RESUMEN
Microcystins are potent phosphatase inhibitors and cellular toxins. They require active transport by OATP1B1 and OATP1B3 transporters for uptake into human cells, and the high expression of these transporters in the liver accounts for their selective hepatic toxicity. Several human tumors have been shown to have high levels of expression of OATP1B3 but not OATP1B1, the main transporter in liver cells. We hypothesized that microcystin variants could be isolated that are transported preferentially by OATP1B3 relative to OATP1B1 to advance as anticancer agents with clinically tolerable hepatic toxicity. Microcystin variants have been isolated and tested for cytotoxicity in cancer cells stably transfected with OATP1B1 and OATP1B3 transporters. Microcystin variants with cytotoxic OATP1B1/OATP1B3 IC50 ratios that ranged between 0.2 and 32 were found, representing a 150-fold range in transporter selectivity. As microcystin structure has a significant impact on transporter selectivity, it is potentially possible to develop analogs with even more pronounced OATP1B3 selectivity and thus enable their development as anticancer drugs.
Asunto(s)
Microcistinas/farmacología , Neoplasias/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/antagonistas & inhibidores , Transportadores de Anión Orgánico/antagonistas & inhibidores , Línea Celular Tumoral , Humanos , Concentración 50 Inhibidora , Transportador 1 de Anión Orgánico Específico del Hígado , Microcistinas/química , Transportadores de Anión Orgánico/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Péptidos Cíclicos/química , Miembro 1B3 de la Familia de los Transportadores de Solutos de Aniones OrgánicosRESUMEN
AR-67 (7-t-butyldimethylsilyl-10-hydroxycamptothecin) is a lipophilic camptothecin analog, currently under early stage clinical trials. Transporters are known to have an impact on the disposition of camptothecins and on the response to chemotherapeutics in general due to their expression in tumor tissues. Therefore, we investigated the interplay between the breast cancer resistance protein (BCRP), multidrug resistant protein 1 (MDR1), and organic anion-transporting polypeptide (OATP) 1B1/1B3 transporters and AR-67 and their impact on the toxicity profile of AR-67. Using cell lines expressing the aforementioned transporters, we showed that the lipophilic AR-67 lactone form is a substrate for efflux transporters BCRP and MDR1. Additionally, OATP1B1 and OATP1B3 facilitated the uptake of AR-67 carboxylate in SLCO1B1- and SLCO1B3-transfected cell systems compared with the mock-transfected ones. Notably, both BCRP and MDR1 conferred resistance to AR-67 lactone. Prompted by recent studies showing increased OATP1B3 expression in certain cancer types, we investigated the effect of OATP1B3 expression on cell viability after exposure to AR-67 carboxylate. OATP1B3-expressing cells had increased carboxylate uptake as compared with mock-transfected cells but were not sensitized because the intracellular amount of lactone was 50-fold higher than that of carboxylate and comparable between OATP1B3-expressing and OATP1B3-nonexpressing cells. In conclusion, BCRP- and MDR1-mediated efflux of AR-67 lactone confers resistance to AR-67, but OATP1B3-mediated uptake of the AR-67 carboxylate does not sensitize OATP1B3-expressing tumor cells.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Transportadoras de Casetes de Unión a ATP/fisiología , Antineoplásicos/farmacología , Camptotecina/análogos & derivados , Proteínas de Neoplasias/fisiología , Transportadores de Anión Orgánico Sodio-Independiente/fisiología , Compuestos de Organosilicio/farmacología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Animales , Camptotecina/farmacocinética , Camptotecina/farmacología , Línea Celular , Perros , Resistencia a Antineoplásicos , Células HeLa , Humanos , Transportador 1 de Anión Orgánico Específico del Hígado , Transportadores de Anión Orgánico/fisiología , Compuestos de Organosilicio/farmacocinética , Miembro 1B3 de la Familia de los Transportadores de Solutos de Aniones OrgánicosRESUMEN
