RESUMO
Toxoplasmosis, an infectious zoonotic disease caused by the apicomplexan parasite Toxoplasma gondii (T. gondii), is a major worldwide health problem. However, there are currently no effective options (chemotherapeutic drugs or prophylactic vaccines) for treating chronic latent toxoplasmosis infection. Accordingly, seeking more effective and safer chemotherapeutics for combating this disease remains a long-term and challenging objective. In this paper, we summarize possible molecular biotargets, with an emphasis on those that are druggable and promising, including, without limitation, calcium-dependent protein kinase 1, bifunctional thymidylate synthase-dihydrofolate reductase, and farnesyl diphosphate synthase. Meanwhile, as important components of medicinal chemistry, the binding modes and structure-activity relationship profiles of the corresponding inhibitors were also illuminated. We anticipate that this information will be helpful for further identification of more effective chemotherapeutic interventions to prevent and treat zoonotic infections caused by T. gondii.
Assuntos
Antiprotozoários/uso terapêutico , Toxoplasmose/tratamento farmacológico , Animais , Inibidores Enzimáticos/farmacologia , Geraniltranstransferase/efeitos dos fármacos , Geraniltranstransferase/metabolismo , Humanos , Complexos Multienzimáticos/efeitos dos fármacos , Complexos Multienzimáticos/metabolismo , Proteínas Quinases/efeitos dos fármacos , Proteínas Quinases/metabolismo , Relação Estrutura-Atividade , Tetra-Hidrofolato Desidrogenase/efeitos dos fármacos , Tetra-Hidrofolato Desidrogenase/metabolismo , Timidilato Sintase/efeitos dos fármacos , Timidilato Sintase/metabolismo , Toxoplasma/enzimologiaRESUMO
The emergence of new drug-resistant strains of bacteria necessitates the development of principally new antibacterial agents. One of the novel classes of antibacterial agents is nucleoside analogs. We have developed a fast and simple one-pot method for preparation of α- and ß-anomers of 5-modified 6-aza- and 2-thio-6-aza-2'-deoxyuridine derivatives in high yields. 2-Thio derivatives demonstrated moderate activity against Mycobacterium smegmatis (MIC = 0.2-0.8 mM), Staphylococcus aureus (MIC = 0.03-0.9 mM) and some other Gram-positive bacteria. 2'-Deoxy-2-thio-5-phenyl-6-azauridine (2b) effectively suppressed the growth of Gram-negative bacteria Pseudomonas aeruginosa ATCC 27853 (MIC = 0.03 mM)-the one that causes diseases difficult to treat due to high resistance to antibiotics. 5'-Monophosphates of compounds 2a, b and 3a, b were docked into a binding site of Mycobacterium tuberculosis flavin-dependent thymidylate synthase (ThyX) enzyme. The molecular modeling demonstrates the possibility of binding of the 5-modified 2-thio-6-aza-2'-deoxyuridine 5'-monophosphates within the active site of the enzyme and thereby inhibiting the growth of the bacteria.
Assuntos
Antibacterianos/síntese química , Azauridina/análogos & derivados , Azauridina/síntese química , Animais , Antibacterianos/farmacologia , Azauridina/farmacologia , Domínio Catalítico , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Estrutura Molecular , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Timidilato Sintase/efeitos dos fármacosRESUMO
OBJECTIVE: Present in silico study was carried out to explore the mode of inhibition of Leishmania donovani dihydrofolate reductase-thymidylate synthase (Ld DHFR-TS) enzyme by Withaferin-A, a withanolide isolated from Withania somnifera. Withaferin-A (WA) is known for its profound multifaceted properties, but its antileishmanial activity is not well understood. The parasite's DHFR-TS enzyme is diverse from its mammalian host and could be a potential drug target in parasites. RESULTS: A 3D model of Ld DHFR-TS enzyme was built and verified using Ramachandran plot and SAVES tools. The protein was docked with WA-the ligand, methotrexate (MTX)-competitive inhibitor of DHFR, and dihydrofolic acid (DHFA)-substrate for DHFR-TS. Molecular docking studies reveal that WA competes for active sites of both Hu DHFR and TS enzymes whereas it binds to a site other than active site in Ld DHFR-TS. Moreover, Lys 173 residue of DHFR-TS forms a H-bond with WA and has higher binding affinity to Ld DHFR-TS than Hu DHFR and Hu TS. The MD simulations confirmed the H-bonding interactions were stable. The binding energies of WA with Ld DHFR-TS were calculated using MM-PBSA. Homology modelling, molecular docking and MD simulations of Ld DHFR-TS revealed that WA could be a potential anti-leishmanial drug.
Assuntos
Inibidores Enzimáticos/farmacologia , Ácido Fólico/análogos & derivados , Leishmania donovani/enzimologia , Metotrexato/farmacologia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Complexos Multienzimáticos/efeitos dos fármacos , Extratos Vegetais/farmacologia , Tetra-Hidrofolato Desidrogenase/efeitos dos fármacos , Timidilato Sintase/efeitos dos fármacos , Withania , Vitanolídeos/farmacologia , Ácido Fólico/farmacologia , HumanosRESUMO
BACKGROUND: This prospective study was conducted to evaluate the feasibility and safety of customized chemotherapy regimens based on the gene characteristics of salivary gland tumors. METHODS: Patients were enrolled with histologically confirmed intermediate or high grade, stage T3-4, N1-3 disease, and T1-2, N0 patients with a close (≤1âmm) or microscopically positive surgical margin were also enrolled in the study. All patients received radical surgery and postoperative concurrent chemoradiotherapy. To evaluate the responsiveness of therapies, the chemotherapy regimen was based on gene targets, ß-tubulin III, ABCB1, STMN1, and CYP1B1 (for docetaxel) and TYMS (for pemetrexed). The primary endpoints were treatment compliance and acute toxicities. RESULTS: A total of 20 patients were enrolled between September 2013 and January 2016. The median age was 46 years (range: 23-70 years). Genetic testing showed that 8 patients may have been sensitive to docetaxel, 5 patients may have been sensitive to pemetrexed, and 7 patients sensitive to either docetaxel or pemetrexed. All patients received the full dose of radiation. A total of 19 patients (95%) completed 2 cycles of concurrent chemotherapy (CCT). One patient treated concurrently with pemetrexed experienced grade 3 neutropenia. Three patients experienced grade 3 oral mucositis, and 2 patients experienced grade 3 dermatitis. CONCLUSION: Our study demonstrated that a CCT selecting method based on the gene targets associated with drug sensitivity was clinically feasible and safe. Further studies enrolled more patients with longer follow-up times are needed to confirm the clinical efficacy of this CCT selecting method.
Assuntos
Antineoplásicos/uso terapêutico , Marcação de Genes/métodos , Testes Genéticos/métodos , Seleção de Pacientes , Neoplasias das Glândulas Salivares/terapia , Subfamília B de Transportador de Cassetes de Ligação de ATP/análise , Subfamília B de Transportador de Cassetes de Ligação de ATP/efeitos dos fármacos , Adulto , Idoso , Antineoplásicos/administração & dosagem , Quimiorradioterapia/métodos , Citocromo P-450 CYP1B1/análise , Citocromo P-450 CYP1B1/efeitos dos fármacos , Docetaxel , Estudos de Viabilidade , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pemetrexede/administração & dosagem , Estudos Prospectivos , Neoplasias das Glândulas Salivares/genética , Estatmina/análise , Estatmina/efeitos dos fármacos , Taxoides/administração & dosagem , Timidilato Sintase/análise , Timidilato Sintase/efeitos dos fármacos , Tubulina (Proteína)/análise , Tubulina (Proteína)/efeitos dos fármacos , Adulto JovemRESUMO
The mode of action of many antimalarial drugs is unknown. Chemogenomic profiling is a powerful method to address this issue. This experimental approach entails disruption of gene function and phenotypic screening for changes in sensitivity to bioactive compounds. Here, we describe the application of reverse genetics for chemogenomic profiling in Plasmodium. Plasmodium falciparum parasites harbouring a transgenic insertion of the glmS ribozyme downstream of the dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene were used for chemogenomic profiling of antimalarial compounds to identify those which target DHFR-TS. DHFR-TS expression can be attenuated by exposing parasites to glucosamine. Parasites with attenuated DHFR-TS expression were significantly more sensitive to antifolate drugs known to target DHFR-TS. In contrast, no change in sensitivity to other antimalarial drugs with different modes of action was observed. Chemogenomic profiling was performed using the Medicines for Malaria Venture (Switzerland) Malaria Box compound library, and two compounds were identified as novel DHFR-TS inhibitors. We also tested the glmS ribozyme in Plasmodium berghei, a rodent malaria parasite. The expression of reporter genes with downstream glmS ribozyme could be attenuated in transgenic parasites comparable with that obtained in P. falciparum. The chemogenomic profiling method was applied in a P. berghei line expressing a pyrimethamine-resistant Toxoplasma gondii DHFR-TS reporter gene under glmS ribozyme control. Parasites with attenuated expression of this gene were significantly sensitised to antifolates targeting DHFR-TS, but not other drugs with different modes of action. In conclusion, these data show that the glmS ribozyme reverse genetic tool can be applied for identifying primary targets of antimalarial compounds in human and rodent malaria parasites.
Assuntos
Antimaláricos/farmacologia , Antagonistas do Ácido Fólico/farmacologia , Plasmodium berghei/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Tetra-Hidrofolato Desidrogenase/efeitos dos fármacos , Timidilato Sintase/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Eritrócitos/parasitologia , Feminino , Expressão Gênica , Humanos , Concentração Inibidora 50 , Camundongos , Camundongos Endogâmicos BALB C , Organismos Geneticamente Modificados , Plasmídeos , Plasmodium berghei/enzimologia , Plasmodium berghei/genética , Plasmodium falciparum/enzimologia , Plasmodium falciparum/genética , RNA Catalítico/efeitos dos fármacos , Organismos Livres de Patógenos Específicos , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo , Timidilato Sintase/antagonistas & inibidores , Timidilato Sintase/genética , Timidilato Sintase/metabolismo , TransfecçãoRESUMO
PURPOSE: Despite several therapeutic options renal cell carcinoma is associated with a poor clinical outcome. Therefore, we investigated whether combining 5-fluorouracil with the histone deacetylase inhibitor belinostat would exert a synergistic effect on renal cell carcinoma cells in vitro and in vivo. MATERIALS AND METHODS: We used SN12C cells treated with 5-fluorouracil and/or belinostat in vitro and in xenograft experiments in vivo. Cell viability and death mechanisms were assessed by MTS assay and Western blot. To investigate the role of reactive oxygen species we used H2DCF-DA, reactive oxygen species scavengers and the roGFP2 construct. RESULTS: Belinostat potentiated the anticancer effect of 5-fluorouracil. It synergistically induced apoptosis by activating caspases and increasing the subG1 cell population. Effects on reactive oxygen species mediated DNA damage included decreased thioredoxin expression and increased levels of TBP-2, γ-H2AX and Ac-H3. Furthermore, belinostat attenuated the 5-fluorouracil mediated induction of thymidylate synthase via HSP90 hyperacetylation. Co-administration of 5-fluorouracil with belinostat similarly reduced tumor volume and weight, and increased γ-H2AX and Ac-H3 levels in the SN12C xenograft model. CONCLUSIONS: In combination with 5-fluorouracil the targeted inhibitor of histone deacetylase synergistically inhibited renal cancer cell growth by the blockade of thymidylate synthase induction and the induction of reactive oxygen species mediated DNA damage in vitro and in vivo. Our results suggest that combined treatment with belinostat and 5-fluorouracil may represent a promising new approach to renal cancer.
Assuntos
Antimetabólitos Antineoplásicos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/metabolismo , Dano ao DNA/efeitos dos fármacos , Fluoruracila/administração & dosagem , Proteínas de Choque Térmico HSP90/efeitos dos fármacos , Inibidores de Histona Desacetilases/administração & dosagem , Ácidos Hidroxâmicos/administração & dosagem , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Sulfonamidas/administração & dosagem , Timidilato Sintase/efeitos dos fármacos , Animais , Quimioterapia Combinada , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico Sintase , Células Tumorais CultivadasRESUMO
Chemotherapy for colorectal cancer has become more complicated and diversified with the appearance of molecular-targeting agents. 5-Fluorouracil (5-FU) has been a mainstay of chemotherapy for colorectal cancer, but it is still unknown whether the combining of 5-FU with novel molecular-targeting agents is effective. Thymidylate synthase (TS) is a direct target of 5-FU, and the low TS level has been generally supposed to sensitize 5-FU's efficacy. We therefore hypothesized that RB-reactivating agents could enhance the efficacy of 5-FU, because the RB-reactivating agents could suppress the function of transcription factor E2F of TS gene promoter. We used three RB-reactivating agents, trametinib/GSK1120212 (MEK inhibitor), fenofibrate (PPARα agonist), and LY294002 (PI3K inhibitor), with 5-FU against human colon cancer HT-29 and HCT15 cells. Trametinib induced p15 and p27 expression and reduced cyclin D1 levels in HT-29 cells. Fenofibrate also dephosphorlated ERK1/2 and reduced cyclin D1 levels in HT-29 cells. LY294002 induced p27 expression in HCT15 cells. All three agents caused dephosphorylation of RB protein and G1-phase arrest with a reduction of TS expression. As a consequence, the combination of 5-FU with each of the agents resulted in a significant decrease of colony numbers in HT-29 or HCT15 cells. These results suggest "RB-reactivation therapy" using molecular-targeting agents to be a new strategy for 5-FU-based chemotherapy. In particular, we strongly expect trametinib, which was discovered in Japan and was recently submitted to FDA for approval, to be used together with established regimens for colorectal cancer.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias do Colo/metabolismo , Fluoruracila/administração & dosagem , Piridonas/administração & dosagem , Pirimidinonas/administração & dosagem , Apoptose/efeitos dos fármacos , Western Blotting , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Inibidores Enzimáticos/administração & dosagem , Genes do Retinoblastoma/efeitos dos fármacos , Células HT29 , Humanos , MAP Quinase Quinase Quinases/antagonistas & inibidores , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Timidilato Sintase/efeitos dos fármacos , Timidilato Sintase/metabolismoRESUMO
PURPOSE: Downstaging (DS) of rectal cancers is achieved in approximately 45% of patients with neoadjuvant fluorouracil (FU) -based chemoradiotherapy (CRT). Polymorphisms in the thymidylate synthase gene (TYMS) had previously defined two risk groups associated with disparate tumor DS rates (60% v 22%). We conducted a prospective single-institution phase II study using TYMS genotyping to direct neoadjuvant CRT for patients with rectal cancer. PATIENTS AND METHODS: Patients with T3/T4, N0-2, M0-1 rectal adenocarcinoma were evaluated for germline TYMS genotyping. Patients with TYMS *2/*2, *2/*3, or *2/*4 (good risk) were treated with standard chemoradiotherapy using infusional FU at 225 mg/m²/d. Patients with TYMS *3/*3 or *3/*4 (poor risk) were treated with FU/RT plus weekly intravenous irinotecan at 50 mg/m². The primary end point was pathologic DS. Secondary end points included complete tumor response (ypT0), toxicity, recurrence rates, and overall survival. RESULTS: Overall, 135 patients were enrolled, of whom 27.4% (37 of 135) were considered poor risk. The prespecified statistical goals were achieved, with DS and ypT0 rates reaching 64.4% and 20% for good-risk and 64.5% and 42% for poor-risk patients, respectively. CONCLUSION: To our knowledge, this is the first study to prospectively use TYMS genotyping to direct neoadjuvant CRT in patients with rectal cancer. High rates of DS and ypT0 were achieved among both risk groups when personalized treatment was based on TYMS genotype. These results are encouraging, and further evaluation of this genotype-based strategy using a randomized study design for locally advanced rectal cancer is warranted.
