Insulin on hydrogen peroxide-induced oxidative stress involves ROS/Ca2+ and Akt/Bcl-2 signaling pathways

Abstract

Oxidative stress is induced by excess accumulation of reactive oxygen and nitrogen species (RONS). Astrocytes are metabolically active cells in the brain and understanding astrocytic responses to oxidative stress is essential to understand brain pathologies. In addition to direct oxidative stress, exogenous hydrogen peroxide (H₂O₂) can penetrate biological membranes and enhance formation of other RONS. The present study was carried out to examine the role of insulin in H₂O₂-induced oxidative stress in rat astrocytic cells. To measure changes in the viability of astrocytes at different concentrations of H₂O₂ for 3 h, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)-based assay was used and 500 μM H₂O₂ was selected to establish a model of H₂O₂-induced oxidative stress. Further assays showed that 3 h of 500 μM H₂O₂-induced significant changes in the levels of lactate dehydrogenase (LDH), reactive oxygen species (ROS) and calcium ion (Ca²⁺) in C6 cells, with insulin able to effectively diminish H₂O₂-induced oxidative damage to C6 cells. Western blotting studies showed that insulin treatment of astrocytes increased the levels of phosphorylated Akt and magnified the decrease in total Bcl-2 protein. The protective effect of insulin treatment on H₂O₂-induced oxidative stress in astrocytes by reducing apoptosis may relate to the PI3K/Akt pathway.     

D(+) galactosamine induced oxidative and nitrosative stress-mediated renal damage in rats via NF-κB and inducible nitric oxide synthase (iNOS) pathways is ameliorated by a polyphenol xanthone,mangiferin

The present study investigated the possible protective effect of mangiferin against D(+) galactosamine (DGal)-induced nephrotoxicity. DGal intoxication increased reactive oxygen species (ROS), reactive nitrogen species and tumor necrosis factor-α (TNF-α) production and disturbed the antioxidant machineries in the kidney tissue. Mangiferin treatment post to DGal exposure reduced all these DGal-induced adverse effects. Signal transduction studies showed that DGal significantly increased the protein expression of Bax, cytochrome c, caspase 3/9 and inducible nitric oxide synthase (iNOS) in the cytosol and NF-κB in nuclear fraction. The same exposure, on the other hand, reduced the protein expression of Bcl-2 in the cytosol. Mangiferin treatment could, however, reduce the DGal-induced up-regulation of cytochrome c, NF-κB, iNOS, caspase 3/9 and alter the reciprocal regulation of Bcl-2 family proteins. Histological studies also revealed the nephroprotective effect of mangiferin against DGal induced nephrotoxicity. Combining, results suggest that mangiferin protects rat's kidney in DGal-induced oxidative/nitrosative stress and acute nephrotoxicity via its antioxidant activities.     

Critical roles of reactive oxygen species in mitochondrial permeability transition in mediating evodiamine-induced human melanoma A375-S2 cell apoptosis

Previous studies have shown that evodiamine could trigger apoptosis in human malignant melanoma A375-S2 cells within 24 h. To further investigate the biochemical basis of this activity, the roles of reactive oxygen species (ROS) and mitochondrial permeability transition (MPT) were evaluated. Exposure to evodiamine led to a rapid increase in intracellular ROS followed by an onset of mitochondrial depolarization. ROS scavenger rescued the ΔΨm dissipation and cell death induced by evodiamine, whilst MPT inhibitor blocked the second-time ROS formation as well as cell death. Expressions of key proteins in Fas- and mitochondria-mediated pathways were furthermore examined. Both pathways were activated and regulated by ROS and MPT and were converged to a final common pathway involving the activation of caspase-3. These data suggested that a phenomenon termed ROS-induced ROS release (RIRR) was involved in evodiamine-treated A375-S2 cells and greatly contributed to the apoptotic process through both extrinsic and intrinsic pathways.     

Reactive oxygen species and nitric oxide regulate mitochondria-dependent apoptosis and autophagy in evodiamine-treated human cervix carcinoma HeLa cells

The redox environment of the cell is currently thought to be extremely important to control either apoptosis or autophagy. This study reported that reactive oxygen species (ROS) and nitric oxide (NO) generations were induced by evodiamine time-dependently; while they acted in synergy to trigger mitochondria-dependent apoptosis by induction of mitochondrial membrane permeabilization (MMP) through increasing the Bax/Bcl-2 or Bcl-x_L ratio. Autophagy was also stimulated by evodiamine, as demonstrated by the positive autophagosome-specific dye monodansylcadaverine (MDC) staining as well as the expressions of autophagy-related proteins, Beclin 1 and LC3. Pre-treatment with 3-MA, the specific inhibitor for autophagy, dose-dependently decreased cell viability, indicating a survival function of autophagy. Importantly, autophagy was found to be promoted or inhibited by ROS/NO in response to the severity of oxidative stress. These findings could help shed light on the complex regulation of intracellular redox status on the balance of autophagy and apoptosis in anti-cancer therapies.     

