ABSTRACT
Recent studies have demonstrated that statins reduce cell viability and induce apoptosis in various types of cancer cells. The molecular mechanisms underlying these effects are poorly understood. The JAK/STAT pathway plays an important role in the regulation of proliferation and apoptosis in many tissues, and its deregulation is believed to be involved in tumorigenesis and cancer. The physiological activation of STAT proteins by GH is rapid but transient in nature and its inactivation is regulated mainly by the expression of SOCS proteins. UMR-106 osteosarcoma cells express a GH-responsive JAK2/STAT5 signaling pathway, providing an experimental model to study the influence of statins on this system. In this study we investigated the actions of simvastatin on cell proliferation, migration, and invasion on UMR-106 cells and examined whether alterations in GH-stimulated JAK/STAT/SOCS signaling may be observed. Results showed that treatment of osteosarcoma cells with simvastatin at 3 to 10 µM doses decreases cell proliferation, migration, and invasion in a time- and dose-dependent manner. At the molecular level, although the mechanisms used by simvastatin are not entirely clear, the effect of the statin on the reduction of JAK2 and STAT5 phosphorylation levels may partially explain the decrease in the GH-stimulated STAT5 transcriptional activity. This effect correlated with a time- and dose-dependent increase of SOCS-3 expression levels in cells treated with simvastatin, a regulatory role that has not been previously described. Furthermore, the finding that simvastatin is capable of inducing SOCS-3 and CIS genes expression shows the potential of the JAK/STAT pathway as a therapeutic target, reinforcing the efficacy of simvastatin as chemotherapeutic drug for the treatment of osteosarcoma.
Subject(s)
Antineoplastic Agents/pharmacology , Growth Hormone/physiology , STAT5 Transcription Factor/metabolism , Signal Transduction/drug effects , Simvastatin/pharmacology , Animals , Bone Neoplasms , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Drug Screening Assays, Antitumor , Gene Expression Regulation, Neoplastic/drug effects , Humans , Janus Kinases/metabolism , Osteosarcoma , Rats , Rats, Inbred BUF , Suppressor of Cytokine Signaling 3 Protein , Suppressor of Cytokine Signaling Proteins/genetics , Suppressor of Cytokine Signaling Proteins/metabolism , Transcription, Genetic , Transcriptional ActivationABSTRACT
BACKGROUND: Angiotensin receptor 1 blockers (ARB) are standard nephroprotective drugs in chronic kidney disease. There is less evidence for a nephroprotective effect of HMG-CoA reductase inhibitors (statins) and much less is known about potential benefits of combination therapy. We evaluated the therapeutic potential of a statin alone or in combination with an ARB in experimental chronic kidney disease. METHODS: Subtotally nephrectomized (5/6 Nx) rats were treated early with vehicle, losartan, cerivastatin or losartan/cerivastatin. Expression of messenger RNA (mRNA) was assessed by real-time reverse transcription-polymerase chain reaction. Tissue proteins were localized by immunohistochemistry. Nuclear factor-κB (NF-κB) activation was measured in whole kidneys. RESULTS: In contrast to the sham group, at 6 weeks, vehicle-treated 5/6 Nx rats displayed renal lesions, albuminuria and increased blood pressure, serum creatinine and total kidney NF-κB p65 DNA-binding activity and preproendothelin-1, fibronectin and type I and III collagen mRNA. NF-κB activation correlated with albuminuria and histological renal injury. Losartan or combination therapy preserved renal function, abrogated albuminuria and improved glomerular and interstitial histology. Cerivastatin alone preserved renal function and improved interstitial injury but did not influence albuminuria, glomerular histology or NF-κB activation. Losartan/cerivastatin normalized kidney NF-κB activation and extracellular matrix mRNA expression pattern. The effect of losartan alone on these parameters was less intense. All treatments decreased preproendothelin-1 mRNA and preserved interstitial capillaries. CONCLUSIONS: In a chronic kidney disease model, early treatment with either an ARB or a statin preserved renal function although the mechanisms differed. Combination therapy with an ARB and a statin did not confer clear-cut advantages on biochemical and histological parameters over ARB alone, although it further improved the kidney NF-κB and gene expression profile.
Subject(s)
Angiotensin Receptor Antagonists/therapeutic use , Hydroxymethylglutaryl-CoA Reductase Inhibitors/therapeutic use , Kidney Diseases/etiology , Kidney Diseases/prevention & control , Losartan/therapeutic use , Nephrectomy/adverse effects , Pyridines/therapeutic use , Angiotensin II Type 1 Receptor Blockers/antagonists & inhibitors , Animals , Blotting, Western , Collagen Type I/genetics , Collagen Type I/metabolism , Collagen Type III/genetics , Collagen Type III/metabolism , Drug Therapy, Combination , Fibronectins/genetics , Fibronectins/metabolism , Kidney Diseases/pathology , Male , NF-kappa B/genetics , NF-kappa B/metabolism , RNA, Messenger/genetics , Rats , Rats, Sprague-Dawley , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The C677T transition of methylenetetrahydrofolate reductase (MTHFR) gene causes a moderate increase in total plasma homocysteine (tHcy). We studied the effect of MTHFR TT homozygosity and mild hyperhomocysteinemia on arterial hypertension. Normotensive controls (n = 223) and hypertensive subjects (n = 235) were matched for age, gender, and history of cardiovascular disease. Homocysteine levels were measured by a polarization immunoassay method. Methylenetetrahydrofolate reductase we determined by polymerase chain reaction and restriction fragment analysis. Hypertensives showed elevated tHcy compared to normotensive group in men (P = 0.039). Homocysteine values higher than 15 micromol/L were associated with increased hypertensive risk in the male population [odds ratios (OR) = 1.63; 95% confidence interval (CI) = 1.06-2.52; P = 0.027]. In multivariate analysis, TT genotype was associated with an increased risk of hypertension in males (OR = 2.27; 95% CI = 1.12-4.60; P = 0.022) An increased hypertensive risk was observed in those TT males with tHcy levels higher than 15 micromol/L (OR = 2.78; 95% CI = 1.05-7.3; P = 0.032) but not in those non-TT males with tHcy levels higher than 15 micromol/L (P = 0.33). Our findings do not support the possibility that mild hyperhomocysteinemia my solely account for the hypertensive risk associated to the TT genotype.
Subject(s)
Homocysteine/blood , Hypertension/epidemiology , Hypertension/genetics , Oxidoreductases Acting on CH-NH Group Donors/genetics , Point Mutation , Aged , Blood Pressure/genetics , Female , Genetic Predisposition to Disease/epidemiology , Genotype , Humans , Hyperhomocysteinemia/blood , Hyperhomocysteinemia/epidemiology , Hyperhomocysteinemia/genetics , Hypertension/blood , Male , Methylenetetrahydrofolate Reductase (NADPH2) , Middle Aged , Multivariate Analysis , Polymorphism, Genetic , Risk FactorsABSTRACT
Statins improve vascular functions by mechanisms independent from their cholesterol-lowering effect. Rho GTPases are emerging as key targets for the vascular effects of statins. RhoB is a short-lived, early-response inducible protein involved in receptor endocytosis, apoptosis, and gene expression. Here we show that statins regulate RhoB expression by acting at multiple levels. Simvastatin increased RhoB protein levels by 8- to 10-fold. This effect was related to a depletion of isoprenoid intermediates, as deduced from the observation that several metabolites of the cholesterol biosynthetic pathway, namely, mevalonate and geranylgeranyl-pyrophosphate, attenuated simvastatin-induced RhoB up-regulation. Moreover, prenyltransferase inhibitors mimicked simvastatin effect. Cholesterol supplementation did not prevent simvastatin-elicited up-regulation but increased RhoB levels per se. Simvastatin moderately augmented RhoB transcript levels, but markedly impaired the degradation of RhoB protein, which accumulated in the cytosol in its non-isoprenylated form. Inhibition of RhoB isoprenylation was apparently required for simvastatin-induced up-regulation, because levels of an isoprenylation-deficient RhoB mutant were not affected by simvastatin. Moreover, this mutant was found to be markedly more stable than the wild-type protein. These results show that RhoB isoprenylation is necessary for rapid turnover of this protein and identify a novel link between the cholesterol biosynthetic pathway and the regulation of G-protein expression.
