Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.604
Filtrar
1.
J Enzyme Inhib Med Chem ; 34(1): 1439-1450, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31409157

RESUMO

Leishmaniasis is a tropical disease found in more than 90 countries. The drugs available to treat this disease have nonspecific action and high toxicity. In order to develop novel therapeutic alternatives to fight this ailment, pteridine reductase 1 (PTR1) and dihydrofolate reductase-thymidylate synthase (DHF-TS) have been targeted, once Leishmania is auxotrophic for folates. Although PTR1 and DHFR-TS from other protozoan parasites have been studied, their homologs in Leishmania chagasi have been poorly characterized. Hence, this work describes the optimal conditions to express the recombinant LcPTR1 and LcDHFR-TS enzymes, as well as balanced assay conditions for screening. Last but not the least, we show that 2,4 diaminopyrimidine derivatives are low-micromolar competitive inhibitors of both enzymes (LcPTR1 Ki = 1.50-2.30 µM and LcDHFR Ki = 0.28-3.00 µM) with poor selectivity index. On the other hand, compound 5 (2,4-diaminoquinazoline derivative) is a selective LcPTR1 inhibitor (Ki = 0.47 µM, selectivity index = 20).


Assuntos
Inibidores Enzimáticos/farmacologia , Leishmania infantum/enzimologia , Complexos Multienzimáticos/antagonistas & inibidores , Oxirredutases/antagonistas & inibidores , Timidilato Sintase/antagonistas & inibidores , Catálise , Cromatografia de Afinidade , Clonagem Molecular , Avaliação Pré-Clínica de Medicamentos , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Concentração Inibidora 50 , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/isolamento & purificação , Complexos Multienzimáticos/metabolismo , Oxirredutases/genética , Oxirredutases/isolamento & purificação , Oxirredutases/metabolismo , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/isolamento & purificação , Tetra-Hidrofolato Desidrogenase/metabolismo , Timidilato Sintase/genética , Timidilato Sintase/isolamento & purificação , Timidilato Sintase/metabolismo
2.
Int J Mol Sci ; 20(15)2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31370354

RESUMO

Methylenetetrahydrofolate reductase (MTHFR) is a pivotal enzyme in the one-carbon metabolism, a metabolic pathway required for DNA synthesis and methylation reactions. MTHFR hypermethylation, resulting in reduced gene expression, can contribute to several human disorders, but little is still known about the factors that regulate MTHFR methylation levels. We performed the present study to investigate if common polymorphisms in one-carbon metabolism genes contribute to MTHFR methylation levels. MTHFR methylation was assessed in peripheral blood DNA samples from 206 healthy subjects with methylation-sensitive high-resolution melting (MS-HRM); genotyping was performed for MTHFR 677C>T (rs1801133) and 1298A>C (rs1801131), MTRR 66A>G (rs1801394), MTR 2756A>G (rs1805087), SLC19A1 (RFC1) 80G>A (rs1051266), TYMS 28-bp tandem repeats (rs34743033) and 1494 6-bp ins/del (rs34489327), DNMT3A -448A>G (rs1550117), and DNMT3B -149C>T (rs2424913) polymorphisms. We observed a statistically significant effect of the DNMT3B -149C>T polymorphism on mean MTHFR methylation levels, and particularly CT and TT carriers showed increased methylation levels than CC carriers. The present study revealed an association between a functional polymorphism of DNMT3B and MTHFR methylation levels that could be of relevance in those disorders, such as inborn defects, metabolic disorders and cancer, that have been linked to impaired DNA methylation.


Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , Epigênese Genética , Redes e Vias Metabólicas/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Polimorfismo de Nucleotídeo Único , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/genética , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Idoso , Idoso de 80 Anos ou mais , DNA (Citosina-5-)-Metiltransferases/metabolismo , Feminino , Ferredoxina-NADP Redutase/genética , Ferredoxina-NADP Redutase/metabolismo , Ácido Fólico/metabolismo , Genótipo , Voluntários Saudáveis , Humanos , Masculino , Metionina/metabolismo , Metilenotetra-Hidrofolato Redutase (NADPH2)/metabolismo , Pessoa de Meia-Idade , Proteína Carregadora de Folato Reduzido/genética , Proteína Carregadora de Folato Reduzido/metabolismo , Timidilato Sintase/genética , Timidilato Sintase/metabolismo
3.
Gene ; 716: 144034, 2019 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-31377317