Assuntos
Adenocarcinoma/genética , Adenocarcinoma/terapia , Terapia Neoadjuvante/métodos , Neoplasias Retais/genética , Neoplasias Retais/terapia , Timidilato Sintase/genética , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Quimioterapia Adjuvante , Terapia Combinada , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Genótipo , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Invasividade Neoplásica/patologia , Estadiamento de Neoplasias , Seleção de Pacientes , Prognóstico , Estudos Prospectivos , Radioterapia Adjuvante , Neoplasias Retais/mortalidade , Neoplasias Retais/patologia , Medição de Risco , Análise de Sobrevida , Timidilato Sintase/efeitos dos fármacos , Resultado do TratamentoRESUMO
To clarify the molecular interaction of irinotecan (CPT-11) and oxaliplatin (l-OHP) in combination with 5-fluorouracil (5-FU), the antitumor effects of CPT-11 and l-OHP combined with the oral 5-FU prodrug, S-1 composed by tegafur, gimeracil and potassium oteracil, were investigated on human colon cancer KM12C xenografts sensitive or resistant to 5-FU in nude mice. In parental KM12C tumor xenografts, combined treatment of CPT-11 with oral S-1 significantly augmented the antitumor activity compared with those of CPT-11 and S-1 alone. Interestingly, combined therapy of CPT-11 with S-1 was markedly effective with almost 90% of inhibition of tumor growth on 5-FU-resistant tumors (KM12C/ 5-FU), and its potency likely corresponded to that in parental tumors. In contrast, combined administration of l-OHP with S-1 did not shown an effect on KM12C/5-FU tumor xenografts. To investigate why only CPT-11 potentiated the anti-tumor activity in combination with 5-FU pro-drugs against 5-FU-resistant colon tumors, the activities or expression levels of thymidylate synthase (TS), ribonucleotide reductase (RNR) and other enzymes in 5-FU-metabolism in both tumors were measured following administration of CPT-11 and/or l-OHP. CPT-11, but not l-OHP, induced a decrease in activities and protein levels of TS and an increase in those of RNR in KM12C/5-FU tumors only, which was likely related to decreased expressions of several proteins in G1/S phase of the cells including CDK4, pRB, and E2F1 in these tumors. These findings suggest that CPT-11, but not l-OHP, would overcome the resistance to 5-FU in combination with 5-FU pro-drugs on 5-FU-resistant colon tumors.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Camptotecina/análogos & derivados , Neoplasias do Colo/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/fisiologia , Timidilato Sintase/metabolismo , Animais , Antineoplásicos Fitogênicos/administração & dosagem , Western Blotting , Camptotecina/administração & dosagem , Neoplasias do Colo/enzimologia , Regulação para Baixo , Combinação de Medicamentos , Fluoruracila , Humanos , Irinotecano , Camundongos , Camundongos Nus , Compostos Organoplatínicos/administração & dosagem , Oxaliplatina , Ácido Oxônico/administração & dosagem , Pró-Fármacos/administração & dosagem , Tegafur/administração & dosagem , Timidilato Sintase/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Auger electron emitting radiopharmaceuticals are attractive for targeted nanoirradiation therapy, provided that DNA of malignant cells is selectively addressed. Here, we examine 5-[123/125/131I]iodo-4'-thio-2'-deoxyuridine (ITdU) for targeting DNA in tumor cells in a HL60 xenograft severe combined immunodeficient mouse model. EXPERIMENTAL DESIGN: Thymidine kinase and phosphorylase assays were done to determine phosphorylation and glycosidic bond cleavage of ITdU, respectively. The biodistribution and DNA incorporation of ITdU were determined in severe combined immunodeficient mice bearing HL60 xenografts receiving pretreatment with 5-fluoro-2'-deoxyuridine (FdUrd). Organ tissues were dissected 0.5, 4, and 24 h after radioinjection and uptake of [131I]ITdU (%ID/g tissue) was determined. Cellular distribution of [125I]ITdU was imaged by microautoradiography. Apoptosis and expression of the proliferation marker Ki-67 were determined by immunohistologic staining using corresponding paraffin tissue sections. RESULTS: ITdU is phosphorylated by thymidine kinase 1 and stable toward thymidylate phosphatase-mediated glycosidic bond cleavage. Thymidylate synthase-mediated deiodination of [123/125/131I]ITdU was inhibited with FdUrd. Pretreatment with FdUrd increased preferentially tumor uptake of ITdU resulting in favorable tumor-to-normal tissue ratios and tumor selectivity. ITdU was exclusively localized within the nucleus and incorporated into DNA. In FdUrd-pretreated animals, we found in more than 90% of tumor cells apoptosis induction 24 h postinjection of ITdU, indicating a highly radiotoxic effect in tumor cells but not in cells of major proliferating tissues. CONCLUSION: ITdU preferentially targets DNA in proliferating tumor cells and leads to apoptosis provided that the thymidylate synthase is inhibited.