Subject(s)
Mevalonic Acid/metabolism , rhoB GTP-Binding Protein/metabolism , 3T3 Cells , Animals , Anticholesteremic Agents/pharmacology , Blotting, Northern , Blotting, Western , Cattle , Cells, Cultured , Cholesterol/metabolism , Cholesterol/pharmacology , DNA, Complementary/metabolism , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Endothelium, Vascular/cytology , Gene Expression Regulation , HeLa Cells , Humans , Immunoblotting , In Situ Hybridization , Mice , Models, Biological , Mutation , Pertussis Toxin/pharmacology , Plasmids/metabolism , Protein Binding , Protein Prenylation , RNA, Messenger/metabolism , Simvastatin/pharmacology , Subcellular Fractions , Time Factors , Transfection , Up-Regulation , rhoB GTP-Binding Protein/chemistryABSTRACT
INTRODUCTION AND OBJECTIVES: Previous studies angiotensin-converting enzyme gene insertion/deletion polymorphism ACE (I/D), angiotensinogen gene polymorphism, and angiotensin II AT1 receptor polymorphism in relation to coronary heart disease controversial results. This study was designed to analyze the association between these gene polymorphisms and the first coronary event in individuals residing on Grand Canary Island, Spain. PATIENTS AND METHOD: Case-control study. Case subjects (n = 304) were recruited at the first coronary event; age-matched controls (n = 315) were randomly selected from the Grand Canary population. Participants were examined for the usual risk factors. Blood samples were obtained for biochemical analyses and DNA extraction. Genotyping was performed by PCR and restriction analysis. RESULTS: Neither ACE (I/D) nor AT1 receptor polymorphism was associated with coronary heart disease, whereas the frequency distribution of AGT M235T genotypes among patients and control subjects (TT: 29% and 19%; MT: 48% and 50%; MM: 22% and 31%, respectively) was statistically different (p = 0.003). Multiple logistic regression analysis identified the TT genotype of the angiotensinogen gene (OR = 1.9; 95% CI 1.1-3.4), diabetes (OR = 4.4; 95% CI 2.0-9.4) and hypertension (OR = 2.1; 95% CI 1.3-3.3) as risk factors predicting the coronary event. CONCLUSIONS: Our results provide no evidence of an association between ACE (I/D) or AT1 receptor polymorphism and coronary heart disease. However, homozygosity for the T allele of the angiotensinogen gene, diabetes and hypertension independently place individuals at higher risk of experiencing a coronary event on Grand Canary Island.
Subject(s)
Coronary Disease/genetics , Polymorphism, Genetic , Renin-Angiotensin System/genetics , Case-Control Studies , Female , Humans , Life Style , Male , Middle Aged , Multivariate Analysis , Risk FactorsABSTRACT
Introducción y objetivos. Estudios previos sobre la relación de la enfermedad coronaria y el polimorfismo de inserción/deleción de la enzima conversiva de la angiotensina (ECA [I/D]), el polimorfismo del gen del angiotensinógeno AGT M235T, o del receptor AT1 de la angiotensina II (A1166C) han demostrado resultados controvertidos. El objetivo de este estudio fue determinar la asociación entre estos polimorfismos génicos y el primer acontecimiento coronario en la población de Gran Canaria. Pacientes y método. Estudio de casos y controles ajustados según edad. Los casos (n = 304) se seleccionaron al padecer un primer acontecimiento coronario; los controles constituyen una muestra aleatoria poblacional (n = 315).Todos los sujetos fueron evaluados para los factores de riesgo clásicos. Se tomaron muestras sanguíneas para determinaciones analíticas y extracción de ADN. Las genotipificaciones se realizaron por PCR y análisis de restricción. Resultados. No se encontró asociación entre el polimorfismo ECA (I/D), AT1R (A1166C) y la enfermedad coronaria, mientras que la distribución de frecuencias de los genotipos del angiotensinógeno entre pacientes y controles (TT: 29 y 19 por ciento; MT: 48 y 50 por ciento; MM: 22 y 31 por ciento, respectivamente) resultaron estadísticamente diferentes (p = 0,003). El análisis multivariado identificó como factores predictores de acontecimiento coronario al genotipo TT del gen del angiotensinógeno (OR = 1,9; IC del 95 por ciento, 1,13,4), la diabetes (OR = 4,4; IC del 95 por ciento, 2,0-9,4) y la hipertensión (OR = 2,1; IC del 95 por ciento, 1,3-3,3).Conclusiones. No se ha observado asociación entre el polimorfismo ECA (I/D), AT1R (A1166C) y la enfermedad coronaria. Sin embargo, la homocigosis TT del gen del angiotensinógeno, la diabetes y la hipertensión arterial predisponen de manera independiente a la aparición de un primer acontecimiento coronario en la población canaria (AU)
