RESUMO
We tested the effect of substituents at the (1) C3´, C3´N, (2) C10, and (3) C2-meta-benzoate positions of taxane derivatives on their activity against sensitive versus counterpart paclitaxel-resistant breast (MCF-7) and ovarian (SK-OV-3) cancer cells. We found that (1) non-aromatic groups at both C3´ and C3´N positions, when compared with phenyl groups at the same positions of a taxane derivative, significantly reduced the resistance of ABCB1 expressing MCF-7/PacR and SK-OV-3/PacR cancer cells. This is, at least in the case of the SB-T-1216 series, accompanied by an ineffective decrease of intracellular levels in MCF-7/PacR cells. The low binding affinity of SB-T-1216 in the ABCB1 binding cavity can elucidate these effects. (2) Cyclopropanecarbonyl group at the C10 position, when compared with the H atom, seems to increase the potency and capability of the derivative in overcoming paclitaxel resistance in both models. (3) Derivatives with fluorine and methyl substituents at the C2-meta-benzoate position were variously potent against sensitive and resistant cancer cells. All C2 derivatives were less capable of overcoming acquired resistance to paclitaxel in vitro than non-substituted analogs. Notably, fluorine derivatives SB-T-121205 and 121,206 were more potent against sensitive and resistant SK-OV-3 cells, and derivatives SB-T-121405 and 121,406 were more potent against sensitive and resistant MCF-7 cells. (4) The various structure-activity relationships of SB-T derivatives observed in two cell line models known to express ABCB1 favor their complex interaction not based solely on ABCB1.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP , Resistencia a Medicamentos Antineoplásicos , Humanos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Células MCF-7 , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Relação Estrutura-Atividade , Taxoides/farmacologia , Taxoides/química , Linhagem Celular Tumoral , Paclitaxel/farmacologia , Feminino , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Antineoplásicos/farmacologia , Antineoplásicos/química , Benzoatos/farmacologia , Benzoatos/química , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologiaRESUMO
AIMS: Patients with heart failure (HF) display metabolic alterations, including heightened ketogenesis, resulting in increased beta-hydroxybutyrate (ß-OHB) formation. We aimed to investigate the determinants and prognostic impact of circulating ß-OHB levels in patients with advanced HF and reduced ejection fraction (HFrEF). METHODS AND RESULTS: A total of 867 patients with advanced HFrEF (age 57 ± 11 years, 83% male, 45% diabetic, 60% New York Heart Association class III), underwent clinical and echocardiographic examination, circulating metabolite assessment, and right heart catheterization (n = 383). The median ß-OHB level was 64 (interquartile range [IQR] 33-161) µmol/L (normal 0-74 µmol/L). ß-OHB levels correlated with increased markers of lipolysis (free fatty acids [FFA]), higher natriuretic peptides, worse pulmonary haemodynamics, and lower humoral regulators of ketogenesis (insulin/glucagon ratio). During a median follow-up of 1126 (IQR 410-1781) days, there were 512 composite events, including 324 deaths, 81 left ventricular assist device implantations and 107 urgent cardiac transplantations. In univariable Cox regression, increased ß-OHB levels (T3 vs. T1: hazard ratio [HR] 1.39, 95% confidence interval [CI] 1.13-1.72, p = 0.002) and elevated FFA levels (T3 vs. T1: HR 1.39, 95% CI 1.09-1.79, p = 0.008) were both predictors of a worse prognosis. In multivariable Cox analysis evaluating the simultaneous associations of FFA and ß-OHB levels with outcomes, only FFA levels remained significantly associated with adverse outcomes. CONCLUSIONS: In patients with advanced HFrEF, increased plasma ß-OHB correlate with FFA levels, worse right ventricular function, greater neurohormonal activation and other markers of HF severity. The association between plasma ß-OHB and adverse outcomes is eliminated after accounting for FFA levels, suggesting that increased ß-OHB is a consequence reflecting heightened lipolytic state, rather than a cause of worsening HF.
