RESUMO
Epoxyeicosatrienoic acids, substrates for soluble epoxide hydrolase (sEH), exhibit vasodilatory and antihypertrophic activities. Inhibitors of sEH might therefore hold promise as heart failure therapeutics. We examined the ability of sEH inhibitors GSK2188931 and GSK2256294 to modulate cardiac hypertrophy, fibrosis, and function after transverse aortic constriction (TAC) in rats and mice. GSK2188931 administration was initiated in rats 1 day before TAC, whereas GSK2256294 treatment was initiated in mice 2 weeks after TAC. Four weeks later, cardiovascular function was assessed, plasma was collected for drug and sEH biomarker concentrations, and left ventricle was isolated for messenger RNA and histological analyses. In rats, although GSK2188931 prevented TAC-mediated increases in certain genes associated with hypertrophy and fibrosis (α-skeletal actin and connective tissue growth factor), the compound failed to attenuate TAC-induced increases in left ventricle mass, posterior wall thickness, end-diastolic volume and pressure, and perivascular fibrosis. Similarly, in mice, GSK2256294 did not reverse cardiac remodeling or systolic dysfunction induced by TAC. Both compounds increased the sEH substrate/product (leukotoxin/leukotoxin diol) ratio, indicating sEH inhibition. In summary, sEH inhibition does not prevent cardiac remodeling or dysfunction after TAC. Thus, targeting sEH seems to be insufficient for reducing pressure overload hypertrophy.
Assuntos
Aorta/efeitos dos fármacos , Cicloexilaminas/farmacologia , Inibidores Enzimáticos/farmacologia , Epóxido Hidrolases/antagonistas & inibidores , Piperidinas/farmacologia , Triazinas/farmacologia , Animais , Aorta/patologia , Cardiomegalia/tratamento farmacológico , Cardiomegalia/patologia , Constrição Patológica , Modelos Animais de Doenças , Fibrose , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie , Remodelação Ventricular/efeitos dos fármacosRESUMO
Soluble guanylate cyclase (sGC), the primary mediator of nitric oxide (NO) bioactivity, exists as reduced (NO-sensitive) and oxidized (NO-insensitive) forms. We tested the hypothesis that the cardiovascular protective effects of NO-insensitive sGC activation would be potentiated under conditions of oxidative stress compared to those of NO-sensitive sGC stimulation. The cardiovascular effects of the NO-insensitive sGC activator GSK2181236A [a low, non-depressor dose, and a high dose which lowered mean arterial pressure (MAP) by 5-10 mmHg] and those of equi-efficacious doses of the NO-sensitive sGC stimulator BAY 60-4552 were assessed in (1) Sprague Dawley rats during coronary artery ischemia/reperfusion (I/R) and (2) spontaneously hypertensive stroke prone rats (SHR-SP) on a high salt/fat diet (HSFD). In I/R, neither compound reduced infarct size 24 h after reperfusion. In SHR-SP, HSFD increased MAP, urine output, microalbuminuria, and mortality, caused left ventricular hypertrophy with preserved ejection fraction, and impaired endothelium-dependent vasorelaxation. The low dose of BAY 60-4552, but not that of GSK2181236A, decreased urine output, and improved survival. Conversely, the low dose of GSK2181236A, but not that of BAY 60-4552, attenuated the development of cardiac hypertrophy. The high doses of both compounds similarly attenuated cardiac hypertrophy and improved survival. In addition to these effects, the high dose of BAY 60-4552 reduced urine output and microalbuminuria and attenuated the increase in MAP to a greater extent than did GSK2181236A. Neither compound improved endothelium-dependent vasorelaxation. In SHR-SP isolated aorta, the vasodilatory responses to the NO-dependent compounds carbachol and sodium nitroprusside were attenuated by HSFD. In contrast, the vasodilatory responses to both GSK2181236A and BAY 60-4552 were unaltered by HSFD, indicating that reduced NO-bioavailability and not changes in the oxidative state of sGC is responsible for the vascular dysfunction. In summary, GSK2181236A and BAY 60-4552 provide partial benefit against hypertension-induced end-organ damage. The differential beneficial effects observed between these compounds could reflect tissue-specific changes in the oxidative state of sGC and might help direct the clinical development of these novel classes of therapeutic agents.
