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Solute carrier 40A1 (SLC40A1) encodes ferroportin, which is the only known transmembrane protein that exports elemental iron from mammalian cells and is essential for iron homeostasis. Mutations in SLC40A1 are associated with iron-overload disorders. In addition to ferroportin diseases, SLC40A1 expression is downregulated in various cancer types. Despite the clinical significance of the SLC40A1 transporter, only a few studies have investigated genetic variants in SLC40A1. The present study was performed to identify genetic variations in the SLC40A1 promoter and functionally characterize each variant using in vitro assays. We investigated four haplotypes and five variants in the SLC40A1 promoter. We observed that haplotype 3 (H3) had significantly lower promoter activity than H1, whereas the activity of H4 was significantly higher than that of H1. Luciferase activity of H2 was comparable to that of H1. In addition, four variants of SLC40A1, c.-1355G>C, c.-662C>T, c.-98G>C, and c.-8C>G, showed significantly increased luciferase activity compared to the wild type (WT), whereas c.-750G>A showed significantly decreased luciferase activity compared to the WT. Three transcription factors, cAMP response element-binding protein-1 (CREB-1), chicken ovalbumin upstream promoter transcription factor 1, and hepatic leukemia factor (HLF), were predicted to bind to the promoter regions of SLC40A1 near c.-662C>T, c.-98G>C, and c.-8C>G, respectively. Among these, CREB-1 and HLF bound more strongly to the variant sequences than to the WT and functioned as activators of SLC40A1 transcription. Collectively, our findings indicate that the two SLC40A1 promoter haplotypes affect SLC40A1 transcription, which is regulated by CREB-1 and HLF.
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BACKGROUND: Micro RNA of Marsupenaeus japonicas has been known to promote apoptosis of tumor cells. However, the detailed mechanisms are not well understood. RESULTS: Using tomographic microscope, which can detect the internal structure of cells, we observed breast tumor cells following treatment of the miRNA. Intriguingly, we found that mitochondria migrate to an adjacent tumor cells through a tunneling nanotube. To recapitulate this process, we engineered a microfluidic device through which mitochondria were transferred. We show that this mitochondrial transfer process released endonuclease G (Endo G) into tumor cells, which we referred to herein as unsealed mitochondria. Importantly, Endo G depleted mitochondria alone did not have tumoricidal effects. Moreover, unsealed mitochondria had synergistic apoptotic effects with subtoxic dose of doxorubicin thereby mitigating cardiotoxicity. CONCLUSIONS: Together, we show that the mitochondrial transfer through microfluidics can provide potential novel strategies towards tumor cell death.
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Promyelocytic leukemia (PML) gene, through alternative splicing of its C-terminal region, generates several PML isoforms that interact with specific partners and perform distinct functions. The PML protein is a tumor suppressor that plays an important role by interacting with various proteins. Herein, we investigated the effect of the PML isoforms on oncostatin M (OSM)-induced signal transducer and activator of transcription-3 (STAT-3) transcriptional activity. PML influenced OSM-induced STAT-3 activity in a cell type-specific manner, which was dependent on the p53 status of the cells but regardless of PML isoform. Interestingly, overexpression of PML exerted opposite effects on OSM-induced STAT-3 activity in p53 wild-type and mutant cells. Specifically, overexpression of PML in the cell lines bearing wild-type p53 (NIH3T3 and U87-MG cells) decreased OSM-induced STAT-3 transcriptional activity, whereas overexpression of PML increased OSM-induced STAT-3 transcriptional activity in mutant p53-bearing cell lines (HEK293T and U251-MG cells). When wild-type p53 cells were co-transfected with PML-IV and R273H-p53 mutant, OSM-mediated STAT-3 transcriptional activity was significantly enhanced, compared to that of cells which were transfected with PML-IV alone; however, when cells bearing mutant p53 were co-transfected with PML-IV and wild-type p53, OSM-induced STAT-3 transcriptional activity was significantly decreased, compared to that of transfected cells with PML-IV alone. In conclusion, PML acts together with wild-type or mutant p53 and influences OSM-mediated STAT-3 activity in a negative or positive manner, resulting in the aberrant activation of STAT-3 in cancer cells bearing mutant p53 probably might occur through the interaction of mutant p53 with PML.