RESUMO
A mathematical model of myocardial perfusion based on the lattice Boltzmann method (LBM) is proposed and its applicability is investigated in both healthy and diseased cases. The myocardium is conceptualized as a porous material in which the transport and mass transfer of a contrast agent in blood flow is studied. The results of myocardial perfusion obtained using LBM in 1D and 2D are confronted with previously reported results in the literature and the results obtained using the mixed-hybrid finite element method. Since LBM is not suitable for simulating flow in heterogeneous porous media, a simplified and computationally efficient 1D-analog approach to 2D diseased case is proposed and its applicability discussed.
Assuntos
Análise de Elementos Finitos , Modelos Cardiovasculares , Humanos , Circulação Coronária/fisiologia , Simulação por Computador , Meios de ContrasteRESUMO
Decades after most countries banned hexachlorocyclohexane, HCH isomers still pollute the environment. Many studies described HCH as a pro-diabetic factor; nevertheless, the effect of HCH isomers on pancreatic beta-cells remains unexplored. This study investigated the effects of a one-month exposure to α-HCH, ß-HCH, and γ-HCH on protein expression in human (NES2Y) and rat (INS1E) pancreatic beta-cell lines. α-HCH and γ-HCH increased proinsulin and insulin levels in INS1E cells, while ß-HCH showed the opposite trend. α-HCH altered the expression of PKA, ATF3, and PLIN2. ß-HCH affected the expression of GLUT1, GLUT2, PKA, ATF3, p-eIF2α, ATP-CL, and PLIN2. γ-HCH altered the expression of PKA, ATF3, PLIN2, PLIN5, and IDH1. From the tested proteins, PKA, ATF3, and PLIN-2 were the most sensitive to HCH exposure and have the potential to be used as biomarkers.
Assuntos
Poluentes Ambientais , Hexaclorocicloexano , Humanos , Animais , Ratos , Hexaclorocicloexano/toxicidade , Hexaclorocicloexano/metabolismo , Poluentes Ambientais/toxicidade , IsomerismoRESUMO
A limited number of studies are devoted to regulating TRIP6 expression in cancer. Hence, we aimed to unveil the regulation of TRIP6 expression in MCF-7 breast cancer cells (with high TRIP6 expression) and taxane-resistant MCF-7 sublines (manifesting even higher TRIP6 expression). We found that TRIP6 transcription is regulated primarily by the cyclic AMP response element (CRE) in hypomethylated proximal promoters in both taxane-sensitive and taxane-resistant MCF-7 cells. Furthermore, in taxane-resistant MCF-7 sublines, TRIP6 co-amplification with the neighboring ABCB1 gene, as witnessed by fluorescence in situ hybridization (FISH), led to TRIP6 overexpression. Ultimately, we found high TRIP6 mRNA levels in progesterone receptor-positive breast cancer and samples resected from premenopausal women.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP , Resistencia a Medicamentos Antineoplásicos , Proteínas com Domínio LIM , Neoplasias , Feminino , Humanos , Proteínas Adaptadoras de Transdução de Sinal/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , AMP Cíclico , Resistencia a Medicamentos Antineoplásicos/genética , Hibridização in Situ Fluorescente , Proteínas com Domínio LIM/genética , Células MCF-7 , Neoplasias/genética , Elementos de Resposta , Taxoides , Fatores de Transcrição/genéticaRESUMO
The role of metformin (MET) in the treatment of patients with advanced HFrEF and type 2 diabetes mellitus (DM) is not firmly established. We studied the impact of MET on metabolic profile, quality of life (QoL) and survival in these patients. A total of 847 stable patients with advanced HFrEF (57.4 ± 11.3 years, 67.7% NYHA III/IV, LVEF 23.6 ± 5.8%) underwent clinical and laboratory evaluation and were prospectively followed for a median of 1126 (IQRs 410; 1781) days for occurrence of death, urgent heart transplantation or mechanical circulatory support implantation. A subgroup of 380 patients (44.9%) had DM, 87 of DM patients (22.9%) were treated with MET. Despite worse insulin sensitivity and more severe DM (higher BMI, HbA1c, worse insulin resistance), MET-treated patients exhibited more stable HF marked by lower BNP level (400 vs. 642 ng/l), better LV and RV function, lower mitral and tricuspid regurgitation severity, were using smaller doses of diuretics (all p < 0.05). Further, they had higher eGFR (69.23 vs. 63.34 ml/min/1.73 m2) and better QoL (MLHFQ: 36 vs. 48 points, p = 0.002). Compared to diabetics treated with other glucose-lowering agents, MET-treated patients had better event-free survival even after adjustment for BNP, BMI and eGFR (p = 0.035). Propensity score-matched analysis with 17 covariates yielded 81 pairs of patients and showed a significantly better survival for MET-treated subgroup (p = 0.01). MET treatment in patients with advanced HFrEF and DM is associated with improved outcome by mechanisms beyond the improvement of blood glucose control.
