Bj-PRO-10c, as an allosteric regulator of argininosuccinate synthase, is a potential therapy for neuroblastoma metastasis.

Cancer is a global public health issue. Neuroblastoma (NB) originates from any tissue of the sympathetic nervous system, and the most affected site is the abdomen. The adrenal gland is the primary site in 38% of cases. Approximately 50% of patients have metastatic disease at diagnosis, and bone marrow is often affected. Metastatic disease is characterized by the spreading of cancer cells that are frequently resistant to chemotherapy and radiotherapy from the primary tumor to other specific parts of the body and is responsible for 90% of cancer-related deaths. Increasing evidence has indicated that nitric oxide (NO) signaling is implicated in the pathophysiology of many types of cancer, particularly in tumorigenesis and cancer progression. However, the effect of NO on metastasis cannot be easily classified as prometastatic or antimetastatic. An understanding at the molecular level of the role of NO in cancer will have profound therapeutic implications for the diagnosis and treatment of disease. Here, the proline-rich decapeptide isolated from Bothrops jararaca venom (Bj-PRO-10c) that enhances and sustains the generation of NO was used to unravel the role of metabolic NO in steps of metastasis. Bj-PRO-10c showed an antimetastatic effect, mainly by interfering with actin cytoskeleton rearrangement, controlling cell proliferation, and decreasing the seeding efficiency of NB in metastatic niches. Therefore, we proposed that an approach for controlled NO induction with the right molecular strategies can hopefully inhibit metastasis and increase the lifespan of NB patients.

The lectin DrfL inhibits cell migration, adhesion and triggers autophagy-dependent cell death in glioma cells.

Glioblastoma multiforme (GBM) is the most aggressive type of glioma, displaying atypical glycosylation pattern that may modulate signaling pathways involved in tumorigenesis. Lectins are glycan binding proteins with antitumor properties. The present study was designed to evaluate the antitumor capacity of the Dioclea reflexa lectin (DrfL) on glioma cell cultures. Our results demonstrated that DrfL induced morphological changes and cytotoxic effects in glioma cell cultures of C6, U-87MG and GBM1 cell lines. The action of DrfL was dependent upon interaction with glycans, and required a carbohydrate recognition domain (CRD), and the cytotoxic effect was apparently selective for tumor cells, not altering viability and morphology of primary astrocytes. DrfL inhibited tumor cell migration, adhesion, proliferation and survival, and these effects were accompanied by activation of p38MAPK and JNK (p46/54), along with inhibition of Akt and ERK1/2. DrfL also upregulated pro-apoptotic (BNIP3 and PUMA) and autophagic proteins (Atg5 and LC3 cleavage) in GBM cells. Noteworthy, inhibition of autophagy and caspase-8 were both able to attenuate cell death in GBM cells treated with DrfL. Our results indicate that DrfL cytotoxicity against GBM involves modulation of cell pathways, including MAPKs and Akt, which are associated with autophagy and caspase-8 dependent cell death.

Structural Prediction and Characterization of Canavalia grandiflora (ConGF) Lectin Complexed with MMP1: Unveiling the Antiglioma Potential of Legume Lectins.

A glioblastoma (GBM) is a highly malignant primary brain tumor with a poor prognosis because of its invasiveness and high resistance to current therapies. In GBMs, abnormal glycosylation patterns are associated with malignancy, which allows for the use of lectins as tools for recognition and therapy. More specifically, lectins can interact with glycan structures found on the malignant cell surface. In this context, the present work aimed to investigate the antiglioma potential of ConGF, a lectin purified from Canavalia grandiflora seeds, against C6 cells. The treatment of C6 cells with ConGF impaired the mitochondrial transmembrane potential, reduced cell viability, and induced morphological changes. ConGF also induced massive autophagy, as evaluated by acridine orange (AO) staining and LC3AB-II expression, but without prominent propidium iodide (PI) labeling. The mechanism of action appears to involve the carbohydrate-binding capacity of ConGF, and in silico studies suggested that the lectin can interact with the glycan structures of matrix metalloproteinase 1 (MMP1), a prominent protein found in malignant cells, likely explaining the observed effects.

Protective effect of (-)-α-bisabolol on rotenone-induced toxicity in Drosophila melanogaster.