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BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a debilitating lung disease. To date, a large number of clinical studies have been conducted to investigate the association between genetic variations and COPD. However, little is known regarding the genetic susceptibility of Koreans to this disease. MER receptor tyrosine kinase (MERTK) plays important roles in the inhibition of inflammation and in the clearance of apoptotic cells. Here, we investigated the association between genetic variations in MERTK and the development of COPD in Koreans. METHODS: We conducted genetic analysis of MERTK using genomic DNA samples from 87 patients with COPD and 88 healthy controls and compared the frequency of each variation or haplotype between the patient and control groups. Subsequently, the effect of each variation was evaluated using in vitro assays. RESULTS: Ten variations were identified in this study, four of them for the first time. In addition, we found that the frequency of each variation or haplotype was comparable between the patient and control groups. However, we observed that the frequency for the wild-type haplotype was higher in the control group, compared to that in the group of patients with COPD, in the subgroup analysis of current smokers, although the difference was not statistically significant (P = 0.080). In in vitro assays, we observed that none of the variations affected the activity of the promoter or the expression of MERTK. CONCLUSION: Our findings indicate that the susceptibility to COPD is not related to the genetic variations or haplotypes of MERTK in Koreans.
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Povo Asiático/genética , Doença Pulmonar Obstrutiva Crônica/patologia , c-Mer Tirosina Quinase/genética , Idoso , Estudos de Casos e Controles , Feminino , Volume Expiratório Forçado , Predisposição Genética para Doença , Variação Genética , Genótipo , Células HCT116 , Haplótipos , Humanos , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , Doença Pulmonar Obstrutiva Crônica/genética , República da Coreia , Fumar , c-Mer Tirosina Quinase/metabolismoRESUMO
Crohn's disease (CD) is a chronic inflammatory bowel disease with multifactorial causes including environmental and genetic factors. Several studies have demonstrated that the organic cation/carnitine transporter 1 (OCTN1) non-synonymous variant L503F is associated with susceptibility to CD. However, it was reported that L503F is absent in Asian populations. Previously, we identified and functionally characterized genetic variants of the OCTN1 promoter region in Koreans. In that study, four variants demonstrated significant changes in promoter activity. In the present study, we determined whether four functional variants of the OCTN1 promoter play a role in the susceptibility to or clinical course of CD in Koreans. To examine it, the frequencies of the four variants of the OCTN1 promoter were determined by genotyping using DNA samples from 194 patients with CD and 287 healthy controls. Then, associations between genetic variants and the susceptibility to CD or clinical course of CD were evaluated. We found that susceptibility to CD was not associated with OCTN1 functional promoter variants or haplotypes showing altered promoter activities in in vitro assays. However, OCTN1 functional promoter haplotypes showing decreased promoter activities were significantly associated with a penetrating behavior in CD patients (HR=2.428, p=0.009). Our results suggest that the OCTN1 functional promoter haplotypes can influence the CD phenotype, although these might not be associated with susceptibility to this disease.
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Human organic cation/carnitine transporter 1 (OCTN1) plays an important role in the transport of drugs and endogenous substances. It is known that a missense variant of OCTN1 is significantly associated with Crohn's disease susceptibility. This study was performed to identify genetic variants of the OCTN1 promoter in Korean individuals and to determine their functional effects. First, the promoter region of OCTN1 was directly sequenced using genomic DNA samples from 48 healthy Koreans. OCTN1 promoter activity was then measured using a luciferase reporter assay in HCT-116 cells. Seven variants of the OCTN1 promoter were identified, two of which were novel. There were also four major OCTN1 promoter haplotypes. Three haplotypes (H1, H3, and H4) showed decreased transcriptional activity, which was reduced by 22.9%, 23.0%, and 44.6%, respectively (p<0.001), compared with the reference haplotype (H2). Transcription factor binding site analyses and gel shift assays revealed that NF-Y could bind to the region containing g.-1875T>A, a variant present in H3, and that the binding affinity of NF-Y was higher for the g.-1875T allele than for the g.-1875A allele. NF-Y could also repress OCTN1 transcription. These data suggest that three OCTN1 promoter haplotypes could regulate OCTN1 transcription. To our knowledge, this is the first study to identify functional variants of the OCTN1 promoter.
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Solute carrier family (SLC) transporters are expressed in the digestive system and play important roles in maintaining physiological functions in the body. In addition, SLC transporters act as oncoproteins or tumor-suppressor proteins during the development, progression, and metastasis of various digestive system cancers. SLC22A18, a member of the SLC22 gene family, is an orphan transporter with an unknown endogenous substrate. Previous study revealed that SLC22A18 is downregulated in colorectal cancer tissues and that it acts as a suppressor in colorectal cancer, although the effects of SLC22A18 variants on colon cancer cell proliferation, migration, and invasion are unknown. Therefore, in this study, we identified SLC22A18 variants found in multiple populations by searching public databases and determined the in vitro effects of these missense variations on transporter expression and cancer progression. Our results indicated that three missense SLC22A18 variants-p.Ala6Thr, p.Arg12Gln, and p.Arg86His-had significantly lower cell expression than the wild type, possibly owing to intracellular degradation. Furthermore, these three variants caused significantly higher proliferation, migration, and invasion of colon cancer cells than the wild type. Our findings suggest that missense variants of SLC22A18 can potentially serve as biomarkers or prognostic tools that enable clinicians to predict colorectal cancer progression.
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Neoplasias do Colo , Proteínas de Transporte de Cátions Orgânicos , Humanos , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Neoplasias do Colo/genética , Regulação Neoplásica da Expressão Gênica , Prognóstico , Proteínas Supressoras de Tumor/genética , Proteínas de Transporte de Cátions Orgânicos/genéticaRESUMO
OBJECTIVE: Human multidrug and toxin extrusion member 2 (MATE2-K, SLC47A2) plays an important role in the renal elimination of various clinical drugs including the antidiabetic drug metformin. The goal of this study was to characterize genetic variants of MATE2-K and determine their association with the pharmacokinetics of metformin. METHODS: We screened DNA samples from 48 healthy Koreans for variants in the promoter and coding regions of MATE2-K and examined the function of common haplotypes in the promoter region using in-vitro luciferase assays. Then, the metformin pharmacokinetic study was carried out to determine the association between MATE2-K promoter haplotypes and metformin pharmacokinetics. RESULTS: Nine variants in the promoter region of MATE2-K and one nonsynonymous variant, p.G211V, were identified. The MATE2-K promoter haplotype 1 containing a known functional polymorphism, g.-130G>A and haplotype 2 containing two polymorphisms, g.-609G>A and g.-396G>A showed a significant increase in reporter activity. Among the 45 individuals who participated in the metformin pharmacokinetic study, 12 healthy Koreans who were homozygous for haplotype 1 or 2 showed a significant increase in renal clearance [539 ± 76 (reference group) vs. 633 ± 102 (variant group) ml/min; P=0.006] and secretion clearance [439 ± 81 (reference group) vs. 531 ± 102 (variant group) ml/min; P=0.007] of metformin compared with that shown by the reference group. CONCLUSION: Our study suggests that common promoter haplotypes of MATE2-K are associated with the pharmacokinetics of metformin.