Assuntos
Desoxiuridina/análogos & derivados , Neoplasias Experimentais/tratamento farmacológico , Compostos Radiofarmacêuticos/farmacocinética , Compostos Radiofarmacêuticos/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Autorradiografia , DNA/efeitos dos fármacos , Desoxiuridina/farmacocinética , Desoxiuridina/uso terapêutico , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Camundongos SCID , Timidilato Sintase/antagonistas & inibidores , Timidilato Sintase/efeitos dos fármacos , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Resistance to chemotherapy is believed to be a major cause of treatment failure in pancreatic cancer. Thus, it is necessary to explore alternative therapeutic modalities to overcome drug resistance in pancreatic cancer treatment. We tested the hypothesis that Src tyrosine kinase inhibition could augment the chemosensitivity of 5-fluorouracil (5-FU)-resistant human pancreatic cancer cells to 5-FU. As detected by MTT proliferation assay, propidium iodide and annexin V staining, a combination of 5-FU+Src kinase inhibitor PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) reflected the chemotherapeutic sensitivity and restored the 5-FU-induced apoptosis in 5-FU-resistant cells. Furthermore, when small-interfering RNA approach to silence Src gene expression was applied, the degree of 5-FU-induced apoptosis was increased in all cell lines independently of the chemoresistance status. Western blotting and RT-PCR analysis revealed that the expression of thymidylate synthase (TS) was higher in 5-FU-resistant cells, however, decreased significantly after pretreatment with PP2. Furthermore, the combination of 5-FU+PP2 decreased the 5-FU-induced activation of epidermal growth factor receptor (EGFR)-AKT pathway. Finally, PP2 in combination with 5-FU substantially decreased the in vivo tumor growth and inhibited distant metastases. Taken together, 5-FU chemoresistance can be reversed through indirect TS regulation by inhibiting Src tyrosine kinase. A potential mechanism of action of Src kinase inhibitors on 5-FU chemosensitivity might be linked to the inhibition of 5-FU-induced EGFR-AKT activation.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Receptores ErbB/metabolismo , Fluoruracila/administração & dosagem , Neoplasias Pancreáticas/metabolismo , Quinases da Família src/metabolismo , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Inibidores Enzimáticos/administração & dosagem , Citometria de Fluxo , Humanos , Masculino , Camundongos , Neoplasias Pancreáticas/tratamento farmacológico , Pirimidinas/administração & dosagem , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Timidilato Sintase/efeitos dos fármacos , Timidilato Sintase/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Immunotherapy is one of the most effective treatments against metastatic renal cell carcinoma (RCC). However, the response rate is not high. Therefore, more effective therapies are necessary for patients with metastatic RCC. We previously reported on the significant antitumor activity of cationic multilamellar liposome containing human interferon-beta (huIFN-beta) gene (IAB-1) against RCC. We then examined the antitumor effect of IAB-1 in combination with anticancer drugs against RCC. The cytotoxicity of IAB-1 alone, and in combination with anticancer drugs, cisplatin, adriamycin, 5-fluorouracil, gemcitabine, paclitaxel and irinotecan hydrochloride against the human RCC cell line NC65 was examined by the colorimetric method using tetrazolium salt. For the in vivo study, we used NC65 cells inoculated into the severe combined immunodeficiency mouse. The results showed that the in vitro combination therapy with IAB-1 and 5-FU was more cytotoxic than IAB-1 alone. However, synergistic cytotoxicity was not observed when combined with IAB-1 and other anticancer drugs. NC65 tumors transfected with IAB-1 in mice were smaller than those receiving an injection of empty liposome or the recombinant huIFN-beta protein. Treatment with IAB-1 in combination with 5-FU resulted in significant anticancer activity. IAB-1 enhanced the activity of thymidine phosphorylase (TP), which converts 5-FU to the active metabolite, FdUMP. In contrast, IAB-1 decreased the activity of thymidylate synthase (TS), which is a target enzyme of 5-FU. In conclusion, these findings indicate that a combination of IAB-1 and 5-FU may have enhanced antitumor activity against human RCC, suggesting its potential clinical application. The mechanism of enhanced cytotoxicity by combination therapy with IAB-1 and 5-FU may up-regulate TP activity and down-regulate TS activity.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma de Células Renais/tratamento farmacológico , Fluoruracila/administração & dosagem , Imunoterapia/métodos , Interferon beta/administração & dosagem , Neoplasias Renais/tratamento farmacológico , Animais , Camptotecina/administração & dosagem , Camptotecina/análogos & derivados , Cátions , Cisplatino/administração & dosagem , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Doxorrubicina/administração & dosagem , Feminino , Humanos , Interferon beta/genética , Irinotecano , Lipossomos , Camundongos , Camundongos SCID , Paclitaxel/administração & dosagem , Timidina Fosforilase/efeitos dos fármacos , Timidina Fosforilase/metabolismo , Timidilato Sintase/efeitos dos fármacos , Timidilato Sintase/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
BACKGROUND: S-1 is the most effective oral fluoropyrimidine derivative widely used for patients with gastric carcinoma in Japan. Although S-1 plus taxane has been a promising candidate as an effective chemotherapeutic regimen, the mechanisms of its additive or synergistic anticancer effects and changes in gene expression after the administration of these agents have not yet been fully elucidated. METHODS: Experimental chemotherapy was performed using human gastric carcinoma xenografts, MKN-45 and TMK-1, to examine anticancer effects and gene expressions of fluoropyrimidine metabolism-related enzymes including thymidine phosphorylase (TP), thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), orotate phosphoribosyltransferase (OPRT), and uridine phosphorylase (UP). Nude mice were treated with S-1, paclitaxel, and their combination. After treatment, in vivo antitumor effects of S-1, paclitaxel alone, and their combination and the effects on gene expressions of enzymes involved in 5-fluorouracil metabolism were examined using the RT-PCR method. RESULTS: The combined use of S-1 and paclitaxel showed additive to synergistic antitumor effects on both gastric cancer xenografts. While consistent upregulation of dThPase and DPD gene expression was exhibited after administration of S-1, no further increase of dThPase gene expression after combined use of S-1 with paclitaxel was observed. There was no increase in TS gene expression after the administration of either S-1 alone or paclitaxel alone. CONCLUSION: These results provide some insight into the mechanism and/or rationale underlying the additive to synergistic effect of combined administration of S-1 and paclitaxel in gastric carcinoma.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica , Ácido Oxônico/farmacologia , Paclitaxel/farmacologia , Neoplasias Gástricas/tratamento farmacológico , Tegafur/farmacologia , Animais , Di-Hidrouracila Desidrogenase (NADP)/efeitos dos fármacos , Combinação de Medicamentos , Sinergismo Farmacológico , Quimioterapia Combinada , Feminino , Fluoruracila/farmacologia , Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Orotato Fosforribosiltransferase/efeitos dos fármacos , Neoplasias Gástricas/enzimologia , Timidina Fosforilase/efeitos dos fármacos , Timidilato Sintase/efeitos dos fármacos , Uridina Fosforilase/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Despite presenting bioavailability problems, tea catechins have emerged as promising chemopreventive agents because of their observed efficacy in various animal models. To improve the stability and cellular absorption of tea polyphenols, we developed a new catechin-derived compound, 3- O-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin (TMECG), which has shown significant antiproliferative activity against several cancer cell lines, especially melanoma. The presence of methoxy groups in its ester-bound gallyl moiety drastically decreased its antioxidant and prooxidant properties without affecting its cell-antiproliferative effects, and the data indicated that the 3-gallyl moiety was essential for its biological activity. As regards its action mechanism, we demonstrated that TMECG binds efficiently to human dihydrofolate reductase and down-regulates folate cycle gene expression in melanoma cells. Disruption of the folate cycle by TMECG is a plausible explanation for its observed biological effects and suggests that, like other antifolate compounds, TMECG could be of clinical value in cancer therapy.
