Involvement Of Gene Polymorphisms Of Thymidylate Synthase And Chemosensitivity Testing In Pancreatic Cancer

BackgroundPancreatic cancer is considered one of the most deadly forms of cancer. The prognosis of patients with pancreatic cancer is very poor and 85% of the patients with unresectable pancreatic cancer die within 1 year.Even after curative resection, the 5-year survival rate is only 0.4-5%.Accordingly, in order to improve the outcome of treatment for pancreatic cancer, effective adjuvant chemotherapy is necessary in the treatment of pancreatic cancer. 5-fluorouracil (5-FU) is widely used in the chemotherapy of pancreatic cancer and is considered to be one of the most important agent against pancreatic cancer. However, a substantial number of patients with pancreatic cancer are resistant to the currently used adjuvant 5-FU-based chemotherapy, and the benefit of chemotherapy is limited to a number of patients. Therefore, predictive markers are needed in order to discriminate between responsive and nonresponsive patients. Thymidylate synthase (TS) is a central enzyme in DNA synthesis and is a potentially valuable marker since it is the molecular target of 5-FU. Recently, a variable number of tandem repeat (VNTR) polymorphism consisting of multiple repeats of a 28bp sequence in the 5-untranslated region (5-UTR) of the TS gene, has also been identified, which may have an effect on the TS mRNA stability and translation, perhaps affecting TS protein expression level as well. Several recent studies have show that the polymorphisms of TS may predict the sensitivity to 5-FU and other TS inhibitor-based chemotherapy and may also be an important prognostic marker in colorectal cancer patients. The chemotherapy sensitivity test for carcinoma used in past years was based on single-layer cultured cells in two-dimensional model, while the pancreatic cancer cell in the human body survive in a three-dimensional growing model. Single-layer cultured cells model neglects the different characteristics between a whole tumor and its individual tumor cells, the interaction between tumor cells and the impact of microenvironment. Single-layer cultured cells model can not simulate the environment in vivo, and it can not provide a reliable platform for pancreatic cancer chemotherapy sensitivity test in vitro studies. A new three-dimensional culture model is established in these series of experiment to simulate the environment and tumor cell growth status in vivo. This model is Collagen gel droplet culture drug-sensitivity test (CD-DST), a new tumor in vitro chemosensitivity test technique is established with rat tail collagen. The chemosensitivity test results of CD-DST are more authentic.ObjectiveIn this study, we investigated correlation between the polymorphic tandem repeat sequences of the TS gene and its implications regarding the efficacy of chemotherapy with 5-FU using pancreatic carcinoma cell lines, peripheral blood lymphocytes of pancreatic cancer patients, pancreatic cancer cells of patients, tumor tissue of pancreatic cancer patients and prognostic factors in pancreatic cancer patients treated with 5-FU-based adjuvant chemotherapy. We examined an association between TS genotypes and its chemosensitivity. The purpose of our study tried to provide individualized treatment of pancreatic cancer in a new way. MethodsThe number of the 28bp tandem repeat polymoiphisms of TS were assessed in fresh blood and cell lines. Polymerase chain reactions (PCR) were performed on genomic DNA from blood samples and cell lines. After PCR products were electrophoresed onto a capillary electrophoresis. The SNP of TS was determined by DNA sequencing. Quantification of the mRNA levels was carried out using a real-time fluorescence detection method. Analysis of TS mRNA was performed using a two-step procedure. The quantities of TS mRNA were expressed as ratios relative to that of gene GAPDH mRNA.Western blotting analysis was performed to detect TS protein expression in seven cell lines. Chemosensitivity test of cells were added into 96-well multi-plates with CCK-8.Pancreatic cancer cells of patients with pancreatic cancer were isolated and tested using CD-DST.Immunohistochemical staining of TS was performed on 4 lm sections obtained from paraffin-embedded tumor tissues using the TS-106 antibody. Data are presented as the means±SD. Statistical significance was determined using one-way ANOVA and Student-Newman-Keuls analysis. The correlations between TS expression and in vitro chemosensitivity were analyzed by Pearson's correlation coefficient test. All statistical tests were two-sided and differences were considered significant at p<0.05.Results1. Seven pancreatic carcinoma cell lines had different genotypes in terms of the 28-bp TS tandem repeat, as follows:homozygous 2R/2R (T3M4 and BxPC-3 cells), heterozygous 2R/3R (ASPC-1, Capan-1, and SU86.86), and homozygous 3R/3R (PANC-1 and COLO357). 