(-)-α-Bisabolol (BISA) is a sesquiterpene alcohol, which has several recognized biological activities, including anti-inflammatory, anti-irritant, and antibacterial properties. In the present study, we investigated the influence of BISA (5, 25, and 250 µmol/L) on rotenone (500 µmol/L)-induced toxicity in Drosophila melanogaster for 7 days. BISA supplementation significantly decreased rotenone-induced mortality and locomotor deficits. The loss of motor function induced by rotenone correlated with a significant change in stress response factors; it decreased thiol levels, inhibited mitochondria complex I, and increased the mRNA expression of antioxidant marker proteins such as superoxide dismutase (SOD), catalase (CAT), and the keap1 gene product. Taken together, our findings indicate that the toxicity of rotenone is likely due to the direct inhibition of complex I activity, resulting in a high level of oxidative stress. Dietary supplementation with BISA affected the expression of SOD mRNA only at a concentration of 250 µmol/L, and did not affect any other parameter measured. Our results showed a protective effect of BISA on rotenone-induced mortality and locomotor deficits in Drosophila; this effect did not correlate with mitochondrial complex I activity, but may be related to the antioxidant protection afforded by eliminating superoxide generated as a result of rotenone-induced mitochondrial dysfunction.

High-sucrose diet induces diabetic-like phenotypes and oxidative stress in Drosophila melanogaster: Protective role of Syzygium cumini and Bauhinia forficata.

Diet is a key component for development and longevity of organisms. Here, the fruit fly was used to evaluate the detrimental effects caused by consumption of high-sucrose diets (HSD), namely phenotypic responses linked to insulin signaling and oxidative stress. The protective effects of extracts from medicinal plants Syzygium cumini and Bauhinia forficata were investigated. HSD intake (15% and 30%) delayed the time to pupation and reduced the number of white pupae. In adult flies, the intake of diets was associated with mortality and increased levels of glucose+trehalose, triacylglycerols and hydrogen peroxide. Indeed, 30% HSD induced body-weight loss, mitochondrial dysfunction and changes in acetylcholinesterase, δ-aminolevulinate dehydratase and antioxidant enzymes activity. Catalase, superoxide dismutase, keap1, HSP70, dILP-5 and Insulin receptor mRNA levels were over-expressed in flies emerged from 30% HSD. The extract treatments blunted the developmental alterations elicited by diets. Syzygium cumini extract was more efficient than B. forficata in reducing hyperglycaemia, redox disturbances and the changes in mRNA expression of insulin receptor.

Effect of Syzygium cumini and Bauhinia forficata aqueous-leaf extracts on oxidative and mitochondrial parameters in vitro.

Aqueous-leaf extract of Syzygium cumini and Bauhinia forficata are traditionally used in the treatment of diabetes and cancer, especially in South America, Africa, and Asia. In this study, we analyzed the effects of these extracts on oxidative and mitochondrial parameters in vitro, as well as their protective activities against toxic agents. Phytochemical screenings of the extracts were carried out by HPLC analysis. The in vitro antioxidant capacities were compared by DPPH radical scavenging and Fe(2+) chelating activities. Mitochondrial parameters observed were swelling, lipid peroxidation and dehydrogenase activity. The major chemical constituent of S. cumini was rutin. In B. forficata were predominant quercetin and gallic acid. S. cumini reduced DPPH radical more than B. forficata, and showed iron chelating activity at all tested concentrations, while B. forficata had not similar property. In mitochondria, high concentrations of B. forficata alone induced a decrease in mitochondrial dehydrogenase activity, but low concentrations of this extract prevented the effect induced by Fe(2+)+H2O2. This was also observed with high concentrations of S. cumini. Both extracts partially prevented the lipid peroxidation induced by Fe(2+)/citrate. S. cumini was effective against mitochondrial swelling induced by Ca(2+), while B. forficata alone induced swelling more than Ca(2+). This study suggests that leaf extract of S. cumini might represent a useful therapeutic for the treatment of diseases related with mitochondrial dysfunctions. On the other hand, the consumption of B. forficata should be avoided because mitochondrial damages were observed, and this possibly may pose risk to human health.

Parkia biglobosa improves mitochondrial functioning and protects against neurotoxic agents in rat brain hippocampal slices.

OBJECTIVE: Methanolic leaf extracts of Parkia biglobosa, PBE, and one of its major polyphenolic constituents, catechin, were investigated for their protective effects against neurotoxicity induced by different agents on rat brain hippocampal slices and isolated mitochondria. METHODS: Hippocampal slices were preincubated with PBE (25, 50, 100, or 200 µg/mL) or catechin (1, 5, or 10 µg/mL) for 30 min followed by further incubation with 300 µM H2O2, 300 µM SNP, or 200 µM PbCl2 for 1 h. Effects of PBE and catechin on SNP- or CaCl2-induced brain mitochondrial ROS formation and mitochondrial membrane potential (ΔΨm) were also determined. RESULTS: PBE and catechin decreased basal ROS generation in slices and blunted the prooxidant effects of neurotoxicants on membrane lipid peroxidation and nonprotein thiol contents. PBE rescued hippocampal cellular viability from SNP damage and caused a significant boost in hippocampus Na(+), K(+)-ATPase activity but with no effect on the acetylcholinesterase activity. Both PBE and catechin also mitigated SNP- or CaCl2-dependent mitochondrial ROS generation. Measurement by safranine fluorescence however showed that the mild depolarization of the ΔΨm by PBE was independent of catechin. CONCLUSION: The results suggest that the neuroprotective effect of PBE is dependent on its constituent antioxidants and mild mitochondrial depolarization propensity.

