Mucin overexpression limits the effectiveness of 5-FU by reducing intracellular drug uptake and antineoplastic drug effects in pancreatic tumours

Immunohistochemical staining of tumour specimens using anti-MUC1 antibody (NCL-MUC1). A: Relative extent of MUC1 levels (brown staining) in (a) Capan-1, (b) HPAF-II and (c) U-87 MG tumours. B: Effects of benzyl-α-GalNAc on MUC1 O-glycosylation levels in pancreatic tumours. Tumours in treatment group (b,d) received four intratumoural injections of benzyl-α-GalNAc (10mg/ml, 0.1cc) at an interval of 48h, whereas control tumours (a,c) received saline injections (20× magnification; bar: 50μm).

Effects of inhibiting mucin on antitumour activity to 5-fluorouracil. Capan-1 tumours were established in s.c. dorsa of female SCID mice. When tumour size reached ∼50–70mm³, intratumoural injections of benzyl-α-GalNAc (0.1ml, 10mg/ml) were administered on days 4, 6, 8 and 10; the control groups received comparable injections of saline. 5-FU therapy (↑) began when tumour size was ∼100mm³. (A) Percent change in body weight on day 14. (B) Tumour growth in response to 5-FU treatment was significantly lower (^∗P⩽0.05) compared to control groups (saline and benzyl-α-GalNAc). Tumour volumes of group exposed to benzyl-α-GalNAc followed by 5-FU treatment were significantly lower (^#P⩽0.05) compared to 5-FU treatment alone. (C) Histochemical staining of tumour sections showing the arrangement of neoplastic cells within (a) saline, (b) benzyl-α-GalNAc, (c) 5-FU and (d) benzyl-α-GalNAc+5-FU treated tumours (20× magnification; bar: 50μm). (D) Quantification of neoplatic cell density using bioquant imaging software. Neoplastic cell density was significantly lower (^∗P⩽0.05) for 5-FU treated groups as compared to controls. The group exposed to benzyl-α-GalNAc followed by 5-FU treatment had significantly lower (^#P⩽0.05) neoplastic cells compared to 5-FU treatment alone. The values represent mean±s.d. of four animals.

Effects of inhibiting O-glycosylation on 5-FU uptake by Capan-1 cells. Approximately 2×10⁴cells/ml were exposed to benzyl-α-GalNAc for 48h followed by (A) 1h or (B) 4h exposure to 5-FU. Intracellular uptake of 5-FU was determined by immunofluorescence staining using anti-5-FU antibody. (A) Following 1h of 5-FU exposure, higher staining of cells was observed when exposed to benzyl-α-GalNAc (+) as compared to cells not exposed to benzyl-α-GalNAc (–). The insets show cytoplasmic localisation of 5-FU and higher accumulation observed around the nucleus (blue) in cells exposed to benzyl-α-GalNAc (+) compared to control (–) cells. (B) The 5-FU staining following 4h of drug exposure was not altered in the presence (+) or absence (–) of benzyl-α-GalNAc (20× magnification; bar: 100μm). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Effects of inhibiting O-glycosylation on 5-FU uptake by U-87 MG cells. Approximately 2×10⁴cells/ml were exposed to benzyl-α-GalNAc for 48h followed by (A) 1h or (B) 4h exposure to 5-FU. Intracellular uptake of 5-FU was determined by immunofluorescence staining using anti-5-FU antibody. The intracellular staining of cells following 1h or 4h of 5-FU exposure was not altered in the presence (+) or absence (–) of benzyl-α-GalNAc (20× magnification; bar: 100μm).

Schematic illustrates the enhanced intracellular accumulation of 5-FU following reduction of the mucin glycation mesh.