2. The distribution and categorization of the 3 different genotypes is depicted. The majority of the pancreatic adenocarcinoma patients(n=15,48.4%) were found to be homozygous the 2R/2R genotype. There were 7 pancreatic adenocarcinoma patients showed the heterozygous 2R/3R genotype (22.6%).9 patients with homozygous the 3R/3R genotype (29.0%)3. Quantification of the mRNA levels was carried out using a real-time fluorescence detection method, which described in Methods. TS mRNA with 3R/3R genotypes are higher than 2R/3R or 2R/2R,but results showed no statistically significant differences.4. The optical density ratio of genotypes 2R/2R,2R/3R and 3R/3R was 0.568±0.032, 0.561±0.056 and 1.393±0.374, respectively. Cells possessing the 2R/2R and 2R/3R genotypes of TS had a reduced TS protein expression as compared with those with the 3R/3R.5. Cells with the 2R/2R and 2R/3R genotypes of TS were hypersensitive to 5-FU in vitro as compared with those with the 3R/3R cells. The IRs for cells to 10,25 and 50μg/ml 5-FU were 0.811±0.090,0.880±0.067 and 0.922±0.038, respectively, for cells with the 2R/2R genotype,0.595±0.065,0.695±0.070 and 0.780±0.075, respectively, for 2R/3R, and 0.370±0.157,0.548±0.018 and 0.638±0.020, respectively, for 3R/3R. The extent of growth inhibition greatly varied depending on the cell lines used. However, the IRs to 5-FU tended to be lower in cells with the 3R/3R genotype than in those with 2R/2R and 2R/3R genotypes. The IRs to 5-FU were significantly different between each concentration (P<0.05)6. Pancreatic cancer cell lines (COLO357,AsPC-1,BxPC-3) were treated with 50ug/ml concentrations of 5-FU for 72h, cells were harvested and processed for Annexin V-FITC/PI double staining. There was significant difference of 5-FU apoptosis for Annexin V-FITC/PI double staining of pancreatic cancer cells between 3R/3R genotype and those with 2R/2R,2R/3R genotype. The apoptotic rates(early apoptotic and late apoptotic cells) of 3R/3R was lower than 2R/2R,2R/3R.7. CD-DST:The IRs for cells to 10μg/ml 5-FU was 24.93±4.88 for 3R/3R (PI) 40.70±3.33,35.95±3.94 and 47.94±4.38respectively, for cells with the 2R/2R genotype (P2,P3,P4). the IRs to 5-FU tended to be lower in cells with the 3R/3R genotype than in those with 2R/2R (P<0.05)8. Immunohistochemical staining of TS was performed in patients, which described in Methods. TS protin level with 3R/3R genotypes are higher than 2R/3R or 2R/2R, the results showed statistically significant differences.9. The OS in our entire patients with 5-fluorouracil-based Chemotherapy and in that of patients with the 3R/3R genotype (n=9) was lower the median value than those with 2R/2R or 2R/3R genotypes(p=0.007). The PFS was also different in patients between the 3R/3R genotype and 2R/2R or 2R/3R genotypes patients. There were consistent differences in the OS and PFS were observed patients between 2R/2R 2R/3R and 3R/3R genotypes.Conclusions1. In this study, the distribution and categorization of the TS genotypes is different. The majority of the pancreatic adenocarcinoma patients were found to be homozygous the 2R/2R genotype. Then, pancreatic adenocarcinoma patients showed homozygous the 3R/3R genotype are more than patients with heterozygous 2R/3R genotype. 2. We found that TS polymorphisms were correlated with its protein expression levels. Cells with a 3R/3R genotype had higher TS protein expression than cells with the 2R/2R or 2R/3R genotypes.3. TS polymorphisms were correlated with the IRs to 5-FU in these cell lines. The IRs to 5-FU tended to be lower in cells with the 3R/3R genotype than in cells with the 2R/2R or 2R/3R genotypes4. Our results of the reporter as says using 2R,3R repeat sequences prompted us to classify 3R/3R as a high TS protein expression genotype, and 2R/2R and 2R/3R as low TS protein expression genotype. The cells with low TS protein expression genotype were shown to exhibit significantly higher 5-FU sensitivity in vitro than the cells with high TS protein expression genotype.5. Genotypes based on the number of the 28bp tandem repeat of TS appear to be potential prognostic factors in pancreatic cancer patients treated with 5-FU-based adjuvant chemotherapy. The pancreatic cancer patients with the 3R/3R genotype may be not sensitivity from 5-FU chemotherapy. The data might warrant further large scale clinical study of the role of the gene polymorphisms of TS for the prediction of efficacy using 5-FU based chemotherapy in pancreatic cancer patients.