African eggplant (Solanum anguivi Lam.) fruit with bioactive polyphenolic compounds exerts in vitro antioxidant properties and inhibits Ca(2+)-induced mitochondrial swelling.

OBJECTIVE: To evaluate the antioxidant and radical scavenging activities of Solanum anguivi fruit (SAG) and its possible effect on mitochondrial permeability transition pore as well as mitochondrial membrane potential (ΔΨm) isolated from rat liver. METHODS: Antioxidant activity of SAG was assayed by using 2,2-diphenyl-1-picrylhydrazyl (DPPH), reducing power, iron chelation and ability to inhibit lipid peroxidation in both liver and brain homogenate of rats. Also, the effect of SAG on mitochondrial membrane potential and mitochondrial swelling were determined. Identification and quantification of bioactive polyphenolics was done by HPLC-DAD. RESULTS: SAG exhibited potent and concentration dependent free radical-scavenging activity (IC50/DPPH=275.03±7.8 µg/mL). Reductive and iron chelation abilities also increase with increase in SAG concentration. SAG also inhibited peroxidation of cerebral and hepatic lipids subjected to iron oxidative assault. SAG protected against Ca(2+) (110 µmol/L)-induced mitochondrial swelling and maintained the ΔΨm. HPLC analysis revealed the presence of gallic acid [(17.54±0.04) mg/g], chlorogenic acid (21.90±0.02 mg/g), caffeic acid (16.64±0.01 mg/g), rutin [(14.71±0.03) mg/g] and quercetin [(7.39±0.05) mg/g]. CONCLUSIONS: These effects could be attributed to the bioactive polyphenolic compounds present in the extract. Our results suggest that SAG extract is a potential source of natural antioxidants that may be used not only in pharmaceutical and food industry but also in the treatment of diseases associated with oxidative stress.

Valeriana officinalis attenuates the rotenone-induced toxicity in Drosophila melanogaster.

In this study, we investigated the potential protective effects of Valeriana officinalis (V. officinalis) against the toxicity induced by rotenone in Drosophila melanogaster (D. melanogaster). Adult wild-type flies were concomitantly exposed to rotenone (500 µM) and V. officinalis aqueous extract (10mg/mL) in the food during 7 days. Rotenone-fed flies had a worse performance in the negative geotaxis assay (i.e. climbing capability) and open-field test (i.e. mobility time) as well as a higher incidence of mortality when compared to control group. V. officinalis treatment offered protection against these detrimental effects of rotenone. In contrast, the decreased number of crossings observed in the flies exposed to rotenone was not modified by V. officinalis. Rotenone toxicity was also associated with a marked decrease on the total-thiol content in the homogenates and cell viability of flies, which were reduced by V. officinalis treatment. Indeed, rotenone exposure caused a significant increase in the mRNA expression of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) and also in the tyrosine hydroxylase (TH) gene. The expression of SOD and CAT mRNAs was normalized by V. officinalis treatment. Our results suggest that V. officinalis extract was effective in reducing the toxicity induced by rotenone in D. melanogaster as well as confirm the utility of this model to investigate potential therapeutic strategies on movement disorders, including Parkinson disease (PD).

Mitochondrial electron transfer chain complexes inhibition by different organochalcogens.

Mitochondrial dysfunction plays a pivotal role in the cell toxicology and death decision. The aim of the present study was to investigate the effect of three organocompounds (ebselen [Ebs], diphenyl diselenide [(PhSe)(2)] and diphenyl ditelluride [(PhTe)(2)]) on mitochondrial complexes (I, II, I-III, II-III and IV) activity from rat liver and kidney to determine their potential role as molecular targets of organochalcogens. All studied organochalcogens caused a statistically significant inhibition of the mitochondrial complex I activity. Ebs and (PhTe)(2) caused a statistically significant inhibition of the mitochondrial complex II activity in both hepatic and renal membranes. Hepatic mitochondrial complex II activity was practically unchanged by (PhSe)(2), whereas it significantly inhibited renal complex II activity. Mitochondrial complex IV activity was practically unchanged by the organochalcogens. Furthermore, organochalcogens inhibited the mitochondrial respiration supported by complex I or complex II substrates. The inhibitory effect of Ebs, (PhSe)(2) and (PhTe)(2) on mitochondrial complex I was prevented by NADH, but it was not prevented by catalase (CAT) and/or superoxide dismutase (SOD). Additionally, the organochalcogens-induced inhibition of complex I and II was completely reversed by reduced glutathione (GSH). In conclusion, Ebs, (PhSe)(2) and (PhTe)(2) were more effective inhibitors of renal and hepatic mitochondrial complex I than complex II, whereas complexes III and IV were little modified by these compounds. Taking into account the presented results, we suggest that organochalcogen-induced mitochondrial complexes I and II inhibition can be mediated by their thiol oxidation activity, i.e., Ebs, (PhSe)(2) and (PhTe)(2) can oxidize critical thiol groups from mitochondrial complexes I and II. So, mitochondrial dysfunction can be considered an important factor in the toxicity of Ebs, (PhSe)(2) and (PhTe)(2).

Organochalcogens inhibit mitochondrial complexes I and II in rat brain: possible implications for neurotoxicity.

Organochalcogens, such as organoselenium and organotellurium compounds, can be neurotoxic to rodents. Since mitochondrial dysfunction plays a pivotal role in neurological disorders, the present study was designed to test the hypothesis that rat brain mitochondrial complexes (I, II, I-III, II-III and IV) could be molecular targets of organochalcogens. The results show that organochalcogens caused statistically significant inhibition of mitochondrial complex I activity, which was prevented by preincubation with NADH and fully blunted by reduced glutathione (GSH). Mitochondrial complex II activity remained unchanged in response to (PhSe)2 treatment. Ebs and (PhTe)2 caused a significant concentration-dependent inhibition of complex II that was also blunted by GSH. Mitochondrial complex IV activity was not modified by organochalcogens. Collectively, Ebs, (PhSe)2 and (PhTe)2 were more effective inhibitors of brain mitochondrial complex I than of complex II, whereas they did not affect complex IV. These observations are consistent with organochalcogens inducing mitochondrial complex I and II inhibition via their thiol-oxidase-like activity, with Ebs, (PhSe)2 and (PhTe)2 effectively oxidising critical thiol groups of these complexes.

Role of calcium and mitochondria in MeHg-mediated cytotoxicity.

Methylmercury (MeHg) mediated cytotoxicity is associated with loss of intracellular calcium (Ca²âº) homeostasis. The imbalance in Ca²âº physiology is believed to be associated with dysregulation of Ca²âº intracellular stores and/or increased permeability of the biomembranes to this ion. In this paper we summarize the contribution of glutamate dyshomeostasis in intracellular Ca²âº overload and highlight the mitochondrial dysfunctions induced by MeHg via Ca²âº overload. Mitochondrial disturbances elicited by Ca²âº may involve several molecular events (i.e., alterations in the activity of the mitochondrial electron transport chain complexes, mitochondrial proton gradient dissipation, mitochondrial permeability transition pore (MPTP) opening, thiol depletion, failure of energy metabolism, reactive oxygen species overproduction) that could culminate in cell death. Here we will focus on the role of oxidative stress in these phenomena. Additionally, possible antioxidant therapies that could be effective in the treatment of MeHg intoxication are briefly discussed.

Antioxidant effect of Stryphnodendron rotundifolium Martius extracts from Cariri-Ceará State (Brazil): potential involvement in its therapeutic use.

Stryphnodendron rotundifolium is a phytotherapic used in the northeast of Brazil for the treatment of inflammatory processes which normally are associated with oxidative stress. Consequently, we have tested the antioxidant properties of hydroalcoholic (HAB) and aqueous extracts (AB) from the bark and aqueous extract (AL) from the leaves of Stryphnodendron rotundifolium to determine a possible association between antioxidant activity and the popular use of this plant. Free radical scavenger properties were assessed by the quenching of 1',1'-diphenil-2-picrylhydrazyl (DPPH) and the calculated IC(50) were: HAB = 5.4 ± 0.7, AB = 12.0 ± 2.6, and AL = 46.3 ± 12.3 µg/mL. Total phenolic contents were: HAB = 102.7 ± 2.8, AB = 114.4 ± 14.6, and AL = 93.8 ± 9.1 µg/mg plant). HPLC/DAD analyses indicated that gallic acid, catechin, rutin and caffeic acid were the major components of the crude extracts of S. rotundifolium. Plant extracts inhibited Fe(II)-induced lipid peroxidation in brain homogenates. Iron chelation was also investigated and only HBA exhibited a weak activity. Taken together, the results suggest that S. rotundifolium could be considered an effective agent in the prevention of diseases associated with oxidative stress.