RESUMO
L-type amino acid transporter 1 (LAT1, SLC7A5) is an amino acid transporter expressed in various carcinomas, and it is postulated to play an important role in the proliferation of cancer cells through the uptake of essential amino acids. Cabazitaxel is a widely used anticancer drug for treating castration-resistant prostate cancer (CRPC); however, its effectiveness is lost when cancer cells acquire drug resistance. In this study, we investigated the expression of LAT1 and the effects of a LAT1-specific inhibitor, JPH203, in cabazitaxel-resistant prostate cancer cells. LAT1 was more highly expressed in the cabazitaxel-resistant strains than in the normal strains. Administration of JPH203 inhibited the growth, migration, and invasive ability of cabazitaxel-resistant strains in vitro. Phosphoproteomics using liquid chromatography-mass spectrometry to comprehensively investigate changes in phosphorylation due to JPH203 administration revealed that cell cycle-related pathways were affected by JPH203, and that JPH203 significantly reduced the kinase activity of cyclin-dependent kinases 1 and 2. Moreover, JPH203 inhibited the proliferation of cabazitaxel-resistant cells in vivo. Taken together, the present study results suggest that LAT1 might be a valuable therapeutic target in cabazitaxel-resistant prostate cancer.
Assuntos
Benzoxazóis , Transportador 1 de Aminoácidos Neutros Grandes , Neoplasias da Próstata , Taxoides , Tirosina/análogos & derivados , Masculino , Humanos , Fosforilação , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Quinases Ciclina-Dependentes/metabolismo , Linhagem Celular TumoralRESUMO
L-type amino acid transporter 1 (LAT1) is specifically expressed in many malignancies, contributes to the transport of essential amino acids, such as leucine, and regulates the mammalian target of rapamycin (mTOR) signaling pathway. We investigated the expression profile and functional role of LAT1 in prostate cancer using JPH203, a specific inhibitor of LAT1. LAT1 was highly expressed in castration-resistant prostate cancer (CRPC) cells, including C4-2 and PC-3 cells, but its expression level was low in castration-sensitive LNCaP cells. JPH203 significantly inhibited [14C] leucine uptake in CRPC cells but had no effect in LNCaP cells. JPH203 inhibited the proliferation, migration, and invasion of CRPC cells but not of LNCaP cells. In C4-2 cells, Cluster of differentiation (CD) 24 was identified by RNA sequencing as a novel downstream target of JPH203. CD24 was downregulated in a JPH203 concentration-dependent manner and suppressed activation of the Wnt/ß-catenin signaling pathway. Furthermore, an in vivo study showed that JPH203 inhibited the proliferation of C4-2 cells in a castration environment. The results of this study indicate that JPH203 may exert its antitumor effect in CRPC cells via mTOR and CD24.
Assuntos
Antígeno CD24 , Movimento Celular , Proliferação de Células , Transportador 1 de Aminoácidos Neutros Grandes , Neoplasias de Próstata Resistentes à Castração , Masculino , Humanos , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Linhagem Celular Tumoral , Animais , Proliferação de Células/efeitos dos fármacos , Antígeno CD24/metabolismo , Camundongos , Movimento Celular/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Benzoxazóis/farmacologia , Leucina/farmacologia , Leucina/análogos & derivados , Camundongos Nus , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Tirosina/análogos & derivadosRESUMO
P-glycoprotein (P-gp) is an ATP-binding cassette transporter known for its roles in expelling xenobiotic compounds from cells and contributing to cellular drug resistance through multidrug efflux. This mechanism is particularly problematic in cancer cells, where it diminishes the therapeutic efficacy of anticancer drugs. P-gp inhibitors, such as elacridar, have been developed to circumvent the decrease in drug efficacy due to P-gp efflux. An earlier study reported the cryo-EM structure of human P-gp-Fab (MRK-16) complex bound by two elacridar molecules, at a resolution of 3.6 Å. In this study, we have obtained a higher resolution (2.5 Å) structure of the P-gp- Fab (UIC2) complex bound by three elacridar molecules. This finding, which exposes a larger space for compound-binding sites than previously acknowledged, has significant implications for the development of more selective inhibitors and enhances our understanding of the compound recognition mechanism of P-gp.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Acridinas , Tetra-Hidroisoquinolinas , Humanos , Acridinas/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Microscopia CrioeletrônicaRESUMO
