Modulation of uridine phosphorylase gene expression by tumor necrosis factor-alpha enhances the antiproliferative activity of the capecitabine intermediate 5'-deoxy-5-fluorouridine in breast cancer cells.
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-alpha (TNF-alpha) on UPase gene expression and 5'-DFUR antiproliferative activity and elucidated the involved signal transduction pathway. Our data indicate that TNF-alpha 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-alpha-induced UPase expression, we first observed the effect of TNF-alpha on the UPase promoter activity with a series of 5'-deleted promoter-luciferase constructs. Transient transfection analysis showed that the TNF-alpha-inductive pattern in EMT6 cells was consistent with the presence of a nuclear factor-kappaB (NF-kappaB) 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-alpha. Finally, the induction of UPase by TNF-alpha could be suppressed by PS-341, a NF-kappaB inhibitor. In summary, TNF-alpha efficiently induces UPase gene expression through a NF-kappaB 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.
Wan
Laxiang
L
Department of Internal Medicine (Oncology), Yale University School of Medicine, 333 Cedar Street, SHM I 220, New Haven, CT 06520, USA.
Cao
Deliang
D
Zeng
Jianmin
J
Yan
Ruilan
R
Pizzorno
Giuseppe
G
D000818:Animals; D001483:Base Sequence; D000069287:Capecitabine; D017931:DNA Primers; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D004357:Drug Synergism; D024202:Electrophoretic Mobility Shift Assay; D005467:Floxuridine / Q000494:pharmacology*; D005472:Fluorouracil / Q000031:analogs & derivatives; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D008325:Mammary Neoplasms, Experimental / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D014543:Uridine Phosphorylase / Q000235:genetics*
D017931:DNA Primers; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D005467:Floxuridine; D003841:Deoxycytidine; D000069287:Capecitabine; D014543:Uridine Phosphorylase; D005472:Fluorouracil; C025034:doxifluridine