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Alzheimer's disease (AD) is thought to be a series of neuroinflammatory diseases caused by abnormal deposits of amyloid-ß (Aß) and tau protein in the brain as part of its etiology. We focused on Aß aggregation and M1 and M2 microglial polarity in microglia to search for novel therapeutic agents. It has been reported that the inhibition of choline uptake via choline transporter-like protein 1 (CTL1) in microglia preferentially induces M2 microglial polarity. However, the role of the choline transport system on the regulation of microglial M1/M2 polarity in AD is not fully understood. Licochalcones (Licos) A-E, flavonoids extracted from licorice, have been reported to have immunological anti-inflammatory effects, and Lico A inhibits Aß aggregation. In this study, we compared the efficacy of five Licos, from Lico A to E, at inhibiting Aß1-42 aggregation. Among the five Licos, Lico E was selected to investigate the relationship between the inhibition of choline uptake and microglial M1/M2 polarization using the immortalized mouse microglial cell line SIM-A9. We newly found that Lico E inhibited choline uptake and Aß1-42 aggregation in SIM-A9 cells in a concentration-dependent manner, suggesting that the inhibitory effect of Lico E on choline uptake is mediated by CTL1. The mRNA expression of tumor necrosis factor (TNF-α), a marker of M1 microglia, was increased by Aß1-42, and its effect was inhibited by choline deprivation and Lico E in a concentration-dependent manner. In contrast, the mRNA expression of arginase-1 (Arg-1), a marker of M2 microglia, was increased by IL-4, and its effect was enhanced by choline deprivation and Lico E. We found that Lico E has an inhibitory effect on Aß aggregation and promotes polarity from M1 to M2 microglia via inhibition of the CTL1 function in microglia. Thus, Lico E may become a leading compound for a novel treatment of AD.
Asunto(s)
Enfermedad de Alzheimer , Microglía , Animales , Ratones , Microglía/metabolismo , Péptidos beta-Amiloides/metabolismo , Enfermedad de Alzheimer/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Colina/metabolismo , ARN Mensajero/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismoRESUMEN
BACKGROUND: Microglia are key cells of the immune system in the central nervous system and are suggested to be deeply involved in the development of neurodegenerative diseases. It is well known that microglia have functional plasticity, with an inflammatory M1 phenotype and an anti-inflammatory M2 phenotype. Inhibition of choline transport in macrophages has been reported to suppress the secretion of inflammatory cytokines. However, the role of the choline transport system in regulating microglial M1/M2 polarization has not been fully elucidated to date. In this study, we investigated the mechanism of choline uptake in microglia, and its association with microglial M1/M2 polarization. METHODS: The immortalized mouse microglial cell line SIM-A9 was used for [3H]choline uptake and expression analysis of choline transporters. The association between the choline uptake system and the M1/M2 polarization of microglia was also analyzed. RESULTS: Choline transporter-like protein (CTL) 1 and CTL2 were highly expressed in SIM-A9 cells, and CTL1 and CTL2 were localized in the plasma membrane and mitochondria, respectively. Functional analysis of choline uptake demonstrated the existence of Na+-independent, pH-dependent, and intermediate-affinity choline transport systems. Choline uptake was concentration-dependently inhibited by hemicholinium-3 (HC-3), an inhibitor of choline uptake, and increased by lipopolysaccharide (LPS) and interleukin-4 (IL-4). Expression of the mRNA of M1 microglia markers IL-1ß and IL-6 was increased by LPS, and their effects were suppressed by choline deprivation and HC-3. In contrast, mRNA expression of the M2 microglial marker arginase-1 (Arg-1) was increased by IL-4, and the effect was enhanced by choline deprivation and HC-3. CONCLUSIONS: Our results suggest that inhibition of CTL1-mediated choline uptake in microglia preferentially induces M2 microglia polarization, which is a potential therapeutic approach for inflammatory brain diseases.
