RESUMO
Here we present a novel hyperpolarization method, Chemical Reaction-Induced Multi-molecular Polarization (CRIMP), which could be applied to the study of several in vivo processes simultaneously including glycolysis, TCA cycle, fatty acid synthesis and pH mapping. Through the use of non-enzymatic decarboxylation, we generate four hyperpolarized imaging agents from hyperpolarized 1,2-(13)C pyruvic acid.
Assuntos
Espectroscopia de Ressonância Magnética , Acetatos/química , Isótopos de Carbono/química , Ciclo do Ácido Cítrico , Ácidos Graxos/síntese química , Ácidos Graxos/química , Glicólise , Concentração de Íons de Hidrogênio , Imageamento por Ressonância Magnética , Ácido Pirúvico/químicaRESUMO
Proteasome inhibitors (PIs), namely bortezomib, have become a cornerstone therapy for multiple myeloma (MM), potently reducing tumor burden and inhibiting pathologic bone destruction. In clinical trials, carfilzomib, a next generation epoxyketone-based irreversible PI, has exhibited potent anti-myeloma efficacy and decreased side effects compared with bortezomib. Carfilzomib and its orally bioavailable analog oprozomib, effectively decreased MM cell viability following continual or transient treatment mimicking in vivo pharmacokinetics. Interactions between myeloma cells and the bone marrow (BM) microenvironment augment the number and activity of bone-resorbing osteoclasts (OCs) while inhibiting bone-forming osteoblasts (OBs), resulting in increased tumor growth and osteolytic lesions. At clinically relevant concentrations, carfilzomib and oprozomib directly inhibited OC formation and bone resorption in vitro, while enhancing osteogenic differentiation and matrix mineralization. Accordingly, carfilzomib and oprozomib increased trabecular bone volume, decreased bone resorption and enhanced bone formation in non-tumor bearing mice. Finally, in mouse models of disseminated MM, the epoxyketone-based PIs decreased murine 5TGM1 and human RPMI-8226 tumor burden and prevented bone loss. These data demonstrate that, in addition to anti-myeloma properties, carfilzomib and oprozomib effectively shift the bone microenvironment from a catabolic to an anabolic state and, similar to bortezomib, may decrease skeletal complications of MM.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Reabsorção Óssea/tratamento farmacológico , Mieloma Múltiplo/tratamento farmacológico , Osteogênese/efeitos dos fármacos , Inibidores de Proteassoma/uso terapêutico , Administração Oral , Animais , Western Blotting , Medula Óssea/efeitos dos fármacos , Medula Óssea/metabolismo , Medula Óssea/patologia , Reabsorção Óssea/etiologia , Ácidos Borônicos/administração & dosagem , Bortezomib , Calcificação Fisiológica/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Compostos de Epóxi/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Mieloma Múltiplo/complicações , Oligopeptídeos/administração & dosagem , Osteoblastos/efeitos dos fármacos , Osteoblastos/patologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/patologia , Pirazinas/administração & dosagem , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Células Estromais/patologia , Células Tumorais Cultivadas , Microambiente Tumoral/efeitos dos fármacosRESUMO
Nucleophosmin (NPM/B23) is a multifunctional oncoprotein whose protein expression levels dictate cellular growth and proliferation rates. NPM is translationally responsive to hyperactive mammalian target of rapamycin (mTOR) signals, but the mechanism of this regulation is not understood. Using chimeric translational reporters, we found that the 3' untranslated region (UTR) of the NPM messenger (m)RNA is sufficient to mediate its translational modulation by mTOR signalling. We show that far upstream element (FUSE)-binding protein 1 (FBP1) interacts specifically with the 3' UTR of NPM to repress translation. Overexpression of FBP1 resulted in translational repression of NPM mRNAs, whereas depletion of FBP1 caused a dramatic increase in NPM translation and resulted in enhanced overall cell proliferation. Thus, we propose that FBP1 is a key regulator of cell growth and proliferation through its ability to selectively bind the NPM 3' UTR and repress NPM translation.