The L-type amino acid transporter (LAT) family contains four members, LAT1~4, which are important amino acid transporters. They mainly transport specific amino acids through cell membranes, provide nutrients to cells, and are involved in a variety of metabolic pathways. They regulate the mTOR signaling pathway which has been found to be strongly linked to cancer in recent years. However, in the field of prostate cancer (PCa), the LAT family is still in the nascent stage of research, and the importance of LATs in the diagnosis and treatment of prostate cancer is still unknown. Therefore, this article aims to report the role of LATs in prostate cancer and their clinical significance and application. LATs promote the progression of prostate cancer by increasing amino acid uptake, activating the mammalian target of rapamycin (mTOR) pathway and downstream signals, mediating castration-resistance, promoting tumor angiogenesis, and enhancing chemotherapy resistance. The importance of LATs as diagnostic and therapeutic targets for prostate cancer was emphasized and the latest research results were introduced. In addition, we introduced selective LAT1 inhibitors, including JPH203 and OKY034, which showed excellent inhibitory effects on the proliferation of various tumor cells. This is the future direction of amino acid transporter targeting therapy drugs.
Assuntos
Neoplasias da Próstata , Masculino , Humanos , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/genética , Neoplasias da Próstata/terapia , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Aminoácidos/metabolismo , Transdução de Sinais , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/metabolismoRESUMO
Ingestion of amino acids is fundamental for cellular activity. Amino acids are important components for protein synthesis but are also crucial for intracellular metabolic reactions and signal transduction. Following activation, immune cells induce metabolic reprogramming to generate adequate energy and constitutive substances. Hence, the delivery of amino acids by transporters is necessary for the progression of metabolic rewiring. In this review, we discuss how amino acids and their transporters regulate immune cell functions, with emphasis on LAT1, a transporter of large neutral amino acids. Furthermore, we explore the possibility of targeting amino acid transporters to improve immune disorders and cancer immune therapies.
Assuntos
Expressão Gênica , Imunoterapia/métodos , Inflamação/genética , Inflamação/imunologia , Transportador 1 de Aminoácidos Neutros Grandes/fisiologia , Neoplasias/genética , Neoplasias/imunologia , Linfócitos T/imunologia , Humanos , Inflamação/terapia , Transportador 1 de Aminoácidos Neutros Grandes/genética , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Terapia de Alvo Molecular , Neoplasias/terapiaRESUMO
Currently, the emergence of drug resistance is an important issue in the treatment of hepatitis B virus (HBV). Recently, our collaborating group developed a novel long-acting anti-HBV drug, E-CFCP. However, until this study, the effects of E-CFCP in the kidney have remained unclarified. Using cell viability and uptake assays, we examined the effects of E-CFCP on the function of renal organic anion transporters (OATs). No cytotoxicity was shown related to the E-CFCP in the renal OATs in either assay. Thus, this study suggested that E-CFCP may be a novel, excellent candidate drug for the treatment of drug-resistant HBV.
Assuntos
Hepatite B , Transportadores de Ânions Orgânicos , Humanos , Hepatite B/tratamento farmacológico , Vírus da Hepatite B , Rim , Proteínas de Membrana Transportadoras , Antivirais/farmacologia , Antivirais/uso terapêutico , Farmacorresistência ViralRESUMO
Amino acid transporters are responsible for the uptake of amino acids, critical for cell proliferation. L-type amino acid transporters play a major role in the uptake of essential amino acids. L-type amino acid transporter 1 (LAT1) exerts its functional properties by forming a dimer with 4F2hc. Utilizing this cancer-specificity, research on diagnostic imaging and therapeutic agents for malignant tumors targeting LAT1 progresses in various fields. In hormone-sensitive prostate cancer, the up-regulation of L-type amino acid transporter 3 (LAT3) through the androgen receptor (AR) has been identified. On the other hand, in castration-resistant prostate cancer, the negative regulation of LAT1 through AR has been determined. Furthermore, 4F2hc: a binding partner of LAT1, was identified as the specific downstream target of Androgen Receptor Splice Variant 7: AR-V7. LAT1 has been suggested to contribute to acquiring castration resistance in prostate cancer, making LAT1 a completely different therapeutic target from anti-androgens and taxanes. Increased expression of LAT1 has also been found in renal and bladder cancers, suggesting a contribution to acquiring malignancy and progression. In Japan, clinical trials of LAT1 inhibitors for solid tumors are in progress, and clinical applications are now underway. This article will summarize the relationship between LAT1 and urological malignancies.
Assuntos
Transportador 1 de Aminoácidos Neutros Grandes , Neoplasias da Próstata , Neoplasias Urológicas , Humanos , Masculino , Sistemas de Transporte de Aminoácidos , Transportador 1 de Aminoácidos Neutros Grandes/genética , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Receptores Androgênicos/genética , Neoplasias Urológicas/tratamento farmacológico , Neoplasias Urológicas/genéticaRESUMO
Organ bath experiments are conventionally used to investigate the physiological actions and effects of hormones and drugs on organ responses. We developed an experimental method to reproduce insulin secretion from isolated rat pancreas preparations, to investigate substances that promote insulin secretion ex vivo. 1,5-anhydro-D-glucitol (1,5-AG) is found in foods, and exists in humans and rodents; however, whether 1,5-AG stimulates insulin secretion remains unclear. This study aimed to assess the effects of short-term 1,5-AG stimulation on insulin secretion in both ex vivo and in INS-1E (rat-derived) cells in vitro. Our results indicated that 1,5-AG had no potency to increase the proportion of insulin outflow both in ex vivo and in vitro experiments. Insulin outflow significantly increased upon stimulation with 10 µM glimepiride, a member of the sulfonylurea class of drugs, ex vivo. Glucose-stimulated insulin secretion was observed not only in INS-1E cells but also in rat pancreatic preparations. Our findings demonstrated that short-term exposure to 1,5-AG had no effect on insulin secretion in rats.
Assuntos
Insulina , Sorbitol , Animais , Desoxiglucose , Glucose/metabolismo , Insulina/metabolismo , Secreção de Insulina , Pâncreas/metabolismo , Ratos , Sorbitol/metabolismoRESUMO
A sub-lethal ischemic episode (preconditioning [PC]) protects neurons against a subsequent lethal ischemic injury. This phenomenon is known as ischemic tolerance. PC itself does not cause brain damage, but affects glial responses, especially astrocytes, and transforms them into an ischemia-resistant phenotype. P2X7 receptors (P2X7Rs) in astrocytes play essential roles in PC. Although P2X7Rs trigger inflammatory and toxic responses, PC-induced P2X7Rs in astrocytes function as a switch to protect the brain against ischemia. In this review, we focus on P2X7Rs and summarize recent developments on how astrocytes control P2X7Rs and what molecular mechanisms they use to induce ischemic tolerance.
Assuntos
Astrócitos , Isquemia Encefálica , Isquemia Encefálica/genética , Humanos , Isquemia , Neurônios , Receptores Purinérgicos P2X7/genéticaRESUMO
We previously showed that noninvasive mild ischemia (preconditioning; PC) induced ischemic tolerance by upregulation of P2X7 receptors in astrocytes via a hypoxia inducible factor-1α (HIF-1α)-dependent mechanism. The P2X7 receptor is known as a low-sensitivity P2 receptor that requires a high extracellular ATP (eATP) concentration for activation. PC increased the eATP level but was not sufficient to activate P2X7 receptors. Here, we show that astrocytes possess an elaborate mechanism for activation of P2X7 receptors, thus contributing to ischemic tolerance. Nicotinamide adenine dinucleotide (NAD+ ) was shown to increase the sensitivity of P2X7 receptors to eATP via ecto-ADP-ribosyltransferase 2 (ARTC2)-catalyzed ADP-ribosylation in peripheral immune cells. Although ARTC2-positive signals were mostly absent in the naïve brain, they were selectively increased in astrocytes by PC. The spatiotemporal pattern of PC-evoked ARTC2 was well associated with that of P2X7 receptors. In the in vitro experiments, NAD+ increased the sensitivity of P2X7 receptors to ATP, and at higher concentrations, NAD+ itself activated P2X7 receptors without eATP in cultured astrocytes. In the in vivo experiments using middle cerebral artery occlusion model mice, the PC-evoked increase in HIF-1α in astrocytes was abolished by the ARTC2 inhibitor S + 16a. S + 16a also abolished PC-evoked ischemic tolerance. Taken together, the results suggested that P2X7 receptors can be sensitized to ATP by NAD+ /ARTC2-catalyzed ADP-ribosylation, which allows astrocytes to drive P2X7 receptor-mediated ischemic tolerance even though PC only slightly increases the amount of eATP.
Assuntos
Astrócitos , Receptores Purinérgicos P2X7 , ADP Ribose Transferases/metabolismo , Trifosfato de Adenosina , Animais , Astrócitos/metabolismo , Células Cultivadas , Infarto da Artéria Cerebral Média , Camundongos , NAD/metabolismoRESUMO
L-type amino acid transporter 3 (LAT3, SLC43A1) is abundantly expressed in prostate cancer (PC) and is thought to play an essential role in PC progression through the cellular uptake of essential amino acids. Here, we analyzed the expression, function, and downstream target of LAT3 in PC. LAT3 was highly expressed in PC cells expressing androgen receptor (AR), and its expression was increased by dihydrotestosterone treatment and decreased by bicalutamide treatment. In chromatin immunoprecipitation sequencing of AR, binding of AR to the SLC43A1 region was increased by dihydrotestosterone stimulation. Knockdown of LAT3 inhibited cell proliferation, migration, and invasion, and the phosphorylation of p70S6K and 4EBP-1. Separase (ESPL1) was identified as a downstream target of LAT3 by RNA sequencing analysis. In addition, immunostaining of prostatectomy specimens was performed. In the multivariate analysis, high expression of LAT3 was an independent prognostic factor for recurrence-free survival (hazard ratio: 3.24; P = .0018). High LAT3 expression was correlated with the pathological T stage and a high International Society of Urological Pathology grade. In summary, our results suggest that LAT3 plays an important role in the progression of PC.
Assuntos
Sistema y+L de Transporte de Aminoácidos/metabolismo , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Separase/metabolismo , Transdução de Sinais/genética , Idoso , Sistemas de Transporte de Aminoácidos Básicos/genética , Movimento Celular/genética , Proliferação de Células/genética , Sobrevivência Celular/genética , Estudos de Coortes , Di-Hidrotestosterona/farmacologia , Progressão da Doença , Técnicas de Silenciamento de Genes , Humanos , Masculino , Pessoa de Meia-Idade , Células PC-3 , Prognóstico , Prostatectomia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Neoplasias da Próstata/cirurgia , Ligação Proteica/efeitos dos fármacos , Receptores Androgênicos/genética , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , TransfecçãoRESUMO
Islatravir (ISL; 4'-ethynyl-2-fluoro-2'-deoxyadenosine or EFdA) is a novel reverse transcriptase translocation inhibitor and has a unique structure and high antiviral activity against wild-type and multidrug resistant HIV strains. In this study, we investigated whether islatravir (ISL) can cause kidney damage compared to tenofovir disoproxil fumarate (TDF) and tenofovir (TFV). We also investigated interactions of these drugs with organic anion transporters (OATs). There is a large gap in ISL concentration between the pharmacological dose to proximal tubular cells and the clinical dose. ISL is unlikely to be taken up via OAT1 or OAT3; therefore, OAT1 and OAT3 may not be involved in the injury to tubular cells. Present data strongly suggests that ISL is not toxic to proximal tubules because blood levels of ISL are not high enough to cause kidney damage in the clinical setting.