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Lipopolisacáridos , Microglía , Animales , Polaridad Celular , Colina/metabolismo , Interleucina-4/metabolismo , Lipopolisacáridos/metabolismo , Lipopolisacáridos/farmacología , Proteínas de Transporte de Membrana , Ratones , Microglía/metabolismo , Proteínas de Transporte de Catión Orgánico/genética , Proteínas de Transporte de Catión Orgánico/metabolismo , ARN Mensajero/metabolismoRESUMEN
BACKGROUND: Despite recent advances in the early detection and treatment of TSCC patients, recurrence rates and survival rates have not improved. The high frequency of lymph node metastasis is one of the causes, and the drug development of new therapeutic mechanisms such as metastasis control is desired. Choline transporter-like protein 1 (CTL1) has attracted attention as a target molecule in cancer therapy. In this study, we examined the antitumor effects of Amb544925, a plant-derived CTL1 inhibitor. METHODS: The TSCC cell line HSC-3 was used to measure [3H]choline uptake, cell survival, caspase activity, and cell migration. Xenograft model mice were prepared to verify the antitumor effect of Amb544925. RESULTS: Amb544925 inhibited cell viability and increased caspase-3/7 activity at concentrations that inhibited choline uptake. Amb544925 and ceramide increased SMPD4 expression and suppressed surivivin expression. Furthermore, Amb544925 and ceramide inhibited the migration of HSC-3 cells. In the xenograft model mice, Amb544925 suppressed tumor growth and CTL1 mRNA expression. CONCLUSIONS: The plant-derived CTL1 inhibitor Amb544925 is a lead compound of a new anticancer agent exhibiting antitumor effects and inhibition of cell migration through the ceramide/survivin pathway.
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Choline and choline metabolites are essential for all cellular functions. They have also been reported to be crucial for neural development. In this work, we studied the functional characteristics of the choline uptake system in human neural stem cells (hNSCs). Additionally, we investigated the effect of extracellular choline uptake inhibition on the cellular activities in hNSCs. We found that the mRNAs and proteins of choline transporter-like protein 1 (CTL1) and CTL2 were expressed at high levels. Immunostaining showed that CTL1 and CTL2 were localized in the cell membrane and partly in the mitochondria, respectively. The uptake of extracellular choline was saturable and performed by a single uptake mechanism, which was Na+-independent and pH-dependent. We conclude that CTL1 is responsible for extracellular choline uptake, and CTL2 may uptake choline in the mitochondria and be involved in DNA methylation via choline oxidation. Extracellular choline uptake inhibition caused intracellular choline deficiency in hNSCs, which suppressed cell proliferation, cell viability, and neurite outgrowth. Our findings contribute to the understanding of the role of choline in neural development as well as the pathogenesis of various neurological diseases caused by choline deficiency or choline uptake impairment.
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Proteínas de Transporte de Membrana/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Proyección Neuronal , Línea Celular , Proliferación Celular , Supervivencia Celular , Colina/metabolismo , Espacio Extracelular/metabolismo , Regulación de la Expresión Génica , Humanos , Proteínas de Transporte de Membrana/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Fracciones Subcelulares/metabolismo , Tritio/metabolismoRESUMEN
Choline, an organic cation, is one of the biofactors that play an important role in the structure and the function of biological membranes, and it is essential for the synthesis of phospholipids. Choline positron emission tomography-computed tomography (PET/CT) provides useful information for the imaging diagnosis of cancers, and increased choline accumulation has been identified in a variety of tumors. However, the molecular mechanisms of choline uptake and choline transporters in pancreatic cancer have not been elucidated. Here, we examined molecular and functional analyses of choline transporters in human pancreatic-cancer cell line MIA PaCa-2 and the elucidation of the action mechanism behind the antitumor effect of novel choline-transporter-like protein 1 (CTL1) inhibitors, Amb4269951 and its derivative Amb4269675. CTL1 and CTL2 mRNAs were highly expressed in MIA PaCa-2 cells, and CTL1 and CTL2 proteins were localized in the plasma membrane and the intracellular compartments, respectively. Choline uptake was characterized by Na+-independence, a single-uptake mechanism, and inhibition by choline-uptake inhibitor HC-3, similar to the function of CTL1. These results suggest that the uptake of extracellular choline in MIA PaCa-2 cells is mediated by CTL1. Choline deficiency and HC-3 treatment inhibited cell viability and increased caspase 3/7 activity, suggesting that the inhibition of CTL1 function, which is responsible for choline transport, leads to apoptosis-induced cell death. Both Amb4269951 and Amb4269675 inhibited choline uptake and cell viability and increased caspase-3/7 activity. Ceramide, which is increased by inhibiting choline uptake, also inhibited cell survival and increased caspase-3/7 activity. Lastly, both Amb4269951 and Amb4269675 significantly inhibited tumor growth in a mouse-xenograft model without any adverse effects such as weight loss. CTL1 is a target molecule for the treatment of pancreatic cancer, and its inhibitors Amb4269951 and Amb4269675 are novel lead compounds.