RESUMO

BACKGROUND: Outcome in adjuvant chemotherapy of gastric cancer (GC) has considerable stage-independent variability, which underscores the need for prognostic or predictive molecular markers. CHAF1A promotes tumor growth while its impact on chemotherapy outcome remains unknown. METHODS: CHAF1A protein expression was measured in independent discovery and validation sets that included 86 and 325 patients respectively who received fluoropyrimidines-based adjuvant chemotherapy after radical gastrectomy. The chemosensitizing effect of CHAF1A knockdown was investigated in vitro. Bioinformatics analysis based on RNA-seq and proteome data from public database was performed to investigate the potential mechanisms and further validation was conducted. RESULTS: In both the discovery and validation sets, CHAF1A expression level was an independent predictor for disease-free survival (HR = 4.25; 95% CI: 2.31-7.79; P < 0.001; and HR = 1.91; 95% CI: 1.03-3.54; P = 0.039, respectively) and overall survival (HR = 3.25; 95% CI: 1.75-6.05; P < 0.001; and HR = 2.42; 95% CI: 1.12-5.20; P = 0.024, respectively) in patients with non-cardia GC but not in those with cardia GC. In GC cells, CHAF1A knockdown significantly decreased the IC50 of 5-FU. Bioinformatics analyses indicated that CHAF1A correlated with folate metabolism and the expression of thymidylate synthetase (TS). Furthermore, CHAF1A knockdown significantly reduced TS expression in GC cells and CHAF1A positively correlated with TS protein expression in tumor tissues. Finally, ten proteins potentially relevant to the regulation of TS expression by CHAF1A were identified using online tools based on RNA-seq and proteome data. CONCLUSIONS: CHAF1A may impact adjuvant chemotherapy outcome of GC by regulating the expression of TS.


Assuntos
Antineoplásicos/uso terapêutico , Chaperonas de Histonas/metabolismo , Pirimidinas/uso terapêutico , Neoplasias Gástricas/tratamento farmacológico , Timidilato Sintase/metabolismo , Idoso , Linhagem Celular Tumoral , Quimioterapia Adjuvante , Fator 1 de Modelagem da Cromatina/metabolismo , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos , Feminino , Ácido Fólico/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Gástricas/enzimologia , Neoplasias Gástricas/mortalidade , Análise de Sobrevida , Timidilato Sintase/genética , Resultado do Tratamento
4.
Artigo em Inglês | MEDLINE | ID: mdl-31159295

RESUMO

One of the most interesting features of Staphylococcus aureus is its ability to switch to a small colony variant (SCV). This switch allows the pathogen to survive periods of antibiotic treatment or pressure from the immune system of the host and further enables it to start the infection once again after the environmental stress declines. However, so far only little is known about this reversion back to the more virulent wild type phenotype. Therefore, this study aimed to analyze the frequency of reversion to the wild type phenotype of thymidine auxotroph S. aureus SCV isolates (TD-SCVs) obtained from patients with cystic fibrosis (CF). With the use of single cell starting cultures, the occurrence of the thymidine prototroph revertants was monitored. The underlying mutational cause of the SCVs and subsequent revertants were analyzed by sequencing the gene coding for thymidylate synthase (ThyA), whose mutations are known to produce thymidine auxotroph S. aureus SCV. In our study, the underlying mutational cause for the switch to the TD-SCV phenotype was primarily point mutations. Out of twelve isolates, seven isolates showed an occurrence of revertants with a frequency ranging from 90.06% to 0.16%. This high variability in the frequency of reversion to the wild type was not expected. However, this variability in the frequency of reversion may also be the key to successful re-infection of the host. Sometimes quick reversion to the wild type proves necessary for survival, whereas other times, staying hidden for a bit longer leads to success in re-colonization of the host.


Assuntos
Fibrose Cística/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Humanos , Mutação , Fenótipo , Timidilato Sintase/genética , Combinação Trimetoprima e Sulfametoxazol
5.
Genes Genomics ; 41(8): 983-991, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31209768

RESUMO

BACKGROUND: Hyperhomocysteinemia is a potential risk factor for the development of metabolic syndrome (MetS). Among genes involved in homocysteine metabolism, polymorphisms of methylenetetrahydrofolate reductase (MTHFR) gene are known to be associated with MetS incidence. However, effects of polymorphisms of other folate metabolism-related genes on MetS susceptibility are not well known yet. OBJECTIVE: This study was to determine whether methionine synthase (MTR) 2756A > G, methionine synthase reductase (MTRR) 66A > G, and thymidylate synthase enhancer region (TSER) 2R/3R polymorphisms might be associated with risks of MetS development in the Korean population. METHODS: Genotype analysis of the three polymorphisms was performed for a total of 483 subjects including 236 MetS patients and 247 unrelated healthy controls using polymerase chain reaction-restriction fragment length polymorphism technique. RESULTS: The present study revealed that MTRR and TSER polymorphisms were associated with susceptibility to MetS. Several genotypes and allele combinations from the three polymorphisms were also related to the MetS prevalence. When polymorphism data were stratified according to the risk components of MetS, MTR polymorphism was significantly associated with an increased risk of MetS in subjects with systolic blood pressure < 132.7 mmHg (AOR 1.842, 95% CI 1.039-3.266, P = 0.037) and fasting blood glucose level < 106.3 mg/dL (AOR 1.772, 95% CI 1.069-2.937, P = 0.027). MTRR polymorphism was significantly associated with a decreased risk of MetS in subjects with triglyceride level < 216.3 mg/dL (AOR 0.616, 95% CI 0.399-0.951, P = 0.029). To the best of our knowledge, this is the first to provide reliable evidence about the association of other folate metabolism-related gene polymorphisms besides MTHFR with MetS susceptibility and its risk factors. CONCLUSION: Results of this study suggest that MTRR and TSER polymorphisms might be potential genetic markers for the risk of MetS development in Korean population.