We have previously shown that the bacterial enzyme thiaminase 1 has antitumor activity. In an attempt to make thiaminase I a more effective pharmaceutical agent, we have modified it by adding polyethylene glycol (PEG) chains of various lengths. We were surprised to find that 5k-PEGylation eliminated thiaminase cytotoxic activity in all cell lines tested. Both native thiaminase and 5k-PEGylated thiaminase efficiently depleted thiamine from cell culture medium, and both could use intracellular phosphorylated thiamine as substrates. However, native enzyme more effectively depleted thiamine and thiamine diphosphate in RS4 leukemia cell cytosol, and native thiaminase depressed cellular respiration, whereas PEGylated thiaminase did not. Despite the lack of in vitro cytotoxicity, PEGylation markedly increased the in vivo toxicity of the enzyme. Pharmacokinetic studies revealed that the half-life of native thiaminase was 1.5 h compared with 34.4 h for the 5k-PEGylated enzyme. Serum thiamine levels were depleted by both native and 5k-PEGylated enzyme. Despite superior pharmacokinetics, 5k-PEGylated thiaminase showed no antitumor effect against an RS4 leukemia xenograft, in contrast to native thiaminase, which showed antitumor activity. PEGylation of thiaminase I has demonstrated that depression of mitochondrial function contributes, at least in part, to its anticancer activity. PEGylation also enhances plasma retention time, which increased its vivo toxicity and decreased its activity against a leukemia xenograft, the opposite of the desired effects. These studies suggest that the mechanism of anticancer cytotoxicity of thiaminase requires acute depression of cellular respiration, whereas systemic toxicity is related to the duration of extracellular thiamine depletion.
Asunto(s)
Transferasas Alquil y Aril/farmacología , Antineoplásicos/farmacología , Bacillus/enzimología , Polietilenglicoles/farmacología , Tiamina/metabolismo , Transferasas Alquil y Aril/farmacocinética , Animales , Antineoplásicos/farmacocinética , Línea Celular Tumoral/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Medios de Cultivo , Femenino , Semivida , Humanos , Immunoblotting , Ratones , Ratones Desnudos , Microscopía Fluorescente , Mitocondrias/metabolismo , Consumo de Oxígeno/fisiología , Polietilenglicoles/farmacocinéticaRESUMEN
We have previously described the down-regulation of thiamine transporter gene expression in breast cancer, and others have shown an epidemiologic relationship between obesity and breast cancer. To further explore the relationship of thiamine, fat, and breast cancer, we exposed FVB/N-Tg(MMTVneu)202Mul/J female mice to four diets that varied in fat and thiamine content (15 mice per group). The high-fat (HF) diet contained 60 % of calories from fat and the normal-fat (NF) diet contained 10 % of calories from fat. The normal-thiamine (NT) diet contained 6 mg thiamine per 4057 kcal and the low-thiamine (LT) diet contained 2 mg thiamine/4057 kcal. Tumor latency was 203 days from date of birth for the HF/NT group, 210 days for the HF/LT group, 225 days for the NF/NT group, and 295 days for the NF/LT group (p = 0.01). The time to endpoint of a mammary tumor volume > 1000 mm3 was 231 days for the HF/NT group, 238 days for the HF/LT group, 257 days for the NF/NT group, and undefined (>310 days) for the NF/LT group (p < 0.001). The high-fat groups were heavier than the normal-fat groups, and the low-thiamine group had a lower serum thiamine level than the normal-thiamine group. There were no differences in the number of pulmonary metastases between groups. This study demonstrates a potential role for dietary thiamine, and an interaction between thiamine and fat, in breast cancer progression.
Asunto(s)
Dieta Alta en Grasa/efectos adversos , Dieta , Neoplasias Mamarias Experimentales/patología , Neoplasias Mamarias Experimentales/prevención & control , Tiamina/administración & dosificación , Animales , Grasas de la Dieta/administración & dosificación , Femenino , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Neoplasias Mamarias Experimentales/etiología , Ratones , Ratones Transgénicos , Tiamina/sangreRESUMEN
Cancer cells acquire abnormalities in energy metabolism, collectively known as the Warburg effect, affecting substrate availability of thiamine-dependent enzymes. To investigate a strategy to exploit abnormal cancer-associated metabolism related to thiamine, we tested the cytotoxicity of native Bacillus thiaminolyticus thiaminase I enzyme, which digests thiamine, in the NCI60 cell line drug cytotoxicity screening program and found that leukemia cell lines were among the most sensitive to thiaminase I. We obtained additional lymphoid leukemia cell lines and confirmed that native thiaminase I and linear chain PEGylated thiaminase I enzyme (LCPTE) have cytotoxic activity in these cell lines. In addition, the IC(50) of 3 of the 5 leukemia cell lines (Reh, RS4, and Jurkat) were at least 1,000-fold more sensitive than Molt-4 cells, which in turn, were among the most sensitive in the NCI60 panel. The 3 LCPTE-sensitive leukemia cell lines were also sensitive to removal of thiamine from the medium, thus suggesting the mechanism of action of LCPTE involves extracellular thiamine starvation. Surprisingly, rapamycin showed a protective effect against LCPTE toxicity in the 3 LCPTE-sensitive cell lines but not in the other 2 cell lines, suggesting involvement of an mTOR-dependent pathway. Immunoblot analysis of the LCPTE-sensitive cell lines after LCPTE exposure revealed changes in mTOR pathway phosphorylation. Nude mice bearing RS4 leukemia xenografts showed both tumor growth delay and prolonged survival after a single dose of LCPTE. Therefore, disruption of thiamine-dependent metabolism may be a novel therapeutic approach to target altered energy metabolism in leukemia and other cancers.
Asunto(s)
Antineoplásicos/toxicidad , Bacillus/enzimología , Hidrolasas/toxicidad , Leucemia Linfoide/tratamiento farmacológico , Animales , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Células HL-60 , Humanos , Hidrolasas/uso terapéutico , Células Jurkat , Leucemia Linfoide/metabolismo , Leucemia Linfoide/mortalidad , Ratones , Ratones Desnudos , Proteínas Recombinantes/uso terapéutico , Proteínas Recombinantes/toxicidad , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Tiamina/metabolismo , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Solute transporters that are selectively expressed on tumor cell membranes could be targeted with small molecule toxins that are selective substrates for these transporters. HeLa cells transfected to express the solute transporter OATP1B1 are exquisitely sensitive in vitro to microcystin LR (MCLR) and its analogs, and undergo rapid morphologic changes after exposure to MCLR. Immunoblot analyses revealed HSP27 phosphorylation increased prior to the rapid MCLR-induced morphologic changes. However, transfection of OATP1B1-expressing cells with HSP27 dominant negative mutants did not reverse MCLR toxicity. Although the MAP kinase p38 inhibitor SB202190 partially reversed MCLR cytotoxicity, the control molecule, SB202474, had similar effects. Unexpectedly, both SB202190 and SB202474 inhibited OATP1B1 uptake activity, indicating an alternative explanation for cytotoxicity reversal that did not involve p38 MAP kinase. Similarly, although the potassium chloride co-transporter (KCC) inhibitor (dihydro-indenyl)oxyalkanoic acid (DIOA), and the anti-oxidant, N-acetyl cysteine (NAC) both reversed MCLR cytotoxicity, both were also found to be unexpected OATP1B1 transport inhibitors. Therefore, the mechanism of MCLR-induced cytotoxicity is obscured by the inhibition of OATP1B1 uptake activity by MAP kinase inhibitors, DIOA, and NAC. Finally, growth of OATP1B1-expressing HeLa xenografts was inhibited by MCLR, suggesting that MCLR structural analogs selected for a broader therapeutic index could target OATP-expressing tumors.
Asunto(s)
Acetilcisteína/farmacología , Microcistinas/farmacología , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Transportadores de Anión Orgánico/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Western Blotting , Células HeLa , Humanos , Transportador 1 de Anión Orgánico Específico del Hígado , Transportadores de Anión Orgánico/metabolismoRESUMEN
A number of therapeutic strategies for treating Alzheimer's disease have focused on reducing amyloid burden in the brain. Among these approaches, the expression of amyloid beta peptide (Abeta)-degrading enzymes in the brain has been shown to be effective but to date not practical for treating patients. We report here a novel strategy for lowering amyloid burden in the brain by peripherally expressing the Abeta-degrading enzyme neprilysin on leukocytes in the 3xTg-AD mouse model of Alzheimer's disease. Through transplantation of lentivirus-transduced bone marrow cells, the Abeta-degrading protease neprilysin was expressed on the surface of leukocytes. This peripheral neprilysin reduced soluble brain Abeta peptide levels by approximately 30% and lowered the accumulation of amyloid beta peptides by 50-60% when transplantation was performed at both young and early adult age. In addition, peripheral neprilysin expression reduced amyloid-dependent performance deficits as measured by the Morris water maze. Unlike other methods designed to lower Abeta levels in blood, which cause a net increase in peptide, neprilysin expression results in the catabolism of Abeta to small, innocuous peptide fragments. These findings demonstrate that peripherally expressed neprilysin, and likely other Abeta-degrading enzymes, has the potential to be utilized as a therapeutic approach to prevent and treat Alzheimer's disease and suggest that this approach should be explored further.