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Antígenos CD/metabolismo , Antineoplásicos/farmacología , Hemicolinio 3/farmacología , Isoquinolinas/farmacología , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismo , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Animales , Antígenos CD/genética , Apoptosis , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Ceramidas/farmacología , Colina/metabolismo , Hemicolinio 3/química , Humanos , Isoquinolinas/química , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana/genética , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas de Transporte de Catión Orgánico/antagonistas & inhibidores , Proteínas de Transporte de Catión Orgánico/genética , Neoplasias Pancreáticas/enzimología , Neoplasias Pancreáticas/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Choline transporter-like protein 1 (CTL1) is highly expressed in glioma cells, and inhibition of CTL1 function induces apoptotic cell death. Therefore, CTL1 is a potential target molecule for glioma therapy. Here, we investigated the therapeutic mechanism underlying the antitumor effects of Amb4269951, a recently discovered novel CTL1 inhibitor, in the human glioma cell line U251MG, and evaluated its in vivo effects in a mouse xenograft model. Amb4269951 inhibited choline uptake and cell viability and increased caspase-3/7 activity. CTL1-mediated choline uptake is associated with cell viability, and the functional inhibition of CTL1 by Amb4269951 may promote apoptotic cell death via ceramide-induced suppression of the expression of survivin, an apoptotic inhibitory factor. Finally, Amb4269951 demonstrated an antitumor effect in a mice xenograft model by significantly inhibiting tumor growth without any weight loss. Amb4269951 is the lead compound in the treatment of glioma and exhibits a novel therapeutic mechanism. These results may lead to the development of novel anticancer drugs targeting the choline transporter CTL1, which has a different mechanism of action than conventional anticancer drugs against gliomas.
RESUMEN
Choline is used to synthesize phospholipids and a lack of choline induces a number of liverrelated diseases, including nonalcoholic steatohepatitis. The current study characterized the choline uptake system, at molecular and functional levels, in the immortalized human hepatic cell line, Fa2N4, to identify the specific choline transporter involved in choline uptake. The present study also assesed whether choline deficiency or the inhibited choline uptake affected cell viability and apoptosis. Reverse transcriptionquantitative polymerase chain reaction (PCR) revealed choline transporterlike protein 1 (CTL1) and CTL2 mRNA and protein expression in Fa2N4 cells. [Methyl3H]choline studies revealed choline uptake was saturable and mediated by a single transport system that functioned in a Na+independent but pHdependent manner, which was similar to CTL1. Hemicholinium3 (HC3), which is a choline uptake inhibitor, and choline deficiency inhibited cell viability, increased caspase3 and 7 activities, and increased fluorescein isothiocyanateAnnexin V immunofluorescent staining indicated apoptosis. Immunofluorescent staining also revealed CTL1 and CTL2 localized in plasma and mitochondrial membranes, respectively. [Methyl3H]choline uptake was enhanced by a protein kinase C (PKC) activator, phorbol12myristate 13acetate (PMA). Immunofluorescence staining and western blot analysis demonstrated increased CTL1 expression on the cell membrane following PMA treatment. The results of current study indicated that extracellular choline is primarily transported via CTL1, relying on a direct H+ gradient that functions as a driving force in Fa2N4 cells. Furthermore, it was hypothesized that CTL1 and the choline uptake system are strongly associated with cell survival, and that the choline uptake system is modulated by PKC signaling via increased CTL1 expression on the cell surface. These findings provide further insights into the pathogenesis of liver disease involving choline metabolism.
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Antígenos CD/metabolismo , Membrana Celular/metabolismo , Hígado/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismo , Proteína Quinasa C/metabolismo , Antígenos CD/genética , Apoptosis , Transporte Biológico , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Línea Celular Transformada , Supervivencia Celular/genética , Colina/metabolismo , Humanos , Proteínas de Transporte de Catión Orgánico/genética , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Prostate cancer is one of the most common cancers in men. Choline PET or PET/CT has been used to visualize prostate cancer, and high levels of choline accumulation have been observed in tumors. However, the uptake system for choline and the functional expression of choline transporters in prostate cancer are not completely understood. In this study, the molecular and functional aspects of choline uptake were investigated in the LNCaP prostate cancer cell line along with the correlations between choline uptake and cell viability in drug-treated cells. Choline transporter-like protein 1 (CTL1) and CTL2 mRNA were highly expressed in LNCaP cells. CTL1 and CTL2 were located in the plasma membrane and mitochondria, respectively. [3H]Choline uptake was mediated by a single Na+-independent, intermediate-affinity transport system in the LNCaP cells. The anticancer drugs, flutamide and bicalutamide, inhibited cell viability and [3H]choline uptake in a concentration-dependent manner. The correlations between the effects of these drugs on cell viability and [3H]choline uptake were significant. Caspase-3/7 activity was significantly increased by both flutamide and bicalutamide. Furthermore, these drugs decreased CTL1 expression in the prostate cancer cell line. These results suggest that CTL1 is functionally expressed in prostate cancer cells and are also involved in abnormal proliferation. Identification of this CTL1-mediated choline transport system in prostate cancer cells provides a potential new therapeutic target for the treatment of this disease.
