Separate and Combined Effects of DNMT and HDAC Inhibitors in Treating Human Multi-Drug Resistant Osteosarcoma HosDXR150 Cell Line

Understanding the molecular mechanisms underlying multi-drug resistance (MDR) is one of the major challenges in current cancer research. A phenomenon which is common to both intrinsic and acquired resistance, is the aberrant alteration of gene expression in drug-resistant cancers. Although such dysregulation depends on many possible causes, an epigenetic characterization is considered a main driver. Recent studies have suggested a direct role for epigenetic inactivation of genes in determining tumor chemo-sensitivity. We investigated the effects of the inhibition of DNA methyltransferase (DNMT) and hystone deacethylase (HDAC), considered to reverse the epigenetic aberrations and lead to the re-expression of de novo methylated genes in MDR osteosarcoma (OS) cells. Based on our analysis of the HosDXR150 cell line, we found that in order to reduce cell proliferation, co-treatment of MDR OS cells with DNMT (5-Aza-dC, DAC) and HDAC (Trichostatin A, TSA) inhibitors is more effective than relying on each treatment alone. In re-expressing epigenetically silenced genes induced by treatments, a very specific regulation takes place which suggests that methylation and de-acetylation have occurred either separately or simultaneously to determine MDR OS phenotype. In particular, functional relationships have been reported after measuring differential gene expression, indicating that MDR OS cells acquired growth and survival advantage by simultaneous epigenetic inactivation of both multiple p53-independent apoptotic signals and osteoblast differentiation pathways. Furthermore, co-treatment results more efficient in inducing the re-expression of some main pathways according to the computed enrichment, thus emphasizing its potential towards representing an effective therapeutic option for MDR OS.     

Synthesis and biological activity of a novel class nicotinic acetylcholine receptors (nAChRs) ligands structurally related to anatoxin-a

The introduction of the isoxazole ring as bioisosteric replacement of the acetyl group of anatoxin-a led to a new series of derivatives binding to nicotinic acetylcholine receptors. Bulkier substitutions than methyl at the 3 position of isoxazole were shown to be detrimental for the activity. The binding potency of the most interesting compounds with α1, α7 and α3β4 receptor subtypes, was, anyway, only at micromolar level. Moreover, differently from known derivatives with pyridine, isoxazole condensed to azabicyclo ring led to no activity.     

Catalytic properties of thioredoxin immobilized on superparamagnetic nanoparticles

Thioredoxin (Trx1), a very important protein for regulating intracellular redox reactions, was immobilized on iron oxide superparamagnetic nanoparticles previously coated with 3-aminopropyltriethoxysilane (APTS) via covalent coupling using the EDC (1-ethyl-3-{3-dimethylaminopropyl}carbodiimide) method. The system was extensively characterized by atomic force microscopy, vibrational and magnetic techniques. In addition, gold nanoparticles were also employed to probe the exposed groups in the immobilized enzyme based on the SERS (surface enhanced Raman scattering) effect, confirming the accessibility of the cysteines residues at the catalytic site. For the single coated superparamagnetic nanoparticle, by monitoring the enzyme activity with the Ellman reagent, DTNB = 5,5′-dithio-bis(2-15 nitrobenzoic acid), an inhibitory effect was observed after the first catalytic cycle. The inhibiting effect disappeared after the application of an additional silicate coating before the APTS treatment, reflecting a possible influence of unprotected iron-oxide sites in the redox kinetics. In contrast, the doubly coated system exhibited a normal in-vitro kinetic activity, allowing a good enzyme recovery and recyclability.     

New Insights into the Relationship between mIGF-1-Induced Hypertrophy and Ca2+ Handling in Differentiated Satellite Cells

Muscle regeneration involves the activation of satellite cells, is regulated at the genetic and epigenetic levels, and is strongly influenced by gene activation and environmental conditions. The aim of this study was to determine whether the overexpression of mIGF-1 can modify functional features of satellite cells during the differentiation process, particularly in relation to modifications of intracellular Ca²⁺ handling.Satellite cells were isolated from wild-type and MLC/mIGF-1 transgenic mice. The cells were differentiated in vitro, and morphological analyses, intracellular Ca²⁺ measurements, and ionic current recordings were performed.mIGF-1 overexpression accelerates satellite cell differentiation and promotes myotube hypertrophy. In addition, mIGF-1 overexpression-induced potentiation of myogenesis triggers both quantitative and qualitative changes to the control of intracellular Ca²⁺ handling. In particular, the differentiated MLC/mIGF-1 transgenic myotubes have reduced velocity and amplitude of intracellular Ca²⁺ increases after stimulation with caffeine, KCl and acetylcholine. This appears to be due, at least in part, to changes in the physico-chemical state of the sarcolemma (increased membrane lipid oxidation, increased output currents) and to increased expression of dihydropyridine voltage-operated Ca²⁺ channels. Interestingly, extracellular ATP and GTP evoke intracellular Ca²⁺ mobilization to greater extents in the MLC/mIGF-1 transgenic satellite cells, compared to the wild-type cells.These data suggest that these MLC/mIGF-1 transgenic satellite cells are more sensitive to trophic stimuli, which can potentiate the effects of mIGF-1 on the myogenic programme.     