Asunto(s)
Enfermedad de Alzheimer/terapia , Péptidos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Terapia Genética , Leucocitos/metabolismo , Neprilisina/genética , Neprilisina/metabolismo , Envejecimiento , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/genética , Animales , Presión Sanguínea , Trasplante de Médula Ósea , Trastornos del Conocimiento/terapia , Modelos Animales de Enfermedad , Humanos , Lentivirus , Aprendizaje por Laberinto , Ratones , Ratones Transgénicos , Transducción GenéticaRESUMEN
Dementia associated with human immunodeficiency virus (HIV) infection occurs commonly in the aging population and amyloid depositions are noted in the brains of patients with HIV infection in younger age groups. This suggests a dysregulation of amyloid processing in the setting of HIV infection. The Tat protein of HIV has been implicated in the neuropathogenesis of HIV infection due to its neurotoxic and glial activation properties. However, Tat protein and Tat-derived peptides were recently also shown to inhibit neprilysin, the major amyloid beta peptide degrading enzyme in brain, in a cell aggregate system. This effect could contribute to the observed accumulation of amyloid in the brain of HIV-infected patients. The authors report here that peptides derived from the Tat protein, but not Tat protein itself, inhibit homogeneous recombinant neprilysin. This inhibition was found to be competitive and reversible and therefore does not involve covalent bond formation. Tat peptides and Tat protein were slowly hydrolyzed by neprilysin. Thus the accumulation of Tat-derived proteolytic fragments may serve to inhibit neprilysin and increase amyloid beta peptide levels.
Asunto(s)
Complejo SIDA Demencia/metabolismo , Complejo SIDA Demencia/patología , Productos del Gen tat/metabolismo , Neprilisina/metabolismo , Complejo SIDA Demencia/virología , Secuencia de Aminoácidos , Péptidos beta-Amiloides/metabolismo , Amiloidosis/metabolismo , Amiloidosis/patología , Amiloidosis/virología , Secuencia de Consenso , Cisteína/metabolismo , Activación Enzimática , Productos del Gen tat/genética , Humanos , Hidrólisis , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
The active site glutamate (Glu(111)) and the active site histidine (His(112)) of insulin-degrading enzyme (IDE) were mutated. These mutant enzymes exhibit, in addition to a large decrease in catalytic activity, a change in the substrate-velocity response from a sigmoidal one seen with the native enzyme (Hill coefficient > 2), to a hyperbolic response. With 2-aminobenzoyl-GGFLRKHGQ-N-(2,4-dinitrophenyl)ethylenediamine as substrate, ATP and triphosphate increase the reaction rate of the wild type enzyme some 50-80-fold. This effect is dampened with glutamate mutants to no effect or less than a 3-fold increase in activity and changed to inhibition with the histidine mutants. Sedimentation equilibrium shows the IDE mutants exhibit a similar oligomeric distribution as the wild type enzyme, being predominantly monomeric, with triphosphate having little if any effect on the oligomeric state. Triphosphate did induce aggregation of many of the IDE mutants. Thus, the oligomeric state of IDE does not correlate with kinetic properties. The His(112) mutants were shown to bind zinc, but with a lower affinity than the wild type enzyme. The glutamate mutants displayed an altered cleavage profile for the peptide beta-endorphin. Wild type IDE cleaved beta-endorphin at Leu(17)-Phe(18) and Phe(18)-Lys(19), whereas the glutamate mutants cleaved at these sites, but in addition at Lys(19)-Asn(20) and at Met(5)-Thr(6). Thus, active site mutations of IDE are suggested to not only reduce catalytic activity but also cause local conformational changes that affect the allosteric properties of the enzyme.