Apoptosis through Bcl-2/Bax and Cleaved Caspase Up-Regulation in Melanoma Treated by Boron Neutron Capture Therapy

Boron neutron capture therapy (BNCT) is a binary treatment involving selective accumulation of boron carriers in a tumor followed by irradiation with a thermal or epithermal neutron beam. The neutron capture reaction with a boron-10 nucleus yields high linear energy transfer (LET) particles, alpha and ⁷Li, with a range of 5 to 9 µm. These particles can only travel very short distances and release their damaging energy directly into the cells containing the boron compound. We aimed to evaluate proliferation, apoptosis and extracellular matrix (ECM) modifications of B16F10 melanoma and normal human melanocytes after BNCT. The amounts of soluble collagen and Hsp47, indicating collagen synthesis in the ECM, as well as the cellular markers of apoptosis, were investigated. BNCT decreased proliferation, altered the ECM by decreasing collagen synthesis and induced apoptosis by regulating Bcl-2/Bax in melanoma. Additionally, BNCT also increased the levels of TNF receptor and the cleaved caspases 3, 7, 8 and 9 in melanoma. These results suggest that multiple pathways related to cell death and cell cycle arrest are involved in the treatment of melanoma by BNCT.     

Fluorinated Cannabidiol Derivatives: Enhancement of Activity in Mice Models Predictive of Anxiolytic,Antidepressant and Antipsychotic Effects

Cannabidiol (CBD) is a major Cannabis sativa constituent, which does not cause the typical marijuana psychoactivity. However, it has been shown to be active in a numerous pharmacological assays, including mice tests for anxiety, obsessive-compulsive disorder, depression and schizophrenia. In human trials the doses of CBD needed to achieve effects in anxiety and schizophrenia are high. We report now the synthesis of 3 fluorinated CBD derivatives, one of which, 4''-F-CBD (HUF-101) (1), is considerably more potent than CBD in behavioral assays in mice predictive of anxiolytic, antidepressant, antipsychotic and anti-compulsive activity. Similar to CBD, the anti-compulsive effects of HUF-101 depend on cannabinoid receptors.     

Biochemical characterization of ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP, E.C. 3.1.4.1) from rat heart left ventricle

In the present study we investigate the biochemical properties of the members of NPP family in synaptosomes prepared from rat heart left ventricles. Using p-nitrophenyl-5′-thymidine monophosphate (p-Nph-5′-TMP) as substrate for E-NPPs in rat cardiac synaptosomes, we observed an alkaline pH dependence, divalent cation dependence and the K _M value corresponded to 91.42 ± 13.97 μM and the maximal velocity (V _max ) value calculated was 63.79 ± 3.59 nmol p-nitrophenol released/min/mg of protein (mean ± SD, n = 4). Levamisole (1 mM), was ineffective as inhibitor of p-Nph-5′-TMP hydrolysis in pH 8.9 (optimum pH for the enzyme characterized). Suramin (0.25 mM) strongly reduced the hydrolysis of p-Nph-5′-TMP by about 46%. Sodium azide (10 and 20 mM) and gadolinium chloride (0.3 and 0.5 mM), E-NTPases inhibitors, had no effects on p-Nph-5′-TMP hydrolysis. RT-PCR analysis of left ventricle demonstrated the expression of NPP2 and NPP3 enzymes, but excluded the presence of NPP1 member. By quantitative real-time PCR we identified the NPP3 as the enzyme with the highest expression in rat left ventricle. The demonstration of the presence of the E-NPP family in cardiac system, suggest that these enzymes could contribute with the fine-tuning control of the nucleotide levels at the nerve terminal endings of left ventricles that are involved in several cardiac pathologies.     

1-(7-Chloroquinolin-4-yl)-N-(4-Methoxybenzyl)-5-Methyl-1H-1,2, 3-Triazole-4- carboxamide Reduces Aβ Formation and Tau Phosphorylation in Cellular Models of Alzheimer’s Disease

1-(7-Chloroquinolin-4-yl)-N-(4-methoxybenzyl)-5-methyl-1H-1,2,3-triazole-4- carboxamide (QTC-4-MeOBnE) is a new multi-target directed ligand (MTDL) rationally designed to have affinity with β-secretase (BACE), Glycogen Synthase Kinase 3β (GSK3β) and acetylcholinesterase, which are considered promising targets on the development of disease-modifying therapies against Alzheimer’s Disease (AD). Previously, QTC-4-MeOBnE treatment showed beneficial effects in preclinical AD-like models by influencing in vivo neurogenesis, oxidative and inflammatory pathways. However, the biological effect and mechanism of action exerted by QTC-4-MeOBnE in AD cellular models have not been elucidated yet. Hereby we investigate the acute effect of QTC-4-MeOBnE on neuronal cells overexpressing Amyloid Protein Precursor (APP) or human tau protein, the two main features of the AD pathophysiology. When compared to the control group, QTC-4-MeOBnE treatment prevented amyloid beta (Aβ) formation through the downregulation of APP and BACE levels in APPswe-expressing cells. Furthermore, in N2a cells overexpressing human tau, QTC-4-MeOBnE reduced the levels of phosphorylated forms of tau via the modulation of the GSK3β pathway. Taken together, our findings provide new insights into the mechanism of action exerted by QTC-4-MeOBnE in AD cellular models, and further support its potential as an interesting therapeutic strategy against AD.