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Variação Genética , Hipoglicemiantes/farmacocinética , Metformina/farmacocinética , Proteínas de Transporte de Cátions Orgânicos/genética , Povo Asiático , Haplótipos , Homozigoto , Humanos , Polimorfismo Genético , Regiões Promotoras GenéticasRESUMO
Multidrug resistance 3 (MDR3) is present on the canalicular membrane of the hepatocyte and plays an important role in protecting the liver from bile acids. In this study, we characterized the transcriptional effects of four common haplotypes and four polymorphic variants in the promoter region of MDR3 that were identified in 126 DNA samples from Koreans. We measured the luciferase activities of the four MDR3 promoter haplotypes using in vitro reporter assays. Among them, two haplotypes showed a significant decrease in reporter activity compared to the reference. One of the mechanisms by which these haplotypes might decrease MDR3 transcriptional activity was determined: one of the polymorphisms that are present in haplotype 3, was associated with a significant reduction in the promoter activity of MDR3, and the transcription factor NF-Y was predicted to bind to the promoter in the region of g.-1584C>T. Electrophoretic mobility shift assays showed that the g.-1584C allele exhibited greater binding to NF-Y than did the g.-1584T allele. Through the measurement of promoter activity after the overexpression of NF-Y, we found that NF-Y can act as a transcriptional activator of MDR3. These data suggest that the reduced transcriptional activity of g.-1584C>T results from a reduction in the binding affinity of the activator NF-Y to the MDR3 promoter region. Our study suggests that two common haplotypes of MDR3 can regulate the transcriptional rate of MDR3 and that NF-Y may be one of the transcriptional factors involved in this regulation.
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Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Fator de Ligação a CCAAT/metabolismo , Regulação da Expressão Gênica , Regiões Promotoras Genéticas/genética , Alelos , Povo Asiático/genética , Ensaio de Desvio de Mobilidade Eletroforética , Genes Reporter , Variação Genética , Células HEK293 , Haplótipos , Células HeLa , Células Hep G2 , Humanos , República da CoreiaRESUMO
Multidrug and toxin extrusion 1 (MATE1, SLC47A1), an organic cation transporter, plays an important role in the renal and biliary elimination of various clinical drugs, including the anti-diabetic drug metformin. The goal of this study was to identify and characterize novel genetic variants of MATE1. Five variants in the promoter region and two nonsynonymous variants, p.D64G and p.L125F, were identified in 48 DNA samples from healthy Koreans. MATE1 promoter haplotype 3 containing g.-1975C>A showed a significant increase in reporter activity. Three transcription factors, Nkx-2.5, SREBP-1, and USF-1 were predicted to bind to the promoter in the region of g.-1975C>A. Results from electrophoretic mobility shift assays showed that the g.-1975A allele exhibits greater binding affinity to all of these transcription factors than the g.-1975C allele. In particular, we found that Nkx-2.5 and USF-1 induce MATE1 transcription. Our study suggests that the common promoter haplotype of MATE1 changes MATE1 transcriptional activity regulated by Nkx-2.5, SREBP-1, and USF-1.
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Povo Asiático/genética , Variação Genética , Proteínas de Transporte de Cátions Orgânicos/genética , Alelos , Ensaio de Desvio de Mobilidade Eletroforética , Frequência do Gene , Genética Populacional/métodos , Genoma Humano , Células HCT116 , Haplótipos , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/genética , Humanos , Regiões Promotoras Genéticas , Ligação Proteica , República da Coreia , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Fatores de Transcrição/genética , Transcrição Gênica , Transfecção , Fatores Estimuladores Upstream/genéticaRESUMO
Mer receptor tyrosine kinase (Mer) signaling plays a central role in the intrinsic inhibition of the inflammatory response to Toll-like receptor activation. Previously, we found that lung Mer protein expression decreased after lipopolysaccharide (LPS) treatment due to enhanced Mer cleavage. The purpose of the present study was to examine whether pharmacologically restored membrane-bound Mer expression upregulates the Mer signaling pathways and suppresses lung inflammatory responses. Pretreatment with the ADAM17 (a disintegrin and metalloproteinase-17) inhibitor TAPI-0 (tumor necrosis factor alpha protease inhibitor-0) reduced LPS-induced production of soluble Mer protein in bronchoalveolar lavage (BAL) fluid, restored membrane-bound Mer expression, and increased Mer activation in alveolar macrophages and lungs after LPS treatment. TAPI-0 also enhanced Mer downstream signaling, including phosphorylation of protein kinase b, focal adhesion kinase, and signal transducer and activator of transcription 1. As expected from enhanced Mer signaling, TAPI-0 also augmented suppressor of cytokine signaling-1 and -3 mRNA and protein levels and inhibited nuclear factor κB activation at 4 and 24 hours after LPS treatment. TAPI-0 suppressed LPS-induced inflammatory cell accumulation, total protein level elevation in BAL fluid, and production of inflammatory mediators, including tumor necrosis factor-α, interleukin-1ß, and macrophage inflammatory protein-2. Additionally, the effects of TAPI-0 on the activation of Mer signaling and the production of inflammatory responses could be reversed by cotreatment with specific Mer-neutralizing antibody. Restored Mer protein expression by treatment with TAPI-0 efficiently prevents the inflammatory cascade during acute lung injury.
