Predictive value of Ki67 and p53 in locally advanced rectal cancer:Correlation with thymidylate synthase and histopathological tumor regression after neoadjuvant 5-FU-based chemoradiotherapy

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.

Polymorphism of thymidylate synthase gene associated with its protein expression in human colon cancer

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.

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

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

Expression of thymidylate synthase and glutathione-stransferase π in patients with esophageal squamous cell carcinoma

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.

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

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

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

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.

Bcl-2 expression significantly correlates with thymidylate synthase expression in colorectal cancer patients

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.

Structural analysis of a shrimp thymidylate synthase reveals species-specific interactions with dUMP and raltitrexed

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.

Thymidylate synthase confers pemetrexed resistance of non-small cell lung cancer cells by EGFR/PI3K/AKT pathway

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.

Herbal formula enhances 5-fluorouracil activity through suppression of thymidylate synthase

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.

Correlation between Thymidylate Synthase Genotype and Susceptibility to Gastric Carcinoma

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.

The Role of Thymidylate Synthase in Pemetrexed-Resistant Malignant Pleural Mesothelioma Cells

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.

Good Response to Pemetrexed after Treatment Failure with Another Thymidylate Synthase Inhibitor in Lung Adenocarcinoma: A Case Report

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.

Expression, purification and activity assay of recombinant human thymidylate synthase

Thymidylate synthase （TS, E.C.2.1.1.45） catalyzes a critical reaction in the only pathway of de novo synthesis of thymidylate （dTMP） in human cells, and is an important target of chemotherapy. To evaluate the inhibitory activities of novel compounds to TS, a convenient method of activity assay using 6x His-tagged recombinant human TS （rhTS） was established and 49 novel synthetic folate analogues were screened to discover potential TS inhibitors. During the process, 4 novel compounds were found to effectively inhibit TS, while the IC 50 of a positive control raltitrexed was 3.4 μM in this assay.

Associations between gene polymorphisms of thymidylate synthase with its protein expression and chemosensitivity to 5-fluorouracil in pancreatic carcinoma cells

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.

Thymidylate synthase genetic polymorphisms and cancer risk： a meta-analysis of 37 case-control studies

Background Several studies have evaluated the association between polymorphisms of thymidylate synthase （TS） and cancer risk in diverse populations but with conflicting results. By pooling the relatively small samples in each study, it is possible to evaluate the association using a meta-analysis. Methods A comprehensive search was conducted to identify all case-control studies on TS on a 28-bp tandem repeats in 5＇untranslated region （5UTR） and a 6-bp insertion （ins） and deletion （del） mutation in 3＇UTR of the gene and cancer risk. Meta-analysis was conducted using a fixed and random effect model. Results Our meta-analysis on a total of 13307 cancer cases and 18226 control subjects from 37 published case-control studies showed no significant association between the risk of cancer and the 5＇UTR 28-bp tandem repeats polymorphism （3R/3R vs. 2R/2R： 0R=1.06, 95% CI, 0.93-1.20） or the 3＇UTR 6-bp ins/del polymorphism （del6/del6 vs. ins6/ins6： OR=0.93, 95% CI, 0.81-1.08） with significant between-study heterogeneity. In the cancer type- and ethnic subgroup-stratification analyses, we did not find any association between TS polymorphisms and cancer risk either. Conclusion TS 5＇UTR 28-bp tandem repeats and 3＇UTR 6-bp ins/del polymorphisms may not be associated with cancer risk.

In vitro selected peptides bind with thymidylate synthase mRNA and inhibit its translation

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.

UGT1A1 predicts outcome in colorectal cancer treated with irinotecan and fluorouracil

AIM:To evaluate effects of UDP-glucuronosyltransferase1A1(UGT1A1) and thymidylate synthetase(TS) gene polymorphisms on irinotecan in metastatic colorectal cancer(mCRC).METHODS:Two irinotecan-and fluorouracil-based regimens,FOLFIRI and IFL,were selected as second-line therapy for 138 Chinese mCRC patients.Genomic DNA was extracted from peripheral blood samples before treatment.UGT1A1 and TS gene polymorphisms were determined by direct sequencing and restriction fragment length polymorphism,respectively.Gene polymorphisms of UGT1A1*28,UGT1A1*6 and promoter enhancer region of TS were analyzed.The relationship between genetic polymorphisms and clinical outcome,that is,response,toxicity and survival were assessed.Pharmacokinetic analyses were performed in a subgroup patients based on different UGT1A1 genotypes.Plasma concentration of irinotecan and its active metabolite SN-38 and inactive metabolite SN-38G were determined by high performance liquid chromatography.Differences in irinotecan and its metabolites between UGT1A1 gene variants were compared.RESULTS:One hundred and eight patients received the FOLFIRI regimen,29 the IFL regimen,and one irinotecan monotherapy.One hundred and thirty patients were eligible for toxicity and 111 for efficacy evaluation.One hundred and thirty-six patients were tested for UGT1A1*28 and *6 genotypes and 125 for promoter enhancer region of TS.Patients showed a higher frequency of wild-type UGT1A1*28(TA6/6) compared with a Caucasian population(69.9% vs 45.2%).No significant difference was found between response rates and UGT1A1 genotype,although wild-type showed lower response rates compared with other variants(17.9% vs 24.2% for UGT1A1*28,15.7% vs 26.8% for UGT1A1*6).When TS was considered,the subgroup with homozygous UGT1A1*28(TA7/7) and non-3RG genotypes showed the highest response rate(33.3%),while wild-type UGT1A1*28(TA6/6) with non-3RG only had a 13.6% response rate,but no significant difference was found.Logistic regression showed treatment duration was closely linked to clinical response.In toxicity comparison,UGT1A1*28 TA6/6 was associated with lower incidence of grade 2-4 diarrhea(27.8% vs 100%),and significantly reduced the risk of grade 4 neutropenia compared with TA7/7(7.8% vs 37.5%).Wild-type UGT1A1*6(G/G) tended to have a lower incidence of grade 3/4 diarrhea vs homozygous mutant(A/A) genotype(13.0% vs 40.0%).Taking UGT1A1 and TS genotypes together,lower incidence of grade 2-4 diarrhea was found in patients with non-3RG TS genotypes,when TA6/6 was compared with TA7/7(35.3% vs 100.0%).No significant association with time to progression(TTP) and overall survival(OS) was observed with either UGT1A1 or TS gene polymorphisms,although slightly longer TTP and OS were found with UGT1A1*28(TA6/6).Irinotecan PK was investigated in 34 patients,which showed high area under concentration curve(AUC) of irinotecan and SN-38,but low AUC ratio(SN-38G /SN-38) in those patients with UGT1A1*28 TA7/7.CONCLUSION:A distinct distribution pattern of UGT1A1 genotypes in Chinese patients might contribute to relatively low toxicity associated with irinotecan and 5-fluorouracil in mCRC patients.

