5-FU is phosphorylated in cells to become an active metabolite that inhibits DNA synthesis and induces RNA dysfunction[5,8
]. Intra-tumoral gene expression and activities of several enzymes related to 5-FU metabolism correlate with sensitivity to this drug for the treatment of several cancers[11-14
]. Of the genes we studied, OPRT seems to have predictive power and may be a promising marker. High enzymatic activity of OPRT in tumor tissues is associated with high sensitivity of urinary bladder cancer[17
] and colorectal cancer[18
] to 5-FU. In addition, OPRT mRNA or the ratio of OPRT/DPD mRNA is associated with prolonged survival of metastatic colon cancer patients receiving oral tegafur-uracil and leucovorin[11
] and colorectal liver metastasis patients receiving intra-arterial chemotherapy with 5-FU[19
]. In addition, combined expression of OPRT and TS in pre-chemotherapeutic fresh-frozen samples obtained from primary tumors may predict the response to S-1, an oral DPD-inhibiting fluoropyrimidine, in metastatic gastric cancer patients[20
]. Consistent with the previous studies, we observed here that intratumoral expression of OPRT mRNA in the responder group was significantly higher than expression in the non-responder group. These results suggest that the OPRT mRNA level in CCA tissues may be a promising predictor of an in vitro
sensitivity to 5-FU using the HDRA technique. It should be noted that we observed overlapping expression levels of each gene among responder and non-responder tumors.
We observed moderate variability in the expression levels of TS, TP, and OPRT mRNA among individual samples (24-fold, 33-fold and 17-fold, respectively). The moderate differences in OPRT and TS expression levels observed in the present study were similar to those previously observed in gastric carcinoma and colorectal tissues[21,22
]. Similar to the variability reported in colorectal[22
] and esophageal carcinoma[23
], we observed high variability in the expression of DPD among CCA tissues. Genetic polymorphisms of genes encoding TP, DPD, TS and OPRT are well known[24-27
]. Variable expression of DPD, TP, TS and OPRT may explained by genetic polymorphisms in these genes.
TP is the first enzyme involved in the metabolic activation pathway of 5-FU to 5-fluorodeoxyuridine (FUdR), which can be phosphorylated by thymidine kinase to form FdUMP[28
]. In this study, no relationship was found between the intratumoral expression of TP mRNA and the sensitivity to 5-FU. In addition to TP, that the rate of conversion of 5-FU to FUdR could be influenced by the availability of the TP cofactor, deoxyribose-1-phosphate (dRib-1-P) in cells[29
]. It has been previously demonstrated that the addition of dRib-1-P greatly increases the incorporation of thymidine into DNA and increases the potency of the growth-inhibitory actions of 5-FU[30
]. Thus, it may be possible that the amount of dRib-1-P in CCA tissues may be limited. Similar observations have been reported in colorectal tumor that there was no relationship between TP expression and 5-FU sensitivity[31,32
TS is a key enzyme that catalyzes the methylation of deoxyuridine monophosphate to deoxythymidine monophosphate, an important step in DNA synthesis[33
]. Colon cancer patients with high TS expression are reportedly un-responsive to 5-FU and show poor prognosis[34
]. In contrast, tumoral expression of TS mRNA is associated with response to protracted infusions of 5-FU-based chemotherapy and survival in patients with disseminated colorectal cancer[35
]. Consistent with a report on colorectal tumors[36
], no relationship between TS expression and sensitivity to 5-FU in CCA in vitro
was observed in our present study.
DPD is a major enzyme in the metabolism of 5-FU to an inactive metabolite. Some reports have suggested a negative correlation between 5-FU sensitivity and DPD activity in human stomach cancer cells[37
]. In patients with advanced colorectal cancer treated with 5-FU/leucovorin, patients who responded to the treatment exhibited low levels and a narrow distribution range of DPD mRNA expression compared to the non-responder group[32
]. In the present study, DPD mRNA expression was apparently higher in the responders than in the non-responders. However, no clear relationship was found between DPD mRNA expression and 5-FU sensitivity.
In conclusion, we developed HDRA as an in vitro screening of the response of CCA tissues to 5-FU. We found that the level of OPRT mRNA may be a promising predictor of CCA sensitivity to 5-FU. Whether the OPRT mRNA level could be used as a predictor of the success of 5-FU chemotherapy in CCA patients needs to be confirmed further in patients.