Assuntos
Ferredoxina-NADP Redutase/genética , Síndrome Metabólica/genética , Polimorfismo de Nucleotídeo Único , Timidilato Sintase/genética , Adulto , Elementos Facilitadores Genéticos , Feminino , Ácido Fólico/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade
6.
EBioMedicine ; 43: 473-486, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31060905

RESUMO

BACKGROUND: Exposure to anesthetics during early life may impair cognitive functions. However, the underlying mechanisms remain largely unknown. We set out to determine effects of sevoflurane anesthesia on folate metabolism and myelination in young non-human primates, mice and children. METHODS: Young rhesus macaque and mice received 2.5 to 3% sevoflurane daily for three days. DNA and RNA sequencing and immunohistochemistry among others were used in the studies. We performed unbiased transcriptome profiling in prefrontal cortex of rhesus macaques and mice after the sevoflurane anesthesia. We constructed a brain blood barrier-crossing AAV-PHP.EB vector to harbor ERMN expression in rescue studies. We measured blood folate levels in children after anesthesia and surgery. FINDINGS: We found that thymidylate synthase (TYMS) gene was downregulated after the sevoflurane anesthesia in both rhesus macaque and mice. There was a reduction in blood folate levels in children after the anesthesia and surgery. Combined with transcriptome and genome-wide DNA methylation analysis, we identified that ERMN was the primary target of the disrupted folate metabolism. Myelination was compromised by the anesthesia in the young mice, which was rescued by systematic administration of folic acid or expression of ERMN in the brain through brain-specific delivery of the adeno-associated virus. Moreover, folic acid and expression of ERMN alleviated the cognitive impairment caused by the sevoflurane anesthesia in the mice. INTERPRETATION: General anesthesia leads to disrupted folate metabolism and subsequently defects in myelination in the developmental brain, and ERMN is the important target affected by the anesthesia via epigenetic mechanisms.


Assuntos
Anestesia/efeitos adversos , Biomarcadores , Doenças Desmielinizantes/genética , Doenças Desmielinizantes/metabolismo , Epigênese Genética , Ácido Fólico/metabolismo , Regulação da Expressão Gênica , Animais , Disfunção Cognitiva/genética , Disfunção Cognitiva/metabolismo , Biologia Computacional/métodos , Doenças Desmielinizantes/patologia , Doenças Desmielinizantes/fisiopatologia , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Ontologia Genética , Genoma , Genômica/métodos , Macaca mulatta , Masculino , Aprendizagem em Labirinto , Camundongos , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Transdução de Sinais , Timidilato Sintase/genética , Timidilato Sintase/metabolismo
7.
Anticancer Res ; 39(5): 2483-2491, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31092443

RESUMO

BACKGROUND/AIM: It remains unclear which chemotherapeutic regimens are better for the addition of bevacizumab. We conducted an exploratory randomized phase II trial comparing first-line S-1 plus cisplatin with bevacizumab and pemetrexed plus cisplatin with bevacizumab in patients with advanced non-squamous non-small cell lung cancer (NSCLC). PATIENTS AND METHODS: Chemotherapy-naïve patients received S-1 (80 mg/m2) from day 1 to day 14 plus cisplatin (80 mg/m2) on day 1 with bevacizumab (15 mg/kg) on day 1, followed by maintenance with bevacizumab plus S-1 (SCB) on day 1 every 3 weeks and pemetrexed (500 mg/m2) on day 1 plus cisplatin (75 mg/m2) on day 1 with bevacizumab (15 mg/kg) on day 1 followed by maintenance bevacizumab plus pemetrexed (PCB) on day 1 every 3 weeks. The expression of thymidylate synthase (TS) was analyzed using immunohistochemistry. RESULTS: Forty-eight patients were enrolled in this study, and eligible patients were randomly assigned at 1:1 ratio to receive SCB (n=24) or PCB (n=24). The median number of chemotherapy and maintenance therapy for SCB and PCB was 4 (range, 1-6 cycles) and 4 (range, 2-6 cycles), and 5 (range, 0-39 cycles) and 5 (range, 0-28 cycles), respectively. The overall response rate (ORR) for PCB and SCB were 54.2% and 83.3%, respectively (p=0.06). The median progression-free survival (PFS) and overall survival (OS) for PCB and SCB were 406 and 351 days, (p=0.96), and 678 and 1190 days, respectively (p=0.23). The mild adverse events were observed in both regimens. TS expression was more predictive of the chemotherapeutic response in SCB compared to PCB, but not for PFS. CONCLUSION: The combination regimen of SCB was identified as having a similar activity and tolerability to that of PCB in patients with advanced non-squamous NSCLC.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Bevacizumab/administração & dosagem , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Ácido Oxônico/administração & dosagem , Tegafur/administração & dosagem , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Bevacizumab/efeitos adversos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Cisplatino/administração & dosagem , Cisplatino/efeitos adversos , Esquema de Medicação , Combinação de Medicamentos , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Ácido Oxônico/efeitos adversos , Pemetrexede/administração & dosagem , Pemetrexede/efeitos adversos , Intervalo Livre de Progressão , Tegafur/efeitos adversos , Timidilato Sintase/genética
8.
Biomolecules ; 9(4)2019 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-30987202

RESUMO

In human cells, thymidylate synthase (TS) provides the only source of 2'-deoxythymidyne-5'-monophosphate (dTMP), which is required for DNA biosynthesis. Because of its pivotal role, human TS (hTS) represents a validated target for anticancer chemotherapy. Nonetheless, the efficacy of drugs blocking the hTS active site has limitations due to the onset of resistance in cancer cells, requiring the identification of new strategies to effectively inhibit this enzyme. Human TS works as an obligate homodimer, making the inter-subunit interface an attractive targetable area. Here, we report the design and investigation of a new hTS variant, in which Gln62, located at the dimer interface, has been replaced by arginine in order to destabilize the enzyme quaternary assembly. The hTS Q62R variant has been characterized though kinetic assay, thermal denaturation analysis and X-ray crystallography. Our results provide evidence that hTS Q62R has a reduced melting temperature. The effective destabilization of the TS quaternary structure is also confirmed by structural analysis, showing that the introduced mutation induces a slight aperture of the hTS dimer. The generation of hTS variants having a more accessible interface area can facilitate the screening of interface-targeting molecules, providing key information for the rational design of innovative hTS interface inhibitors.


Assuntos
Mutação de Sentido Incorreto , Multimerização Proteica , Timidilato Sintase/química , Estabilidade Enzimática , Humanos , Desnaturação Proteica , Domínios Proteicos , Timidilato Sintase/genética
9.
Molecules ; 24(7)2019 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-30959951

RESUMO

Human thymidylate synthase (hTS) is pivotal for cell survival and proliferation, indeed it provides the only synthetic source of dTMP, required for DNA biosynthesis. hTS represents a validated target for anticancer chemotherapy. However, active site-targeting drugs towards hTS have limitations connected to the onset of resistance. Thus, new strategies have to be applied to effectively target hTS without inducing resistance in cancer cells. Here, we report the generation and the functional and structural characterization of a new hTS interface variant in which Arg175 is replaced by a cysteine. Arg175 is located at the interface of the hTS obligate homodimer and protrudes inside the active site of the partner subunit, in which it provides a fundamental contribution for substrate binding. Indeed, the R175C variant results catalytically inactive. The introduction of a cysteine at the dimer interface is functional for development of new hTS inhibitors through innovative strategies, such as the tethering approach. Structural analysis, performed through X-ray crystallography, has revealed that a cofactor derivative is entrapped inside the catalytic cavity of the hTS R175C variant. The peculiar binding mode of the cofactor analogue suggests new clues exploitable for the design of new hTS inhibitors.


Assuntos
Timidilato Sintase/química , Timidilato Sintase/metabolismo , Substituição de Aminoácidos , Antineoplásicos/química , Antineoplásicos/farmacologia , Sítios de Ligação , Domínio Catalítico , Descoberta de Drogas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Variação Genética , Humanos , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Relação Estrutura-Atividade , Timidilato Sintase/antagonistas & inibidores , Timidilato Sintase/genética
10.
Int J Clin Oncol ; 24(9): 1020-1029, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30993483

RESUMO

BACKGROUND: Methotrexate (MTX) is used in first-line treatment of primary central nervous system lymphoma (PCNSL), but most cases result in relapse-acquired resistance to MTX. However, only few studies have reported on internal changes and chemotherapies in PCNSL. METHODS: In this study, we generated two MTX-resistant PCNSL cell lines, designated MTX-HKBML and MTX-TK, in addition to a MTX-resistant Burkitt lymphoma cell line, designated MTX-RAJI. We examined gene expression changes and drug sensitivity to a proteasome inhibitor, bortezomib, in these cells. RESULTS: Cytotoxic tests revealed that the 50% inhibitory concentration for MTX in MTX-HKBML is markedly higher than that in the other two cell lines. Expression of the genes in MTX and folate metabolisms, including gamma-glutamyl hydrolase and dihydrofolate reductase, are upregulated in both MTX-HKBML and MTX-TK, whereas the gene expression of folylpolyglutamate synthetase, thymidylate synthase, and methylenetetrahydrofolate dehydrogenase 1 were upregulated and downregulated in MTX-HKBML and MTX-TK, respectively, on the other hand, bortezomib sensitivity was observed in MTX-TK, as compared with control TK, but not in MTX-HKBML. CONCLUSION: These results indicate the cell-type-specific changes downstream of metabolic pathways for MTX and folate, bortezomib sensitivity, and purine and pyrimidine syntheses, in each PCNSL cell line. The MTX-resistant lymphoma cell lines established may be useful for in vitro relapse models for MTX and development of salvage chemotherapy and drug discovery.


Assuntos
Bortezomib/farmacologia , Neoplasias do Sistema Nervoso Central/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Linfoma/tratamento farmacológico , Metotrexato/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Bortezomib/administração & dosagem , Linhagem Celular Tumoral , Neoplasias do Sistema Nervoso Central/genética , Neoplasias do Sistema Nervoso Central/patologia , Ácido Fólico/genética , Ácido Fólico/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Linfoma/genética , Linfoma/patologia , Linfoma não Hodgkin/tratamento farmacológico , Linfoma não Hodgkin/patologia , Metotrexato/administração & dosagem , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Antígenos de Histocompatibilidade Menor/genética , Terapia de Alvo Molecular , Peptídeo Sintases/genética , Timidilato Sintase/genética
11.
Int J Pharm ; 564: 256-262, 2019 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-31015002

RESUMO

RNA interference (RNAi) is one of the most promising strategies for cancer therapeutics. The successful translation of RNAi therapeutics to a clinic setting requires a delivery system that is efficient and simple to upscale. In this study, we devised a simple industrial method to manufacture lipoplex, which includes short hairpin RNA against the expression of thymidylate synthase (TS shRNA) - a key molecule for DNA biosynthesis. An aqueous solution of TS shRNA was gently mixed with either a precursor of cationic liposome (Presome DF-1) or a cationic lipid mixture in an o/w emulsion. This solution was subsequently lyophilized under optimal conditions to obtain either FD-lipoplex-1 or FD-lipoplex-2, respectively. With this method, a lipoplex in activated form was obtained via a simple "one-step" hydration with saline. Both forms of FD-lipoplex showed physicochemical properties comparable to those of conventional lipoplex. FD-lipoplexes stably retained TS shRNA within their formulations in the presence of tumor ascites fluid. Intraperitoneal treatment with either FD-lipoplex-1 or FD-lipoplex-2 provided a therapeutic level of efficacy comparable to that of conventional lipoplex in the treatment of a peritoneal disseminated gastric cancer mouse model. Collectively, established freeze-drying-based methods for RNAi-therapeutic preparation could realistically be used in a clinical setting for the treatment of patients with peritoneal disseminated cancer.


Assuntos
Neoplasias Peritoneais/terapia , RNA Interferente Pequeno/administração & dosagem , Neoplasias Gástricas/terapia , Timidilato Sintase/genética , Animais , Linhagem Celular Tumoral , Liofilização , Humanos , Lipossomos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Peritoneais/genética , Interferência de RNA , RNA Interferente Pequeno/química , Terapêutica com RNAi , Neoplasias Gástricas/genética
12.
J Steroid Biochem Mol Biol ; 190: 139-151, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30923017

RESUMO

5-Fluorouracil (5-FU) is an anticancer drug that is most frequently used to treat colorectal cancer (CRC) patients, but unfortunately it shows limited efficacy. We recently demonstrated that vitamin D analogs (VDAs), particularly tacalcitol (coded as PRI-2191), potentiate its anticancer activity in an in vivo mouse and human CRC model. The purpose of this study was to explain the mechanism underlying the enhancement of 5-FU efficacy by PRI-2191 towards human HT-29 CRC cells. We showed that PRI-2191 induces the CDKN1A (gene encoding p21Waf1/Cip1) expression directly through vitamin D receptor (VDR) in a p53-independent manner and thus decreases the thymidylate synthase expression both at the mRNA and protein level. It is the main mechanism by which PRI-2191 improves the anticancer efficacy of 5-FU towards HT-29 cells. Additionally, we indicated that the VDR also participates in 5-FU mechanism of action. 5-FU significantly increased TYMS (gene encoding thymidylate synthase (TS)) and BIRC5 (gene encoding survivin) level in HT-29 cells with silenced VDR. Furthermore, PRI-2191 induced E-cadherin and ZO-1 expression and thus reduced the level of BIRC5 in HT-29 cells. The induction of E-cadherin expression may also contribute to the reduction of c-Myc level and consequently the downregulation of TS. Our results also indicate that calcium-sensing receptor (CaSR) plays a role in the activity of PRI-2191 but has no influence on the 5-FU mechanism of action. In conclusion, we suggest that both VDR and CaSR might be useful as molecular markers for predicting treatment outcomes and identifying the CRC patient subgroups who might benefit from 5-FU-based chemotherapy combined with vitamin D analog.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Di-Hidroxicolecalciferóis/farmacologia , Fluoruracila/farmacologia , Timidilato Sintase/genética , Neoplasias Colorretais/genética , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Células HT29 , Humanos
13.
Chin Clin Oncol ; 8(3): 28, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30823845

RESUMO

BACKGROUND: The objective is to investigate whether thymidylate synthase gene TS 5'-UTR polymorphism of peripheral blood mononuclear cells are associated with clinical outcomes of patients with stage II-III rectal adenocarcinoma treated with adjuvant 5-fluorouracil (5-FU) chemotherapy in Chinese population. METHODS: One hundred and seventeen pathologically diagnosed colorectal adenocarcinoma patients with stage II-III, who underwent curative resection and received 5-fluoropyrimidine-based adjuvant chemotherapy were enrolled to this study. The 5'-TSER polymorphisms determined from the peripheral blood mononuclear cells were measured by Direct Sequencing. Kaplan-Meier curves and log-rank tests were used for survival analysis. The independent prognostic factors influencing DFS and OS were estimated by Cox proportional hazards model. RESULTS: The distribution of TS 5'-UTR polymorphisms ware 2.6% 2R/2R, 31.6% 2R/3R and 65.8% 3R/3R respectively, which was fitted with Hard-Weinberg equilibrium (χ2=0.345, P=0.558). Stage, N stage, number of mesenteric lymph node metastasis, KPS, and 5'-UTR polymorphisms (2R/2R/2R/3R vs. 3R/3R, P<0.001) were significantly associated with DFS. Meanwhile, gender (female vs. male, P=0.025) and adjuvant radiotherapy (yes vs. no, P=0.025) were significantly associated with OS. Multivariate Cox regression showed that KPS score (HR =0.947, P=0.007), TS 5'-UTR polymorphism (HR =0.455, P=0.004) were independent prognostic factors for DFS. Whereas, KPS score was the only independent prognostic factors for OS (HR =0.910, P=0.005). CONCLUSIONS: TS 5'-UTR tandem repeat polymorphisms had potential utilization for personalized therapy in Chinese population.