Assuntos
Antioxidantes/síntese química , Antioxidantes/farmacologia , Catequina/análogos & derivados , Antagonistas do Ácido Fólico/síntese química , Antagonistas do Ácido Fólico/farmacologia , Antioxidantes/química , Catequina/síntese química , Catequina/química , Catequina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Ácido Fólico/efeitos dos fármacos , Ácido Fólico/metabolismo , Antagonistas do Ácido Fólico/química , Perfilação da Expressão Gênica , Humanos , Metilenotetra-Hidrofolato Redutase (NADPH2)/efeitos dos fármacos , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Modelos Moleculares , Estrutura Molecular , NADP/química , NADP/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , Estereoisomerismo , Relação Estrutura-Atividade , Chá/química , Tetra-Hidrofolato Desidrogenase/efeitos dos fármacos , Tetra-Hidrofolato Desidrogenase/genética , Timidilato Sintase/efeitos dos fármacos , Timidilato Sintase/genética , Fatores de TempoRESUMO
BACKGROUND: The active components of Cannabis sativa L., Cannabinoids, traditionally used in the field of cancer for alleviation of pain, nausea, wasting and improvement of well-being have received renewed interest in recent years due to their diverse pharmacologic activities such as cell growth inhibition, anti-inflammatory activity and induction of tumor regression. Here we used several experimental approaches, which identified delta-9-tetrahydrocannabinol (Delta(9)-THC) as an essential mediator of cannabinoid antitumoral action. METHODS AND RESULTS: Administration of Delta(9)-THC to glioblastoma multiforme (GBM) cell lines results in a significant decrease in cell viability. Cell cycle analysis showed G(0/1) arrest and did not reveal occurrence of apoptosis in the absence of any sub-G(1) populations. Western blot analyses revealed a THC altered cellular content of proteins that regulate cell progression through the cell cycle. The cell content of E2F1 and Cyclin A, two proteins that promote cell cycle progression, were suppressed in both U251-MG and U87-MG human glioblastoma cell lines, whereas the level of p16(INK4A), a cell cycle inhibitor was upregulated. Transcription of thymidylate synthase (TS) mRNA, which is promoted by E2F1, also declined as evident by QRT-PCR. The decrease in E2F1 levels resulted from proteasome mediated degradation and was prevented by proteasome inhibitors. CONCLUSIONS: Delta(9)-THC is shown to significantly affect viability of GBM cells via a mechanism that appears to elicit G(1) arrest due to downregulation of E2F1 and Cyclin A. Hence, it is suggested that Delta(9)-THC and other cannabinoids be implemented in future clinical evaluation as a therapeutic modality for brain tumors.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Ciclo Celular/efeitos dos fármacos , Dronabinol/farmacologia , Fator de Transcrição E2F1/efeitos dos fármacos , Fator de Transcrição E2F1/metabolismo , Glioblastoma/tratamento farmacológico , Western Blotting , Neoplasias Encefálicas/metabolismo , Divisão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Inibidor p16 de Quinase Dependente de Ciclina/efeitos dos fármacos , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Regulação para Baixo/efeitos dos fármacos , Fator de Transcrição E2F1/genética , Imunofluorescência , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/metabolismo , Humanos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Timidilato Sintase/efeitos dos fármacos , Timidilato Sintase/metabolismo , Fatores de Tempo , Regulação para CimaRESUMO
BACKGROUND: Colorectal cancer (CRC) cell lines displaying microsatellite instability (MSI) are resistant to 5-fluorouracil (5-FU) in vitro, which can be overcome by restoring DNA mismatch repair (MMR) competence. Thymidylate synthase (TS) is inhibited by 5-FU, being another potential mediator of therapeutic resistance to 5-FU. The clinical relevance of these observations remains unclear. OBJECTIVE: We examined the expression of TS and two MMR proteins (hMLH1 and hMSH2) in advanced CRC patients, to determine a) their mutual relationship, b) association to therapeutic response and c) impact on disease outcome. MATERIAL AND METHODS: Tumour samples from 73 patients CRC who were treated in advanced stage with either irinotecan alone or in combination with 5-FU/leucovorin, were analysed for expression of TS, hMLH1 and hMSH2 using immunohistochemistry (IHC). RESULTS: TS expression was closely correlated with hMLH1 expression (negative-weak/moderate-strong) (p=0.0001). TS-MMR expression was significantly (p=0.029 for whole series; p=0.004 for the 5-FU treated cases) related to response to treatment; tumours with low levels of both TS and MMR responded better (n=14/27, 51.8%) than those with high TS and MMR (n=3/18, 16.6%). Patients with high TS-MMR expression had a significantly longer DFS (47 months vs. 9 months, n=26) than those with low TS-MMR index (p=0.015), while the reverse was true concerning survival with metastases (WMS) (p=0.018) in all the patients (n=73). CONCLUSIONS: The present data suggest that MSI patients with low TS and deficient MMR demonstrate a significantly shorter DFS and longer WMS than patients with high expression of both markers, and they are also more likely to obtain the greatest benefit from 5-FU based chemotherapy.