RESUMO
In this study, we investigated the synergistic effect of L-buthionine sulfoximine (BSO) on the chlorin e6 (Ce6)-based photodynamic therapy (PDT) of cancer cells. Among various cancer cells, HCT116 cells have highest intracellular L-glutathione (GSH) level and SNU478 cells showed the lowest GSH level. BSO alone showed negligible intrinsic cytotoxicity against CCD986sk cells. Since HCT116 and SNU478 cells showed the highest and the lowest intracellular GSH levels, respectively, those were used to test synergistic effect on the Ce6-based PDT. In the absence of light, BSO and Ce6 combination did not practically increase reactive oxygen species (ROS) in either of HCT116 or SNU478 cells, while light irradiation increased ROS level dose-dependently. 10 µM BSO treatment significantly depleted total GSH level in cancer cells, i.e. total GSH level decreased to one-fourth of the control in HCT116 cells while it decreased to two-fifth of the control treatment at SNU478 cell. BSO showed synergistic effect on the ROS production in HCT116 cells while it has practically no benefits in ROS production of SNU478 cells. No synergistic effect was observed in viability of SNU478 cells because BSO itself was cytotoxic to SNU478 cells. However, BSO had negligible cytotoxicity against HCT116 cells and showed synergistic anticancer effect in combination with Ce6-based PDT. Furthermore, the addition of glutathione reduced ethyl ester (GSH-OEt), recovered intracellular GSH level, and cell viability with reduced the intracellular ROS level. We suggest that synergistic effect of BSO in the Ce6-based PDT should be considered with intrinsic intracellular GSH level of cancer cells.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Butionina Sulfoximina/farmacologia , Fotoquimioterapia/métodos , Porfirinas/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Butionina Sulfoximina/uso terapêutico , Sobrevivência Celular/efeitos dos fármacos , Clorofilídeos , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Glutationa/metabolismo , Células HCT116 , Humanos , Camundongos , Porfirinas/uso terapêutico , Espécies Reativas de OxigênioRESUMO
Understanding the structural organization of organs and organisms at the cellular level is a fundamental challenge in biology. This task has been approached by reconstructing three-dimensional structure from images taken from serially sectioned tissues, which is not only labor-intensive and time-consuming but also error-prone. Recent advances in tissue clearing techniques allow visualization of cellular structures and neural networks inside of unsectioned whole tissues or the entire body. However, currently available protocols require long process times. Here, we present the rapid and highly reproducible ACT-PRESTO (active clarity technique-pressure related efficient and stable transfer of macromolecules into organs) method that clears tissues or the whole body within 1 day while preserving tissue architecture and protein-based signals derived from endogenous fluorescent proteins. Moreover, ACT-PRESTO is compatible with conventional immunolabeling methods and expedites antibody penetration into thick specimens by applying pressure. The speed and consistency of this method will allow high-content mapping and analysis of normal and pathological features in intact organs and bodies.
Assuntos
Acrilamida/química , Encéfalo/ultraestrutura , Fixadores/química , Formaldeído/química , Técnicas de Preparação Histocitológica , Polímeros/química , Animais , Encéfalo/anatomia & histologia , Encéfalo/fisiologia , Difusão , Eletroforese , Humanos , Hidrogéis/química , Imageamento Tridimensional , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Pressão , Coelhos , Ratos , Ratos Sprague-Dawley , Fixação de Tecidos/métodos , Xenopus , Peixe-ZebraRESUMO
In this study, the effect of chlorin e6-based photodynamic therapy (Ce6-PDT) was investigated in human intrahepatic (HuCC-T1) and extrahepatic (SNU1196) cholangiocarcinoma (CCA) cells. The amount of intracellular Ce6 increased with increasing Ce6 concentration administered, or with incubation time, in both cell lines. The ability to take up Ce6 and generate reactive oxygen species after irradiation at 1.0 J/cm(2) did not significantly differ between the two CCA cell types. However, after irradiation, marked differences were observed for photodamage and apoptotic/necrotic signals. HuCC-T1 cells are more sensitive to Ce6-PDT than SNU1196 cells. Total glutathione (GSH) levels, glutathione peroxidase and glutathione reductase activities in SNU1196 cells were significantly higher than in HuCC-T1 cells. With inhibition of enzyme activity or addition of GSH, the phototoxic effect could be controlled in CCA cells. The intracellular level of GSH is the most important determining factor in the curative action of Ce6-PDT against tumor cells.
