AIM： To correlate the polymorphisms in the 5＇-untranslated region with thymidylate synthase （TS） protein expression in Han Chinese colonic neoplasms. METHODS： Adenocarcinoma samples were from 68 patients who rece...AIM： To correlate the polymorphisms in the 5＇-untranslated region with thymidylate synthase （TS） protein expression in Han Chinese colonic neoplasms. METHODS： Adenocarcinoma samples were from 68 patients who received no treatment before surgery. Tandem repeat length of TS gene was determined by PCR amplification of genomic DNA. Intratumoral TS protein expression was studied immunohistochemically in corresponding sections from paraffin-embedded primary loci. Immunoreactivity was semiquantitatively evaluated by immunoreactivity score （IRS）. RESULTS： Double-（2R） and triple-repeated （3R） sequences of the TS gene were found in the cancer tissues. Three genotypes of TS were found： 2R/2R （n = 6）, 2R/3R （n = 22） and 3R/3R （n = 40）. Patients who were homozygous for triple-repeated （3R/3R） sequences showed significantly higher IRS of TS than patients who were homozygous for double-repeated （2R/2R） sequences or heterozygous patients （2R/3R）： 5.73 ±3.25 vs 2.17 ± 1.47 or 3.77 ±2.64, P = 0.008 or P = 0.015. But no statistical significance of IRS in cancer tissues was observed between 2R/3R genotype and 2R/2R genotype. CONCLUSION： There is a relationship between TS genotype and TS protein expression in clinical specimens. The data might offer an advantage for selection of Chinese cancer patients to receive fluoropyrimidines treatment.展开更多
AIM： To investigate the expression of thymidylate synthase （TS） and glutathione-s-transferase π （GST-π） in esophageal squamous cell carcinoma and their association with the clinicopathologic characteristics. ME...AIM： To investigate the expression of thymidylate synthase （TS） and glutathione-s-transferase π （GST-π） in esophageal squamous cell carcinoma and their association with the clinicopathologic characteristics. METHODS： Immunohistochemical methods were used to detect the expression of TS and GST-π in surgically resected formalin-fixed, paraffin-embedded esophageal squamous cell carcinoma （ESCC） tissue sections from 102 patients （median age, 58 years） and in 28 normal esophageal mucosa （NEM） samples. The relationship between TS and GST-π expression and clinicopathologic factors was examined. RESULTS： The expression of TS and GST-π was not statistically significantly associated with age of the patients, tumor size, lymph node metastasis, depth of invasion or tumor stage. TS staining was positive in 17.86% of normal esophageal mucosa and in 42.16% of ESCC samples （P 〈 0.05）. The expression level of TS was not only significantly lower in well-differentiated （21.88%） than in poorly-differentiated carcinomas （51.43%, P 〈 0.05）, but was also significantly higher in samples from male patients （46.51%） than from female patients （18.75%, P 〈 0.05）. GST-π was positively stained in 78.57% of normal esophageal mucosa and in 53.92% of ESCC samples （P 〈 0.05）. The expression level of GST-π was also significantly higher in welldifferentiated carcinomas （65.63%） than in poorly- differentiated carcinomas （35.00%, P 〈 0.05）. CONCLUSION： The expression of TS and of GST-π may be used as molecular markers for the characterization of ESCC. Poorly-differentiated cells showed increased expression of T5 and reduced expression of GST-π.展开更多
Objective： Adjuvant 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）, a...Objective： Adjuvant 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 fluoropyfimidines. 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. Methods： We 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. Results： TS 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 IUIII 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. Conclusions： High TS-score in stage III GC was associated with poor DFS in patients treated with fluoropyrimidine-based chemotherapy.展开更多
AIM: To examine the expression of thymidylate synthase (TS) and oncoprotein Bcl-2 in advanced colorectal cancer (CRC) patients,and to determine their mutual relationship,association to therapeutic response and impact ...AIM: To examine the expression of thymidylate synthase (TS) and oncoprotein Bcl-2 in advanced colorectal cancer (CRC) patients,and to determine their mutual relationship,association to therapeutic response and impact on disease outcome.METHODS: Tumor samples from 67 patients with CRC,who were treated at advanced stage with either irinotecan alone or in combination with 5-fluorouracil/ leucovorin,were analyzed for expression of TS and Bcl-2 using immunohistochemistry.RESULTS: A significant linear correlation between lower expression levels of Bcl-2 and lower levels of TS expression was found (P = 0.033).Patients with high levels of both TS and Bcl-2 expression had a significantly longer disease-free survival (DFS) (42.6 mo vs 5.4 mo,n = 25) than those with low TS/Bcl-2 index (P = 0.001).Tumors with low levels of both TS and Bcl-2 were associated with a longer survival with metastasis (WMS) interval in the whole patients group (n = 67,P = 0.035).TS/Bcl-2 index was not significantly related to disease-specific survival.CONCLUSION: The present data suggest that CRC patients with low TS/Bcl-2 demonstrate a significantly shorter DFS and longer WMS.展开更多
