RESUMEN
Technetium-99m-labeled mercaptoacetyltriglycine ([99mTc]MAG3) is widely used for evaluation of transplanted kidneys, diagnosis of tubular necrosis, and scintigraphic studies of tubular function. [99mTc]MAG3 is a substrate for organic anion transporter (OAT)1 and OAT3 on the basolateral membrane side for renal secretion. We investigated the transport mechanism and affinity of [99mTc]MAG3 on the apical membrane of renal proximal tubule cells for renal secretion. Adenosine triphosphate-binding cassette (ABC) transporters for renal secretion of [99mTc]MAG3 were examined using ABC transporter vesicles expressing multiple drug resistance 1 (MDR1), breast cancer resistance protein (BCRP), multidrug resistance-associated protein (MRP)2, and MRP4. MK-571, a MRP inhibitor, was applied to measure the Km and Vmax of MRP2 and MRP4 in a vesicle transport assay. Single photon emission computed tomography (SPECT) was performed in normal rats and MRP2-deficient Eisai hyperbilirubinuria rats (EHBR) using [99mTc]MAG3 with and without MK-571. [99mTc]MAG3 uptake in adenosine triphosphate was significantly higher than that in adenosine monophosphate in vesicles that highly expressed MRP2 and MRP4. The affinity of [99mTc]MAG3 for MRP4 was higher than that for MRP2. Renal secretion via MRP2 and MRP4 was identified by comparing normal and EHBR rats with and without MK-571 on SPECT. [99mTc]MAG3 is transported via MRP2 and MRP4 on the apical membrane of renal proximal tubule cells. The affinity of MRP4 is higher than that of MRP2. SIGNIFICANCE STATEMENT: [99mTc]MAG3, widely used for evaluation of transplanted kidneys, diagnosis of tubular necrosis, and scintigraphic studies of tubular function, is transported via MRP2 and MRP4 on the apical membrane of renal proximal tubule cells. The affinity of MRP4 is higher than that of MRP2.
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Membrana Celular/metabolismo , Túbulos Renales Proximales/citología , Tecnecio Tc 99m Mertiatida/metabolismo , Animales , Transporte Biológico , Túbulos Renales Proximales/diagnóstico por imagen , Ratas , Tomografía Computarizada de Emisión de Fotón ÚnicoRESUMEN
INTRODUCTION: To clarify the difference between system A and L amino acid transport imaging in PET clinical imaging, we focused on the use of α-[N-methyl-11C]-methylaminoisobutyric acid ([11C]MeAIB), and compared it with [S-methyl-11C]-L-methionine ([11C]MET). The aim of this study was to assess the correlation of accumulation of these two radioactive amino acid analogs with expression of amino acid transporters and cell proliferative activity in carcinoma cells. METHODS: Amino acid uptake inhibitor studies were performed in four human carcinoma cells (epidermal carcinoma A431, colorectal carcinoma LS180, and lung carcinomas PC14/GL and H441/GL) using the radioisotope analogs [3H]MET and [14C]MeAIB. MeAIB was used to inhibit the A system and 2-amino-2-norbornane-carboxylic acid (BCH) was used to inhibit the L system. The carcinoma gene expression levels of a number of amino acid transporters were measured by microarray and quantitative polymerase chain reaction. Carcinoma proliferative activity was assessed using accumulation of [methyl-3H]-3'-deoxy-3'-fluorothymidine ([3H]FLT). RESULTS AND CONCLUSION: [14C]MeAIB uptake occurred principally via a Na+-dependent A type mechanism whereas [3H]MET uptake occurred predominantly via a Na+-independent L type mechanism although other transporters were also utilized depending on cell type. There was no correlation between [3H]MET uptake and total system L amino acid transporter (LAT) expression. In contrast, [14C]MeAIB uptake strongly correlated with total system A amino acid transporter (SNAT) expression and proliferative activity in this preliminary study using four human carcinoma cell lines. Carcinoma proliferative activity also correlated with total SNAT expression. Advances in Knowledge and Implications for Patient Care: Because there is a significant correlation between the accumulation of [14C]MeAIB and the gene expression level of total SNAT as well as the accumulation of [3H]FLT, it is suggested that use of the analog [11C]MeAIB in PET may provide an indication of tumor cell proliferative activity. [11C]MeAIB is therefore expected to be very useful in PET imaging.
