2318

Introduction: Mucins are heavily O-glycosylated glycoproteins expressed on apical surface of normal epithelial cells. In cancer cells mucins are overexpressed along the entire cell surface. Clinical studies show a direct correlation between overexpression of MUC1 mucin and poor survival rates of pancreatic cancer patients. Treatment of pancreatic cancer with chemotherapy (involving single agents or combination) has shown limited clinical success. The infusion of 5-fluorouracil (5FU) along with radiation has been the mainstay in treatment of pancreatic cancer. In this study we seek to investigate whether the inhibition of MUC1 O-glycosylation in human pancreatic cancer cells can enhance the antiproliferative activity of 5FU.

Methods: The human pancreatic cancer cell lines Capan-1, HPAF-II and brain cancer cell line U87mg were maintained in their respective growth media. MUC1 mucin expression levels were determined using a combination of fluorescence and DIC microscopy. Nontoxic concentration of benzyl-α-GalNAc (inhibitor of O-glycosylation) was determined 72 hrs following exposure of cells to reagent. The effect of inhibiting O-glycosylation on antiproliferative activity of 5FU was determined by exposing cells to benzyl-α-GalNAc for 48 hrs followed by 5FU treatment. Other studies include detection of relative amounts of MUC1 mucin present on the cell surface using CD227-FITC antibody, and Sulforhodamine B cytotoxicity studies using a fluorescence plate reader.

Results: Capan-1 showed a higher level of MUC1 expression compared to HPAF-II cells. The exposure of Capan-1 and HPAF-II cells to benzyl-α-GalNAc for 72 hrs revealed a maximum non-toxic concentration of 0.4 and 0.8 mg/ml respectively. When Capan-1 and HPAF-II cells were exposed to benzyl-α-GalNAc for 24, 48, or 72 hrs we observed higher association of CD227 with cells compared to control (*P≤0.001). Normal cell proliferation and morphology was not altered 24, 48 and 72 hrs post exposure to benzyl-α-GalNAc. Exposure of cells to benzyl-α-GalNAc prior to 5-FU treatment resulted in enhanced antiproliferative activity of cytotoxic agent. We observed a decrease in cell viability from 66 to 53% (*P<0.01) for Capan-1, and from 82 to 11% (*P<0.01) for HPAF-II cells. In comparison, the effect of 5-FU against U87mg (negative control) was 37% both in the presence and absence of inhibitor.

Conclusions: Our studies show high levels of MUC1 expression on Capan-1 cells compared to moderate levels on HPAF-II cells. Inhibition of MUC1 O-glycosylation was achieved by exposure to benzyl-α-GalNAc. Decreased cell viability after benzyl-α-GalNAc exposure followed by 5FU treatment suggests that the reduction of MUC1 O-glycosylation contributes to the enhanced antiproliferative activity of 5FU. Investigations to understand the specific role of MUC1 O-glycosylation during chemotherapy are currently underway.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA