Enhanced ROS Generation Research Articles

Design of Pluronic-Based Formulation for Enhanced Redaporfin-Photodynamic Therapy against Pigmented Melanoma

The therapeutic outcome of photodynamic therapy (PDT) with redaporfin (a fluorinated sulfonamide bacteriochlorin, F2BMet or LUZ11) was improved using Pluronic-based (P123, F127) formulations. Neither redaporfin encapsulated in Pluronic nor micelles alone exhibited cytotoxicity in a broad concentration range. Comprehensive in vitro studies against B16F10 melanoma cells showed that redaporfin-P123 micelles enhanced cellular uptake and increased oxidative stress compared with redaporfin-F127 or photosensitizer alone after short incubation times. ROS-sensitive fluorescent probes showed that the increased oxidative stress is due, at least in part, to a more efficient formation of hydroxyl radicals, and causes strong light-dose dependent apoptosis and necrosis. Tissue distribution and pharmacokinetic studies in tumor-bearing mice show that the Pluronic P123 formulation of redaporfin increases its bioavailability as well as the tumor-to-muscle and tumor-to-skin ratios, in comparison with Cremophor EL and Pluronic F127 formulations. Redaporfin in P123 was most successful in the PDT of C57BL/6J mice bearing subcutaneously implanted B16F10 melanoma tumors. Vascular-targeted PDT combining 1.5 mg kg(-1) redaporfin in P123 with a light dose of 74 J cm(-2) led to 100% complete cures (i.e., no tumor regrowth over one year post-treatment). This remarkable result reveals that modification of redaporfin with Pluronic block copolymers overcomes the resistance of melanoma cells to PDT possibly via increased tumor selectivity and enhanced ROS generation.

UVB exposure enhanced benzanthrone-induced inflammatory responses in SKH-1 mouse skin by activating the expression of COX-2 and iNOS through MAP kinases/NF-κB/AP-1 signalling pathways

This study was conducted to explore the role of UVB on benzanthrone (BA)-induced skin inflammation and its mechanism/s. SKH-1 hairless mice were topically exposed with BA (25 and 50 mg/kg b.wt) either alone or along with UVB (50 mJ/cm2) for 24 h and estimation of ROS, histopathological analysis, myeloperoxidase (MPO) activity, mast cell staining, immunohistochemistry for COX-2 and iNOS as well as western blotting for MAPKs, p-NF-κB, c-jun, c-fos COX-2 and iNOS were carried out. Enhanced ROS generation, increased epidermal thickness, mast cell number, MPO activity, enhanced expression of COX-2 and iNOS, MAPKs, c-jun, c-fos, NF-κB were found in BA either alone or when followed by UVB treatment, compared to the control groups. Expression of COX-2, iNOS and phosphorylation of ERK1/2 were found to be more enhanced in BA and UVB- exposed group compared to BA and UVB only group, while phosphorylation of JNK1/2, p38, NF-κB and expression of c-jun and c-fos were comparable with BA and UVB only groups. In summary, we suggest that UVB exposure enhanced BA-induced SKH-1 skin inflammation possibly via oxidative stress-mediated activation of MAPKs-NF-κB/AP-1 signalling, which subsequently increased the expression of COX-2 and iNOS and led to inflammation in SKH-1 mouse skin.

High oxidative stress adversely affects NFκB mediated induction of inducible nitric oxide synthase in human neutrophils: Implications in chronic myeloid leukemia

