RESUMO
PURPOSE: De novo synthesis of cholesterol and its rate-limiting enzyme, 3-hydroxy-3-methylglutharyl-coenzyme A reductase (HMGCR), is deregulated in tumors and critical for tumor cell survival and proliferation. However, the role of HMGCR in the induction and maintenance of stem-like states in tumors remains unclear. METHODS: A compiled public database from breast cancer (BC) patients was analyzed with the web application SurvExpress. Cell Miner was used for the analysis of HMGCR expression and statin sensitivity of the NCI-60 cell lines panel. A CRISPRon system was used to induce HMGCR overexpression in the luminal BC cell line MCF-7 and a lentiviral pLM-OSKM system for the reprogramming of MCF-7 cells. Comparisons were performed by two-tailed unpaired t-test for two groups and one- or two-way ANOVA. RESULTS: Data from BC patients showed that high expression of several members of the cholesterol synthesis pathway were associated with lower recurrence-free survival, particularly in hormone-receptor-positive BC. In silico and in vitro analysis showed that HMGCR is expressed in several BC cancer cell lines, which exhibit a subtype-dependent response to statins in silico and in vitro. A stem-like phenotype was demonstrated upon HMGCR expression in MCF-7 cells, characterized by expression of the pluripotency markers NANOG, SOX2, increased CD44 +/CD24low/ -, CD133 + populations, and increased mammosphere formation ability. Pluripotent and cancer stem cell lines showed high expression of HMGCR, whereas cell reprogramming of MCF-7 cells did not increase HMGCR expression. CONCLUSION: HMGCR induces a stem-like phenotype in BC cells of epithelial nature, thus affecting tumor initiation, progression and statin sensitivity.
Assuntos
Neoplasias da Mama , Inibidores de Hidroximetilglutaril-CoA Redutases , Humanos , Feminino , Neoplasias da Mama/patologia , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/metabolismo , Oxirredutases , ColesterolRESUMO
Bark beetles commonly produce de novo terpenoid pheromones using precursors synthesized through the mevalonate pathway. This process is regulated by Juvenile Hormone III (JH III). In this work, the expression levels of mevalonate pathway genes were quantified after phloem feeding-to induce the endogenous synthesis of JH III-and after the topical application of a JH III solution. The mevalonate pathway genes from D. rhizophagus were cloned, molecularly characterized, and their expression levels were quantified. Also, the terpenoid compounds produced in the gut were identified and quantified by Gas Chromatography Mass Spectrometry (GC-MS). The feeding treatment produced an evident upregulation, mainly in acetoacetyl-CoA thiolase (AACT), 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), phosphomevalonate kinase (PMK), and isopentenyl diphosphate isomerase (IPPI) genes, and males reached higher expression levels compared to females. In contrast, the JH III treatment did not present a clear pattern of upregulation in any sex or time. Notably, the genes responsible for the synthesis of frontalin and ipsdienol precursors (geranyl diphosphate synthase/farnesyl diphosphate synthase (GPPS/FPPS) and geranylgeranyl diphosphate synthase (GGPPS)) were not clearly upregulated, nor were these compounds further identified. Furthermore, trans-verbenol and myrtenol were the most abundant compounds in the gut, which are derived from an α-pinene transformation rather than de novo synthesis. Hence, the expression of mevalonate pathway genes in D. rhizophagus gut is not directed to the production of terpenoid pheromones, regardless of their frequent occurrence in the genus Dendroctonus.