Assuntos
Desoxiadenosinas/efeitos adversos , Desoxiadenosinas/metabolismo , Túbulos Renais Proximais/efeitos dos fármacos , Transportadores de Ânions Orgânicos/metabolismo , Inibidores da Transcriptase Reversa/efeitos adversos , Inibidores da Transcriptase Reversa/metabolismo , Injúria Renal Aguda/etiologia , Células Cultivadas , Desoxiadenosinas/sangue , Relação Dose-Resposta a Droga , HumanosRESUMO
JPH203 is a novel anti-cancer drug targeting L-type amino acid transporter 1 (LAT1), which plays a primary role in the uptake of essential amino acids in tumor cells. Although a co-incubation inhibitory effect of JPH203 has been shown in a conventional uptake assay, its preincubation inhibitory effects have remained undetermined. Therefore, we aimed to characterize the preincubation inhibitory effects of JPH203 on LAT1 function using leucine uptake assays in LAT1-positive human colon cancer HT-29 cells. Preincubation of the cells with JPH203 (0.3 µM for 120 min) decreased the activity level to 30% of that in dimethylsulfoxide-treated cells. Similarly, in time-dependency analysis, preincubation of HT-29 cells with 10 µM JPH203 for 30, 60, and 120 min decreased the leucine uptake activity (42%, 32%, and 28% of that in control cells, respectively). Furthermore, the IC50 value of the combination of preincubation and co-incubation effects was lower than that of co-incubation inhibition alone (34.2 ± 3.6 nM vs. 99.2 ± 11.0 nM). In conclusion, we revealed that JPH203 has the capability to inhibit LAT1 function through preincubation effects. Moreover, preincubation synergistically enhances the co-incubation inhibitory effects. These findings provide a novel insight into the anti-cancer effects of JPH203 in cancer therapy.
Assuntos
Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Antineoplásicos/farmacologia , Benzoxazóis/farmacologia , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Tirosina/análogos & derivados , Relação Dose-Resposta a Droga , Células HT29 , Humanos , Transportador 1 de Aminoácidos Neutros Grandes/fisiologia , Leucina/metabolismo , Fatores de Tempo , Tirosina/farmacologiaRESUMO
INTRODUCTION: The kidneys play a central role in eliminating metabolic waste products and drugs through transporter-mediated excretion along the proximal tubule. This task is mostly achieved through a variety of transporters from the solute carrier family 22 (SLC22) family of organic cation and anion transporters. Metabolic acidosis modulates metabolic and renal functions and also affects the clearance of metabolites and drugs from the body. We had previously shown that induction of metabolic acidosis in mice alters a large set of transcripts, among them also many transporters including transporters from the Slc22 family. OBJECTIVE: Here we further investigated the impact of acidosis on Slc22 family members. METHODS: Metabolic acidosis was induced for 2 or 7 days with NH4Cl, some animals also received the uricase inhibitor oxonic acid for comparison. Expression of transporters was studied by qPCR and immunoblotting. RESULTS: NH4Cl induced no significant changes in plasma or urine uric acid levels but caused downregulation of Slc22a1 (Oct1), Slc22a6 (Oat1), Slc22a19 (Oat5), and -Slc22a12 (Urat1) at mRNA level. In contrast, Slc22a4 mRNA (Octn1) was upregulated. On protein level, NH4Cl increased Octn1 (after 7 days) and Urat1 (after 2 days) abundance and decreased Oat1 (after 2 days) and Urat1 (after 7 days). Oxonic acid had no impact on protein abundance of any of the transporters tested. CONCLUSION: In summary, metabolic acidosis alters expression of several transporters involved in renal excretion of metabolic waste products and drugs. This may have implications for drug kinetics and clearance of waste metabolites.
