title: Deficient expression of the DPD gene is caused by epigenetic modification in biliary tract cancer cells, and induces high sensitivity to 5-FU treatment.
abstract: 5-FU is the drug most frequently used to treat biliary tract cancer, while dihydropyrimidine dehydrogenase (DPD) is known to be a principal factor in 5-FU drug resistance. However, whether DPD activity and mRNA levels correlate with response to 5-FU is unknown for biliary tract cancers. The precise mechanism of DPD regulation also remains to be elucidated. In the present study, we quantitatively analyzed DPD mRNA in 8 biliary tract cancer cell lines using real-time RT-PCR, and assessed whether DPD mRNA levels correlate with DPD activity or the sensitivity to 5-FU. Finally, we examined the epigenetic gene silencing of DPD using one of the 8 lines, a gallbladder cancer cell line with deficient DPD expression, KMG-C. Strong correlation was found between DPD activity and DPD mRNA expression in the 8 cancer cell lines (R=0.797, P=0.0148). DPD mRNA expression and DPD activity exhibited positive correlation with the IC50 for 5-FU (R=0.658, R=0.644, respectively), although these relationships were not statistically significant. In the KMGC cells with deficient DPD mRNA levels, restoration of DPD expression was observed by 5-Aza-2' deoxycytidine (5-aza-C) treatment in a dose-dependent manner, suggesting gene suppression by promoter hypermethylation. Combined bisulfite restriction analysis was performed to analyze the methylation on CpG islands around the 5'-flanking region and intron 1 of the DPD gene, however, no methylated CpG sites were identified in these regions. In addition, the restored DPD expression level was more strongly induced by the histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), than 5-aza-C treatment. These findings suggest that other mechanisms, including histone modification, may be important for DPD suppression. In conclusion, these results may aid the selection of 5-FU chemotherapy following determination of DPD expression in biliary tract cancers. Furthermore, epigenetic gene silencing appears to be an important mechanism of DPD suppression in biliary tract cancer.
authors.affiliation: Department of Surgery, Saga University Faculty of Medicine, Saga 849-8501, Japan.
authors.forename: Ken
authors.initials: K
authors.lastname: Sato
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Kitajima
authors.forename: Atsushi
authors.initials: A
authors.lastname: Miyoshi
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Koga
authors.forename: Kohji
authors.initials: K
authors.lastname: Miyazaki
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D001483:Base Sequence; D001661:Biliary Tract Neoplasms / Q000378:metabolism*; D045744:Cell Line, Tumor; D004791:Enzyme Inhibitors / Q000494:pharmacology; D044127:Epigenesis, Genetic; D005472:Fluorouracil / Q000494:pharmacology*; D015972:Gene Expression Regulation, Neoplastic; D056572:Histone Deacetylase Inhibitors; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D008969:Molecular Sequence Data; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology
chemical_list: D000964:Antimetabolites, Antineoplastic; D004791:Enzyme Inhibitors; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D011500:Protein Synthesis Inhibitors; D013778:Tetrazolium Salts; D013844:Thiazoles; C012589:trichostatin A; C022616:thiazolyl blue; D005472:Fluorouracil