Assuntos
Ingestão de Alimentos , Regulação da Expressão Gênica , Redes e Vias Metabólicas/genética , Feromônios/biossíntese , Gorgulhos/genética , Animais , Feminino , Trato Gastrointestinal/metabolismo , Trato Gastrointestinal/fisiologia , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/metabolismo , Masculino , Ácido Mevalônico/metabolismo , Terpenos/metabolismo , Gorgulhos/enzimologia , Gorgulhos/metabolismo , Gorgulhos/fisiologiaRESUMO
Cholesterol, via sterol regulatory element-binding protein (SREBP) transcription factors, activates or represses genes involved in its hepatic biosynthetic pathway, and also modulates the expression of hepatocyte mitochondrial aquaporin-8 (mtAQP8), a channel that can function as peroxiporin by facilitating the transmembrane diffusion of H2O2. Here we tested the hypothesis that mtAQP8 is involved in the SREBP-mediated regulation of hepatocyte cholesterol biosynthesis. Using human hepatocyte-derived Huh-7 cells and primary rat hepatocytes, we found that mtAQP8 knockdown significantly downregulated de novo cholesterol synthesis as well as protein expressions of SREBP-2 and its target gene, a rate-limiting enzyme in cholesterol synthesis 3-Hydroxy-3-Methylglutaryl-CoA Reductase (HMGCR). In contrast, adenovirus-mediated human AQP8 mitochondrial expression significantly increased de novo cholesterol synthesis and protein expressions of SREBP-2 and HMGCR. In mtAQP8-overexpressed hepatocytes, mitochondrial H2O2 release was found to be increased; and a mitochondria-targeted antioxidant prevented the upregulation of mitochondrial H2O2 release and that of cholesterol synthesis. Our results suggest that peroxiporin mtAQP8 plays a role in the SREBP-controlled hepatocyte cholesterogenesis, a finding that might be relevant to cholesterol-related metabolic disorders.
Assuntos
Aquaporinas/genética , Colesterol/biossíntese , Hepatócitos/metabolismo , Hidroximetilglutaril-CoA Redutases/genética , Mitocôndrias/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Animais , Aquaporinas/antagonistas & inibidores , Aquaporinas/metabolismo , Linhagem Celular , Difusão , Regulação da Expressão Gênica , Hepatócitos/citologia , Humanos , Peróxido de Hidrogênio/metabolismo , Hidroximetilglutaril-CoA Redutases/metabolismo , Lipogênese/genética , Fígado/citologia , Fígado/metabolismo , Masculino , Cultura Primária de Células , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismoRESUMO
Hepatocellular carcinoma (HCC) represents 90% of liver tumors. Statins, may reduce the incidence of various tumors, including HCC. Antitumoral activities may be mediated by changes in transforming growth factor-beta (TGF-ß1) and thyroid hormones (TH) regulation. INTRODUCTION AND AIM: Hepatocellular carcinoma (HCC) represents 90% of liver tumors. Statins, may reduce the incidence of various tumors, including HCC. Antitumoral activities may be mediated by changes in transforming growth factor-beta (TGF-ß1) and thyroid hormones (TH) regulation. Aim. The aim of our study is to establish the statins mechanism of action and the potential key molecules involved in an in vivo and in vitro HCC model. MATERIALS AND METHODS: We used two models: in vivo (in rats) using diethylnitrosamine (DEN) and hexachlorobenzene (HCB) to develop HCC. We analyzed cell proliferation parameters (proliferating cel nuclear antigen, PCNA) and cholesterol metabolism (hydroxy-methylglutaryl-CoA reductase, HMGCoAR). In vitro (Hep-G2 cells) we evaluated the effects of different doses of Atorvastatin (AT) and Simvastatin (SM) on HCB induced proliferation and analyzed proliferative parameters, cholesterol metabolism, TGF-ß1 mRNA, c-Src and TH levels. RESULTS: In vivo, we observed that cell proliferation significantly increased as well as cholesterol serum levels in rats treated with HCB. In vitro, we observed the same results on PCNA as in vivo. The statins prevented the increase in HMG-CoAR mRNA levels induced by HCB, reaching levels similar to controls at maximum doses: AT (30 µM), and SM (20 µM). Increases in PCNA, TGF-ß1, and pc-Src, and decreases in deiodinase I mRNA levels induced by HCB were not observed when cells were pre-treated with AT and SM at maximum doses. CONCLUSION: Statins can prevent the proliferative HCB effects on Hep-G2 cells. TGF-ß1, c-Src and TH may be the statins molecular targets in hepatocarcinogenesis.
