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.