RESUMO
Although Hsp90 inhibitors can inhibit multiple tumorigenic pathways in cancer cells, their anticancer activity has been disappointingly modest. However, by forcing Hsp90 inhibitors into the mitochondria with mitochondrial delivery vehicles, they were converted into potent drugs targeting the mitochondrial Hsp90 paralog TRAP1. Here, to improve mitochondrial drug accumulation without using the mitochondrial delivery vehicle, we increased freely available drug concentrations in the cytoplasm by reducing the binding of the drugs to the abundant cytoplasmic Hsp90. After analyzing X-ray cocrystal structures, the purine ring of the Hsp90 inhibitor 2 (BIIB021) was modified to pyrazolopyrimidine scaffolds. One pyrazolopyrimidine, 12b (DN401), bound better to TRAP1 than to Hsp90, inactivated the mitochondrial TRAP1 in vivo, and it exhibited potent anticancer activity. Therefore, the rationale and feasible guidelines for developing 12b can potentially be exploited to design a potent TRAP1 inhibitor.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Pirazóis/química , Pirazóis/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cristalografia por Raios X , Proteínas de Choque Térmico HSP90/metabolismo , Células HeLa , Humanos , Camundongos Nus , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Simulação de Acoplamento Molecular , Neoplasias/metabolismo , Neoplasias/patologia , Pirazóis/farmacocinética , Pirazóis/uso terapêutico , Pirimidinas/farmacocinética , Pirimidinas/uso terapêuticoRESUMO
Mulitpotent mesenchymal stem cells (MSCs) derived from human bone marrow are promising candidates for the development of cell therapeutic strategies. MSC surface protein profiles provide novel biological knowledge concerning the proliferation and differentiation of these cells, including the potential for identifying therapeutic targets. Basic fibroblast growth factor (bFGF) affects cell surface proteins, which are associated with increased growth rate, differentiation potential, as well as morphological changes of MSCs in vitro. Cell surface proteins were isolated using a biotinylation-mediated method and identified using a combination of one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry. The resulting gel lines were cut into 20 bands and digested with trypsin. Each tryptic fragment was analyzed by liquid chromatography-electrospray ionization tandem mass spectrometry. Proteins were identified using the Mascot search program and the International Protein Index human database. Noble MSC surface proteins (n = 1,001) were identified from cells cultured either with (n = 857) or without (n = 667) bFGF-containing medium in three independent experiments. The proteins were classified using FatiGO to elucidate their function. We also confirmed the proteomics results using Western blotting and immunofluorescence microscopic analysis. The nature of the proteins identified makes it clear that MSCs express a wide variety of signaling molecules, including those related to cell differentiation. Among the latter proteins, four Ras-related Rab proteins, laminin-R, and three 14-3-3 proteins that were fractionated from MSCs cultured on bFGF-containing medium are implicated in bFGF-induced signal transduction of MSCs. Consequently, these finding provide insight into the understanding of the surface proteome of human MSCs.
Assuntos
Células-Tronco Mesenquimais/metabolismo , Proteínas/análise , Proteoma/análise , Proteômica/métodos , Proteínas 14-3-3/análise , Proteínas 14-3-3/metabolismo , Diferenciação Celular , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Espectrometria de Massas/métodos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Multipotentes/metabolismo , Proteínas/classificação , Proteínas/metabolismo , Receptores de Laminina/metabolismo , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
BACKGROUND: To identify meningioma-specific proteins, cerebrospinal fluid (CSF) from 4 patients with a meningioma and 4 patients with a non-brain tumorous lesion were analyzed. MATERIAL/METHODS: Two-dimensional electrophoresis and electrospray quadrupole time-of-flight tandem mass spectrometry analyses revealed 10 unique spots, containing 11 independent proteins (spot #2 and #4 each contained 2 proteins and spot #3 was not identified) were evident in CSF associated with human meningioma: serum albumin precursor (3 different isoforms), Apolipoprotein E (Apo E), Apolipoprotein J precursor (Apo J), Transthyretin precursor (TTR), Prostaglandin D2 synthase 21 kDa (PTGDS), proapolipoprotein, Chain D hemoglobin Ypsilanti, alpha-1-antitrypsin (AAT), and beta-2-microglobulin precursor (ß2M). RESULTS: The contents of Apo E, Apo J and AAT were increased, while PTGDS, TTR and ß2M were decreased. CONCLUSIONS: The results observed by 2-dimensional electrophoresis were verified by Western blot analysis. The unique proteins may represent possible candidate biomarkers of meningioma.
Assuntos
Biomarcadores Tumorais/líquido cefalorraquidiano , Neoplasias Meníngeas/líquido cefalorraquidiano , Neoplasias Meníngeas/diagnóstico , Meningioma/líquido cefalorraquidiano , Meningioma/diagnóstico , Proteoma/metabolismo , Proteômica/métodos , Adulto , Idoso , Sequência de Aminoácidos , Western Blotting , Eletroforese em Gel Bidimensional , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Anotação de Sequência Molecular , Dados de Sequência Molecular , Proteínas de Neoplasias/líquido cefalorraquidiano , Proteínas de Neoplasias/química , Proteoma/química , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
BACKGROUND: Current management strategies attempt to diagnose rheumatoid arthritis (RA) at an early stage. Transcription profiling is applied in the search for biomarkers for detecting early-stage disease. Even though gene profiling has been reported using several animal models of RA, most studies were performed after the development of active arthritis, and conducted only on the peripheral blood and joint. Therefore, we investigated gene expression during the initial phase of collagen-induced arthritis (CIA) before the arthritic features developed in the thymus in addition to the peripheral blood and synovium. METHODS: For gene expression analysis using cDNA microarray technology, samples of thymus, blood, and synovium were collected from CIA, rats immunized only with type II collagen (Cll), rats immunized only with adjuvant, and unimmunized rats on days 4 and 9 after the first immunization. Arrays were scanned with an Illumina bead array. RESULTS: Of the 21,910 genes in the array, 1,243 genes were differentially expressed at least 2-fold change in various organs of CIA compared to controls. Among the 1,243 genes, 8 encode T-cell receptors (TCRs), including CD3ζ, CD3δ, CD3ε, CD8α, and CD8ß genes, which were down-regulated in CIA. The synovium was the organ in which the genes were differentially expressed between CIA and control group, and no difference were found in the thymus and blood. Further, we determined that the differential expression was affected by adjuvant more than Cll. The differential expression of genes as revealed by real-time RT-PCR, was in agreement with the microarray data. CONCLUSION: This study provides evidence that the genes encoding TCRs including CD3ζ, CD3δ, CD3ε, CD8α, and CD8ß genes were down-regulated during the initial phase of CIA in the synovium of CIA. In addition, adjuvant played a greater role in the down-regulation of the CD3 complex compared to CII. Therefore, the down-regulation of TCR gene expression occurred dominantly by adjuvant could be involved in the pathogenesis of the early stage at CIA.
RESUMO
Mesenchymal stem cells (MSCs) are multipotent cells, which have the capability to differentiate into various mesenchymal tissues such as bone, cartilage, fat, tendon, muscle, and marrow stroma. However, they lose the capability of multi-lineage differentiation after several passages. It is known that basic fibroblast growth factor (bFGF) increases growth rate, differentiation potential, and morphological changes of MSCs in vitro. In this report, we have used 2-DE coupled to MS to identify differentially expressed proteins at the cell membrane level in MSCs growing in bFGF containing medium. The cell surface proteins isolated by the biotin-avidin affinity column were separated by 2-DE in triplicate experiments. A total of 15 differentially expressed proteins were identified by quadrupole-time of flight tandem MS. Nine of the proteins were upregulated and six proteins were downregulated in the MSCs cultured with bFGF containing medium. The expression level of three actin-related proteins, F-actin-capping protein subunit alpha-1, actin-related protein 2/3 complex subunit 2, and myosin regulatory light chain 2, was confirmed by Western blot analysis. The results indicate that the expression levels of F-actin-capping protein subunit alpha-1, actin-related protein 2/3 complex subunit 2, and myosin regulatory light chain 2 are important in bFGF-induced morphological change of MSCs.
