RESUMEN
The ATP-binding cassette transporter ABCG2 is expressed in various organs, such as the small intestine, liver, and kidney, and influences the pharmacokinetics of drugs that are its substrates. ABCG2 is also expressed by cancer cells and mediates resistance to anticancer agents by promoting the efflux of these drugs. In the present study, we investigated the interactions between epidermal growth factor receptor tyrosine kinase inhibitors and ABCG2 by MTT assay, intracellular drug accumulation assay, and FACS. This study showed that four epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) (gefitinib, erlotinib, lapatinib, and afatinib) were transported from tumor cells as substrates of ABCG2. Q141K is a common single-nucleotide polymorphism of ABCG2 in Asians. We demonstrated that the extracellular efflux of gefitinib, erlotinib, and lapatinib was reduced by Q141K, whereas afatinib transport was not affected. In addition, all four EGFR TKIs inhibited the transport of other substrates by both wild-type and variant ABCG2 at 0.1 µM concentrations. Accordingly, epidermal growth factor receptor tyrosine kinase inhibitors may induce interactions with other drugs that are substrates of ABCG2, and single-nucleotide polymorphisms of ABCG2 may influence both the pharmacokinetics and efficacy of these anticancer agents.
Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Antineoplásicos/farmacología , Proteínas de Neoplasias , Inhibidores de Proteínas Quinasas/farmacología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Transporte Biológico , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Receptores ErbB/antagonistas & inhibidores , Células HEK293 , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Polimorfismo de Nucleótido SimpleRESUMEN
BACKGROUND/AIM: To investigate bioequivalence among generic and brand-name irinotecan products. MATERIALS AND METHODS: Products of Yakult and Daiichi-Sankyo (brand-name products), Sandoz, Nippon Kayaku, Taiho, and Sawai were compared with respect to their composition and antitumor activity. RESULTS: High-performance liquid chromatography demonstrated that related substances were within the acceptable range. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay revealed significant differences in cytotoxicity for four cancer cell lines among the products. The concentration of the active compound SN-38 was highest in Yakult's product (23.82 ng/ml) and lowest in Daiichi-Sankyo's product (8.96 ng/ml). MTT assay data were correlated with the SN-38 concentration, suggesting that it influenced differences in cytocidal activity among products. However, the SN-38 concentration was far lower than that of irinotecan (20 mg/ml), suggesting a negligible clinical effect. Metabolism of irinotecan to SN-38 or open-ring forms did not differ significantly among the products. CONCLUSION: The generic products showed equivalent efficacy and safety to the brand-name products.
Asunto(s)
Antineoplásicos Fitogénicos/farmacocinética , Camptotecina/análogos & derivados , Medicamentos Genéricos/farmacocinética , Camptotecina/farmacocinética , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión , Humanos , Irinotecán , Equivalencia TerapéuticaRESUMEN
PURPOSE: The relationship between plasma concentration and antitumor activity of gefitinib was assessed in patients with advanced non-small cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) mutations. PATIENTS AND METHODS: Plasma trough levels of gefitinib were measured on days 2 (D2) and 8 (D8) by high-performance liquid chromatography in 31 patients. Plasma concentrations of gefitinib were also measured 10 h after the first administration in 21 of these patients to calculate the elimination half-life of gefitinib. RESULTS: The median trough levels were: 197 ng/ml 10 h from the first administration of gefitinib; 113 ng/ml on D2; and 358 ng/ml on D8. The median D8/D2 ratio was 2.709, and the median elimination half-life was 15.7 h. The median progression-free survival (PFS) was 273 days, and the median overall survival (OS) was 933 days. A high D8/D2 ratio was significantly correlated with better PFS, though the plasma trough levels on D2 and D8 were not significantly related to PFS. The elimination half-life was not a significant factor for PFS, but it was significantly correlated with high-grade adverse events. Pharmacokinetic parameters were not significantly correlated with OS. CONCLUSIONS: A high D8/D2 ratio, but not elimination half-life, might be a predictor of better PFS in patients with NSCLC harboring EGFR mutations treated with gefitinib. On the other hand, long elimination half-life was related to high-grade adverse events in these patients. Clinical Trial Registration UMIN000001066.