AIM: To investigate the predictive value of Ki67 and p53 and their correlation with thymidylate synthase (TS) gene expression in a rectal cancer patient cohort treated according to a standardized recommended neoadjuva...AIM: To investigate the predictive value of Ki67 and p53 and their correlation with thymidylate synthase (TS) gene expression in a rectal cancer patient cohort treated according to a standardized recommended neoadjuvant treatment regimen.METHODS: Formalin fixed, paraffin embedded pre-therapeutical tumor biopsies (n = 22) and post-therapeutical resection specimens (n = 40) from patients with rectal adenocarcinoma (clinical UICC stage Ⅱ/Ⅲ) receiving standardized neoadjuvant 5-fluorouracil (5-FU) based chemoradiotherapy were studied for Ki67 and p53 expression by immunohistochemistry and correlated with TS mRNA expression by quantitative TaqMan real-time PCR after laser microdissection. The results were compared with histopathological tumor regression according to a standardized semiquantitative score grading system.RESULTS: Responders (patients with high tumor regression) showed a significantly lower Ki67 expression than non-responders in the pre-therapeutical tumor biopsies (81.2% vs 16.7%; P < 0.05) as well as in the post-therapeutical resection specimens (75.8% vs 14.3%; P < 0.01). High TS mRNA expression was significantly correlated with a high Ki67 index and low TS mRNA expression was significantly correlated with a low Ki67 index in the pre-therapeutical tumor biopsies (corr. coef. = 0.46; P < 0.01) as well as in the post-therapeutical resection specimens (corr. coef. = 0.40; P < 0.05). No significant association was found between p53 and TS mRNA expression or tumor regression.CONCLUSION: Ki67 has, like TS, predictive value in rectal cancer patients after neoadjuvant 5-FU based chemoradiotherapy. The close correlation between Ki67 and TS indicates that TS is involved in active cell cycle processes.展开更多
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...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.展开更多
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-P...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.展开更多
Thymidylate synthase(TS)is a key enzyme in the de novo biosynthesis of thymidine monophosphate,serving as a well-known drug target in chemotherapy against cancers and infectious diseases.Additional to its clinical val...Thymidylate synthase(TS)is a key enzyme in the de novo biosynthesis of thymidine monophosphate,serving as a well-known drug target in chemotherapy against cancers and infectious diseases.Additional to its clinical value,TS is supposed to be a promising drug target in aquatic-disease control.To facilitate designing pathogen-specific TS inhibitors for shrimp-disease control,we report the crystal structures of TS from Litopenaeus vannamei(LvTS)in the apo form,LvTS-dUMP complex and LvTS-dUMP-raltitrexed complex at 2.27Å,1.54Å,and 1.56Åresolution,respectively.LvTS shares a similar fold with known TSs,existing as a dimer in the crystal.The apo LvTS and LvTS-dUMP take an open conformation,and raltitrexed binding induces structural changes into a closed conformation in LvTS-dUMP-raltitrexed.Compared to those in other known TS-dUMP-raltitrexed complexes with the closed conformation,the C-terminal loop in LvTS-dUMP-raltitrexed shifts its position away from the bound raltitrexed;the distance between C6 of dUMP and Sγof the catalytic cysteine is obviously longer than that in the known TS structures with closed conformations,resembling that in the TS structures with open conformations.Other species-specific interactions with dUMP and raltitrexed are also observed.Therefore,LvTS-dUMP-raltitrexed adopts a loosely closed conformation with structural features intermediate between the closed and the open conformations that were reported in other TSs.Our study provides the first crustcean TS structure,and reveals species-specific interactions between TSs and the ligands,which would facilitate designing pathogen-specific TS inhibitors for shrimp-disease control.展开更多
Traditional Chinese herbal medicine(TCM)has been shown to enhance the efficacy of standard anticancer agents.However,there are only a limited number of well-controlled preclinical and clinical studies documenting the ...Traditional Chinese herbal medicine(TCM)has been shown to enhance the efficacy of standard anticancer agents.However,there are only a limited number of well-controlled preclinical and clinical studies documenting the potential benefit of TCM.OBJECTIVE To identify biologically active formulas that were effective against colorectal cancer(CRC)by screening TCM formulas in in vitro and in vivo animal models.METHODS Cell growth assays,cell cycle analysis,immunoblot analysis and qRT-PCR were performed to investigate the mechanism(s)of action of the formulason human CRC cells.In vivo animal models were used to evaluate the antitumor activity of formulasalone and in combination with 5-FU.RESULTS We identified Huangqin Gegen Tang(HQGGT)which suppressed the in vivo growth of human CRC HT-29 xenografts.HQGGT significantly inhibited the growth of CRC cell lines.HQGGT enhanced the cytotoxicity of 5-FU against human 5-FU-resistant cells(H630R1)and mouse colon cancer cells(MC38).This synergy was the result of suppression of thymidylate synthase expression by HQGGT.HQGGT significantly enhanced the antitumor effect of 5-FU in mice bearing MC38 xenografts.Ongoing studies have identified Huangqin as the herb responsible for TS inhibi⁃tion.CONCLUSION These findings provide support for the potential role of HQGGT as a novel modulator of fluoropyrim⁃idine chemotherapy for CRC treatment.展开更多
