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1.
J Pharmacol Sci ; 133(2): 96-102, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28242177

RESUMO

L-type amino acid transporter 1 (LAT1) is known as a cancer-type amino acid transporter. In cancer cells, LAT1 is responsible for the cellular uptake of many essential amino acids including leucine that activates mechanistic/mammalian target of rapamycin (mTOR), regulating cancer cell growth. In this study, we designed a novel series of LAT1 inhibitors, SKN101-105, based on the structure of triiodothyronine (T3), a known LAT1 blocker. The compounds consist of core structure of 2-amino-3-[3,5-dichloro-4-(naphthalene-1-methoxy)-phenyl]-propanoic acid and different modifications on the naphthalene. Among them, the compounds including SKN103 with a modified phenyl group at C-7 position of naphthalene inhibited LAT1-mediated leucine transport, whereas SKN102 with a phenyl group at C-6 position did not, indicating the importance of the position of substituents on the naphthalene for the interaction with LAT1. SKN103 was suggested to be a non-transportable blocker rather than a substrate of LAT1 and inhibited LAT1 in a competitive manner with the Ki value of 2.1 µM. SKN103 suppressed mTOR activity and the growth of cancer cells. Moreover, SKN103 in combination with cisplatin additively enhanced the growth inhibition in cancer cells. This study provides an additional insight into the structure-activity relationship of LAT1 ligands, which could lead to designing desirable LAT1 inhibitors.


Assuntos
Transportador 1 de Aminoácidos Neutros Grandes/química , Tri-Iodotironina/análogos & derivados , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Cisplatino/farmacologia , Humanos , Leucina/metabolismo , Estrutura Molecular , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Serina-Treonina Quinases TOR/metabolismo , Tri-Iodotironina/química
2.
J Virol ; 89(17): 9080-9, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26109726

RESUMO

UNLABELLED: Human T-cell leukemia virus type 1 (HTLV-1) is associated with adult T-cell leukemia (ATL) and transforms T cells in vitro. To our knowledge, the functional role of reactive oxygen species (ROS)-generating NADPH oxidase 5 (Nox5) in HTLV-1 transformation remains undefined. Here, we found that Nox5α expression was upregulated in 88% of 17 ATL patient samples but not in normal peripheral blood T cells. Upregulation of the Nox5α variant was transcriptionally sustained by the constitutive Janus family tyrosine kinase (Jak)-STAT5 signaling pathway in interleukin-2 (IL-2)-independent HTLV-1-transformed cell lines, including MT1 and MT2, whereas it was transiently induced by the IL-2-triggered Jak-STAT5 axis in uninfected T cells. A Nox inhibitor, diphenylene iodonium, and antioxidants such as N-acetyl cysteine blocked proliferation of MT1 and MT2 cells. Ablation of Nox5α by small interfering RNAs abrogated ROS production, inhibited cellular activities, including proliferation, migration, and survival, and suppressed tumorigenicity in immunodeficient NOG mice. The findings suggest that Nox5α is a key molecule for redox-signal-mediated maintenance of the HTLV-1 transformation phenotype and could be a potential molecular target for therapeutic intervention in cancer development. IMPORTANCE: HTLV-1 is the first human oncogenic retrovirus shown to be associated with ATL. Despite the extensive study over the years, the mechanism underlying HTLV-1-induced cell transformation is not fully understood. In this study, we addressed the expression and function of ROS-generating Nox family genes in HTLV-1-transformed cells. Our report provides the first evidence that the upregulated expression of Nox5α is associated with the pathological state of ATL peripheral blood mononuclear cells and that Nox5α is an integral component of the Jak-STAT5 signaling pathway in HTLV-1-transformed T cells. Nox5α-derived ROS are critically involved in the regulation of cellular activities, including proliferation, migration, survival, and tumorigenicity, in HTLV-1-transformed cells. These results indicate that Nox5α-derived ROS are functionally required for maintenance of the HTLV-1 transformation phenotype. The finding provides new insight into the redox-dependent mechanism of HTLV-1 transformation and raises an intriguing possibility that Nox5α serves as a potential molecular target to treat HTLV-1-related leukemia.


Assuntos
Transformação Celular Viral/genética , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Leucemia-Linfoma de Células T do Adulto/patologia , Proteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Acetilcisteína/farmacologia , Linhagem Celular Transformada , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/genética , Transformação Celular Neoplásica/genética , Humanos , Interleucina-2/metabolismo , Janus Quinases/metabolismo , Leucemia-Linfoma de Células T do Adulto/virologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , NADPH Oxidase 5 , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/genética , Oniocompostos/farmacologia , Interferência de RNA , RNA Interferente Pequeno , Fator de Transcrição STAT5/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Regulação para Cima
3.
Int Arch Allergy Immunol ; 161 Suppl 2: 44-51, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23711853

RESUMO

BACKGROUND: Zinc (Zn) affects many aspects of immune function, including thymic development and the activities of immune cells. Zn is also involved in many steps of high-affinity IgE receptor (FcεRI)-induced mast cell (MC) activation, which is required for degranulation and cytokine production. Intracellular Zn levels increase in mouse MCs after FcεRI stimulation. We previously reported that Zn distribution in a human MC line, LAD2, changed dramatically following FcεRI aggregation with synchrotron radiation microbeams. However, the kinetics of Zn distribution and the underlying mechanisms following FcεRI cross-linking remain unknown. METHODS: We used cord-blood-derived MCs and LAD2 cells. Degranulation was assessed by ß-hexosaminidase (ß-hex) release. Extracellular Zn levels were determined by inductively coupled plasma atomic emission spectrometry or based on the fluorescence intensity of a Zn indicator. We also used RNAi to knockdown ZnT1 expression. mRNA expression levels were determined by real-time RT-PCR. RESULTS: Zn was rapidly released from human MCs after FcεRI aggregation. The kinetics and optimal conditions for FcεRI cross-linking for Zn release were different from those for degranulation. Treating LAD2 cells with an intracellular Ca(2+) chelator significantly inhibited IgE-mediated ß-hex release but not Zn release. We investigated IgE-mediated ß-hex and Zn release with specific inhibitors of signaling pathways. Zn and ß-hex release were partly correlated with but also partly independent of IgE. Knockdown of the Zn efflux transporter, ZnT1, significantly inhibited Zn release from human MCs. CONCLUSIONS: Our results indicate that IgE-dependent Zn release from human MCs involves signaling cascades that are distinct from those of degranulation. Thus, Zn may have a unique function as a mediator of allergic inflammation.


Assuntos
Imunoglobulina E/imunologia , Mastócitos/imunologia , Mastócitos/metabolismo , Receptores de IgE/metabolismo , Zinco/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/imunologia , Degranulação Celular/genética , Degranulação Celular/imunologia , Linhagem Celular , Células Cultivadas , Regulação da Expressão Gênica , Liberação de Histamina , Humanos , Interferência de RNA
4.
J Nucl Med ; 64(12): 1949-1955, 2023 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-37827841

RESUMO

Glypican-1 (GPC1) is overexpressed in several solid cancers and is associated with tumor progression, whereas its expression is low in normal tissues. This study aimed to evaluate the potential of an anti-GPC1 monoclonal antibody (GPC1 mAb) labeled with 89Zr or 211At as a theranostic target in pancreatic ductal adenocarcinoma. Methods: GPC1 mAb clone 01a033 was labeled with 89Zr or 211At with a deferoxamine or decaborane linker, respectively. The internalization ability of GPC1 mAb was evaluated by fluorescence conjugation using a confocal microscope. PANC-1 xenograft mice (n = 6) were intravenously administered [89Zr]GPC1 mAb (0.91 ± 0.10 MBq), and PET/CT scanning was performed for 7 d. Uptake specificity was confirmed through a comparative study using GPC1-positive (BxPC-3) and GPC1-negative (BxPC-3 GPC1-knockout) xenografts (each n = 3) and a blocking study. DNA double-strand breaks were evaluated using the γH2AX antibody. The antitumor effect was evaluated by administering [211At]GPC1 mAb (∼100 kBq) to PANC-1 xenograft mice (n = 10). Results: GPC1 mAb clone 01a033 showed increased internalization ratios over time. One day after administration, a high accumulation of [89Zr]GPC1 mAb was observed in the PANC-1 xenograft (SUVmax, 3.85 ± 0.10), which gradually decreased until day 7 (SUVmax, 2.16 ± 0.30). The uptake in the BxPC-3 xenograft was significantly higher than in the BxPC-3 GPC1-knockout xenograft (SUVmax, 4.66 ± 0.40 and 2.36 ± 0.36, respectively; P = 0.05). The uptake was significantly inhibited in the blocking group compared with the nonblocking group (percentage injected dose per gram, 7.3 ± 1.3 and 12.4 ± 3.0, respectively; P = 0.05). DNA double-strand breaks were observed by adding 150 kBq of [211At]GPC1 and were significantly suppressed by the internalization inhibitor (dynasore), suggesting a substantial contribution of the internalization ability to the antitumor effect. Tumor growth suppression was observed in PANC-1 mice after the administration of [211At]GPC1 mAb. Internalization inhibitors (prochlorperazine) significantly inhibited the therapeutic effect of [211At]GPC1 mAb, suggesting an essential role in targeted α-therapy. Conclusion: [89Zr]GPC1 mAb PET showed high tumoral uptake in the early phase after administration, and targeted α-therapy using [211At]GPC1 mAb showed tumor growth suppression. GPC1 is a promising target for future applications for the precise diagnosis of pancreatic ductal adenocarcinoma and GPC1-targeted theranostics.


Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Animais , Camundongos , Glipicanas/metabolismo , Tomografia por Emissão de Pósitrons , Medicina de Precisão , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Linhagem Celular Tumoral , Neoplasias Pancreáticas/diagnóstico por imagem , Neoplasias Pancreáticas/terapia , Neoplasias Pancreáticas/patologia , Carcinoma Ductal Pancreático/diagnóstico por imagem , Carcinoma Ductal Pancreático/terapia , DNA , Zircônio
5.
Biochem Biophys Res Commun ; 416(3-4): 239-45, 2011 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-22033412

RESUMO

Ecotropic viral integration site-1 (EVI1) is an oncogenic transcription factor in human acute myeloid leukemia (AML) associated with poor prognosis. Because the drug-resistance of leukemia cells is partly dependent on cell quiescence in the bone marrow niche, EVI1 may be involved in cell cycle regulation in leukemia cells. As a candidate regulator of the cell cycle in leukemia cells with high EVI1 expression (EVI1(high)), we analyzed angiopoietin1 (Ang1), which is a down-regulated gene in EVI1-deficient mice and is involved in the quiescence of hematopoietic stem cells. The results of real-time PCR analyses showed that Ang1 is highly expressed in leukemia cell lines and primary AML cells with EVI1(high) expression. Introduction of shRNA against EVI1 into EVI1(high) leukemia cells down-regulated Ang1 expression. Moreover, knockdown of Ang1 in EVI1(high) leukemia cells promoted cell cycle progression and down-regulated the CDK inhibitor p18 (INK4c). Treatment with a decoy Tie2/Fc protein also down-regulated the expression of p18. These results suggest that Ang1/Tie2 signaling may suppress cell cycle progression via maintenance of G0/G1 phase through up-regulation of p18 expression. This mechanism may help to maintain EVI1(high) leukemia cells in the bone marrow niche and promote resistance to anti-cancer drugs.