Assuntos
Antineoplásicos/farmacologia , Atorvastatina/farmacologia , Carcinoma Hepatocelular/prevenção & controle , Transformação Celular Neoplásica/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Neoplasias Hepáticas Experimentais/prevenção & controle , Sinvastatina/farmacologia , Animais , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/induzido quimicamente , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Dietilnitrosamina , Feminino , Células Hep G2 , Hexaclorobenzeno , Humanos , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/metabolismo , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Hormônios Tireóideos/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Quinases da Família src/metabolismoRESUMO
The enzyme 3-hydroxy-3-methyl-glutaryl CoA reductase (HMGR) is a glycoprotein of the endoplasmic reticulum that participates in the mevalonate pathway, the precursor of cholesterol in human and ergosterol in fungi. This enzyme has three domains: transmembrane, binding, and soluble. In this study, we expressed and purified the soluble fraction of the HMGR enzyme from Candida glabrata (CgHMGR) in an Escherichia coli heterologous system and used it as a model for studying its inhibitory activity. The soluble fraction of CgHMGR was fused to the maltose binding protein (MBP), purified, and characterized. Optimal pH was 8.0, and its optimal temperature activity was 37 °C. The k m and V max for the HMG-CoA were 6.5 µM and 2.26 × 10-3 µM min-1, respectively. Recombinant CgHMGR was inhibited by simvastatin presenting an IC50 at 14.5 µM. In conclusion, our findings suggest that the recombinant HMGR version from C. glabrata may be used as a study model system for HMGR inhibitors such as statins and newly synthesized inhibitor compounds that might be used in the treatment of hypercholesterolemia or mycosis.
Assuntos
Candida glabrata/enzimologia , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Terapia de Alvo Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Biocatálise , Candida glabrata/genética , Avaliação Pré-Clínica de Medicamentos , Estabilidade Enzimática , Escherichia coli/genética , Humanos , Concentração de Íons de Hidrogênio , Hidroximetilglutaril-CoA Redutases/química , Inibidores de Hidroximetilglutaril-CoA Redutases/síntese química , Cinética , Proteínas Ligantes de Maltose/metabolismo , Modelos Moleculares , Domínios Proteicos , Proteínas Recombinantes/química , Sinvastatina/farmacologia , Solubilidade , Especificidade por Substrato , TemperaturaRESUMO
Many reproductive problems has been described in male and female animals infected by Trypanosoma evansi. Thus, the aim of this study was to evaluate the occurrence of vertical (Experiment I) and venereal (Experiment II) transmission of T. evansi in rats experimentally infected. In the experiment I, eight female Wistar rats were used: three animals as negative controls, and five rats were infected by T. evansi on day ten of gestation. Out of these eight females, half puppies were used for molecular analysis (polymerase chain reaction - PCR) for T. evansi. Two infected females showed delivery problems, such as stillbirth, and fetal death that also led to female death. Three female rats infected had normal delivery of stunted offspring at term that died 2 days after birth. Rats from the control group had normal delivery of healthy offspring. T. evansi PCR was positive for 80% (12/15) of pups in the infected group. For the experiment II, five male rats were infected by T. evansi, and monitored by blood smears to check the parasitemia level. When the male rats showed parasitemia between 2 and 5 parasites per field, they were individually housed with one female adult rat. After approximately 21 days, the females delivered their offspring. Blood sample was collected from the females for blood smears and T. evansi PCR tests, which revealed negative results. Therefore, we were able to prove the occurrence of transplacental transmission of T. evansi and its negative effect on female rats and their offspring.
Assuntos
Transmissão Vertical de Doenças Infecciosas , Infecções Sexualmente Transmissíveis/transmissão , Tripanossomíase/transmissão , Animais , Antígenos CD/genética , Apirase/genética , DNA de Protozoário/isolamento & purificação , Cães , Feminino , Hidroximetilglutaril-CoA Redutases/genética , Masculino , Parasitemia/transmissão , Reação em Cadeia da Polimerase , Gravidez , Ratos , Ratos Wistar , Trypanosoma/genética , Trypanosoma/isolamento & purificação , Trypanosoma/fisiologiaRESUMO