We established new pemetrexed-resistant cells originating from malignant pleural mesothelioma MSTO-211H cells to clarify the mechanism involved in pemetrexed resistance in malignant pleural mesothelioma. In the pemetr...We established new pemetrexed-resistant cells originating from malignant pleural mesothelioma MSTO-211H cells to clarify the mechanism involved in pemetrexed resistance in malignant pleural mesothelioma. In the pemetrexed-resistant cells, only thymidylate synthase (TYMS) mRNA was overexpressed among other well-known molecular targets and chemosensitivity determinants of pemetrexed, and the role of the TYMS gene was ascertained by artificial regulation induced by specific siRNA. Silencing the TYMS expression partially restored the cytotoxicity of pemetrexed. The resistant cells did not display other gene alterations related to folate metabolism. We conclude that the primary mechanism imparting resistance to these cells is specific up-regulation of TYMS function. Further, the TYMS gene may serve as a useful biomarker for the prediction of pemetrexed chemosensitivity in patients with malignant pleural mesothelioma. We also investigated the efficacy of 1-(3-C-ethynyl-β-D-ribo-pento furanosyl)cytosine (ECyd) in overcoming pemetrexed resistance;this compound is presently undergoing clinical trials in the USA as TAS-106. ECyd had a similar antitumor effect on the resistant cells as that on the parental cells. In the clinical treatment of malignant pleural mesothelioma, ECyd promises to emerge as a novel drug.展开更多
Chemotherapy drug resistance is the main cause leading to the relapse and metastasis of non-small cell lung cancer(NSCLC)patients.Our study aimed to investigate the mechanism of pemetrexed resistance in NSCLC.Firstly,...Chemotherapy drug resistance is the main cause leading to the relapse and metastasis of non-small cell lung cancer(NSCLC)patients.Our study aimed to investigate the mechanism of pemetrexed resistance in NSCLC.Firstly,the pemetrexed(PEM)-resistant PC-9 and A549 lung adenocarcinoma cell lines(PC-9/PEM and A549/PEM)were established.The expression of thymidylate synthase(TS)in PC-9/PEM,A549/PEM,A549,and PC-9 cells were analyzed by qRT-PCR and western blot.Then,cell viability,colony formation,migration,and invasion were performed on PEM-resistant cells transfected with TS siRNA.The role of EGFR in PEM resistance of PEM-resistant cells was investigated using EGFR siRNA.The effects of gefitinib and EGFR siRNA on EGFR/PI3K/AKT pathway and downstream signaling Cyclin D1 and E2F1 in PEM-resistant cells were analyzed.Results showed that the protein level of TS was significantly increased in A549/PEM and PC-9/PEM.TS knockdown inhibited the potency of proliferation,colony-forming potential,migration,and invasion in PEM-resistant cells.EGFR knockdown abrogated the resistance to PEM of PEM-resistant cells and suppressed the migration and invasion of PEM-resistant cells.Gefitinib treatment and EGFR knockdown respectively inhibited the EGFR/PI3K/AKT pathway and downregulated Cyclin D1 and E2F1 in PEM-resistant cells.Thus,TS might be a predictive marker for PEM resistance in NSCLC.Inhibition of the EGFR pathway abrogated the resistance to PEM and inhibited the EGFR/PI3K/AKT and downstream signaling of PEM-resistant NSCLC cell lines.展开更多
Pemetrexed is an antimetabolic agent and is well-known as a potent inhibitor of thymidylate synthase (TS). It also inhibits dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyl transferase (GARFT). We ...Pemetrexed is an antimetabolic agent and is well-known as a potent inhibitor of thymidylate synthase (TS). It also inhibits dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyl transferase (GARFT). We reported an intriguing case in which a non-small cell lung cancer patient who was refractory to pretreatment with S-1, a strong TS inhibitor, showed good response to pemetrexed treatment.展开更多
OBJECTIVE To investigate the correlation between polymorphism of the 5′-untranslated region （5′-UTR） of thymidylate synthase genes, as well as the lifestyle, and the susceptibility of gastric carcinoma. METHODS A ...OBJECTIVE To investigate the correlation between polymorphism of the 5′-untranslated region （5′-UTR） of thymidylate synthase genes, as well as the lifestyle, and the susceptibility of gastric carcinoma. METHODS A case-control study, with 60 cases of gastric carcinoma and 170 cases of general risk population-based controls from Nantong, Jiangsu province, China, was conducted. The epidemiological data, such as living habits of the cancer patients, were collected. DNA of peripheral blood leukocytes was obtained from all of the subjects. The TS 5′-UTR tandem repeat genotype was detected using polymerase chain reaction （PCR）. RESULTS There were three TS 5′-UTR genotypes in the group of gastric cancer cases （2R/2R, 2R/3R and 3R/3R） and six TS 5′-UTR genotypes in the group of the controls （2R/2R, 2R/3R, 3W3R, 2R/4R, 2R/5R and 3R/4R）. The genotypic frequencies were respectively 5.0%, 43.3% and 51.7% in the gastric cancer group. Compared with the parameters in the control group, i.e., 4.7%, 31.7%, 60.6%, 1.2%, 1.2% and 0.6%. There were no significant differences between the two groups. Compared with the 3R/3R- genotype individuals who where non-smokers, drank alcohol twice or less each week, drank tea and did not intake pickled food （PF）, the risk of gastric cancer significantly went up in the 2R/2R or 2R/3R-genotype people who had habits of smoking, drinking alcohol more than twice each week, no tea drinking but with frequent intake of PF. The adjusted ORs were as follows, 3.79 （95% CI： 2.45-8.64）, 3.41 （95% CI and 3.61 （95% CI： 1.81-8.78）. CONCLUSION There is 1.21-8.47）, 5.99 （95% Ch 3.01-14.7）, an obvious correlation between the polymorphisms of TS 5′-UTR genotypes and the lifestyle of individuals in the development of gastric carcinoma.展开更多