Assuntos
Diabetes Mellitus Tipo 2 , Insuficiência Cardíaca , Resistência à Insulina , Metformina , Diabetes Mellitus Tipo 2/induzido quimicamente , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/tratamento farmacológico , Humanos , Metformina/uso terapêutico , Qualidade de Vida , Volume Sistólico , Resultado do TratamentoRESUMO
Duodenal biopsies are considered a suitable source of enterocytes for studies of dietary iron absorption. However, the expression level of molecules involved in iron absorption may vary along the length of duodenum. We aimed to determine whether the expression of molecules involved in the absorption of heme and non-heme iron differs depending on the location in the duodenum. Analysis was performed with samples of duodenal biopsies from 10 individuals with normal iron metabolism. Samples were collected at the following locations: (a) immediately post-bulbar, (b) 1-2 cm below the papilla of Vater and (c) in the distal duodenum. The gene expression was analyzed at the mRNA and protein level using real-time PCR and Western blot analysis. At the mRNA level, significantly different expression of HCP1, DMT1, ferroportin and Zip8 was found at individual positions of duodenum. Position-dependent expression of other molecules, especially of FLVCR1, HMOX1 and HMOX2 was also detected but with no statistical significances. At the protein level, we observed statistically significantly decreasing expression of transporters HCP1, FLVCR1, DMT1, ferroportin, Zip14 and Zip8 with advancing positions of duodenum. Our results are consistent with a gradient of diminishing iron absorption along the duodenum for both heme and non-heme iron.
Assuntos
Duodeno , Ferro , Duodeno/metabolismo , Heme/metabolismo , Humanos , Transporte de Íons , Ferro/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
It was evidenced that saturated fatty acids (FAs) have a detrimental effect on pancreatic ß-cells function and survival, leading to endoplasmic reticulum (ER) calcium release, ER stress, and apoptosis. In the present study, we have tested the effect of three calcium influx inhibitors, i.e., diazoxide, nifedipine, and verapamil, on the apoptosis-inducing effect of saturated stearic acid (SA) in the human pancreatic ß-cell lines NES2Y and 1.1B4. We have demonstrated that the application of all three calcium influx inhibitors tested has no inhibitory effect on SA-induced ER stress and apoptosis in both tested cell lines. Moreover, these inhibitors have pro-apoptotic potential per se at higher concentrations. Interestingly, these findings are in contradiction with those obtained with rodent cell lines and islets. Thus our data obtained with human ß-cell lines suggest that the prospective usage of calcium channel blockers for prevention and therapy of type 2 diabetes mellitus, developed with the contribution of the saturated FA-induced apoptosis of ß-cells, seems rather unlikely.