Assuntos
Acidose/metabolismo , Proteínas da Membrana Plasmática de Transporte de Catecolaminas/biossíntese , Nefropatias/patologia , Acidose/sangue , Acidose/patologia , Acidose/urina , Animais , Proteínas da Membrana Plasmática de Transporte de Catecolaminas/genética , Proteínas da Membrana Plasmática de Transporte de Catecolaminas/metabolismo , Nefropatias/sangue , Nefropatias/metabolismo , Nefropatias/urina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator 1 de Transcrição de Octâmero/biossíntese , Fator 1 de Transcrição de Octâmero/genética , Fator 1 de Transcrição de Octâmero/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ácido Úrico/sangue , Ácido Úrico/urinaRESUMO
In light of the recent pandemic, favipiravir (Avigan®), a purine nucleic acid analog and antiviral agent approved for use in influenza in Japan, is being studied for the treatment of coronavirus disease 2019 (COVID-19). Increase in blood uric acid level is a frequent side effect of favipiravir. Here, we discussed the mechanism of blood uric acid elevation during favipiravir treatment. Favipiravir is metabolized to an inactive metabolite M1 by aldehyde oxidase and xanthine oxidase, and excreted into urine. In the kidney, uric acid handling is regulated by the balance of reabsorption and tubular secretion in the proximal tubules. Favipiravir and M1 act as moderate inhibitors of organic anion transporter 1 and 3 (OAT1 and OAT3), which are involved in uric acid excretion in the kidney. In addition, M1 enhances uric acid reuptake via urate transporter 1 (URAT1) in the renal proximal tubules. Thus, favipiravir is thought to decrease uric acid excretion into urine, resulting in elevation of uric acid levels in blood. Elevated uric acid levels were returned to normal after discontinuation of favipiravir, and favipiravir is not used for long periods of time for the treatment of viral infection. Thus, the effect on blood uric acid levels was subclinical in most studies. Nevertheless, the adverse effect of favipiravir might be clinically important in patients with a history of gout, hyperuricemia, kidney function impairment (in which blood concentration of M1 increases), and where there is concomitant use of other drugs affecting blood uric acid elevation.
Assuntos
Amidas/efeitos adversos , Antivirais/efeitos adversos , Infecções por Coronavirus/tratamento farmacológico , Hiperuricemia/induzido quimicamente , Pneumonia Viral/tratamento farmacológico , Pirazinas/efeitos adversos , Ácido Úrico/sangue , Aldeído Oxidase/metabolismo , Amidas/farmacocinética , Amidas/urina , Antivirais/farmacocinética , Biotransformação , COVID-19 , Interações Medicamentosas , Humanos , Hiperuricemia/fisiopatologia , Rim/metabolismo , Nefropatias/metabolismo , Estrutura Molecular , Proteína 1 Transportadora de Ânions Orgânicos/antagonistas & inibidores , Transportadores de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/antagonistas & inibidores , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Pandemias , Pirazinas/farmacocinética , Pirazinas/urina , Xantina Oxidase/metabolismoRESUMO
Brain microvascular endothelial cells (BMEC), together with astrocytes and pericytes, form the blood-brain barrier (BBB) that strictly restricts drug penetration into the brain. Therefore, in central nervous system drug development, the establishment of an in vitro human BBB model for use in studies estimating the in vivo human BBB permeability of drug candidates has long been awaited. The current study developed and characterized a human immortalized cell-based BBB triculture model, termed the "hiBBB" model. To set up the hiBBB model, human immortalized BMEC (HBMEC/ci18) were cocultured with human immortalized astrocytes (HASTR/ci35) and brain pericytes (HBPC/ci37) in a transwell system. The trans-endothelial electrical resistance of the hiBBB model was 134.4 ± 5.5 (Ω × cm2), and the efflux ratios of rhodamine123 and dantrolene were 1.72 ± 0.11 and 1.72 ± 0.45, respectively, suggesting that the hiBBB model possesses essential cellular junction and efflux transporter functions. In BBB permeability assays, the mean value of the permeability coefficients (Pe) of BBB permeable compounds (propranolol, pyrilamine, memantine, and diphenhydramine) was 960 × 10-6 cm/s, which was clearly distinguishable from that of BBB nonpermeable compounds (sodium fluorescein and Lucifer yellow, 18 × 10-6 cm/s). Collectively, this study successfully developed the hiBBB model, which exhibits essential BBB functionality. Taking into consideration the high availability of the immortalized cells used in the hiBBB model, our results are expected to become an initial step toward the establishment of a useful human BBB model to investigate drug penetration into the human brain.