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Sistemas de Transporte de Aminoácidos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Metionina/metabolismo , beta-Alanina/análogos & derivados , Transporte Biológico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Proyectos Piloto , Tomografía de Emisión de Positrones , beta-Alanina/metabolismo , beta-Alanina/farmacologíaRESUMEN
[18F]Fluciclovine (trans-1-amino-3-[18F]fluorocyclobutanecarboxylic acid; anti-[18F]FACBC), a positron emission tomography tracer used for the diagnosis of recurrent prostate cancer, is transported via amino acid transporters (AATs) with high affinity (Km: 97-230 µM). However, the mechanism underlying urinary excretion is unknown. In this study, we investigated the involvement of AATs and drug transporters in renal [18F]fluciclovine reuptake. [14C]Fluciclovine (trans-1-amino-3-fluoro[1-14C]cyclobutanecarboxylic acid) was used because of its long half-life. The involvement of AATs in [14C]fluciclovine transport was measured by apical-to-basal transport using an LLC-PK1 monolayer as model for renal proximal tubules. The contribution of drug transporters herein was assessed using vesicles/cells expressing the drug transporters P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), multidrug resistance-associated protein 4 (MRP4), organic anion transporter 1 (OAT1), organic anion transporter 3 (OAT3) , organic cation transporter 2 (OCT2), organic anion transporting polypeptide 1B1 (OATP1B1), and organic anion transporting polypeptide 1B3 (OATP1B3). The apical-to-basal transport of [14C]fluciclovine was attenuated by l-threonine, the substrate for system alanine-serine-cysteine (ASC) AATs. [14C]Fluciclovine uptake by drug transporter-expressing vesicles/cells was not significantly different from that of control vesicles/cells. Fluciclovine inhibited P-gp, MRP4, OAT1, OCT2, and OATP1B1 (IC50 > 2.95 mM). Therefore, system ASC AATs may be partly involved in the renal reuptake of [18F]fluciclovine. Further, given that [18F]fluciclovine is recognized as an inhibitor with millimolar affinity for the tested drug transporters, slow urinary excretion of [18F]fluciclovine may be mediated by system ASC AATs, but not by drug transporters.
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Sistemas de Transporte de Aminoácidos/metabolismo , Ácidos Carboxílicos/metabolismo , Ciclobutanos/metabolismo , Riñón/metabolismo , Tomografía de Emisión de Positrones/métodos , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Transporte Biológico , Línea Celular , Células HEK293 , Humanos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Transportadores de Anión Orgánico/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismoRESUMEN
INTRODUCTION: trans-1-Amino-3-[(18)F]fluorocyclobutanecarboxylic acid ([(18)F]fluciclovine, also known as anti-[(18)F]FACBC), is a tracer for positron emission tomography (PET) imaging for detection of tumors such as prostate cancer (PCa). Our previous study showed that ASCT2 (Na(+)-dependent amino acid transporter (AAT)) mediates fluciclovine uptake in androgen-dependent PCa cells; its expression is influenced by androgen, a key hormone in the progression of primary PCa and castration-resistant prostate cancer (CRPC). In this study, we investigated the uptake mechanisms and feasibility of [(18)F]fluciclovine for CRPC in the androgen-dependent PCa cell line LNCaP and LNCaP-derivatives LNCaP-SF and LN-REC4. METHODS: LNCaP-SF was established after long-term cultivation of LNCaP in steroid-free conditions, and LN-Pre and LN-REC4 were established from LNCaP inoculated in intact and castrated severe combined immunodeficient mice, respectively. Uptake and competitive inhibition experiments were performed with trans-1-amino-3-fluoro[1-(14)C]cyclobutanecarboxylic acid ([(14)C]fluciclovine) to characterize the involvement of AATs in androgen-dependent PCa (LNCaP and LN-Pre) and CRPC-like (LNCaP-SF and LN-REC4) cell lines. AAT expression was analyzed by Western blotting, and [(14)C]fluciclovine uptake in androgen-dependent PCa and CRPC-like cell lines were investigated in the presence or absence of dihydrotestosterone (DHT). RESULTS: The contribution of Na(+)-dependent AATs to [(14)C]fluciclovine uptake in all cell lines was 88-98%, and [(14)C]fluciclovine uptake was strongly inhibited by L-glutamine and L-serine, the substrates for Na(+)-dependent alanine-serine-cysteine (system ASC) AATs, in the presence of Na(+). DHT enhanced ASCT2 expression in LNCaP, LN-Pre, and LN-REC4, but not in LNCaP-SF, and the responses of ASCT2 expression to DHT correlated with [(14)C]fluciclovine uptake. CONCLUSIONS: System ASC, especially ASCT2, could play a major role in [(14)C]fluciclovine uptake into CRPC-like and androgen-dependent PCa cells, suggesting [(18)F]fluciclovine-PET is applicable to the detection of CRPC as well as androgen-dependent PCa. ADVANCE IN KNOWLEDGE: [(18)F]fluciclovine-PET may be applied for the detection of CRPC. IMPLICATION FOR PATIENT CARE: [(18)F]fluciclovine-PET may permit early intervention for CRPC treatment.