High molecular weight RNA containing histone messenger in the sea urchin Paracentrotus lividus

Sea urchin RNA extracted from early and mesenchyme blastula embryos and oocytes and fractionated on denaturing sucrose density gradients, was hybridized with histone DNA recombinants of Psammechinus miliaris (clone λh22) and of Paracentrotus lividus (clone pPH70). Histone sequences are found in the 9 S and larger than 9 S regions of the formamide/sucrose density gradients. The melting of the RNA-DNA duplexes obtained by hybridization of polysomal and high molecular weight RNA of embryos of P. lividus at the stage of early blastula, suggests a degree of heterogeneity in the high M_r RNA. The high M_r RNA contains at least four of the five histone gene sequences covalently linked.     

Sympathovagal Balance,Nighttime Blood Pressure,and QT Intervals in Normotensive Obese Women

Objective : We describe associations among the heart‐rate‐corrected QT (QTc) interval, QTc dispersion (QTc‐d), circadian BP variation, and autonomic function in obese normotensive women and the effect of sustained weight loss. Research Methods and Procedures : In 71 obese (BMI = 37.14 ± 2.6 kg/m²) women, 25 to 44 years of age, circadian BP variations (24‐hour ambulatory BP monitoring), autonomic function (power spectral analysis of RR interval oscillations), and cardiac repolarization times (QTc‐d and QTc interval) were recorded at baseline and after 1 year of a multidisciplinary program of weight reduction. Results : Compared with nonobese age‐matched women (n = 28, BMI = 23 ± 2.0 kg/m²), obese women had higher values of QTc‐d (p < 0.05) and QTc (p < 0.05), an altered sympathovagal balance (ratio of low‐frequency/high‐frequency power, p < 0.01), and a blunted nocturnal drop in BP (p < 0.01). In obese women, QTc‐d and the QTc interval correlated with diastolic nighttime BP (p < 0.01) and sympathovagal balance (p < 0.01). Waist‐to‐hip ratio, free fatty acids, and plasma insulin levels correlated with QT intervals and reduced nocturnal drops in both systolic and diastolic BP and sympathovagal balance (p < 0.01). After 1 year, obese women lost at least 10% of their original weight, which was associated with decrements of QTc‐d (p < 0.02), the QTc interval (p < 0.05), nighttime BP (p < 0.01), and sympathovagal balance (p < 0.02). Discussion : Sustained weight loss is a safe method to ameliorate diastolic nighttime BP drop and sympathetic overactivity, which may reduce the cardiovascular risk in obese women.     

Plasma glutamine decreases immediately after surgery and is related to incisiveness

Glutamine (gln) is the most abundant free amino acid in the blood. It is involved in important metabolic and biochemical processes, like cell proliferation and oxidative stress. Previous studies have demonstrated that gln concentration in human plasma decreases in several conditions such as sepsis, ischemia-reperfusion, trauma, major surgery and burn. The aim of the present work was to compare the acute effects of different types of surgical interventions and of anesthetization on blood gln concentration. Plasma samples from 88 subjects (30 males and 58 females) were collected before and after major or minor surgery and the gln concentration was analyzed with high-performance liquid chromatography. The results showed that plasma gln concentration after surgery was lower than pre-surgery values and that in major surgery the decrease of gln was higher than in minor surgery. No significant effect was shown for sex or type of anesthesia. These results demonstrate the importance of a gln supplementation before a surgical intervention and show that the amount of gln supplementation should also be adjusted based on the type of surgery.     

Impaired myocardial metabolic reserve and substrate selection flexibility during stress in patients with idiopathic dilated cardiomyopathy

Under resting conditions, the failing heart shifts fuel use toward greater glucose and lower free fatty acid (FFA) oxidation. We hypothesized that chronic metabolic abnormalities in patients with dilated cardiomyopathy (DCM) are associated with the absence of the normal increase in myocardial glucose uptake and maintenance of cardiac mechanical efficiency in response to pacing stress. In 10 DCM patients and 6 control subjects, we measured coronary flow by intravascular ultrasonometry and sampled arterial and coronary sinus blood. Myocardial metabolism was determined at baseline, during atrial pacing at 130 beats/min, and at 15 min of recovery by infusion of [(3)H]oleate and [(13)C]lactate and measurement of transmyocardial arteriovenous differences of oxygen and metabolites. At baseline, DCM patients showed depressed coronary flow, reduced uptake and oxidation of FFA, and preferential utilization of carbohydrates. During pacing, glucose uptake increased by 106% in control subjects but did not change from baseline in DCM patients. Lactate release increased by 122% in DCM patients but not in control subjects. Cardiac mechanical efficiency in DCM patients was not different compared with control subjects at baseline but was 34% lower during stress. Fatty acid uptake and oxidation did not change with pacing in either group. Our results show that in DCM there is preferential utilization of carbohydrates, which is associated with reduced flow and oxygen consumption at rest and an impaired ability to increase glucose uptake during stress. These metabolic abnormalities might contribute to progressive cardiac deterioration and represent a target for therapeutic strategies aimed at modulating cardiac substrate utilization.