Nitric oxide (•NO) generation but not ROS plays a major role in silibinin-induced autophagic and apoptotic death in human epidermoid carcinoma A431 cells

Abstract

Silibinin, a major active constituent of silymarin, is clinically used as a hepatoprotectant, and in recent years, it has been used for the treatment of cancer in China. Because the mechanism of silibinin action on cancer cells was still unclear, we investigated the contribution of silibinin to the induction of apoptosis and autophagy via generation of reactive oxygen species (ROS) and nitric oxide (?NO) in human epidermoid carcinoma A431 cells. Silibinin inhibited the cell growth in a dose‐and time-dependent manner. Obvious autophagy was observed after treatment with different doses of silibinin. At a high dose (400 μM), silibinin induced apoptosis through both the intrinsic and extrinsic apoptotic pathways. Loss of mitochondrial membrane potential by silibinin led to mitochondrial dysfunction and decreased ROS levels, suggesting that silibinin might act as an antioxidant in this process. Furthermore, silibinin induced ?NO generation in a time‐and dose-dependent manner. The ?NO scavenger PTIO could effectively clear ?NO and exerted a minor cell protection effect through partial inhibition of silibinin-induced apoptosis and autophagy.     

DNA damage in response to an Ironman triathlon

The major aims of this study were to investigate the effect of an Ironman triathlon on DNA migration in the single cell gel electrophoresis assay, apoptosis and necrosis in the cytokinesis-block micronucleus cytome assay with lymphocytes and on changes of total antioxidant capacity in plasma. Blood samples were taken 2 days (d) before, within 20 min, 1 d, 5 d and 19 d post-race. The level of strand breaks decreased (p<0.05) immediately after the race, then increased (p<0.01) 1 d post-race and declined (p<0.01) until 19 d post-race. Apoptotic and necrotic cells decreased (p<0.01) and the total antioxidant status increased (p<0.01) immediately after the race. The results indicate that ultra-endurance exercise does not cause prolonged DNA damage in well-trained male athletes.     

Silencing of cytosolic NADP+-dependent isocitrate dehydrogenase by small interfering RNA enhances the sensitivity of HeLa cells toward staurosporine

Staurosporine induces the production of reactive oxygen species, which play an important causative role in apoptotic cell death. Recently, it was demonstrated that the control of cellular redox balance and the defense against oxidative damage is one of the primary functions of cytosolic NADP⁺-dependent isocitrate dehydrogenase (IDPc) by supplying NADPH for antioxidant systems. The present report shows that silencing of IDPc expression in HeLa cells greatly enhances apoptosis induced by staurosporine. Transfection of HeLa cells with an IDPc small interfering RNA (siRNA) markedly decreased activity of IDPc, enhancing the susceptibility of staurosporine-induced apoptosis reflected by DNA fragmentation, cellular redox status and the modulation of apoptotic marker proteins. These results indicate that IDPc may play an important role in regulating the apoptosis induced by staurosporine and the sensitizing effect of IDPc siRNA on the apoptotic cell death of HeLa cells offers the possibility of developing a modifier of cancer chemotherapy.     

Repression of apurinic/apyrimidinic endonuclease by p53-dependent apoptosis in hydronephrosis-induced rat kidney

Abstract

p53 plays a major role in apoptosis through activation of pro-apoptotic gene Bax. It also regulates apurinic/apyrimidinic endonuclease (APE) expression in the base excision repair pathway against oxidative DNA damages. This study investigated whether p53-dependent apoptosis is correlated with APE using an experimental rat model of hydronephrosis. Hydronephrosis was induced by partial ligation of the right ureter. Animals were sacrificed on scheduled time after unilateral ureteral obstruction and the expression of 8-OHdG, γ-H2AX, apoptotic proteins and APE was determined. The accumulated p53 activated Bax and caspase-3 7 days after hydronephrosis induction and the resulting high levels of p53-dependent apoptotic proteins and γ-H2AX tended to decrease APE. The intensities of 8-OHdG and caspase-3 immunolocalization significantly increased in obstructed kidneys than in sham-operated kidneys, although APE immunoreactivity increased after hydronephrosis induction. These results suggest that oxidative DNA damages in obstructed kidneys may trigger p53-dependent apoptosis through repression of APE.     

Cytoprotective effect of dieckol on human endothelial progenitor cells (hEPCs) from oxidative stress-induced apoptosis

Abstract

Although endothelial progenitor cells (EPCs) have been used to promote revascularization after peripheral or myocardial ischemia, excess amounts of reactive oxygen species (ROS) are often involved in senescence and apoptosis of EPCs, thereby causing defective neovascularization and reduced or failed recovery. Here, we examined the cytoprotective effect of Ecklonia cava-derived antioxidant dieckol (DK) on oxidative stress-induced apoptosis in EPCs to improve EPC bioactivity for vessel repair. Although H₂O₂ (10 ^{? 3} M) increased the intracellular ROS level in EPCs, DK (10ug/ml) pretreatment suppressed the H₂O₂-induced ROS increase and drastically reduced the ratios of apoptotic cells. H₂O₂-induced ROS increased the phosphorylation of p38 MAPK and JNK; this was inhibited by DK pretreatment. H₂O₂ treatment increased the phosphorylation of NF-κB, which was blocked by pretreatment with SB 203580, a p38 MAPK inhibitor, or SP 600125, a JNK inhibitor. H₂O₂ decreased the cellular levels of Bcl-2 and c-IAPs, cellular inhibitors of apoptosis proteins, but increased caspase-3 activation. However, all these effects were inhibited by pretreatment with DK. Injection of DK-mixed EPCs (DK + EPCs) into myocardial ischemic sites in vivo induced cellular proliferation and survival of cells at the ischemic sites and, thereby, enhanced the secretion of angiogenic cytokines at the ischemic sites. These results show that DK + EPC exhibit markedly enhanced anti-apoptotic and antioxidative capabilities, unlike that shown by EPCs alone; thus, they contribute to improved repair of ischemic myocardial injury through cell survival and angiogenic cytokine production.