Objective:Hepatocellular carcinoma(HCC)is a leading cause of mortality worldwide.Huachansu(Cinobufacini)is active extract isolated from the dry skin of Bufo Bufo gargarizans.It has now been widely used in clinical tre...Objective:Hepatocellular carcinoma(HCC)is a leading cause of mortality worldwide.Huachansu(Cinobufacini)is active extract isolated from the dry skin of Bufo Bufo gargarizans.It has now been widely used in clinical treatment of cancer,this study is to clarify the material basis of down-regulation of thymidylate synthase(TYMS)induced by Huachansu.Methods:Our study utilized UPLC-MS/MS to identify major bioactive components from Huachansu.Cell Counting Kit 8(CCK-8)assay and clone formation assay were used to examine the cell viability of tumor cells.TYMS and y-H2AX level were detected by using quantitative real-time RT-PCR and/or western blotting.Small interfering RNA(siRNA)transfection was used to explore whether inhibition of TYMS could enhance the suppressive effect of Huachansu on cell growth of HCC cells.Results:In our study,firstly,we identify 21 major bioactive components from Huachansu.CCK-8 assay results showed that Huachansu and its bioactive bufadienolides(Bufalin,Bufotalin,Cinobufotalin,Desacetylcinobufagin,Arenobufagin,Telocinobu fagin,and Resibu fogenin)significantly inhibited the proliferation of HepG2 and SK-HEP-1 cells in a dose-and time-dependent manner.Further molecular mechanistic investigation demonstrates that Huachansu significantly suppresses thymidylate synthase(TYMS),the enzyme which provides the sole de novo source of thymidylate for DNA synthesis.The inhibition of TYMS could lead to cell-cycle block and DNA damage of HCC cells.Furthermore,we identified that Huachansu markedly increased y-H2AX expression,which indicated the presence of DNA damage.Moreover,we confirmed that transfection of cells with small interfering RNA specific to TYMS could increase the suppressive effects of Huachansu on the HCC cells proliferation.Quantitative RT-PCR analysis showed that Huachansu treatment had no effect on the transcription level of TYMS.Fu rthermore,proteasomal inhibitor MG132 could block TYMS inhibition induced by Huachansu,and concomita nt administration of protein synthesis inhibitor cycloheximide(CHX)with Huachansu could further suppress the protein level of TYMS,indicating that Huachansu promotes proteasome-dependent degradation of TYMS in liver cancer cells.More importantly,the bioactive bufadienolides of Huachansu such as Bufalin,Bufotalin,Cinobufotalin,Desacetylcinobufagin,Arenobufagin,Telocinobufagin,and Resibufogenin could also significantly restrain the protein level of TYMS,revealing the material basis of inhibition of TYMS exposed to Huachansu.5-Fluorouracil(5-FU)is a TYMS inhibitor,we also evaluate the effects of the combined treatment of Huachansu with 5-FU,the results show that interactions between Huachansu and 5-FU are synergistic or antagonistic.Thus,in clinical,attention should be paid to the dosage of Huachansu in combination with 5-FU.Conclusion:Huachansu inhibits the growth and induces DNA damage of human HCC cells through proteasome-dependent degradation of TYMS,bioactive bufadienolides are the material basis of downregulation of TYMS induced by Huachansu.展开更多
Background Thymidylate synthase （TS） is a key regulatory enzyme for de novo DNA synthesis.TS activity is also an important determinant of the response to chemotherapy with fluoropyrimidine prodrugs,and its expressio...Background Thymidylate synthase （TS） is a key regulatory enzyme for de novo DNA synthesis.TS activity is also an important determinant of the response to chemotherapy with fluoropyrimidine prodrugs,and its expression may be affected by gene polymorphisms.In this study,we investigated the associations between polymorphisms of the TS gene and its protein expression,and the implications on the efficacy of 5-fluorouracil （5-FU） in pancreatic cancer cells.Methods Genotypes based on the 28-bp TS tandem repeat for pancreatic cell lines were determined by electrophoretic analysis of PCR products.A single nucleotide polymorphism （SNP） at nucleotide 12 of the second 28-bp repeat of the 3R allele was determined by nucleotide sequencing.The chemosensitivity of pancreatic carcinoma cells to 5-FU in vitro was evaluated using Cell Counting Kit-8 （CCK-8）.TS protein expression was analyzed by Western blotting.Results 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）.The optical density ratio of genotypes 3R/3R,2R/2R and 2R/3R was 1.393±0.374,0.568±0.032 and 0.561±0.056,respectively.Cells with the 2R/3R or 3R/3R genotypes were further analyzed for the G to C SNP at nucleotide 12 of the second 28-bp repeat of the 3R allele,yielding heterozygous 2R/3Rc （AsPC-1,Capan-1,and SU86.86）,homozygous 3Rg/3Rg （COLO357） and homozygous 3Rc/3Rc （PANC-1）.The optical density ratio of homozygous 3Rg/3Rg cells and homozygous 3Rc/3Rc cells was 1.723±0.062 and 1.063±0.134,respectively,and this difference was statistically significant （P 〈0.05）.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.Conclusions Polymorphisms in the TS gene influenced its protein expression and affected sensitivity of 5-FU in seven pancreatic cancer cell lines.Cells with the 3R/3R genotype had higher TS protein expression than the 2R/2R or 2R/3R genotypes.Cells of the 3R/3R genotype with high TS protein expression were shown lower 5-FU sensitivity than cells with the 2R/2R or 2R/3R genotypes.These data warrant large-scale clinical studies to assess the role of polymorphisms in the TS gene on its protein expression and chemosensitivity to 5-FU in pancreatic cancer.展开更多