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In adipocytes, intracellular Ca2+ and Mg2+ modulates physiological functions, such as insulin action and the secretion of adipokines. TRPM7 is a Ca2+ /Mg2+ -permeable non-selective cation channel. TRPM7 mRNA is highly expressed in adipose tissue, however, its functional expression in adipocytes remains to be elucidated. In this study, we demonstrated for the first time that TRPM7 was functionally expressed in both freshly isolated white adipocytes and in 3T3-L1 adipocytes differentiated from a 3T3-L1 pre-adipocyte cell line by whole-cell patch-clamp recordings. Consistent with known properties of TRPM7 current, the current in adipocytes was activated by the elimination of extracellular divalent cations and the reduction of intracellular free Mg2+ concentrations, and was inhibited by the TRPM7 inhibitors, 2-aminoethyl diphenylborinate (2-APB), hydrogen peroxide (H2 O2 ), N-methyl maleimide (NMM), NS8593, and 2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol (FTY720). Treatment with small-interfering (si) RNA targeting TRPM7 resulted in a reduction in the current to 23 ± 7% of nontargeting siRNA-treated adipocytes. Moreover a TRPM7 activator, naltriben, increased the TRPM7-like current and [Ca2+ ]i in 3T3-L1 adipocytes but not in TRPM7-knockdown adipocytes. These findings indicate that TRPM7 is functionally expressed, and plays a role as a Ca2+ influx pathway in adipocytes.
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Adipocitos/metabolismo , Calcio/metabolismo , Canales Catiónicos TRPM/metabolismo , Células 3T3-L1/metabolismo , Animales , Ratones , Técnicas de Placa-Clamp , Canales Catiónicos TRPM/genéticaRESUMEN
Cholinergic neurons in the central nervous system play a vital role in higher brain functions, such as learning and memory. Choline is essential for the synthesis of the neurotransmitter acetylcholine by cholinergic neurons. The synthesis and metabolism of acetylcholine are important mechanisms for regulating neuronal activity. Choline is a positively charged quaternary ammonium compound that requires transporters to pass through the plasma membrane. Currently, there are three groups of choline transporters with different characteristics, such as affinity for choline, tissue distribution, and sodium dependence. They include (I) polyspecific organic cation transporters (OCT1-3: SLC22A1-3) with a low affinity for choline, (II) high-affinity choline transporter 1 (CHT1: SLC5A7), and (III) choline transporter-like proteins (CTL1-5: SLC44A1-5). Brain microvascular endothelial cells, which comprise part of the blood-brain barrier, take up extracellular choline via intermediate-affinity choline transporter-like protein 1 (CTL1) and low-affinity CTL2 transporters. CTL2 is responsible for excreting a high concentration of choline taken up by the brain microvascular endothelial cells on the brain side of the blood-brain barrier. CTL2 is also highly expressed in mitochondria and may be involved in the oxidative pathway of choline metabolism. Therefore, CTL1- and CTL2-mediated choline transport to the brain through the blood-brain barrier plays an essential role in various functions of the central nervous system by acting as the rate-limiting step of cholinergic neuronal activity.
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Antígenos CD/fisiología , Barrera Hematoencefálica/metabolismo , Sistema Nervioso Central/metabolismo , Glicoproteínas de Membrana/fisiología , Proteínas de Transporte de Membrana/fisiología , Proteínas de Transporte de Catión Orgánico/fisiología , Animales , Encéfalo/citología , Membrana Celular/metabolismo , Colina/metabolismo , Células Endoteliales/metabolismo , Humanos , Proteínas de Transporte de Catión Orgánico/metabolismoRESUMEN
In this study, we examined the molecular and functional characterization of choline uptake in the human esophageal cancer cells. In addition, we examined the influence of various drugs on the transport of [3H]choline, and explored the possible correlation between the inhibition of choline uptake and apoptotic cell death. We found that both choline transporter-like protein 1 (CTL1) and CTL2 mRNAs and proteins were highly expressed in esophageal cancer cell lines (KYSE series). CTL1 and CTL2 were located in the plasma membrane and mitochondria, respectively. Choline uptake was saturable and mediated by a single transport system, which is both Na+-independent and pH-dependent. Choline uptake and cell viability were inhibited by various cationic drugs. Furthermore, a correlation analysis of the potencies of 47 drugs for the inhibition of choline uptake and cell viability showed a strong correlation. Choline uptake inhibitors and choline deficiency each inhibited cell viability and increased caspase-3/7 activity. We conclude that extracellular choline is mainly transported via a CTL1. The functional inhibition of CTL1 by cationic drugs could promote apoptotic cell death. Furthermore, CTL2 may be involved in choline uptake in mitochondria, which is the rate-limiting step in S-adenosylmethionine (SAM) synthesis and DNA methylation. Identification of this CTL1- and CTL2-mediated choline transport system provides a potential new target for esophageal cancer therapy.
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OBJECTIVES: In this study, we examined the functional characteristics of choline uptake and sought to identify the transporters in rheumatoid arthritis synovial fibroblasts (RASFs). METHODS: The expression of choline transporters was evaluated by quantitative real-time PCR, western blotting, and immunocytochemistry. Time course, Na+-dependency, and kinetics of [3H]choline uptake were investigated. Effects of cationic drugs on the uptake of [3H]choline, cell viability, and caspase-3/7 activity were also examined. Finally, we investigated the influence of choline uptake inhibitor, hemicholinium-3 (HC-3), and choline deficiency on cell viability and caspase-3/7 activity. RESULTS: Choline transporter-like protein 1 (CTL1) and CTL2 mRNA and protein were highly expressed in RASFs and were localized to the plasma membrane. [3H]Choline uptake occurred via a Na+-independent and pH-dependent transport system. The cells have two different [3H]choline transport systems, high- and low-affinity. Various organic cations, HC-3 and choline deficiency inhibited both [3H]choline uptake and cell viability, and enhanced the caspase-3/7 activity. The functional inhibition of choline transporters could promote apoptotic cell death. In RASFs, [3H]choline uptake was significantly increased compared with that in OASFs without a change in gene expression. CONCLUSIONS: These results suggest that CTL1 (high-affinity) and CTL2 (low-affinity) are highly expressed in RASFs and choline may be transported by a choline/H+ antiport system. Identification of this CTL1- and CTL2-mediated choline transport system should provide a potential new target for RA therapy.
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Artritis Reumatoide/metabolismo , Fibroblastos/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Transporte Biológico , Supervivencia Celular , Células Cultivadas , Colina/metabolismo , Fibroblastos/efectos de los fármacos , Humanos , Proteínas de Transporte de Membrana/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Membrana Sinovial/citologíaRESUMEN
Autophagy, a self-digestive system for cytoplasmic components, is required to maintain the amino acid pool for cellular homeostasis. We previously reported that the macrolide antibiotics azithromycin (AZM) and clarithromycin (CAM) have an inhibitory effect on autophagy flux, and they potently enhance the cytocidal effect of various anticancer reagents in vitro. This suggests that macrolide antibiotics can be used as an adjuvant for cancer chemotherapy. Since cancer cells require a larger metabolic demand than normal cells because of their exuberant growth, upregulated autophagy in tumor cells has now become the target for cancer therapy. In the present study, we examined whether macrolides exhibit cytotoxic effect under an amino acid-starving condition in head and neck squamous cancer cell lines such as CAL 27 and Detroit 562 as models of solid tumors with an upregulated autophagy in the central region owing to hypovascularity. AZM and CAM induced cell death under the amino acid-depleted (AAD) culture condition in these cell lines along with CHOP upregulation, although they showed no cytotoxicity under the complete culture medium. CHOP knockdown by siRNA in the CAL 27 cells significantly suppressed macrolide-induced cell death under the AAD culture condition. CHOP-/- murine embryonic fibroblast (MEF) cell lines also attenuated AZM-induced cell death compared with CHOP+/+ MEF cell lines. Using a tet-off atg5 MEF cell line, knockout of atg5, an essential gene for autophagy, also induced cell death and CHOP in the AAD culture medium but not in the complete culture medium. This suggest that macrolide-induced cell death via CHOP induction is dependent on autophagy inhibition. The cytotoxicity of macrolide with CHOP induction was completely cancelled by the addition of amino acids in the culture medium, indicating that the cytotoxicity is due to the insufficient amino acid pool. These data suggest the possibility of using macrolides for "tumor-starving therapy".
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Antibacterianos/farmacología , Autofagia/efectos de los fármacos , Azitromicina/farmacología , Carcinoma de Células Escamosas/patología , Muerte Celular/efectos de los fármacos , Claritromicina/farmacología , Neoplasias de Cabeza y Cuello/patología , Macrólidos/farmacología , Animales , Línea Celular Tumoral , Humanos , RatonesRESUMEN
We examined the functional characteristics of choline uptake in human tongue carcinoma using the cell line HSC-3. Furthermore, we explored the possible correlation between the inhibition of choline uptake and apoptotic cell death. Both choline transporter-like protein 1 (CTL1) and CTL2 mRNAs and proteins were expressed, and were located in plasma membrane and mitochondria, respectively. Choline uptake was saturable and mediated by a single transport system, which is pH-dependent. Several cationic drugs inhibited cell viability and [(3)H]choline uptake. Choline uptake inhibitors and choline deficiency inhibited cell viability and increased caspase-3/7 activity. We conclude that extracellular choline is mainly transported via a CTL1 that relies on a directed H(+) gradient as a driving force. The functional inhibition of CTL1 by cationic drugs could promote apoptotic cell death. Furthermore, CTL2 may be the major site for the control of choline oxidation in mitochondria and hence for the supply of endogenous betaine and S-adenosyl methionine, which serves as a major methyl donor. Identification of this CTL1- and CTL2-mediated choline transport system provides a potential new target for tongue cancer therapy.
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Antígenos CD/genética , Glicoproteínas de Membrana/genética , Proteínas de Transporte de Membrana/genética , Neoplasias de la Lengua/metabolismo , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Colina/metabolismo , Humanos , ARN Mensajero/metabolismoRESUMEN
In this study, we examined the molecular and functional characterization of choline transporter in human brain microvascular endothelial cells (hBMECs). Choline uptake into hBMECs was a saturable process that was mediated by a Na(+)-independent, membrane potential and pH-dependent transport system. The cells have two different [(3)H]choline transport systems with Km values of 35.0 ± 4.9 µM and 54.1 ± 8.1 µM, respectively. Choline uptake was inhibited by choline, acetylcholine (ACh) and the choline analog hemicholinium-3 (HC-3). Various organic cations also interacted with the choline transport system. Choline transporter-like protein 1 (CTL1) and CTL2 mRNA were highly expressed, while mRNA for high-affinity choline transporter 1 (CHT1) and organic cation transporters (OCTs) were not expressed in hBMECs. CTL1 and CTL2 proteins were localized to brain microvascular endothelial cells in human brain cortical sections. Both CTL1 and CTL2 proteins were expressed on the plasma membrane and mitochondria. CTL1 and CTL2 proteins are mainly expressed in plasma membrane and mitochondria, respectively. We conclude that choline is mainly transported via an intermediate-affinity choline transport system, CTL1 and CTL2, in hBMECs. These transporters are responsible for the uptake of extracellular choline and organic cations. CTL2 participate in choline transport mainly in mitochondria, and may be the major site for the control of choline oxidation.
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Antígenos CD/metabolismo , Encéfalo/irrigación sanguínea , Endotelio Vascular/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Microvasos/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismo , Antígenos CD/genética , Encéfalo/citología , Encéfalo/metabolismo , Células Cultivadas , Colina/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Cinética , Glicoproteínas de Membrana/genética , Potenciales de la Membrana , Proteínas de Transporte de Membrana/genética , Microvasos/citología , Proteínas de Transporte de Catión Orgánico/genética , ARN Mensajero/genética , Sodio/metabolismo , Fracciones Subcelulares/metabolismoRESUMEN
The pathophysiology of sepsis-associated encephalopathy (SAE) is complex and remains incompletely elucidated. Dysregulated reactive oxygen species (ROS) production and mitochondrial-mediated necrotic-apoptotic pathway have been proposed as part of the pathogenesis. The present study aimed at analyzing the preventive effect of the free radical scavenger edaravone on sepsis-induced brain alterations. Sepsis was induced by cecal ligation and puncture (CLP) and the mice were divided into three groups-CLP vehicle (CLPV), CLP and edaravone (MCI-186, 3-methyl-1-phenyl-2-pyrazolin-5-one) (CLPE), and sham-operated (Sham). Mice in CLPV and CLPE were injected with saline or edaravone intraperitoneally at a dose of 10âmg/kg twice daily. The treatments were initiated 4 days prior to the surgical procedure. Mortality, histological changes, electron microscopy (EM), and expression of Bcl-2 family genes (Bcl-2 and Bax) were analyzed in selected brain regions. CLPE showed significant improvement in survival compared with CLPV 18âh postinduction of sepsis (Pâ<â0.05). At the same time point, pathohistological analysis also showed marked reduction of neuronal cell death in both parietal cortex and hippocampus in the CLPE (Pâ<â0.05). RT-PCR and immunoblotting directed at the Bcl-2 family revealed increased Bax mRNA levels in hippocampus at 12âh in CLPV as well as an increased Bax/Bcl-2 protein ratio, changes that were significantly suppressed in CLPE. In conclusion, our study suggests that sepsis induced by cecal ligation alters cerebral redox status and supports a proapoptotic phenotype. The free radical scavenger edavarone reduces mortality of septic mice and protects against sepsis-induced neuronal cell death.
