.01 compared to the control. "A" is the representative of three independent and reproducible experiments. (C) Immunohistochemical staining for Lewis y antigen. (a) RMG-I-H cells; (b) RMG-I-pcDNA3.1 cells; (c) RMG-I cells; (d) RMG-I-H-A cells; (e) RMG-I-A cells. Meanwhile, a, b and c represents cells without α-L-fucosidase treatmeant; d and e represents cells with α-L-fucosidase treatmeant.

Figure 2

The growth curves of each group of cells before and after the transfection.

Figure 3

Effects of α-L-fucosidase on the proliferation of the cells before and after the transfection. (A) The cell growth curves of each group before and after the process by α-L-fucosidase (B) The colony formation rates of each group before and after the process by α-L-fucosidase. *p < 0.01 compared to the control.

Figure 4

The cell growth curves of each group before and after the process by anti-Lewis y antibody.

Figure 5

The cell growth curves of each group before and after the process of LY294002.

Figure 6

PI3K/Akt signaling is required for Lewis y-enhanced growth of RMG-I cells. (A) Western blot profiles of Akt and p-Akt in non- and α1,2-FT transfected cells, as well as in the absence and presence of anti-Lewis y antibody and LY294002. (B) Densitometric quantification of protein expression of A (n = 3). (C) Western blot profiles of PCNA in non- and α1,2-FT transfected cells, as well as in the absence and presence of anti-Lewis y antibody and LY294002. (D) Densitometric quantification of protein expression of C (n = 3).* p < 0.01 compared to RMG-I. # p < 0.01 compared to RMG-I-H cells without anti-Lewis y antibody or LY294002 treatment. "A" and "C" are the representative of three independent and reproducible experiments.