RESUMO
BACKGROUND: The use of ß-blockers for the management of hypertension has been recently associated with significant clinical benefits in cancer patients. Herein, we investigated whether ß-blockers could be used in combination with chemotherapy for the treatment of neuroblastoma. METHODS: Seven ß-blockers were tested for their antiproliferative and anti-angiogenic properties alone, and in combination with chemotherapy in vitro; the most potent drug combinations were evaluated in vivo in the TH-MYCN mouse model of neuroblastoma. RESULTS: Three ß-blockers (i.e., carvedilol, nebivolol and propranolol) exhibited potent anticancer properties in vitro and interacted synergistically with vincristine, independently of P-glycoprotein expression. ß-blockers potentiated the anti-angiogenic, antimitochondrial, antimitotic and ultimately pro-apoptotic effects of vincristine. In vivo, ß-blockers alone transiently slowed tumour growth as compared with vehicle only (P<0.01). More importantly, when used in combination, ß-blockers significantly increased the tumour regression induced by vincristine (P<0.05). This effect was associated with an increase in tumour angiogenesis inhibition (P<0.001) and ultimately resulted in a four-fold increase in median survival, as compared with vincristine alone (P<0.01). CONCLUSION: ß-blockers can increase treatment efficacy against neuroblastoma, and their combination with chemotherapy may prove beneficial for the treatment of this disease and other drug-refractory cancers.
Assuntos
Neoplasias Abdominais/tratamento farmacológico , Antagonistas Adrenérgicos beta/uso terapêutico , Inibidores da Angiogênese/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neuroblastoma/tratamento farmacológico , Neoplasias Abdominais/irrigação sanguínea , Neoplasias Abdominais/patologia , Inibidores da Angiogênese/administração & dosagem , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Humanos , Camundongos , Camundongos Transgênicos , Neovascularização Patológica/tratamento farmacológico , Neuroblastoma/irrigação sanguínea , Neuroblastoma/patologiaAssuntos
Proteínas de Membrana Transportadoras/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Polimorfismo Genético , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Criança , Homozigoto , Humanos , Reação em Cadeia da Polimerase , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Proteína Carregadora de Folato Reduzido , Resultado do TratamentoRESUMO
Resistance to the antifolate methotrexate (MTX) can cause treatment failure in childhood acute lymphoblastic leukemia (ALL). This may result from defective MTX accumulation due to alterations in the human reduced folate carrier (hRFC) gene. We have identified an hRFC gene point mutation in a transport-defective CCRF-CEM human T-ALL cell line resulting in a lysine to glutamic acid substitution at codon 45 (E45K), which has been identified in other antifolate-resistant sublines (JBC 273:30 189, 1998; JBC 275:30 855, 2000). To characterize the role of this mutation in MTX resistance, transfection experiments were performed using hRFC-null CCRF-CEM cells. E45K transfectants demonstrated an initial rate of MTX influx that was approximately 0.5-fold that of CCRF-CEM cells, despite marked protein overexpression. Cytotoxicity studies revealed partial reversal of MTX and raltitrexed resistance in E45K transfectants, while trimetrexate resistance was significantly increased. Kinetic analysis indicated only minor differences in MTX kinetics between wild-type and E45K hRFCs, however, K(i)s for folic acid and 5-formyltetrahydrofolate were markedly reduced for E45K hRFC. This was paralleled by increased folic acid transport and reduced synthesis of MTX polyglutamates. Collectively, the results demonstrate that expression of E45K hRFC leads to increased MTX resistance due to decreased membrane transport and, secondarily, from alterations in binding affinities and transport of folate substrates. However, despite these findings, we could find no evidence of this mutation in 121 childhood ALL samples, suggesting that it does not contribute to clinical MTX resistance in this disease.
