Thymidylate Synthase Levels Research Articles

Association of expression and genotypes of thymidylate synthase in non-small cell lung cancer patients with different clinicopathological characteristics

Abstract Objective To explore the expression and genotypes of thymidylate synthase (TS) in patients of non-small cell lung cancer (NSCLC) with different clinicopathological characteristics. Methods The expression profiles of TS were examined by immunohistochemical staining and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) in 160 patients with NSCLC. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to detect TS-5′UTR tandem repeats, G/C nucleotide polymorphisms, and 3′UTR 6 bp deletion/insertion polymorphisms. The relationships between clinicopathological characteristics and TS expression or genotypes were investigated through χ 2 test. Kaplan–Meier survival analysis was used to analyze the association between TS expression and overall survival (OS) and disease-free survival (DFS) of NSCLC patients. Results The expression levels of TS protein and TS gene in NSCLC tissues were significantly higher than that in paracancerous tissues (P < 0.05). Furthermore, high expression of TS protein and 5′UTR polymorphism of TS gene showed significant correlation with differentiation, TNM stage, and lymph node metastases. The frequency of −6 bp/−6 bp genotypes in patients with NSCLC was 43.13% (69/160), which was higher than others. In addition, the rate of TS protein overexpression in NSCLC patients with 3R/3R was 79.79%, which was higher than others. Interestingly, high expression of TS protein predicted shorter DFS and OS and lower 3-year DFS rate and 3-year OS rate. Conclusions The expression levels of TS in NSCLC were significantly increased and may help to predict the prognosis of NSCLC, and high expression of TS protein and 5′UTR polymorphism of TS gene were significantly related to differentiation, TNM stage, and lymph node metastases.

Pyrotinib Sensitizes 5-Fluorouracil-Resistant HER2+ Breast Cancer Cells to 5-Fluorouracil.

5-Fluorouracil (5-FU) is a widely used chemotherapeutic agent for breast cancer. However, acquired chemoresistance leads to a loss of its efficacy; methods to reverse are urgently needed. Some studies have shown that pyrotinib, an ErbB receptor tyrosine kinase inhibitor, is effective against HER2+ breast cancer. However, whether pyrotinib sensitizes 5-FU-resistant breast cancer cells to 5-FU is unknown. We hypothesized that the combination of pyrotinib and 5-FU would show synergistic antitumor activity, and pyrotinib could reverse 5-FU resistance in HER2+ breast cancer cells in vitro and in vivo. Our data showed that pyrotinib inhibited the growth of 5-FU-resistant SKBR-3/FU and MDA-MB-453/FU cell lines and the parental cell lines. 5-FU remarkably suppressed the growth of SKBR-3 and MAD-MB-453 cells. However, SKBR-3/FU and MAD-MB-453/FU cells showed resistance to 5-FU. A combination of pyrotinib and 5-FU resulted in the synergistic inhibition of the growth of the 5-FU-resistant SKBR-3/FU and MDA-MB-453/FU cell lines and the parental cell lines. Pyrotinib decreased significantly the IC50 values of 5-FU and the thymidylate synthase (TS) mRNA expression levels in the 5-FU-resistant SKBR-3/FU and MDA-MB-453/FU cell lines and the parental cell lines and increased significantly the intracellular concentration of 5-FU in SKBR-3/FU and MDA-MB-453/FU cells. In addition, pyrotinib reduced the ABCG2 mRNA and protein expression levels in SKBR-3/FU and MDA-MB-453/FU cells and downregulated the protein expression levels of pAKT, pHER2, and pHER4 in all four cell lines. After TS or ABCG2 in 5-FU-resistant breast cancer cells was knocked down, the sensitivity of SKBR-3/FU and MDA-MB-453/FU cells to 5-FU was restored. Moreover, in vivo experiments demonstrated that pyrotinib in combination with 5-FU more effectively inhibited SKBR-3/FU tumor growth than either pyrotinib or 5-FU alone. In conclusion, our findings suggest that pyrotinib could restore sensitivity of 5-FU-resistant HER2+ breast cancer cells to 5-FU through downregulating the expression levels of TS and ABCG2.

Plasma thymidylate synthase and dihydrofolate reductase mRNA levels as potential predictive biomarkers of pemetrexed sensitivity in patients with advanced non-small cell lung cancer.

