A Possible Inhibitory Role of Sialic Acid on MUC1 in Peritoneal Dissemination of Clear Cell-Type Ovarian Cancer Cells

Abstract

The role of sialic acids on MUC1 in peritoneal dissemination of ovarian cancer cells was investigated. A human ovarian carcinoma cell line, ES-2, was transfected with full-length MUC1 containing 22 or 42 tandem repeats. These transfectants were less adherent to monolayers of patient-derived mesothelial cells than ES-2/mock transfectants. When these cells were inoculated into the abdominal cavity of female nude mice, mice that had received the transfectants showed better survival. When the transfectants were mixed with sialidase and injected, the survival was poorer, whereas when they were mixed with N-acetyl-2,3-dehydro-2-deoxyneuraminic acid, a sialidase inhibitor, the survival was significantly prolonged. These behaviors, concerned with peritoneal implantation and dissemination observed in vitro and in vivo, were dependent on the expression of MUC1. Therefore, sialic acid linked to MUC1 in the form, at least in part, of sialyl-T, as shown to be recognized by monoclonal antibody MY.1E12, is responsible for the suppression of adhesion of these cells to mesothelial cells and the suppression of peritoneal implantation and dissemination.

Keywords: mucins; neuraminidase; ovarian neoplasms; peritoneal dissemination; sialic acid.

Figure 1

Adhesion of ES-2/mock cells, ES-2/T-22 cells, and ES-2/T-42 cells to peritoneal mesothelial cells. The Y-axis indicates the number of cells attached to the mesothelial cell monolayer 30 min after seeding. Data shown are mean ± SEM. Kruskal-Wallis rank sum test. p < 0.05.

Figure 2

Patterns of dissemination of ES-2/mock cells and ES-2/T-42 cells in vivo. Nude mice were inoculated with ES-2/mock cells, ES-2/T-22 cells, or ES-2/T-42 cells. (A) Macroscopic and intraperitoneal findings of nude mice inoculated with ES-2/mock cells. Small red arrows indicate the position of tumors. (B) Macroscopic and intraperitoneal findings of nude mice inoculated with ES-2/T-42 cells. The big red arrow indicates a bloated abdomen due to accumulated ascites. (C) Tumor burden in mice intraperitoneally injected with ES-2/mock or ES-2/T-42 cells three weeks after cell inoculation. Mesentery, omentum, peritoneum, ovary, and uterus of the mice were resected together with implanted tumors, and their cumulative weights were measured. Each dot represents one mouse, and mean ± SEM are shown. Unpaired Student’s t-test. * p < 0.05.

Figure 3

Effect of sialidase treatment of ES-2/mock, ES-2/T-22, and ES-2/T-42 cells on their adhesion to mesothelial cells. Data shown are mean ± SEM. Mann-Whitney U-test. * p < 0.05.

Figure 4

Kaplan-Meier survival curves of nude mice after abdominal inoculation with ES-2/T-42 cells alone (black dashed line), ES-2/T-42 cells together with sialidase (red solid line), and ES-2/T-42 cells together with N-acetyl-2,3-dehydro-2-deoxyneuraminic acid (NADNA), a sialidase inhibitor, (dotted blue line). Ten mice were used in each group. Kaplan-Meier method log rank (Mantel-Cox) test. p < 0.0001.

Figure 5

Effect of sialic acids expressed on the surface of peritoneal mesothelial cells on the binding of MUC1 transfected ovarian cancer cells. (A) Bindings of lectins specific for sialylated carbohydrate chains and antibodies specific for sialylated epitopes of MUC1 to the cell surfaces of peritoneal mesothelial cells derived from ovarian cancer patients. (A1) Maackia amurensis leucoagglutinin (specific for α2,3-linked sialic acid), (A2) Sambucus sieboldiana agglutinin (specific for α2,6-linked sialic acid), (A3) mAb KM93 (specific for sialyl Lewis X), and (A4) mAb MY.1E12 (specific for sialyl-T MUC1). Flow cytometric analysis. No shading: negative control (secondary antibody only); black shading: antibody/lectin bound cells. (B) Effect of sialidase treatment of mesothelial cells on the adhesion of ES-2/mock, ES-2/T-22, and ES-2/T-42 cells to the peritoneal mesothelial cell layer. Data shown are mean ± SEM. Mann-Whitney U-test. * p < 0.05.