RESUMO
We describe the casuistry of a homozygous familial hypercholesterolemia female patient with a biallelic missense variant (NM_000527.4:c.1775G>A, p.Gly592Glu) in the LDLR gene, severe hypertriglyceridemia and late manifestation of coronary heart disease not earlier than at the age of 45 years. An atypical phenotype led to a delayed diagnosis.
Assuntos
Homozigoto , Hiperlipoproteinemia Tipo II/genética , Fenótipo , Feminino , Testes Genéticos/métodos , Humanos , Hiperlipoproteinemia Tipo II/diagnóstico , Hiperlipoproteinemia Tipo II/fisiopatologia , Lipídeos/sangue , Lipídeos/classificação , Pessoa de Meia-Idade , Herança Multifatorial , Mutação de Sentido Incorreto , Polimorfismo de Nucleotídeo Único , Índice de Gravidade de DoençaRESUMO
Pancreatic ß-cell failure and death contribute significantly to the pathogenesis of type 2 diabetes. One of the main factors responsible for ß-cell dysfunction and subsequent cell death is chronic exposure to increased concentrations of FAs (fatty acids). The effect of FAs seems to depend particularly on the degree of their saturation. Saturated FAs induce apoptosis in pancreatic ß-cells, whereas unsaturated FAs are well tolerated and are even capable of inhibiting the pro-apoptotic effect of saturated FAs. Molecular mechanisms of apoptosis induction by saturated FAs in ß-cells are not completely elucidated. Saturated FAs induce ER stress, which in turn leads to activation of all ER stress pathways. When ER stress is severe or prolonged, apoptosis is induced. The main mediator seems to be the CHOP transcription factor. Via regulation of expression/activity of pro- and anti-apoptotic Bcl-2 family members, and potentially also through the increase in ROS production, CHOP switches on the mitochondrial pathway of apoptosis induction. ER stress signalling also possibly leads to autophagy signalling, which may activate caspase-8. Saturated FAs activate or inhibit various signalling pathways, i.e., p38 MAPK signalling, ERK signalling, ceramide signalling, Akt signalling and PKCδ signalling. This may lead to the activation of the mitochondrial pathway of apoptosis, as well. Particularly, the inhibition of the pro-survival Akt signalling seems to play an important role. This inhibition may be mediated by multiple pathways (e.g., ER stress signalling, PKCδ and ceramide) and could also consequence in autophagy signalling. Experimental evidence indicates the involvement of certain miRNAs in mechanisms of FA-induced ß-cell apoptosis, as well. In the rather rare situations when unsaturated FAs are also shown to be pro-apoptotic, the mechanisms mediating this effect in ß-cells seem to be the same as for saturated FAs. To conclude, FA-induced apoptosis rather appears to be preceded by complex cross talks of multiple signalling pathways. Some of these pathways may be regulated by decreased membrane fluidity due to saturated FA incorporation. Few data are available concerning molecular mechanisms mediating the protective effect of unsaturated FAs on the effect of saturated FAs. It seems that the main possible mechanism represents a rather inhibitory intervention into saturated FA-induced pro-apoptotic signalling than activation of some pro-survival signalling pathway(s) or metabolic interference in ß-cells. This inhibitory intervention may be due to an increase of membrane fluidity.