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Antipirina/análogos & derivados , Encéfalo/patología , Depuradores de Radicales Libres/uso terapéutico , Oxidación-Reducción , Sepsis/fisiopatología , Animales , Antipirina/uso terapéutico , Apoptosis , Encéfalo/efectos de los fármacos , Ciego/cirugía , Muerte Celular , Edaravona , Ligadura , Metabolómica , Ratones , Microscopía Electrónica , Neuronas/patología , Oxígeno/química , Fenotipo , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Obesity is a major global public health problem, and understanding its pathogenesis is critical for identifying a cure. In this study, a gene knockout strategy was used in post-neonatal mice to delete synoviolin (Syvn)1/Hrd1/Der3, an ER-resident E3 ubiquitin ligase with known roles in homeostasis maintenance. Syvn1 deficiency resulted in weight loss and lower accumulation of white adipose tissue in otherwise wild-type animals as well as in genetically obese (ob/ob and db/db) and adipose tissue-specific knockout mice as compared to control animals. SYVN1 interacted with and ubiquitinated the thermogenic coactivator peroxisome proliferator-activated receptor coactivator (PGC)-1ß, and Syvn1 mutants showed upregulation of PGC-1ß target genes and increase in mitochondrion number, respiration, and basal energy expenditure in adipose tissue relative to control animals. Moreover, the selective SYVN1 inhibitor LS-102 abolished the negative regulation of PGC-1ß by SYVN1 and prevented weight gain in mice. Thus, SYVN1 is a novel post-translational regulator of PGC-1ß and a potential therapeutic target in obesity treatment.
Asunto(s)
Peso Corporal/genética , Mitocondrias/fisiología , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/fisiología , Células 3T3-L1 , Animales , Células Cultivadas , Regulación hacia Abajo , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Obesos , Obesidad/genética , Obesidad/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación/genéticaRESUMEN
The inhibitory effects of macrolide antibiotics including clarithromycin (CAM) on autophagy flux have been reported. Although a macrolide antibiotic exhibits no cytotoxicity, its combination with bortezomib (BZ), a proteasome inhibitor, for the simultaneous blocking of the ubiquitin (Ub)proteasome and autophagylysosome pathways leads to enhanced multiple myeloma (MM) cell apoptosis induction via stress overloading of the endoplasmic reticulum (ER). As misfolded protein cargo is recruited by histone deacetylase 6 (HDAC6) to dynein motors for aggresome transport, serving to sequester misfolded proteins, we further investigated the cellular effects of targeting proteolytic pathways and aggresome formation concomitantly in MM cells. Pronounced apoptosis was induced by the combination of vorinostat [suberoylanilide hydroxamic acid (SAHA); potently inhibits HDAC6] with CAM and BZ compared with each reagent or a 2reagent combination. CAM/BZ treatment induced vimentin positiveaggresome formation along with the accumulation of autolysosomes in the perinuclear region, whereas they were inhibited in the presence of SAHA. The SAHA/CAM/BZ combination treatment maximally upregulated genes related to ER stress including C/EBP homologous protein (CHOP). Similarly to MM cell lines, enhanced cytotoxicity with CHOP upregulation following SAHA/CAM/BZ treatment was shown by a wildtype murine embryonic fibroblast (MEF) cell line; however, a CHOPdeficient MEF cell line almost completely canceled this pronounced cytotoxicity. Knockdown of HDAC6 with siRNA exhibited further enhanced CAM/BZinduced cytotoxicity and CHOP induction along with the cancellation of aggresome formation. Targeting the integrated networks of aggresome, proteasome, and autophagy is suggested to induce efficient ER stressmediated apoptosis in MM cells.