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Dipeptídeos/uso terapêutico , Ácidos Hidroxâmicos/uso terapêutico , Lipopolissacarídeos/farmacologia , Pulmão/efeitos dos fármacos , Pneumonia Bacteriana/tratamento farmacológico , Proteínas Proto-Oncogênicas/biossíntese , Receptores Proteína Tirosina Quinases/biossíntese , Transdução de Sinais/efeitos dos fármacos , Proteínas ADAM/antagonistas & inibidores , Proteína ADAM17 , Animais , Western Blotting , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Contagem de Células , Células Cultivadas , Dipeptídeos/administração & dosagem , Ensaio de Desvio de Mobilidade Eletroforética , Ativação Enzimática , Indução Enzimática , Ensaio de Imunoadsorção Enzimática , Ácidos Hidroxâmicos/administração & dosagem , Pulmão/enzimologia , Pulmão/metabolismo , Pulmão/patologia , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/enzimologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fosforilação , Pneumonia Bacteriana/enzimologia , Pneumonia Bacteriana/patologia , Proteínas Proto-Oncogênicas/metabolismo , RNA/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima , c-Mer Tirosina QuinaseRESUMO
Multidrug resistance 3 (MDR3) is expressed on the canalicular membrane of the hepatocytes and plays an important role in protecting the liver from bile acids. Altered ABCB4 gene expression can lead to a rare hepatic disease, low phospholipid-associated cholelithiasis (LPAC). In this study, we characterized 3 ABCB4 mutations in LPAC patients using various in vitro assay systems. We first measured the ability of each mutant to transport paclitaxel and then the mechanisms by which these mutations might change MDR3 transport activity were determined using immunoblotting, cell surface protein biotinylation, and immunofluorescence. Through a membrane vesicular transport assay, we observed that the uptake of paclitaxel was significantly reduced in membrane vesicles expressing 2 ABCB4 mutations, F165I and S320F. Both mutants showed significantly decreased total and cell surface MDR3 expression. These data suggest two missense mutations of ABCB4 may alter function of MDR3 and ultimately can be determined as LPAC-causing mutations.
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Aquaporin 4 (AQP4), the most abundant water channel protein in the brain, is involved in brain edema induced by ischemic insults. To evaluate whether the neuroprotective effects of estrogen are associated with AQP4 expression and edema formation, changes in AQP levels and ischemic edema were examined in the brains of male and female mice subjected to transient middle cerebral artery occlusion. Infarct volume and edema formation were markedly less in females than in males. AQP4 expression in the ischemic cortex of females was relatively well preserved, whereas it was significantly decreased in males. These effects disappeared in ovariectomized females but were reversed by estrogen replacement. Furthermore, AQP4 expression was decreased with increased brain edema in females treated with ICI182,780, an estrogen receptor antagonist. These findings suggest that the estrogen effect on the reduction of ischemic brain edema is associated with the preserved level of AQP4 that is partly mediated by estrogen receptors.