     

MicroRNA 155 Control of p53 Activity Is Context Dependent and Mediated by Aicda and Socs1

In biological processes, the balance between positive and negative inputs is critical for an effective physiological response and to prevent disease. A case in point is the germinal center (GC) reaction, wherein high mutational and proliferation rates are accompanied by an obligatory suppression of the DNA repair machinery. Understandably, when the GC reaction goes awry, loss of immune cells or lymphoid cancer ensues. Here, we detail the functional interactions that make microRNA 155 (miR-155) a key part of this process. Upon antigen exposure, miR-155^−/− mature B cells displayed significantly higher double-strand DNA break (DSB) accumulation and p53 activation than their miR-155^+/+ counterparts. Using B cell-specific knockdown strategies, we confirmed the role of the miR-155 target Aicda (activation-induced cytidine deaminase) in this process and, in combination with a gain-of-function model, unveiled a previously unappreciated role for Socs1 in directly modulating p53 activity and the DNA damage response in B lymphocytes. Thus, miR-155 controls the outcome of the GC reaction by modulating its initiation (Aicda) and termination (Socs1/p53 response), suggesting a mechanism to explain the quantitative defect in germinal center B cells found in mice lacking or overexpressing this miRNA.     

Fractal characteristics of May-Grünwald-Giemsa stained chromatin are independent prognostic factors for survival in multiple myeloma

Background

The use of computerized image analysis for the study of nuclear texture features has provided important prognostic information for several neoplasias. Recently fractal characteristics of the chromatin structure in routinely stained smears have shown to be independent prognostic factors in acute leukemia. In the present study we investigated the influence of the fractal dimension (FD) of chromatin on survival of patients with multiple myeloma.

Methodology

We analyzed 67 newly diagnosed patients from our Institution treated in the Brazilian Multiple Myeloma Study Group. Diagnostic work-up consisted of peripheral blood counts, bone marrow cytology, bone radiograms, serum biochemistry and cytogenetics. The International Staging System (ISS) was used. In every patient, at least 40 digital nuclear images from diagnostic May-Grünwald-Giemsa stained bone marrow smears were acquired and transformed into pseudo-3D images. FD was determined by the Minkowski-Bouligand method extended to three dimensions. Goodness-of-fit of FD was estimated by the R² values in the log-log plots. The influence of diagnostic features on overall survival was analyzed in Cox regressions. Patients that underwent autologous bone marrow transplantation were censored at the day of transplantation.

Principal Findings

Median age was 56 years. According to ISS, 14% of the patients were stage I, 39% were stage II and 47% were stage III. Additional features of a bad prognosis were observed in 46% of the cases. When stratifying for ISS, both FD and its goodness-of-fit were significant prognostic factors in univariate analyses. Patients with higher FD values or lower goodness-of-fit showed a worse outcome. In the multivariate Cox-regression, FD, R², and ISS stage entered the final model, which showed to be stable in a bootstrap resampling study.

Conclusions

Fractal characteristics of the chromatin texture in routine cytological preparations revealed relevant prognostic information in patients with multiple myeloma.     

Ectopic expression of an amino acid transporter (VfAAP1) in seeds of Vicia narbonensis and pea increases storage proteins

Storage protein synthesis is dependent on available nitrogen in the seed, which may be controlled by amino acid import via specific transporters. To analyze their rate-limiting role for seed protein synthesis, a Vicia faba amino acid permease, VfAAP1, has been ectopically expressed in pea (Pisum sativum) and Vicia narbonensis seeds under the control of the legumin B4 promoter. In mature seeds, starch is unchanged but total nitrogen is 10% to 25% higher, which affects mainly globulin, vicilin, and legumin, rather than albumin synthesis. Transgenic seeds in vitro take up more [14C]-glutamine, indicating increased sink strength for amino acids. In addition, more [14C] is partitioned into proteins. Levels of total free amino acids in growing seeds are unchanged but with a shift toward higher relative abundance of asparagine, aspartate, glutamine, and glutamate. Hexoses are decreased, whereas metabolites of glycolysis and the tricarboxylic acid cycle are unchanged or slightly lower. Phosphoenolpyruvate carboxylase activity and the phosphoenolpyruvate carboxylase-to-pyruvate kinase ratios are higher in seeds of one and three lines, indicating increased anaplerotic fluxes. Increases of individual seed size by 20% to 30% and of vegetative biomass indicate growth responses probably due to improved nitrogen status. However, seed yield per plant was not altered. Root application of [15N] ammonia results in significantly higher label in transgenic seeds, as well as in stems and pods, and indicates stimulation of nitrogen root uptake. In summary, VfAAP1 expression increases seed sink strength for nitrogen, improves plant nitrogen status, and leads to higher seed protein. We conclude that seed protein synthesis is nitrogen limited and that seed uptake activity for nitrogen is rate limiting for storage protein synthesis.