Assuntos
Apoptose , Ácidos Graxos/metabolismo , Células Secretoras de Insulina/metabolismo , Animais , Apoptose/genética , Sobrevivência Celular/genética , Diabetes Mellitus Tipo 2/etiologia , Diabetes Mellitus Tipo 2/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Metabolismo dos Lipídeos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Modelos Biológicos , Transdução de Sinais , Estresse FisiológicoRESUMO
The main problem precluding successful therapy with conventional taxanes is de novo or acquired resistance to taxanes. Therefore, novel experimental taxane derivatives (Stony Brook taxanes; SB-Ts) are synthesized and tested as potential drugs against resistant solid tumors. Recently, we reported alterations in ABCC3, CPS1, and TRIP6 gene expression in a breast cancer cell line resistant to paclitaxel. The present study aimed to investigate gene expression changes of these three candidate molecules in the highly resistant ovarian carcinoma cells in vitro and corresponding in vivo models treated with paclitaxel and new experimental Stony Brook taxanes of the third generation (SB-T-121605 and SB-T-121606). We also addressed their prognostic meaning in ovarian carcinoma patients treated with taxanes. We estimated and observed changes in mRNA and protein profiles of ABCC3, CPS1, and TRIP6 in resistant and sensitive ovarian cancer cells and after the treatment of resistant ovarian cancer models with paclitaxel and Stony Brook taxanes in vitro and in vivo. Combining Stony Brook taxanes with paclitaxel caused downregulation of CPS1 in the paclitaxel-resistant mouse xenograft tumor model in vivo. Moreover, CPS1 overexpression seems to play a role of a prognostic biomarker of epithelial ovarian carcinoma patients' poor survival. ABCC3 was overexpressed in EOC tumors, but after the treatment with taxanes, its up-regulation disappeared. Based on our results, we can suggest ABCC3 and CPS1 for further investigations as potential therapeutic targets in human cancers.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Carbamoil-Fosfato Sintase (Amônia)/genética , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas com Domínio LIM/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Taxoides/uso terapêutico , Fatores de Transcrição/genética , Animais , Biomarcadores Tumorais/genética , Carcinoma Epitelial do Ovário/tratamento farmacológico , Carcinoma Epitelial do Ovário/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Feminino , Humanos , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Paclitaxel/uso terapêuticoRESUMO
Many compounds have the potential to harm pancreatic beta-cells; organochlorine pollutants belong to those compounds. In this work, we aimed to find markers of acute toxicity of p,p'-DDT exposure among proteins expressed in NES2Y human pancreatic beta-cells employing 2-D electrophoresis. We exposed NES2Y cells to a high concentration (150 µM, LC96 after 72 hours) of p,p'-DDT for 24 and 30 hours and determined proteins with changed expression using 2-D electrophoresis. We have found 22 proteins that changed their expression. They included proteins involved in ER stress (GRP78, and endoplasmin), mitochondrial proteins (GRP75, ECHM, IDH3A, NDUS1, and NDUS3), proteins involved in the maintenance of the cell morphology (EFHD2, TCPA, NDRG1, and ezrin), and some other proteins (HNRPF, HNRH1, K2C8, vimentin, PBDC1, EF2, PCNA, biliverdin reductase, G3BP1, FRIL, and HSP27). The proteins we have identified may serve as indicators of p,p'-DDT toxicity in beta-cells in future studies, including long-term exposure to environmentally relevant concentrations.
Assuntos
Biomarcadores/metabolismo , DDT/toxicidade , Células Secretoras de Insulina/citologia , Proteômica/métodos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Eletroforese em Gel Bidimensional , Chaperona BiP do Retículo Endoplasmático , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Espectrometria de MassasRESUMO
Microtubule-targeting agents (MTAs) constitute a diverse group of chemical compounds that bind to microtubules and affect their properties and function. Disruption of microtubules induces various cellular responses often leading to cell cycle arrest or cell death, the most common effect of MTAs. MTAs have found a plethora of practical applications in weed control, as fungicides and antiparasitics, and particularly in cancer treatment. Here we summarize the current knowledge of MTAs, the mechanisms of action and their role in cancer treatment. We further outline the potential use of MTAs in anti-metastatic therapy based on inhibition of cancer cell migration and invasiveness. The two main problems associated with cancer therapy by MTAs are high systemic toxicity and development of resistance. Toxic side effects of MTAs can be, at least partly, eliminated by conjugation of the drugs with various carriers. Moreover, some of the novel MTAs overcome the resistance mediated by both multidrug resistance transporters as well as overexpression of specific ß-tubulin types. In anti-metastatic therapy, MTAs should be combined with other drugs to target all modes of cancer cell invasion.