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Aquaporina 4/biossíntese , Edema Encefálico/metabolismo , Isquemia Encefálica/fisiopatologia , Estrogênios/farmacologia , Acidente Vascular Cerebral/metabolismo , Animais , Edema Encefálico/prevenção & controle , Infarto Encefálico/patologia , Estradiol/análogos & derivados , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Feminino , Fulvestranto , Infarto da Artéria Cerebral Média/complicações , Infarto da Artéria Cerebral Média/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Estrogênio/fisiologia , Fatores Sexuais , Acidente Vascular Cerebral/complicaçõesRESUMO
The ATP-binding cassette subfamily 4 (ABCA4), a transporter, is localized within the photoreceptors of the retina, and its genetic variants cause retinal dystrophy. Despite the clinical importance of the ABCA4 transporter, a few studies have investigated the function of each variant. In this study, we functionally characterized ABCA4 variants found in Korean patients with Stargardt disease or variants of the ABCA4 promoter region. We observed that four missense variants-p.Arg290Gln, p.Thr1117Ala, p.Cys1140Trp, and p.Asn1588Tyr-significantly decreased ABCA4 expression on the plasma membrane, which could be due to intracellular degradation. There are four major haplotypes in the ABCA4 proximal promoter. We observed that the H1 haplotype (c.-761C>A) indicated significantly increased luciferase activity compared to that of the wild-type, whereas the H3 haplotype (c.-1086A>C) indicated significantly decreased luciferase activity (P < 0.01 and 0.001, respectively). In addition, c.-900A>T in the H2 haplotype exhibited significantly increased luciferase activity compared with that of the wild-type. Two transcription factors, GATA-2 and HLF, were found to function as enhancers of ABCA4 transcription. Our findings suggest that ABCA4 variants in patients with Stargardt disease affect ABCA4 expression. Furthermore, common variants of the ABCA4 proximal promoter alter the ABCA4 transcriptional activity, which is regulated by GATA-2 and HLF transcription factors.
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Transportadores de Cassetes de Ligação de ATP , Doença de Stargardt , Humanos , Transportadores de Cassetes de Ligação de ATP/genética , Mutação de Sentido Incorreto , Retina/patologia , Distrofias Retinianas/genética , Doença de Stargardt/genéticaRESUMO
The signal transducer and activator of transcription 6 (STAT6) transcription factor promotes activation of the peroxisome proliferator-activated receptor gamma (PPARγ) pathway in macrophages. Little is known about the effect of proximal signal transduction leading to PPARγ activation for the resolution of acute inflammation. Here, we studied the role of STAT6 signaling in PPARγ activation and the resolution of acute sterile inflammation in a murine model of zymosan-induced peritonitis. First, we showed that STAT6 is aberrantly activated in peritoneal macrophages after zymosan injection. Utilizing STAT6-/- and wild-type (WT) mice, we found that STAT6 deficiency further enhanced zymosan-induced proinflammatory cytokines, such as tumor necrosis factor-α, interleukin (IL)-6, and macrophage inflammatory protein-2 in peritoneal lavage fluid (PLF) and serum, neutrophil numbers and total protein amount in PLF, but reduced proresolving molecules, such as IL-10 and hepatocyte growth factor, in PLF. The peritoneal macrophages and spleens of STAT6-/- mice exhibited lower mRNA and protein levels of PPARγ and its target molecules over the course of inflammation than those of WT mice. The deficiency of STAT6 was shown to impair efferocytosis by peritoneal macrophages. Taken together, these results suggest that enhanced STAT6 signaling results in PPARγ-mediated macrophage programming, contributing to increased efferocytosis and inflammation resolution.
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Inflamação/genética , Inflamação/metabolismo , PPAR gama/metabolismo , Fator de Transcrição STAT6/metabolismo , Doença Aguda , Animais , Camundongos , Transdução de SinaisRESUMO
OBJECTIVES: CYP3A4 receives electrons from P450 oxidoreductase (POR) to metabolize about 50% of clinically used drugs. There is substantial inter-individual variation in CYP3A4 catalytic activity that is not explained by CYP3A4 genetic variants. CYP3A4 is flexible and distensible, permitting it to accommodate substrates varying in shape and size. To elucidate the mechanisms of variability in CYP3A4 catalysis, we examined the effects of genetic variants of POR, and explored the possibility that substrate-induced conformational changes in CYP3A4 differentially affect the ability of POR variants to support catalysis. METHODS: We expressed human CYP3A4 and four POR variants (Q153R, A287P, R457H, A503 V) in bacteria, reconstituted them in vitro and measured the Michaelis constant and maximum velocity with testosterone, midazolam, quinidine and erythromycin as substrates. RESULTS: POR A287P and R457H had low activity with all substrates; Q153R had 76-94% of wild-type (WT) activity with midazolam and erythromycin, but 129-150% activity with testosterone and quinidine. The A503 V polymorphism reduced the CYP3A4 activity to 61-77% of WT with testosterone and midazolam, but had nearly WT activity with quinidine and erythromycin. CONCLUSION: POR variants affect CYP3A4 activities. The impact of a POR variant on catalysis by CYP3A4 is substrate-specific, probably because of substrate-induced conformational changes in CYP3A4.