Thymidylate synthase (TS),an essential enzyme for catalyzing the biosynthesisof thymidylate,is a critical therapeutic target in cancer therapy.Recent studies have shown that TSfunctions as an RNA-binding protein by in...Thymidylate synthase (TS),an essential enzyme for catalyzing the biosynthesisof thymidylate,is a critical therapeutic target in cancer therapy.Recent studies have shown that TSfunctions as an RNA-binding protein by interacting with two different sequences on its ownmRNA,thus,repressing translational efficiency.In this study,peptides binding TS RNA with highaffinity were isolated using mRNA display from a large peptide library (】10^(13) differentsequences).The randomized library was subjected up to twelve rounds of in vitro selection andamplification.Comparing the amino acid composition of the selected peptides (12th round,R12) withthose from the initial random library (round zero,R0),the basic and aromatic residues in theselected peptides were enriched significantly,suggesting that these peptide regions might beimportant in the peptide-TS mRNA interaction.Categorizing the amino acids at each random positionbased on their physicochemical properties and comparing the distributions with those of the initialrandom pool,an obvious basic charge characteristic was found at positions 1,12,17 and 18,suggestingthat basic side chains participate in RNA binding.Secondary structure prediction showed that theselected peptides of R12 pool represented a helical propensity compared with R0 pool,and the regionswere rich in basic residues.The electrophoretic gel mobility shift and in vitro translation assaysshowed that the peptides selected using mRNA display could bind TS RNA specifically and inhibit thetranslation of TS mRNA.Our results suggested that the identified peptides could be used as new TSinhibitors and developed to a novel class of anticancer agents.展开更多
文摘AIM： To correlate the polymorphisms in the 5＇-untranslated region with thymidylate synthase （TS） protein expression in Han Chinese colonic neoplasms. METHODS： Adenocarcinoma samples were from 68 patients who received no treatment before surgery. Tandem repeat length of TS gene was determined by PCR amplification of genomic DNA. Intratumoral TS protein expression was studied immunohistochemically in corresponding sections from paraffin-embedded primary loci. Immunoreactivity was semiquantitatively evaluated by immunoreactivity score （IRS）. RESULTS： Double-（2R） and triple-repeated （3R） sequences of the TS gene were found in the cancer tissues. Three genotypes of TS were found： 2R/2R （n = 6）, 2R/3R （n = 22） and 3R/3R （n = 40）. Patients who were homozygous for triple-repeated （3R/3R） sequences showed significantly higher IRS of TS than patients who were homozygous for double-repeated （2R/2R） sequences or heterozygous patients （2R/3R）： 5.73 ±3.25 vs 2.17 ± 1.47 or 3.77 ±2.64, P = 0.008 or P = 0.015. But no statistical significance of IRS in cancer tissues was observed between 2R/3R genotype and 2R/2R genotype. CONCLUSION： There is a relationship between TS genotype and TS protein expression in clinical specimens. The data might offer an advantage for selection of Chinese cancer patients to receive fluoropyrimidines treatment.
基金Supported by The Key Medical Talent Foundation of Jiangsu Province,China,No.RC2007036
文摘AIM： To investigate the expression of thymidylate synthase （TS） and glutathione-s-transferase π （GST-π） in esophageal squamous cell carcinoma and their association with the clinicopathologic characteristics. METHODS： Immunohistochemical methods were used to detect the expression of TS and GST-π in surgically resected formalin-fixed, paraffin-embedded esophageal squamous cell carcinoma （ESCC） tissue sections from 102 patients （median age, 58 years） and in 28 normal esophageal mucosa （NEM） samples. The relationship between TS and GST-π expression and clinicopathologic factors was examined. RESULTS： The expression of TS and GST-π was not statistically significantly associated with age of the patients, tumor size, lymph node metastasis, depth of invasion or tumor stage. TS staining was positive in 17.86% of normal esophageal mucosa and in 42.16% of ESCC samples （P 〈 0.05）. The expression level of TS was not only significantly lower in well-differentiated （21.88%） than in poorly-differentiated carcinomas （51.43%, P 〈 0.05）, but was also significantly higher in samples from male patients （46.51%） than from female patients （18.75%, P 〈 0.05）. GST-π was positively stained in 78.57% of normal esophageal mucosa and in 53.92% of ESCC samples （P 〈 0.05）. The expression level of GST-π was also significantly higher in welldifferentiated carcinomas （65.63%） than in poorly- differentiated carcinomas （35.00%, P 〈 0.05）. CONCLUSION： The expression of TS and of GST-π may be used as molecular markers for the characterization of ESCC. Poorly-differentiated cells showed increased expression of T5 and reduced expression of GST-π.
基金supported by Fundacao de Amparo a Pesquisa do Estado de Sao Paulo(FAPESP agency)(No2016/25524-0)
文摘Objective： Adjuvant 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 fluoropyfimidines. 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. Methods： We 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. Results： TS 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 IUIII 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. Conclusions： High TS-score in stage III GC was associated with poor DFS in patients treated with fluoropyrimidine-based chemotherapy.
文摘AIM: To examine the expression of thymidylate synthase (TS) and oncoprotein Bcl-2 in advanced colorectal cancer (CRC) patients,and to determine their mutual relationship,association to therapeutic response and impact on disease outcome.METHODS: Tumor samples from 67 patients with CRC,who were treated at advanced stage with either irinotecan alone or in combination with 5-fluorouracil/ leucovorin,were analyzed for expression of TS and Bcl-2 using immunohistochemistry.RESULTS: A significant linear correlation between lower expression levels of Bcl-2 and lower levels of TS expression was found (P = 0.033).Patients with high levels of both TS and Bcl-2 expression had a significantly longer disease-free survival (DFS) (42.6 mo vs 5.4 mo,n = 25) than those with low TS/Bcl-2 index (P = 0.001).Tumors with low levels of both TS and Bcl-2 were associated with a longer survival with metastasis (WMS) interval in the whole patients group (n = 67,P = 0.035).TS/Bcl-2 index was not significantly related to disease-specific survival.CONCLUSION: The present data suggest that CRC patients with low TS/Bcl-2 demonstrate a significantly shorter DFS and longer WMS.
