Abstract
Uridine phosphorylase (UPase) has been shown to play an important role in the antineoplastic activity of 5-fluorouracil (5-FU) and in the anabolism of its oral prodrug, capecitabine, through the conversion of 5′-deoxy-5-fluorouridine (5′-DFUR) into 5-FU. In this study, we investigated the effect of tumor necrosis factor-α (TNF-α) on UPase gene expression and 5′-DFUR antiproliferative activity and elucidated the involved signal transduction pathway. Our data indicate that TNF-α significantly induced UPase mRNA expression and its enzymatic activity in EMT6 murine breast cancer cells, leading to an enhanced cytotoxicity of 5′-DFUR. This is further confirmed by an increased incorporation of 5′-DFUR-originated 5-FU nucleotides into nucleic acids. To clarify the mechanism of TNF-α-induced UPase expression, we first observed the effect of TNF-α on the UPase promoter activity with a series of 5′-deleted promoter-luciferase constructs. Transient transfection analysis showed that the TNF-α-inductive pattern in EMT6 cells was consistent with the presence of a nuclear factor-κB (NF-κB) binding element (-1332/-1312 bp) in the UPase promoter region. Furthermore, electrophoretic mobility shift assays, supershift, and cotransfection assays revealed that the activation of p65 was responsible for UPase induction by TNF-α. Finally, the induction of UPase by TNF-α could be suppressed by PS-341, a NF-κB inhibitor. In summary, TNF-α efficiently induces UPase gene expression through a NF-κB subunit p65-dependent pathway enhancing cell sensitivity to 5′-DFUR. The elucidation of this regulation mechanism may aid in the clinical use of 5-FU-based chemotherapy.
- Received August 30, 2005.
- Accepted January 3, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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