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/análise , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/análise , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/genética , Instabilidade de Microssatélites , Proteína 2 Homóloga a MutS/análise , Proteínas Nucleares/análise , Timidilato Sintase/análise , Proteínas Adaptadoras de Transdução de Sinal/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/genética , Adulto , Idoso , Biomarcadores Tumorais/genética , Camptotecina/administração & dosagem , Camptotecina/análogos & derivados , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Neoplasias Colorretais/terapia , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Intervalo Livre de Doença , Europa (Continente)/epidemiologia , Feminino , Fluoruracila/administração & dosagem , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Irinotecano , Estimativa de Kaplan-Meier , Masculino , Instabilidade de Microssatélites/efeitos dos fármacos , Pessoa de Meia-Idade , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS/efeitos dos fármacos , Proteína 2 Homóloga a MutS/genética , Estadiamento de Neoplasias , Proteínas Nucleares/efeitos dos fármacos , Proteínas Nucleares/genética , Timidilato Sintase/efeitos dos fármacos , Timidilato Sintase/genética , Resultado do TratamentoRESUMO
Non-small-cell lung cancer (NSCLC) represents approximately 80% of all lung cancers. With modern platinum- based combination regimens, overall median survival has reached 9-12 months. Antifolates are active against several solid tumors and hematologic malignancies. The cytotoxic action of antifolates is mainly related to their ability to inhibit several different folate-dependent enzymes involved in DNA synthesis. Pemetrexed is a novel multitargeted antifolate that inhibits at least 3 of the enzymes involved in purine and pyrimidine synthesis: thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT). Pemetrexed was approved for the treatment of relapsed NSCLC as it produced equivalent response and survival rates and less toxicity compared with docetaxel. Pemetrexed in combination with platinum analogues or with gemcitabine showed equivalent clinical impact compared with standard combinations of platinum plus third-generation agents. We analyze the potential implications of pemetrexed's role in first-line chemotherapy of NSCLC as well as hints of differential cytotoxic action according to histology, new schedules of vitamin supplementation, and target enzymes expression levels. Issues of pharmacogenomics are becoming relevant in defining pemetrexed efficacy. Chemosensitivity was significantly linked to low levels of TS, GARFT, and DHFR in preclinical models. Consequently, the differential expression of TS according to histology might explain the different activity of pemetrexed according to histology, as recently postulated.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Antagonistas do Ácido Fólico/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Recidiva Local de Neoplasia/tratamento farmacológico , Antimetabólitos Antineoplásicos/uso terapêutico , Carboplatina/uso terapêutico , Cisplatino/uso terapêutico , Reagentes de Ligações Cruzadas/uso terapêutico , Quimioterapia Combinada , Glutamatos/uso terapêutico , Guanina/análogos & derivados , Guanina/uso terapêutico , Humanos , Pemetrexede , Timidilato Sintase/antagonistas & inibidores , Timidilato Sintase/efeitos dos fármacos , Falha de TratamentoRESUMO
The purpose of this study was to investigate the utility of plasma pharmacokinetic and pharmacodynamic measures including plasma deoxynucleosides, homocysteine and methylmalonic acid concentrations in understanding the time course and extent of the inhibition of thymidylate synthase (TS) by pemetrexed in the context of a phase I/II combination study with vinorelbine. Eighteen patients received supplementation with folic acid and Vitamin B(12) 1 week before beginning treatment with pemetrexed and vinorelbine administered in a dose-escalating manner on a 21-day cycle. Heparinised blood samples were collected from consenting patients in the first cycle for pharmacokinetic analyses and in the first two cycles for determination of plasma thymidine, deoxyuridine, homocysteine and methylmalonic acid concentrations. These values were correlated with response and toxicity. Plasma deoxyuridine was used as a measure of TS inhibition, and concentrations of deoxyuridine were significantly elevated relative to baseline on days 1 (P<0.01), 2 (P<0.001) and 3 (P<0.05) after treatment at all pemetrexed dose levels (400-700 mg m(-2)). The magnitude of deoxyuridine elevation correlated with pemetrexed area under the plasma concentration-time curve (AUC) (r(2)=0.23, P<0.05). However, deoxyuridine concentrations returned to baseline between 8 and 15 days after treatment with pemetrexed, suggesting that inhibition of TS was not durable. Pemetrexed AUC correlated with the percentage decline (relative to baseline) in both platelets (r(2)=0.58, P<0.001) and leucocytes (r(2)=0.26, P<0.05) at day 8. Baseline homocysteine was also significantly correlated with these measures of haematological toxicity (r(2)=0.37, P<0.01 and r(2)=0.39, P<0.01, respectively). In addition, there was a significant reduction of plasma homocysteine on days 8 (P<0.005) and 15 (P<0.05) in cycle 1 compared to baseline values. The results suggest that the TS inhibitory effects of pemetrexed are short-lived and make the case for a more frequent schedule of administration such as every 2 weeks. The lack of protracted TS inhibition may be due to concomitant vitamin administration, and this may be the mechanism by which vitamins prevent life-threatening toxicity from pemetrexed. Baseline homocysteine concentration remains a predictive marker for haematological toxicity even following folate supplementation.