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Biocatálise , Citocromo P-450 CYP3A/metabolismo , Variação Genética , NADPH-Ferri-Hemoproteína Redutase/genética , Polimorfismo de Nucleotídeo Único/genética , Eritromicina/química , Eritromicina/metabolismo , Humanos , Cinética , Midazolam/química , Midazolam/metabolismo , Proteínas Mutantes/metabolismo , Quinidina/química , Quinidina/metabolismo , Especificidade por Substrato , Testosterona/química , Testosterona/metabolismoRESUMO
Organic cation transporter 1 (OCT1; SLC22A1) seems to play a role in the efficacy and disposition of the widely used antidiabetic drug metformin. Genetic variants in OCT1 have been identified largely in European populations. Metformin is increasingly being used in Asian populations where the incidence of type 2 diabetes (T2D) is on the rise. The goal of this study is to identify genetic variants of OCT1 in Chinese and Japanese populations, which may potentially modulate response to metformin. We used recent data from the 1000 Genomes Project (Chinese and Japanese) and direct sequencing of selected amplicons of OCT1 in 66 DNA samples from Japanese patients with T2D. A total of six nonsynonymous variants were identified. Three of them (Q97K, P117L, and R206C) had not been functionally characterized previously and had allele frequencies of 0.017, 0.023 and 0.008, respectively. The uptake of metformin in cells expressing Q97K, P117L, and R206C was significantly reduced relative to the OCT1 reference (62 ± 4.3, 55 ± 6.8, and 22 ± 1.5% for Q97K, P117L, and R206C, respectively). Kinetic studies indicated that P117L and R206C exhibited a reduced V(max), whereas Q97K showed an increased K(m). The green fluorescent protein (GFP)-tagged Q97K and P117L variants localized to the plasma membrane, whereas the GFP-tagged R206C was retained mainly in the endoplasmic reticulum. Replacement of the highly conserved R206 with different amino acids modulated the subcellular localization and function of the transporter. This study suggests that nonsynonymous variants of OCT1 in Chinese and Japanese populations may affect the differential response to metformin.
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Transportador 1 de Cátions Orgânicos/genética , Transportador 1 de Cátions Orgânicos/metabolismo , Idoso , Arginina/genética , Transporte Biológico Ativo , Biotinilação , Western Blotting , Linhagem Celular , China/epidemiologia , DNA/genética , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Citometria de Fluxo , Variação Genética , Genótipo , Proteínas de Fluorescência Verde/genética , Humanos , Hipoglicemiantes/metabolismo , Japão/epidemiologia , Cinética , Masculino , Metformina/metabolismo , Pessoa de Meia-Idade , Polimorfismo Genético , Polimorfismo de Nucleotídeo Único , Alinhamento de SequênciaRESUMO
Circulating tumor cells (CTCs) are cancer cells that have been shed from a primary tumor and circulate in the bloodstream during progression of cancer. They may thus serve as circulating biomarkers that can predict, diagnose and guide therapy. Moreover, phenotypic and genotypic analysis of CTCs can facilitate prospective assessment of mutations and enable personalized treatment. A number of methodologies based on biological and physical differences between circulating tumor and non-tumor cells have been developed over the past few years. However, these methods did not have sufficient sensitivity or specificity. In this work, a remote analysis protocol was designed using motion microscopy that amplifies cellular micro motions in a captured video by re-rendering small motions to generate extreme magnified visuals to detect dynamic motions that are not otherwise visible by naked eye. Intriguingly, motion microscopy demonstrated fluctuations around breast tumor cells, which we referred to herein as cellular trail. Phenomena of cellular trail mostly emerged between 0.5 and 1.5 Hz on amplified video images. Interestingly, cellular trails were associated with cell surface proteins and flow rates rather than mitochondrial activity. Moreover, cellular trails were present only around circulating tumor cells from individuals with breast cancer under conditions of 20-30 µm/s and 0.5-1.5 Hz. Thus, motion microscopy based CTC detection method can offer a valuable supplementary diagnostic tool for assessment of drug efficacy and identifying physical characteristics of tumor cells for further research.
