Nicotinamide Inhibits Vasculogenic Mimicry,an Alternative Vascularization Pathway Observed in Highly Aggressive Melanoma

Vasculogenic mimicry (VM) describes functional vascular channels composed only of tumor cells and its presence predicts poor prognosis in melanoma patients. Inhibition of this alternative vascularization pathway might be of clinical importance, especially as several anti-angiogenic therapies targeting endothelial cells are largely ineffective in melanoma. We show the presence of VM structures histologically in a series of human melanoma lesions and demonstrate that cell cultures derived from these lesions form tubes in 3D cultures ex vivo. We tested the ability of nicotinamide, the amide form of vitamin B3 (niacin), which acts as an epigenetic gene regulator through unique cellular pathways, to modify VM. Nicotinamide effectively inhibited the formation of VM structures and destroyed already formed ones, in a dose-dependent manner. Remarkably, VM formation capacity remained suppressed even one month after the complete withdrawal of Nicotimamid. The inhibitory effect of nicotinamide on VM formation could be at least partially explained by a nicotinamide-driven downregulation of vascular endothelial cadherin (VE-Cadherin), which is known to have a central role in VM. Further major changes in the expression profile of hundreds of genes, most of them clustered in biologically-relevant clusters, were observed. In addition, nicotinamide significantly inhibited melanoma cell proliferation, but had an opposite effect on their invasion capacity. Cell cycle analysis indicated moderate changes in apoptotic indices. Therefore, nicotinamide could be further used to unravel new biological mechanisms that drive VM and tumor progression. Targeting VM, especially in combination with anti-angiogenic strategies, is expected to be synergistic and might yield substantial anti neoplastic effects in a variety of malignancies.     

In vitro and in vivo investigation of the efficacy of arylimidamide DB1831 and its mesylated salt form--DB1965--against Trypanosoma cruzi infection

Chagas disease is caused by infection with the intracellular protozoan parasite Trypanosoma cruzi. At present, nifurtimox and benznidazole, both compounds developed empirically over four decades ago, represent the chemotherapeutic arsenal for treating this highly neglected disease. However, both drugs present variable efficacy depending on the geographical area and the occurrence of natural resistance, and are poorly effective against the later chronic stage. As a part of a search for new therapeutic opportunities to treat chagasic patients, pre-clinical studies were performed to characterize the activity of a novel arylimidamide (AIA--DB1831 (hydrochloride salt) and DB1965 (mesylate salt)) against T. cruzi. These AIAs displayed a high trypanocidal effect in vitro against both relevant forms in mammalian hosts, exhibiting a high selectivity index and a very high efficacy (IC(50) value/48 h of 5-40 nM) against intracellular parasites. DB1965 shows high activity in vivo in acute experimental models (mouse) of T. cruzi, showing a similar effect to benznidazole (Bz) when compared under a scheme of 10 daily consecutive doses with 12.5 mg/kg. Although no parasitological cure was observed after treating with 20 daily consecutive doses, a combined dosage of DB1965 (5 mg/kg) with Bz (50 mg/kg) resulted in parasitaemia clearance and 100% animal survival. In summary, our present data confirmed that aryimidamides represent promising new chemical entities against T. cruzi in therapeutic schemes using the AIA alone or in combination with other drugs, like benznidazole.     

Continuous and High-Intensity Interval Training: Which Promotes Higher Pleasure?

Objectives

To compare the psychological responses to continuous (CT) and high-intensity interval training (HIT) sessions.

Methods

Fifteen men attended one CT session and one HIT session. During the first visit, the maximum heart rate, VO_2Peak and respiratory compensation point (RCP) were determined through a maximal cardiopulmonary exercise test. The HIT stimulus intensity corresponded to 100% of VO_2Peak, and the average intensity of both sessions was maintained at 15% below the RCP. The order of the sessions was randomized. Psychological and physiological variables were recorded before, during and after each session.

Results

There were no significant differences between the average percentages of VO₂ during the two exercise sessions (HIT: 73.3% vs. CT: 71.8%; p = 0.779). Lower responses on the feeling scale (p≤0.01) and higher responses on the felt arousal scale (p≤0.001) and the rating of perceived exertion were obtained during the HIT session. Despite the more negative feeling scale responses observed during HIT and a greater feeling of fatigue (measured by Profile of Mood States) afterwards (p<0.01), the physical activity enjoyment scale was not significantly different between the two conditions (p = 0.779).

Conclusion

Despite the same average intensity for both conditions, similar psychological responses under HIT and CT conditions were not observed, suggesting that the higher dependence on anaerobic metabolism during HIT negatively influenced the feeling scale responses.     

Cellular Growth and Mitochondrial Ultrastructure of Leishmania (Viannia) braziliensis Promastigotes Are Affected by the Iron Chelator 2,2-Dipyridyl

Background

Iron is an essential element for the survival of microorganisms in vitro and in vivo, acting as a cofactor of several enzymes and playing a critical role in host-parasite relationships. Leishmania (Viannia) braziliensis is a parasite that is widespread in the new world and considered the major etiological agent of American tegumentary leishmaniasis. Although iron depletion leads to promastigote and amastigote growth inhibition, little is known about the role of iron in the biology of Leishmania. Furthermore, there are no reports regarding the importance of iron for L. (V.) braziliensis.

Methodology/Principal Findings

In this study, the effect of iron on the growth, ultrastructure and protein expression of L. (V.) braziliensis was analyzed by the use of the chelator 2,2-dipyridyl. Treatment with 2,2-dipyridyl affected parasites'' growth in a dose- and time-dependent manner. Multiplication of the parasites was recovered after reinoculation in fresh culture medium. Ultrastructural analysis of treated promastigotes revealed marked mitochondrial swelling with loss of cristae and matrix and the presence of concentric membranar structures inside the organelle. Iron depletion also induced Golgi disruption and intense cytoplasmic vacuolization. Fluorescence-activated cell sorting analysis of tetramethylrhodamine ester-stained parasites showed that 2,2-dipyridyl collapsed the mitochondrial membrane potential. The incubation of parasites with propidium iodide demonstrated that disruption of mitochondrial membrane potential was not associated with plasma membrane permeabilization. TUNEL assays indicated no DNA fragmentation in chelator-treated promastigotes. In addition, two-dimensional electrophoresis showed that treatment with the iron chelator induced up- or down-regulation of proteins involved in metabolism of nucleic acids and coordination of post-translational modifications, without altering their mRNA levels.

Conclusions

Iron chelation leads to a multifactorial response that results in cellular collapse, starting with the interruption of cell proliferation and culminating in marked mitochondrial impairment in some parasites and their subsequent cell death, whereas others may survive and resume proliferating.     

Nandrolone decanoate impairs exercise-induced cardioprotection: role of antioxidant enzymes

The beneficial effects of exercise in reducing the incidence of cardiovascular diseases are well known and the abuse of anabolic androgenic steroids (AAS) has been associated to cardiovascular disorders. Previous studies showed that heart protection to ischemic events would be mediated by increasing the antioxidant enzyme activities. Here, we investigated the impact of exercise and high doses of the AAS nandrolone decanoate (DECA), 10 mg kg⁻¹ body weight during 8 weeks, in cardiac tolerance to ischemic events as well as on the activity of antioxidant enzymes in rats. After a global ischemic event, hearts of control trained (CT) group recovered about 70% of left ventricular developed pressure, whereas DECA trained (DT), control sedentary (CS) and DECA sedentary (DS) animals recovered only about 20%. Similarly, heart infarct size was significantly lower in the CT group compared to animals of the three other groups. The activities of the antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GR) were significantly higher in CT animals than in the other three groups, whereas catalase activity was not affected in any group. Together, these results indicate that chronic treatment with DECA cause an impairment of exercise induction of antioxidant enzyme activities, leading to a reduced cardioprotection upon ischemic events.     

Persister-promoting bacterial toxin TisB produces anion-selective pores in planar lipid bilayers

We studied membrane activity of the bacterial peptide TisB involved in persister cell formation. TisB and its analogs form multi-state ion-conductive pores in planar lipid bilayers with all states displaying similar anionic selectivity. TisB analogs differing by ±1 elementary charges show corresponding changes in selectivity. Probing TisB pores with poly-(ethylene glycol)s reveals only restricted partitioning even for the smallest polymers, suggesting that the pores are characterized by a relatively small diameter. These findings allow us to suggest that TisB forms clusters of narrow pores that are essential for its mechanism of action.     

Suppression of GATA-3 Nuclear Import and Phosphorylation: A Novel Mechanism of Corticosteroid Action in Allergic Disease

Background

GATA-3 plays a critical role in regulating the expression of the cytokines interleukin (IL)-4, IL-5, and IL-13 from T helper-2 (Th2) cells and therefore is a key mediator of allergic diseases. Corticosteroids are highly effective in suppressing allergic inflammation, but their effects on GATA-3 are unknown. We investigated the effect of the corticosteroid fluticasone propionate on GATA-3 regulation in human T-lymphocytes in vitro and in vivo.

Methods and Findings

In a T lymphocyte cell line (HuT-78) and peripheral blood mononuclear cells stimulated by anti-CD3 and anti-CD28 in vitro we demonstrated that fluticasone inhibits nuclear translocation of GATA-3 and expression of Th2 cytokines via a mechanism independent of nuclear factor-κB and is due, in part, to competition between GATA-3 and the ligand-activated glucocorticoid receptor for nuclear transport through the nuclear importer importin-α. In addition, fluticasone induces the expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1), the endogenous inhibitor of p38 MAPK, which is necessary for GATA-3 nuclear translocation. These inhibitory effects of fluticasone are rapid, potent, and prolonged. We also demonstrated that inhaled fluticasone inhibits GATA-3 nuclear translocation in peripheral blood lymphocytes of patients with asthma in vivo.

Conclusions

Corticosteroids have a potent inhibitory effect on GATA-3 via two interacting mechanisms that potently suppress Th2 cytokine expression. This novel mechanism of action of corticosteroids may account for the striking clinical efficacy of corticosteroids in the treatment of allergic diseases. Please see later in the article for Editors'' Summary     

Synergistic Induction of Endothelin-1 by Tumor Necrosis Factor �� and Interferon �� Is due to Enhanced NF-��B Binding and Histone Acetylation at Specific ��B Sites

Endothelin-1 (ET-1) is a potent vasoconstrictor and co-mitogen for vascular smooth muscle and is implicated in pulmonary vascular remodeling and the development of pulmonary arterial hypertension. Vascular smooth muscle is an important source of ET-1. Here we demonstrate synergistic induction of preproET-1 message RNA and release of mature peptide by a combination of tumor necrosis factor α (TNFα) and interferon γ (IFNγ) in primary human pulmonary artery smooth muscle cells. This induction was prevented by pretreatment with the histone acetyltransferase inhibitor anacardic acid. TNFα induced a rapid and prolonged pattern of nuclear factor (NF)-κB p65 subunit activation and binding to the native preproET-1 promoter. In contrast, IFNγ induced a delayed activation of interferon regulatory factor-1 without any effect on NF-κB p65 nuclear localization or consensus DNA binding. However, we found cooperative p65 binding and histone H4 acetylation at distinct κB sites in the preproET-1 promoter after stimulation with both TNFα and IFNγ. This was associated with enhanced recruitment of RNA polymerase II to the ATG start site and read-through of the ET-1 coding region. Understanding such mechanisms is crucial in determining the key control points in ET-1 release. This has particular relevance to developing novel treatments targeted at the inflammatory component of pulmonary vascular remodeling.Endothelin-1 is a 21-amino acid peptide which is known to be both a potent vasoconstrictor and mitogen for vascular smooth muscle (1, 2). It is released as a 38-amino acid precursor (Big ET-1²) before cleavage to the mature ET-1 form. As such it has been implicated in the pathogenesis of vascular disease and is particularly associated with pulmonary arterial hypertension (3). Indeed, several endothelin receptor antagonists are now approved for the treatment of pulmonary arterial hypertension (4). However, endothelin receptor antagonists as a class are associated with potentially serious side effects (4), making new treatments aimed at blocking ET-1 synthesis an attractive alternative.Although endothelial cells are thought to be the main source of ET-1 release, several groups including our own have shown that ET-1 can be released from the more numerous vascular smooth muscle cells (5–10). The vascular pathology observed in pulmonary arterial hypertension is propagated by inflammation, and circulating levels of cytokines including tumor necrosis factor α (TNFα) are elevated in patients with pulmonary arterial hypertension (11–15). In many cell types cytokines mediate their biological effects at least in part by the activation of the nuclear factor κB (NF-κB) pathway (16), and a role for NF-κB in pulmonary arterial hypertension has been proposed (17). In addition, we have shown previously that a combination of TNFα and interferon γ (IFNγ) stimulates human pulmonary artery smooth muscle (HPASM) cells to release ET-1 (18). However, the mechanisms underlying this effect are unknown.The preproET-1 promoter region has been shown experimentally to possess binding sites for nuclear factor (NF)-1 and phorbol ester-sensitive c-Fos and c-Jun complexes (19), acute phase reactant regulatory proteins, and binding sites for AP-1 and GATA-2 (20–22). In addition, binding sites for interferon regulatory factor-1 (IRF-1) and NF-κB are predicted by Transfac analysis (23). The close proximity of the IRF-1 site and one of the NF-κB sites is characteristic of genes that are regulated by the synergistic action of TNFα and IFNγ, such as interleukin-6 (IL-6) and intercellular adhesion molecule-1 (24, 25), although ET-1 has not previously been recognized in this group.Our aims were, therefore, to investigate the role of NF-κB in ET-1 release by primary HPASM cells. In addition, we were interested in the role of histone acetylation in the epigenetic control of the ET-1 production. Understanding these novel mechanisms will allow a greater understanding of the pathogenesis of vascular remodeling in pulmonary vessels and aid in the development of new treatment strategies aimed at blocking synthesis of ET-1.