Asunto(s)
Antineoplásicos/administración & dosificación , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Receptores ErbB/genética , Neoplasias Pulmonares/tratamiento farmacológico , Quinazolinas/administración & dosificación , Anciano , Anciano de 80 o más Años , Antineoplásicos/efectos adversos , Antineoplásicos/farmacocinética , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Cromatografía Líquida de Alta Presión/métodos , Supervivencia sin Enfermedad , Femenino , Gefitinib , Semivida , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Mutación , Quinazolinas/efectos adversos , Quinazolinas/farmacocinética , Tasa de SupervivenciaRESUMEN
An efficient and versatile synthesis of 5-N-acetylardeemin (1a) and sixteen 2-, 3- and 13-substituted derivatives 1b-q was achieved through Ugi three-component reaction of 3,3a,8,8a-tetrahydropyrrolo[2,3-b]indole and cyclization/epimerization. Their inhibitory activity on the drug efflux of breast cancer resistance protein (ABCG2) was evaluated by flow cytometric analysis of accumulation of Hoechst 33342 stain in Flp-In-293/ABCG2 cells. Most of the derivatives exhibited a stronger ABCG2 inhibitory effect compared with natural product 1a. The derivative 1m with a 4-tolyl substituent at the C-13 position exhibited the most potent ABCG2 inhibition. This preliminary structure-activity relationship study indicates that an electron-rich aryl moiety as the 13-substituent is key to increasing the inhibitory activity.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Proteínas de Neoplasias/antagonistas & inhibidores , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Pirimidinonas/síntesis química , Pirimidinonas/química , Pirimidinonas/farmacología , Relación Estructura-ActividadRESUMEN
Primary brain tumors occur in around 250,000 people per year globally. Survival rates in primary brain tumors depend on the type of tumor, patient's age, the extent of surgical tumor removal, and other factors. Photodynamic diagnosis (PDD) is a practical tool currently used in surgical operation of aggressive brain tumors, such as glioblastoma and meningiomas, whereas clinical application of photodynamic therapy (PDT) to brain tumor therapy has just recently started. Both PDD and PDT are achieved by a photon-induced physicochemical reaction, which is induced by the excitation of porphyrins exposed to light. In fluorescence-guided gross-total resection, PDD can be achieved by the administration of 5-aminolevulinic acid (5-ALA) as the precursor of protoporphyrin IX (PpIX). Exogenously administered ALA induces biosynthesis and accumulation of PpIX, a natural photosensitizer, in cancer cells. However, ATP-binding cassette transporter ABCG2 plays a critical role in regulating the cellular accumulation of porphyrins in cancer cells and thereby its expression and function can affect the efficacy of PDD and PDT. In response to the photoreaction of porphyrins leading to oxidative stress, the nuclear factor erythroid-derived 2-related transcription factor can transcriptionally upregulate ABCG2, which may reduce the efficacy of PDD and PDT. On the other hand, certain protein kinase inhibitors potentially enhance the efficacy of PDD and PDT by blocking ABCG2-mediated porphyrin efflux from cancer cells. In this context, it is of great interest to develop ABCG2 inhibitors that can be applied to PDD or PDT for the therapy of brain tumor and other tumors.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/tratamiento farmacológico , Proteínas de Neoplasias/metabolismo , Fotoquimioterapia/métodos , Porfirinas/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Ácido Aminolevulínico/metabolismo , Animales , Antineoplásicos/uso terapéutico , Transporte Biológico , Gefitinib , Humanos , Ratones , Factor 2 Relacionado con NF-E2/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Estrés Oxidativo , Porfirinas/biosíntesis , Inhibidores de Proteínas Quinasas/uso terapéutico , Protoporfirinas/biosíntesis , Quinazolinas/uso terapéutico , Transcripción Genética/genética , Resultado del TratamientoRESUMEN
