RESUMO
We investigated the effects of the antifolate methotrexate on intracellular folate pools of human myeloid precursor cells (MPCs). Immature MPCs, representing 3.2% of the original marrow population, were selected from normal human bone marrow by immune rosetting. The intracellular folate pools were labeled by incubation with 5 X 10(-8) M [3H]5-formyl-FH4 and were quantitated by high performance liquid chromatography. The predominant folates were 5-methyl-tetrahydrofolate (5-methyl-FH4) (36%), 10-formyl-FH4 (41.4%), 5-formyl-FH4 (12.3%), and FH4 (10.3%). A 12-h exposure to 1 microM methotrexate (MTX) resulted in a 34% reduction in the intracellular concentration of 10-formyl-FH4, a 61% decrease in 5-formyl-FH4, and a 62% decrease in 5-methyl-FH4, as well as the appearance and progressive expansion of the FH2 and 10-formyl-FH2 pools. These changes were maximal after 4 h of incubation with MTX. Paralleling the changes in folates, particularly the increase in FH2, were a 64% reduction in myeloid colony formation and a 77% depression of de novo purine synthesis after 4 h of MTX. We conclude that MTX does not produce quantitative depletion of 10-formyl-FH4 and that its antipurine effect may be mediated by direct inhibition of de novo purine synthesis by FH2 and, at later time points, by MTX polyglutamates.
Assuntos
Células da Medula Óssea , Granulócitos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Metotrexato/farmacologia , Tetra-Hidrofolatos/metabolismo , Separação Celular , Sobrevivência Celular/efeitos dos fármacos , Formiltetra-Hidrofolatos/metabolismo , Granulócitos/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Cinética , Purinas/biossínteseRESUMO
PURPOSE: The follow-up of patients with malignant brain tumors after surgery, radiation, and/or chemotherapy has been inadequate for evaluating brain tumor burden using computerized tomography (CT) or magnetic resonance imaging (MRI). Thallium-201 has been shown to concentrate in viable tumor, and Tl-201 single photon emission computerized tomography (SPECT) imaging can identify tumor burden more accurately than CT. METHODS AND MATERIALS: Thirty-one patients with glioblastoma and three patients with low grade astrocytoma were studied with Tl-201 SPECT. Histololgic diagnosis was obtained in all patients by biopsy and all patients had CT scans within 2 weeks of the SPECT study. Seventeen patients were followed with one or more SPECT and CT evaluations. RESULTS: Single photon emission computerized tomography studies, after surgery, radiotherapy, and/or chemotherapy, were more accurate than CT in identifying progression or regression of disease. Twenty-three patients had evidence of disease and 11 patients had no evidence of recurrent disease in the initial Tl-201 SPECT study following therapy. Computerized tomography identified 20 of the 23 patients with disease and 6 of 11 patients with no recurrent disease. Follow-up with Tl-201 SPECT in 17 patients suggested progression of disease in 9 patients, while CT showed progression in only 3 patients. Clinical examinations and repeat CT studies confirmed the accuracy of Tl-201 SPECT images. CONCLUSION: We found Tl-201 SPECT more accurate than CT scans in a prospective evaluation of 34 patients with brain tumor. Follow-up studies with both Tl-201 SPECT and CT imaging in 17 patients demonstrated that SPECT was more reliable than CT in identifying progression, improvement, or no change in brain tumor burden.
Assuntos
Astrocitoma/diagnóstico por imagem , Neoplasias Encefálicas/diagnóstico por imagem , Glioblastoma/diagnóstico por imagem , Recidiva Local de Neoplasia/diagnóstico por imagem , Radioisótopos de Tálio , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Adulto , Idoso , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Sensibilidade e Especificidade , Tomografia Computadorizada por Raios XRESUMO
Previous studies have suggested that metabolic inhibition by methotrexate (MTX) is multifactorial and that cytotoxicity can be reversed by the reduced folate leucovorin. In this report we investigated the mechanism of leucovorin rescue in the MCF-7 human breast cancer cell line. Cells were exposed to various concentrations of MTX (0.5, 1.0, 3.0, and 10.0 microM) for 24 hr followed by rescue with labelled leucovorin (0.5 to 50 microM). The changes in the intracellular folate pools 24 hr following the addition of leucovorin were quantitated by high-pressure liquid chromatographic methods. The changes in the folate pools during rescue were compared with the ability of various concentrations of leucovorin to affect cellular rescue from MTX using a cloning assay. Our studies show that the total labelled intracellular folate pools increased in a log-linear fashion with respect to leucovorin exposure concentrations up to 100 microM. The degree of accumulation at a given leucovorin concentration was not significantly different in the absence or presence of MTX over the concentration range of 0.5 to 10 microM. Individual folate pool levels (tetrahydrofolate, 10-formyl tetrahydrofolate, 5-formyl tetrahydrofolate, 5-methyl tetrahydrofolate, and 5,10-methylene tetrahydrofolate) reached those present in cells not exposed to MTX at concentrations of leucovorin that were not adequate to rescue the MTX-treated cells. With exposure to concentrations of leucovorin capable of rescue, the individual folate pool levels were up to twelve times greater than those found in untreated cells, consistent with competition for catalytic activity at folate-dependent enzymes in addition to dihydrofolate reductase. The dihydrofolate pool also increased with increasing leucovorin concentration: but, unlike the reduced folates, this oxidized folate reached a maximal level that was dependent on the MTX concentration to which the cells had been exposed. This suggests that competition between MTX and leucovorin occurs at the level of dihydrofolate reductase via a competitive interaction with dihydrofolate in this intact cell system. The ability of leucovorin and its metabolites to compete with direct inhibitors of dihydrofolate reductase and other metabolically important folate-dependent enzymes appears to be associated with leucovorin rescue.
Assuntos
Ácido Fólico/metabolismo , Leucovorina/farmacologia , Metotrexato/antagonistas & inibidores , Neoplasias da Mama , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ácido Fólico/análogos & derivados , Humanos , Técnicas In Vitro , Metotrexato/toxicidade , Ácidos Pteroilpoliglutâmicos/metabolismo , Tetra-Hidrofolato Desidrogenase/metabolismo , Células Tumorais CultivadasRESUMO
5 patients diagnosed as having extraskeletal Ewing's sarcoma have been referred to our adult oncology unit since 1980. All were men, ranging in age from 18-57 (mean 32 years). The primary tumor was located on the trunk in 4 and in an extremity in 1. Wide tumor excision was feasible in only 2. 3 died within 27 months and 2 are alive, 13 and 67 months, respectively, following diagnosis. This study demonstrates the highly aggressive nature of extraskeletal Ewing's sarcoma and the need for early diagnosis and efficient chemotherapy.
Assuntos
Sarcoma de Ewing , Neoplasias de Tecidos Moles , Adulto , Humanos , Masculino , Pessoa de Meia-Idade , Sarcoma de Ewing/diagnóstico , Sarcoma de Ewing/mortalidade , Neoplasias de Tecidos Moles/diagnóstico , Neoplasias de Tecidos Moles/mortalidadeAssuntos
Metotrexato/metabolismo , Peptídeos/metabolismo , Ácido Poliglutâmico/metabolismo , Animais , Medula Óssea/efeitos dos fármacos , Linhagem Celular , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Preparações de Ação Retardada , Humanos , Mucosa Intestinal/efeitos dos fármacos , Cinética , Metotrexato/análogos & derivados , Metotrexato/farmacologia , Metotrexato/toxicidade , Peptídeo Sintases/metabolismo , Ácido Poliglutâmico/análogos & derivados , Tetra-Hidrofolato Desidrogenase/metabolismo , Timidilato Sintase/antagonistas & inibidoresAssuntos
Neoplasias da Mama/enzimologia , Ácido Fólico/análogos & derivados , Trifosfato de Adenosina/farmacologia , Cromatografia Líquida de Alta Pressão , Citoplasma/enzimologia , Ácido Fólico/metabolismo , Formiatos/farmacologia , Humanos , Cinética , Magnésio/farmacologia , Metotrexato/metabolismo , Células Tumorais CultivadasAssuntos
Aciltransferases/antagonistas & inibidores , Antagonistas do Ácido Fólico/farmacologia , Hidroximetil e Formil Transferases , Metotrexato/análogos & derivados , Timidilato Sintase/antagonistas & inibidores , Humanos , Metotrexato/farmacologia , Fosforribosilaminoimidazolcarboxamida Formiltransferase , Ácido Poliglutâmico/análogos & derivados , Ácido Poliglutâmico/farmacologiaAssuntos
Neoplasias da Mama/enzimologia , Antagonistas do Ácido Fólico , Ácido Fólico/farmacologia , Metotrexato/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Linhagem Celular , Ácido Fólico/metabolismo , Humanos , Líquido Intracelular/análise , Metotrexato/análogos & derivados , Metotrexato/metabolismo , Proteínas de Neoplasias/metabolismo , Ácido Poliglutâmico/análogos & derivados , Ácido Poliglutâmico/farmacologia , Tetra-Hidrofolato Desidrogenase/metabolismoAssuntos
Antimetabólitos Antineoplásicos/farmacologia , Animais , Azacitidina/farmacologia , Citarabina/farmacocinética , Citarabina/farmacologia , Floxuridina/farmacologia , Fluoruracila/farmacologia , Antagonistas do Ácido Fólico/farmacologia , Humanos , Mercaptopurina/farmacologia , Metotrexato/farmacocinética , Metotrexato/farmacologia , Purinas/farmacologia , Tioguanina/farmacologiaRESUMO
We have studied the roles of 5,10-methylenetetrahydrofolate (5,10-methylene-H4PteGlu) depletion and dihydrofolate (H2PteGlu) accumulation in the inhibition of de novo thymidylate synthesis by methotrexate (MTX) in human MCF-7 breast cancer cells. Using both a high pressure liquid chromatography system and a modification of the 5-fluoro-2'-deoxyuridine-5'-monophosphate radioenzymatic binding assay, we determined that the 5,10-methylene-H4PteGlu pool is 50-60% depleted in human MCF-7 breast cancer cells following exposure to 1 micron MTX for up to 21 h. Similar alterations in the 5,10-methylene-H4PteGlu pools were obtained when human promyelocytic HL-60 leukemia cells and normal human myeloid precursor cells were incubated with 1 micron MTX. The H2PteGlu pools within the MCF-7 cells increased significantly after 15 min of 1 micron MTX exposure, reaching maximal levels by 60 min. Thymidylate synthesis, as measured by labeled deoxyuridine incorporation into DNA, decreased to less than 20% of control activity within 30 min of 1 micron MTX exposure. The inhibition of thymidylate synthesis coincided temporally with the rapid intracellular accumulation of H2PteGlu, a known inhibitor of thymidylate synthase. Furthermore, inhibition of this pathway was associated in a log-linear fashion with the intracellular level of dihydrofolate. These studies provide further evidence that depletion of the thymidylate synthase substrate 5,10-methylene-H4PteGlu is inadequate to account completely for diminished thymidylate synthesis resulting from MTX treatment. Our findings suggest that acute inhibition of de novo thymidylate synthesis is a multifactorial process consisting of partial substrate depletion and direct enzymatic inhibition by H2PteGlu polyglutamates.
Assuntos
Células-Tronco Hematopoéticas/enzimologia , Metotrexato/farmacologia , Timidilato Sintase/antagonistas & inibidores , Células Tumorais Cultivadas/enzimologia , Neoplasias da Mama , Linhagem Celular , Feminino , Ácido Fólico/metabolismo , Humanos , Cinética , Leucemia Promielocítica AgudaRESUMO
Recently, folate-dependent enzymes in the de novo thymidine and purine biosynthetic pathways have come under scrutiny as potential sites for chemotherapeutic exploitation by antifolates. In this manuscript we report on the progress that has been made in designing inhibitors of these pathways. In addition, a molecular model is proposed for the design of new antifolates directed against thymidylate synthase (TS).
Assuntos
Antagonistas do Ácido Fólico/metabolismo , Purinas/biossíntese , Timidilato Sintase/antagonistas & inibidores , Fluordesoxiuridilato/metabolismo , Humanos , Timidina/biossínteseRESUMO
We have investigated the role of dihydrofolate (H2PteGlu) accumulation in the inhibition of de novo purine synthesis by methotrexate (MTX) in human MCF-7 breast cancer cells. Previous studies have shown that cytotoxic concentrations of MTX that inhibit dihydrofolate reductase produce only minimal depletion of the reduced folate cofactor, 10-formyltetrahydrofolate, required for purine synthesis. At the same time, de novo purine synthesis is totally inhibited. In these studies, we show that 10 microM MTX causes inhibition of purine synthesis at the step of phosphoribosylaminoimidazolecarboxamide (AICAR) transformylase, as reflected in a 2-3-fold expansion of the intracellular AICAR pool. The inhibition of purine synthesis coincides with the rapid intracellular accumulation of H2PteGlu, a known inhibitor of AICAR transformylase. When the generation of H2PteGlu is blocked by pretreatment with 50 microM 5-fluorodeoxyuridine (FdUrd), an inhibitor of thymidylate synthase, MTX no longer causes inhibition of purine synthesis. Intermediate levels of H2PteGlu produced in the presence of lower (0.1-10 microM) concentrations of FdUrd led to proportional inhibition of purine biosynthesis, and the exogenous addition of H2PteGlu to breast cells in culture re-established the block in purine synthesis in the presence of FdUrd and MTX. The early phases of inhibition of purine biosynthesis could be ascribed only to H2PteGlu accumulation. MTX polyglutamates, also known to inhibit AICAR transformylase, were present in breast cells only after 6 h of incubation with the parent compounds and were not formed in cells preincubated with FdUrd. The lipid-soluble antifolate trimetrexate, which does not form polyglutamates, produced modest 10-formyltetrahydrofolate depletion, but caused marked H2PteGlu accumulation and a parallel inhibition of purine biosynthesis. This evidence leads to the conclusion that MTX and the lipid-soluble analog trimetrexate cause inhibition of purine biosynthesis through the accumulation of H2PteGlu behind the blocked dihydrofolate reductase reaction.
Assuntos
Aciltransferases/metabolismo , Hidroximetil e Formil Transferases , Metotrexato/farmacologia , Purinas/biossíntese , Antineoplásicos/farmacologia , Neoplasias da Mama , Radioisótopos de Carbono , Linhagem Celular , Feminino , Floxuridina/farmacologia , Ácido Fólico/metabolismo , Glicina/metabolismo , Humanos , Cinética , Metotrexato/análogos & derivados , Metotrexato/biossíntese , Metotrexato/isolamento & purificação , Fosforribosilaminoimidazolcarboxamida Formiltransferase , Ácido Poliglutâmico/análogos & derivados , Ácido Poliglutâmico/biossíntese , Ácido Poliglutâmico/isolamento & purificação , Purinas/antagonistas & inibidores , Quinazolinas/farmacologia , TrimetrexatoRESUMO
The folate compound 10-formyldihydrofolate (H2folate) has not been found as a component of intracellular folates in normal tissues but has been identified in the cytosol of methotrexate (MTX)-treated MCF-7 breast cancer cells and normal human myeloid precursor cells. Its identity was verified by coelution of this compound with a synthetic marker on high pressure liquid chromatography, its reduction to 10-formyltetrahydrofolate (H4folate) in the presence of dihydrofolate reductase, and its enzymatic deformylation to dihydrofolate in the presence of aminoimidazolecarboxamide ribonucleotide (AICAR) transformylase. Chemically synthesized monoglutamated or pentaglutamated 10-formyl-H2folate was examined for its interaction with three folate-dependent enzymes: AICAR transformylase, glucinamide ribotide (GAR) transformylase, and thymidylatesynthase. 10-Formyl-H2folate-Glu5 was a competitive inhibitor of thymidylate synthase (Ki = 0.16 microM with 5,10-methylene-H4folate-Glu1 as substrate and 1.6 microM with 5,10-methylene-H4folate-Glu5) and inhibited GAR transformylase (Ki = 2.0 microM). It acted as a substrate for AICAR transformylase (Km = 5.3 microM), and its efficiency was equal to that of the natural substrate 10-formyl-H4folate-Glu5. The inhibition of thymidylate synthase by 10-formyl-H2folate was highly dependent on the inhibitor's polyglutamation state, the -Glu5 derivative having a 52-85-fold greater affinity as compared to the affinity of -Glu1. Polyglutamation of 10-formyl-H2folate did not affect its inhibition of GAR transformylase. While the actual role of 10-formyl-H2folate contributing to the cytotoxicity of MTX has not been determined, this compound has the potential to enhance inhibition of GAR transformylase and thymidylate synthase, and at the same time provides additional substrate for AICAR transformylase. The MTX-induced intracellular accumulation of 10-formyl-H2folate and H2folate may play a role in the drug-related cytotoxicity through the contribution of these folates to the inhibition of thymidylate synthase and de novo purine synthesis.