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Sistema de Transporte de Aminoácidos ASC/metabolismo , Ácidos Carboxílicos/metabolismo , Ciclobutanos/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Próstata Resistentes a la Castración/patología , Andrógenos/metabolismo , Animales , Unión Competitiva , Transporte Biológico/efectos de los fármacos , Línea Celular Tumoral , Dihidrotestosterona/farmacología , Estudios de Factibilidad , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Ratones , Antígenos de Histocompatibilidad Menor , Neoplasias de la Próstata Resistentes a la Castración/diagnóstico , Neoplasias de la Próstata Resistentes a la Castración/metabolismoRESUMEN
UNLABELLED: Anti-1-amino-3-(18)F-fluorocyclobutane-1-carboxylic acid ((18)F-FACBC) is a synthetic amino acid analog PET radiotracer undergoing clinical trials for the evaluation of prostate and other cancers. We aimed to describe common physiologic uptake patterns, incidental findings, and variants in patients who had undergone (18)F-FACBC PET. METHODS: Sixteen clinical trials involving 611 (18)F-FACBC studies from 6 centers, which included dosimetry studies on 12 healthy volunteers, were reviewed. Qualitative observations of common physiologic patterns, incidental uptake, and variants that could simulate disease were recorded and compared with similar observations in studies of the healthy volunteers. Quantitative analysis of select data and review of prior published reports and observations were also made. RESULTS: The liver and pancreas demonstrated the most intense uptake. Moderate salivary and pituitary uptake and variable mild to moderate bowel activity were commonly visualized. Moderate bone marrow and mild muscle activity were present on early images, with marrow activity decreasing and muscle activity increasing with time. Brain and lungs demonstrated activity less than blood pool. Though (18)F-FACBC exhibited little renal excretion or bladder uptake during the clinically useful early imaging time window, mild to moderate activity might accumulate in the bladder and interfere with evaluation of adjacent prostate bed and seminal vesicles in 5%-10% of patients. Uptake might also occur from benign processes such as infection, inflammation, prostatic hyperplasia, and metabolically active benign bone lesions such as osteoid osteoma. CONCLUSION: Common physiologic uptake patterns were similar to those noted in healthy volunteers. The activity in organs followed the presence of amino acid transport and metabolism described with other amino acid-based PET radiotracers. As with other PET radiotracers such as (18)F-FDG, focal nonphysiologic uptake may represent incidental malignancy. Uptake due to benign etiologies distinct from physiologic background also occurred and could lead to misinterpretations if the reader is unaware of them.
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Ácidos Carboxílicos , Ciclobutanos , Hallazgos Incidentales , Tomografía de Emisión de Positrones/métodos , Radiofármacos , Adulto , Anciano , Artefactos , Ácidos Carboxílicos/farmacocinética , Sistema Nervioso Central/diagnóstico por imagen , Ciclobutanos/farmacocinética , Femenino , Humanos , Procesamiento de Imagen Asistido por Computador , Masculino , Persona de Mediana Edad , Tomografía de Emisión de Positrones/normas , Radiofármacos/farmacocinética , Estándares de Referencia , Reproducibilidad de los ResultadosRESUMEN
PURPOSE: Androgens play a crucial role in prostate cancer progression, and trans-1-amino-3-[(18)F]fluorocyclobutanecarboxylic acid (anti-[(18) F]FACBC) are used for visualization of prostate cancer. We examined the effect of androgen on the expression of amino acid transporters related to anti-[(18)F]FACBC transport and uptake of trans-1-amino-3-fluoro-[1-(14)C]cyclobutanecarboxylic acid (anti-[(14)C]FACBC). PROCEDURES: Expression of amino acid transporters and uptake of anti-[(14)C]FACBC in androgen receptor (AR)-positive LNCaP and AR-negative DU145 human prostate cancer cells cultured with/without 5α-dihydrotestosterone (DHT) and the effect of bicalutamide, an AR antagonist, on DHT-associated changes were investigated. RESULTS: DHT stimulated the expression of amino acid transporters ASCT2, SNAT5, 4F2 heavy chain, and LAT3 in LNCaP but not in DU145 cells. Anti-[(14)C]FACBC uptake was enhanced, in a DHT-dependent manner, in LNCaP cells only. CONCLUSIONS: DHT enhanced the expression of ASCT2, the transporter responsible for anti-[(18)F]FACBC uptake, thereby increasing anti-[(14)C]FACBC uptake in AR-positive LNCaP cells. Androgen-mediated induction may contribute to the distinct anti-[(18)F]FACBC accumulation pattern in prostate cancer.