RESUMO
UNLABELLED: Pro-inflammatory cytokines like TNF-α activate sphingosine kinase (SK). Therefore, inhibition of SK is a potential molecular target for the treatment of rheumatoid arthritis. AIMS: The primary goal of this study was to assess the efficacy of ABC249640 (a selective SK-2 inhibitor) in two models of rodent arthritis. A secondary goal was to evaluate the pharmacological profile of ABC294640, when given in combination with methotrexate. METHODS: The efficacy of ABC294640 was determined by paw diameter/volume measurements, histological evaluations, and micro-CT analyses. RESULTS: ABC294640 attenuated both collagen-induced arthritis in mice, as well as adjuvant-induced arthritis in rats. With the adjuvant arthritis model, the prophylactic efficacy profile of ABC294640 was similar to indomethacin. Of note, ABC294640 reduced the bone and cartilage degradation, associated with adjuvant-induced arthritis. Rats treated with a suboptimal dose of MTX (50 µg/kg/day) in combination with ABC249640 (100 mg/kg/day) had better anti-arthritis effects in the adjuvant model, than treatment with either agent alone. CONCLUSION: Our results suggest that ABC249640 is an orally available drug candidate with a good pre-clinical efficacy profile for the prevention and/or treatment of RA.
Assuntos
Adamantano/análogos & derivados , Artrite Experimental/tratamento farmacológico , Artrite Experimental/prevenção & controle , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/uso terapêutico , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Piridinas/administração & dosagem , Piridinas/uso terapêutico , Adamantano/administração & dosagem , Adamantano/farmacologia , Adamantano/uso terapêutico , Administração Oral , Animais , Tornozelo/patologia , Articulação do Tornozelo/efeitos dos fármacos , Articulação do Tornozelo/patologia , Artrite Experimental/patologia , Peso Corporal/efeitos dos fármacos , Quimioterapia Combinada , Edema/patologia , Inibidores Enzimáticos/farmacologia , Feminino , Pé/patologia , Indometacina/administração & dosagem , Indometacina/farmacologia , Indometacina/uso terapêutico , Inflamação/patologia , Metotrexato/administração & dosagem , Metotrexato/farmacologia , Metotrexato/uso terapêutico , Camundongos , Camundongos Endogâmicos DBA , Piridinas/farmacologia , Ratos , Ratos Endogâmicos Lew , Esplenomegalia/induzido quimicamente , Esplenomegalia/tratamento farmacológico , Esplenomegalia/patologia , Esplenomegalia/prevenção & controle , Microtomografia por Raio-XRESUMO
Sphingolipid-metabolizing enzymes control the dynamic balance of the cellular levels of important bioactive lipids, including the apoptotic compound ceramide and the proliferative compound sphingosine 1-phosphate (S1P). Many growth factors and inflammatory cytokines promote the cleavage of sphingomyelin and ceramide leading to rapid elevation of S1P levels through the action of sphingosine kinases (SK1 and SK2). SK1 and SK2 are overexpressed in a variety of human cancers, making these enzymes potential molecular targets for cancer therapy. We have identified an aryladamantane compound, termed ABC294640 [3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide], that selectively inhibits SK2 activity in vitro, acting as a competitive inhibitor with respect to sphingosine with a K(i) of 9.8 muM, and attenuates S1P formation in intact cells. In tissue culture, ABC294640 suppresses the proliferation of a broad panel of tumor cell lines, and inhibits tumor cell migration concomitant with loss of microfilaments. In vivo, ABC294640 has excellent oral bioavailability, and demonstrates a plasma clearance half-time of 4.5 h in mice. Acute and chronic toxicology studies indicate that ABC294640 induces a transient minor decrease in the hematocrit of rats and mice; however, this normalizes by 28 days of treatment. No other changes in hematology parameters, or gross or microscopic tissue pathology, result from treatment with ABC294640. Oral administration of ABC294640 to mice bearing mammary adenocarcinoma xenografts results in dose-dependent antitumor activity associated with depletion of S1P levels in the tumors and progressive tumor cell apoptosis. Therefore, this newly developed SK2 inhibitor provides an orally available drug candidate for the treatment of cancer and other diseases.