BACKGROUND: Orofacial clefts (OFCs) are common birth defects, which include a range of disorders with a complex etiology affecting formation of craniofacial structures. Some forms of syndromic OFCs are produced by defects in the cholesterol pathway. The principal enzyme of the cholesterol pathway is the 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR). Our aim is to study whether defects of HMGCR function would produce orofacial malformation similar to those found in disorders of cholesterol synthesis. METHODS: We used zebrafish hmgcrb mutants and HMGCR inhibition assay using atorvastatin during early and late stages of orofacial morphogenesis in zebrafish. To describe craniofacial phenotypes, we stained cartilage and bone and performed in situ hybridization using known craniofacial markers. Also, we visualized neural crest cell migration in a transgenic fish. RESULTS: Our results showed that mutants displayed loss of cartilage and diminished orofacial outgrowth, and in some cases palatal cleft. Late treatments with statin show a similar phenotype. Affected-siblings displayed a moderate phenotype, whereas early-treated embryos had a minor cleft. We found reduced expression of the downstream component of Sonic Hedgehog-signaling gli1 in ventral brain, oral ectoderm, and pharyngeal endoderm in mutants and in late atorvastatin-treated embryos. CONCLUSION: Our results suggest that HMGCR loss-of-function primarily affects postmigratory cranial neural crest cells through abnormal Sonic Hedgehog signaling, probably induced by reduction in metabolites of the cholesterol pathway. Malformation severity correlates with the grade of HMGCR inhibition, developmental stage of its disruption, and probably with availability of maternal lipids. Together, our results might help to understand the spectrum of orofacial phenotypes found in cholesterol synthesis disorders. Birth Defects Research (Part A) 106:814-830, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Anormalidades Induzidas por Medicamentos , Atorvastatina/efeitos adversos , Fenda Labial , Fissura Palatina , Hidroximetilglutaril-CoA Redutases , Mutação , Proteínas de Peixe-Zebra , Peixe-Zebra , Anormalidades Induzidas por Medicamentos/enzimologia , Anormalidades Induzidas por Medicamentos/genética , Animais , Atorvastatina/farmacologia , Fenda Labial/induzido quimicamente , Fenda Labial/enzimologia , Fenda Labial/genética , Fenda Labial/patologia , Fissura Palatina/induzido quimicamente , Fissura Palatina/enzimologia , Fissura Palatina/genética , Fissura Palatina/patologia , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Lipid-lowering response to statin therapy shows large interindividual variability. At a genome-wide significance level, single nucleotide polymorphisms (SNPs) in PCSK9 and HMGCR have been implicated in this differential response. However, the influence of these variants is uncertain in the Chilean population. Hence, we aimed to evaluate the contribution of PCSK9 rs7552841 and HMGCR rs17671591 SNPs as genetic determinants of atorvastatin response in Chilean hypercholesterolaemic individuals. One hundred and one hypercholesterolaemic patients received atorvastatin 10 mg/day for 4 weeks. Plasma lipid profile (TC, HDL-C, LDL-C and TG) was determined before and after statin treatment, and SNPs were identified by allelic discrimination using TaqMan(®) SNP Genotyping Assays. Adjusted univariate and multivariate analyses' models were used for statistical analyses, and a p-value <0.05 was considered significant. From baseline (week 0) to the study end-point (week 4), significant reductions were observed in plasma TC, LDL-C and TG (p < 0.001), while HDL-C levels were increased (p < 0.001). Multivariate analysis showed no association between lipid levels and atorvastatin therapy for the PCSK9 variant. However, the HMGCR rs17671591 T allele contributed to basal HDL-C concentration variability along with a higher increase in this lipid fraction after statin medication. In addition, this allele determined greater plasma LDL-C reductions after therapy with atorvastatin. Our data suggest that the HMGCR rs17671591 polymorphism can constitute a genetic marker of lower plasma LDL-C and enhanced HDL-C concentration after atorvastatin therapy in the Chilean population.