Assuntos
Adenocarcinoma/genética , Fluoruracila/uso terapêutico , Polimorfismo Genético/genética , Neoplasias Retais/tratamento farmacológico , Neoplasias Retais/genética , Timidilato Sintase/genética , Adenocarcinoma/tratamento farmacológico , Adulto , Idoso , Quimioterapia Adjuvante , Intervalo Livre de Doença , Feminino , Fluoruracila/administração & dosagem , Fluoruracila/farmacologia , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias Retais/patologia , Estudos Retrospectivos , Adulto Jovem
14.
Curr Microbiol ; 76(5): 613-619, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30863882

RESUMO

Black rot is a cruciferous disease caused by Xanthomonas campestris pv. campestris (Xcc) and results in significant economic losses worldwide; therefore, elucidation of the mechanism of Xcc pathogenesis is urgently required. In this study, we aimed to select optimized reference genes to verify the relative quantification of virulent genes in Xcc. Xcc strains were cultured in three different media [basic medium (MMX), hrp-inducing medium (MMXC) and rich medium (NYG)] and the expression stability of five candidate genes [thymidylate synthase (thyA), DNA gyrase subunit B (gyrB), DNA-directed RNA polymerase subunit beta, glyceraldehyde-3-phosphate dehydrogenase and 16S ribosomal RNA (16S rRNA)] was evaluated using BestKeeper, GeNorm, and NormFinder software programs. Quantitative real-time PCR (qRT-PCR) analysis confirmed that two Xcc effector genes were hrpX/hrpG-regulated in MMXC using selected genes as controls. Finally, gyrB and thyA were validated as the optimized reference genes of Xcc cultured in MMXC, and qRT-PCR analysis was demonstrated to be an efficient alternative to Gus-activity detection for the analysis of Xcc expression. This information will be useful in the future studies of Xcc, especially those seeking new functional genes.


Assuntos
Meios de Cultura/química , Genes Bacterianos , Reação em Cadeia da Polimerase em Tempo Real , Xanthomonas campestris/genética , Proteínas de Bactérias/genética , Técnicas Bacteriológicas , DNA Girase/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Ribossômico 16S/genética , Timidilato Sintase/genética , Xanthomonas campestris/crescimento & desenvolvimento
15.
J Chemother ; 31(2): 95-104, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30739598

RESUMO

Pemetrexed is an antimetabolite approved for treatment of non-small cell lung cancer. Harbouring interindividual variability in both the pharmacokinetic and pharmacogenetic profiles may lead to life-threatening toxicities. A prospective cohort study of adult patients initiating treatment with pemetrexed in combination with platinum between 2013 and 2015 were follow up. Primary exposure were the methylenetetrahydrofolate reductase (MTHFR) single base polymorphisms in exon 4 and 7 and 5'-UTR- thymidylate synthase (TYMS) VNTR genotypes, in addition to baseline clinical and demographic variables. We used a Cox regression model to evaluate patient's survival and toxicity experience and its association with both baseline characteristics, and a-priori determined genetic polymorphisms. Seventy two patients were included, 52.7% developed severe hematologic toxicity during follow-up. None of the tested genotypes were significantly associated with the main outcome on multivariate analysis, nor other basal clinical variables. Overall survival between patients experiencing the outcome was not different from those without it, but hospital admissions were more frequent. MTHFR and 5'-UTR-TYMS genotypes were not useful for predicting high grade toxicity events in patients under treatment with pemetrexed.


Assuntos
Antineoplásicos/efeitos adversos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/diagnóstico , Neoplasias Pulmonares/tratamento farmacológico , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Pemetrexede/efeitos adversos , Índice de Gravidade de Doença , Timidilato Sintase/genética , Adulto , Idoso , Antineoplásicos/administração & dosagem , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/etiologia , Feminino , Seguimentos , Genótipo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Pemetrexede/administração & dosagem , Polimorfismo de Nucleotídeo Único , Valor Preditivo dos Testes
16.
Cancer Chemother Pharmacol ; 83(4): 755-762, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30684021

RESUMO

BACKGROUND: Acute lymphoblastic leukemia (ALL) is one of the major malignancies affecting children in Jordan. Methotrexate (MTX) is the cornerstone of chemotherapy for ALL, and works by targeting enzymes involved in the folate pathway. We hypothesize that genetic polymorphisms of the folate pathway are associated with MTX toxicity in children with ALL. METHODS: A total of 64 children with ALL were included in this study; 31 (48.4%) boys and 33 (51.6%) girls aged 2-16 years. The folate pathway genes were genotyped using polymerase chain reaction followed by sequencing and studying the association between genetic polymorphisms and MTX toxicity. RESULTS: The immunophenotype was B-lineage in 55 patients (85.9%) and T-lineage in nine patients (14.1%). All genetic polymorphisms, except for dihydropyrimidine dehydrogenase polymorphisms, were associated with hematological toxicities and did not appear to precipitate any non-hematological adverse events. Patients with ALL carrying dominant alleles of methylene tetrahydrofolate (MTHFR) C677T and dihydrofolate reductase 19 bp deletion were at a higher risk of developing severe leucopenia [OR (95% CI) = 4.5 (1.2-17), p = 0.03; 5.4 (1.6-17.8); p = 0.006] while minor allele carriers of MTHFR A1298C were more likely to develop neutropenia [OR (95% CI) = 6.1 (1.3-29.5); 0.04]. Furthermore, dominant allele carriers of thymidylate synthase 1494 del6 were at a higher risk of developing neutropenia [OR (95% CI) = 6 (1.2-31.1); p = 0.04]. CONCLUSION: Genetic polymorphisms of the folate pathway may modulate MTX-induced toxicity in childhood ALL.