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Sistemas de Transporte de Aminoácidos/metabolismo , Ácidos Carboxílicos/farmacocinética , Ciclobutanos/farmacocinética , Dihidrotestosterona/farmacología , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Radiofármacos/farmacocinética , Sistemas de Transporte de Aminoácidos/genética , Andrógenos/metabolismo , Anilidas/farmacología , Ácidos Carboxílicos/análisis , Línea Celular Tumoral , Ciclobutanos/análisis , Radioisótopos de Flúor , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Nitrilos/farmacología , Neoplasias de la Próstata Resistentes a la Castración/diagnóstico por imagen , Trazadores Radiactivos , Cintigrafía , Radiofármacos/análisis , Receptores Androgénicos/metabolismo , Compuestos de Tosilo/farmacologíaRESUMEN
INTRODUCTION: In clinical hepatobiliary scintigraphy, (99m)Tc-N-pyridoxyl-5-methyltryptophan ((99m)Tc-PMT) is an effective radiotracer among the (99m)Tc-pyridoxylaminates. However, the mechanisms of human hepatic uptake and bile excretion transport of (99m)Tc-PMT have not been determined. We thus investigated the transport mechanisms of human hepatic uptake and bile excretion in hepatobiliary scintigraphy with (99m)Tc-PMT. METHODS: Four solute carrier (SLC) transporters involved in hepatic uptake were evaluated using human embryonic kidney (HEK) and HeLa cells with high expression of SLC transporters (organic anion transporting polypeptide (OATP)1B1, OATP1B3, OATP2B1, organic anion transporters (OAT)2 and organic cation transporters (OCT)1) after 5 min of (99m)Tc-PMT incubation. Metabolic analysis of (99m)Tc-PMT was performed using pooled human liver S9. Adenosine triphosphate (ATP)-binding cassette (ABC) transporters for bile excretion were examined using hepatic ABC transporter vesicles human expressing multiple drug resistance 1 (MDR1), multidrug resistance-associated protein 2 (MRP2), breast cancer resistance protein or bile salt export pump. (99m)Tc-PMT was incubated for 1, 3 and 5 min with ATP or adenosine monophosphate and these vesicles. SPECT scans were performed in normal and Eisai hyperbilirubinemic (EHBR) model rats, deficient in Mrp2 transporters, without and with verapamil (rat Mdr1 and human MDR1 inhibitor) after intravenous injection of (99m)Tc-PMT. RESULTS: Uptake of (99m)Tc-PMT in HEK293/OATP1B1 and HeLa/OATP1B3 was significantly higher than that in HEK293- and HeLa-mock cells. (99m)Tc-PMT was not metabolized in the human liver S9. In vesicles with high expression of ABC transporters, uptake of MDR1 or MRP2 was significantly higher at all incubation times. Bile excretion of (99m)Tc-PMT was also identified by comparison between normal and EHBR rats with and without verapamil on in-vivo imaging. CONCLUSIONS: Human hepatic uptake of (99m)Tc-PMT was transferred by OATP1B1 and OATP1B3, and excretion into bile canaliculi via MDR1 and MRP2. (99m)Tc-PMT hepatobiliary scintigraphy may be a useful ligand as a noninvasive method of visualizing and quantifying hepatobiliary transporter functionality, which could predict drug pharmacokinetics.
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Bilis/diagnóstico por imagen , Bilis/metabolismo , Hígado/diagnóstico por imagen , Hígado/metabolismo , Compuestos de Organotecnecio , Piridoxal/análogos & derivados , Triptófano/análogos & derivados , Animales , Transporte Biológico , Células HEK293 , Células HeLa , Humanos , Masculino , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Cintigrafía , RatasRESUMEN
PURPOSE: We aimed to elucidate trans-1-amino-3-[(18)F]fluorocyclobutanecarboxylic acid (anti-[(18)F]FACBC) uptake mechanisms in inflammatory and tumor cells, in comparison with those of L-[methyl-(11)C]methionine ([(11)C]Met) and 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG). PROCEDURES: Using carbon-14-labeled tracers, in vitro time-course, pH dependence, and competitive inhibition uptake experiments were performed in rat inflammatory (T cells, B cells, granulocytes, macrophages), prostate cancer (MLLB2), and glioma (C6) cells. RESULTS: Anti-[(14)C]FACBC uptake ratios of T/B cells to tumor cells were comparable, while those of granulocytes/macrophages to tumor cells were lower than those for [(14)C]FDG. Over half of anti-[(14)C]FACBC uptake by T/B and tumor cells was mediated by Na(+)-dependent amino acid transporters (system ASC), whereas most [(14)C]Met transport in all cells was mediated by Na(+)-independent carriers (system L). CONCLUSIONS: The low anti-[(18)F]FACBC accumulation in granulocytes/macrophages may be advantageous in discriminating inflamed regions from tumors. The significant anti-[(18)F]FACBC uptake in T/B cells may cause false-positives in some cancer patients who undergo FACBC-positron emission tomography (PET).
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Ácidos Carboxílicos/farmacocinética , Ciclobutanos/farmacocinética , Radioisótopos de Flúor/farmacocinética , Fluorodesoxiglucosa F18/farmacocinética , Inflamación/metabolismo , Metionina/análogos & derivados , Neoplasias de la Próstata/metabolismo , Animales , Línea Celular Tumoral , Humanos , Concentración de Iones de Hidrógeno , Masculino , Metionina/farmacocinética , RatasRESUMEN
Positron emission tomography (PET) with amino acid tracers is useful for the visualization and assessment of therapeutic effects on gliomas. Our purpose is to elucidate the transport mechanisms of trans-1-amino-3-[¹8F]fluorocyclobutanecarboxylic acid (anti-[¹8F]FACBC) and L-[methyl-¹¹C]methionine ([¹¹C]Met) in normal human astrocytes (NHA), low-grade (Hs683, SW1088), and high-grade (U87MG, T98G) human glioma cell lines. Because the short half-lives of fluorine-18 and carbon-11 are inconvenient for in vitro experiments, trans-1-amino-3-fluoro[1-¹4C]cyclobutanecarboxylic acid (anti-[¹4C]FACBC) and L-[methyl-¹4C]methionine ([¹4C]Met) were used instead of the PET tracers. Time-course uptake experiments showed that uptake of anti-[¹4C]FACBC was 1.4-2.6 times higher than that of [¹4C]Met in NHA and low-grade glioma cells, and was almost equal to that of [¹4C]Met in high-grade glioma cells. To identify the amino acid transporters (AATs) involved in the transport of anti-[¹4C]FACBC and [¹4C]Met, we carried out competitive inhibition experiments using synthetic/naturally-occurring amino acids as inhibitors. We found that anti-[¹4C]FACBC uptake in the presence of Na⺠was strongly inhibited by L-glutamine and L-serine (the substrates for ASC system AATs), whereas L-phenylalanine and 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid (BCH, the substrates for L system AATs) robustly inhibited Naâº-independent anti-[¹4C]FACBC uptake. Regardless of Naâº, [¹4C]Met uptake was inhibited strongly by L-phenylalanine and BCH. Moreover, the exchange transport activity of L-glutamine for anti-[¹4C]FACBC was stronger than that of BCH in the presence of Naâº, whereas that for [¹4C]Met was almost equal to BCH. These results demonstrate that ASC and L are important transport systems for anti-[¹8F]FACBC uptake, while system L is predominantly involved in [¹¹C]Met transport in human astrocytes and glioma cells.
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Radioisótopos de Carbono/farmacocinética , Ácidos Carboxílicos/farmacocinética , Ciclobutanos/farmacocinética , Glioma/metabolismo , Metionina/farmacocinética , Transporte Biológico/efectos de los fármacos , Línea Celular Tumoral , Glioma/diagnóstico , HumanosRESUMEN
INTRODUCTION: Trans-1-amino-3-[(18)F]fluorocyclobutanecarboxylic acid (anti-[(18)F]FACBC) is a promising amino acid positron emission tomography (PET) radiotracer for visualizing prostate cancer. We previously showed that anti-FACBC is transported by amino acid transporters, especially by alanine-serine-cysteine transporter 2 (ASCT2), which is associated with tumor growth. We studied this affinity to assess the mechanism of anti-FACBC transport in prostate cancer cells. METHODS: Kinetic assays for trans-1-amino-3-fluoro-[1-(14)C]cyclobutanecarboxylic acid ([(14)C]FACBC) were performed in Xenopus laevis oocytes over-expressing either ASCT2 or sodium-coupled neutral amino acid transporter 2 (SNAT2), both of which are highly expressed in prostate cancer cells. We also examined the kinetics of [(14)C]FACBC uptake using mammalian cell lines over-expressing system L amino acid transporter 1 or 2 (LAT1 or LAT2). Results: ASCT2 and SNAT2 transported [14C]FACBC with MichaelisMenten kinetics Km values of 96.7 ± 45.2 µM and 196.5 ± 19.7 µM, respectively. [correted]. LAT1 and LAT2 transported [(14)C]FACBC with Michaelis-Menten Km values of 230.4 ± 184.5 µM and 738.5 ± 87.6 µM, respectively. CONCLUSIONS: Both ASCT2 and SNAT2 recognize anti-FACBC as a substrate. Anti-FACBC has higher affinity for ASCT2 than for SNAT2, LAT1, or LAT2. The ASCT2-preferential transport of anti-[(18)F]FACBC in cancer cells could be used for more effective prostate cancer imaging.
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Sistema de Transporte de Aminoácidos ASC/genética , Sistema de Transporte de Aminoácidos ASC/metabolismo , Sistema de Transporte de Aminoácidos A/genética , Sistema de Transporte de Aminoácidos A/metabolismo , Ácidos Carboxílicos/metabolismo , Ciclobutanos/metabolismo , Oocitos/metabolismo , Xenopus laevis/genética , Animales , Transporte Biológico , Línea Celular , Expresión Génica , Humanos , Cinética , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Ratones , Antígenos de Histocompatibilidad MenorRESUMEN
The role of polyamines at the G1/S boundary and in the G2/M phase of the cell cycle was studied using synchronized HeLa cells treated with thymidine or with thymidine and aphidicolin. Synchronized cells were cultured in the absence or presence of α-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, plus ethylglyoxal bis(guanylhydrazone) (EGBG), an inhibitor of S-adenosylmethionine decarboxylase. When polyamine content was reduced by treatment with DFMO and EGBG, the transition from G1 to S phase was delayed. In parallel, the level of p27(Kip1) was greatly increased, so its mechanism was studied in detail. Synthesis of p27(Kip1) was stimulated at the level of translation by a decrease in polyamine levels, because of the existence of long 5'-untranslated region (5'-UTR) in p27(Kip1) mRNA. Similarly, the transition from the G2/M to the G1 phase was delayed by a reduction in polyamine levels. In parallel, the number of multinucleate cells increased by 3-fold. This was parallel with the inhibition of cytokinesis due to an unusual distribution of actin and α-tubulin at the M phase. Since an association of polyamines with chromosomes was not observed by immunofluorescence microscopy at the M phase, polyamines may have only a minor role in structural changes of chromosomes at the M phase. In general, the involvement of polyamines at the G2/M phase was smaller than that at the G1/S boundary.
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Poliaminas Biogénicas/metabolismo , División Celular/fisiología , Fase G1/fisiología , Fase G2/fisiología , Fase S/fisiología , Adenosilmetionina Descarboxilasa/antagonistas & inhibidores , Adenosilmetionina Descarboxilasa/metabolismo , División Celular/efectos de los fármacos , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Eflornitina/farmacología , Inhibidores Enzimáticos/farmacología , Fase G1/efectos de los fármacos , Fase G2/efectos de los fármacos , Células HeLa , Humanos , Mitoguazona/análogos & derivados , Mitoguazona/farmacología , Ornitina Descarboxilasa/metabolismo , Inhibidores de la Ornitina Descarboxilasa , Fase S/efectos de los fármacosRESUMEN
INTRODUCTION: We investigated the mechanisms of trans-1-amino-3-fluoro[1-(14)C]cyclobutanecarboxylic acid (anti-[(14)C]FACBC) transport by human-derived prostate cancer (PCa) cells and normal human prostatic epithelial cells (PrECs). METHODS: Using PCa cells (DU145, PC-3, LNCaP) and PrECs, we performed the following in vitro experiments: time-course, kinetics, competitive inhibition by synthetic/naturally occurring amino acids (AAs), exchange transport with synthetic/naturally occurring AAs and pH-dependency of anti-[(14)C]FACBC uptake. We also examined the amino acid transporter (AAT) expression using flow cytometry. RESULTS: The uptake of anti-[(14)C]FACBC by LNCaP and DU145 cells was higher than that by PC-3 and PrECs. The K(m) values for anti-[(14)C]FACBC were 64.4 and 191.7 µmol/L in the DU145 cells and PrECs, respectively. Total levels of anti-[(14)C]FACBC uptake were positively correlated with the expression level of system ASC in PCa cells. The contributions of Na(+)-dependent AATs to anti-[(14)C]FACBC uptake were greater than those of Na(+)-independent AATs, especially in PCa cells. In the presence of Na(+), glutamine and serine showed the strongest inhibitory effect against anti-[(14)C]FACBC uptake, suggesting that system ASC, especially ASCT2, is an important AAT for anti-[(14)C]FACBC. In contrast, phenylalanine and 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid, but not N-ethylmaleimide, almost completely inhibited the anti-[(14)C]FACBC uptake in the absence of Na(+), indicating the contribution of LAT1. In the exchange transport experiments, glutamine showed the strongest transstimulation of intracellular anti-[(14)C]FACBC efflux in DU145 cells. Furthermore, the contributions of Na(+)-independent AATs to the uptake of anti-[(14)C]FACBC in DU145 and PrECs were greater under acidic pH conditions than under neutral or alkaline pH conditions. CONCLUSIONS: Total uptake of anti-[(14)C]FACBC by PCa cells correlates with the expression level of system ASC in PCa cells. Furthermore, LAT1 is an important transport system for anti-[(14)C]FACBC uptake, especially in an acidic environment, such as the intra-tumoural environment.