BackgroundHigh levels of thymidylate synthase (TS) and dihydrofolate reductase (DHFR) expression in tumour tissues are an indicator of ineffective responses to pemetrexed-based chemotherapy in various tumours, including non-small cell lung cancer (NSCLC). However, tumour tissues are highly heterogeneous, so a single biopsy may not reflect genetic alterations during disease progression. This study investigated the potential use of plasma TS and DHFR mRNA levels as biomarkers for predicting sensitivity to pemetrexed-based chemotherapy.MethodsPlasma samples were obtained from 245 patients with advanced NSCLC and 30 healthy donors. Total RNA was extracted from the plasma samples, and TS and DHFR mRNA levels were determined via real-time PCR. TS and DHFR mRNA levels between cancer patients and healthy controls were compared. The association between plasma TS and DHFR mRNA levels and tumour response to pemetrexed/cisplatin chemotherapy was analysed.ResultsThe plasma TS and DHFR mRNA levels decreased in patients with advanced NSCLC compared with healthy controls. Moreover, plasma TS and DHFR mRNA levels negatively correlated with tumour response to pemetrexed/cisplatin chemotherapy in patients with advanced NSCLC. Overall survival time was prolonged in patients with low TS mRNA expression compared with those with high TS mRNA expression, although the difference was not statistically significant.ConclusionsLow expression levels of plasma TS and DHFR mRNA confer increased tumour sensitivity to pemetrexed/cisplatin chemotherapy in patients with advanced NSCLC. The results suggested that plasma TS and DHFR mRNA levels are promising biomarkers for choosing patients who are likely to respond and benefit the most from pemetrexed-based chemotherapy.

MiR-192 overexpression effect on DHFR and TYMS in acute lymphoblastic leukemia

PurposeNon-coding RNAs play a critical role in gene regulation in cancer cells. Reduced expression of microRNA-192 has been detected in many cancers. Here, we investigated the role of miR-192 in Dihydrofolate reductase (DHFR) and Thymidylate Synthase (TYMS) expression level and in an acute lymphoblastic leukemia cell line. Basic procedures20 patients diagnosed with acute lymphoblastic leukemia (ALL) were studied for the level of (micro RNA-192) miR-192, DHFR and TYMS expression level by qRT-PCR. NALM-6 cells were transduced using recombinant lentiviruses for overexpression of miR-192 and its backbone, then the relative changes in DHFR and TYMS genes expression were studied by qRT-PCR. DHFR Protein level changes were analyzed by Western blotting. Main findingsALL patients with relapse, experience lower levels of miR-192 and higher levels of DHFR and TYMS in comparison with treated patients. Overexpression of miR-192 in NALM-6 cells resulted in decreased expression of DHFR and TYMS. Moreover, the protein level of DHFR was decreased after overexpression of miR-192. Principal conclusionsThese results suggest the tumor suppression effects of miR-192 and its correlation with decreased DHFR and TYMS level in acute lymphoblastic leukemia cells for the first time.

Molecular determinants of response to 5-fluorouracil-based chemotherapy in colorectal cancer: The undisputable role of micro-ribonucleic acids

5-flurouracil (5-FU)-based chemotherapy is the main pharmacological therapy for advanced colorectal cancer (CRC). Despite significant progress in the treatment of CRC during the last decades, 5-FU drug resistance remains the most important cause of failure in CRC therapy. Resistance to 5-FU is a complex and multistep process. Different mechanisms including microsatellite instability, increased expression level of key enzyme thymidylate synthase and its polymorphism, increased level of 5-FU-activating enzymes and mutation of TP53 are proposed as the main determinants of resistance to 5-FU in CRC cells. Recently, micro-ribonucleic acids (miRNA) and their alterations were found to have a crucial role in 5-FU resistance. In this regard, the miRNA-mediated mechanisms of 5-FU drug resistance reside among the new fields of pharmacogenetics of CRC drug response that has not been completely discovered. Identification of the biological markers that are related to response to 5-FU-based chemotherapy is an emerging field of precision medicine. This approach will have an important role in defining those patients who are most likely to benefit from 5-FU-based chemotherapy in the future. Thereby, the identification of 5-FU drug resistance mechanisms is an essential step to predict and eventually overcome resistance. In the present comprehensive review, we will summarize the latest knowledge regarding the molecular determinants of response to 5-FU-based chemotherapy in CRC by emphasizing the role of miRNAs.