Assuntos
Adamantano/análogos & derivados , Antineoplásicos/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Piridinas/farmacologia , Adamantano/farmacocinética , Adamantano/farmacologia , Adamantano/uso terapêutico , Administração Oral , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Apoptose , Disponibilidade Biológica , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Piridinas/farmacocinética , Piridinas/uso terapêutico , Ratos , Ratos Sprague-DawleyRESUMO
A critical step in the mechanism of action of inflammatory cytokines is the stimulation of sphingolipid metabolism, including activation of sphingosine kinase (SK), which produces the mitogenic and proinflammatory lipid sphingosine 1-phosphate (S1P). We have developed orally bioavailable compounds that effectively inhibit SK activity in vitro in intact cells and in cancer models in vivo. In this study, we assessed the effects of these SK inhibitors on cellular responses to tumor necrosis factor alpha (TNFalpha) and evaluated their efficacy in the dextran sulfate sodium (DSS) model of ulcerative colitis in mice. Using several cell systems, it was shown that the SK inhibitors block the ability of TNFalpha to activate nuclear factor kappa B (NFkappaB), induce expression of adhesion proteins, and promote production of prostaglandin E(2) (PGE(2)). In an acute model of DSS-induced ulcerative colitis, SK inhibitors were equivalent to or more effective than Dipentum in reducing disease progression, colon shortening, and neutrophil infiltration into the colon. The effects of SK inhibitors were associated with decreased colonic levels of inflammatory cytokines TNFalpha, interleukin (IL)-1beta, interferon gamma (IFN)-gamma, IL-6, and reduction of S1P levels. A similar reduction in disease progression was provided by SK inhibitors in a chronic model of ulcerative colitis in which the mice received 3-week-long cycles of DSS interspaced with week-long recovery periods. In the chronic model, immunohistochemistry for SK showed increased expression in DSS-treated mice (compared with water-treated controls) that was reduced by drug treatment. S1P levels were also elevated in the DSS group and significantly reduced by drug treatment. Together, these data indicate that SK is a critical component in inflammation and that inhibitors of this enzyme may be useful in treating inflammatory bowel diseases.
Assuntos
Colite Ulcerativa/tratamento farmacológico , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Administração Oral , Animais , Colite Ulcerativa/metabolismo , Colite Ulcerativa/patologia , Citocinas/metabolismo , Sulfato de Dextrana , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/administração & dosagem , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Sphingosine kinase (SK) is an oncogenic sphingolipid-metabolizing enzyme that catalyzes the formation of the mitogenic second messenger sphingosine-1-phosphate (S1P) at the expense of proapoptotic ceramide. Thus, SK is an attractive target for cancer therapy because blockage of S1P formation leads to inhibition of proliferation, as well as the induction of apoptosis in cancer cells. We have recently identified novel SK inhibitors with nanomolar to low micromolar potencies toward recombinant human SK. This study describes the continuing analysis of these inhibitors through in vitro and in vivo experiments. All three structurally diverse SK inhibitors tested showed antitumor activity in mice without exhibiting toxicity. Blood and tumor inhibitor concentrations exceeded in vitro potency levels. Cell signaling analyses in vitro revealed mixed inhibition of mitogen-activated protein kinase kinase and Akt phosphorylation by the SK inhibitors. Importantly, 4-[4-(4-chloro-phenyl)-thiazol-2-ylamino]-phenol (SKI-II) is orally bioavailable, detected in the blood for at least 8 h, and showed a significant inhibition of tumor growth in mice. These compounds are the first examples of nonlipid selective inhibitors of SK with in vivo antitumor activity and provide leads for further development of inhibitors of this important molecular target.
Assuntos
Antineoplásicos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacocinética , Disponibilidade Biológica , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Inibidores Enzimáticos/farmacocinética , Feminino , Meia-Vida , Lisofosfolipídeos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Quinases Ativadas por Mitógeno/biossíntese , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas Proto-Oncogênicas c-akt/biossíntese , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Tiazóis/farmacologiaRESUMO
N-myristoyltransferases (NMT) add myristate to the NH(2) termini of certain proteins, thereby regulating their localization and/or biological function. Using RNA interference, this study functionally characterizes the two NMT isozymes in human cells. Unique small interfering RNAs (siRNA) for each isozyme were designed and shown to decrease NMT1 or NMT2 protein levels by at least 90%. Ablation of NMT1 inhibited cell replication associated with a loss of activation of c-Src and its target FAK as well as reduction of signaling through the c-Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase pathway. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assays showed that depletion of either NMT isozyme induced apoptosis, with NMT2 having a 2.5-fold greater effect than NMT1. Western blot analyses revealed that loss of NMT2 shifted the expression of the BCL family of proteins toward apoptosis. Finally, intratumoral injection of siRNA for NMT1 or for both NMT1 and NMT2 inhibited tumor growth in vivo, whereas the same treatment with siRNA for NMT2 or negative control siRNA did not. Overall, the data indicate that NMT1 and NMT2 have only partially overlapping functions and that NMT1 is critical for tumor cell proliferation.