Assuntos
Antimetabólitos Antineoplásicos/efeitos adversos , Ácido Fólico/metabolismo , Metotrexato/efeitos adversos , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Adolescente , Antimetabólitos Antineoplásicos/administração & dosagem , Criança , Pré-Escolar , Estudos Transversais , Feminino , Genótipo , Humanos , Masculino , Metotrexato/administração & dosagem , Polimorfismo Genético , Estudos Prospectivos , Estudos Retrospectivos , Tetra-Hidrofolato Desidrogenase/genética , Timidilato Sintase/genética
17.
Mol Med Rep ; 19(2): 1092-1100, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30535504

RESUMO

Mitogen­activated protein kinase kinase (MEK) small molecule inhibitors have been investigated in preclinical or clinical trials for the treatment of cancer. In the present study the genetic test results of 120 patients with colorectal cancer (CRC) were screened and the mutation rate of MEK1 was identified to be 1.67%. MEK inhibition by U0126 significantly decreased the growth of SW48 cells that harbored the MEK1 Q56P mutation, although it did not evidently affect the growth of NCI­H508 cells with MEK1 wild­type. In addition, U0126 increased the sensitivity of SW48 cells to 5­fluorouracil (5­FU) and oxaliplatin by producing more γH2AX foci and decreasing the expression of excision repair cross­complementation group 1 and thymidylate synthase. The results suggested that MEK inhibitors in combination with oxaliplatin/5­FU may offer an improved therapeutic effect in patients with MEK­mutant CRC.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Butadienos/farmacologia , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica , MAP Quinase Quinase 1/genética , Nitrilos/farmacologia , Oxaliplatina/farmacologia , Idoso , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Sinergismo Farmacológico , Endonucleases/genética , Endonucleases/metabolismo , Feminino , Histonas/genética , Histonas/metabolismo , Humanos , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/metabolismo , Masculino , Pessoa de Meia-Idade , Mutação , Gradação de Tumores , Metástase Neoplásica , Estadiamento de Neoplasias , Transdução de Sinais , Timidilato Sintase/genética , Timidilato Sintase/metabolismo
18.
Cell Death Dis ; 9(12): 1185, 2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30538221

RESUMO

Fluorouracil (5-FU) is the first-line chemotherapeutic drug for cholangiocarcinoma (CCA), but its efficacy has been compromised by the development of resistance. Development of 5-FU resistance is associated with elevated expression of its cellular target, thymidylate synthase (TYMS). E2F1 transcription factor has previously been shown to modulate the expression of FOXM1 and TYMS. Immunohistochemical (IHC) analysis revealed a strong correlated upregulation of FOXM1 (78%) and TYMS (48%) expression at the protein levels in CCA tissues. In agreement, RT-qPCR and western blot analyses of four human CCA cell lines at the baseline level and in response to high doses of 5-FU revealed good correlations between FOXM1 and TYMS expression in the CCA cell lines tested, except for the highly 5-FU-resistant HuCCA cells. Consistently, siRNA-mediated knockdown of FOXM1 reduced the clonogenicity and TYMS expression in the relatively sensitive KKU-D131 but not in the highly resistant HuCCA cells. Interestingly, silencing of TYMS sensitized both KKU-D131 and HuCCA to 5-FU treatment, suggesting that resistance to very high levels of 5-FU is due to the inability of the genotoxic sensor FOXM1 to modulate TYMS expression. Consistently, ChIP analysis revealed that FOXM1 binds efficiently to the TYMS promoter and modulates TYMS expression at the promoter level upon 5-FU treatment in KKU-D131 but not in HuCCA cells. In addition, E2F1 expression did not correlate with either FOXM1 or TYMS expression and E2F1 depletion has no effects on the clonogenicity and TYMS expression in the CCA cells. In conclusion, our data show that FOXM1 regulates TYMS expression to modulate 5-FU resistance in CCA and that severe 5-FU resistance can be caused by the uncoupling of the regulation of TYMS by FOXM1. Our findings suggest that the FOXM1-TYMS axis can be a novel diagnostic, predictive and prognostic marker as well as a therapeutic target for CCA.