Assuntos
Colangiocarcinoma/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/uso terapêutico , Porfirinas/farmacologia , Porfirinas/uso terapêutico , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Clorofilídeos , Colangiocarcinoma/patologia , Relação Dose-Resposta a Droga , Humanos , Fármacos Fotossensibilizantes/administração & dosagem , Porfirinas/administração & dosagem , Relação Estrutura-AtividadeRESUMO
CD137 is a member of the tumor necrosis factor/nerve growth factor receptor superfamily. Interaction of CD137 with its ligand (CD137L) affects the apoptosis, proliferation and differentiation of immune cells. Interestingly, the CD137 receptor/ligand system involves the bi-directional transduction of signals. The expression of CD137 and its ligand is not restricted to immune organs, but can also be detected in a wide variety of tissues such as the brain, kidney, lung and heart. However, its role in brain is largely unknown. This study was performed to determine the role of CD137L reverse signaling in the apoptosis of neural stem cells. We identified the expression of CD137 and its ligand in C17.2 neural stem cells derived from mouse embryonic cerebellum. We found that the activation of CD137L reverse signaling by CD137 resulted in a decrease in cell adhesion to the fibronectin-coated culture basement, thus causing detachment-induced cell death. Furthermore, we showed that the cell death induced by CD137 was completely ameliorated by integrin activators and caspase inhibitors. Therefore we suggest that CD137L reverse signaling exerts a pro-apoptotic effect by suppressing integrin-mediated survival signals in neural stem cells.
Assuntos
Ligante 4-1BB/metabolismo , Apoptose , Cerebelo/citologia , Células-Tronco Neurais/fisiologia , Transdução de Sinais , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Animais , Inibidores de Caspase/farmacologia , Adesão Celular , Diferenciação Celular , Linhagem Celular , Sobrevivência Celular , Cerebelo/embriologia , Fibronectinas/metabolismo , Integrinas/antagonistas & inibidores , Camundongos , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologiaRESUMO
PURPOSE: Nanoparticles based on stimuli-sensitive drug delivery have been extensively investigated for tumor targeting. Among them, pH-responsive drug targeting using pH-sensitive polymers has attracted attention because solid tumors have an acidic environment. A dextran-b-poly(L-histidine) (DexPHS) copolymer was synthesized and pH-responsive nanoparticles were fabricated for drug targeting. METHODS AND RESULTS: A DexPHS block copolymer was synthesized by attaching the reductive end of dextran to the amine groups of poly(L-histidine). pH-responsive nanoparticles incorporating doxorubicin were fabricated and studied in HuCC-T1 cholangiocarcinoma cells. Synthesis of DexPHS was confirmed by 1H nuclear magnetic resonance spectroscopy, with specific peaks of dextran and PHS observed at 2-5 ppm and 7.4-9.0 ppm, respectively. DexPHS nanoparticles showed changes in particle size with pH sensitivity, ie, the size of the nanoparticles increased at an acidic pH and decreased at a basic pH. DexPHS block copolymer nanoparticles incorporating doxorubicin were prepared using the nanoprecipitation dialysis method. The doxorubicin release rate was increased at acidic pH compared with basic pH, indicating that DexPHS nanoparticles have pH-sensitive properties and that drug release can be controlled by variations in pH. The antitumor activity of DexPHS nanoparticles incorporating doxorubicin were studied using HuCC-T1 cholangiocarcinoma cells. Viability was decreased in cells treated with nanoparticles at acidic pH, whereas cell viability in response to treatment with doxorubicin did not vary according to changes of pH. CONCLUSION: Our results indicated that DexPHS polymeric micelles are promising candidates for antitumor drug targeting.
Assuntos
Antineoplásicos/farmacocinética , Dextranos/química , Portadores de Fármacos/farmacocinética , Histidina/química , Nanopartículas/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/química , Doxorrubicina/farmacocinética , Doxorrubicina/farmacologia , Portadores de Fármacos/química , Humanos , Concentração de Íons de Hidrogênio , Nanomedicina , Nanopartículas/toxicidade , Tamanho da Partícula , Polímeros/químicaRESUMO
The aim of this study was to investigate the effect of the combination of vorinostat and epigallocatechin-3-gallate against HuCC-T1 human cholangiocarcinoma cells. A novel chemotherapy strategy is required as cholangiocarcinomas rarely respond to conventional chemotherapeutic agents. Both vorinostat and EGCG induce apoptosis and suppress invasion, migration, and angiogenesis of tumor cells. The combination of vorinostat and EGCG showed synergistic growth inhibitory effects and induced apoptosis in tumor cells. The Bax/Bcl-2 expression ratio and caspase-3 and -7 activity increased, but poly (ADP-ribose) polymerase expression decreased when compared to treatment with each agent alone. Furthermore, invasion, matrix metalloproteinase (MMP) expression, and migration of tumor cells decreased following treatment with the vorinostat and EGCG combination compared to those of vorinostat or EGCG alone. Tube length and junction number of human umbilical vein endothelial cells (HUVECs) decreased as well as vascular endothelial growth factor expression following vorinostat and EGCG combined treatment. These results indicate that the combination of vorinostat and EGCG had a synergistic effect on inhibiting tumor cell angiogenesis potential. We suggest that the combination of vorinostat and EGCG is a novel option for cholangiocarcinoma chemotherapy.
RESUMO
Chitosan was hydrophobically modified with ursodeoxycholic acid (UDCA) to fabricate nano-photosensitizer for photodynamic therapy (PDT) of HuCC-T1 cholangiocarcinoma cells. Synthesis of UDCA-conjugated chitosan (ChitoUDCA) was confirmed using (1)H NMR spectra. Chlorin E6 (Ce6) was used as a photosensitizer and incorporated into ChitoUDCA nanoparticles through formation of ion complexes. Morphology of Ce6-incorporated ChitoUDCA nanoparticles was observed using TEM and their shapes were spherical with sizes around 200-400 nm. The PDT potential of Ce6-incorporated ChitoUDCA nanoparticles were studied with HuCC-T1 human cholangiocarcinoma cells. The results showed that ChitoUDCA nanoparticles enhances of Ce6 uptake into tumor cells, phototoxicity, and ROS generation compared to Ce6 itself. Furthermore, Ce6-incorporated ChitoUDCA nanoparticles showed quenching in aqueous solution and sensing at tumor cells. We suggest that Ce6-incorporated ChitoUDCA nanoparticles are promising candidates for PDT of cholangiocarcinoma cells.
Assuntos
Neoplasias dos Ductos Biliares/patologia , Ductos Biliares Intra-Hepáticos/efeitos dos fármacos , Quitosana/química , Colangiocarcinoma/patologia , Portadores de Fármacos , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Porfirinas/farmacologia , Ácido Ursodesoxicólico/metabolismo , Neoplasias dos Ductos Biliares/metabolismo , Ductos Biliares Intra-Hepáticos/metabolismo , Ductos Biliares Intra-Hepáticos/patologia , Transporte Biológico , Linhagem Celular Tumoral , Química Farmacêutica , Clorofilídeos , Colangiocarcinoma/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Microscopia Eletrônica de Transmissão , Tamanho da Partícula , Fármacos Fotossensibilizantes/química , Porfirinas/química , Espécies Reativas de Oxigênio/metabolismo , Tecnologia Farmacêutica/métodos , Ácido Ursodesoxicólico/químicaRESUMO
5-Aminolevulinic acid (ALA)-based photodynamic therapy (PDT) has the potential to kill cancer cells via apoptotic or necrotic signals that are dependent on the generation of intracellular reactive oxygen species (ROS). Celecoxib is an anti-inflammatory drug that induces intracellular ROS generation. We investigated whether the combined application of celecoxib and ALA-PDT improved the efficacy of PDT in human cholangiocarcinoma cells and in tumor bearing mice. In vitro, combined treatment of celecoxib and ALA-PDT increased phototoxicity and intracellular ROS levels after irradiation with 0.75 J/cm(2) when compared to ALA-PDT alone. Even though ROS levels increased with 0.25 J/cm(2) of irradiation, it did not influence phototoxicity. When heme oxygenase-1, a defensive protein induced by oxidative stress, was inhibited in the combined treatment group, phototoxicity was increased at both 0.25 J/cm(2) and 0.75 J/cm(2) of irradiation. We identified the combined effect of ALA-PDT and celecoxib through the increase of oxidative stress such as ROS. In vivo, about 40% tumor growth inhibition was observed with combined application of ALA-PDT and celecoxib when compared to ALA-PDT alone. The combined application of ALA-PDT and celecoxib could be an effective therapy for human cholangiocarcinoma. Moreover, use of a heme oxygenase-1 inhibitor with PDT could play an important role for management of various tumors involving oxidative stress.