Abstract 353: Expression of astrocyte-elevated gene-1 is associated with prognostic value of adjuvant chemotherapy in resectable stage III colorectal cancer

Abstract Background: Administration of postoperative adjuvant chemotherapy has reduced the risk of tumor recurrence and improved survival for patients with resected colorectal cancer (CRC). Regarding the prognostic factors, including pathologic features and gene mutation have major relevance for stage III disease, it is not well known whether they might help select patients who would fare favorably when received adjuvant chemotherapy. OBJECTIVE: Expression of astrocyte-elevated gene-1 (AEG-1), thymidylate synthase (TS), excision repair cross-complementation group 1 (ERCC1), epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF) were needed to investigate the role involved in adjuvant combination therapy for patients with resectable stage III colon cancer. Methods: AEG-1, TS, ERCC1, EGFR and VEGF were investigated by CRC cell lines and intra-tumor immunohistochemistry stain. Furthermore, this study collected 29 patients with stage III colorectal cancer who underwent surgical resection from 2011 to 2016 and received postoperative adjuvant chemotherapy. The culmination of clinical data registration included surgical procedures, tumor size, number of lymph node metastases, and types of chemotherapy drugs. Result: A significant positive correlation between AEG-1, TS, ERCC1, EGFR, and VEGF gene expression levels were revealed in CRC cell lines. Cell lines with lower AEG-1 and TS expression had higher sensitivity to 5-FU treatment. In clinical studies, we found that higher AEG-1 expression was noted in T4 and also caused CRC recurrence or metastasis. Patients with T4 and stage IIIC CRC had a significantly shorter DFS (T4 v.s. T1-3, DFS, months, median: 4.1 ± 1.2 v.s. non-reach [NR], p < 0.001; IIIC v.s. IIIA or IIIB, DFS, months, median: 10.6 ± 5.3 v.s. NR, p = 0.037; respectively). Patients with higher AEG-1 and TS expression had a shorter DFS (AEG-1, high v.s. low, months, median: 12.8 ± 2.4 v.s. NR, p = 0.003; TS, high v.s. low, months, median: 13.8 ± 2.3 v.s. NR, p = 0.012; respectively). In multivariate Cox Regression analysis, advanced tumor invasion (T4) and high AEG-1 expression could be an independent prognostic factor to cause poor survival in resectable stage III CRC patients treated with adjuvant chemotherapy. Conclusion: Expressions of AEG-1 and tumor grade as a candidate prognostic factor of recurrence in stage III colorectal cancer patients receiving adjuvant fluoropyrimidine-based chemotherapy. A prospective cohort study should be guaranteed to validate these factors to be predictive markers of adjuvant chemotherapy benefit for stage III CRC. Citation Format: Chung-Yu Chen, Peng-Sheng Lai, Ying-Yin Chen, Long-Wei Lin. Expression of astrocyte-elevated gene-1 is associated with prognostic value of adjuvant chemotherapy in resectable stage III colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 353.

Relationship between the Expression of the Thymidylate Synthase and the Prognosis of Gastric Cancer Patients Treated with Combinational Chemotherapy Regimen Including Fluorouracil, Docetaxel and Cisplatin.

Background: Thymidylate synthase is one of the target enzymes of 5-fluorouracil. However, the clinical and prognostic significance of TS expression in gastric cancer has remained controversial. In this study, the expression of thymidylate synthase was evaluated in gastric cancer patients treated with combinational chemotherapy; moreover, the association between TS expression and clinicopathologic characteristics and overall survival of the patients were also assessed. Materials and Methods: In this descriptive study, 89 pathological samples were gathered from patients at Kermanshah hospitals during 2008-2017. The survival status of patients was recorded and their overall survival was evaluated individually. Results: The average survival period for low and high thymidylate synthase groups was 54 and 50 months, respectively, meaning higher survival time in the lower thymidylate group. But this difference was not statistically significant (log Rank=0.88). In addition, sex, stage, recurrence, and survival had no significant difference between the low and high expression of thymidylate synthase groups (p=0.89). Conclusion: The results clearly indicated that the level of thymidylate synthase is not a significant modulator of 5- fluorouracil in gastric cancer patients. Nevertheless, evaluation of the level of the enzymes and markers as well as their effects are highly recommended for accurate selection of chemotherapeutical strategies.

Nine hub genes related to the prognosis of HBV-positive hepatocellular carcinoma identified by protein interaction analysis.