Assuntos
Anticolesterolemiantes/uso terapêutico , Atorvastatina/uso terapêutico , LDL-Colesterol/sangue , Hidroximetilglutaril-CoA Redutases/genética , Hipercolesterolemia/tratamento farmacológico , Polimorfismo de Nucleotídeo Único , Anticolesterolemiantes/administração & dosagem , Anticolesterolemiantes/farmacocinética , Atorvastatina/administração & dosagem , Atorvastatina/farmacocinética , Chile , Feminino , Estudo de Associação Genômica Ampla , Humanos , Hipercolesterolemia/sangue , Hipercolesterolemia/genética , Masculino , Pessoa de Meia-Idade , Pró-Proteína Convertase 9/genética , Resultado do TratamentoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Cyclocarya paliurus Batal., native only to China, is widely consumed as a Chinese traditional folk medicine for the prevention and treatment of hyperlipidemia, obesity, and diabetes. The aim of the study is to investigate the cholesterol-lowering effect and potential mechanisms of different polar extracts from Cyclocarya paliurus leaves in mice fed with high-fat-diet. MATERIALS AND METHODS: Cyclocarya paliurus leaves extracts were orally administered to diet-induced hyperlipidemic mice for 4 weeks. Simvastatin was used as a positive control. Body weight, food intake, histopathology of liver and adipose tissues, hepatic and renal function indices, lipid profiles in the serum and liver were evaluated. Total bile acid concentrations of the liver and feces were also measured. Furthermore, the activities and mRNA expression of cholesterol metabolism-related enzymes including 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, cholesterol 7α-hydroxylase (CYP7A1) and acyl-CoA cholesterol acyltransferase 2 (ACAT2) in the livers of the mice were analyzed. LC-MS detection was performed to identify the components in the active fraction of Cyclocarya paliurus extracts. RESULTS: Different Cyclocarya paliurus polar extracts, especially ChE reduced the levels of serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C) and hepatic TC and TG, enhanced the level of serum high-density lipoprotein cholesterol (HDL-C), restored hepatic and renal function indices and histomorphology. HMG-CoA reductase activity and mRNA expression were decreased, while CYP7A1 activity and mRNA expression as well as the level of fecal and hepatic bile acid were increased by ChE. LC-MS analysis of ChE revealed the presence of six main triterpenoids, which might be responsible for its antihyperlipidemic bioactivity. CONCLUSIONS: Evidently ChE possesses the best antihyperlipidemic activity, and the cholesterol-lowering effect is at least partly attributed to its role in promoting the conversion of cholesterol into bile acids by upgrading the activity and mRNA expression of CYP7A1 and inhibiting those of HMG-CoA reductase to lower the cholesterol biosynthesis.
Assuntos
Hiperlipidemias/tratamento farmacológico , Hipolipemiantes/farmacologia , Hipolipemiantes/uso terapêutico , Juglandaceae , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Animais , Ácidos e Sais Biliares/metabolismo , Linhagem Celular , Colesterol/sangue , Colesterol/metabolismo , Colesterol 7-alfa-Hidroxilase/genética , Colesterol 7-alfa-Hidroxilase/metabolismo , Dieta Hiperlipídica , Fezes/química , Humanos , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/metabolismo , Hiperlipidemias/sangue , Hiperlipidemias/metabolismo , Hiperlipidemias/patologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Fitoterapia , Folhas de Planta , Esterol O-Aciltransferase/genética , Esterol O-Aciltransferase/metabolismo , Triglicerídeos/sangue , Triglicerídeos/metabolismo , Esterol O-Aciltransferase 2RESUMO
In models of metabolic disorders, cinnamon improves glucose and lipid metabolism. This study explores the effect of chronic supplementation with aqueous cinnamon extract (CE) on the lipid metabolism of rats. Male adult Wistar rats were separated into a control group (CTR) receiving water and a CE Group receiving aqueous cinnamon extract (400 mg of cinnamon per kg body mass per day) by gavage for 25 consecutive days. Cinnamon supplementation did not change the food intake or the serum lipid profile but promoted the following changes: lower body mass gain (P = 0.008), lower relative mass of white adipose tissue (WAT) compartments (P = 0.045) and higher protein content (percentage of the carcass) (P = 0.049). The CE group showed lower leptin mRNA expression in the WAT (P = 0.0017) and an important tendency for reduced serum leptin levels (P = 0.059). Cinnamon supplementation induced lower mRNA expression of SREBP1c (sterol regulatory element-binding protein 1c) in the WAT (P = 0.001) and liver (P = 0.013) and lower mRNA expression of SREBP2 (P = 0.002), HMGCoA reductase (3-hydroxy-3-methylglutaryl-CoA reductase) (P = 0.0003), ACAT1 (acetyl-CoA acetyltransferase 1) (P = 0.032) and DGAT2 (diacylglycerol O-acyltransferase 2) (P = 0.03) in the liver. These changes could be associated with the reduced esterified cholesterol and triacylglycerol content detected in this tissue. Our results suggest that chronic ingestion of aqueous cinnamon extract attenuates lipogenic processes, regulating the expression of key enzymes and transcriptional factors and their target genes, which are directly involved in lipogenesis. These molecular changes possibly promote adaptations that would prevent an increase in circulating cholesterol and triacylglycerol levels and prevent lipid accumulation in tissues, such as liver and WAT. Therefore, we speculate that cinnamon may also be useful for preventing or retarding the development of lipid disorders.