Assuntos
Células da Medula Óssea/citologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Proteínas de Membrana/biossíntese , Células-Tronco Mesenquimais/metabolismo , Proteômica/métodos , Actinas/metabolismo , Western Blotting , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células Cultivadas , Meios de Cultura , Eletroforese em Gel Bidimensional , Humanos , Imuno-Histoquímica , Células-Tronco Mesenquimais/efeitos dos fármacos , Proteínas/metabolismo , Reprodutibilidade dos Testes , Espectrometria de Massas em TandemRESUMO
It has been reported that the SW480 human colon cancer cell line consists of E-type and R-type cells. The long-term tumorigenic potential, invasive and metastatic properties of these subclones have not been characterized. E-type and R-type cells were subcloned using limiting dilution methods from parental SW480 cells. The cell growth rate was determined by MTT colorimetric assay, and colony forming efficiency was analyzed using Matrigel-coated plates. The activity of matrix metalloproteinase (MMP) and of urokinase plasminogen activator (uPA) was assessed by zymography. Invasive and locomotive ability was analyzed using transwell chambers. In situ apoptosis detection of these subclones was also performed. In vivo long-term tumorigenicity and nodal metastasis were evaluated using nude mice. E-type cells produced spontaneously regressive tumors in spite of invasion and lymph node metastasis. In contrast, R-type cells revealed progressively growing tumors without invasion or metastasis. E-type cells exhibited increased apoptosis and invasive and motile ability, as well as strong MMP-9 and -2 activity. Although phorbol 12-myristate 13-acetate treatment induced MMP-9 activity in E-type cells, it had no effect on R-type cells. These findings suggest that E- and R-type cells may have different biological properties in terms of colon cancer progression, regression, invasion and nodal metastasis, and might serve as a useful model for these studies.
RESUMO
We have previously shown that 2,4,3',5'-tetramethoxystilbene (TMS), a synthetic trans-stilbene analog, is one of the most potently selective inhibitors of recombinant human cytochrome P450 1B1 (CYP1B1) in vitro. In the present studies, the effects of TMS on CYP1B1 expression were investigated in human cancer cells. TMS significantly inhibited CYP1-mediated 7-ethoxyresorufin O-deethylation activity in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced MCF-7 cells or lung microsomes of Sprague-Dawley rats treated with 7,12-dimethylbenz[a]anthracene. TCDD-stimulated CYP1B1 protein and mRNA expression was significantly suppressed by TMS in a concentration-dependent manner in MCF-7, MCF-10A, and HL-60 cells. Whereas TMS down-regulated TCDD-induced CYP1B1 gene expression, the levels of aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator mRNA expression were not changed by TMS treatment. In human cancer cells, TMS induced apoptotic cell death, and the cytotoxic effects of TMS were significant when the cells were incubated with TCDD. CYP1B1 was able to convert TMS to a metabolite(s) when incubated with NADPH. Metabolic activation of TMS by CYP1B1 induced by TCDD may mediate cellular toxicity of TMS in human cancer cells because the sensitivity to TMS in MCF-7 cells treated with TCDD was more significant than in HL-60 cells treated with TCDD. Taken together, our results indicate that TMS acts as a strong modulator of CYP1B1 gene expression as well as a potent selective inhibitor in vitro. The ability of TMS to induce apoptotic cell death in tumor cells, as well as CYP1B1 inhibition, may contribute to its usefulness for cancer chemoprevention.
Assuntos
Anticarcinógenos/farmacologia , Inibidores das Enzimas do Citocromo P-450 , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Extratos Vegetais/farmacologia , Estilbenos/farmacologia , Apoptose/efeitos dos fármacos , Hidrocarboneto de Aril Hidroxilases , Linhagem Celular Tumoral , Citocromo P-450 CYP1B1 , Sistema Enzimático do Citocromo P-450/genética , Relação Dose-Resposta a Droga , Humanos , Extratos Vegetais/metabolismo , Dibenzodioxinas Policloradas/farmacologia , Estilbenos/metabolismoRESUMO
Previously it was reported that various hydroxystilbene compounds strongly inhibit human cytochrome P-450 1 enzymes and were postulated as candidate chemopreventive agents. In this study, the inhibitory potential of P-450 1 enzyme activities by 3,5,3,4,5-pentamethoxystilbene (PMS), a synthetic stilbene compound, was evaluated with the Escherichia coli (E. coil) membranes of recombinant human cytochrome P-450 1A1, 1A2, or 1B1 coexpressed with human NADPH-P-450 reductase. PMS produced a significant inhibition of ethoxyresorufin O-deethylation (EROD) activities with IC50 values of 0.14, 934, and 3.2 M for 1A1, 1A2, and 1B1, respectively. PMS did not significantly inhibit EROD activities in human liver microsomes. To elucidate the mechanism of inhibition by PMS, kinetic studies were performed. Analysis of the mode of inhibition indicated a mixed-type inhibition of P-450 1A1. The inhibition of P-450 1A1-mediated EROD activity by PMS was not irreversible-mechanism based. The loss of EROD activity of P-450 1A1 with PMS was blocked by trapping agents such as glutathione, N-acetylcysteine, or dithiothreitol. Moreover, PMS significantly suppressed P-450 1A1-mediated EROD activity and P-450 1A1 gene expression in HepG2 cells induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Taken together, the results suggested that PMS is a potent and selective inhibitor of human P-450 1A1 and may be considered for use as a cancer chemopreventive agent in humans.