Assuntos
DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/biossíntese , Regiões 5' não Traduzidas , Processos de Crescimento Celular/genética , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Humanos , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Nucleofosmina , Ligação Proteica , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Transdução de Sinais , Sirolimo/metabolismoRESUMO
The ability to achieve tumor selective expression of therapeutic genes is an area that needs improvement for cancer gene therapy to be successful. One approach to address this is through the use of promoters that can be controlled by external means, such as hyperthermia. In this regard, we constructed a replication-deficient adenovirus that consists of a mutated herpes simplex virus 1 thymidine kinase (mTK) fused to enhanced green fluorescent protein (EGFP) under the control of the full-length human heat shock (HS) 70b promoter. The virus (AdHSmTK-EGFP) was evaluated both in vitro and in vivo in oral squamous cell carcinoma SCC-9 cells for expression of both mTK and EGFP. The in vitro expression of mTK-EGFP was validated using both (3)H-penciclovir and fluorescence-activated cell sorting assays. These studies show that specific expression could be achieved by heating the cells at 41 degrees C for 1 h, whereas little expression was observed using high doses of virus without hyperthermia. The vector was also evaluated in vivo by direct intratumoral injection into mice bearing SCC-9 xenografts. These studies demonstrated tumor expression of mTK-EGFP after ultrasound heating of the tumors by radioactive biodistribution assays, histology and microPET imaging. These in vivo results, which demonstrate HS-inducible transgene expression using PET imaging, provide a means for noninvasive monitoring of heat-induced gene therapy in local tumors, such as oral squamous cell carcinomas.
Assuntos
Regulação da Expressão Gênica , Genes Transgênicos Suicidas/genética , Neoplasias de Cabeça e Pescoço/terapia , Temperatura Alta , Tomografia por Emissão de Pósitrons/métodos , Transplante Heterólogo , Aciclovir/análogos & derivados , Aciclovir/farmacocinética , Adenoviridae/genética , Adenoviridae/metabolismo , Animais , Antivirais/farmacocinética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/terapia , Linhagem Celular Tumoral , Citometria de Fluxo , Vetores Genéticos , Guanina , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Proteínas de Choque Térmico/genética , Humanos , Fígado/patologia , Camundongos , Camundongos SCIDRESUMO
The (1)H MR signal arising from flowing extracellular media in a perfused, microbead-adherent cultured cell system can be suppressed with a slice-selective, spin-echo pulse sequence. The signal from intracellular water can, thus, be selectively monitored. Herein, this technique was combined with pulsed field gradients (PFGs) to quantify intracellular water diffusion in HeLa cells. The intracellular water MR diffusion-signal attenuation at various diffusion times was well described by a biophysical model that characterizes the incoherent displacement of intracellular water as a truncated Gaussian distribution of apparent diffusion coefficients (ADCs). At short diffusion times, the water "free" diffusion coefficient and the surface-to-volume ratio of HeLa cells were estimated and were, 2.0 +/- 0.3 microm(2)/ms and 0.48 +/- 0.1 microm(-1) (mean +/- SD), respectively. At long diffusion times, the cell radius of 10.1 +/- 0.4 microm was inferred and was consistent with that measured by optical microscopy. In summary: 1) intracellular water "free" diffusion in HeLa cells was rapid, two-thirds that of pure water; and 2) the cell radius inferred from modeling the incoherent displacement of intracellular water by a truncated Gaussian distribution of ADCs was confirmed by independent optical microscopy measures.
Assuntos
Células HeLa/metabolismo , Líquido Intracelular/metabolismo , Espectroscopia de Ressonância Magnética , Água/metabolismo , Teorema de Bayes , Difusão , Humanos , Modelos EstatísticosRESUMO
Quantitative characterization of the intracellular water (1)H MR signal from cultured cells will provide critical biophysical insight into the MR signal from tissues in vivo. Microbeads provide a robust immobilization substrate for the many mammalian cell lines that adhere to surfaces and also provide sufficient cell density for observation of the intracellular water MR signal. However, selective observation of the intracellular water MR signal from perfused, microbead-adherent mammalian cells requires highly effective suppression of the extracellular water MR signal. We describe how high-velocity perfusion of microbead-adherent cells results in short apparent (1)H MR longitudinal and transverse relaxation times for the extracellular water in a thin slice selected orthogonal to the direction of flow. When combined with a spin-echo pulse sequence, this phenomenon provides highly effective suppression of the extracellular water MR signal. This new method is exploited here to quantify the kinetics of water exchange from the intracellular to extracellular spaces of HeLa cells. The time constant describing water exchange from intracellular to extracellular spaces, also known as the exchange lifetime for intracellular water, is 119 +/- 14 ms.