BackgroundHepatocellular carcinoma (HCC) represents the second highest cause of cancer-associated deaths worldwide, and hepatitis B virus (HBV) infection is a major risk factor. Here, we aimed to identify genetic signatures of HBV-positive (HBV+) HCC and uncover potential carcinogenic mechanisms.MethodsGene expression profiles of 124 HBV-positive samples, including tumor and non-tumor tissues were subjected to bioinformatics analysis. The expression levels of thymidylate synthase (TYMS) and CDC45 in patients’ samples were validated by immunohistochemistry (IHC) and their association with patient survival was assessed by the Kaplan-Meier method.ResultsA total of 666 differentially expressed genes (DEGs) were identified. The 137 upregulated genes were mainly enriched in the cell cycle, P53 signaling pathway, and extracellular matrix-receptor interaction, whereas the 529 downregulated genes were enriched in cytochrome P450 xenobiotic and drug metabolism, and cytokine-cytokine receptor interaction. A total of 15 hub genes were identified from the protein-protein interaction (PPI) network and 10 of them were strongly associated with HBV+ HCC. The expression of 9 hub genes (CDK1, NDC80, TYMS, AURKA, FOXM1, CDC45, ZWINT, PBK, and TPX2) was associated with poor overall survival. Validation of TYMS and CDC45 protein expression levels in clinical samples by IHC showed that they were higher in HBV+ HCC than in HBV- HCC or normal tissue and were associated with poor patient survival.ConclusionsHBV may induce HCC through regulation of host gene expression. Among the hub DEGs identified, 9 key genes could be used as new prognostic biomarkers and treatment targets for HBV+ HCC.

Matrix metalloproteinase 1 promotes tumorigenesis and inhibits the sensitivity to 5-fluorouracil of nasopharyngeal carcinoma

Problem and object5-fluorouracil (5-FU) is a pyrimidine-like antimetabolite. It has been widely used in human cancer therapies; however, the drug sensitivity can be very low and the survival outcome can be very poor dependent on tumor types and individual heterogeneity. This research was aimed to study the effects of matrix metalloproteinase 1 (MMP1) gene on nasopharyngeal carcinoma (NPC) cell proliferation, viability, invasiveness, apoptosis, and sensitivity to 5-FU. MethodsBioinformatics method was used to screen out the differentiated expressed genes in nasopharyngeal carcinomas compared with normal nasopharyngeal epithelial tissues. qRT-PCR was used to determine the expression of MMP1 mRNA, and western blot was used to determine the protein expression of MMP1, thymidylate synthase (TS), and dihydropyrimidine dehydrogenase (DPD). Colony foci formation assay, CCK8 assay, and BrdU incorporation assay were used to study the cell proliferation. Transwell invasion assay was carried out to determine cell invasion. Flow cytometry and caspase 3/7 activation assay were used to detect cell apoptosis. ResultsMMP1 gene was the most significantly upregulated gene in nasopharyngeal carcinomas according to the bioinformatics analysis. And the upregulation of MMP1 gene was confirmed in both NPC tissues and cell lines using RT-QPCR and western blot technique. When 5-FU was not a player, the forced overexpression of MMP1 gene led to enhanced growth and invasion of CNE1 and HNE1 cell lines, whereas MMP1 gene knockdown resulted in the opposite outcome. When 5-FU was added, MMP1 gene knockdown led to significantly suppressed cell proliferation and enhanced cell apoptosis. Also, MMP1 gene knockdown caused significantly lower level of TS and DPD enzymes. ConclusionNot only the knockdown of MMP1 gene led to suppressed proliferation and invasion but also increased the sensitivity to 5-FU of CNE1 and HNE1 cells. Our results provided convincing evidence that MMP1 gene knockdown could offer a favorable sensitivity approach for NPC with 5-FU treatment.

