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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 correlation between overexpression of MUC1 mucin and poor survival rates of pancreatic cancer patients. Treatment of pancreatic cancer with chemotherapy 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 mucin O-glycosylation in human pancreatic adenocarcinoma can enhance response to 5FU therapy. Methods: The human pancreatic cancer cell lines Capan-1, HPAF-II were maintained in their respective growth media. MUC1 expression levels were determined using fluorescence and DIC microscopy. Nontoxic concentration of benzyl-α-GalNAc (inhibitor of O-glycosylation) was determined 72 h following exposure of cells to reagent. The effect of inhibiting O-glycosylation on 5FU cytotoxicity was determined by exposing cells to benzyl-α-GalNAc for 48 h followed by 5FU treatment. MUC1 levels in tumors were determined by immunohistochemistry. Intratumoral injections of benzyl-α-GalNAc were administered to inhibit O-glycosylation in tumors. 5FU was administered intravenously (125 mg/kg). Animal bodyweight and tumor volumes were monitored during the experiment.
 Results: Human pancreatic cancer cells revealed high and moderate MUC1 levels for Capan-1 and HPAF-II respectively. The exposure of Capan-1 and HPAF-II cells to benzyl-α-GalNAc for 72 h revealed a maximum non-toxic concentration of 0.4 and 0.8 mg/ml respectively. Normal cell proliferation and morphology were not altered 24, 48 and 72 h post removal of inhibitor. We observed significantly higher association of Capan-1 and HPAF-II cells to anti-MUC1 antibody (CD227) compared to control cells when pretreated with inhibitor (*P≤0.001). Pretreatment also enhanced 5FU activity. Capan-1 viability reduced from 65 to 54% (*P≤0.001) whereas HPAF-II viability lowered from 77 to 56% (*P≤0.001). Immunohistochemical staining revealed high MUC1 levels in Capan-1 compared to HPAF-II tumors. Intratumoral injections of inhibitor reduced MUC1 O-glycosylation in tumor cells. Pretreatment of Capan-1 tumors with inhibitor followed by 5FU resulted in significantly lower tumor volumes (*P≤0.05) as compared 5FU treatment alone.Conclusions: Our studies show high levels of MUC1 expression on Capan-1 compared to moderate levels on HPAF-II cells. Inhibition of MUC1 O-glycosylation was achieved in vitro and in vivo by exposure to benzyl-α-GalNAc. Inhibition of mucin O-glycosylation enhanced the therapeutic activity of 5FU in both cellular and tumor models of pancreatic cancer. Our experimental findings suggest that overexpression of mucins by human pancreatic tumors may limit the effectiveness of chemotherapy.

99th AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA