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1.
J Biol Chem ; 290(20): 12547-57, 2015 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-25829491

RESUMO

It is widely known that ion channels are expressed in the plasma membrane. However, a few studies have suggested that several ion channels including voltage-gated K(+) (Kv) channels also exist in intracellular organelles where they are involved in the biochemical events associated with cell signaling. In the present study, Western blot analysis using fractionated protein clearly indicates that Kv1.3 channels are expressed in the nuclei of MCF7, A549, and SNU-484 cancer cells and human brain tissues. In addition, Kv1.3 is located in the plasma membrane and the nucleus of Jurkat T cells. Nuclear membrane hyperpolarization after treatment with margatoxin (MgTX), a specific blocker of Kv1.3 channels, provides evidence for functional channels at the nuclear membrane of A549 cells. MgTX-induced hyperpolarization is abolished in the nuclei of Kv1.3 silenced cells, and the effects of MgTX are dependent on the magnitude of the K(+) gradient across the nuclear membrane. Selective Kv1.3 blockers induce the phosphorylation of cAMP response element-binding protein (CREB) and c-Fos activation. Moreover, Kv1.3 is shown to form a complex with the upstream binding factor 1 in the nucleus. Chromatin immunoprecipitation assay reveals that Sp1 transcription factor is directly bound to the promoter region of the Kv1.3 gene, and the Sp1 regulates Kv1.3 expression in the nucleus of A549 cells. These results demonstrate that Kv1.3 channels are primarily localized in the nucleus of several types of cancer cells and human brain tissues where they are capable of regulating nuclear membrane potential and activation of transcription factors, such as phosphorylated CREB and c-Fos.


Assuntos
Encéfalo/metabolismo , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Canal de Potássio Kv1.3/metabolismo , Potenciais da Membrana/fisiologia , Encéfalo/citologia , Membrana Celular/genética , Núcleo Celular/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Inativação Gênica , Humanos , Células Jurkat , Canal de Potássio Kv1.3/antagonistas & inibidores , Canal de Potássio Kv1.3/genética , Potenciais da Membrana/efeitos dos fármacos , Fosforilação , Venenos de Escorpião/farmacologia , Fator de Resposta Sérica/genética , Fator de Resposta Sérica/metabolismo , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo
2.
Anal Chem ; 84(5): 2172-8, 2012 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-22280519

RESUMO

We investigated in vitro and in vivo glutathione (GSH)-induced intracellular thiopurine anticancer drug release on gold nanoparticle (Au NP) surfaces by means of label-free confocal Raman spectroscopy. Direct monitoring of GSH-triggered release of 6-mercaptopurine (6MP) and 6-thioguanine (6TG) was achieved in real time. Live cell imaging technique provides a nanomolar range release of 6MP and 6TG from Au NP surfaces after the injection of external GSH. In vivo SERS spectra of 6TG were obtained from the subcutaneous sites in living mice after GSH treatment. GSH-triggered releases of Cy5-dye assembled on 6TG-capped Au NPs were also compared using independent fluorescence measurements. Our work demonstrates that the time-lapse Raman spectroscopic tools are useful for monitoring of the controlled release of thiopurine drug molecules in vitro and in vivo.


Assuntos
Antineoplásicos/análise , Glutationa/metabolismo , Mercaptopurina/análise , Análise Espectral Raman , Tioguanina/análise , Animais , Carbocianinas/química , Linhagem Celular Tumoral , Ouro/química , Humanos , Masculino , Nanopartículas Metálicas/química , Camundongos , Camundongos Nus , Pele/química
4.
Anticancer Res ; 32(12): 5279-84, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23225427

RESUMO

Voltage-gated K(+) (Kv) channels are known to be associated with the proliferation of several types of cancer cells, including lung adenocarcinoma cells, and certain Kv channel blockers inhibit cancer cell proliferation. In the present study, we investigated the effects of Kv channel blockers in gefitinib-resistant H460 non-small cell lung cancer (NSCLC) cells. Treatment with dendrotoxin-κ (DTX-κ), which is a Kv1.1-specific blocker, reduced H460 cell viability and arrested cells in G(1)/S transition during cell-cycle progression. We administered DTX-κ in a xenograft model using nude mice. The tumor volume was reduced by the injection of DTX-κ into the tumor tissues compared to the control group. These results indicate that DTX-κ has antitumor effects in gefitinib-resistant H460 cells through the pathway governing the G(1)/S transition both in vitro and in vivo. These findings suggest that Kv1.1 could serve as a novel therapeutic target for gefitinib-resistant NSCLC.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Canal de Potássio Kv1.1/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Peptídeos/farmacologia , Quinazolinas/farmacologia , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Ciclo Celular/efeitos dos fármacos , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Gefitinibe , Humanos , Canal de Potássio Kv1.1/biossíntese , Canal de Potássio Kv1.1/genética , Canal de Potássio Kv1.1/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Nus , Peptídeos/administração & dosagem , Quinazolinas/administração & dosagem , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Talanta ; 88: 631-7, 2012 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-22265551