A predictive factor for the response to S-1 plus cisplatin in gastric cancer

AIM:To prove that the protein expression level of thymidylate synthase is a predictive factor for the response to S-1/cisplatin(CDDP)chemotherapy in gastric cancer.METHODS:We measured the protein expression levels of thymidylate synthase(TS),dihydropyrimidine dehydrogenase(DPD),and orotate phosphoribosyltrans-ferase(OPRT)in advanced gastric cancer.Before S-1/CDDP chemotherapy,tumor specimens from primary sites were obtained by endoscopic biopsy and analyzed by enzyme-linked immunosorbent assay.The chemo-therapeutic effects on the primary sites were evaluated by endoscopic biopsy performed more than once after S-1/CDDP chemotherapy.The effects are a predictive factor for the response to S-1/CDDP chemotherapy inpatients with advanced gastric cancer,as evaluated by endoscopic biopsy over time.RESULTS:The protein expression level of TS was significantly higher(P<0.05)in the tumor than in the normal tissue,and significantly lower(P<0.05)in the responders than in the non-responders.We were able to evaluate the correlation between changes in the protein expression levels of TS,DPD and OPRT and chemotherapeutic responses in 7 patients by assessing tumor tissues more than twice.In the responders,the protein expression level of TS was<40 ng/mg protein.However,there were significant increases in the protein expression levels of TS(P<0.01)and DPD(P<0.05)after chemotherapy in 3 patients.In these cases,the patient assessment changed from"responder"to"non-responder".In the non-responders,the protein expression level of TS was>40 ng/mg protein.CONCLUSION:We have confirmed that the protein expression level of TS is a predictive factor for the response to S-1/CDDP chemotherapy in patients with advanced gastric cancer.

TYMS/KRAS/BRAF molecular profiling predicts survival following adjuvant chemotherapy in colorectal cancer

BACKGROUND Patients with stage II-III colorectal cancer (CRC) treated with adjuvant chemotherapy, gain a 25% survival benefit. In the context of personalized medicine, there is a need to identify patients with CRC who may benefit from adjuvant chemotherapy. Molecular profiling could guide treatment decisions in these patients. Thymidylate synthase (TYMS) gene polymorphisms, KRAS and BRAF could be included in the molecular profile under consideration. AIM To investigate the association of TYMS gene polymorphisms, KRAS and BRAF mutations with survival of CRC patients treated with chemotherapy.METHODS A retrospective study studied formalin-fixed paraffin-embedded tissues (FFPEs) of consecutive patients treated with adjuvant chemotherapy during January/2005-January/2007. FFPEs were analysed with PCR for the detection of TYMS polymorphisms, mutated KRAS (mKRAS) and BRAF (mBRAF). Patients were classified into three groups (high, medium and low risk) according to 5’UTR TYMS polymorphisms Similarly, based on 3’UTR polymorphism ins/loss of heterozygosity (LOH) patients were allocated into two groups (high and low risk of relapse, respectively). Cox regression models examined the associated 5- year survival outcomes. RESULTS One hundred and thirty patients with early stage CRC (stage I-II: 55 patients;stage III 75 patients;colon: 70 patients;rectal: 60 patients) were treated with surgery and chemotherapy. The 5-year disease free survival and overall survival rate was 61.6% and 73.9% respectively. 5’UTR polymorphisms of intermediate TYMS polymorphisms (2RG/3RG, 2RG/LOH, 3RC/LOH) were associated with lower risk for relapse [hazard ratio (HR) 0.320, P = 0.02 and HR 0.343, P = 0.013 respectively] and death (HR 0.368, P = 0.031 and HR 0.394, P = 0.029 respectively). The 3’UTR polymorphism ins/LOH was independently associated with increased risk for disease recurrence (P = 0.001) and death (P = 0.005). mBRAF (3.8% of patients) was associated with increased risk of death (HR 4.500, P = 0.022) whereas mKRAS (39% of patients) not. CONCLUSION Prospective validating studies are required to confirm whether 2RG/3RG, 2RG/LOH, 3RC/LOH, absence of ins/LOH and wild type BRAF may indicate patients at lower risk of relapse following adjuvant chemotherapy.