Assuntos
Microtúbulos/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , HumanosRESUMO
An unknown intense signal (Pun ) with a mean chemical shift of 5.3 ppm was observed in 31 P MR spectra from the calf muscles of patients with the diabetic foot syndrome. The aim of the study was to identify the origin of this signal and its potential as a biomarker of muscle injury. Calf muscles of 68 diabetic patients (66.3 ± 8.6 years; body mass index = 28.2 ± 4.3 kg/m2 ) and 12 age-matched healthy controls were examined by (dynamic) 31 P MRS (3 T system, 31 P/1 H coil). Phantoms (glucose-1-phosphate, Pi and PCr) were measured at pH values of 7.05 and 7.51. At rest, Pun signals with intensities higher than 50% of the Pi intensity were observed in 10 of the 68 examined diabetic subjects. We tested two hypothetical origins of the Pun signal: (1) phosphorus from phosphoesters and (2) phosphorus from extra- and intracellular alkaline phosphate pools. 2,3-diphosphoglycerate and glucose-1-phosphate are the only phosphoesters with signals in the chemical shift region close to 5.3 ppm. Both compounds can be excluded: 2,3-diphosphoglycerate due to the missing second signal component at 6.31 ppm; glucose-1-phosphate because its chemical shifts are about 0.2 ppm downfield from the Pi signal (4.9 ppm). If the Pun signal is from phosphate, it represents a pH value of 7.54 ± 0.05. Therefore, it could correspond to signals of Pi in mitochondria. However, patients with critical limb ischemia have rather few mitochondria and so the Pun signal probably originates from interstitia. Our data suggest that the increased Pun signal observed in patients with the diabetic foot syndrome is a biomarker of severe muscular damage.
Assuntos
Extremidades/diagnóstico por imagem , Extremidades/patologia , Isquemia/diagnóstico por imagem , Espectroscopia de Ressonância Magnética , Fósforo/química , Processamento de Sinais Assistido por Computador , Idoso , Humanos , Concentração de Íons de Hidrogênio , Imagens de Fantasmas , DescansoRESUMO
The history of studying hypertriglyceridemia as a risk factor for atherosclerosis has been going on for a half a century. The significance of this parameter as measured in fasting state is not entirely clear, since the statistical significance between triglyceride concentration and cardiovascular risk is lost after adjustment to HDL-cholesterol concentration. Remnant particles of chylomicrons and very low density lipoproteins measured postprandially appear to be responsible for the risk associated with hypertriglyceridemia. As the concentration of non-fasting triglycerides increases, the risk of myocardial infarction increases gradually up to five times.
Assuntos
Aterosclerose , Hiperlipidemias , Hipertrigliceridemia , Aterosclerose/complicações , HDL-Colesterol , Humanos , Hipertrigliceridemia/complicações , Fatores de Risco , TriglicerídeosRESUMO
Persistent organochlorine pollutants (POPs) gradually accumulate in the human organism due to their presence in the environment. Some studies have described a correlation between the level of POPs in the human body and the incidence of diabetes, but we know little about the direct effect of POPs on pancreatic beta-cells. We exposed pancreatic beta-cells INS1E to non-lethal concentrations of p,p'-DDT (1,1'-(2,2,2-Trichloroethane-1,1-diyl)bis(4-chlorobenzene)) and p,p'-DDE (1,1'-(2,2-dichloroethene-1,1-diyl)bis(4-chlorobenzene)) for 1 month, and assessed changes in protein expression and the intracellular insulin level. 2-D electrophoresis revealed 6 proteins with changed expression in cells exposed to p,p'-DDT or p,p'-DDE. One of the detected proteins - vitamin D-binding protein (VDBP) - was upregulated in both cells exposed to p,p'-DDT, and cells exposed to p,p'-DDE. Both exposures to pollutants reduced the intracellular level of insulin mRNA, proinsulin, and insulin monomer; p,p'-DDT also slightly reduced the level of hexameric insulin. Overexpression of VDBP caused by the stable transfection of beta-cells with the gene for VDBP decreased both the proinsulin and hexameric insulin level in beta-cells similarly to the reduction detected in cells exposed to p,p'-DDT. Our data suggest that in the cells exposed to p,p'-DDT and p,p'-DDE, the increased VDBP protein level decreased the proinsulin expression in an unknown mechanism.