Assuntos
Citocromo P-450 CYP1A1/biossíntese , Citocromo P-450 CYP1A1/farmacologia , Inibidores Enzimáticos/farmacologia , Estilbenos/farmacologia , Carcinoma Hepatocelular/patologia , Membrana Celular , Escherichia coli , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/patologia , Microssomos Hepáticos/enzimologia , Células Tumorais CultivadasRESUMO
Cytochrome P450 (P450) 1 enzymes such as P450 1A1, 1A2, and 1B1 are known to be involved in the oxidative metabolism of various procarcinogens and are regarded as important target enzymes for cancer chemoprevention. Previously, several hydroxystilbene compounds were reported to inhibit P450 1 enzymes and were rated as candidate chemopreventive agents. In this study, we investigated the inhibitory effect of 2-[2-(3,5-dimethoxyphenyl)vinyl]-thiophene (DMPVT), produced from the chemical modification of oxyresveratrol, on the activities of P450 1 enzymes. The inhibitory potential by DMPVT on the P450 1 enzyme activity was evaluated with the Escherichia coli membranes of the recombinant human cytochrome P450 1A1, 1A2, or 1B1 coexpressed with human NADPH-P450 reductase. DMPVT significantly inhibited ethoxyresorufin O-deethylation (EROD) activities with IC50 values of 61, 11, and 2 nM for 1A1, 1A2, and 1B1, respectively. The EROD activity in DMBA-treated rat lung microsomes was also significantly inhibited by DMPVT in a dose-dependent manner. The modes of inhibition by DMPVT were non-competitive for all three P450 enzymes. The inhibition of P450 1B1-mediated EROD activity by DMPVT did not show the irreversible mechanism-based effect. The loss of EROD activity in P450 1B1 with DMPVT incubation was not blocked by treatment with the trapping agents such as glutathione, N-acetylcysteine, or dithiothreitol. Taken together, the results suggested DMPVT to be a strong noncompetitive inhibitor of human P450 1 enzymes that should be considered as a good candidate for a cancer chemopreventive agent in humans.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Inibidores das Enzimas do Citocromo P-450 , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Tiofenos/síntese química , Tiofenos/farmacologia , Animais , Citocromo P-450 CYP1A1/metabolismo , Relação Dose-Resposta a Droga , Escherichia coli/metabolismo , Humanos , Técnicas In Vitro , Isoenzimas/antagonistas & inibidores , Cinética , Pulmão/efeitos dos fármacos , Pulmão/enzimologia , Microssomos/efeitos dos fármacos , Microssomos/enzimologia , NADP/metabolismo , Ratos , Proteínas Recombinantes/antagonistas & inibidoresRESUMO
A series of trans-stilbene derivatives containing a 3,5-dimethoxyphenyl moiety were prepared through a new efficient solution phase synthetic pathway, and their inhibitory activities were evaluated on human cytochrome P450s (CYP) 1A1, 1A2, and 1B1 to find a potent and selective CYP1B1 inhibitor. We found that a substituent at the 2-position of the stilbene skeleton plays a very important role in discriminating between CYP1As and CYP1B1. Among the compounds tested, the most selective and potent CYP1B1 inhibitor was 2,3',4,5'-tetramethoxystilbene. Compound 7j, 2-[2-(3,5-dimethoxy-phenyl)vinyl]thiophene, showed greater inhibitory activities but had a lower selectivity toward all of the CYP1s tested.