RESUMO
Transforming growth factor (TGF)-ß is a pro-oncogenic cytokine that induces the epithelial-mesenchymal transition (EMT), a crucial event in tumor progression. During TGF-ß-mediated EMT in NMuMG mouse mammary epithelial cells, we observed sustained increases in reactive oxygen species (ROS) levels in the cytoplasm and mitochondria with a concomitant decrease in mitochondrial membrane potential and intracellular glutathione levels. In pseudo ρ0 cells, whose respiratory chain function was impaired, the increase in intracellular ROS levels was abrogated, suggesting an important role of mitochondrial activity as a trigger for TGF-ß-stimulated ROS generation. In line with this, TGF-ß-mediated expression of the EMT marker fibronectin was inhibited not only by chemicals that interfere with ROS signaling but also by exogenously expressed mitochondrial thioredoxin (TXN2) independent of Smad signaling. Of note, TGF-ß-mediated induction of HMGA2, a central mediator of EMT and metastatic progression, was similarly impaired by TXN2 expression, revealing a novel mechanism involving a thiol oxidation reaction in mitochondria, which regulates TGF-ß-mediated gene expression associated with EMT.
Assuntos
Transição Epitelial-Mesenquimal/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Tiorredoxinas/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Animais , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Células HEK293 , Proteínas HMGA/metabolismo , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Glândulas Mamárias Animais/citologia , Camundongos , Mitocôndrias/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
Radionuclide imaging and therapy are promising methods for controlling systemic cancers; however, their clinical application has been limited by excessive radionuclide accumulation in healthy tissues. To minimize radionuclide accumulation in non-cancerous tissues while ensuring sufficient build up in tumors, we aimed to develop a method that controlled the in vivo dynamics of radionuclides post-administration. To this end, we describe a novel strategy that combines liposomes, a potent carrier system for drug delivery, with unique radionuclide-ligand complexes based on 111In-ethylenedicysteine. Conventional 111In-ligand-complexes-carrying liposomes delivered substantial amounts of radionuclides to tumors; however, they also accumulated in the liver and spleen. In contrast, 111In-ethylenedicysteine-carrying liposomes greatly reduced non-specific accumulation, while being retained selectively at high doses within tumors. Liposomes were rapidly broken down in the liver, releasing encapsulated 111In-ligand complexes. Among the chelates used, only 111In-ethylenedicysteine could escape from the liver and be excreted in the urine. Instead, most liposomes remained intact in tumors, retaining the radionuclide-ligand complexes within them. Therefore, high tumor accumulation was obtained regardless of the type of 111In-ligand complexes in the liposomes. In vivo single photon emission computed tomography/computed tomography imaging with 111In-ethylenedicysteine-carrying liposomes accurately revealed tumor-selective radionuclide retention with little background. Hence, our new strategy could greatly enhance tumor-to-healthy tissue ratios, improve diagnostic imaging, boost therapeutic efficacy, reduce toxicity to healthy tissues, and facilitate radionuclide imaging and therapy.
Assuntos
Lipossomos , Neoplasias , Humanos , Ligantes , Radioisótopos , Neoplasias/diagnóstico por imagem , Neoplasias/tratamento farmacológicoRESUMO
Although ex vivo expanded mesenchymal stem cells (MSC) have been used in numerous studies, the molecular signature and in vivo distribution status of MSC remain unknown. To address this matter, we identified numerous human MSC-characteristic genes--including nine transcription factor genes--using DNA microarray and real-time RT-PCR analyses: Most of the MSC-characteristic genes were down-regulated 24 h after incubation with osteogenesis-, chondrogenesis- or adipogenesis-induction medium, or 48-72 h after knockdown of the nine transcription factors. Furthermore, knockdowns of ETV1, ETV5, FOXP1, GATA6, HMGA2, SIM2 or SOX11 suppressed the self-renewal capacity of MSC, whereas those of FOXP1, SOX11, ETV1, SIM2 or PRDM16 reduced the osteogenic- and/or adipogenic potential. In addition, immunohistochemistry using antibodies for the MSC characteristic molecules--including GATA6, TRPC4, FLG and TGM2--revealed that MSC-like cells were present near the endosteum and in the interior of bone marrow of adult mice. These findings indicate that MSC synthesize a set of MSC markers in vitro and in vivo, and that MSC-characteristic transcription factors are involved in MSC stemness regulation.
Assuntos
Expressão Gênica , Células-Tronco Mesenquimais/metabolismo , Fatores de Transcrição/genética , Fibroblastos/citologia , Proteínas Filagrinas , Técnicas de Silenciamento de Genes , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Pele/citologiaRESUMO
Hypoxia plays important roles in the prognosis of malignant brain tumors such as glioblastoma because it causes drug delivery deficiencies and the induction of hypoxia-inducible factor-1α in tumor cells. Extensive hypoxic areas are associated with poor prognosis of these fatal diseases. We previously reported that multiple administrations of the hypoxia-targeted internal radiotherapy agent 64Cu-diacetyl-bis(N4-methylthiosemicarbazone) (64Cu-ATSM), four times at intervals of 1 or 2 weeks, show antitumor effects in glioblastoma without treatment-related adverse events. Before initiating clinical trials, preclinical safety studies using Cu-ATSM composed of stable isotopes and its precursor ATSM were required to understand the potential risks of systemic and repeated chemical exposure of our investigational drug. In this study, the concentrations of Cu-ATSM and ATSM in mouse plasma after intravenous administration were determined by liquid chromatography-tandem mass spectrometry, and the half-lives were estimated to be 21.5 and 22.4â¯minutes for Cu-ATSM and ATSM, respectively. Based on this result, approach 2 of the current ICH M3 [R2] guideline was adopted, and a 7-day intravenous toxicity study was conducted in mice. Cu-ATSM and ATSM in a ratio of 2:25 mimicking our current investigational drug was used, and no adverse effects were observed when Cu-ATSM and ATSM were administered at 81⯵g/kg. These results and those of previous studies suggest that our current investigational drug formulation containing Cu-ATSM and ATSM at a dose of 15⯵g can be safely administered to patients once per week for 4 weeks for treatment with 64Cu-ATSM.
RESUMO
99mTc-labeled mannosyl-neoglycoalbumin (NMA) was prepared and evaluated as a radiopharmaceutical for sentinel lymph node (SLN) identification, since 99mTc-labeled human serum albumin (HSA) rapidly cleared from injection sites. NMA was conjugated with 6-hydrazinopyridine-3-carboxylic acid (HYNIC) and reacted with [99mTc](tricine)2 to prepare [99mTc](HYNIC-NMA)(tricine)2. After subcutaneous injection of [99mTc](HYNIC-NMA)(tricine)2 from murine foot pad, radioactivity levels in the popliteal and lumbar lymph nodes, the injection site and other tissues were compared with those of [99mTc](HYNIC-HSA)(tricine)2 and 99mTc-labeled colloidal rhenium sulfate ([99mTc]colloid). [99mTc](HYNIC-NMA)(tricine)2 demonstrated significantly higher radioactivity levels in the popliteal lymph node, the SLN in this model, than did [99mTc](HYNIC-HSA)(tricine)2 and [99mTc]colloid at 0.5, 1, and 6 h post-injection. [99mTc](HYNIC-NMA)(tricine)2 showed a dose-dependent decrease in the popliteal accumulation while the radioactivity levels in the blood, liver and spleen increased with an increase in the molar dose of NMA. [99mTc]colloid registered a decrease in the radioactivity levels in the popliteal lymph node, blood, liver, and spleen with dilution. However, the radioactivity levels at the injection site increased with dilution of [99mTc] colloid. Both [99mTc](HYNIC-NMA)(tricine)2 and [99mTc](HYNIC-HSA)(tricine)2 showed the radioactivity levels at the injection site similar each other. These findings indicated that an addition of a macrophage binding function to 99mTc-labeled HSA provided high and selective accumulation of the radioactivity in the SLN without affecting the elimination rate from the injection site. Such characteristics render [99mTc](HYNIC-NMA)(tricine)2 attractive as a radiopharmaceutical for SLN identification. This study also demonstrated that the number of non-radiolabeled colloidal particles and the molar dose of mannosylated compounds play a crucial role in the SLN accumulation.