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Glutamatos/administração & dosagem , Guanina/análogos & derivados , Neoplasias Pulmonares/tratamento farmacológico , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Área Sob a Curva , Desoxiuridina/sangue , Suplementos Nutricionais , Relação Dose-Resposta a Droga , Feminino , Ácido Fólico/uso terapêutico , Glutamatos/efeitos adversos , Glutamatos/farmacocinética , Guanina/administração & dosagem , Guanina/efeitos adversos , Guanina/farmacocinética , Homocisteína/sangue , Humanos , Masculino , Ácido Metilmalônico/sangue , Pessoa de Meia-Idade , Pemetrexede , Timidina/sangue , Timidilato Sintase/efeitos dos fármacos , Vimblastina/administração & dosagem , Vimblastina/efeitos adversos , Vimblastina/análogos & derivados , Vimblastina/farmacocinética , Vinorelbina , Vitamina B 12/uso terapêuticoRESUMO
BACKGROUND: S-1 is an oral fluorouracil antitumor drug that combines three pharmacological agents: tegafur, a prodrug of 5-fluorouracil (5-FU); 5-chloro-2,4-dihydroxypyridine (CDHP), an inhibitor of dihydropyrimidine dehydrogenase (DPD); and potassium oxonate, an agent included to reduce gastrointestinal toxicity. S-1 has a potent antitumor effect on gastric cancer, even in 5-FU-refractory cases. However, there is a lack of basic information to account for this clinical outcome. This study was performed to determine the differences in antitumor effects of combined administration of 5-FU and CDHP between NUGC-3 cells and NUGC-3/5FU/L cells, which are resistant to 5-FU (established by repeated cultures of NUGC-3 with escalating concentrations of 5-FU), and to determine the mechanisms involved. METHODS: Both cell lines were incubated with various concentrations of 5-FU and/or CDHP. The antitumor effect was assessed using an MTS assay and cell counts. DPD levels were assayed by using enzyme-linked immunosorbent assay. Expression of DPD and thymidylate synthase (TS) mRNA was quantified using real-time quantitative polymerase chain reaction analysis. RESULTS: The combination of 5-FU (IC15) with CDHP exerted a synergistic antitumor effect on NUGC-3/5FU/L, but not on NUGC-3, while CDHP by itself did not affect cell growth in either cell line. Expression of DPD was not detected in NUGC-3/5FU/L. In NUGC-3/5FU/L, 5-FU-enhanced expression of TS mRNA was inhibited by the addition of CDHP. In contrast, in NUGC-3, administration of 5-FU with or without CDHP did not alter TS mRNA expression. CONCLUSIONS: The inhibitory mechanism of CDHP, which is independent of DPD, may in part contribute to the antitumor effect of S-1 even in 5-FU-resistant gastric cancer cases.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Di-Hidrouracila Desidrogenase (NADP)/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Neoplasias Gástricas/tratamento farmacológico , Linhagem Celular Tumoral , Di-Hidrouracila Desidrogenase (NADP)/genética , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Ensaio de Imunoadsorção Enzimática , Fluoruracila/administração & dosagem , Fluoruracila/farmacologia , Humanos , Ácido Oxônico/farmacologia , Piridinas/administração & dosagem , Piridinas/farmacologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Gástricas/genética , Tegafur/farmacologia , Timidilato Sintase/efeitos dos fármacos , Timidilato Sintase/genéticaRESUMO
Colorectal cancer (CRC) resistance to fluoropyrimidines and other inhibitors of thymidylate synthase (TS) is a serious clinical problem often associated with increased intracellular levels of TS. Since the tumour suppressor gene p53, which is mutated in 50% of CRC, regulates the expression of several genes, it may modulate TS activity, and changes in the status of p53 might be responsible for chemoresistance. Therefore, this study was aimed to investigate TS levels and sensitivity to TS inhibitors in wild-type (wt) and mutant (mt) p53 CRC cells, Lovo and WiDr, respectively, transfected with mt and wt p53. Lovo 175X2 cells (transfected with mt p53) were more resistant to 5-fluorouracil (5-FU; 2-fold), nolatrexed (3-fold), raltitrexed (3-fold) and pemetrexed (10-fold) in comparison with the wt p53 parental cells Lovo 92. Resistance was associated with an increase in TS protein expression and catalytic activity, which might be caused by the loss of the inhibitory effect on the activity of TS promoter or by the lack of TS mRNA degradation, as suggested by the reversal of TS expression to the levels of Lovo 92 cells by adding actinomycin. In contrast, Lovo li cells, characterized by functionally inactive p53, were 3-13-fold more sensitive to nolatrexed, raltitrexed and pemetrexed, and had a lower TS mRNA, protein expression and catalytic activity than Lovo 92. However, MDM-2 expression was significantly higher in Lovo li, while no significant differences were observed in Lovo 175X2 cells with respect to Lovo 92. Finally, mt p53 WiDr transfected with wt p53 were not significantly different from mt p53 WiDr cells with respect to sensitivity to TS inhibitors or TS levels. Altogether, these results indicate that changes in the status of p53, can differently alter sensitivity to TS inhibitors by affecting TS levels, depending on activity or cell line, and might explain the lack of clear correlation between mutations in p53 and clinical outcome after chemotherapy with TS inhibitors.