Assuntos
Poluentes Ambientais/toxicidade , Células Secretoras de Insulina/efeitos dos fármacos , Insulina/metabolismo , Proteína de Ligação a Vitamina D/metabolismo , Animais , Linhagem Celular , DDT/toxicidade , Diclorodifenil Dicloroetileno/toxicidade , Células Secretoras de Insulina/metabolismo , Ratos , Testes de Toxicidade Subcrônica , Regulação para Cima/efeitos dos fármacosRESUMO
Saturated fatty acids (FAs) induce apoptosis in the human pancreatic NES2Y ß-cell line while unsaturated FAs have nearly no detrimental effect. Moreover, unsaturated FAs are capable of inhibiting the pro-apoptotic effect of saturated FAs. Hypoxia is also known to have deleterious effects on ß-cells function and viability. In the present study, we have tested the modulatory effect of hypoxia on the effect of FAs on the growth and viability of the human pancreatic NES2Y ß-cells. This study represents the first study testing hypoxia effect on effects of FAs in pancreatic ß-cells as well as in other cell types. We showed that hypoxia increased the pro-apoptotic effect of saturated stearic acid (SA). Endoplasmic reticulum stress signaling seemed to be involved while redistribution of FA transporters fatty acid translocase/cluster of differentiation 36 (FAT/CD36) and fatty acid-binding protein (FABP) do not seem to be involved in this effect. Hypoxia also strongly decreased the protective effect of unsaturated oleic acid (OA) against the pro-apoptotic effect of SA. Thus, in the presence of hypoxia, OA was unable to save SA-treated ß-cells from apoptosis induction. Hypoxia itself had only a weak detrimental effect on NES2Y cells. Our data suggest that hypoxia could represent an important factor in pancreatic ß-cell death induced and regulated by FAs and thus in the development of type 2 diabetes mellitus.
Assuntos
Ácidos Graxos/metabolismo , Hipóxia/metabolismo , Células Secretoras de Insulina/metabolismo , Biomarcadores , Caspases/metabolismo , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Estresse do Retículo Endoplasmático , Humanos , Transdução de Sinais/efeitos dos fármacosRESUMO
Identification of novel proteins with changed expression in resistant cancer cells could be helpful in elucidation mechanisms involved in the development of acquired resistance to paclitaxel. In this study, we carried out a 2D-PAGE using the mitochondrial-enriched fraction from paclitaxel-resistant MCF7/PacR cells compared to original paclitaxel-sensitive MCF7 breast cancer cells. Differentially expressed proteins were identified employing mass spectrometry. We found that lysosomal cathepsin D and mitochondrial abhydrolase-domain containing protein 11 (ABHD11) had decreased expression in MCF7/PacR cells. On the other hand, mitochondrial carbamoyl-phosphate synthetase 1 (CPS1) and ATPase family AAA-domain containing protein 3A and 3B (ATAD3A, ATAD3B) were overexpressed in MCF7/PacR cells. Further, we showed that there was no difference in localization of CPS1 in MCF7 and MCF7/PacR cells. We demonstrated a significant increase in the number of CPS1 positive MCF7/PacR cells, using FACS analysis, compared to the number of CPS1 positive MCF7 cells. Silencing of CPS1 expression by specific siRNA had no significant effect on the resistance of MCF7/PacR cells to paclitaxel. To summarize, we identified several novel proteins of a mitochondrial fraction whose role in acquired resistance to paclitaxel in breast cancer cells should be further assessed.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Paclitaxel/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Carbamoil-Fosfato Sintase (Amônia)/genética , Carbamoil-Fosfato Sintase (Amônia)/metabolismo , Fracionamento Celular , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Células MCF-7 , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteoma , Proteômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em TandemRESUMO