文摘AIM: To investigate the predictive value of Ki67 and p53 and their correlation with thymidylate synthase (TS) gene expression in a rectal cancer patient cohort treated according to a standardized recommended neoadjuvant treatment regimen.METHODS: Formalin fixed, paraffin embedded pre-therapeutical tumor biopsies (n = 22) and post-therapeutical resection specimens (n = 40) from patients with rectal adenocarcinoma (clinical UICC stage Ⅱ/Ⅲ) receiving standardized neoadjuvant 5-fluorouracil (5-FU) based chemoradiotherapy were studied for Ki67 and p53 expression by immunohistochemistry and correlated with TS mRNA expression by quantitative TaqMan real-time PCR after laser microdissection. The results were compared with histopathological tumor regression according to a standardized semiquantitative score grading system.RESULTS: Responders (patients with high tumor regression) showed a significantly lower Ki67 expression than non-responders in the pre-therapeutical tumor biopsies (81.2% vs 16.7%; P < 0.05) as well as in the post-therapeutical resection specimens (75.8% vs 14.3%; P < 0.01). High TS mRNA expression was significantly correlated with a high Ki67 index and low TS mRNA expression was significantly correlated with a low Ki67 index in the pre-therapeutical tumor biopsies (corr. coef. = 0.46; P < 0.01) as well as in the post-therapeutical resection specimens (corr. coef. = 0.40; P < 0.05). No significant association was found between p53 and TS mRNA expression or tumor regression.CONCLUSION: Ki67 has, like TS, predictive value in rectal cancer patients after neoadjuvant 5-FU based chemoradiotherapy. The close correlation between Ki67 and TS indicates that TS is involved in active cell cycle processes.
文摘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.
基金supported by a grant from the National Natural Sciences Foundation of China (No. 30973184)
文摘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.
基金Supported by the National Natural Science Foundation of China(Nos.31572660,31872600)the“1000 Talents Program”,and the Qingdao Innovation Leadership Project(No.18-1-2-12-zhc)。
文摘Thymidylate synthase(TS)is a key enzyme in the de novo biosynthesis of thymidine monophosphate,serving as a well-known drug target in chemotherapy against cancers and infectious diseases.Additional to its clinical value,TS is supposed to be a promising drug target in aquatic-disease control.To facilitate designing pathogen-specific TS inhibitors for shrimp-disease control,we report the crystal structures of TS from Litopenaeus vannamei(LvTS)in the apo form,LvTS-dUMP complex and LvTS-dUMP-raltitrexed complex at 2.27Å,1.54Å,and 1.56Åresolution,respectively.LvTS shares a similar fold with known TSs,existing as a dimer in the crystal.The apo LvTS and LvTS-dUMP take an open conformation,and raltitrexed binding induces structural changes into a closed conformation in LvTS-dUMP-raltitrexed.Compared to those in other known TS-dUMP-raltitrexed complexes with the closed conformation,the C-terminal loop in LvTS-dUMP-raltitrexed shifts its position away from the bound raltitrexed;the distance between C6 of dUMP and Sγof the catalytic cysteine is obviously longer than that in the known TS structures with closed conformations,resembling that in the TS structures with open conformations.Other species-specific interactions with dUMP and raltitrexed are also observed.Therefore,LvTS-dUMP-raltitrexed adopts a loosely closed conformation with structural features intermediate between the closed and the open conformations that were reported in other TSs.Our study provides the first crustcean TS structure,and reveals species-specific interactions between TSs and the ligands,which would facilitate designing pathogen-specific TS inhibitors for shrimp-disease control.
文摘Traditional Chinese herbal medicine(TCM)has been shown to enhance the efficacy of standard anticancer agents.However,there are only a limited number of well-controlled preclinical and clinical studies documenting the potential benefit of TCM.OBJECTIVE To identify biologically active formulas that were effective against colorectal cancer(CRC)by screening TCM formulas in in vitro and in vivo animal models.METHODS Cell growth assays,cell cycle analysis,immunoblot analysis and qRT-PCR were performed to investigate the mechanism(s)of action of the formulason human CRC cells.In vivo animal models were used to evaluate the antitumor activity of formulasalone and in combination with 5-FU.RESULTS We identified Huangqin Gegen Tang(HQGGT)which suppressed the in vivo growth of human CRC HT-29 xenografts.HQGGT significantly inhibited the growth of CRC cell lines.HQGGT enhanced the cytotoxicity of 5-FU against human 5-FU-resistant cells(H630R1)and mouse colon cancer cells(MC38).This synergy was the result of suppression of thymidylate synthase expression by HQGGT.HQGGT significantly enhanced the antitumor effect of 5-FU in mice bearing MC38 xenografts.Ongoing studies have identified Huangqin as the herb responsible for TS inhibi⁃tion.CONCLUSION These findings provide support for the potential role of HQGGT as a novel modulator of fluoropyrim⁃idine chemotherapy for CRC treatment.
文摘We established new pemetrexed-resistant cells originating from malignant pleural mesothelioma MSTO-211H cells to clarify the mechanism involved in pemetrexed resistance in malignant pleural mesothelioma. In the pemetrexed-resistant cells, only thymidylate synthase (TYMS) mRNA was overexpressed among other well-known molecular targets and chemosensitivity determinants of pemetrexed, and the role of the TYMS gene was ascertained by artificial regulation induced by specific siRNA. Silencing the TYMS expression partially restored the cytotoxicity of pemetrexed. The resistant cells did not display other gene alterations related to folate metabolism. We conclude that the primary mechanism imparting resistance to these cells is specific up-regulation of TYMS function. Further, the TYMS gene may serve as a useful biomarker for the prediction of pemetrexed chemosensitivity in patients with malignant pleural mesothelioma. We also investigated the efficacy of 1-(3-C-ethynyl-β-D-ribo-pento furanosyl)cytosine (ECyd) in overcoming pemetrexed resistance;this compound is presently undergoing clinical trials in the USA as TAS-106. ECyd had a similar antitumor effect on the resistant cells as that on the parental cells. In the clinical treatment of malignant pleural mesothelioma, ECyd promises to emerge as a novel drug.