BACKGROUND: We conducted a phase II trial of erlotinib in patients with advanced non-small-cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) mutations and evaluated the relationship between plasma concentration and efficacy of erlotinib. METHODS: Patients who were previously treated but naive to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs), with advanced NSCLC harboring EGFR mutations, were enrolled. Erlotinib was given at 150 mg once daily until disease progression. The primary end point was objective response rate (ORR). Plasma trough levels of erlotinib were measured on Days 2 (D2) and 8 (D8) by high-performance liquid chromatography. RESULTS: In total, 29 patients were enrolled from September 2008 to January 2011. ORR was 61.5 % (95 % confidence interval [CI] 40.5779.8) of 26 assessable patients. The median progression-free survival (PFS) and overall survival (OS) were 6.3 months and 16.9 months, respectively. Skin rash was observed in 24 patients, mostly at grade 1 or 2. Grade 2 pneumonitis was observed in one patient. We collected blood samples from 16 patients. The median PFS of the high and low D8/D2 ratio group was 11.2 months and 5.7 months, respectively (p = 0.044, hazard ratio = 0.301, 95 % CI 0.0940.968). CONCLUSION: Erlotinib showed an ORR comparable to that seen in previous studies for patients with NSCLC harboring EGFR mutations, although response, the primary end point, did not reach the predetermined threshold level. The D8/D2 ratio of erlotinib plasma trough levels might be a predictive factor for PFS.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Receptores ErbB/genética , Neoplasias Pulmonares/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Quinazolinas/uso terapéutico , Anciano , Anciano de 80 o más Años , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Cromatografía Líquida de Alta Presión , Supervivencia sin Enfermedad , Clorhidrato de Erlotinib , Femenino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Mutación , Estudios Prospectivos , Inhibidores de Proteínas Quinasas/efectos adversos , Inhibidores de Proteínas Quinasas/farmacocinética , Quinazolinas/efectos adversos , Quinazolinas/farmacocinética , Tasa de Supervivencia , Resultado del TratamientoRESUMEN
Photodynamic therapy (PDT) is a recently developed anticancer treatment that utilizes the generation of singlet oxygen and other reactive oxygen species in cancer tissue. In response to oxidative stress, NF-E2-related transcription factor (Nrf2) encoded by the NFE2L2 gene plays a key role in transcriptional upregulation of many target genes, including those for metabolizing enzymes and transporters essential for cellular defense. Recent studies have provided evidence that Nrf2 regulates the transcription of the human ABC transporter ABCG2 gene, which is critically involved in the cellular accumulation of porphyrins in PDT. Nrf2 interacts with the antioxidant responsive element (ARE) located in the promoter region of human ABCG2 gene. Nrf2-specific siRNA treatments suppressed the induction of ABCG2 expression after the photoactivation of porphyrins in vitro. One SNP (-617C>A; rs6721961) in the ARE-like loci of the human Nrf2 gene is considered to affect the positive feedback loop of transcriptional activation of the Nrf2 gene as well as its target genes including ABCG2. Since patients have demonstrated individual differences in their response to PDT, Nrf2-mediated transcriptional activation of the ABCG2 gene in cancer may affect patients' responses to PDT as well as chemotherapy.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamiento farmacológico , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Animales , Humanos , Factor 2 Relacionado con NF-E2/genética , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/metabolismo , Fotoquimioterapia , Polimorfismo de Nucleótido Simple , Porfirinas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Activación TranscripcionalRESUMEN
BACKGROUND: Despite the potent antitumor activity of CPT-11, late-onset diarrhea owing to enterohepatic circulation of SN-38 is a critical issue. METHODS: We aimed to evaluate the inhibitory potency of gefitinib against the ABCB1- or ABCG2-mediated excretion of CPT-11 and its active metabolite SN-38 in vitro and in vivo. RESULTS: Gefitinib dose-dependently enhanced the antiproliferation activity of SN-38 in vitro by inhibiting ABCG2. The inhibitory effect of gefitinib on ABCB1 was marginal. When both CPT-11 and gefitinib were administered orally to nude mice bearing human lung cancer PC-6 cells, tumor growth was markedly suppressed. By gefitinib coadministration, the lactone forms of both CPT-11 and SN-38 in the tumor tissue increased more than 2-fold. CONCLUSIONS: Gefitinib significantly enhances the antitumor efficacy of CPT-11 and its tumor distribution in vivo. Coadministration of gefitinib may provide a new means to reduce the dose of CPT-11 and to circumvent the gastrointestinal toxicity risk.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Antineoplásicos/farmacología , Camptotecina/análogos & derivados , Proteínas de Neoplasias/metabolismo , Quinazolinas/farmacología , Subfamilia B de Transportador de Casetes de Unión a ATP , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/genética , Administración Oral , Animales , Antineoplásicos/efectos adversos , Antineoplásicos/uso terapéutico , Camptotecina/efectos adversos , Camptotecina/farmacología , Camptotecina/uso terapéutico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Diarrea/etiología , Sinergismo Farmacológico , Quimioterapia Combinada , Femenino , Gefitinib , Células HEK293 , Humanos , Irinotecán , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Quinazolinas/efectos adversos , Quinazolinas/uso terapéutico , ARN Mensajero/metabolismo , Trasplante HeterólogoRESUMEN
