RESUMO
Bioluminescence (BL) imaging based on d-luciferin (d-luc)-luciferase reaction allows noninvasive and real-time monitoring of luciferase-expressing cells. Because BL intensity depends on photons generated through the d-luc-luciferase reaction, an approach to increase intracellular levels of d-luc could improve the detection sensitivity. In the present study, we showed that organic anion transporter 1 (OAT1) is useful, as a d-luc transporter, in boosting the BL intensity in luciferase-expressing cells. Functional screening of several transporters showed that the expression of OAT1 in HEK293â¯cells stably expressing Pyrearinus termitilluminans luciferase (HEK293/eLuc) markedly enhanced BL intensity in the presence of d-luc. When OAT1 was transiently expressed in HEK293â¯cells, intracellular accumulation of d-luc was higher than that in control cells, and the specific d-luc uptake mediated by OAT1 was saturable with a Michaelis constant (Km) of 0.23⯵M. The interaction between OAT1 and d-luc was verified using 6-carboxyfluorescein, a typical substrate of OAT1, which showed that d-luc inhibited the uptake of 6-carboxyfluorescein mediated by OAT1. BL intensity was concentration-dependent at steady states in HEK293/eLuc cells stably expressing OAT1, and followed Michaelis-Menten kinetics with an apparent Km of 0.36⯵M. In addition, the enhanced BL was significantly inhibited by OAT1-specific inhibitors. Thus, OAT1-mediated transport of d-luc could be a rate-limiting step in the d-luc-luciferase reaction. Furthermore, we found that expressing OAT1 in HEK293/eLuc cells implanted subcutaneously in mice also significantly increased the BL after intraperitoneal injection of d-luc. Our findings suggest that because OAT1 is capable of transporting d-luc, it can also be used to improve visualization and monitoring of luciferase-expressing cells.
Assuntos
Benzotiazóis/metabolismo , Aumento da Imagem/métodos , Luciferases/metabolismo , Medições Luminescentes/métodos , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Genes Reporter/genética , Células HEK293 , Humanos , Luciferases/genética , Imagem Molecular/métodos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
A suicide gene therapy using herpes simplex virus thymidine kinase (HSV-TK) with ganciclovir (GCV) has been under development as a tumor-targeted therapy; however, the mechanism of cellular GCV uptake, which is prerequisite in the therapy, has not been clarified. In an attempt to resolve this situation and gain information to optimize HSV-TK/GCV system for cancer therapy, we found that human equilibrative nucleobase transporter 1 (ENBT1) can transport GCV with a Michaelis constant of 2.75 mM in Madin-Darby canine kidney II (MDCKII) cells stably transfected with this transporter. In subsequent experiments using green fluorescent protein (GFP)-tagged ENBT1 (GFP-ENBT1) and HSV-TK, the uptake of GCV (30 µM), which was minimal in MDCKII cells and unchanged by their transfection with HSV-TK alone, was increased extensively by their transfection with GFP-ENBT1, together with HSV-TK. Accordingly, cytotoxicity, which was assessed by the WST-8 cell viability assay after the treatment of those cells with GCV (30 µM) for 72 hours, was induced in those transfected with GFP-ENBT1, together with HSV-TK but not in those transfected with HSV-TK alone. These results suggest that ENBT1 could facilitate GCV uptake and thereby enhance cytotoxicity in HSV-TK/GCV system. We also identified Helacyton gartleri (HeLa) and HepG2 as cancer cell lines that are rich with ENBT1 and A549, HCT-15 and MCF-7 as those poor with ENBT1. Accordingly, the HSV-TK/GCV system was effective in inducing cytotoxicity in the former but not in the latter. Thus, ENBT1 was found to be a GCV transporter that could enhance the performance of HSV-TK/GCV suicide gene therapy.
Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Apoptose/genética , Ganciclovir/metabolismo , Ganciclovir/farmacologia , Terapia Genética , Simplexvirus/enzimologia , Timidina Quinase/genética , Animais , Transporte Biológico , Linhagem Celular , Cães , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Simplexvirus/genéticaRESUMO
We previously demonstrated that differentiated enterocytes from human induced pluripotent stem (iPS) cells exhibited drug-metabolizing activities and cytochrome P450 CYP3A4 inducibility. The aim of this study was to apply human iPS cell-derived enterocytes in pharmacokinetic studies by investigating the characteristics of drug transport into enterocyte-like cells. Human iPS cells cultured on feeder cells were differentiated into endodermal cells using activin A. These endodermal-like cells were then differentiated into intestinal stem cells by fibroblast growth factor 2. Finally, epidermal growth factor and small-molecule compounds induced the maturation of the intestinal stem cell-like cells. After differentiation, we performed transepithelial electrical resistance (TEER) measurements, immunofluorescence staining, and transport studies. TEER values increased in a time-dependent manner and reached approximately 100 Ω × cm(2) Efflux transport of Hoechst 33342, a substrate of breast cancer resistance protein (BCRP), was observed and inhibited by the BCRP inhibitor Ko143. The uptake of peptide transporter 1 substrate glycylsarcosine was also confirmed and suppressed when the temperature was lowered to 4°C. Using immunofluorescence staining, villin and Na(+)-K(+) ATPase were expressed. These results suggest that human iPS cell-derived enterocytes had loose tight junctions, polarity, as well as uptake and efflux transport functions. In addition, the rank order of apparent membrane permeability coefficient (Papp) values of these test compounds across the enterocyte-like cell membrane corresponded to the fraction absorbance (Fa) values. Therefore, differentiated enterocytes from human iPS cells may provide a useful comprehensive evaluation model of drug transport and metabolism in the small intestine.
Assuntos
Enterócitos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Mucosa Intestinal/metabolismo , Benzimidazóis/metabolismo , Transporte Biológico , Imunofluorescência , Humanos , Intestinos/citologiaRESUMO
Proton-coupled folate transporter (PCFT), which is responsible for the intestinal uptake of folates and analogs, is expressed only in the proximal region in the small intestine. The present study was to examine its transcriptional regulation, which may be involved in such a unique expression profile and potentially in its alteration, using dual-luciferase reporter assays in human embryonic kidney (HEK) 293 cells. The luciferase activity derived from the reporter construct containing the 5'-flanking sequence of -1695/+96 of the human PCFT gene was enhanced most extensively by the introduction of Krüppel-like factor 4 (KLF4). The KLF4-induced luciferase activity was further enhanced by hepatocyte nuclear factor 4α (HNF4α) synergistically. To the contrary, caudal-type homeobox transcription factor 2 (CDX2) and CCAAT/enhancer-binding protein α (C/EBPα) extensively suppressed the luciferase activity induced by KLF4 alone and also that induced by KLF4 and HNF4α. Western blot analysis using the rat small intestine indicated uniform expression of KLF4 along the intestinal tract, proximal-oriented expression of HNF4α, distal-oriented expression of CDX2 and C/EBPα. These results suggest that the activity of PCFT promoter is basically induced by KLF4 and the gradiented expression profile of PCFT may be at least in part accounted for by those of HNF4α, CDX2 and C/EBPα.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Fator 4 Nuclear de Hepatócito/metabolismo , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Transportador de Folato Acoplado a Próton/genética , Ativação Transcricional , Animais , Fator de Transcrição CDX2 , Metilação de DNA , Genes Reporter , Células HEK293 , Humanos , Fator 4 Semelhante a Kruppel , Luciferases/genética , Masculino , Regiões Promotoras Genéticas , Ratos , Ratos WistarRESUMO
Nucleobases are important compounds that constitute nucleosides and nucleic acids. Although it has long been suggested that specific transporters are involved in their intestinal absorption and uptake in other tissues, none of their molecular entities have been identified in mammals to date. Here we describe identification of rat Slc23a4 as the first sodium-dependent nucleobase transporter (rSNBT1). The mRNA of rSNBT1 was expressed highly and only in the small intestine. When transiently expressed in HEK293 cells, rSNBT1 could transport uracil most efficiently. The transport of uracil mediated by rSNBT1 was sodium-dependent and saturable with a Michaelis constant of 21.2 microM. Thymine, guanine, hypoxanthine, and xanthine were also transported, but adenine was not. It was also suggested by studies of the inhibitory effect on rSNBT1-mediated uracil transport that several nucleobase analogs such as 5-fluorouracil are recognized by rSNBT1, but cytosine and nucleosides are not or only poorly recognized. Furthermore, rSNBT1 fused with green fluorescent protein was mainly localized at the apical membrane, when stably expressed in polarized Madin-Darby canine kidney II cells. These characteristics of rSNBT1 were almost fully in agreement with those of the carrier-mediated transport system involved in intestinal uracil uptake. Therefore, it is likely that rSNBT1 is its molecular entity or at least in part responsible for that. It was also found that the gene orthologous to the rSNBT1 gene is genetically defective in humans. This may have a biological and evolutional meaning in the transport and metabolism of nucleobases. The present study provides novel insights into the specific transport and metabolism of nucleobases and their analogs for therapeutic use.
