Is Nox the source of ROS involved in Glut1 activity in B1647 cells?

The discovery of a family of superoxide-generating enzymes, homologues of ABSTRACT phagocyte oxidase, has led to the concept that ROS are “intentionally” generated with biological functions in various cell types. We have recently shown that, in two leukaemic cell lines (M07e and B1647), there is a correlation between ROS and an important physiological activity, like glucose uptake, which is up-regulated in leukaemic cells. In this study, we tried to elucidate the sources of ROS generation and the mechanisms by which ROS are involved in the regulation of glucose uptake in B1647 cells. In particular, we investigated the presence and the role of a member of the NAD(P)H oxidase family (Nox). Data obtained in the presence of Nox inhibitors suggest that ROS involved in glucose uptake could be generated by this membrane-bound enzymatic complex. The effects of tyrosine kinase inhibitors and antioxidants show the importance of phosphorylation processes in the regulation of glucose uptake. PI3-kinase seems to be involved in ROS generation, possibly through Rac, which binds to Nox. The activation of tyrosine kinase receptor by vascular endothelium growth factor (VEGF), produced by an autocrine pathway in this cell line, seems to be an important step of this pathway

Natural Compounds as a Strategy to Optimize " In Vitro" Expansion of Stem Cells

The efficient use of stem cells for transplantation is often limited by the relatively low number of stem cells collected. The ex vivo expansion of human stem cells for clinical use is a potentially valuable approach to increase stem cell number. Currently, most of the procedures used to expand stem cells are carried out using a 21% oxygen concentration, which is about 4- to 10-fold greater than the concentration characteristic of their natural niches. Hyperoxia might cause oxidative stress with a deleterious effect on the physiology of cultured stem cells. In this review, we investigate and critically examine the available information on the ability of natural compounds to counteract hyperoxia-induced damage in different types of stem cells ex vivo. In particular, we focused on proliferation and stemness maintenance in an attempt to draw up useful indications to define new culture media with a promoting activity on cell expansion in vitro

Combination of Epigallocatechin Gallate and Sulforaphane Counteracts In Vitro Oxidative Stress and Delays Stemness Loss of Amniotic Fluid Stem Cells

Amniotic fluid stem cells (AFSCs) are characterized in vivo by a unique niche guarantying their homeostatic role in the body. Maintaining the functionality of stem cells ex vivo for clinical applications requires a continuous improvement of cell culture conditions. Cellular redox status plays an important role in stem cell biology as long as reactive oxygen species (ROS) concentration is finely regulated and their adverse effects are excluded. The aim of this study was to investigate the protective effect of two antioxidants, sulforaphane (SF) and epigallocatechin gallate (EGCG), against in vitro oxidative stress due to hyperoxia and freeze-thawing cycles in AFSCs. Human AFSCs were isolated and characterized from healthy subjects. Assays of metabolic function and antioxidant activity were performed to investigate the effect of SF and EGCG cotreatment on AFSCs. Real-time PCR was used to investigate the effect of the cotreatment on pluripotency, senescence, osteogenic and adipogenic markers, and antioxidant enzymes. Alkaline phosphatase assays and Alizarin Red staining were used to confirm osteogenic differentiation. The cotreatment with SF and EGCG was effective in reducing ROS production, increasing GSH levels, and enhancing the endogenous antioxidant defences through the upregulation of glutathione reductase, NAD(P)H:quinone oxidoreductase-1, and thioredoxin reductase. Intriguingly, the cotreatment sustained the stemness state by upregulating pluripotency markers such as OCT4 and NANOG. Moreover, the cotreatment influenced senescence-associated gene markers in respect to untreated cells. The cotreatment upregulated osteogenic gene markers and promoted osteogenic differentiation in vitro. SF and EGCG can be used in combination in AFSC culture as a strategy to preserve stem cell functionality

Sulforaphane Modulates AQP8-Linked Redox Signalling in Leukemia Cells

Sulforaphane, a biologically active isothiocyanate compound extracted from cruciferous vegetables, has been shown to exert cytotoxic effects on many human cancer cells, including leukemia. However, the exact molecular mechanisms behind the action of sulforaphane in hematological malignancies are still unclear. Like other cancer cells, leukemia cells produce high level of reactive oxygen species; in particular, hydrogen peroxide derived from Nox family is involved in various redox signal transduction pathways, promoting cell proliferation and survival. Recent evidence show that many tumour cell types express elevated level of aquaporin isoforms, and we previously demonstrated that aquaporin-8 acts as H2O2 transport facilitator across the plasma membrane of B1647 cells, a model of acute myeloid human leukemia. Thus, the control of AQP8-mediated H2O2 transport could be a novel strategy to regulate cell signalling and survival. To this purpose, we evaluated whether sulforaphane could somehow affect aquaporin-8-mediated H2O2 transport and/or Nox-mediated H2O2 production in B1647 cell line. Results indicated that sulforaphane inhibited both aquaporin-8 and Nox2 expression, thus decreasing B1647 cells viability. Moreover, the data obtained by coimmunoprecipitation technique demonstrated that these two proteins are linked to each other; thus, sulforaphane has an important role in modulating the downstream events triggered by the axis Nox2-aquaporin-8. Cell treatment with sulforaphane also reduced the expression of peroxiredoxin-1, which is increased in almost all acute myeloid leukemia subtypes. Interestingly, sulforaphane concentrations able to trigger these effects are achievable by dietary intake of cruciferous vegetables, confirming the importance of the beneficial effect of a diet rich in bioactive compounds

Protective Effect of Wheat Derived Non-specific lipid-transfer Protein 2 on Vascular Endothelium Inflammation

The important functions of the endothelium and the relationship between cardiovascular risk factors and endothelial dysfunction suggest the primary role of this tissue as a target for dietary strategies aimed at the prevention from related diseases. Cereals are key component of a healthy and balanced diet, and the presence of non-specific lipid-transfer protein 2 (nsLTP2) in wheat represents an added value to contribute to maintain the functionality of the vascular endothelium and consequently of the cardiovascular system. Indeed, nsLTP2 downregulates the expression of the main cell adhesion molecules induced by a pro-inflammatory cytokine and, meanwhile, upregulates heme oxigenase-1, exerting a cytoprotective/anti-inflammatory activity. Therefore, nsLTP2 might represent a food-derived tool to protect the vascular system against several pathological condition

Identification of Anti-Neuroinflammatory Bioactive Compounds in Essential Oils and Aqueous Distillation Residues Obtained from Commercial Varieties of Cannabis sativa L

Neuroinflammation, which is mainly triggered by microglia, is a key contributor to multiple neurodegenerative diseases. Natural products, and in particular Cannabis sativa L., due to its richness in phytochemical components, represent ideal candidates to counteract neuroinflammation. We previously characterized different C. sativa commercial varieties which showed significantly different chemical profiles. On these bases, the aim of this study was to evaluate essential oils and aqueous distillation residues from the inflorescences of three different hemp varieties for their anti-neuroinflammatory activity in BV-2 microglial cells. Cells were pretreated with aqueous residues or essential oils and then activated with LPS. Unlike essential oils, aqueous residues showed negligible effects in terms of anti-inflammatory activity. Among the essential oils, the one obtained from 'Gorilla Glue' was the most effective in inhibiting pro-inflammatory mediators and in upregulating anti-inflammatory ones through the modulation of the p38 MAPK/NF-kappa B pathway. Moreover, the sesquiterpenes (E)-caryophyllene, alpha-humulene, and caryophyllene oxide were identified as the main contributors to the essential oils' anti-inflammatory activity. To our knowledge, the anti-neuroinflammatory activity of alpha-humulene has not been previously described. In conclusion, our work shows that C. sativa essential oils characterized by high levels of sesquiterpenes can be promising candidates in the prevention/counteraction of neuroinflammation

Role of Plasma Membrane Caveolae/Lipid Rafts in VEGF-Induced Redox Signaling in Human Leukemia Cells

Caveolae/lipid rafts are membrane-rich cholesterol domains endowed with several functions in signal transduction and caveolin-1 (Cav-1) has been reported to be implicated in regulating multiple cancer-associated processes, ranging from tumor growth to multidrug resistance and angiogenesis. Vascular endothelial growth factor receptor-2 (VEGFR-2) and Cav-1 are frequently colocalized, suggesting an important role played by this interaction on cancer cell survival and proliferation. Thus, our attention was directed to a leukemia cell line (B1647) that constitutively produces VEGF and expresses the tyrosine-kinase receptor VEGFR-2. We investigated the presence of VEGFR-2 in caveolae/lipid rafts, focusing on the correlation between reactive oxygen species (ROS) production and glucose transport modulation induced by VEGF, peculiar features of tumor proliferation. In order to better understand the involvement of VEGF/VEGFR-2 in the redox signal transduction, we evaluated the effect of different compounds able to inhibit VEGF interaction with its receptor by different mechanisms, corroborating the obtained results by immunoprecipitation and fluorescence techniques. Results here reported showed that, in B1647 leukemia cells, VEGFR-2 is present in caveolae through association with Cav-1, demonstrating that caveolae/lipid rafts act as platforms for negative modulation of VEGF redox signal transduction cascades leading to glucose uptake and cell proliferation, suggesting therefore novel potential targets

Impact of castor meal on root-knot and free-living nematodes

Soil amendment may enhance soil quality as well as reduce plant-parasitic nematode. Despite the many applications already undertaken using castor meal, its efficiency in controlling root-knot nematodes (RKN, Meloidogyne incognita) when applied to melon (Cucumis melo) is still not clear. Three different amounts of castor meal (Ricinus communis) applied were evaluated in microplots planted with melon either with or without RKN. The impact of castor meal on soil free-living nematode communities was also determined. Total nematode genera richness was estimated as 37 for the entire set of microplots sampled across both sampling dates. Rarefaction analysis resulted in 12 collector's curves out of the total of 30 that reached the horizontal asymptote. Univariate ANOVA with two factors yielded differences (p < 0.05) only with regard to the time factor. Simpson, Shannon, Evenness and Equitability indices showed a trend toward moderate increases by the end of the experiment, while the other indices were higher for tomato in pre-transplant sampling compared to harvest. Nematode community and diversity changed during the course of the experiment, although there was substantial confounding heterogeneity within and between the factorial combinations from the beginning. Root knot population was not reduced by the castor meal but increased throughout the period, regardless of treatment. RKN reduced melon yield, number and weight of melon