Abstract 2082: NUC 3373 induces a cytoplasmic translocation of thymidylate synthase in colorectal carcinoma cell lines

Abstract BACKGROUND: Although 5-fluorouracil-based (5-FU) chemotherapies remain the cornerstone of combination therapies for colorectal cancer (CRC), their clinical utility is limited by key cancer resistance mechanisms associated with breakdown, transport, and activation. This includes the intracellular conversion of 5-FU into its active metabolite, fluorodeoxyuridine-monophosphate (FUDR-MP), before core anti-cancer activity can be exerted via inhibition of the enzyme thymidylate synthase (TS). Although widely studied, there are mixed reports as to whether high or low basal tumor TS levels are prognostic of response to 5-FU therapy. NUC-3373, a phosphoramidate transformation of FUDR-MP, is designed to bypass the key resistance mechanisms associated with 5-FU, resulting in a potentially more effective and safer treatment option. This study aimed to determine the prognostic value of cancer cell TS protein expression in predicting therapeutic response to NUC-3373 and to further explore the effects of NUC-3373 on TS biology. METHODS: IC50 values for NUC-3373 were established in nine CRC cell lines by sulforhodamine B assay, after which one sensitive (HCT116) and one resistant (SW480) cell line were selected for further characterization. In these two cell lines, endogenous, induced, and ternary complex TS expression was measured by Western blot analysis and the cellular localization of TS was confirmed by immunocytochemistry and assessed by stereological methods. RESULTS: TS protein expression was measured in nine CRC cell lines treated with NUC-3373 and no correlation was found between TS protein levels and the IC50 for NUC-3373. This indicates that the sensitivity of these cell lines to NUC-3373 is not dependent on basal TS protein expression levels. When the two cell lines selected for their sensitivity/resistance to NUC-3373 were treated with NUC-3373, TS expression was induced after 12 hours and remained elevated at 48 hours. In addition, treatment with NUC-3373 was associated with a translocation of TS from the nucleus to the cell cytoplasm, which was more pronounced in the NUC-3373 sensitive cells. Translocated TS did not localize to vesicles or the Golgi apparatus; the cytoplasmic localization remains to be determined. CONCLUSIONS: These results suggest that basal TS levels are not prognostic of response to NUC-3373 in CRC cells, instead NUC-3373 induces TS expression and causes nuclear to cytoplasmic translocation. This novel finding is indicative of an alternative mechanism of action for FUDR-MP in promoting anti-cancer activity that is independent of the DNA damage pathway. Citation Format: Fiona G. McKissock, In Hwa Um, Peter Mullen, David J. Harrison. NUC 3373 induces a cytoplasmic translocation of thymidylate synthase in colorectal carcinoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2082.

Predictive factors for long-term responders of pemetrexed maintenance treatment in non-small cell lung cancer.

BackgroundWe determined the clinical characteristics and predictive factors of long‐term response to pemetrexed maintenance therapy as first‐line treatment for non‐small cell lung cancer (NSCLC).MethodsA total of 950 advanced NSCLC patients received pemetrexed (500 mg/m2) plus cisplatin (60 mg/m2) (Pem‐Cis) induction chemotherapy every three weeks as first‐line treatment between January 2010 and August 2018. Patients who did not show progression after four cycles of Pem‐Cis and received at least one cycle of pemetrexed maintenance were recruited (n = 199).ResultsPatients were divided into subgroups according to total cycles of pemetrexed: ≤ 10 (F10, n = 134) and > 10 (M10, n = 65). The M10 group had a higher proportion of patients with stage M1a (intrathoracic metastasis alone) and exhibited lower levels of thymidylate synthase (TS) than the F10 group (median H‐score 10.0% vs. 60.0%; P = 0.031). Further subgrouping identified extreme responders: ≤ 7 (F7, n = 101) and ≥ 20 (M20, n = 26) cycles. The M20 group showed lower mean serum CEA levels before (17.5 vs. 147.0; P = 0.099) and after (6.9 vs. 53.2; P = 0.001) Pem‐Cis treatment, and a higher incidence of normalization after Pem‐Cis (abnormal 41.7% vs. 68.5%; P = 0.015). M1a stage, normalization of CEA levels after Pem‐Cis, and lower TS H‐score were predictors of progression‐free survival in patients administered pemetrexed maintenance.ConclusionM1a stage and lower TS expression were predictors of long‐term response to pemetrexed maintenance. CEA normalization after Pem‐Cis could be an additional surrogate marker of positive response to long‐term treatment.