Assuntos
Albuminas/farmacocinética , Linfonodos/diagnóstico por imagem , Linfonodos/metabolismo , Compostos de Organotecnécio/farmacocinética , Tomografia por Emissão de Pósitrons/métodos , Biópsia de Linfonodo Sentinela/métodos , Albuminas/química , Animais , Masculino , Taxa de Depuração Metabólica , Camundongos , Camundongos Endogâmicos , Especificidade de Órgãos , Compostos de Organotecnécio/química , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/farmacocinética , Distribuição TecidualRESUMO
Since the expiration of patent protection, a number of new recombinant erythropoietin (rEPO) biosimilars have appeared on the worldwide market. In 2010, epoetin kappa, which is biosimilar to epoetin alfa, was clinically approved in Japan. Currently, both isoelectric focusing polyacrylamide gel electrophoresis (IEF-PAGE) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) are approved by the World Anti-Doping Agency (WADA) for detection of rEPO doping. Because it was unclear whether epoetin kappa could be detected by WADA-accredited detection methods, intravenous administration studies of epoetin kappa, epoetin alfa, and epoetin beta were performed to test the applicability of these methods. The isoform bands of epoetin kappa expanded more widely towards the basic area and the profile appeared to be composed of at least eight bands, which were clearly different from those of other epoetins. The results showed that epoetin kappa also contains isoforms of higher molecular masses than those of originator epoetins on SDS-PAGE; the mass distribution was confirmed by electrospray ionization time-of-flight mass spectrometry. We clearly detected epoetin kappa after its administration up to 10 h by IEF-PAGE and 24 h by SDS-PAGE; the detection window of the SDS-PAGE is longer than that of the IEF-PAGE. SDS-PAGE compensates for the disadvantages of IEF-PAGE in detecting urinary epoetin kappa. We also concluded that athletes abusing rEPO might move to intravenous injections for shorter clearance times instead of subcutaneous injections. In conclusion, out-of-competition tests need to be applied more frequently to improve the effectiveness of the rEPO detection.
Assuntos
Medicamentos Biossimilares/sangue , Medicamentos Biossimilares/urina , Dopagem Esportivo , Eritropoetina/sangue , Eritropoetina/urina , Detecção do Abuso de Substâncias , Adulto , Medicamentos Biossimilares/administração & dosagem , Medicamentos Biossimilares/análise , Eletroforese em Gel de Poliacrilamida , Eritropoetina/administração & dosagem , Eritropoetina/análise , Humanos , Injeções Intravenosas , Focalização Isoelétrica , Masculino , Espectrometria de Massas , Isoformas de Proteínas/administração & dosagem , Isoformas de Proteínas/análise , Isoformas de Proteínas/sangue , Isoformas de Proteínas/urina , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/análise , Proteínas Recombinantes/sangue , Proteínas Recombinantes/urina , Fatores de Tempo , Adulto JovemAssuntos
Agonistas de Receptores Adrenérgicos alfa 2/farmacologia , Agonistas de Receptores Adrenérgicos alfa 2/uso terapêutico , Quinoxalinas/farmacologia , Quinoxalinas/uso terapêutico , Agonistas de Receptores Adrenérgicos alfa 2/efeitos adversos , Tartarato de Brimonidina , Ensaios Clínicos como Assunto , Glaucoma/tratamento farmacológico , Humanos , Pressão Intraocular/efeitos dos fármacos , Soluções Oftálmicas , Quinoxalinas/efeitos adversosRESUMO
The nuclear receptors liver X receptor (LXR) alpha and LXRbeta serve as oxysterol receptors and regulate the expression of genes involved in lipid metabolism. LXR activation induces the expression of ATP-binding cassette (ABC) transporters, such as ABCG5 and ABCG8, which inhibit intestinal absorption of cholesterol and phytosterols. Although several synthetic LXR agonists have been generated, these compounds have limited clinical application, because they cause hypertriglycemia by inducing the expression of lipogenic genes in the liver. We synthesized derivatives of phytosterols and found some of them to act as LXR agonists. Among them, YT-32 [(22E)-ergost-22-ene-1alpha,3beta-diol], which is related to ergosterol and brassicasterol, is the most potent LXR agonist. YT-32 directly bound to LXRalpha and LXRbeta and induced the interaction of LXRalpha with cofactors, such as steroid receptor coactivator-1, as effectively as the natural ligands, 22(R)-hydroxycholesterol and 24(S),25-epoxycholesterol. Although the nonsteroidal synthetic LXR agonist T0901317 induced the expression of intestinal ABC transporters and liver lipogenic genes, oral administration of YT-32 selectively activated intestinal ABC transporters in mice. Unlike T0901317 treatment, YT-32 inhibited intestinal cholesterol absorption without increasing plasma triglyceride levels. The phytosterol-derived LXR agonist YT-32 might selectively modulate intestinal cholesterol metabolism.