Thymidylate synthase and thymidine phosphorylase gene expression as predictive parameters for the efficacy of 5-fluorouraci-based adjuvant chemotherapy for gastric cancer

AIM： To investigate the prognostic role of thymidylate synthase （TS） and thymidine phosphorylase （TP） mRNA levels in T3 or T4 gastric cancer treated with 5-fluorouraci-based adjuvant chemotherapy. METHODS： Fifty-one patients with T3 or T4 gastric cancer received systemic 5-fluorouraci-based adjuvant chemotherapy, and intratumoral expression of TS and TP in 51 gastric cancer tissue samples was tested by realtime quantitative PCR.RESULTS： The median disease-free survival （DFS） time was 10.2 mo in the patients. There were no significant differences in DFS between the groups with high and low levels of TP. However, the group with low level of TS had a longer DFS （14.4 mo vs 8.3 mo, P = 0.017）. The median overall survival （OS） time was 18.5 mo, and there were significant differences in OS between the groups with high and low levels of TS or TP （for TS, 17.0 mo vs 21.3 mo, P = 0.010; for TP, 16.6 mo vs 22.5 too, P = 0.009）. Moreover, the coupled low expression of these two genes was strongly associated with a longer survival time of patients as compared with that of a single gene.CONCLUSION： Expression of TS and TP mRNA is a useful predictive parameter for the survival of postoperative gastric cancer patients after 5-fluorouracilbased adjuvant chemotherapy.

Relationship between the Expression of Thymidylate Synthase,Thymidine Phosphorylase and Dihydropyrimidine Dehydrogenase and Survival in Epithelial Ovarian Cancer

The mRNA and protein expression of thymidylate synthase (TS), thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) and their relationship with prognosis were investigated. Real-time quantitative RT-PCR (Taqman) was used to detect the mRNA expression of TS, TP and DPD in formalin-fixed and paraffin-embedded 106 samples of epithelial ovarian cancer and 29 normal ovaries. A TATA box-binding protein (TBP) was used as an endogenous reference gene. A relationship between TS, TP, DPD expression and clinicopathologic features was investigated. The protein location and expression of TS, TP and DPD was examined in the same patients by an avidin-biotin-peroxidase immunohistochemistry. TS and TP mRNA expression levels were significantly higher in tumor group than in normal controls, with the average value of TS and TP mRNA being 6.14±0.62 and 0.59±0.06 in tumor tissue, and 0.71±0.14 and 0.16±0.04 in normal tissue, respectively. DPD mRNA expression levels were significantly lower in tumor group (0.11±0.02) than in normal controls (0.38±0.05). There was statistically significant difference in TS and TP mRNA expression levels among different pathological grades and clinical stages (P<0.05), but histological subtype was not significantly associated with TS and TP mRNA expression. DPD gene expression was not significantly associated with any clinicopathological parameters. Immunohistochemistry revealed that TP protein was mainly distributed in nucleus, and TS and DPD mainly in cytoplasm. The protein expression intensity of TS, TP and DPD was coincided with the mRNA expression levels. It was concluded that TS, TP mRNA and protein expression levels were significantly higher in epithelial ovarian cancer, and DPD mRNA and protein expression levels were significantly lower. The expression levels of TS and DPD were related to the patients’ prognosis and survival. Combined gene expression levels of TS, TP and DPD represent a new variable to predict the clinical outcome in ovarian cancer. The association of TS, TP and DPD expression levels with survival suggests an importance of these genes for tumor occurrence and progression.

Effects of thymidine phosphorylase on tumor aggressiveness and 5-fluorouracil sensitivity in cholangiocarcinoma

AIM: To evaluate the role of thymidine phosphorylase (TP) in cholangiocarcinoma using small interfering RNA (siRNA). METHODS: A human cholangiocarcinoma-derived cell line KKU-M139, which has a naturally high level of endogenous TP, had TP expression transiently knocked down using siRNA. Cell growth, migration, in vitro angiogenesis, apoptosis, and cytotoxicity were assayed in TP knockdown and wild-type cell lines. RESULTS: TP mRNA and protein expression were decreased by 87.1% ± 0.49% and 72.5% ± 3.2%, respectively, compared with control cells. Inhibition of TP significantly decreased migration of KKU-M139, and suppressed migration and tube formation of human umbilical vein endothelial cells. siRNA also reduced the ability of TP to resist hypoxia-induced apoptosis, while suppression of TP reduced the sensitivity of KKU-M139 to 5-fluorouracil. CONCLUSION: Inhibition of TP may be beneficial in decreasing angiogenesis-dependent growth and migration of cholangiocarcinoma but may diminish the response to 5-fluorouracil chemotherapy.

Intraperitoneal distribution of alginate microcapsules in mice

Introduction: Improved drug delivery mechanisms for the treatment of residual peritoneal cancer cells following cytoreduction surgery are needed. Alginate microcapsules are a potentially useful mechanism for delivery of bioengineered cells, but when injected into the peritoneum, their distribution and properties are not well described. Methods: Aliquots of 300, 600 or 1200 microcapsules were injected into the peritoneum of 81 mice. Mice were sacrificed at 6, 12, 18, and 48 days and laparotomy was performed to quantify the distribution of microspheres. Results: The injections were well tolerated for up to 48 days. No peritoneal adherence or inflammatory reaction was noted to the microcapsules. Injection of 1200 microcapsules resulted in a better overall persistence and widespread peritoneal distribution at up to 48 days. The volume of fluid used for injection of the microcapsules did not affect their distribution or persistence. Conclusion: The intraperitoneal injection of alginate microspheres allows wide and persistent distribution throughout the abdominal cavity. The next step is to test the distribution of microcapsules when delivered following surgery in a rodent model.

Protective autophagy by thymidine causes resistance to rapamycin in colorectal cancer cells in vitro

Aim:Thynidine phosphorylase(TP)acts as a proangiogenic growth factor which may regulate mammalian Target of Rapamycin(mTOR).We investigated whether the TP substrate thymidine and overexpression of TP affected mTOR signaling by comparing Colo320(TP deficient)cells and its TP-transfected variant(Colo320TP1).Methods:Drug resistance was assessed with the sulforhodamine B assay,protein expression with Western blotting,cell cycle distribution and cell death with Fluorescence-activated cell sorting analysis,and autophagy with immunofluorescence.Results:Colo320 and Colo320TP1 cells had comparable levels of sensitivity to the mTOR inhibitor rapamycin.Thymidine treatment led to 13-and 50-fold resistance to rapamycin in Colo320 and Colo320TP1 cells,respectively.In Colo320TP1 cells,the thymidine phosphorylase inhibitor(TPI)reversed the thymidine induced resistance to rapamycin,but not in Colo320 cells,indicating a role for TP in the protection.Thymidine increased p70/S6k-phosphorylation(downstream of mTOR)in Colo320TP1,but it was not affected in Colo320.As a mechanism behind resistance,we studied the levels of autophagy and found that,in Colo320TP1 cells,autophagy was highly induced by thymidine-rapamycin,which was decreased by TPI.In addition,the autophagy inhibitor 3-methyl-adenine completely inhibited autophagy and its protection.Conclusion:Rapamycin resistance in TP-expressing cancer cells may therefore be related to thymidine-mediated autophagy activation.