Assuntos
Aciltransferases/química , Apoptose , Animais , Antineoplásicos/farmacologia , Sequência de Bases , Northern Blotting , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células , Relação Dose-Resposta a Droga , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Proteínas de Fluorescência Verde/metabolismo , Humanos , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Dados de Sequência Molecular , Ácidos Mirísticos/química , Isoformas de Proteínas , Proteínas Proto-Oncogênicas c-raf/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Fatores de Tempo , Transfecção , Quinases da Família src/metabolismoRESUMO
Protein palmitoyltransferases (PATs) represent an exciting new target for anticancer drug design due to their pivotal roles in the subcellular localization of a number of oncogenes. We show that the Huntingtin interacting protein 14 (HIP14) is a PAT with a preference for the farnesyl-dependent palmitoylation motif found in H- and N-RAS. Characterization of HIP14 in mouse cells has revealed that it has the ability to induce colony formation in cell culture, anchorage-independent growth, and tumors in mice. Activity of the enzyme and its ability to transform cells is dependent on critical residues in the active site of the enzyme.
Assuntos
Proteínas de Transporte/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Aciltransferases , Proteínas Adaptadoras de Transdução de Sinal , Animais , Sequência de Bases , Transformação Celular Neoplásica , Primers do DNA , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células NIH 3T3RESUMO
Microarray analysis of human tissue is frequently hindered by the limited amount of RNA available. Although amplification protocols can be utilized, the relative representation of transcripts present in the starting material must remain unaltered. In this study, 200 ng of total RNA derived from cultured renal epithelial cells from tuberous sclerosis complex (TSC) carriers and control individuals was amplified by in vitro transcription with T7 RNA polymerase. The resulting Cy-labeled cDNAs (from total or amplified RNA (aRNA)) were analyzed as direct replicates and dye-flips on slides containing 10,000 human cDNAs. The Pearson correlation coefficients for the direct replicate experiments were 0.80 (20 microg total RNA), 0.85 (40 microg total RNA), and 0.93 (2 microg of aRNA). Comparisons between the array data revealed that the majority of genes expressed in total RNA (97% for 20 microg and 85% for 40 microg) were also detected in aRNA. The correlation coefficient of the expression ratios for genes detected in both total RNA (40 microg) and aRNA was 0.63. Further, Student's t-test indicated no significant difference (P = 0.83) between these ratios. These results indicate that the number of expressed genes detected with total RNA is proportional to the amount of RNA used and underscore the requirement of large amounts of total RNA for a comprehensive characterization of gene expression profiles. RNA amplification allows the detection of a large number of genes expressed in the starting RNA population without altering their relative intensities significantly. Thus, an RNA amplification step improves the quality of gene expression results obtained by microarray analysis. This study indicates that high quality microarray data can be generated from small amounts of RNA, including those extracted from limiting clinical samples and microdissected histological specimens.
Assuntos
Perfilação da Expressão Gênica/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Análise de Sequência com Séries de Oligonucleotídeos , RNA/análise , RNA/biossíntese , Humanos , RNA/genética , Reprodutibilidade dos TestesRESUMO
Analysis of cell-specific gene expression patterns using microarrays can reveal genes that are differentially expressed in diseased and normal tissue, as well as identify genes associated with specialized cellular functions. However, the cellular heterogeneity of the tissues precludes the resolution of expression profiles of specific cell types. While laser capture microdissection (LCM) can be used to obtain purified cell populations, the limited quantity of RNA isolated makes it necessary to perform an RNA amplification step prior to microarray analysis. The linearity and reproducibility of two RNA amplification protocols--the Baugh protocol (Baugh et al., 2001, Nucleic Acids Res 29:E29) and an in-house protocol have been assessed by conducting microarray analyses. Cy3-labeled total RNA from the colorectal cell line Colo-205 was compared to Cy5-labeled Colo-205 amplified RNA (aRNA) generated with each of the two protocols, using a human 10K cDNA array. The correlation of the gene intensities between amplified and total RNA measured in the two channels of each microarray was 0.72 and 0.61 for the Baugh protocol and the in-house protocol, respectively. The two protocols were further evaluated using aRNA obtained from normal colonic crypt cross-sections isolated via LCM. In both cases a microarray profile representative of colonic mucosa was obtained; statistically, the Baugh protocol was superior. Furthermore, a substantial overlap between highly expressed genes in the Colo-205 cells and colonic crypts underscores the reliability of the microarray analysis of LCM-derived material. Taken together, these results demonstrate that LCM-derived tissue from histological specimens can generate abundant amounts of high-quality aRNA for subsequent microarray analysis.