Thymidylate synthase maintains the de-differentiated state of triple negative breast cancers

Cancer cells frequently boost nucleotide metabolism (NM) to support their increased proliferation, but the consequences of elevated NM on tumor de-differentiation are mostly unexplored. Here, we identified a role for thymidylate synthase (TS), a NM enzyme and established drug target, in cancer cell de-differentiation and investigated its clinical significance in breast cancer (BC). In vitro, TS knockdown increased the population of CD24+ differentiated cells, and attenuated migration and sphere-formation. RNA-seq profiling indicated repression of epithelial-to-mesenchymal transition (EMT) signature genes upon TS knockdown, and TS-deficient cells showed an increased ability to invade and metastasize in vivo, consistent with the occurrence of a partial EMT phenotype. Mechanistically, TS enzymatic activity was found essential for maintenance of the EMT/stem-like state by fueling a dihydropyrimidine dehydrogenase—dependent pyrimidine catabolism. In patient tissues, TS levels were found significantly higher in poorly differentiated and in triple negative BC, and strongly correlated with worse prognosis. The present study provides the rationale to study in-depth the role of NM at the crossroads of proliferation and differentiation, and depicts new avenues for the design of novel drug combinations for the treatment of BC.

Study of Thymidylate Synthase (TS) and Dihydropyrimidine Dehydrogenase (DPD) Expressions on 5-Fluorouracil in Oral Squamous Cell Carcinoma

Background:The study aims to analyze Thymidylate Synthase (TS) and Dihydropyrimidine Dehydrogenase (DPD) Expressions on 5-Fluorouracil in Oral Squamous Cell Carcinoma (OSCC). Methods:50 oral squamous cell carcinoma samples were taken from non-treated cancer patients at Hiroshima University Dental Hospital. The patients were investigated for TS, that included 36 males and 14 females. Additionally, 31 patients were evaluated for DPD that included 22 males and 9 females.Results:The samples had also undergone clinical and pathological evaluation, immunohistochemical staining, evaluation of immune-staining, enzymatic expression, and statistical analyses. Mean age of the population was 62.1 years. Conclusion: Over-expression of TS contributes significantly to the resistance of 5-FU treatment; while inhibition of intra-tumoral DPD increases the sensitivity level. TS levels are not only predictive of 5-FU response, but also prognostic in clinical value of non-treated cancer patients.

Thymidine phosphorylase affects clinical outcome following surgery and mRNA expression levels of four key enzymes for 5‑fluorouracil metabolism in patients with stage�I and II non‑small cell lung cancer

The expression levels of thymidine phosphorylase (TP), dihydropyrimidine dehydrogenase (DPD), thymidylate synthase (TS) and orotate phosphoribosyltransferase (OPRT) may predict the clinical efficacy of 5-fluorouracil-based chemotherapy in patients with cancer. We herein investigated the differences in the mRNA levels of these enzymes in non-small-cell lung cancer (NSCLC) and evaluated their prognostic value for NSCLC treated by surgical resection. The intratumoral mRNA levels of TP, DPD, TS, and OPRT were quantified in 66 patients with pathological stage I and II NSCLC (adenocarcinoma or squamous cell carcinoma) following complete resection according to the Danenberg Tumor Profile method. The TP level was the only significant prognostic factor for disease-specific survival (DSS) following complete resection; the mean TP mRNA level differed significantly between the high and low mRNA expression groups. The DSS at 5 years was significantly higher in the low TP mRNA compared with that in the high TP mRNA expression group (83.4 vs. 58.6%, respectively; P=0.005). A Cox proportional hazards model revealed that pathological stage, sex, and TP expression were independent prognostic factors for DSS in patients with stage I and II NSCLC following complete resection. Thus, TP level may be used to monitor treatment efficacy and predict the outcome of NSCLC patients.

Effect of Simultaneous Inhibition of Protein Kinase CK2 and Thymidylate Synthase in Leukemia and Breast Cancer Cells.

Protein kinase CK2 was recently identified as a promising therapeutic target for combination therapy. Our study aims to investigate the anticancer effect of a simultaneous inhibition of thymidylate synthase (TS) and CK2 in MCF-7 breast cancer and CCRF-CEM leukemia cells. The type of interaction between CK2 inhibitors: CX-4945, 4,5,6,7-tetrabromo-1H-benzimidazole (TBBi), or recently obtained 4,5,6,7-tetrabromo-2-methyl-1H-benzimidazol-1-yl)acetonitrile (2b) and TS-directed anticancer drug, 5-fluorouracil (5-FU) was determined using the MTT assay and a combination index method. The influence of the combined treatment on apoptosis in leukemia cells, as well as on cell-cycle progression and the levels of TS, CK2α and P-Ser529-p65 were determined in both cell lines, using flow cytometry and western blot analysis, respectively. The best synergistic effect was observed in CCRF-CEM cell line with the combination of 5-FU and 2b which correlated with a decrease in the endocellular CK2 activity and enhancement of the pro-apoptotic effect. The obtained results demonstrate the ability of CK2 inhibitors to enhance the efficacy of 5-FU in anticancer treatment, indicating a different molecular mechanism of the studied CK2 inhibitors interaction with 5-FU.