文摘Chemotherapy drug resistance is the main cause leading to the relapse and metastasis of non-small cell lung cancer(NSCLC)patients.Our study aimed to investigate the mechanism of pemetrexed resistance in NSCLC.Firstly,the pemetrexed(PEM)-resistant PC-9 and A549 lung adenocarcinoma cell lines(PC-9/PEM and A549/PEM)were established.The expression of thymidylate synthase(TS)in PC-9/PEM,A549/PEM,A549,and PC-9 cells were analyzed by qRT-PCR and western blot.Then,cell viability,colony formation,migration,and invasion were performed on PEM-resistant cells transfected with TS siRNA.The role of EGFR in PEM resistance of PEM-resistant cells was investigated using EGFR siRNA.The effects of gefitinib and EGFR siRNA on EGFR/PI3K/AKT pathway and downstream signaling Cyclin D1 and E2F1 in PEM-resistant cells were analyzed.Results showed that the protein level of TS was significantly increased in A549/PEM and PC-9/PEM.TS knockdown inhibited the potency of proliferation,colony-forming potential,migration,and invasion in PEM-resistant cells.EGFR knockdown abrogated the resistance to PEM of PEM-resistant cells and suppressed the migration and invasion of PEM-resistant cells.Gefitinib treatment and EGFR knockdown respectively inhibited the EGFR/PI3K/AKT pathway and downregulated Cyclin D1 and E2F1 in PEM-resistant cells.Thus,TS might be a predictive marker for PEM resistance in NSCLC.Inhibition of the EGFR pathway abrogated the resistance to PEM and inhibited the EGFR/PI3K/AKT and downstream signaling of PEM-resistant NSCLC cell lines.
文摘Pemetrexed is an antimetabolic agent and is well-known as a potent inhibitor of thymidylate synthase (TS). It also inhibits dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyl transferase (GARFT). We reported an intriguing case in which a non-small cell lung cancer patient who was refractory to pretreatment with S-1, a strong TS inhibitor, showed good response to pemetrexed treatment.
基金The work was supported by a grant from Nantong Municipal Bureau of Public Health,Jiangsu Province, China(2006[No.29])
文摘OBJECTIVE To investigate the correlation between polymorphism of the 5′-untranslated region （5′-UTR） of thymidylate synthase genes, as well as the lifestyle, and the susceptibility of gastric carcinoma. METHODS A case-control study, with 60 cases of gastric carcinoma and 170 cases of general risk population-based controls from Nantong, Jiangsu province, China, was conducted. The epidemiological data, such as living habits of the cancer patients, were collected. DNA of peripheral blood leukocytes was obtained from all of the subjects. The TS 5′-UTR tandem repeat genotype was detected using polymerase chain reaction （PCR）. RESULTS There were three TS 5′-UTR genotypes in the group of gastric cancer cases （2R/2R, 2R/3R and 3R/3R） and six TS 5′-UTR genotypes in the group of the controls （2R/2R, 2R/3R, 3W3R, 2R/4R, 2R/5R and 3R/4R）. The genotypic frequencies were respectively 5.0%, 43.3% and 51.7% in the gastric cancer group. Compared with the parameters in the control group, i.e., 4.7%, 31.7%, 60.6%, 1.2%, 1.2% and 0.6%. There were no significant differences between the two groups. Compared with the 3R/3R- genotype individuals who where non-smokers, drank alcohol twice or less each week, drank tea and did not intake pickled food （PF）, the risk of gastric cancer significantly went up in the 2R/2R or 2R/3R-genotype people who had habits of smoking, drinking alcohol more than twice each week, no tea drinking but with frequent intake of PF. The adjusted ORs were as follows, 3.79 （95% CI： 2.45-8.64）, 3.41 （95% CI and 3.61 （95% CI： 1.81-8.78）. CONCLUSION There is 1.21-8.47）, 5.99 （95% Ch 3.01-14.7）, an obvious correlation between the polymorphisms of TS 5′-UTR genotypes and the lifestyle of individuals in the development of gastric carcinoma.