Assuntos
Neoplasias dos Ductos Biliares/tratamento farmacológico , Colangiocarcinoma/tratamento farmacológico , Proteína Forkhead Box M1/genética , Timidilato Sintase/genética , Apoptose/efeitos dos fármacos , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Colangiocarcinoma/genética , Colangiocarcinoma/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , RNA Interferente Pequeno/genética
19.
Zhonghua Gan Zang Bing Za Zhi ; 26(9): 666-669, 2018 Sep 20.
Artigo em Chinês | MEDLINE | ID: mdl-30481863

RESUMO

Objective: To investigate epidermal growth factor receptor (EGFR) and thymidylate synthase (TS) expression in primary liver cancer, and analyze its clinicopathological features and prognostic significance. Methods: Immunohistochemistry was performed using EnVision method to detect EGFR and TS expression in 41 cases of liver cancer. Correlation coefficient between EGFR and TS was calculated by Spearman method. Fisher's exact probability method or χ(2) test was used to analyze the clinicopathological features of EGFR and TS. Kaplan-Meier method was used to calculate the survival rate of patients in conjunction with the log-rank test.COX proportional hazard regression model was used to analyze the prognostic factors of patients. ROC curve was used to analyze the predictive accuracy of EGFR and TS for prognosis. Results: The positive rates of EGFR and TS in liver cancer tissues were 34.15% and 39.02%, respectively. There was a positive correlation between EGFR and TS expressions, and the difference was statistically significant (P < 0.05). EGFR was associated with tumor size and tissue differentiation (P < 0.05) in HCC patients, whereas TS was associated with tissue differentiation (P < 0.05). There was no significant difference in prognostic effect of EGFR on survival rate (P > 0.05). TS prognostic effect on survival rate was statistically significant (P < 0.05). HR of EGFR was 0.210 with 95% CI, 0.052-0.852, P = 0.029; indicating that the risk of death in patients with negative EGFR was 0.210 times higher than that in patients with positive EGFR. HR of TS was 2.496, with 95% CI, 1.325-4.701, P = 0.005, indicating that the risk of death increased by 2.496 times with the same level of EGFR. The area under the EGFR curve was 0.553 and its approximate reference confidence interval was 95% (0.355, 0.751), indicating that EGFR was a risk factor for death and the area under the TS curve was 0.695, and its approximate reference confidence interval was 95% (0.513, 0.878), indicating that TS was a risk factor for death. Conclusion: EGFR and TS were equally expressed in primary liver cancer, and EGFR and TS expressions were positively correlated. EGFR and TS had an effect on the degree of tissue differentiation in patients with liver cancer. EGFR and TS were risk factors for prognosis, and TS may assist EGFR.


Assuntos
Receptores ErbB/genética , Neoplasias Hepáticas/metabolismo , Timidilato Sintase/genética , Adulto , Biomarcadores Tumorais , Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Prognóstico , Timidilato Sintase/metabolismo
20.
Pharmacogenet Genomics ; 28(10): 223-229, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30222710

RESUMO

OBJECTIVE: Methotrexate (MTX) is an important drug in the treatment of pediatric acute lymphoblastic leukemia (ALL). MTX is cytotoxic as it impairs DNA and RNA synthesis by inhibiting the enzymes dihydrofolate reductase (DHFR) and thymidylate synthase (TYMS). The association between genetic variants within the TYMS gene and MTX-induced toxicity has been studied, but results are inconsistent. We determined the role of three previously described variants within the TYMS gene and MTX-induced oral mucositis in a prospective cohort of Dutch children with ALL and performed a meta-analysis of the previous results. MATERIALS AND METHODS: We analyzed the presence of a 28-base pair tandem repeat (rs34743033; 2R3R), a single nucleotide polymorphism present within the 28-base pair repeat on the 3R allele (rs2853542; 3RG>C) and a 6-base pair deletion (rs15126436; TTAAAG) within the TYMS gene in germline DNA of 117 pediatric patients with ALL. Oral mucositis was defined as grade≥3 according to the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) v.3.0. Data were analyzed for the individual rs34743033 (2R3R) and rs151264360 (6 bp deletion) polymorphisms, whereas rs2853542 (3RG>C) was combined with rs34743033 (2R3R) and analyzed according to predicted expression levels of TYMS: low expression (2R/2R, 2R/3RC and 3RC/3RC), median expression (2R/3RG and 3RC/3RG) and high expression (3RG/3RG). We performed a meta-analysis of the current literature on these polymorphisms in relation to oral mucositis using a fixed effects model. RESULTS: The 2R2R genotype (rs34743033) was not significantly associated with developing MTX-induced oral mucositis compared with the 2R3R/3R3R genotypes, which was confirmed in a meta-analysis [odds ratio (OR): 1.17 (0.62-2.19)]. Patients carrying the low-expression TYMS genotype (2R2R, 2R3RC, 3RC3RC) had an increased odds of developing MTX-induced oral mucositis [OR: 2.42 (0.86-6.80)], which did not reach statistical significance. The 6-bp deletion [rs151264360, OR: 0.79 (0.20-3.19)] was not associated with the development of MTX-induced oral mucositis. CONCLUSION: The TYMS 6-bp deletion and 2R3R polymorphism were not associated with MTX-induced oral mucositis. Validation studies in prospective cohorts are necessary to assess the possible role of the low-expression TYMS genotypes in relation to MTX-induced oral mucositis.


Assuntos
Estudos de Associação Genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Estomatite/genética , Timidilato Sintase/genética , Alelos , Criança , Pré-Escolar , Feminino , Regulação da Expressão Gênica/genética , Genótipo , Humanos , Masculino , Metotrexato/administração & dosagem , Metotrexato/efeitos adversos , Polimorfismo de Nucleotídeo Único/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/complicações , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Estomatite/induzido quimicamente , Estomatite/patologia , Sequências de Repetição em Tandem/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA