RESUMEN
BACKGROUND: Expression of proton-coupled folate transporter (PCFT) is associated with survival of mesothelioma patients treated with pemetrexed, and is reduced by hypoxia, prompting studies to elucidate their correlation. METHODS: Modulation of glycolytic gene expression was evaluated by PCR arrays in tumour cells and primary cultures growing under hypoxia, in spheroids and after PCFT silencing. Inhibitors of lactate dehydrogenase (LDH-A) were tested in vitro and in vivo. LDH-A expression was determined in tissue microarrays of radically resected malignant pleural mesothelioma (MPM, N = 33) and diffuse peritoneal mesothelioma (DMPM, N = 56) patients. RESULTS: Overexpression of hypoxia marker CAIX was associated with low PCFT expression and decreased MPM cell growth inhibition by pemetrexed. Through integration of PCR arrays in hypoxic cells and spheroids and following PCFT silencing, we identified the upregulation of LDH-A, which correlated with shorter survival of MPM and DMPM patients. Novel LDH-A inhibitors enhanced spheroid disintegration and displayed synergistic effects with pemetrexed in MPM and gemcitabine in DMPM cells. Studies with bioluminescent hypoxic orthotopic and subcutaneous DMPM athymic-mice models revealed the marked antitumour activity of the LDH-A inhibitor NHI-Glc-2, alone or combined with gemcitabine. CONCLUSIONS: This study provides novel insights into hypoxia/PCFT-dependent chemoresistance, unravelling the potential prognostic value of LDH-A, and demonstrating the preclinical activity of LDH-A inhibitors.
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Antígenos de Neoplasias/genética , Anhidrasa Carbónica IX/genética , Inhibidores Enzimáticos/administración & dosificación , L-Lactato Deshidrogenasa/genética , Mesotelioma Maligno/tratamiento farmacológico , Neoplasias Peritoneales/tratamiento farmacológico , Neoplasias Pleurales/tratamiento farmacológico , Transportador de Folato Acoplado a Protón/genética , Animales , Antígenos de Neoplasias/metabolismo , Anhidrasa Carbónica IX/metabolismo , Técnicas de Cultivo de Célula , Hipoxia de la Célula , Línea Celular Tumoral , Desoxicitidina/administración & dosificación , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Sinergismo Farmacológico , Inhibidores Enzimáticos/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Mesotelioma Maligno/genética , Mesotelioma Maligno/metabolismo , Ratones , Pemetrexed/administración & dosificación , Pemetrexed/farmacología , Neoplasias Peritoneales/genética , Neoplasias Peritoneales/metabolismo , Neoplasias Pleurales/genética , Neoplasias Pleurales/metabolismo , Transportador de Folato Acoplado a Protón/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
PURPOSE: Many patients with Crohn's disease (CD) and ulcerative colitis (UC) who have a high 6-methylmercaptopurine/6-thioguanine (6-MMP/6-TGN) ratio receive allopurinol 100 mg in addition to thiopurines to optimize metabolite concentrations. However, some patients do not tolerate allopurinol at this dosage. The aim of this study was to determine the intra-patient effect of reducing the allopurinol dosage from 100 to 50 mg, in terms of metabolite concentrations, enzyme activities, efficacy, and tolerability. METHODS: A prospective non-inferiority one-way crossover study was performed. CD and UC patients with stable disease using a thiopurine and allopurinol 100 mg were switched to 50 mg for 1 month. Primary outcomes were thiopurine metabolite concentrations. Secondary outcomes were enzyme activities of xanthine oxidase, thiopurine methyltransferase and hypoxanthine-guanine phosphoribosyltransferase, disease activity, and tolerability. RESULTS: Twenty-two patients were included. Treatment with allopurinol 50 mg compared with 100 mg resulted in a significant decrease in mean 6-TGN levels (761 to 625 pmol/8 × 108 RBC; p = 0.005) and a significant increase in mean 6-MMP levels (451 to 665 pmol/8 × 108 RBC; p = 0.01). However, the mean metabolite concentrations were still therapeutic. Enzyme activities, disease activity scores, and patient experiences did not alter significantly. Generally, UC patients were more positive about their improved treatment than CD patients. CONCLUSION: Combination therapy with 50 mg allopurinol led to a decrease of 6-TGN levels compared with 100 mg allopurinol. Disease activity, side effects, and patient experience, however, were similar between allopurinol 100 and 50 mg. UC patients seem to benefit and prefer lower doses whereas the contrary is seen in CD patients. TRIAL REGISTRATION: EudraCT trial registry - number 2016-001638-84.
Asunto(s)
Alopurinol/administración & dosificación , Azatioprina/administración & dosificación , Colitis Ulcerosa/tratamiento farmacológico , Enfermedad de Crohn/tratamiento farmacológico , Mercaptopurina/análogos & derivados , Adulto , Anciano , Alopurinol/efectos adversos , Colitis Ulcerosa/metabolismo , Enfermedad de Crohn/metabolismo , Estudios Cruzados , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Femenino , Humanos , Masculino , Mercaptopurina/administración & dosificación , Mercaptopurina/metabolismo , Metiltransferasas/metabolismo , Persona de Mediana Edad , Estudios Prospectivos , Tioguanina/metabolismoRESUMEN
A novel cytidine analog fluorocyclopentenylcytosine (RX-3117; TV-1360) was characterized for its cytotoxicity in a 59-cell line panel and further characterized for cytotoxicity, metabolism and mechanism of action in 15 additional cancer cell lines, including gemcitabine-resistant variants. In both panels sensitivity varied 75-fold (IC50: 0.4- > 30 µM RX-3117). RX-3117 showed a different sensitivity profile compared to cyclopentenyl-cytosine (CPEC) and azacytidine, substrates for uridine-cytidine-kinase (UCK). Dipyridamole, an inhibitor of the equilibrative-nucleoside-transporter protected against RX-3117. Uridine and cytidine protected against RX-3117, but deoxycytidine (substrate for deoxycytidine-kinase [dCK]) not, although it protected against gemcitabine, demonstrating that RX-3117 is a substrate for UCK and not for dCK. UCK activity was abundant in all cell lines, including the gemcitabine-resistant variants. RX-3117 was a very poor substrate for cytidine deaminase (66,000-fold less than gemcitabine). RX-3117 was rapidly metabolised to its nucleotides predominantly the triphosphate, which was highest in the most sensitive cells (U937, A2780) and lowest in the least sensitive (CCRF-CEM). RX-3117 did not significantly affect cytidine and uridine nucleotide pools. Incorporation of RX-3117 into RNA and DNA was higher in sensitive A2780 and low in insensitive SW1573 cells. In sensitive U937 cells 1 µM RX-3117 resulted in 90% inhibition of RNA synthesis but 100 µM RX-3117 was required in A2780 and CCRF-CEM cells. RX-3117 at IC50 values did not affect the integrity of RNA. DNA synthesis was completely inhibited in sensitive U937 cells at 1 µM, but in other cells even higher concentrations only resulted in a partial inhibition. At IC50 values RX-3117 downregulated the expression of DNA methyltransferase. In conclusion, RX-3117 showed a completely different sensitivity profile compared to gemcitabine and CPEC, its uptake is transporter dependent and is activated by UCK. RX-3117 is incorporated into RNA and DNA, did not affect RNA integrity, depleted DNA methyltransferase and inhibited RNA and DNA synthesis. Nucleotide formation is related with sensitivity.
Asunto(s)
Antineoplásicos/farmacología , Citidina/análogos & derivados , Línea Celular Tumoral , Citidina/farmacología , Citidina Desaminasa/metabolismo , ADN/metabolismo , Metilasas de Modificación del ADN/metabolismo , Humanos , ARN/metabolismo , Uridina Quinasa/metabolismoRESUMEN
Cytarabine (ara-C) and gemcitabine (dFdC) are commonly used anticancer drugs, which depend on the equilibrative (ENT) and concentrative-nucleoside-transporters to enter the cell. To bypass transport-related drug resistance, lipophilic derivatives elacytarabine (CP-4055), ara-C-5'elaidic-acid-ester, and CP-4126, (CO 1.01) gemcitabine-5'elaidic-acid-ester, were investigated for the entry into the cell, distribution, metabolism and retention. The leukemic CEM-cell-line and its deoxycytidine-kinase deficient variant (CEM/dCK-) were exposed for 30 and 60 min to the radiolabeled drugs; followed by culture in drug-free medium in order to determine drug retention in the cell. The cellular fractions were analyzed with thin-layer-chromatography and HPLC. Elacytarabine and CP-4126 were converted to the parent compounds both inside and outside the cell (35-45%). The ENT-inhibitor dipyridamole did not affect their uptake or retention. Inside the cell Elacytarabine and CP-4126 predominantly localized in the membrane and cytosolic fraction, leading to a long retention after removal of the medium. In contrast, in cells exposed to the parent drugs ara-C and dFdC, intracellular drug concentration increased during exposure but decreased to undetectable levels after drug removal. In the dCK- cell line, no metabolism was observed. The concentrations of ara-CTP and dFdCTP reached a peak at the end of the incubation with the drugs, and decreased after drug removal; peak levels of dFdCTP were 35 times higher than ara-CTP and was retained better. In contrast, after exposure to elacytarabine or CP-4126, ara-CTP and dFdCTP levels continued to increase not only during exposure but also during 120 min after removal of the elacytarabine and CP-4126. Levels of ara-CTP and dFdCTP were higher than after exposure to the parent drugs. In conclusion, the lipophilic derivatives elacytarabine and CP-4126 showed a nucleoside-transporter independent uptake, with long retention of the active nucleotides. These lipophilic nucleoside analogues are new chemical entities suitable for novel clinical applications.
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Antineoplásicos/farmacocinética , Citarabina/análogos & derivados , Desoxicitidina/análogos & derivados , Nucleotidasas/metabolismo , Antineoplásicos/metabolismo , Línea Celular , Citarabina/metabolismo , Citarabina/farmacocinética , Desoxicitidina/metabolismo , Desoxicitidina/farmacocinética , Desoxicitidina Quinasa/metabolismo , Dipiridamol/metabolismo , Resistencia a Antineoplásicos , Humanos , Leucemia/metabolismo , Proteínas de Transporte de Nucleósidos/metabolismo , GemcitabinaRESUMEN
Prodrugs can have the advantage over parent drugs in increased activation and cellular uptake. The multidrug ETC-L-FdUrd and the duplex drug ETC-FdUrd are composed of two different monophosphate-nucleosides, 5-fluoro-2'deoxyuridine (FdUrd) and ethynylcytidine (ETC), coupled via a glycerolipid or phosphodiester, respectively. The aim of the study was to determine cytotoxicity levels and mode of drug cleavage. Moreover, we determined whether a liposomal formulation of ETC-L-FdUrd would improve cytotoxic activity and/or cleavage. Drug effects/cleavage were studied with standard radioactivity assays, HPLC and LC-MS/MS in FM3A/0 mammary cancer cells and their FdUrd resistant variants FM3A/TK(-). ETC-FdUrd was active (IC(50) of 2.2 and 79 nM) in FM3A/0 and TK(-) cells, respectively. ETC-L-FdUrd was less active (IC(50): 7 nM in FM3A/0 vs 4500 nM in FM3A/TK(-)). Although the liposomal formulation was less active than ETC-L-FdUrd in FM3A/0 cells (IC(50):19.3 nM), resistance due to thymidine kinase (TK) deficiency was greatly reduced. The prodrugs inhibited thymidylate synthase (TS) in FM3A/0 cells (80-90%), but to a lower extent in FM3A/TK(-) (10-50%). FdUMP was hardly detected in FM3A/TK(-) cells. Inhibition of the transporters and nucleotidases/phosphatases resulted in a reduction of cytotoxicity of ETC-FdUrd, indicating that this drug was cleaved outside the cells to the monophosphates, which was verified by the presence of FdUrd and ETC in the medium. ETC-L-FdUrd and the liposomal formulation were neither affected by transporter nor nucleotidase/phosphatase inhibition, indicating circumvention of active transporters. In vivo, ETC-FdUrd and ETC-L-FdURd were orally active. ETC nucleotides accumulated in both tumor and liver tissues. These formulations seem to be effective when a lipophilic linker is used combined with a liposomal formulation.
Asunto(s)
Citidina/análogos & derivados , Floxuridina/farmacología , Animales , Línea Celular Tumoral , Citidina/administración & dosificación , Citidina/química , Citidina/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Floxuridina/administración & dosificación , Floxuridina/química , Fluorodesoxiuridilato/metabolismo , Humanos , Concentración 50 Inhibidora , Liposomas/metabolismo , Ratones , Proteínas de Transporte de Nucleósidos/metabolismo , Nucleósidos/metabolismo , Timidilato Sintasa/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The pyrimidine trifluorothymidine (TFT) inhibits thymidylate synthase (TS) and can be incorporated into the DNA. TFT, as part of TAS-102, is clinically evaluated in phase II studies as an oral chemotherapeutic agent. Erlotinib is a tyrosine kinase inhibitor of the epidermal growth factor receptor (EGFR) that is often deregulated in colorectal cancer. This study investigated molecular mechanisms underlying the cytotoxic actions of the combination of an EGFR-tyrosine kinase inhibitor with TFT in colorectal cancer cells Caco2, WiDR, Lovo92, and Colo320. Drug interactions were examined by the sulforhodamine B assay and subsequent combination index (CI) analyses, cell cycle effects by FACS analysis of propidium iodide stained cells, Akt, MAPK and EGFR phosphorylation and expression levels by Western blotting and TS activity by the TS in situ assay. All combination schedules were synergistic in wt-EGFR expressing (but with mutated downstream pathways) WiDR and Lovo92 (CI 0.4-0.8) and very synergistic in Caco2 cells (with wt-EGFR and functional downstream pathways; CI 0.1-0.3), but in EGFR-lacking Colo320 cells, no additional activity was found (CI 1.0-1.2). Synergism was mostly related to the induction of cell cycle arrest and an erlotinib-mediated inhibition of the pro-survival signaling through Akt and MAPK that was activated (phosphorylated) by TFT. Erlotinib inhibited TS activity in EGFR-expressing cell lines, probably due to cell cycle arrest in the G(1) phase. TS activity was slightly lower in the combinations, probably due to cell cycle interference. Taken together, the combination of erlotinib with TFT seems to present a potential strategy in the field of molecular therapeutics.
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Neoplasias Colorrectales/tratamiento farmacológico , Receptores ErbB/antagonistas & inhibidores , Quinazolinas/administración & dosificación , Trifluridina/administración & dosificación , Línea Celular Tumoral , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Daño del ADN , Sinergismo Farmacológico , Clorhidrato de Erlotinib , Fase G1/efectos de los fármacos , Genes ras , Humanos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Timidilato Sintasa/antagonistas & inhibidoresRESUMEN
Because the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib and the multitargeted antifolate pemetrexed are registered in the treatment of second-line non-small-cell lung cancer (NSCLC), empirical combinations of these drugs are being tested. This study investigated molecular mechanisms underlying their combination in six NSCLC cell lines. Cells were characterized by heterogeneous expression of pemetrexed determinants, including thymidylate synthase (TS) and dihydrofolate reductase (DHFR), and mutations potentially affecting chemosensitivity. Pharmacological interaction was studied using the combination index (CI) method, whereas cell cycle, apoptosis induction, and EGFR, extracellular signal-regulated kinases 1 and 2, and Akt phosphorylation were studied by flow cytometry, fluorescence microscopy, and enzyme-linked immunosorbent assays. Reverse-transcriptase polymerase chain reaction (RT-PCR), Western blot, and activity assays were performed to assess whether erlotinib influenced TS. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assays demonstrated that EGFR and k-Ras mutations were related to erlotinib sensitivity, whereas TS and DHFR expression were related to pemetrexed sensitivity. Synergistic cytotoxicity was found in all cells, most pronounced with pemetrexed + erlotinib (24 h) --> erlotinib (48 h) sequence (CI, 0.09-0.40), which was associated with a significant induction of apoptosis. Pemetrexed increased EGFR phosphorylation and reduced Akt phosphorylation, which was additionally reduced by drug combination (-70.6% in H1650). Erlotinib significantly reduced TS expression and activity, possibly via E2F-1 reduction, as detected by RT-PCR and Western blot, and the combination decreased TS in situ activity in all cells. Erlotinib and pemetrexed showed a strong synergism in NSCLC cells, regardless of their genetic characteristics. Induction of apoptosis, modulation of EGFR and Akt phosphorylation, and changes in the expression of critical genes involved in pemetrexed activity contribute to this synergistic interaction and support the clinical investigation of these markers.
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Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/patología , Receptores ErbB/antagonistas & inhibidores , Antagonistas del Ácido Fólico/farmacología , Glutamatos/farmacología , Guanina/análogos & derivados , Neoplasias Pulmonares/patología , Quinazolinas/farmacología , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Carcinoma de Pulmón de Células no Pequeñas/genética , Catálisis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Extractos Celulares , Línea Celular Tumoral , Cromonas/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Factor de Transcripción E2F1/genética , Factor de Transcripción E2F1/metabolismo , Clorhidrato de Erlotinib , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Guanina/farmacología , Humanos , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/genética , Morfolinas/farmacología , Pemetrexed , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/metabolismo , Timidilato Sintasa/genética , Timidilato Sintasa/metabolismoRESUMEN
Multidrugs have the potential to bypass resistance. We investigated the in vitro activity and resistance circumvention of the multidrug cytarabine-L-fluorodeoxyuridine (AraC-L-5FdU), linked via a glycerophospholipid linkage. Cytotoxicity was determined using sensitive (A2780, FM3A/0) and resistant (AG6000, AraC resistant, deoxycytidine kinase deficient; FM3A/TK-, 5FdU resistant, thymidine kinase deficient) cell lines. Circumvention of nucleoside transporter and activating enzymes was determined using specific inhibitors, HPLC analysis and standard radioactivity assays. AraC-L-5FdU was active (IC50: 0.03 microM in both A2780 and FM3A/0), had some activity in AG6000 (IC50: 0.28 microM), but no activity in FM3A/TK(-) (IC50: 18.3 microM). AraC-nucleotides were not detected in AG6000. 5FdU-nucleotides were detected in all cell lines. AraC-L-5FdU did not inhibit TS in FM3A/TK(-) (5%). Since phosphatase/nucleotidase-inhibition reduced cytotoxicity 7-70-fold, cleavage seems to be outside the cell, presumably to nucleotides, and then to nucleosides. The multidrug was orally active in the HT-29 colon carcinoma xenografts which are resistant toward the single drugs.
Asunto(s)
Antineoplásicos/farmacología , Desoxiuridina/análogos & derivados , Administración Oral , Animales , Antineoplásicos/química , Antineoplásicos/metabolismo , Línea Celular Tumoral , Desoxiuridina/química , Desoxiuridina/metabolismo , Desoxiuridina/farmacología , Femenino , Concentración 50 Inhibidora , Ratones , Ratones Endogámicos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Gemcitabine is a deoxycytidine (dCyd) analogue with activity against several solid cancers. Gemcitabine is activated by dCyd kinase (dCK) and interferes, as its triphosphate dFdCTP, with tumor growth through incorporation into DNA. Alternatively, the metabolite gemcitabine diphosphate (dFdCDP) can interfere with DNA synthesis and thus tumor growth through inhibition of ribonucleotide reductase. Gemcitabine can be inactivated by the enzyme dCyd deaminase (dCDA). In most in vitro models, resistance to gemcitabine was associated with a decreased dCK activity. In all these models, resistance was established using continuous exposure to gemcitabine with increasing concentrations; however, these in vitro models have limited clinical relevance. To develop in vivo resistance to gemcitabine, we treated mice bearing a moderately sensitive tumor Colon 26-A (T/C = 0.25) with a clinically relevant schedule (120 mg/kg every 3 days). By repeated transplant of the most resistant tumor and continuation of gemcitabine treatment for >1 year, the completely resistant tumor Colon 26-G (T/C = 0.96) was created. Initial studies focused on resistance mechanisms known from in vitro studies. In Colon 26-G, dCK activity was 1.7-fold decreased; dCDA and DNA polymerase were not changed; and Colon 26-G accumulated 1.5-fold less dFdCTP, 6 hours after a gemcitabine injection, than the parental tumor. Based on in vitro studies, these relative minor changes were considered insufficient to explain the completely resistant phenotype. Therefore, an expression microarray was done with Colon 26-A versus Colon 26-G. Using independently grown nonresistant and resistant tumors, a striking increase in expression of the RRM1 subunit gene was found in Colon 26-G. The expression of RRM1 mRNA was 25-fold increased in the resistant tumor, as measured by real-time PCR, which was confirmed by Western blotting. In contrast, RRM2 mRNA was 2-fold decreased. However, ribonucleotide reductase enzyme activity was only moderately increased in Colon 26-G. In conclusion, this is the first model with in vivo induced resistance to gemcitabine. In contrast to most in vitro studies, dCK activity was not the most important determinant of gemcitabine resistance. Expression microarray identified RRM1 as the gene with the highest increase in expression in the Colon 26-G, which might clarify its complete gemcitabine-resistant phenotype. This study is the first in vivo evidence for a key role for RRM1 in acquired gemcitabine resistance.
Asunto(s)
Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/enzimología , Desoxicitidina/análogos & derivados , Ribonucleótido Reductasas/biosíntesis , Animales , Western Blotting , Neoplasias del Colon/genética , Citidina Desaminasa , Desoxicitidina/farmacología , Desoxicitidina Quinasa/biosíntesis , Desoxicitidina Quinasa/genética , Nucleótidos de Desoxicitosina/metabolismo , Resistencia a Antineoplásicos , Femenino , Perfilación de la Expresión Génica , Ratones , Ratones Endogámicos BALB C , Nucleósido Desaminasas/biosíntesis , Nucleósido Desaminasas/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Subunidades de Proteína , Ribonucleótido Reductasas/genética , GemcitabinaRESUMEN
Gemcitabine is a commonly used therapy for many solid tumors. Acquired resistance to this nucleoside analogue, however, diminishes the long-term effectiveness in a majority of patients. To better define the molecular background of gemcitabine resistance, a mouse colon tumor was selected during successive rounds of transplantation with continued treatment of gemcitabine. Expression microarray analysis was applied to determine which genes are consistently and highly overexpressed or underexpressed in the resistant versus the nonresistant tumor. For the statistical interpretation of the microarray data, a parametric model was implemented, which returns model-based differential gene expression (log-) ratios and their uncertainties. This defined a set of 13 genes, putatively responsible for the gemcitabine resistance in solid tumors. One of these, RRM1, was previously identified as an important marker for gemcitabine resistance in human cell lines. Five of the 13 genes, including RRM1, are located within a 3 Mb region at chromosome 7E1 of which four are highly overexpressed, suggesting a chromosomal amplification. Therefore, chromosomal copy number changes were measured, using oligo array comparative genomic hybridization. A narrow and high amplification area was identified on 7E1 that encompassed all five genes. In addition, reduced RNA expression of two other genes at 8E1 encoding COX4I1 and RPL13 could be explained by a decrease in chromosomal copy number on chromosome 8. In conclusion, the array comparative genomic hybridization biologically validates our statistical approach and shows that gemcitabine is capable to select for chromosomally aberrant tumor cells, where changed gene expression levels lead to drug resistance.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Aberraciones Cromosómicas/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/genética , Desoxicitidina/análogos & derivados , Animales , Replicación del ADN/genética , Desoxicitidina/farmacología , Resistencia a Antineoplásicos , Expresión Génica/efectos de los fármacos , Ratones , Hibridación de Ácido Nucleico , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , GemcitabinaRESUMEN
Gemcitabine (dFdC) can increase the sensitivity of both cisplatin (CDDP)-sensitive and -resistant cell lines. It has been postulated that both formation and repair of platinum-(Pt)-DNA adducts are related to these effects. Therefore, we investigated the effects of dFdC on the formation and repair of Pt-DNA adducts in the human ovarian cancer cell line, A2780, and its CDDP- or dFdC-resistant variants, ADDP and AG6000, which have a different expression of various repair enzymes. Cells were exposed for 1 h to CDDP alone or combined with dFdC in IC50 concentrations, followed by a 1-h exposure to thiourea and, subsequently, by a drug-free period of 1, 3 or 23 h (i.e. 2, 4 or 24 h after CDPP +/- dFdC removal). Pt-DNA adducts were quantified with 32P-post-labeling. The gene expression of the repair enzymes, XPA and XRCC1, was the same in all 3 cell lines but ERCC1, ERCC3 and XPC were 2-6 times higher in AG6000 compared to A2780 cells. In contrast, both ERCC1 and ERCC3 were 10- and 1.5-fold lower in ADDP cells compared to A2780. The mismatch enzyme, MLH1, was lower in ADDP cells. At equally toxic CDDP concentrations, all cell lines formed comparable peak levels of total Pt-DNA adducts (36-48 fmol/microg DNA). However, the time at which peak levels were reached showed large variation. The repair of the adducts was very efficient in the resistant cell lines whereas, in A2780 cells, plateau levels were retained until 24 h after CDDP exposure. In A2780 cells, dFdC shifted the adduct peaks from 4 h to directly after CDDP exposure and increased peak levels by >3.9-fold. dFdC also enhanced the repair of adducts by >1.7-fold and increased the Pt-GG:Pt-AG ratio compared to CDDP alone by >1.4-fold. Overall, dFdC decreased the area under the Pt-DNA adduct-time curve (AUA0-25 h) in A2780 cells by 2.7-fold. In ADDP cells, dFdC shifted the adduct peaks from 2 to 4 h and increased them by >2.2-fold. dFdC also increased the Pt-GG:Pt-AG ratio during the repair process by 1.4-fold. Overall, dFdC increased the AUA0-25 h in ADDP cells by 1.7-fold. In AG6000 cells, dFdC increased the Pt-GG:Pt-AG ratio by 1.6-fold directly after exposure but did not clearly affect the AUA0-25 h. In conclusion, dFdC can affect both Pt-DNA adduct formation and repair, depending on the initial sensitivity of the cells.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Aductos de ADN , Reparación del ADN/efectos de los fármacos , Desoxicitidina/análogos & derivados , Neoplasias Ováricas/patología , Cisplatino/farmacología , Desoxicitidina/farmacología , Resistencia a Antineoplásicos , Femenino , Humanos , Células Tumorales Cultivadas , GemcitabinaRESUMEN
Fluorocyclopentenylcytosine (RX-3117) is an orally available cytidine analog, currently in Phase I clinical trial. RX-3117 has promising antitumor activity in various human tumor xenografts including gemcitabine resistant tumors. RX-3117 is activated by uridine-cytidine kinase (UCK). Since UCK exists in two forms, UCK1 and UCK2, we investigated which form is responsible for RX-3117 phosphorylation. For that purpose we transfected A549 and SW1573 cell lines with UCK-siRNAs. Transfection of UCK1-siRNA efficiently downregulated UCK1-mRNA, but not UCK2-mRNA expression, and did not affect sensitivity to RX-3117. However, transfection of UCK2-siRNA completely downregulated UCK2-mRNA and protein and protected both A549 and SW1573 against RX-3117. UCK enzyme activity in two panels of tumor cell lines and xenograft cells correlated only with UCK2-mRNA expression (r = 0.803 and 0.915, respectively), but not with UCK1-mRNA. Moreover, accumulation of RX-3117 nucleotides correlated with UCK2 expression. In conclusion, RX-3117 is activated by UCK2 which may be used to select patients potentially sensitive to RX-3117.
Asunto(s)
Citidina/análogos & derivados , Uridina Quinasa/metabolismo , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Citidina/química , Citidina/farmacología , Regulación hacia Abajo/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Fosforilación/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Análisis de Regresión , Reproducibilidad de los Resultados , Especificidad por Sustrato/efectos de los fármacos , Transfección , Uridina Quinasa/genéticaRESUMEN
2',2'-Difluoro-2'-deoxycytidine (dFdC, gemcitabine) is a cytidine analogue active against several solid tumor types, such as ovarian, pancreatic and non-small cell lung cancer. The compound has a complex mechanism of action. Because of the structural similarity of one metabolite of dFdC, dFdUMP, with the natural substrate for thymidylate synthase (TS) dUMP, we investigated whether dFdC and its deamination product 2',2'-difluoro-2'-deoxyuridine (dFdU) would inhibit TS. This study was performed using two solid tumor cell lines: the human ovarian carcinoma cell line A2780 and its dFdC-resistant variant AG6000. The specific TS inhibitor Raltitrexed (RTX) was included as a positive control. Using the in situ TS activity assay measuring the intracellular conversion of [5-(3)H]-2'-deoxyuridine or [5-(3)H]-2'-deoxycytidine to dTMP and tritiated water, it was observed that dFdC and dFdU inhibited TS. In A2780 cells after a 4h exposure to 1 µM dFdC tritium release was inhibited by 50% but did not increase after 24h, Inhibition was also observed following dFdU at 100 µM. No effect was observed in the dFdC-resistant cell line AG6000; in this cell line only RTX had an inhibitory effect on TS activity. In the A2780 cell line RTX inhibited TS in a time dependent manner. In addition, DNA specific compounds such as 2'-C-cyano-2'-deoxy-1-beta-D-arabino-pentafuranosylcytosine and aphidicoline were utilized to exclude DNA inhibition mediated down regulation of the thymidine kinase. Inhibition of the enzyme resulted in a relative increase of mis-incorporation of [5-(3)H]-2'-deoxyuridine into DNA. In an attempt to elucidate the mechanism of in situ TS inhibition the ternary complex formation and possible inhibition in cellular extracts of A2780 cells, before and after exposure to dFdC, were determined. With the applied methods no proof for formation of a stable complex was found. In simultaneously performed experiments with 5FU such a complex formation could be demonstrated. However, using purified TS it was demonstrated that dFdUMP and not dFdCMP competitively inhibited TS with a Ki of 130 µM, without ternary complex formation. In conclusion, in this paper we reveal a new target of dFdC: thymidylate synthase.
Asunto(s)
Desoxicitidina/análogos & derivados , Floxuridina/análogos & derivados , Timidilato Sintasa/metabolismo , Línea Celular Tumoral , Daño del ADN/efectos de los fármacos , Desoxicitidina/farmacología , Activación Enzimática/efectos de los fármacos , Floxuridina/farmacología , Humanos , GemcitabinaRESUMEN
Folate based inhibitors of thymidylate synthase (TS) might facilitate binding of 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) to TS similar to the natural reduced folate 5,10-methylenetetrahydrofolate (CH(2)-H(4)-folate). We studied the lipophilic, non-polyglutamatable antifolates Nolatrexed (NTX) and AG331 and antifolates, that can have a polyglutamate side chain like the natural folate CH(2)-H(4)-folate; GW1843U89, Raltitrexed (RTX) and Multi-targetted antifolate (MTA) and pentaglutamates (RTX-Glu(5) and MTA-Glu(5)). The capacity of these compounds to facilitate the binding of [(3)H]FdUMP to Lactobacillus casei TS and an ammoniumsulphate precipitate of human TS was investigated. Only NTX, RTX-Glu(5) and MTA-Glu(5) facilitated FdUMP binding to L. casei TS and their dissociation constant K(d) (0.2-0.7 microM) was low compared to CH(2)-H(4)-folate (2.0 microM). The small lipophilic molecule NTX was favorable to the larger AG331. Polyglutamylation, as indicated by the difference in effect of RTX vs. RTX-Glu(5) and MTA vs. MTA-Glu(5), seems to be important for a classical antifolate to facilitate binding of FdUMP to bacterial TS. Effects of antifolates on FdUMP binding to human TS were different. At a low concentration (0.05 microM) NTX, RTX-Glu(5) and MTA-Glu(5) facilitated 3-5 times higher binding of [(3)H]FdUMP to TS than CH(2)-H(4)-folate. At higher concentrations (0.3-5 microM) of NTX, RTX-Glu(5) and MTA-Glu(5) the FdUMP binding decreased. The complex remained stable in the absence of (anti)folate for at least 24hr. The K(d) values of the antifolates for human TS varied from 19 to 387 nM, while the K(d) of CH(2)-H(4)-folate for human TS was 351 nM. The Hill coefficients, which indicated the type of cooperativity of the antifolates in the binding of FdUMP to TS were positive (0.58-0.99) at low concentrations (<0.3 microM) and negative (-0.35 to -0.81) at concentrations >0.3 microM except for GW1843U89, which only showed negative cooperativity (-1.70). It was shown with [(14)C]NTX that when the binding of FdUMP decreased at high NTX concentrations, the binding of NTX to TS still increased. This also held for the natural substrate dUMP. The negative cooperativity of the antifolates was clearly concentration dependent. The difference between human and L. casei TS in the FdUMP binding assays with antifolates can possibly be explained by interaction of the two subunits of human TS, which was absent in L. casei TS. The binding of antifolates to one of the two subunits induced a conformational change of the other subunit. This change no longer allowed the binding of FdUMP or dUMP at the active site. In conclusion this study showed that antifolates enhanced the binding of FdUMP to TS, especially at low antifolate concentrations, that are also clinically achievable, e.g. in human plasma.
Asunto(s)
Fluorodesoxiuridilato/metabolismo , Antagonistas del Ácido Fólico/farmacología , Timidilato Sintasa/metabolismo , Unión Competitiva , Sistema Libre de Células , Interacciones Farmacológicas , Humanos , Cinética , Lacticaseibacillus casei/enzimología , Timidilato Sintasa/química , Timidilato Sintasa/efectos de los fármacosRESUMEN
Thymidine phosphorylase (platelet-derived-endothelial-cell-growth-factor) catalyzes the reversible phosphorolysis of thymidine to thymine and 2-deoxyribose-1-phosphate, activates 5'-deoxy-5-fluorouridine (5'DFUR) and inactivates trifluorothymidine (TFT). The effect of 5'DFUR and TFT with or without a specific thymidine phosphorylase inhibitor (TPI) on thymidine phosphorylase mRNA, protein expression and activity was studied, in three human colon cancer cell lines, WiDR, HT29 and Lovo exposed for 72 h at IC50 concentrations. In Lovo cells TFT plus TPI only increased thymidine phosphorylase-protein expression 1.7-fold; 5'DFUR and TFT treatment increased thymidine phosphorylase mRNA levels 5- and 1.4-fold, respectively. In WiDR cells, 5'DFUR plus TPI significantly decreased thymidine phosphorylase-protein. TFT and TFT plus TPI increased thymidine phosphorylase-protein 2- and 3-fold, respectively. TPI and 5'DFUR decreased thymidine phosphorylase-mRNA levels significantly. In HT29 cells, 5'DFUR and 5'DFUR plus TPI decreased both thymidine phosphorylase-protein and thymidine phosphorylase-mRNA. In all cell lines 5'DFUR and TFT did not affect thymidine phosphorylase activity, but treatment with TPI (alone or in combination) eliminated thymidine phosphorylase activity. This demonstrated that regulation is drug and cell line dependent.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Flúor/farmacología , Pirimidinas/farmacología , Timidina Fosforilasa/antagonistas & inhibidores , Timidina Fosforilasa/biosíntesis , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/fisiología , HumanosRESUMEN
UNLABELLED: We aimed to determine whether the multidrug-resistance-proteins MRP4 (ABCC4) and MRP5 (ABCC5) confer resistance to the antimetabolites cytarabine (Ara-C), gemcitabine (GEM), and the L-nucleoside analog troxacitabine. For this purpose we used HEK293 and the transfected HEK/MRP4 (59-fold increased MRP4) or HEK/MRP5i (991-fold increased MRP5) as model systems and tested the cells for drug sensitivity using a proliferation test. Drug accumulation was performed by using radioactive Ara-C, and for GEM and troxacitabine with HPLC with tandem-MS or UV detection. At 4-hr exposure HEK/MRP4 cells were 2-4-fold resistant to troxacitabine, ara-C and 9-(2-phosphonylmethoxyethyl)adenine (PMEA), and HEK/MRP5i to ara-C and PMEA, but none to GEM. The inhibitors probenecid and indomethacin reversed resistance. After 4-hr exposure ara-C-nucleotides were 2-3-fold lower in MRP4/5 cells, in which they decreased more rapidly after washing with drug-free medium (DFM). Trocacitabine accumulation was similar in the 3 cell lines, but after the DFM period troxacitabine decreased 2-4-fold faster in MRP4/5 cells. Troxacitabine-nucleotides were about 25% lower in MRP4/5 cells and decreased rapidly in MRP4, but not in MRP5 cells. Accumulation of GEM-nucleotides was higher in the MRP4/5 cells. IN CONCLUSION: MRP4 and MRP5 overexpression confer resistance to troxacitabine and ara-C, but not to GEM, which was associated with a rapid decline of the ara-C and troxacitabine-nucleotides in HEK/MRP4-5 cells.
RESUMEN
RX-3117 (fluorocyclopentenylcytosine) is a cytidine analog and this class of drugs, including gemcitabine, has been widely used for the treatment of various types of cancers. However, there is no oral formulation of gemcitabine and drug resistance to gemcitabine is common. In this study, the efficacy of orally-administered RX-3117 was examined in 9 different human tumor xenograft models (colon, non-small cell lung, small cell lung, pancreatic, renal and cervical), grown subcutaneously in athymic nude mice. In the Colo 205, H460, H69 and CaSki models, gemcitabine treatment resulted in 28%, 30%, 25% and 0% tumor growth inhibition (TGI), respectively, whereas oral treatment with RX-3117 induced 100%, 78%, 62% and 66% TGI, respectively. This indicates that RX-3117 may have the potential to be used for the treatment of tumors that do not respond to gemcitabine. RX-3117 was also evaluated in a single primary low-passage human pancreatic Tumorgraft™CTG-0298 (TGI 76%), which is relatively resistant to gemcitabine (TGI 38%) and has a favorable RX-3117-activating enzyme profile. These studies demonstrated the therapeutic potential and anticancer efficacy of RX-3117.
Asunto(s)
Antimetabolitos Antineoplásicos/uso terapéutico , Citidina/análogos & derivados , Desoxicitidina/análogos & derivados , Resistencia a Antineoplásicos , Neoplasias/tratamiento farmacológico , Administración Oral , Animales , Línea Celular Tumoral , Citidina/administración & dosificación , Citidina/uso terapéutico , Desoxicitidina/administración & dosificación , Desoxicitidina/uso terapéutico , Modelos Animales de Enfermedad , Femenino , Células HCT116 , Células HT29 , Humanos , Ratones , Ratones Desnudos , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
For decades 5-fluorouracil (5-FU) has remained the treatment of choice in the adjuvant and palliative setting of colorectal cancer (CRC). The combinations of 5-FU or its oral prodrug capecitabine with irinotecan/oxaliplatin and the novel agents bevacizumab/cetuximab increased responses. However, the overall prognosis is poor, and predictive biomarkers of cytotoxic drugs activity are missing. Pharmacogenetic studies focused on candidate determinants of drug activity/metabolism, such as thymidylate synthase or dihydropyrimidine dehydrogenase, but reported controversial results. Given the heterogeneous and complex nature of CRC, it is likely that many aberrations underlying its progression can also affect therapeutic response. Therefore, high-throughput arrays for genome-wide-DNA aberrations play a pivotal role for new markers discovery by moving from hypothesis-driven, targeted-research to unbiased screening of the whole genetic spectrum. Chromosomal aberrations are critical events in tumorigenesis, and genomic regions harbouring DNA gains/losses have been identified in 85% of CRC patients. These aberrations change the expression of many genes, which might explain the differential effects of specific chemotherapeutic agents. In particular, recent studies reported correlations between DNA copy-number profiles and response to fluoropyrimidine-based regimens, such as leucovorin-modulated-5-FU+irinotecan (FOLFIRI), capecitabine+irinotecan (CAPIRI) and pemetrexed+irinotecan (ALIRI). Genome-wide profiling by oligonucleotide-based array-comparative-genomic-hybridization (aCGH) revealed genomic loci, of which the copy-number status may serve as marker for outcome after FOLFIRI and CAPIRI. Larger randomized and prospective trials of these aCGH platforms in CRC patients treated with fluoropyrimidine-based regimens are ongoing, and will ultimately demonstrate whether these findings can be of actual value to predict clinical outcome and direct the choice of therapy.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Colorrectales/genética , Variaciones en el Número de Copia de ADN , Antineoplásicos/administración & dosificación , Camptotecina/análogos & derivados , Camptotecina/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapéutico , Fluorouracilo/análogos & derivados , Fluorouracilo/uso terapéutico , Antagonistas del Ácido Fólico/administración & dosificación , Glutamatos/administración & dosificación , Guanina/administración & dosificación , Guanina/análogos & derivados , Humanos , Leucovorina/uso terapéutico , Pemetrexed , Resultado del TratamientoRESUMEN
Thiopurines are crucial in the treatment of inflammatory bowel disease. The phenotype of pivotal metabolic enzymes determines whether thioguanine nucleotides (6-TGN) are generated in clinically sufficiently high levels. The first step in activation of thiopurine prodrugs to 6-TGN is catalysis by hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Often, patients exhibit a clinically unfavorable metabolism, leading to discontinuation of conventional thiopurine therapy. The combination of allopurinol and low-dose thiopurine therapy may optimize this variant metabolism, presumably by affecting enzyme activities. We performed a prospective pharmacodynamic study to determine the effect of combination therapy on the activity of HGPRT. The activity of HGPRT and 6-TGN concentrations was measured in red blood cells during thiopurine monotherapy and after 4 weeks of combination therapy. The activity of HGPRT was also measured after 12 weeks of combination therapy. From the results, we conclude that combination therapy increases the activity of HGPRT and subsequently 6-TGN concentrations.
Asunto(s)
Alopurinol/uso terapéutico , Supresores de la Gota/uso terapéutico , Hipoxantina Fosforribosiltransferasa/metabolismo , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Enfermedades Inflamatorias del Intestino/enzimología , Mercaptopurina/análogos & derivados , Adolescente , Adulto , Alopurinol/administración & dosificación , Azatioprina/uso terapéutico , Niño , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Femenino , Supresores de la Gota/administración & dosificación , Humanos , Masculino , Mercaptopurina/administración & dosificación , Mercaptopurina/metabolismo , Mercaptopurina/uso terapéutico , Persona de Mediana Edad , Adulto JovenRESUMEN
Trifluorothymidine (TFT) is part of the novel oral formulation TAS-102, which is currently evaluated in phase II studies. Drug resistance is an important limitation of cancer therapy. The aim of the present study was to induce resistance to TFT in H630 colon cancer cells using two different schedules and to analyze the resistance mechanism. Cells were exposed either continuously or intermittently to TFT, resulting in H630-cTFT and H630-4TFT, respectively. Cells were analyzed for cross-resistance, cell cycle, protein expression, and activity of thymidine phosphorylase (TP), thymidine kinase (TK), thymidylate synthase (TS), equilibrative nucleoside transporter (hENT), gene expression (microarray), and genomic alterations. Both cell lines were cross-resistant to 2'-deoxy-5-fluorouridine (>170-fold). Exposure to IC(75)-TFT increased the S/G(2)-M phase of H630 cells, whereas in the resistant variants, no change was observed. The two main target enzymes TS and TP remained unchanged in both TFT-resistant variants. In H630-4TFT cells, TK protein expression and activity were decreased, resulting in less activated TFT and was most likely the mechanism of TFT resistance. In H630-cTFT cells, hENT mRNA expression was decreased 2- to 3-fold, resulting in a 5- to 10-fold decreased TFT-nucleotide accumulation. Surprisingly, microarray-mRNA analysis revealed a strong increase of secretory phospholipase-A2 (sPLA2; 47-fold), which was also found by reverse transcription-PCR (RT-PCR; 211-fold). sPLA2 inhibition reversed TFT resistance partially. H630-cTFT had many chromosomal aberrations, but the exact role of sPLA2 in TFT resistance remains unclear. Altogether, resistance induction to TFT can lead to different mechanisms of resistance, including decreased TK protein expression and enzyme activity, decreased hENT expression, as well as (phospho)lipid metabolism. Mol Cancer Ther; 9(4); 1047-57. (c)2010 AACR.