基金financially supported by National Natural Science Foundation of China Grant(No.82204693)Taishan scholar engineering youth expert program of Shandong Province(No.tsqn202103110)+2 种基金Young and Creative Team for Talent Introduction of Shandong Province(No.10073004)Binzhou Medical University Scientific Research Fund for High-level Talents(NO.BY2020KYQD13,2019KYQD06)Standard study on Chinese medicinal materials and Decoction pieces in Shandong Province(No.2020-201)。
文摘Objective:Hepatocellular carcinoma(HCC)is a leading cause of mortality worldwide.Huachansu(Cinobufacini)is active extract isolated from the dry skin of Bufo Bufo gargarizans.It has now been widely used in clinical treatment of cancer,this study is to clarify the material basis of down-regulation of thymidylate synthase(TYMS)induced by Huachansu.Methods:Our study utilized UPLC-MS/MS to identify major bioactive components from Huachansu.Cell Counting Kit 8(CCK-8)assay and clone formation assay were used to examine the cell viability of tumor cells.TYMS and y-H2AX level were detected by using quantitative real-time RT-PCR and/or western blotting.Small interfering RNA(siRNA)transfection was used to explore whether inhibition of TYMS could enhance the suppressive effect of Huachansu on cell growth of HCC cells.Results:In our study,firstly,we identify 21 major bioactive components from Huachansu.CCK-8 assay results showed that Huachansu and its bioactive bufadienolides(Bufalin,Bufotalin,Cinobufotalin,Desacetylcinobufagin,Arenobufagin,Telocinobu fagin,and Resibu fogenin)significantly inhibited the proliferation of HepG2 and SK-HEP-1 cells in a dose-and time-dependent manner.Further molecular mechanistic investigation demonstrates that Huachansu significantly suppresses thymidylate synthase(TYMS),the enzyme which provides the sole de novo source of thymidylate for DNA synthesis.The inhibition of TYMS could lead to cell-cycle block and DNA damage of HCC cells.Furthermore,we identified that Huachansu markedly increased y-H2AX expression,which indicated the presence of DNA damage.Moreover,we confirmed that transfection of cells with small interfering RNA specific to TYMS could increase the suppressive effects of Huachansu on the HCC cells proliferation.Quantitative RT-PCR analysis showed that Huachansu treatment had no effect on the transcription level of TYMS.Fu rthermore,proteasomal inhibitor MG132 could block TYMS inhibition induced by Huachansu,and concomita nt administration of protein synthesis inhibitor cycloheximide(CHX)with Huachansu could further suppress the protein level of TYMS,indicating that Huachansu promotes proteasome-dependent degradation of TYMS in liver cancer cells.More importantly,the bioactive bufadienolides of Huachansu such as Bufalin,Bufotalin,Cinobufotalin,Desacetylcinobufagin,Arenobufagin,Telocinobufagin,and Resibufogenin could also significantly restrain the protein level of TYMS,revealing the material basis of inhibition of TYMS exposed to Huachansu.5-Fluorouracil(5-FU)is a TYMS inhibitor,we also evaluate the effects of the combined treatment of Huachansu with 5-FU,the results show that interactions between Huachansu and 5-FU are synergistic or antagonistic.Thus,in clinical,attention should be paid to the dosage of Huachansu in combination with 5-FU.Conclusion:Huachansu inhibits the growth and induces DNA damage of human HCC cells through proteasome-dependent degradation of TYMS,bioactive bufadienolides are the material basis of downregulation of TYMS induced by Huachansu.
文摘Background Thymidylate synthase （TS） is a key regulatory enzyme for de novo DNA synthesis.TS activity is also an important determinant of the response to chemotherapy with fluoropyrimidine prodrugs,and its expression may be affected by gene polymorphisms.In this study,we investigated the associations between polymorphisms of the TS gene and its protein expression,and the implications on the efficacy of 5-fluorouracil （5-FU） in pancreatic cancer cells.Methods Genotypes based on the 28-bp TS tandem repeat for pancreatic cell lines were determined by electrophoretic analysis of PCR products.A single nucleotide polymorphism （SNP） at nucleotide 12 of the second 28-bp repeat of the 3R allele was determined by nucleotide sequencing.The chemosensitivity of pancreatic carcinoma cells to 5-FU in vitro was evaluated using Cell Counting Kit-8 （CCK-8）.TS protein expression was analyzed by Western blotting.Results 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）.The optical density ratio of genotypes 3R/3R,2R/2R and 2R/3R was 1.393±0.374,0.568±0.032 and 0.561±0.056,respectively.Cells with the 2R/3R or 3R/3R genotypes were further analyzed for the G to C SNP at nucleotide 12 of the second 28-bp repeat of the 3R allele,yielding heterozygous 2R/3Rc （AsPC-1,Capan-1,and SU86.86）,homozygous 3Rg/3Rg （COLO357） and homozygous 3Rc/3Rc （PANC-1）.The optical density ratio of homozygous 3Rg/3Rg cells and homozygous 3Rc/3Rc cells was 1.723±0.062 and 1.063±0.134,respectively,and this difference was statistically significant （P 〈0.05）.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.Conclusions Polymorphisms in the TS gene influenced its protein expression and affected sensitivity of 5-FU in seven pancreatic cancer cell lines.Cells with the 3R/3R genotype had higher TS protein expression than the 2R/2R or 2R/3R genotypes.Cells of the 3R/3R genotype with high TS protein expression were shown lower 5-FU sensitivity than cells with the 2R/2R or 2R/3R genotypes.These data warrant large-scale clinical studies to assess the role of polymorphisms in the TS gene on its protein expression and chemosensitivity to 5-FU in pancreatic cancer.
基金Supported by the National Natural Science Foundation of China (Grant No. 30472043)the Department of Science and Technology of Shandong Province (Grant No. 2004C07)
文摘Thymidylate synthase (TS),an essential enzyme for catalyzing the biosynthesisof thymidylate,is a critical therapeutic target in cancer therapy.Recent studies have shown that TSfunctions as an RNA-binding protein by interacting with two different sequences on its ownmRNA,thus,repressing translational efficiency.In this study,peptides binding TS RNA with highaffinity were isolated using mRNA display from a large peptide library (】10^(13) differentsequences).The randomized library was subjected up to twelve rounds of in vitro selection andamplification.Comparing the amino acid composition of the selected peptides (12th round,R12) withthose from the initial random library (round zero,R0),the basic and aromatic residues in theselected peptides were enriched significantly,suggesting that these peptide regions might beimportant in the peptide-TS mRNA interaction.Categorizing the amino acids at each random positionbased on their physicochemical properties and comparing the distributions with those of the initialrandom pool,an obvious basic charge characteristic was found at positions 1,12,17 and 18,suggestingthat basic side chains participate in RNA binding.Secondary structure prediction showed that theselected peptides of R12 pool represented a helical propensity compared with R0 pool,and the regionswere rich in basic residues.The electrophoretic gel mobility shift and in vitro translation assaysshowed that the peptides selected using mRNA display could bind TS RNA specifically and inhibit thetranslation of TS mRNA.Our results suggested that the identified peptides could be used as new TSinhibitors and developed to a novel class of anticancer agents.