Assuntos
Ácido Aminolevulínico/farmacologia , Colangiocarcinoma , Estresse Oxidativo/efeitos dos fármacos , Fotoquimioterapia/métodos , Pirazóis/farmacologia , Sulfonamidas/farmacologia , Ácido Aminolevulínico/química , Análise de Variância , Animais , Neoplasias dos Ductos Biliares , Ductos Biliares Intra-Hepáticos , Celecoxib , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Heme Oxigenase-1/análise , Heme Oxigenase-1/metabolismo , Humanos , Masculino , Camundongos Endogâmicos BALB C , Pirazóis/química , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/metabolismo , Sulfonamidas/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Cholangiocarcinoma is a malignant tumor arising from the epithelium of the bile ducts. In this study, we prepared sorafenib-loaded biliary stents for potential application as drug-delivery systems for localized treatment of extrahepatic cholangiocarcinoma. METHODS: A sorafenib-coated metal stent was prepared using an electrospray system with the aid of poly(ε-caprolactone) (PCL), and then its anticancer activity was investigated using human cholangiocellular carcinoma (HuCC)-T1 cells in vitro and a mouse tumor xenograft model in vivo. Anticancer activity of sorafenib against HuCC-T1 cells was evaluated by the proliferation test, matrix metalloproteinase (MMP) activity, cancer cell invasion, and angiogenesis assay in vitro and in vivo. RESULTS: The drug-release study showed that the increased drug content on the PCL film induced a faster drug-release rate. The growth of cancer cells on the sorafenib-loaded PCL film surfaces decreased in a dose-dependent manner. MMP-2 expression of HuCC-T1 cells gradually decreased according to sorafenib concentration. Furthermore, cancer cell invasion and tube formation of human umbilical vein endothelial cells significantly decreased at sorafenib concentrations higher than 10 mM. In the mouse tumor xenograft model with HuCC-T1 cells, sorafenib-eluting PCL films significantly inhibited the growth of tumor mass and induced apoptosis of tumor cells. Various molecular signals, such as B-cell lymphoma (Bcl)-2, Bcl-2-associated death promoter, Bcl-x, caspase-3, cleaved caspase-3, Fas, signal transducer and activator of transcription 5, extracellular signal-regulated kinases, MMP-9 and pan-janus kinase/stress-activated protein kinase 1, indicated that apoptosis, inhibition of growth and invasion was cleared on sorafenib-eluting PCL films. CONCLUSION: These sorafenib-loaded PCL films are effective in inhibiting angiogenesis, proliferation and invasion of cancer cells. We suggest that sorafenib-loaded PCL film is a promising candidate for the local treatment of cholangiocarcinoma.
Assuntos
Antineoplásicos/farmacologia , Neoplasias dos Ductos Biliares , Ductos Biliares Intra-Hepáticos , Colangiocarcinoma , Stents Farmacológicos , Niacinamida/análogos & derivados , Compostos de Fenilureia/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Camundongos Nus , Niacinamida/química , Niacinamida/farmacocinética , Niacinamida/farmacologia , Compostos de Fenilureia/química , Compostos de Fenilureia/farmacocinética , Poliésteres , Sorafenibe , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Hexyl-aminolevulinic acid (HALA) was compared with aminolevulinic acid (ALA) in terms of improving ALA-based photodynamic therapy (PDT) for human intra- and extrahepatic cholangiocarcinoma (CCA) HuCC-T1 and SNU1196 cells. Because of the different uptake mechanisms of HALA, a relatively higher amount of protoporphyrin IX (PpIX) was induced in the both CCA cell types at low concentrations of HALA. Furthermore, higher expression of porphobilinogen deaminase, coproporphyrinogen III oxidase, and protoporphyrinogen oxidase, the key enzymes for synthesizing PpIX in the heme biosynthetic pathway, facilitated the exuberant generation of PpIX in HuCC-T1 cells. PpIX accumulation with ALA was markedly different between the two CCA cell types. Even at lower concentrations of ALA, SNU1196 cell successfully synthesized PpIX, due to the higher expression of the ALA transporter, mammalian H (+)/peptide co-transporter PEPT1. Considering the difference of PEPT1 or key enzyme expression, HALA could be a very effective substitute for ALA in doing PDT for cure of CCA.
Assuntos
Ácido Aminolevulínico/análogos & derivados , Colangiocarcinoma/tratamento farmacológico , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Ácido Aminolevulínico/administração & dosagem , Ácido Aminolevulínico/química , Ácido Aminolevulínico/farmacologia , Neoplasias dos Ductos Biliares/tratamento farmacológico , Neoplasias dos Ductos Biliares/patologia , Ductos Biliares Intra-Hepáticos/patologia , Linhagem Celular Tumoral , Colangiocarcinoma/patologia , Relação Dose-Resposta a Droga , Humanos , Transportador 1 de Peptídeos , Fármacos Fotossensibilizantes/administração & dosagem , Fármacos Fotossensibilizantes/química , Protoporfirinas/metabolismo , Simportadores/genéticaRESUMO
We have recently found that thyroid transcription factor-1 (TTF-1), a homeodomain-containing transcription factor, is postnatally expressed in discrete areas of the hypothalamus and closely involved in neuroendocrine functions. We now report that transcription of cyclooxygenase-2 (COX-2), the rate limiting enzyme in prostaglandin biosynthesis, was inhibited by TTF-1. Double immunohistochemistry demonstrated that TTF-1 was expressed in the astrocytes and endothelial cells of blood vessel in the hypothalamus. Promoter assays and electrophoretic mobility shift assays showed that TTF-1 inhibited COX-2 transcription by binding to specific binding domains in the COX-2 promoter. Furthermore, blocking TTF-1 synthesis by intracerebroventricular injection of an antisense oligomer induced an increase of COX-2 synthesis in non-neuronal cells of the rat hypothalamus, and resulted in animals' hyperthermia. These results suggest that TTF-1 is physiologically involved in the control of thermogenesis by regulating COX-2 transcription in the brain.