RESUMO

The anticancer drug doxorubicin (DOX) appeared to adsorb efficiently on TiO(2) nanoparticles (NPs) as evidenced by visible absorption and diffuse reflectance infrared spectroscopy data. The adsorbed drugs were found released in a controlled way by external glutathione (GSH). Fluorescence of DOX appeared to be quenched substantially by TiO(2) NPs. The fabrication and release of DOX on TiO(2) NPs were checked by monitoring the fluorescence. We could monitor real-time drug release in the live cell using fluorescence imaging techniques. By these methods, we were able to monitor up to a nanomolar amount of DOX release in vitro from TiO(2) NPs triggered by external GSH. In vivo fluorescence images of DOX were obtained from the subcutaneous site in living mice after GSH treatment. On the basis of label-free fluorescence quenching measurements, a real-time release of DOX on TiO(2) NPs can be monitored in vitro and in vivo after an external trigger of GSH.


Assuntos
Antineoplásicos/farmacocinética , Doxorrubicina/farmacocinética , Titânio , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Preparações de Ação Retardada , Portadores de Fármacos , Glutationa/metabolismo , Humanos , Injeções Subcutâneas , Masculino , Camundongos , Camundongos Nus , Microscopia Eletrônica de Transmissão , Imagem Molecular , Nanopartículas , Espectrometria de Fluorescência
6.
J Biomed Mater Res A ; 100(5): 1221-8, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22359274

RESUMO

We investigate the cellular uptake behaviors and efficacy of folate-coated gold nanoparticles (AuNPs) for the targeted drug delivery system in human cancer cells. Folate-conjugated AuNPs embedded with a purine analogue cancer drug of 6-mercaptopurine (6MP) were assembled via a 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) coupling reaction between the amino group of 4-aminobenzenethiol (ABT) and the carboxyl group of folic acid. The assembly of folate and 6MP on AuNPs has been examined by absorption spectroscopy, transmission electron microscopy (TEM), and confocal Raman spectroscopy. The internalization of the conjugated AuNPs inside the folate receptor-positive HeLa and KB cells was checked by TEM and dark-field microscopy (DFM) combined with label-free confocal spectroscopy over the depth variable z at a micrometer resolution. DFM live cell imaging of folate-conjugated AuNPs in HeLa cells indicated that the targeted AuNPs appeared to attach on the cell surfaces and enter into the cell with an hour. The cell viability was also compared to estimate the efficacy of folate-conjugated AuNP delivery systems. Folate receptor-targeted AuNP systems appeared to decrease cancer cell viability both in vitro and in vivo more than did the use of the 6MP-coated AuNPs drug without any targeting systems.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Receptores de Folato com Âncoras de GPI/metabolismo , Ouro/química , Mercaptopurina/farmacologia , Nanopartículas Metálicas/química , Análise Espectral Raman/métodos , Absorção/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Ácido Fólico/metabolismo , Células HeLa , Humanos , Masculino , Nanopartículas Metálicas/ultraestrutura , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus
7.
J Hazard Mater ; 198: 347-55, 2011 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-22088504

RESUMO

Photocytotoxicity of visible-light catalytic Ag/AgBr/TiO(2) nanoparticles (NPs) was examined both in vitro and in vivo. The Ag/AgBr/TiO(2) NPs were prepared by the deposition-precipitation method. Their crystalline structures, atomic compositions, and light absorption property were examined by X-ray diffraction (XRD) patterns, X-ray photoelectron (XPS) intensities, and ultraviolet-visible (UV-vis) diffuse reflectance spectroscopic tools. The Ag/AgBr/TiO(2) NPs appeared to be well internalized in human carcinoma cells as evidenced by transmission electron microscopy (TEM). The cytotoxicity of cetylmethylammonium bromide (CTAB) appeared to be significantly reduced by adsorption of serum proteins in the cellular medium on the NP surfaces. Two types of human cervical HeLa and skin A431 cancer cells were tested to check their viability after the cellular uptake of the Ag/AgBr/TiO(2) NPs and subsequent exposure to an illumination of visible light from a 60 W/cm(2) halogen lamp. Fluorescence images taken to label mitochondria activity suggest that the reactive oxygen species should trigger the photo-destruction of cancer cells. After applying the halogen light illumination for 50-250 min and ∼8 ppm (µg/mL) of photocatalytic Ag/AgBr/TiO(2) NPs, we observed a 40-60% selective decrease of cell viability. Ag/AgBr/TiO(2) NPs were found to eliminate xenograft tumors significantly by irradiating visible light in vivo for 10 min.


Assuntos
Proteínas Sanguíneas/química , Brometos/química , Luz , Nanopartículas Metálicas , Compostos de Prata/química , Prata/química , Titânio/química , Adsorção , Animais , Catálise , Células HeLa , Humanos , Camundongos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Fotoquímica , Difração de Raios X
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