Assuntos
Antimetabólitos Antineoplásicos/farmacocinética , Proteínas de Transporte/genética , Resistencia a Medicamentos Antineoplásicos/genética , Leucemia/tratamento farmacológico , Proteínas de Membrana Transportadoras , Metotrexato/farmacocinética , Mutação Puntual , Substituição de Aminoácidos , Células da Medula Óssea/patologia , Proteínas de Transporte/fisiologia , Criança , Ácido Fólico/farmacocinética , Humanos , Cinética , Leucemia/genética , Leucemia/patologia , Estrutura Terciária de Proteína , Proteína Carregadora de Folato Reduzido , Transfecção , Células Tumorais CultivadasRESUMO
The presence of sequence variants in the human reduced folate carrier (hRFC) was assessed in leukemia blasts from children with acute lymphoblastic leukemia (ALL) and in normal peripheral blood specimens. A CATG frame shift insertion at position 191 was detected in 10-60% of hRFC transcripts from 10 of 16 ALL specimens, by RFLP analysis and direct sequencing of hRFC cDNAs. In genomic DNAs prepared from 105 leukemia (n = 54) and non-leukemia (n = 51) specimens, PCR amplifications and direct sequencing of exon 3 identified a high-frequency G to A single nucleotide polymorphism at position 80 that resulted in a change of arginine-27 to histidine-27. The allelic frequencies of G/A80 were nearly identical for the non-leukemia (42.2% CGC and 57.8% CAC) and leukemia (40.7% CGC and 59.3% CAC) genomic DNAs. In cDNAs prepared from 10 of these ALL patients, identical allelic frequencies (40 and 60%, respectively) were recorded. In up to 62 genomic DNAs, hRFC-coding exons 4-7 were PCR-amplified and sequenced. A high-abundance C/T696 polymorphism was detected with nearly identical frequencies for both alleles, and a heterozygous C/A1242 sequence variant was identified in two ALL specimens. Both C/T696 and C/A1242 were phenotypically silent. In transport assays with [(3)H]methotrexate and [(3)H]5-formyl tetrahydrofolate, nearly identical uptake rates were measured for the arginine-27- and histidine-27-hRFC proteins expressed in transport-impaired K562 cells. Although there were no significant differences between the kinetic parameters for methotrexate transport for the hRFC forms, minor (approximately 2-fold) differences were measured in the K(i)s for other substrates including Tomudex, 5,10-dideazatetrahydrofolate, GW1843U89, and 10-ethyl-10-deazaaminopterin and for 5-formyl tetrahydrofolate.
Assuntos
Proteínas de Transporte/genética , Proteínas de Membrana Transportadoras , Polimorfismo de Nucleotídeo Único , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Substituição de Aminoácidos , Linfócitos B/metabolismo , Sequência de Bases , Transporte Biológico/genética , Criança , Análise Mutacional de DNA , DNA Complementar/química , DNA Complementar/genética , DNA de Neoplasias/química , DNA de Neoplasias/genética , Frequência do Gene , Humanos , Células K562 , Metotrexato/farmacocinética , Mutagênese Insercional , Plasmídeos/genética , Mutação Puntual , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Proteína Carregadora de Folato Reduzido , Células-Tronco/metabolismo , TransfecçãoRESUMO
We have previously described a series of methotrexate (MTX)-selected CCRF-CEM sublines (CEM/MTX R1-3) displaying increased resistance to drugs associated with the multidrug resistance phenotype and have provided evidence that MDR1 P-glycoprotein contributes to multifactorial MTX resistance in these cells. We have also suggested that P-glycoprotein-mediated MTX transport arises in these cells due to a deficiency in the normal MTX transport route, the reduced folate carrier (RFC). We have now determined the nucleotide sequence of the RFC gene in CEM/MTX R1-3 cells and confirm that the carrier is defective in these cells as a result of a premature stop mutation at codon 99, which severely truncates the encoded protein. CEM/MTX R3 cells were removed from MTX, and a series of sublines with increasing MDR1 expression were derived, following selection with vincristine. These cells show increasing cross-resistance to vincristine as well as other drugs associated with the multidrug resistance phenotype. More importantly, the increased P-glycoprotein expression correlates with increased resistance to MTX, supporting the hypothesis that in cells with a defective carrier protein, MTX can become a substrate for P-glycoprotein. Our data have implications for the P-glycoprotein-mediated transport of other hydrophilic drugs in situations where the relevant carrier protein has been functionally inhibited.