Asunto(s)
Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Mieloma Múltiple/tratamiento farmacológico , Animales , Protocolos de Quimioterapia Combinada Antineoplásica , Ácidos Borónicos/administración & dosificación , Bortezomib , Línea Celular Tumoral , Claritromicina/administración & dosificación , Sinergismo Farmacológico , Retículo Endoplásmico/efectos de los fármacos , Histona Desacetilasa 6 , Histona Desacetilasas/efectos de los fármacos , Humanos , Ácidos Hidroxámicos/administración & dosificación , Ratones , Mieloma Múltiple/genética , Mieloma Múltiple/patología , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Pirazinas/administración & dosificación , VorinostatRESUMEN
Choline is essential for the synthesis of the major membrane phospholipid phosphatidylcholine (PC), the methyl donor betaine and the neurotransmitter acetylcholine (ACh). Elevated levels of choline and up-regulated choline kinase activity have been detected in various cancers. Thus, the intracellular accumulation of choline through choline transporters is the rate-limiting step in phospholipid metabolism and a prerequisite for cancer cell proliferation. Previous studies have demonstrated abnormalities in choline uptake and choline phospholipid metabolism in cancer cells using the imaging of cancer with positron emission tomography (PET) and magnetic resonance spectroscopy (MRS). The aberrant choline metabolism in cancer cells is strongly correlated with their malignant progression. Using quantitative real-time PCR, the mRNA expression of choline transporters was measured, and it was found that choline transporter-like proteins CTLs/SLC44 family are highly expressed in various cancer cell lines. Choline uptake through CTLs is associated with cell viability, and the functional inhibition of CTLs could promote apoptotic cell death. Furthermore, non-neuronal cholinergic systems that include CTLs-mediated choline transport are associated with cell proliferation and their inhibition promotes apoptotic cell death in colon cancer, small cell lung cancer and human leukemic T-cells. The identification of this new CTLs-mediated choline transport system provides a potential new target for cancer therapy.
Asunto(s)
Antineoplásicos/uso terapéutico , Moduladores del Transporte de Membrana/uso terapéutico , Modelos Biológicos , Proteínas de Neoplasias/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Proteínas de Transporte de Catión Orgánico/antagonistas & inhibidores , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Transporte Biológico/efectos de los fármacos , Colina/metabolismo , Humanos , Moduladores del Transporte de Membrana/farmacología , Terapia Molecular Dirigida , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Proteínas de Transporte de Catión Orgánico/genética , Proteínas de Transporte de Catión Orgánico/metabolismo , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMEN
Positron emission tomography (PET) and PET/computed tomography (PET-CT) studies with (11)C- or (18)F-labeled choline derivatives are used for PET imaging in glioblastoma patients. However, the nature of the choline transport system in glioblastoma is poorly understood. In this study, we performed a functional characterization of [methyl-(3)H]choline uptake and sought to identify the transporters that mediate choline uptake in the human glioblastoma cell lines A-172 and U-251MG. In addition, we examined the influence of anti-cancer drugs and central nervous system drugs on the transport of [methyl-(3)H]choline. High- and low-affinity choline transport systems were present in A-172 cells, U-251MG cells and astrocytes, and these were Na(+)-independent and pH-dependent. Cell viability in A-172 cells was not affected by choline deficiency. However, cell viability in U-251MG cells was significantly inhibited by choline deficiency. Both A-172 and U-251MG cells have two different choline transporters, choline transporter-like protein 1 (CTL1) and CTL2. In A-172 cells, CTL1 is predominantly expressed, whereas in U-251MG cells, CTL2 is predominantly expressed. Treatment with anti-cancer drugs such as cisplatin, etoposide and vincristine influenced [methyl-(3)H]choline uptake in U-251MG cells, but not A-172 cells. Central nervous system drugs such as imipramine, fluvoxamine, paroxetine, reboxetine, citalopram and donepezil did not affect cell viability or [methyl-(3)H]choline uptake. The data presented here suggest that CTL1 and CTL2 are functionally expressed in A-172 and U-251MG cells and are responsible for [methyl-(3)H]choline uptake that relies on a directed H(+) gradient as a driving force. Furthermore, while anti-cancer drugs altered [methyl-(3)H]choline uptake, central nervous system drugs did not affect [methyl-(3)H]choline uptake.