Measurement of Nucleic Acid Metabolizing Enzymes in Stage III Colorectal Cancer Adds Precision to Adjuvant Fluorouracil and Leucovorin Therapy

1. Abstract 1.1. <span style=font-size:10.0pt;mso-fareast-font-family: ms= mincho=>Background <span style=font-size:10.0pt;mso-fareast-font-family: ms= mincho=>: Recurrence of colorectal cancer has been shown to be inhibited by adjuvant chemotherapy. To achieve effective adjuvant chemotherapy, a target molecule is necessary . Therefore, we performed a prospective clinical trial to determine the relationship between adjuvant chemotherapy outcomes and prognostic factors in colorectal cancer based on nucleic acid metabolizing enzyme levels. 1.2. Methods: A total of 147 patients with stage IIIA, IIIB, and IIIC cancer were selected for adjuvant chemotherapy. We analyzed Disease-Free Survival (DFS) as the primary endpoint and Overall Survival (OS) as the secondary endpoint in relation to the Thymidylate Synthase (TS) and Dihydropyrimidine Dehydrogenase (DPD) levels as measured by enzyme-linked immunosorbent assay. According <span style=letter-spacing: -.15pt>to the levels of the mean preliminary TS of 21.0 ng/mg protein and DPD of 115 ng/mg protein, the patients were divided into 4 groups. They were then administered postoperative adjuvant chemotherapy that primarily included fluorouracil and Leucovorin (FL) or Uracil/Tegafur (UFT) and Leucovorin (UZEL) for 6 months. 1.3. Results: The 5-year DFS in advanced stage III cancer was 63.9%. A significant difference was observed between the LL (low TS and low DPD levels) and HL (high TS and low DPD levels) groups and the LH (low TS and high DPD levels) and HH (high TS and high DPD levels) groups. Multivariate<span style=letter-spacing: -.5pt> analysis revealed a significant difference<span style=letter-spacing: -.1pt> in stage, TS level, and TS and DPD levels. The OS was 79.6% for all the groups. A significant difference was observed between the LL, LH, and HL groups and the HH groups. Multivariate analysis revealed a significant difference<span style=letter-spacing: -.5pt> in the stage, age, TS level, and<span style=letter-spacing: -.55pt> TS and DPD levels. <span style=font-size:10.0pt;letter-spacing: -.15pt>1.4. Conclusions: Measurement of the TS and DPD levels of a primary colorectal cancer lesion as target molecules may be helpful for achieving an effective postoperative adjuvant chemotherapy. TS and DPD can potentially serve as more accurate predictors of OS than age and cancer stage. 2. Keywords: Adjuvant Chemotherapy; Colorectal Cancer; Metabolizing <span style=font-size: 10.0pt;mso-fareast-font-family: ms= mincho=> <span style=font-size: 10.0pt>Enzyme; Precision Medicine

Study on the correlation between thymidylate synthase expression level and postoperative chemotherapy in gastric cancer

Objective To investigate the expression of thymidylate synthase (TS), and the correlations with the clinicopathologcal factors and the prognosis of postoperative fluorouracil chemo-therapy in gastric cancer. Methods 42 patients with gastric cancer who underwent radical gastrectomy were collected from April 2011 to April 2014 in Lu′an Hospital of Anhui Medical University, and the chemotherapy regimen was Cisplatin+ Capecitabine or Cisplatin+ Teggio. The levels of TS in tumor tissues of 42 patients with gastric cancer were detected by immunohistochemical method. The relationships between TS expression level, clinical features and the prognosis of patients treated with fluorouracil chemotherapy were analyzed. Results In 42 patients with gastric cancer, 19 patients (45.2%) have TS high expressions, 23 patients (54.8%) have TS low expressions. There were no significant correlations between the expression of TS and age, sex, clinical stage, differentiation degree, tumor location, depth of invasion, lymph node metastasis, vascular tumor thrombus and nerve bundle invasion (all P>0.05). There were significant differences between low TS expression and high TS expression in median progression-free survival and 3-year survival rate of patients [35 months vs. 17 months, 73.9% (17/23) vs. 42.1% (8/19)], and the differences were statistically significant (χ2=4.788, P=0.029; χ2=4.369, P=0.037). In patients with stage Ⅲ gastric cancer, the median progression-free survival of patients with low TS expression was significantly prolonged compared to that with high TS expression (33 months vs. 9 months), and the difference was statistically significant (χ2=4.731, P=0.030). Conclusion TS expression levels have no significant correlations with the clinicopatho-logcal factors, but closely relate to the prognosis of gastric cancer patients treatment with fluorouracil chemotherapy. The chemotherapy prognosis of TS high expression is worse than that of TS low expression in gastric cancer. Key words: Thymidylate synthase; Stomach neoplasms; Chemotherapy, adjuvant; Fluorouracil