Assuntos
Transporte Biológico/fisiologia , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Preparações Farmacêuticas/metabolismo , Astrócitos/metabolismo , Linhagem Celular , Técnicas de Cocultura/métodos , Células Endoteliais/metabolismo , Humanos , Pericitos/metabolismo , PermeabilidadeRESUMO
Ion exchange in the renal tubules is fundamental to the maintenance of physiological ion levels. Claudin-16 (CLDN16) regulates the paracellular reabsorption of Mg2+ in the thick ascending limb of Henle's loop in the kidney, with dephosphorylation of CLDN16 increasing its intracellular distribution and decreasing paracellular Mg2+ permeability. CLDN16 is located in the tight junctions, but the mechanism regulating its localization is unclear. Using yeast two-hybrid systems, we found that CLDN16 binds to PDZRN3, a protein containing both RING-finger and PDZ domains. We also observed that the carboxyl terminus of the cytoplasmic CLDN16 region was required for PDZRN3 binding. PZDRN3 was mainly distributed in the cytosol of rat kidney cells and upon cell treatment with the protein kinase A inhibitor H-89, colocalized with CLDN16. H-89 also increased mono-ubiquitination and the association of CLDN16 with PDZRN3. Mono-ubiquitination levels of a K275A mutant were lower, and its association with PDZRN3 was reduced compared with wild-type (WT) CLDN16 and a K261A mutant, indicating that Lys-275 is the major ubiquitination site. An S217A mutant, a dephosphorylated form of CLDN16, localized to the cytosol along with PDZRN3 and the endosomal marker Rab7. PDZRN3 siRNA increased cell-surface localization of WT CLDN16 in H-89-treated cells or containing the S217A mutant and also suppressed CLDN16 endocytosis. Of note, H-89 decreased paracellular Mg2+ flux in WT CLDN16 cells, and PDZRN3 siRNA increased Mg2+ flux in the H-89-treated WT CLDN16 and S217A mutant cells. These results suggest that PDZRN3 mediates endocytosis of dephosphorylated CLDN16 and represents an important component of the CLDN16-trafficking machinery in the kidney.
Assuntos
Claudinas/metabolismo , Endocitose , Túbulos Renais/metabolismo , Processamento de Proteína Pós-Traducional , Junções Íntimas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Substituição de Aminoácidos , Animais , Proteínas de Transporte/metabolismo , Claudinas/química , Claudinas/genética , Cães , Endocitose/efeitos dos fármacos , Humanos , Túbulos Renais/citologia , Túbulos Renais/efeitos dos fármacos , Lisina/metabolismo , Células Madin Darby de Rim Canino , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Fosforilação/efeitos dos fármacos , Mutação Puntual , Domínios e Motivos de Interação entre Proteínas , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Interferência de RNA , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/enzimologia , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/efeitos dos fármacosRESUMO
The sodium-glucose cotransporter SGLT2 inhibitor empagliflozin (plasma protein binding ~88%) may reach its target in the brush border of the early proximal tubule by glomerular filtration and tubular secretion. Here we determined whether empagliflozin is secreted by renal tubules in mice and whether genetic knockout of the basolateral organic anion transporter 3 ( Oat3-/-) affects its tubular secretion or glucosuric effect. Renal clearance studies in wild-type (WT) mice showed that tubular secretion accounted for 50-70% of empagliflozin urinary excretion. Immunostaining indicated that SGLT2 and OAT3 localization partially overlapped in proximal tubule S1 and S2 segments. Glucosuria in metabolic cage studies was reduced in Oat3-/- vs. WT mice for acute empagliflozin doses of 1, 3, and 10 mg/kg, whereas 30 mg/kg induced similar maximal glucosuria in both genotypes. Chronic application of empagliflozin (~25 mg·kg-1 ·day-1) in Oat3-/- mice was associated with lower urinary glucose-to-creatinine ratios despite maintaining slightly higher blood glucose levels than WT. On a whole kidney level, renal secretion of empagliflozin was largely unchanged in Oat3-/- mice. However, the absence of OAT3 attenuated the influence of empagliflozin on fractional glucose excretion; higher levels of plasma or filtered empagliflozin were needed to induce similar increases in fractional renal glucose excretion. We conclude that empagliflozin is excreted into the urine to similar extent by glomerular filtration and tubular secretion. The latter can occur largely independent of OAT3. However, OAT3 increases the glucosuric effect of empagliflozin, which may relate to the partial overlap of its localization with SGLT2 and thus OAT3-mediated tubular secretion of empagliflozin in the early proximal tubule.
Assuntos
Compostos Benzidrílicos/farmacologia , Glicemia/efeitos dos fármacos , Glucosídeos/farmacologia , Glicosúria/metabolismo , Túbulos Renais Proximais/efeitos dos fármacos , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Eliminação Renal , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Transportador 2 de Glucose-Sódio/efeitos dos fármacos , Animais , Compostos Benzidrílicos/farmacocinética , Compostos Benzidrílicos/urina , Glicemia/metabolismo , Taxa de Filtração Glomerular , Glucosídeos/farmacocinética , Glucosídeos/urina , Glicosúria/genética , Glicosúria/prevenção & controle , Túbulos Renais Proximais/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 1 Transportadora de Ânions Orgânicos/genética , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/deficiência , Transportadores de Ânions Orgânicos Sódio-Independentes/genética , Transportador 2 de Glucose-Sódio/metabolismo , Inibidores do Transportador 2 de Sódio-Glicose/farmacocinética , Inibidores do Transportador 2 de Sódio-Glicose/urinaRESUMO
An integrated analysis was performed with data from 4 phase 2 and phase 3 studies of tofogliflozin in which patients with type 2 diabetes mellitus received the sodium-glucose cotransporter 2 inhibitor tofogliflozin for up to 24 weeks. Sex differences, baseline haemoglobin A1c (HbA1c) and serum uric acid (UA) levels, and log10 -transformed urinary N-acetyl-ß-D-glucosaminidase ratio were significantly correlated with the reduction in serum UA levels at both 4 and 24 weeks in multivariate analysis (respectively, P < .0001). The decrease in HbA1c levels was greatest in the group with the highest baseline HbA1c level (quartile 4; HbA1c > 8.6%) and lowest in the group with the lowest baseline HbA1c level (quartile 1; HbA1c ≤ 7.4%). The decrease in serum UA levels was greatest in the quartile 1 group and lowest in the quartile 4 group. In most groups, the maximum decrease in serum UA levels was seen in the first 4 weeks, while the maximum decrease in HbA1c was seen at week 24. Thus, serum UA levels were significantly decreased in patients with moderate HbA1c levels.
Assuntos
Compostos Benzidrílicos/uso terapêutico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Glucosídeos/uso terapêutico , Hemoglobinas Glicadas/efeitos dos fármacos , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico , Ácido Úrico/sangue , Adulto , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Ensaios Clínicos Fase II como Assunto/estatística & dados numéricos , Ensaios Clínicos Fase III como Assunto/estatística & dados numéricos , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/epidemiologia , Regulação para Baixo/efeitos dos fármacos , Feminino , Hemoglobinas Glicadas/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Resultado do TratamentoRESUMO
p-Boronophenylalanine (l-BPA) is applied in clinical settings as a boron carrier for boron neutron capture therapy (BNCT) to cure malignant melanomas. Structural modification or derivatization of l-BPA, however, to improve its uptake efficiency into tumor cells has scarcely been investigated. We successfully synthesized (S)-2-amino-3-(4-boronophenyl)-2-methylpropanoic acid in enantioenriched form as a novel candidate molecule for BNCT. Key steps to enhance the efficiency of this synthesis were enantioselective alkylation of N-protected alanine tert-butyl ester with a Maruoka catalyst and Miyaura borylation reaction to install the boron functionality.