Human ATP-binding cassette (ABC) transporter ABCG2 (BCRP) is critically involved in multidrug resistance of human cancer. This transporter exhibits broad substrate specificity toward structurally diverse compounds, as do other ABC transporters, such as ABCB1 (P-glycoprotein/MDR1), ABCC1 (MRP1/GS-X pump), and ABCC2 (MRP2/cMOAT). To gain insight into the relationship between the molecular structure of compounds and the ABCG2-mediated transport activity, we have developed a high-speed screening method to analyze the substrate specificity of ABCG2. In addition, we have developed an algorithm that analyzes QSAR to evaluate ABCG2-drug interactions. This chapter presents our strategy of transport mechanism-based molecular design to circumvent multidrug resistance of cancer.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Diseño de Fármacos , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Proteínas de Neoplasias/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Algoritmos , Protocolos de Quimioterapia Combinada Antineoplásica/química , Ensayos Analíticos de Alto Rendimiento , Humanos , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Relación Estructura-Actividad CuantitativaRESUMEN
BACKGROUND: Gefitinib competes with topoisomerase I inhibitors at drug efflux pumps in vitro. We evaluated the effects of oral gefitinib on pharmacokinetic parameters of orally coadministered irinotecan. METHODS: We measured the serum pharmacokinetic parameters and biliary excretion of irinotecan, SN-38 and its glucuronide after irinotecan (50 or 100 mg/kg) was orally administered with or without gefitinib 100 mg/kg to rats. We measured the concentrations of irinotecan and SN-38 in the small intestine, liver, lungs and kidneys in each rat. RESULTS: The plasma area under the curve (0-24 h) of irinotecan and SN-38 was increased significantly, while the apparent elimination constant of irinotecan was decreased significantly. Gefitinib significantly increased the biliary cumulative amounts of irinotecan, but not of SN-38, and also influenced the enterohepatic circulation of irinotecan, but not of SN-38. CONCLUSIONS: Gefitinib increased the serum concentrations of irinotecan and SN-38 following oral administration of irinotecan without increasing the biliary excretion of SN-38 in vivo.
Asunto(s)
Sistema Biliar , Camptotecina/análogos & derivados , Quinazolinas/farmacología , Administración Oral , Animales , Sistema Biliar/metabolismo , Camptotecina/administración & dosificación , Camptotecina/metabolismo , Gefitinib , Irinotecán , Masculino , Ratas , Ratas WistarRESUMEN
Impacts of genetic polymorphisms of the ATP-binding cassette (ABC) transporter BCRP/MXR1/ABCP (ABCG2) on drug response have been implicated; however, the hitherto reported data involve some inconsistencies. To re-evaluate the effect of single nucleotide polymorphisms (SNP) of ABCG2 in vitro, we created a total of seven variant cDNAs (V12M, Q141K, F208S, S248P, F431L, S441N and F489L) by site-directed mutagenesis and stably expressed each of them in Flp-In-293 cells using the Flp recombinase system. Multicolor fluorescence in situ hybridization mapping analysis revealed that one single copy of ABCG2 cDNA was incorporated into the telomeric region of chromosome 12p. It was proven that mRNAs of those integrated ABCG2 variants were expressed evenly in Flp-In-293 cells. However, the protein expression levels varied among those variants. In particular, expression of the F208S and S441N variants was markedly low, suggesting the instability of these variant proteins. Drug resistance profiles of Flp-In-293 cells expressing two major SNP variants (V12M and Q141K) toward the drug SN-38 demonstrated that the IC50 value (drug concentrations producing a 50% reduction of cell growth) for Q141K was approximately 50% of that for wild type. The contributions of the minor SNP variants (F208S, S248P, F431L, S441N and F489L) to drug resistance toward SN-38, mitoxantrone, doxorubicin, daunorubicin or etoposide were significantly lower than wild type. Based on our functional validation, the above-mentioned non-synonymous polymorphisms as well as acquired mutants (R482G and R482T) of ABCG2 were classified into four groups. Furthermore, new camptothecin analogs synthesized by our research group had potent effects in circumventing ABCG2-mediated drug resistance without any influence from major non-synonymous polymorphisms.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Polimorfismo de Nucleótido Simple/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/clasificación , Adenosina Trifosfatasas/metabolismo , Camptotecina/química , Camptotecina/farmacología , Línea Celular , Cromosomas Humanos/genética , ADN Complementario/genética , Resistencia a Medicamentos , Expresión Génica/efectos de los fármacos , Genoma Humano/genética , Glutamina/genética , Glutamina/metabolismo , Humanos , Hibridación Fluorescente in Situ , Estructura Molecular , Mutación/genética , Proteínas de Neoplasias/clasificación , Valina/genética , Valina/metabolismoRESUMEN
Acquired and intrinsic drug resistance in cancer is the major obstacle to long-term, sustained patient response to chemotherapy. Irinotecan (CPT-11) is a widely-used potent antitumor drug that inhibits mammalian DNA topoisomerase I (Topo I). However, overexpression of ABCG2 (BCRP/MXR/ABCP) reportedly confers cancer cells resistance to SN-38, the active form of CPT-11. To circumvent the ABCG2-associated drug resistance, we have synthesized and characterized a total of fourteen new camptothecin (CPT) analogues with respect to both the inhibition of Topo I and the substrate specificity of ABCG2. While the lactone E ring is a prerequisite for anticancer activity, modifications of the A or B rings do not significantly affect Topo I inhibition activity. In this context, we have synthesized new CPT analogues with different substitutions at positions 10 or 11 of the A ring. All of the tested CPT analogues strongly inhibited the Topo I activity in a cell-free system. Accordingly, we have examined ATP-dependent transport of those CPT analogues by using plasma membrane vesicles prepared from ABCG2-overexpressing cells. Based on the substrate specificity of ABCG2 thus evaluated, it is strongly suggested that CPT analogues with a hydroxyl group at position 10 or 11 of the A ring are good substrates for ABCG2 and therefore effectively extruded from cancer cells. Thus, hydrogen bond formation is considered to be involved in substrate recognition and/or transport processes of ABCG2. The present study provides a practical approach to discover new CPT-based drugs for the chemotherapy of drug-resistant human cancer.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Antineoplásicos Fitogénicos/farmacología , Camptotecina/análogos & derivados , Camptotecina/farmacología , Diseño de Fármacos , Resistencia a Antineoplásicos/genética , Proteínas de Neoplasias/genética , Neoplasias/tratamiento farmacológico , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Animales , Antineoplásicos Fitogénicos/síntesis química , Camptotecina/síntesis química , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Humanos , Neoplasias/genética , Inhibidores de Topoisomerasa IRESUMEN
Irinotecan (CPT-11) is a widely used potent antitumor drug that inhibits mammalian DNA topoisomerase I (Topo I). However, overexpression of ABCG2 (BCRP/MXR/ABCP) reportedly confers cancer cells resistance to SN-38, the active form of CPT-11. To circumvent the ABCG2-associated drug resistance, the structure-activity-relationship (SAR) of 14 new camptothecin (CPT) analogues has been studied with respect to the substrate specificity of ABCG2. While the lactone E ring is a prerequisite for anticancer activity, modifications of the A or B rings do not significantly affect Topo I inhibition. Based on the substrate specificity of ABCG2, it is strongly suggested that CPT analogues with a hydroxyl group at position 10 or 11 of the A ring are recognized by ABCG2 and are thereby effectively extruded from cancer cells. To develop a platform for the molecular modeling to circumvent anticancer drug resistance, we have carried out quantum chemical calculations and neural network SAR analysis. Electrostatic potential iso-surfaces generated by ab initio MO calculations using restricted Hartree-Fock method have revealed that negative potential localized at positions 10 or 11 in the A ring is important for recognition by ABCG2.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Antineoplásicos Fitogénicos/farmacología , Camptotecina/análogos & derivados , Resistencia a Antineoplásicos/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamiento farmacológico , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Animales , Camptotecina/farmacología , Diseño de Fármacos , Humanos , Modelos Moleculares , Neoplasias/genéticaRESUMEN
Gefitinib ("Iressa", ZD1839) is an orally active, selective epidermal growth factor receptor tyrosine kinase inhibitor, and the single agent is clinically effective in non-small cell lung cancer. Although gefitinib combined with various cytotoxic agents has been reported to enhance cytotoxicity in vitro and in mouse models, the mechanism remains undetermined. Here, to explore the mechanism with topoisomerase I inhibitors, we focused on the efflux pump of the breast cancer resistance protein (BCRP/ABCG2), and then examined whether gefitinib restored drug sensitivity in multidrug-resistant cancer cells overexpressing BCRP. We used PC-6 human small cell lung cancer cells and multidrug-resistant PC-6/SN2-5H cells selected with SN-38 of the active metabolite of irinotecan, and BCRP-overexpressing MCF-7/MX cells selected with mitoxantrone and BCRP cDNA transfectant MCF-7/clone 8 cells. Drug sensitivity against anticancer drugs was determined by tetrazolium dye assay, and intracellular topotecan accumulation by FACScan. The topotecan transport study was done using the plasma membrane vesicles of PC-6/SN2-5H cells. The resistant PC-6/SN2-5H cells overexpressed BCRP but not epidermal growth factor receptor mRNA. Ten micromoles of gefitinib reversed topotecan, SN-38, and mitoxantrone resistance, and increased the intracellular topotecan accumulation in the resistant cells but not in the parental cells. Furthermore, gefitinib inhibited the topotecan transport into the vesicles, and the K(i) value was 1.01 +/- 0.09 micromol/L in the Dixon plot analysis, indicating direct inhibition of BCRP by gefitinib. However, gefitinib was not transported into the vesicles with the high-performance liquid chromatography method. These results indicate that gefitinib reverses BCRP-mediated drug resistance by direct inhibition other than competitive inhibition as a BCRP substrate. Combination of gefitinib and topoisomerase I inhibitors could be clinically effective in cancers expressing BCRP.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Neoplasias de la Mama/tratamiento farmacológico , Camptotecina/análogos & derivados , Carcinoma de Células Pequeñas/tratamiento farmacológico , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Receptores ErbB/antagonistas & inhibidores , Neoplasias Pulmonares/tratamiento farmacológico , Proteínas de Neoplasias/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/farmacología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/biosíntesis , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/metabolismo , Transporte Biológico Activo , Neoplasias de la Mama/metabolismo , Camptotecina/farmacología , Carcinoma de Células Pequeñas/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales , Factor de Crecimiento Epidérmico/biosíntesis , Gefitinib , Humanos , Concentración 50 Inhibidora , Irinotecán , Neoplasias Pulmonares/metabolismo , Mitoxantrona/farmacología , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/metabolismo , Topotecan/farmacocinética , Topotecan/farmacologíaRESUMEN
Irinotecan (CPT-11) is a widely-used potent anticancer drug that inhibits mammalian DNA topoisomerase I, however overexpression of the ATP-binding cassette transporter ABCG2 can confer cancer cells resistance to SN-38, the active form of CPT-11. In the present study, we have examined the contribution of three variant forms of ABCG2 to SN-38 resistance. Exogenous expression of the Arg482 (wild type), Gly482, and Thr482 variants of ABCG2 conferred HEK293 cells resistance to SN-38 by 15.0-, 5.0-, and 5.3-fold, respectively. In plasma membrane vesicles prepared from the ABCG2 variant cDNA-transfected HEK293 cells, [Arg482]ABCG2 transported SN-38 and its glucuronide conjugate in an ATP-dependent manner; however, only minimal transport activities were observed with the other variants (Gly482 and Thr482). In addition, we have screened natural flavonoids to find potent inhibitors of [Arg482]ABCG2. Quercetin was found to be the strongest inhibitor (Ki = 0.28 microM) among natural flavonoids tested in the plasma membrane system in this study. When [Arg482]ABCG2-transfected HEK293 cells were incubated with SN-38 in the presence of 20 microM quercetin, cellular resistance to SN-38 was partly reversed. In this context, certain flavonoid derivatives are considered to be good candidates for development of ABCG2 inhibitors.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/metabolismo , Antineoplásicos Fitogénicos/metabolismo , Camptotecina/metabolismo , Mutación/genética , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Quercetina/farmacología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Adenosina Trifosfato/metabolismo , Transporte Biológico , Camptotecina/análogos & derivados , Membrana Celular , Células Cultivadas , Resistencia a Medicamentos , Glucurónidos/metabolismo , Humanos , IrinotecánRESUMEN
Irinotecan (7-ethyl-10-[4-(1-piperidino)-1-piperidino]-carbonyloxycamptothecin; CPT-11) is a widely used potent antitumor drug that inhibits mammalian DNA topoisomerase I (Topo I); however, overexpression of ABCG2 (BCRP/MXR/ABCP) can confer cancer cell resistance to SN-38, the active form of CPT-11. We have recently demonstrated that plasma membrane vesicles prepared from ABCG2-overexpressing PC-6/SN2-5H cells transported SN-38 and its glucuronide conjugate in an ATP-dependent manner (Nakatomi et al., Biochem Biophys Res Commun 2001;288:827-32). In the present study, we have characterized a total of 14 new camptothecin (CPT) analogues with respect to both the inhibition of Topo I and the substrate specificity of ABCG2. All of the tested CPT analogues, which have different substitutions at positions 10 and 11, strongly inhibited the Topo I activity in a cell-free system, as did SN-38. Their antitumor activities in the SN-38-resistant PC-6/SN2-5H2 cell line greatly varied, however, being correlated with intracellular accumulation levels. We have examined ATP-dependent transport of those CPT analogues by using plasma membrane vesicles prepared from both PC-6/SN2-5H2 cells and ABCG2-transfected HEK-293 cells. Based on the substrate specificity of ABCG2 thus evaluated, it is strongly suggested that CPT analogues with high polarity are good substrates for ABCG2 and are therefore effectively extruded from cancer cells. In this context, to circumvent ABCG2-associated drug resistance, low-polarity CPT analogues are considered to be potent lead compounds. The present study provides a practical approach to discover new CPT-based drugs for the chemotherapy of drug-resistant human cancer.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Camptotecina/análogos & derivados , Camptotecina/toxicidad , Resistencia a Antineoplásicos/efectos de los fármacos , Proteínas de Neoplasias/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Adenosina Trifosfato/metabolismo , Antineoplásicos/toxicidad , Transporte Biológico , Camptotecina/farmacocinética , Carcinoma de Células Pequeñas , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Clonación Molecular , Humanos , Neoplasias Pulmonares , Proteínas Recombinantes/metabolismo , Relación Estructura-ActividadRESUMEN
Remarkable advances have been made in cancer chemotherapy by developing new anticancer drugs and pharmacogenomics strategies. However, multidrug resistance in human cancers is the major obstacle to long-term, sustained patient response to chemotherapy. Several ATP-binding cassette (ABC) transporters cause multidrug resistance in cancer cells by actively extruding the clinically administered chemotherapeutic drugs. P-glycoprotein (ABCB1/MDR1/P-gp) and MRP1 (ABCC1/GS-X pump) have been well characterized in terms of their molecular structure and function. In addition, ABCG2/breast cancer resistance protein (BCRP) is the most recently identified/ABC transporter, and is also reportedly associated with cellular resistance against chemotherapeutic agents, such as DNA topoisomerase I, II inhibitor. It is important to note that these ABC transporters are expressed not only in cancer cells but also in normal tissues to play a pivotal role in the absorption, distribution, and excretion of endogenous substances as well as xenobiotics. ABC transporters are key factors that can affect the pharmacokinetic profiles of drugs. Recent studies have revealed that many single nucleotide polymorphisms (SNPs) reside in these ABC transporter genes. Functional analysis of the genetic polymorphism of ABC transporters would greatly contribute to our understanding of individual differences in the drug response and also to the development of personalized medicine in the near future.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/fisiología , Resistencia a Múltiples Medicamentos , Genes MDR , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/biosíntesis , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Resistencia a Antineoplásicos , Humanos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/fisiología , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiología , Polimorfismo GenéticoRESUMEN
Breast cancer resistance protein (BCRP/ABCG2) of an ATP-binding cassette half-transporter confers resistance against mitoxantrone and camptothecin derivatives of topotecan and irinotecan. Novobiocin, a coumermycin antibiotic, is known to enhance anticancer drug sensitivity of cancer cells in vitro and in vivo, the mechanism of which remains undetermined. Here we focused on drug efflux pump and examined whether novobiocin reversed drug resistance in multidrug-resistant cells highly expressing BCRP. To explore the reversal mechanisms, intracellular drug accumulation was measured by flow cytometry, and a topotecan transport study using plasma membrane vesicles was performed. We used PC-6/SN2-5H2 small cell lung cancer and MCF-7/MX breast cancer cells selected with SN-38 of the active irinotecan metabolite and mitoxantrone, respectively, and the BCRP cDNA transfectant MCF-7/clone 8 cells. These cells expressed high levels of BCRP mRNA but not other known transporters. Compared to the parental PC-6 cells, PC-6/SN2-5H2 cells were 141-, 173- and 57.2-fold resistant to topotecan, SN-38 and mitoxantrone, respectively. Novobiocin at 60 microM decreased the degree of the above resistance by approximately 26-fold in PC-6/SN2-5H2 cells, and similarly reversed resistance in MCF-7/MX, MCF-7/clone 8 and un-selected NCI-H460 cells highly expressing BCRP. Furthermore, novobiocin increased the intracellular topotecan accumulation in these cells and inhibited the topotecan transport into the membrane vesicles of PC-6/SN2-5H2 cells. No effects of novobiocin in these assay were observed in the parental PC-6 and MCF-7 cells. The kinetic parameters in the transport study indicated that novobiocin was a inhibitor for BCRP, resulting in competitive inhibition of BCRP-mediated topotecan transport. These findings suggest that novobiocin effectively overcomes BCRP-mediated drug resistance at acceptable concentrations.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Camptotecina/análogos & derivados , Resistencia a Antineoplásicos/efectos de los fármacos , Proteínas de Neoplasias/metabolismo , Novobiocina/farmacología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Aminocumarinas , Camptotecina/farmacología , Cumarinas/farmacología , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Humanos , Irinotecán , Neoplasias Pulmonares/tratamiento farmacológico , Mitoxantrona/farmacología , Proteínas de Neoplasias/antagonistas & inhibidores , Inhibidores de Topoisomerasa I , Topotecan/metabolismo , Topotecan/farmacología , Células Tumorales CultivadasRESUMEN
SN-38 (7-ethyl-10-hydroxycamptothecin) is an active metabolite derived from the semi-synthetic compound camptothecin (CPT) named Irinotecan (CPT-11). The antitumor activity of SN-38 is 1000-fold more potent than the parent CPT-11. Fourteen new derivatives of camptothecin have recently been developed by Yakult Honsha (Tokyo, Japan). Here we describe a simple and cost-effective high-performance liquid chromatography (HPLC) method without an ion-pairing agent, which allows the simultaneous determination of both lactone and carboxylate forms of SN-38 and other camptothecin derivatives. A weak linear relationship between the HPLC retention factors (ln k') and the cellular concentrations of these compounds was observed. These results suggest that low-polarity compounds easily accumulate in cancer cells and may circumvent drug resistance. The HPLC analysis herein described is expected to greatly assist in derivative synthesis and chemical modification of camptothecin-based antitumor drugs.
Asunto(s)
Camptotecina/aislamiento & purificación , Ácidos Carboxílicos/análisis , Cromatografía Líquida de Alta Presión/métodos , Lactonas/análisis , Camptotecina/química , Cromatografía Líquida de Alta Presión/instrumentación , Concentración de Iones de Hidrógeno , Reproducibilidad de los Resultados , Espectrometría de FluorescenciaRESUMEN
Overexpression of the ATP-binding cassette transporter ABCG2 reportedly causes multidrug resistance, whereas altered drug-resistance profiles and substrate specificity are implicated for certain variant forms of ABCG2. At least three variant forms of ABCG2 have been hitherto documented on the basis of their amino acid moieties (i.e., arginine, glycine and threonine) at position 482. In the present study we have generated those ABCG2 variants by site-directed mutagenesis and expressed them in HEK-293 cells. Exogenous expression of the Arg(482), Gly(482), and Thr(482) variant forms of ABCG2 conferred HEK-293 cell resistance toward mitoxantrone 15-, 47- and 54-fold, respectively, as compared with mock-transfected HEK-293 cells. The transport activity of those variants was examined by using plasma-membrane vesicles prepared from ABCG2-overexpressing HEK-293 cells. [Arg(482)]ABCG2 transports [(3)H]methotrexate in an ATP-dependent manner; however, no transport activity was observed with the other variants (Gly(482) and Thr(482)). Transport of methotrexate by [Arg(482)]ABCG2 was significantly inhibited by mitoxantrone, doxorubicin and rhodamine 123, but not by S -octylglutathione. Furthermore, ABCG2 was found to exist in the plasma membrane as a homodimer bound via cysteinyl disulphide bond(s). Treatment with mercaptoethanol decreased its apparent molecular mass from 140 to 70 kDa. Nevertheless, ATP-dependent transport of methotrexate by [Arg(482)]ABCG2 was little affected by such mercaptoethanol treatment. It is concluded that Arg(482) is a critical amino acid moiety in the substrate specificity and transport of ABCG2 for certain drugs, such as methotrexate.