Assuntos
Absorção Intestinal , Proteínas de Transporte de Nucleobases/metabolismo , Purinas/metabolismo , Pirimidinas/metabolismo , Animais , Linhagem Celular , Cães , Humanos , Cinética , Dados de Sequência Molecular , Ratos , Sódio , Especificidade da Espécie , Especificidade por Substrato , Uracila/metabolismoRESUMO
BACKGROUND/AIMS: Although aquaglyceroporins have been generally believed to operate in a channel mode, which is of nonsaturable nature, for glycerol as well as for water, we recently found that human aquaporin 9 (hAQP9) operates in a carrier-mediated mode, which is of saturable nature, for glycerol. Based on the finding, we assumed that such a characteristic might be shared by the other aquaglyceroporins and examined the functional characteristics of hAQP10, which is an intestine-specific aquaglyceroporin. METHODS: Transport assays were conducted using Xenopus laevis oocytes expressing hAQP10 derived from the microinjected cRNA. RESULTS: The transport of glycerol by hAQP10 was found to be highly saturable with a Michaelis constant of 10.4 µM and specifically inhibited by several glycerol analogs such as monoacetin. Furthermore, when glycerol was preloaded in hAQP10-expressing oocytes, its efflux was trans-stimulated by extracellular glycerol. These results indicate the involvement of a carrier-mediated mechanism in glycerol transport by hAQP10. Interestingly, a channel mechanism was also found to be involved in part in hAQP10-mediated glycerol transport. CONCLUSION: The present study unveiled the uniquely dual functional characteristic of hAQP10 as a carrier/channel for solute transport, providing a novel insight into its operation mechanism, which would help further elucidate its physiological role.
Assuntos
Aquaporinas/fisiologia , Animais , Aquaporinas/genética , Sequência de Bases , Transporte Biológico , Primers do DNA , DNA Complementar , Feminino , Glicerol/metabolismo , Humanos , Cinética , Xenopus laevisRESUMO
Multidrug and toxin extrusion protein 1 (MATE1) and MATE2-K are organic cation/H(+) antiporters that have recently been identified and suggested to be responsible for the brush border secretory transport of many cationic drugs in renal tubules. We here report our finding that 4',6-diamidino-2-phenylindole (DAPI) can be used as a probe substrate for rapid assays of the functionality of the human MATEs, hMATE1, and hMATE2-K, by taking advantage of its fluorescent nature. The specific cellular uptakes of DAPI by cloned hMATE1 and hMATE2-K, which were assessed by fluorescence intensity, were found to be rapid and saturable with the Michaelis constants of 1.13 and 3.16 microM, respectively, indicating that DAPI is a good substrate of both hMATEs. It was found that many organic cations inhibit the specific uptake of DAPI by hMATE1 and hMATE2-K, and the extents of inhibition are in good correlation with those of inhibition of the specific uptake of [(3)H]cimetidine as a typical substrate, indicating comparable performances of both substrates as probes in identifying inhibitors. Thus, DAPI can be an alternative probe substrate that enables fluorometric rapid assays of the functionality of both hMATEs. It was also found that the other major renal organic cation transporters, human organic cation transporter 2 (hOCT2), hOCT3, human novel organic cation transporter 1 (hOCTN1), and hOCTN2, cannot transport DAPI, although hOCT1, which is mainly expressed in the liver, can. Therefore, the DAPI uptake assay can be a method specific to the hMATEs among organic cation transporters in the human kidney.
Assuntos
Proteínas de Transporte/metabolismo , Corantes Fluorescentes , Indóis , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Animais , Cátions/metabolismo , Linhagem Celular , Cimetidina/metabolismo , DNA Complementar/biossíntese , DNA Complementar/genética , Cães , Antagonistas dos Receptores H2 da Histamina/metabolismo , Humanos , Rim/metabolismo , Microscopia de Fluorescência , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas de Transporte de Cátions Orgânicos/genética , Transportador 1 de Cátions Orgânicos/metabolismo , Transportador 2 de Cátion Orgânico , RNA/biossíntese , RNA/genéticaRESUMO
Riboflavin transporter (RFT) 2 has recently been identified as a transporter that may be, mainly based on the functional characteristics of its rat ortholog (rRFT2), involved in the intestinal absorption of riboflavin. The present study was conducted to further examine such a possible role of RFT2, focusing on the functional characteristics of its human ortholog (hRFT2) and the response of rRFT2 expression in the small intestine to deprivation of dietary riboflavin. When transiently expressed in human embryonic kidney 293 cells, hRFT2 could transport riboflavin efficiently in a pH-sensitive manner, favoring acidic pH and without requiring Na(+). Riboflavin transport by hRFT2 was saturable with a Michaelis constant of 0.77 µmol/L at pH 6.0, and inhibited by some riboflavin derivatives, such as lumiflavin. It was also inhibited, to a lesser extent, by some cationic compounds, such as ethidium. Thus, hRFT2 was suggested to, together with a finding that its mRNA is highly expressed in the small intestine, have characteristics as an intestinal RFT. Furthermore, feeding rats a riboflavin-deficient diet caused an upregulation of the expression of rRFT2 mRNA in the small intestine, presumably as an adaptive response to enhance riboflavin absorption, which would involve rRFT2, and its apically localized characteristic was suggested by the observation of rRFT2 tagged with green fluorescent protein stably expressed in polarized Madin-Darby canine kidney II cells. All these results combined indicate that RFT2 is a transporter involved in the epithelial uptake of riboflavin in the small intestine for its nutritional utilization.
Assuntos
Expressão Gênica/efeitos dos fármacos , Intestino Delgado/química , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/fisiologia , Riboflavina/farmacologia , Animais , Linhagem Celular , Proteínas de Ligação a DNA , Dieta , Cães , Embrião de Mamíferos , Etídio/farmacologia , Proteínas de Fluorescência Verde/genética , Humanos , Concentração de Íons de Hidrogênio , Absorção Intestinal/fisiologia , Rim , Masculino , Azul de Metileno/farmacologia , RNA Mensageiro/análise , Ratos , Ratos Wistar , Receptores Acoplados a Proteínas G , Proteínas Recombinantes de Fusão/genética , Riboflavina/administração & dosagem , Riboflavina/metabolismo , Deficiência de Riboflavina/metabolismo , Fatores de TranscriçãoRESUMO
Equilibrative nucleobase transporter 1 (ENBT1/SLC43A3) has recently been identified as a purine-selective nucleobase transporter. Although it is highly expressed in the liver, its role in nucleobase transport has not been confirmed yet in hepatocytes or any relevant cell models. We, therefore, examined its role in adenine transport in the HepG2 cell line as a human hepatocyte model. The uptake of [3H]adenine in HepG2 cells was highly saturable, indicating the involvement of carrier-mediated transport. The carrier-mediated transport component, for which the Michaelis constant was estimated to be 0.268 µM, was sensitive to decynium-22, an ENBT1 inhibitor, with the half maximal inhibitory concentration of 2.59 µM, which was comparable to that of 2.30 µM for [3H]adenine uptake by ENBT1 in its transient transfectant human embryonic kidney 293 cells. Although equilibrative nucleoside transporter 1 (ENT1/SLC29A1) and ENT2/SLC29A2 are also known to be able to transport adenine, [3H]adenine uptake in HepG2 cells was not inhibited by the ENT1/2-specific inhibitor of either dipyridamole or nitrobenzylthioinosine. Finally, [3H]adenine uptake was extensively reduced by silencing of ENBT1 by RNA interference in the hepatocyte model. All these results, taken together, suggest the predominant role of ENBT1 in the uptake of adenine in HepG2 cells.
Assuntos
Transportador Equilibrativo 1 de Nucleosídeo , Transportador Equilibrativo 2 de Nucleosídeo , Adenina , Sistemas de Transporte de Aminoácidos/metabolismo , Transporte Biológico , Transportador Equilibrativo 1 de Nucleosídeo/genética , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Transportador Equilibrativo 2 de Nucleosídeo/genética , Transportador Equilibrativo 2 de Nucleosídeo/metabolismo , Células Hep G2 , HumanosRESUMO
BACKGROUND/AIM: The aim of the present study was to investigate whether idarubicin (IDR) induces oxidative DNA damage in the presence of copper (II). MATERIALS AND METHODS: DNA damage was evaluated by pBR322 plasmid DNA cleavage. The formation of oxidative stress markers [O2 â¢- and 8-hydroxy-2'-deoxyguanosine (8-OHdG)] was analysed. RESULTS: IDR induced DNA damage and O2 â¢- and 8-OHdG generation in the presence of copper (II). CONCLUSION: IDR induced oxidative DNA damage in the presence of copper (II). Since it has been reported that the concentration of copper in the serum of cancer patients is higher than that in healthy groups, IDR-induced oxidative DNA damage in the presence of copper (II) may play an important role in anticancer therapeutic strategies.
Assuntos
Antraciclinas/farmacologia , Idarubicina/farmacologia , Neoplasias/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Antraciclinas/química , Cobre/química , Dano ao DNA/efeitos dos fármacos , Humanos , Idarubicina/química , Neoplasias/genética , Neoplasias/patologia , Espécies Reativas de Oxigênio/química , Superóxido Dismutase/genéticaRESUMO
Many fluoroquinolones are mainly eliminated by urinary excretion, in which tubular secretion by carrier-mediated transport systems has been suggested to be involved. In the present study, we examined the possibility that multidrug and toxin extrusion protein (MATE) 1, which is abundantly expressed in the kidney, might be involved in that, using rat MATE (rMATE) 1 expressed in MDCKII cells. It was found that rMATE1 can transport fluoroquinolones such as ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, norfloxacin (NFX), pazufloxacin, and tosufloxacin. Although rMATE1 has been known as an apical organic cation/H(+) antiporter, detailed investigation of rMATE1-mediated uptake of NFX has revealed that it is not sensitive to intracellular acidification by treatments using NH(4)Cl or nigericin, suggesting that the transmembrane proton gradient is not involved in its transport as a driving force. However, it was dependent on extracellular pH, being greatest at pH 7.0 and smaller at both acidic and basic pH in agreement with the profile of zwitterionization of NFX. The basal-to-apical transcellular transport of NFX in rMATE1-expressing MDCKII cells was greater than that in mock cells and insensitive to acidification of the apical medium, demonstrating proton gradient-independent functionality of rMATE1 in NFX efflux. Finally, rMATE1-mediated NFX uptake at pH 7.4 was saturable with the Michaelis constant of 55.3 microM and inhibited by cationic compounds, such as TEA and cimetidine. These results suggest that rMATE1 mediates the transport of NFX by a facilitative manner. MATE1 may play a key role in the renal tubular secretion of fluoroquinolones.
Assuntos
Antiporters/metabolismo , Fluoroquinolonas/farmacocinética , Rim/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Toxinas Biológicas/farmacocinética , Animais , Transporte Biológico/fisiologia , Linhagem Celular , Concentração de Íons de Hidrogênio , Íons/metabolismo , Rim/citologia , RatosRESUMO
Cimetidine is known to cause drug-drug interactions (DDIs) with organic cations in the kidney, and a previous clinical study showed that coadministration of cimetidine or probenecid with fexofenadine (FEX) decreased its renal clearance. FEX was taken up into human kidney by human organic anion transporter (hOAT) 3 (SLC22A8), but the mechanism of its luminal efflux has not been clarified. The present study examined the molecular mechanism of these DDIs. Saturable uptake of FEX was observed in human kidney slices, with K(m) and V(max) values of 157+/-7 microM and 418+/-16 nmol/15 min/g kidney, respectively. Cimetidine only slightly inhibited its uptake even at 100 microM, far greater than its clinically relevant concentration, whereas 10 microM probenecid markedly inhibited its uptake. As candidate transporters for the luminal efflux of FEX, we focused on human multidrug and toxin extrusions MATE1 (SLC47A1) and MATE2-K (SLC47A2). Saturable uptake of FEX could be observed in human embryonic kidney 293 cells expressing human MATE1 (hMATE1), whereas hMATE2-K-specific uptake of FEX was too small to conduct its further kinetic analysis. The hMATE1-mediated uptake clearance of FEX was inhibited by cimetidine in a concentration-dependent manner, and it was decreased to 60% of the control value in the presence of 3 microM cimetidine. Taken together, our results suggest that the DDI of FEX with probenecid can be explained by the inhibition of renal uptake mediated by hOAT3, whereas the DDI with cimetidine is mainly caused by the inhibition of hMATE1-mediated efflux of FEX rather than the inhibition of its renal uptake process.