Assuntos
Transporte Biológico , Espectroscopia de Ressonância Magnética/métodos , Microesferas , Prótons , Técnica de Subtração , Água/análise , Água/metabolismo , Anisotropia , Células Imobilizadas/metabolismo , Difusão , Espaço Extracelular/metabolismo , Células HeLa , Humanos , Aumento da Imagem/métodos , Líquido Intracelular/metabolismo , ReologiaRESUMO
Current methodologies for quantifying radiolabeled nucleoside monophosphates and nucleoside analogues result in high retention of unphosphorylated guanosine nucleosides in the case of lanthanum chloride precipitation or inconsistent retention of nucleotides in the case of DEAE cellulose filter papers. This study describes an innovative method for quantifying thymidine kinase (TK) activity that is compatible with both purine and pyrimidine nucleoside analogues by using lanthanum phosphate coprecipitation at pH 4.0. This methodology maintains quantitative precipitation of nucleoside monophosphates and yields minimal background binding from a variety of nucleoside analogues. In addition, use of PCR thermocyclers enhances the temporal precision of TK assays. This method was shown to be useful for assaying TK activity in a broad range of biochemically relevant systems, including purified enzymes, stable cell lines, and virally infected cells. Use of this methodology should aid researchers in the evaluation of novel nucleoside analogues and TK enzymes while decreasing radioactive waste, minimizing assay time, increasing accuracy, and enhancing dynamic range.
Assuntos
Nucleotídeos/análise , Fosfatos/química , Timidina Quinase/metabolismo , Células Cultivadas , Precipitação Química , Herpesvirus Humano 1/enzimologia , Concentração de Íons de Hidrogênio , Lantânio/metabolismo , Reação em Cadeia da Polimerase , Padrões de Referência , Reprodutibilidade dos Testes , Timidina Quinase/isolamento & purificação , Fatores de TempoRESUMO
Transglutaminase 2 (TG2, a.k.a. tissue transglutaminase) belongs to a family of transglutaminase enzymes that stabilize proteins by affecting covalent crosslinking via formation of amide bonds. Cell surface TG2 is directly involved as an adhesive receptor in cell-extracellular matrix (ECM) interactions. Here, we show that TG2 activity is elevated in glioblastomas compared with non-neoplastic brain. Immunofluorescent studies showed increased staining of fibronectin colocalized with TG2 in the ECM in glioblastomas. In addition, small clusters of invading human glioblastoma cells present in non-neoplastic brain parenchyma secrete high levels of TG2 and fibronectin that distinguish them from normal brain stroma. Downregulation of TG2 in U87MG glioblastoma cells with RNAi demonstrated decreased assembly of fibronectin in the ECM. Treatment with KCC009 blocked the remodeling of fibronectin in the ECM in glioblastomas in both in vitro and in vivo studies. KCC009 treatment in mice harboring orthotopic glioblastomas (DBT-FG) sensitized the tumors to N,N'-bis(2-chloroethyl)-N-nitrosourea chemotherapy, as measured by reduced bioluminescence, increased apoptosis and prolonged survival. The ability of KCC009 to interfere with the permissive remodeling of fibronectin in the ECM in glioblastomas suggests a novel target to enhance sensitivity to chemotherapy directed not only at the tumor mass, but also invading glioblastoma cells.