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Biomarcadores Tumorais , Neoplasias da Mama/diagnóstico , Microscopia/métodos , Técnicas de Diagnóstico Molecular , Células Neoplásicas Circulantes/metabolismo , Imagem com Lapso de Tempo , Antígenos de Superfície/metabolismo , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Feminino , Humanos , Microfluídica/instrumentação , Microfluídica/métodos , Microscopia de Fluorescência , Mitocôndrias/metabolismo , Células Neoplásicas Circulantes/patologiaRESUMO
Organic cation transporter 3 (OCT3) is expressed in various organs in humans and plays an important role in the transport of organic cations and drugs including metformin. In this study, we identified genetic variations of the OCT3 promoter and functionally characterized each variant by in vitro assays. Next, the association between the functional haplotype of the OCT3 promoter and pharmacokinetics of metformin was evaluated. In our study population, 7 variations and 2 major haplotypes were identified, of which H2 haplotype yielded a significantly higher luciferase activity than did the wild type. Two variants of H2, c.-1603G > A and c.-1547T > G, yielded significantly lower luciferase activities, whereas the luciferase activity of another variant, c.-29G > A, was significantly higher. Two transcription factors, Sp1 and USF1, were involved in the regulation of OCT3 transcription. Analysis of clinical data revealed that 25 subjects, either homozygous or heterozygous for H2, showed increased AUCinf and Cmax by 17.2% and 15.9%, respectively [P = 0.016 and 0.031, GMR (90% CI) = 1.17 (1.06-1.29) and 1.17 (1.04-1.31), respectively], compared to the 20 subjects in the control group. Our study suggests that an OCT3 promoter haplotype affects the pharmacokinetics of metformin in Koreans as well as the OCT3 transcription rate.
Assuntos
Haplótipos , Metformina/farmacocinética , Proteínas de Transporte de Cátions Orgânicos/genética , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas/genética , Adulto , Alelos , Área Sob a Curva , Feminino , Frequência do Gene , Humanos , Hipoglicemiantes/farmacocinética , Masculino , República da Coreia , Adulto JovemRESUMO
The signal transducer and activator of transcription 6 (STAT6) transcription factor activates peroxisome proliferator-activated receptor gamma (PPAR-γ)-regulated gene expression in immune cells. We investigated proximal membrane signaling that was initiated in macrophages after exposure to apoptotic cells that led to enhanced PPAR-γ expression and activity, using specific siRNAs for ABCA1, STAT6, and PPAR-γ, or their antagonists. The interactions between mouse bone marrow-derived macrophages or RAW 264.7 cells and apoptotic Jurkat cells, but not viable cells, resulted in the induction of STAT6 phosphorylation as well as PPAR-γ expression and activation. Knockdown of ATP-binding cassette transporter A1 (ABCA1) after the transfection of macrophages with ABCA1-specific siRNAs reduced apoptotic cell-induced STAT6 phosphorylation as well as PPAR-γ mRNA and protein expression. ABCA1 knockdown also reduced apoptotic cell-induced liver X receptor α (LXR-α) mRNA and protein expression. Moreover, inhibition of STAT6 with specific siRNAs or the pharmacological inhibitor AS1517499AS reversed the induction of PPAR-γ, LXR-α, and ABCA1 by apoptotic Jurkat cells. PPAR-γ-specific siRNAs or the PPAR-γ antagonist GW9662 inhibited apoptotic cell-induced increases in LXR-α and ABCA1 mRNA and protein levels. Thus, these results indicate that apoptotic cells trigger the ABCA1/STAT6 pathway, leading to the activation of the PPAR-γ/LXR-α/ABCA1 pathway in macrophages.