Assuntos
Angiopoietina-1/genética , Ciclo Celular/genética , Proteínas de Ligação a DNA/metabolismo , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p18/genética , Proteínas de Ligação a DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Ensaios de Seleção de Medicamentos Antitumorais , Técnicas de Silenciamento de Genes , Humanos , Proteína do Locus do Complexo MDS1 e EVI1 , Camundongos , Proto-Oncogenes/genética , Fatores de Transcrição/genética
6.
Sci Rep ; 11(1): 12982, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34155314

RESUMO

Astatine-211 (211At)-labeled phenylalanine is expected to be a promising agent for targeted alpha-particle therapy for the treatment of patients with glioma. The existing reactions to prepare the labeled compound usually require organic solvents and metals that are toxic and hazardous to the environment. In this study, we developed a novel method wherein astatination was realized via the substitution of 211At for a dihydroxyboryl group coupled to phenylalanine. [211At]4-astato-L-phenylalanine was obtained as the carrier-free product in aqueous medium in high radiochemical yields (98.1 ± 1.9%, n = 5). The crude reaction mixture was purified by solid-phase extraction, and the radiochemical purity of the product was 99.3 ± 0.7% (n = 5). The high yield and purity were attributed to the formation of [211At]AtI and AtI2- as the reactive intermediates in the astatination reaction. The reaction did not require any organic solvents or toxic reagents, suggesting that this method is suitable for clinical applications.

7.
PLoS One ; 7(1): e30706, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22295105

RESUMO

Ecotropic viral integration site-1 (EVI1) is one of the candidate oncogenes for human acute myeloid leukemia (AML) with chromosomal alterations at 3q26. High EVI1 expression (EVI1(high)) is a risk factor for AML with poor outcome. Using DNA microarray analysis, we previously identified that integrin α6 (ITGA6) was upregulated over 10-fold in EVI1(high) leukemia cells. In this study, we determined whether the increased expression of ITGA6 is associated with drug-resistance and increased cell adhesion, resulting in poor prognosis. To this end, we first confirmed the expression pattern of a series of integrin genes using semi-quantitative PCR and fluorescence-activated cell sorter (FACS) analysis and determined the cell adhesion ability in EVI1(high) leukemia cells. We found that the adhesion ability of EVI1(high) leukemia cells to laminin increased with the increased expression of ITGA6 and integrin ß4 (ITGB4). The introduction of small-hairpin RNA against EVI1 (shEVI1) into EVI1(high) leukemia cells reduced the cell adhesion ability and downregulated the expression of ITGA6 and ITGB4. In addition, the overexpression of EVI1 in EVI1(low) leukemia cells enhanced their cell adhesion ability and increased the expression of ITGA6 and ITGB4. In a subsequent experiment, the introduction of shRNA against ITGA6 or ITGB4 into EVI1(high) AML cells downregulated their cell adhesion ability; however, the EVI1(high) AML cells transfected with shRNA against ITGA6 could not be maintained in culture. Moreover, treating EVI1(high) leukemia cells with neutralizing antibodies against ITGA6 or ITGB4 resulted in an enhanced responsiveness to anti-cancer drugs and a reduction of their cell adhesion ability. The expression of ITGA6 is significantly elevated in cells from relapsed and EVI1(high) AML cases; therefore, ITGA6 might represent an important therapeutic target for both refractory and EVI1(high) AML.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Integrina alfa6/genética , Integrina alfa6/metabolismo , Leucemia Mieloide Aguda/patologia , Fatores de Transcrição/metabolismo , Células 3T3 , Animais , Anticorpos Neutralizantes/imunologia , Bovinos , Adesão Celular , Moléculas de Adesão Celular/metabolismo , Ciclo Celular , Linhagem Celular Tumoral , Colágeno/metabolismo , Regulação para Baixo , Combinação de Medicamentos , Inativação Gênica , Humanos , Integrina alfa6/imunologia , Integrina beta4/genética , Integrina beta4/imunologia , Integrina beta4/metabolismo , Laminina/metabolismo , Leucemia Mieloide Aguda/genética , Proteína do Locus do Complexo MDS1 e EVI1 , Camundongos , Osteoblastos/citologia , Osteoblastos/metabolismo , Proteoglicanas/metabolismo , Proto-Oncogenes , RNA Interferente Pequeno/genética , Recidiva , Calinina
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