Assuntos
Inibidores Enzimáticos/farmacologia , Matriz Extracelular/efeitos dos fármacos , Fibronectinas/metabolismo , Proteínas de Ligação ao GTP/antagonistas & inibidores , Glioblastoma/tratamento farmacológico , Isoxazóis/farmacologia , Transglutaminases/antagonistas & inibidores , Animais , Encéfalo/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Carmustina/farmacologia , Matriz Extracelular/metabolismo , Feminino , Imunofluorescência , Proteínas de Ligação ao GTP/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteína 2 Glutamina gama-Glutamiltransferase , RNA Interferente Pequeno/farmacologia , Taxa de Sobrevida , Transglutaminases/metabolismo , Células Tumorais CultivadasRESUMO
MDR1 (multidrug resistance) P-glycoprotein (Pgp; ABCB1) decreases intracellular concentrations of structurally diverse drugs. Although Pgp is generally thought to be an efflux transporter, the mechanism of action remains elusive. To determine whether Pgp confers drug resistance through changes in transmembrane potential (E(m)) or ion conductance, we studied electrical currents and drug transport in Pgp-negative MCF-7 cells and MCF-7/MDR1 stable transfectants that were established and maintained without chemotherapeutic drugs. Although E(m) and total membrane conductance did not differ between MCF-7 and MCF-7/MDR1 cells, Pgp reduced unidirectional influx and steady-state cellular content of Tc-Sestamibi, a substrate for MDR1 Pgp, without affecting unidirectional efflux of substrate from cells. Depolarization of membrane potentials with various concentrations of extracellular K(+) in the presence of valinomycin did not inhibit the ability of Pgp to reduce intracellular concentration of Tc-Sestamibi, strongly suggesting that the drug transport activity of MDR1 Pgp is independent of changes in E(m) or total ion conductance. Tetraphenyl borate, a lipophilic anion, enhanced unidirectional influx of Tc-Sestamibi to a greater extent in MCF-7/MDR1 cells than in control cells, suggesting that Pgp may, directly or indirectly, increase the positive dipole potential within the plasma membrane bilayer. Overall, these data demonstrate that changes in E(m) or macroscopic conductance are not coupled with function of Pgp in multidrug resistance. The dominant effect of MDR1 Pgp in this system is reduction of drug influx, possibly through an increase in intramembranous dipole potential.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Transporte Biológico/fisiologia , Resistência a Múltiplos Medicamentos/fisiologia , Potenciais da Membrana/fisiologia , Adenocarcinoma , Neoplasias da Mama , Membrana Celular/metabolismo , Feminino , Humanos , Ionóforos/farmacologia , Compostos de Organotecnécio/metabolismo , Técnicas de Patch-Clamp , Células Tumorais Cultivadas , Valinomicina/farmacologiaRESUMO
IRF9/p48/ISGF3gamma (IRF9) is an IFN regulatory factor that mediates signaling by type I IFNs (IFNalpha and IFNbeta). After single-step selection of breast adenocarcinoma cells in paclitaxel, differential display and single gene analysis demonstrated that transcriptional activation of IRF9 and other IFN-responsive genes, independent of IFN, corresponded with resistance to antimicrotubule agents. Transient overexpression of IRF9 reproduced the drug-resistance phenotype and induced expression of IFN-responsive genes. However, drug resistance was not induced by overexpression of Stat1 or Stat2, or treatment with IFNalpha per se. Using a donor-matched array of cDNA prepared from human tumor and normal tissue from a variety of organs, we observed overexpression of IRF9 in approximately one-half of breast and uterine tumors, which indicated that IRF9 may be important in signaling in these tumor types. These data identify a novel IFN-independent role for IRF9 in the development of resistance to antimicrotubule agents in breast tumor cells and may link downstream mediators of IFN signaling to drug resistance in human cancers.
Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos Fitogênicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Proteínas de Ligação a DNA/fisiologia , Paclitaxel/farmacologia , Fatores de Transcrição/fisiologia , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Antibióticos Antineoplásicos/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Ciclo Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Interferon Tipo I/fisiologia , Fator Gênico 3 Estimulado por Interferon , Fator Gênico 3 Estimulado por Interferon, Subunidade gama , Microtúbulos/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Transfecção , Células Tumorais Cultivadas , Neoplasias Uterinas/genética , Neoplasias Uterinas/metabolismo , Vimblastina/farmacologiaRESUMO
Class I P-glycoproteins [Pgp; MDR1 (ABCB1) in humans, mdr1a and mdr1b in mice] confer resistance to structurally diverse chemotherapeutic drugs in cultured cells and intact animals, but the function of these proteins in normal physiology remains poorly characterized. Based on studies in cell culture, a putative role for class I Pgp in absorption and intracellular trafficking of sterols has been proposed. We examined wild-type and mdr1a(-/)-/1b(-/)- mice to determine whether class I Pgp affects cholesterol absorption and esterification in vivo. Using a dual-isotope protocol, absorption of orally administered radiolabeled cholesterol into serum did not differ between wild-type and mdr1a(-/)-/1b(-/)- mice, demonstrating that class I Pgp is not essential for overall absorption of cholesterol through the intestine. However, the ratio of oral to intravenous labeled cholesterol in liver was decreased significantly in mdr1a(-/)-/1b(-/)- mice. In the liver, but not other tested organs, deletion of class I Pgp enhanced kinetics of esterification of an oral bolus of radiolabeled cholesterol without affecting esterification of cholesterol administered intravenously. Steady-state hepatic content of cholesterol and esterified cholesterol also were unaffected by absence of mdr1a and mdr1b.Thus, in normal animals, class I Pgp functions to kinetically increase hepatic accumulation and decrease esterification of orally administered cholesterol in vivo.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/deficiência , Ésteres do Colesterol/metabolismo , Colesterol/farmacocinética , Fígado/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/fisiologia , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/fisiologia , Absorção , Animais , Radioisótopos de Carbono , Colesterol/administração & dosagem , Colesterol/metabolismo , Ésteres do Colesterol/análise , Colesterol na Dieta/administração & dosagem , Colesterol na Dieta/farmacocinética , Esterificação , Injeções Intravenosas , Cinética , Fígado/química , Masculino , Camundongos , Trítio , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPAssuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Dibenzocicloeptenos/farmacologia , Genes MDR/genética , Quinolinas/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Resistência a Múltiplos Medicamentos , Genes MDR/efeitos dos fármacos , Humanos , Compostos Radiofarmacêuticos , Tecnécio Tc 99m SestamibiRESUMO
Rapid and efficient delivery of radioactive metal complexes to the cell interior would enable novel applications in medical imaging and radiotherapy. Membrane permeant peptide conjugates incorporating HIV-1 Tat transactivation protein sequences (GRKKRRQRRR) and an appropriate peptide-based motif (epsilon-KGC) that provides an N(3)S donor core for chelating technetium and rhenium were synthesized. Oxotechnetium(V) and oxorhenium(V) Tat-peptide complexes were prepared by facile transchelation reactions with permetalates, tin(II) chloride and sodium glucoheptonate. RP-HPLC showed two major [(99m)Tc]Tat-peptide species (4) that differed in retention time by approximately 2 min corresponding to two [Re]Tat-peptide species (7) shown to have identical mass, consistent with formation of two isomers, likely the oxo-metal diastereomers. [(99m)Tc]Tat-peptides were stable to transchelation in vitro. In human Jurkat cells, [(99m)Tc]Tat-peptide 4 showed concentrative cell accumulation (30-fold greater than extracellular concentration) and rapid uptake kinetics (t(1/2) < 2 min) in a diastereomeric-comparable manner. Paradoxically, uptake was enhanced in 4 degrees C buffer compared to 37 degrees C, while depolarization of membrane potential as well as inhibition of microtubule function and vesicular trafficking showed no inhibitory effect. Cells preloaded with 4 showed rapid washout kinetics into peptide-free solution. Modification of [(99m)Tc]Tat-peptide by deletion of the N-terminus Gly with or without biotinylation minimally impacted net cell uptake. In addition, the C-terminus thiol of the prototypic Tat-peptide was labeled with fluorescein-5-maleimide to yield conjugate 8. Fluorescence microscopy directly localized conjugate 8 to the cytosol and nuclei (possibly nucleolus) of human Jurkat, KB 3-1 and KB 8-5 tumor cells. Preliminary imaging studies in mice following intravenous administration of prototypic [(99m)Tc]Tat-peptide 4 showed an initial whole body distribution and rapid clearance by both renal and hepatobiliary excretion. Analysis of murine blood in vivo and human serum ex vivo revealed >95% intact complex, while murine urine in vivo showed 65% parent complex. Thus, these novel Tat-peptide chelate conjugates, capable of forming stable [Tc/Re(V)]complexes, rapidly translocate across cell membranes into intracellular compartments and can be readily derivatized for further targeted applications in molecular imaging and radiotherapy.
Assuntos
Quelantes/metabolismo , Produtos do Gene tat/química , Compostos de Organotecnécio/metabolismo , Fragmentos de Peptídeos/metabolismo , Compostos Radiofarmacêuticos/metabolismo , Radioterapia , Rênio/metabolismo , Sequência de Aminoácidos , Animais , Quelantes/química , HIV-1/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Compostos de Organotecnécio/farmacocinética , Compostos de Organotecnécio/uso terapêutico , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacocinética , Compostos Radiofarmacêuticos/uso terapêutico , Rênio/farmacocinética , Rênio/uso terapêutico , Distribuição Tecidual , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
Multidrug resistance (MDR), whereby tumor cells simultaneously possess intrinsic or acquired cross-resistance to diverse chemotherapeutic agents, hampers the effective treatment of cancer. Molecular investigations in MDR resulted in the isolation and characterization of genes coding for several proteins associated with MDR, including P-glycoprotein (P-gp), the multidrug resistance associated protein (MRP1), the lung resistance protein (LRP), and, more recently, the breast cancer resistance protein (BCRP). These transmembrane proteins cause MDR either by decreasing the total intracellular retention of drugs or redistributing intracellular accumulation of drugs away from target organelles. These proteins are expressed at varying degrees in different neoplasms, including the AIDS-associated non-Hodgkin lymphoma and Kaposi sarcoma and are generally associated with poor prognosis. Several MDR-reversing agents are in various stages of clinical development. First-generation modulators such as verapamil, quinidine, and cyclosporin required high doses of drugs to reverse MDR and were associated with unacceptable toxicities. Second- and third-generation MDR inhibitors include PSC 833, GF120918, VX-710, and LY335979, among others. Limitations to the use of these modulators include multiple and redundant cellular mechanisms of resistance, alterations in pharmacokinetics of cytotoxic agents, and clinical toxicities. Studies to validate the role of MDR reversal in the treatment of various malignancies are underway. A potential use of these agents may be to enhance intestinal drug absorption and increase drug penetration to biologically important protective barriers, such as the blood-brain, blood-cerebrospinal fluid, and the maternal-fetal barriers. The use of MDR modulators with drugs such as the antiviral protease inhibitors and cytotoxics may enhance drug accumulation in sanctuary sites that are traditionally impenetrable to these agents.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/uso terapêutico , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Antineoplásicos/administração & dosagem , Transporte Biológico , Ciclosporinas/uso terapêutico , Proteínas de Ligação a DNA/metabolismo , Humanos , Linfoma Relacionado a AIDS/tratamento farmacológico , Linfoma Relacionado a AIDS/metabolismo , Linfoma não Hodgkin/tratamento farmacológico , Linfoma não Hodgkin/metabolismo , Proteína 3 Homóloga a MutS , Proteínas de Neoplasias/metabolismo , Prognóstico , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismoRESUMO
Multidrug resistance P-glycoprotein (Pgp) has been reported to localize in low-density, cholesterol-enriched membranes. However, effects of low-density membrane domains on function of Pgp remain unexplored in whole cell systems. In cells that express modest levels of the protein endogenously or through drug selection, Pgp predominantly localized to low-density membranes following separation on a sucrose gradient. When highly overexpressed in NIH 3T3 cells, a prominent amount of Pgp also was detected in high-density membranes. Removing cholesterol from cells with beta-methylcyclodextrin (CD), a sterol acceptor molecule, shifted fractions that contained Pgp from low toward high density, and this effect was reversed to a similar extent by restoring sterols with either cholesterol or enantiomeric cholesterol. However, function of human MDR1 Pgp as probed with Tc-Sestamibi, a transport substrate for Pgp, was not dependent on localization of Pgp in cholesterol-enriched membranes. Specific inhibition of MDR1 Pgp with GF120918 or LY335979 also was independent of cholesterol. Cell-type-specific effects of cholesterol content on function of human Pgp were detected by use of daunomycin, another substrate for Pgp, although efficacy of inhibitors remained independent of cholesterol. Conversely, both function and inhibition of hamster Pgp as measured with Tc-Sestamibi and daunomycin were in part dependent on normal cell content of cholesterol. These data show that Pgp preferentially localizes to low-density, cholesterol-enriched membrane domains, but acute depletion of cholesterol impacts Pgp-mediated drug transport in a substrate- and cell-type-specific manner.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , LDL-Colesterol/metabolismo , Colesterol/farmacologia , Células 3T3 , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/efeitos dos fármacos , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Colesterol/química , LDL-Colesterol/química , LDL-Colesterol/efeitos dos fármacos , Daunorrubicina/metabolismo , Humanos , Camundongos , Microscopia de Fluorescência , Estereoisomerismo , Tecnécio Tc 99m Sestamibi/metabolismoRESUMO
Multidrug resistance (MDR1) P-glycoprotein (Pgp), multidrug resistance-associated protein (MRP1), and breast cancer resistance protein (BCRP/MXR/ABCP) are members of the ATP-binding-cassette (ABC) superfamily of membrane transporters and are thought to function as energy-dependent efflux pumps of a variety of structurally diverse chemotherapeutic agents. We herein report the characterization of (99m)Tc-Tetrofosmin, a candidate radiopharmaceutical substrate of ABC transporters. (99m)Tc-Tetrofosmin showed high membrane potential-dependent accumulation in drug-sensitive KB 3-1 cells and low antagonist-reversible accumulation in MDR KB 8-5 and KB 8-5-11 cells in proportion to levels of MDR1 Pgp expression. In KB 8-5 cells, EC(50) values of the potent MDR antagonists N-(4-[2-(1,2,3, 4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9, 10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918), (2R)-anti-5-¿3-[4-(10, 11-difluoromethanodibenzo-suber-5-yl)piperazin-1-yl]-2 -hydroxypropoxy ¿quinoline trihydrochloride (LY335979), and (3'-keto-Bmt')-[Val(2)]-cyclosporin A (PSC 833) were 40, 66, and 986 nM, respectively. Furthermore, only baculoviruses carrying human MDR1, but not MDR3, conferred both a decrease in accumulation of (99m)Tc-Tetrofosmin in host Spodoptera frugiperda (Sf9) cells and a GF120918-induced enhancement. Transport studies with a variety of stably transfected and drug-selected tumor cell lines were performed with (99m)Tc-Tetrofosmin and compared with (99m)Tc-Sestamibi, a previously validated MDR imaging agent. MDR1 Pgp readily transported each agent. To a lesser extent, MRP1 also transported each agent, likely as co-transport substrates with GSH; neither agent was a substrate for the BCRP/MXR/ABCP half-transporter. In mdr1a(-/-) and mdr1a/1b(-/-) mice, (99m)Tc-Tetrofosmin showed approximately 3. 5-fold greater brain uptake and retention compared with wild-type, with no net change in blood pharmacokinetics, consistent with transport in vivo by Pgp expressed at the capillary blood-brain barrier. Molecular imaging of the functional transport activity of ABC transporters in vivo with (99m)Tc-Tetrofosmin and related radiopharmaceuticals may enable non-invasive monitoring of chemotherapeutic and MDR gene therapy protocols.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Neoplasias , Compostos Organofosforados/metabolismo , Compostos de Organotecnécio/metabolismo , Células 3T3 , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Animais , Baculoviridae/fisiologia , Transporte Biológico , Barreira Hematoencefálica , Reações Cruzadas , Humanos , Células KB , Camundongos , Compostos Radiofarmacêuticos/metabolismo , Reprodutibilidade dos Testes , Frações Subcelulares , Células Tumorais CultivadasRESUMO
BACKGROUND: Multidrug resistance (MDR) mediated by expression of MDR1 P-glycoprotein (Pgp) represents one of the best characterized barriers to chemotherapy in cancer patients. Positron emission tomography (PET) agents for analysis of Pgp-mediated drug transport activity in vivo would enable noninvasive assessment of chemotherapeutic regimens and MDR gene therapy. RESULTS: Candidate Schiff-base phenolic gallium(III) complexes were synthesized from their heptadentate precursors and gallium(III)acetylacetonate. Crystal structures demonstrated a hexacoordinated central gallium with overall trans-pseudo-octahedral geometry. Radiolabeled (67)Ga-complexes were obtained in high purity and screened in drug-sensitive (Pgp(-)) and MDR (Pgp(+)) tumor cells. Compared with control, lead compound 6. demonstrated antagonist-reversible 55-fold lower accumulation in Pgp-expressing MDR cells. Futhermore, compared with wild-type control, quantitative pharmacokinetic analysis showed markedly increased penetration and retention of 6. in brain and liver tissues of mdr1a/b((-/-)) gene disrupted mice, correctly mapping Pgp-mediated transport activity at the capillary blood-brain barrier and hepatocellular biliary cannalicular surface in vivo. CONCLUSIONS: These results indicate that gallium(III) complex 6. is recognized by MDR1 Pgp as an avid transport substrate, thereby providing a useful scaffold to generate (68)Ga radiopharmaceuticals for molecular imaging of Pgp transport activity in tumors and tissues in vivo using PET.