Assuntos
Cimetidina/farmacologia , Proteínas de Transporte de Cátions Orgânicos/antagonistas & inibidores , Probenecid/farmacologia , Terfenadina/análogos & derivados , Linhagem Celular , Cimetidina/administração & dosagem , Interações Medicamentosas , Humanos , Rim/metabolismo , Probenecid/administração & dosagem , Terfenadina/administração & dosagem , Terfenadina/farmacocinética , Terfenadina/farmacologiaRESUMO
PURPOSE: Human multidrug and toxin extrusion protein 1 (hMATE1) and hMATE2-K are organic cation/H+ antiporters that have recently been identified and suggested to be involved in the renal brush border secretion of various organic cations. Information about functional characteristics of them has been accumulating, but still insufficient to fully understand their functions and respective roles. The present study was conducted to help clarify them. METHODS: The cDNA of hMATE1 was isolated from human brain cDNA by RT-PCR and hMATE2-K cDNA was from human kidney cDNA. HEK293 cells were stably transfected with hMATE1 and hMATE2-K, and the cellular uptakes of [3H]cimetidine and [14C]tetraethylammonium (TEA) were evaluated. RESULTS: It was first found that both hMATE1 and hMATE2-K can transport cimetidine with high affinities, indicated by small Michaelis constants of 8.00 mM and 18.18 mM, respectively. These were much smaller than those for TEA (366 mM and 375 mM, respectively, for hMATE1 and hMATE2-K). Subsequent investigation using cimetidine as a probe substrate into the profiles of inhibition of the two hMATEs by various compounds indicated that they are similar in principle but different to some extent in substrate recognition, reflecting the modest differences in amino acid sequences between them. In fact, cimetidine transport by hMATE1 was correlated to that by hMATE2-K, which is 65% similar to hMATE1, but not as good as to that by rat MATE1, which is 86% similar. CONCLUSIONS: Cimetidine was demonstrated to be a high affinity substrate of both hMATEs. Subsequent evaluation of the inhibition of hMATEs by various compounds indicated no major difference in function or role between hMATE1 and hMATE2-K.
Assuntos
Cimetidina/metabolismo , Inibidores Enzimáticos/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Tetraetilamônio/metabolismo , Animais , Antiporters/metabolismo , Linhagem Celular Tumoral , Cimetidina/farmacocinética , DNA Complementar/isolamento & purificação , Humanos , Rim/metabolismo , Cinética , Proteínas de Transporte de Cátions Orgânicos/antagonistas & inibidores , Proteínas de Transporte de Cátions Orgânicos/genética , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade da Espécie , TransfecçãoRESUMO
Human proton-coupled folate transporter (hPCFT/SLC46A1) has recently been found to be inhibited by myricetin by a sustained mechanism, raising a concern that the inhibition might lead to malabsorption of folates in the intestine, where hPCFT works for their epithelial uptake. However, rat PCFT (rPCFT) has more recently been found not to be inhibited by myricetin. Prompted by this finding, we attempted to determine the amino acid residue involved in that by analyses comparing between hPCFT and rPCFT. In the initial analysis, chimeric constructs prepared from hPCFT and rPCFT were examined for myricetin sensitivity to determine the hPCFT segment involved in the sensitivity. Focusing on the thereby determined segment from 83rd to 186th amino acid residue, hPCFT mutants having a designated amino acid residue replaced with its counterpart in rPCFT were prepared for the subsequent analysis. Among them, only G158N-substituted hPCFT was found to be transformed to be insensitive to myricetin and, accordingly, oppositely N158G-substituted rPCFT was transformed to be sensitive to myricetin. These results indicate the critical role of Gly158 in the myricetin sensitivity of hPCFT. This finding would help advance the elucidation of the mechanism of the myricetin-induced inhibition of hPCFT and manage the potential risk arising from that.
Assuntos
Flavonoides/farmacologia , Glicina/genética , Transportador de Folato Acoplado a Próton/genética , Substituição de Aminoácidos , Ácido Fólico/farmacocinética , Células HEK293 , Humanos , Absorção Intestinal/efeitos dos fármacos , Absorção Intestinal/fisiologia , Mutagênese Sítio-Dirigida , Transportador de Folato Acoplado a Próton/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
BACKGROUND/AIM: This study aimed to investigate aclarubicin (ACR)-induced oxidative DNA damage and apoptosis. MATERIALS AND METHODS: ACR-induced apoptosis was analyzed using HL-60 leukemia cells and HP100 cells, hydrogen peroxide (H2O2)-resistant cells derived from HL-60 cells. ACR-induced DNA damage was analyzed using plasmid DNA. RESULTS: HL-60 cells were more sensitive to ACR than HP100 cells. In HP100 cells, DNA ladder formation and caspase-3/7 activity induced by ACR were suppressed or delayed in comparison to those in HL-60 cells. ACR-induced DNA damage occurred in the presence of Cu(II), and scavenger experiments showed that the reactive species causing DNA damage appeared to be generated from H2O2 and Cu(I). Moreover, we detected intracellular Cu(I) induced by ACR in HL-60 cells, using CopperGREEN™, a fluorescent probe for detection of Cu(I) ion specifically. CONCLUSION: ACR-induced DNA damage and apoptosis can be accounted for by the involvement of H2O2 and Cu(I).
Assuntos
Aclarubicina/efeitos adversos , Antibióticos Antineoplásicos/efeitos adversos , Apoptose/efeitos dos fármacos , Cobre/farmacologia , Dano ao DNA , Peróxido de Hidrogênio/metabolismo , Linhagem Celular Tumoral , Humanos , Neoplasias/metabolismoRESUMO
Sodium-dependent nucleobase transporter 1 (SNBT1) is a nucleobase-specific transporter identified in our recent study. In an attempt to search for its potential substrates other than nucleobases in this study, we could successfully find urate, a metabolic derivative of purine nucleobases, as a novel substrate, as indicated by its specific Na+ -dependent and saturable transport, with a Michaelis constant of 433 µmol/L, by rat SNBT1 (rSNBT1) stably expressed in Madin-Darby canine kidney II cells. However, urate uptake was observed only barely in the everted tissue sacs of the rat small intestine, in which rSNBT1 operates for nucleobase uptake. These findings suggested that urate undergoes a futile cycle, in which urate transported into epithelial cells is immediately effluxed back by urate efflux transporters, in the small intestine. In subsequent attempts to examine that possibility, such a futile urate cycle was demonstrated in the human embryonic kidney 293 cell line as a model cell system, where urate uptake induced by transiently introduced rSNBT1 was extensively reduced by the co-introduction of rat breast cancer resistance protein (rBCRP), a urate efflux transporter present in the small intestine. However, urate uptake was not raised in the presence of Ko143, a BCRP inhibitor, in the everted intestinal tissue sacs, suggesting that some other transporter might also be involved in urate efflux. The newly found urate transport function of SNBT1, together with the suggested futile urate cycle in the small intestine, should be of interest for its evolutional and biological implications, although SNBT1 is genetically deficient in humans.
Assuntos
Proteínas de Transporte de Nucleobases/metabolismo , Ácido Úrico/metabolismo , Animais , Transporte Biológico , Cães , Células HEK293 , Humanos , Intestino Delgado/metabolismo , Células Madin Darby de Rim Canino , Masculino , Ratos WistarRESUMO
BACKGROUND/AIM: One mechanism of the anticancer action of anthracyclines is believed to be oxidative DNA damage. Previously, we reported that doxorubicin induced oxidative DNA damage in the presence of Cu(II). However, the mechanism of pirarubicin-induced oxidative DNA damage has not been well clarified. MATERIALS AND METHODS: DNA damage by pirarubicin in the presence of Cu(II) was analyzed using pBR322 plasmid DNA. O2â¢- derived from pirarubicin in the presence of Cu(II) was detected by cytochrome c reduction. RESULTS: Pirarubicin induced DNA damage in the presence of Cu(II). Scavenger experiments suggest that reactive species are generated from H2O2 and Cu(I). Pirarubicin induced O2â¢- production in the presence of Cu(II). CONCLUSION: These findings suggest that pirarubicin plus Cu(II) induces oxidative DNA damage in a similar manner to doxorubicin, and Cu(II)-mediated oxidative DNA damage may serve as a common mechanism for antitumor effects of anthracyclines.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Cobre/farmacologia , Dano ao DNA , Doxorrubicina/análogos & derivados , Cátions Bivalentes/farmacologia , Citocromos c/análise , DNA Circular/efeitos dos fármacos , Doxorrubicina/farmacologia , Sinergismo Farmacológico , Eletroforese em Gel de Ágar , Humanos , Estrutura Molecular , Oxirredução , Fenantrolinas/farmacologia , Plasmídeos , Espécies Reativas de Oxigênio/metabolismo , Superóxidos/metabolismoRESUMO
Myricetin is a flavonoid that inhibits human proton-coupled folate transporter (hPCFT) in a transient manner, in which inhibition is manifested in its presence, and also in a sustained manner, in which inhibition induced in its presence persists after its removal. In an effort to elucidate the mechanisms involved in those, we examined if myricetin might or might not act similarly on some other transporters. Transporters examined for that, in comparison with hPCFT, were its rat ortholog (rPCFT) and human riboflavin transporter 3 (hRFVT3). Experiments were conducted, using human embryonic kidney 293 cells transiently expressing the transporter to be examined, to assess the effects of myricetin (100 µM) on the uptake of folate by the PCFTs and riboflavin by hRFVT3. For hPCFT, myricetin was confirmed to induce a transient inhibition and also a sustained inhibition. However, myricetin induced neither transient nor sustained type of rPCFT inhibition. hRFVT3 was inhibited by myricetin in a transient manner, but not in a sustained manner. These results suggest the involvement of a hPCFT-specific mechanism in the sustained inhibition. The transient inhibition may be induced by a mechanism specific to hPCFT and also hRFVT3.
Assuntos
Flavonoides/farmacologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Transportador de Folato Acoplado a Próton/antagonistas & inibidores , Animais , Relação Dose-Resposta a Droga , Flavonoides/metabolismo , Ácido Fólico/farmacocinética , Células HEK293 , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transportador de Folato Acoplado a Próton/metabolismo , Ratos , Receptores Acoplados a Proteínas G , Riboflavina/farmacocinética , Relação Estrutura-AtividadeRESUMO
The intestinal absorption of atenolol has recently been reported to be reduced by simultaneous ingestion of fruit juices, such as apple juice. This finding implies a possibility that an unidentified carrier-mediated transport system, which could be interfered by some components of those juices, might be involved in atenolol absorption. In an attempt to explore that possibility, we successfully identified plasma membrane monoamine transporter (PMAT/SLC29A4) as a transporter that can operate for cellular atenolol uptake in the intestine, using Madin-Darby canine kidney II cells stably expressing PMAT. The specific uptake of atenolol by PMAT was greatest at around pH 6.0 and decreased with an increase in pH. At pH 6.0, the PMAT-specific uptake of atenolol was saturable with a Michaelis constant of 0.907 mM. Moreover, PMAT-specific atenolol uptake was extensively inhibited by phloretin and quercetin, which are the major flavonoids contained in apple juice, with the half maximal inhibitory concentrations of 33.3 and 116.3 µM, respectively. PMAT-specific atenolol uptake was also inhibited by several ß-blockers, suggesting that they may also be recognized and transported by PMAT. These results suggest that PMAT is an atenolol transporter that may be involved in intestinal atenolol absorption and sensitive to flavonoids contained in apple juice.
Assuntos
Atenolol/metabolismo , Proteínas de Transporte de Nucleosídeo Equilibrativas/metabolismo , Flavonoides/metabolismo , Sucos de Frutas e Vegetais/análise , Malus/química , Animais , Atenolol/química , Transporte Biológico , Linhagem Celular , Membrana Celular/metabolismo , Cães , Flavonoides/química , Expressão Gênica , Células HEK293 , Humanos , Absorção Intestinal/fisiologia , Rim/metabolismo , Especificidade por Substrato , TermodinâmicaRESUMO
Atenolol, a ß1-adrenergic receptor blocker, is administered orally and its intestinal absorption has recently been indicated to be mediated by carrier protein and reduced markedly by ingestion of some fruit juices, such as apple and orange juices. This could be postulated to be a problem arising from the interaction of some components of fruit juices with atenolol at a transporter involved in its intestinal uptake, but the responsible transporter and its interacting components have not been identified yet. In an attempt to examine that possibility, we could successfully find that human organic cation transporter 1 (OCT1/SLC22A1), which is suggested to be expressed at the brush border membrane of enterocytes, is highly capable of transporting atenolol. In this attempt, OCT1 was stably expressed in Madin-Darby canine kidney II cells and the specific uptake of atenolol by the transporter was found to be saturable, conforming to the Michaelis-Menten kinetics with the maximum transport rate (Vmax) of 4.00 nmol/min/mg protein and the Michaelis constant (Km) of 3.08 mM. Furthermore, the OCT1-specific uptake was found to be inhibited by various flavonoids, including those contained in fruit juices that have been suggested to interfere with intestinal atenolol absorption. Particularly, phloretin and quercetin, which are major components of apple juice, were potent in inhibiting OCT1-mediated atenolol transport with the inhibition constants of 38.0 and 48.0 µM, respectively. It is also notable that the inhibition by these flavonoids was of the noncompetitive type. These results indicate that OCT1 is an atenolol transporter that may be involved in intestinal atenolol uptake and sensitive to fruit juices, although its physiological and clinical relevance remains to be further examined.