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Aminoácidos/farmacocinética , Radioisótopos de Carbono/farmacocinética , Ácidos Carboxílicos/farmacocinética , Ciclobutanos/farmacocinética , Próstata/metabolismo , Neoplasias de la Próstata/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Transporte Biológico , Células Epiteliales/metabolismo , Citometría de Flujo , Humanos , MasculinoRESUMEN
UNLABELLED: Trans-1-amino-3-(18)F-fluorocyclobutanecarboxylic acid (anti-(18)F-FACBC) is an amino acid PET tracer that has shown promise for visualizing prostate cancer. Therefore, we aimed to clarify the anti-(18)F-FACBC transport mechanism in prostate cancer cells. We also studied the fate of anti-(18)F-FACBC after it is transported into cells. METHODS: For convenience, because of their longer half-lives, (14)C compounds were used instead of (18)F-labeled tracers. Trans-1-amino-3-fluoro-1-(14)C-cyclobutanecarboxylic acid ((14)C-FACBC) uptake was examined in human prostate cancer DU145 cells with the following substrates of amino acid transporters: α-(methylamino) isobutyric acid (a system A-specific substrate) and 2-amino-2-norbornanecarboxylic acid (a system L-specific substrate). The messenger RNA expression of amino acid transporters in human prostate cancer specimens was analyzed by complementary DNA microarray and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Gene expression in DU145 cells was analyzed by qRT-PCR. We also examined the knockdown effect of the amino acid transporters system ASC transporter 2 (ASCT2) and sodium-coupled neutral amino acid transporter 2 (SNAT2) on (14)C-FACBC uptake. In addition, the possibility of (14)C-FACBC incorporation into proteins was examined. RESULTS: (14)C-FACBC uptake by DU145 cells was markedly decreased to approximately 20% in the absence of Na(+), compared with that in its presence, indicating that Na(+)-dependent transporters are mainly responsible for the uptake of this tracer. Moreover, 2-amino-2-norbornanecarboxylic acid inhibited the transport of (14)C-FACBC to the basal level in Na(+)-free buffer. In contrast, α-(methylamino) isobutyric acid did not inhibit (14)C-FACBC accumulation in DU145 cells. Human prostate tumor specimens and DU145 cells had similar messenger RNA expression patterns of amino acid transporter genes. Although SNAT2 and ASCT2 are 2 major amino acid transporters expressed in prostate tumor tissues and DU145 cells, ASCT2 knockdown using small interfering RNA was more effective in lowering (14)C-FACBC transport than SNAT2. Almost all intracellular (14)C-FACBC was recovered from the nonprotein fraction. CONCLUSION: ASCT2, which is a Na(+)-dependent amino acid transporter, and to a lesser extent Na(+)-independent transporters play a role in the uptake of (14)C-FACBC by DU145 cells. Among the Na(+)-independent transporters, system L transporters are also involved in the transport of (14)C-FACBC. Moreover, (14)C-FACBC is not incorporated into proteins in cells. These findings suggest a possible mechanism of anti-(18)F-FACBC PET for prostate cancer.
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Ácidos Carboxílicos/metabolismo , Ciclobutanos/metabolismo , Espacio Intracelular/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Sistema de Transporte de Aminoácidos ASC/deficiencia , Sistema de Transporte de Aminoácidos ASC/genética , Sistema de Transporte de Aminoácidos ASC/metabolismo , Unión Competitiva , Transporte Biológico , Línea Celular Tumoral , Perfilación de la Expresión Génica , Humanos , Masculino , Antígenos de Histocompatibilidad Menor , Análisis de Secuencia por Matrices de Oligonucleótidos , Neoplasias de la Próstata/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genéticaRESUMEN
INTRODUCTION: We examined 3-[(123)I]iodo-alpha-methyl-L-tyrosine ([(123)I]IMT) uptake and inhibition by amino acids and amino acid-like drugs in the human DLD-1 colon cancer cell line, to discuss correlation between the inhibition effect and structure. METHODS: Expression of relevant neutral amino acid transporters was examined by real-time PCR with DLD-1 cells. The time course of [(125)I]IMT uptake, contributions of transport systems, concentration dependence and inhibition effects by amino acids and amino acid-like drugs (1 mM) on [(125)I]IMT uptake were examined. RESULTS: Expression of system L (4F2hc, LAT1 and LAT2), system A (ATA1, ATA2) and system ASC (ASCT1) was strongly detected; system L (LAT3, LAT4) and MCT8 were weakly detected; and B(0)AT was not detected. [(125)I]IMT uptake in DLD-1 cells involved Na(+)-independent system L primarily and Na(+)-dependent system(s). Uptake of [(125)I]IMT in Na(+)-free buffer followed Michaelis-Menten kinetics, with a K(m) of 78 microM and V(max) of 333 pmol/10(6) cells per minute. Neutral D- and L-amino acids with branched or aromatic large side chains inhibited [(125)I]IMT uptake. Tyrosine analogues, tryptophan analogues, L-phenylalanine and p-halogeno-L-phenylalanines, and gamma amino acids [including 3,4-dihydroxy-L-phenylalanine (L-DOPA), DL-threo-beta-(3,4-dihydroxyphenyl)serine (DOPS), 4-[bis(2-chloroethyl)amino]-L-phenylalanine and 1-(aminomethyl)-cyclohexaneacetic acid] strongly inhibited [(125)I]IMT uptake, but L-tyrosine methyl ester and R(+)/S(-)-baclofen weakly inhibited uptake. The substrates of system ASC and A did not inhibit [(125)I]IMT uptake except L-serine and D/L-cysteine. CONCLUSIONS: [(125)I]IMT uptake in DLD-1 cells involves mostly LAT1 and its substrates' (including amino acid-like drugs derived from tyrosine, tryptophan and phenylalanine) affinity to transport via LAT1. Whether transport of gamma amino acid analogues is involved in LAT1 depends on the structure of the group corresponding to the amino acid residue. Beta-hydroxylation may confer reduction of transport affinity of tyrosine analogues via LAT1.
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Aminoácidos/química , Aminoácidos/farmacología , Neoplasias del Colon/patología , Metiltirosinas/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Transporte Biológico , Línea Celular Tumoral , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Relación Estructura-ActividadRESUMEN
OBJECTIVES: Almost all D-glucose (GLU) filtered through the glomerulus is reabsorbed by the renal proximal tubules, whereas a high portion of 2-[18F]fluoro-2-deoxy-D-glucose [(18F)FDG] is excreted through the urine. However, [18F]FDG is not entirely excreted in the urine suggesting that it may be partially reabsorbed by the proximal tubules. The purpose of this study was to compare the time course of transcellular transport of administered [14C] labeled FDG ([14C]FDG) with that of [14C] labeled GLU ([14C]GLU) using the kidney epithelial cell line, LLC-PK1. METHODS: Transcellular transport of [14C]FDG and [14C]GLU by LLC-PK1 cells was measured in Na+-containing or Na+-free Dulbecco's phosphate-buffered saline [PBS(+) and PBS(-), respectively] in the presence or absence of phlorizin, phloretin, probenecid, or tetraethylammonium bromide inhibitors that predominantly inhibit sodium-dependent glucose transporters (SGLTs), sodium-independent glucose transporters, organic anion transporters, and organic cation transporters, respectively. RESULTS: When assayed in PBS(+), less [14C]FDG than [14C]GLU was reabsorbed by the proximal tubular cells over the entire incubation time. Reabsorption of [14C]FDG was mediated mainly by SGLT at early time points in the incubation, whereas high reabsorption of [14C]GLU was mediated by both SGLT and glucose transporter over 90 min of incubation. Secretion of [14C]FDG also tended to be slightly higher than that of [14C]GLU over 90 min of incubation. CONCLUSION: Transcellular transport of [14C]FDG over time by LLC-PK1 cells was clarified. The polarized distribution of transcellular transporters of [14C]FDG and [14C]GLU in LLC-PK1 cells differs.
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Células Epiteliales/metabolismo , Fluorodesoxiglucosa F18/metabolismo , Glucosa/metabolismo , Riñón/citología , Transporte Biológico/efectos de los fármacos , Línea Celular , Células Epiteliales/efectos de los fármacos , Proteínas Facilitadoras del Transporte de la Glucosa/antagonistas & inhibidores , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Túbulos Renales Proximales/citología , Transportadores de Anión Orgánico/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismo , Radioisótopos , Proteínas de Transporte de Sodio-Glucosa/antagonistas & inhibidores , Proteínas de Transporte de Sodio-Glucosa/metabolismo , Factores de TiempoRESUMEN
In Escherichia coli, several proteins whose synthesis is enhanced by polyamines at the level of translation have been identified. We looked for proteins that are similarly regulated in eukaryotes using a mouse mammary carcinoma FM3A cell culture system. Polyamine deficiency was induced by adding an inhibitor of ornithine decarboxylase, alpha-difluoromethylornithine, to the medium. Proteins enhanced by polyamines were determined by comparison of protein levels in control and polyamine-deficient cells using two-dimensional gel electrophoresis, and were identified by Edman degradation and/or LC/MALDI-TOF/TOF tandem mass spectrometry. Polyamine stimulation of the synthesis of these proteins at the level of translation was confirmed by measuring levels of the corresponding mRNAs and proteins, and levels of the [(35)S]methionine pulse-labeled proteins. The proteins identified in this way were T-complex protein 1, beta subunit (Cct2); heterogeneous nuclear ribonucleoprotein L (Hnrpl); and phosphoglycerate mutase 1 (Pgam1). Since Cct2 was most strongly enhanced by polyamines among three proteins, the mechanism of polyamine stimulation of Cct2 synthesis was studied using NIH3T3 cells transiently transfected with genes encoding Cct2-EGFP fusion mRNA with normal or mutated 5'-untranslated region (5'-UTR) of Cct2 mRNA. Polyamines most likely enhanced ribosome shunting on the 5'-UTR of Cct2 mRNA.