BACKGROUND: Diets rich in fat and added sugars (especially fructose) play an important role in the pathogenesis of nonalcoholic liver disease (NAFLD), but there is only limited information on the acute effects of these nutrients on hepatic fat content (HFC). OBJECTIVES: We therefore explored how the administration of high-fat load, glucose, fructose, and combinations thereof affects HFC measured in vivo using proton magnetic resonance spectroscopy (1H-MRS) in healthy subjects. METHODS: Ten healthy nonsteatotic male volunteers (age 38.5 ± 9.6 y, body mass index [BMI, kg/m2] 26.9 ± 2.7) underwent, in random order, 6 experiments, each lasting 8 h, that included: 1) fasting; 2) a high-fat load (150 g of fat [dairy cream] at time 0); 3) glucose (3 doses of 50 g at 0, 2, and 4 h); 4) a high-fat load with glucose; 5) fructose (3 doses of 50 g at 0, 2, and 4 h); and 6) a high-fat load with fructose. HFC was measured using 1H-MRS prior to test meal administration (before time 0) and at 3 and 6 h. Plasma concentrations of triglycerides, nonesterified fatty acids, glucose, and insulin were monitored throughout each experiment. RESULTS: HFC increased to 119 ± 19% (P < 0.05) and 117 ± 17% (P < 0.01) of baseline when subjects consumed a high-fat load alone or a high-fat load with fructose, respectively, but was not affected when glucose was coadministered with a high-fat load. HFC was not affected when subjects had fasted or had consumed repeated doses of fructose. When subjects were administered 3 doses of glucose, HFC dropped to 85 ± 13% (P < 0.05) of baseline. CONCLUSIONS: Our results demonstrate that fructose and glucose have a different immediate impact on HFC in humans in vivo. Clinical trial registry: The study was registered at clinicaltrials.gov and obtained clinicaltrials.gov identifier: NCT03680248.
Assuntos
Frutose/metabolismo , Glucose/metabolismo , Fígado/metabolismo , Adolescente , Adulto , Gorduras/metabolismo , Ácidos Graxos não Esterificados/sangue , Humanos , Insulina/sangue , Masculino , Triglicerídeos/sangue , Adulto JovemRESUMO
PURPOSE: Lipotoxicity is implicated in type 2 diabetes pathogenesis. Its molecular mechanisms are not completely understood. The aim of this study is to identify new suspect proteins involved in pancreatic ß-cell death induction by saturated fatty acids and its inhibition by unsaturated fatty acids. EXPERIMENTAL DESIGN: Employing 2DE analysis and subsequent western blot confirmation, the differences in membrane/membrane-associated protein expression in human ß-cell line NES2Y are assessed during cell death induction by stearate and its inhibition by oleate. RESULTS: Induction of apoptosis by stearate is associated with significantly increased levels of Hsp90ß, peroxiredoxin-1, and 14-3-3γ in the membrane fraction of NES2Y cells and significantly decreased levels of annexin A2, annexin A4, and reticulocalbin-2. All these changes are significantly inhibited by oleate co-application. No expression changes are detected after application of stearate together with oleate. Furthermore, the expression of reticulocalbin-2 is significantly decreased after stearate application also in the whole cell lysate. CONCLUSIONS AND CLINICAL RELEVANCE: Several membrane-associated proteins that could be related to pro- and anti-apoptotic signaling initiated by fatty acids in human pancreatic ß-cells are identified. As far as we know, annexin A4, reticulocalbin-2, and 14-3-3γ represent novel molecules related to the effect of fatty acids on ß-cell viability.