Immunohistochemical expression of thymidylate synthase and prognosis in gastric cancer patients submitted to fluoropyrimidine-based chemotherapy.

ObjectiveAdjuvant chemotherapy with 5-fluorouracil (5-FU) has been widely used in gastric cancer (GC) patients to prevent relapse after curative resection. 5-FU acts by inhibiting thymidylate synthase (TS), and high levels of TS correlate with resistance to treatment with fluoropyrimidines. The aim of this study was to evaluate the expression of TS in GC patients, and its relation with clinicopathological characteristics and prognosis in adjuvant chemotherapy with 5-FU.MethodsWe retrospectively evaluated 285 patients who underwent D2-gastrectomy with curative intent. TS expression was determined by immunohistochemistry (IHC) in tumor cells by tissue microarray (TMA). TS level was evaluated according to the intensity and percentage of cells marked by a score system. Patients were divided in three groups according to their TS-score: negative, low and high.ResultsTS expression was positive in 92.3% of GC. TS-high, TS-low and TS-negative were observed in 46.3%, 46.0% and 7.7% of patients, respectively. High-TS GC were associated with older age (P=0.007), high neutrophil/lymphocyte ratio (P=0.048), well/moderately differentiated histology (P=0.001), intestinal Lauren type (P<0.001) and absence of perineural invasion (P=0.003). Among 285 patients, 133 stage II/III patients (46.7%) received chemotherapy with 5-FU. In survival analysis, TS-high was associated with worse disease-free survival (DFS) in stage III GC patients who received 5-FU-based chemotherapy (P=0.007). Multivariate analysis revealed that total gastrectomy, poorly differentiated tumors and high TS-score were associated with worse DFS in stage III GC patients.ConclusionsHigh TS-score in stage III GC was associated with poor DFS in patients treated with fluoropyrimidine-based chemotherapy.

Predictive Significance of Thymidylate Synthase Expression in Non-small Cell Lung Cancer.

To date, many studies have suggested that thymidylate synthase (TS) could be used as a prognostic and predictive marker in non-small cell lung cancer (NSCLC) patients. However, results have been contradictory. The aim of this study was to evaluate TS mRNA levels in tumor tissue of NSCLC patients who underwent complete surgical resection and to analyze its prognostic and predictive potential. The study group consisted of 64 patients who underwent curative lung resection. Paired lung tissue samples were taken directly from the tumor tissue and from adjacent, histologically cancer-free lung tissue. The quantitative estimation of TS expression was performed by reverse transcription real-time polymerase chain reaction (RT-qPCR). The relationship between TS expression level and disease-free interval (DFI) and overall survival (OS) was analyzed. There was significantly higher TS expression in NSCLC tumor tissue comparing to normal lung tissue. In the group of patients who received adjuvant chemotherapy based on platinum derivatives in combination with paclitaxel or gemcitabine, we found shorter DFI (p=0.0473) and OS (p=0.0053) in those with high expression of TS. Our results demonstrated the relationship of high tumor tissue TS levels to adverse prognosis in patients undergoing adjuvant chemotherapy. TS is a non-specific tumor marker with respect to NSCLC, therefore we think that its best use would be as a member of the panel of predictors of adjuvant treatment efficacy.

Efficacy of platinum-based chemotherapy in EGFR WT nonsquamous advanced non-small cell lung cancer (NSCLC) patients: association with KRAS mutation and thymidylate synthase (TS) levels

Efficacy of platinum-based chemotherapy in EGFR WT nonsquamous advanced non-small cell lung cancer (NSCLC) patients: association with KRAS mutation and thymidylate synthase (TS) levels