Increasing evidence support bimodal action of nitric oxide (NO) both as a promoter and as an impeder of oxygen free radicals in neutrophils (PMNs), however impact of high oxidative stress on NO generation is less explored. In the present study, we comprehensively investigated the effect of high oxidative stress on inducible nitric oxide synthase (iNOS) expression and NO generation in human PMNs. Our findings suggest that PMA or diamide induced oxidative stress in PMNs from healthy volunteers, and high endogenous ROS in PMNs of chronic myeloid leukemia (CML) patients attenuate basal as well as LPS/cytokines induced NO generation and iNOS expression in human PMNs. Mechanistically, we found that under high oxidative stress condition, S-glutathionylation of NFκB (p50 and p65 subunits) severely limits iNOS expression due to its reduced binding to iNOS promoter, which was reversed in presence of DTT. Furthermore, by using pharmacological inhibitors, scavengers and molecular approaches, we identified that enhanced ROS generation via NOX2 and mitochondria, reduced Grx1/2 expression and GSH level associated with NFκB S-glutathionylation in PMNs from CML patients. Altogether data obtained suggest that oxidative status act as an important regulator of NO generation/iNOS expression, and under enhanced oxidative stress condition, NOX2-mtROS-NFκB S-glutathionylation is a feed forward loop, which attenuate NO generation and iNOS expression in human PMNs.

UVB exposure enhanced the dermal penetration of zinc oxide nanoparticles and induced inflammatory responses through oxidative stress mediated by MAPKs and NF-κB signaling in SKH-1 hairless mouse skin.

Besides titanium dioxide (TiO2), zinc oxide (ZnO) nanoparticles (NPs) are commonly used in sunscreen formulations as protective agents against exposure to ultraviolet radiation. Although the majority of prior studies have concluded that NPs do not penetrate healthy skin, compromised skin slightly enhanced metal oxide NP penetration. However, a question arises regarding the possible toxic consequences if consumers who had applied sunscreens containing ZnO-NPs were exposed to environmentally relevant doses of UVB. Considering this, we planned a study where SKH-1 hairless mice were topically exposed to a 5% and/or 10% dose of ZnO-NPs (<50 nm and <100 nm) either alone or along with UVB (50 mJ cm-2). In two additional groups, mice were treated with either bulk ZnO-NPs (<5 μm) or with ZnO-NPs (<5 μm) and subsequently UVB (50 mJ cm-2). Animals of all groups were sacrificed after 6, 24 and 48 h and the Zn ion content in the skin was measured. In addition, estimation of ROS generation, histopathology, myeloperoxidase (MPO) activity, immunohistochemistry for COX-2 and western blot analysis for MAPKs, p-IκBα, p-NF-κB, and COX-2 were also carried out. Significant increases in the Zn ion in exposed skin were seen. Enhanced ROS generation and MPO activity were also found in ZnO-NPs followed by UVB exposed groups at all three time points. Similarly, hyperplasia and over-expression of COX-2 were also greater in ZnO-NPs and UVB exposed groups than in the ZnO-NPs and UVB only groups. The expression of MAPKs, and transcription factors NF-κB along with COX-2 were also enhanced significantly in ZnO-NPs and the UVB treated group. Collectively, our findings suggest that UVB exposure enhanced ZnO-NP penetration in mouse skin and possibly dissolution of these ZnO-NPs takes place during this process, causing significant Zn ion generation leading to oxidative stress by ROS generation which subsequently activates MAPK-NF-κB signaling and increases COX-2 and inflammation.

Acute Exercise-Induced Mitochondrial Stress Triggers an Inflammatory Response in the Myocardium via NLRP3 Inflammasome Activation with Mitophagy.

Increasing evidence has indicated that acute strenuous exercise can induce a range of adverse reactions including oxidative stress and tissue inflammation. However, little is currently known regarding the mechanisms that underlie the regulation of the inflammatory response in the myocardium during acute heavy exercise. This study evaluated the mitochondrial function, NLRP3 inflammasome activation, and mitochondrial autophagy-related proteins to investigate the regulation and mechanism of mitochondrial stress regarding the inflammatory response of the rat myocardium during acute heavy exercise. The results indicated that the mitochondrial function of the myocardium was adaptively regulated to meet the challenge of stress during acute exercise. The exercise-induced mitochondrial stress also enhanced ROS generation and triggered an inflammatory reaction via the NLRP3 inflammasome activation. Moreover, the mitochondrial autophagy-related proteins including Beclin1, LC3, and Bnip3 were all significantly upregulated during acute exercise, which suggests that mitophagy was stimulated in response to the oxidative stress and inflammatory response in the myocardium. Taken together, our data suggest that, during acute exercise, mitochondrial stress triggers the rat myocardial inflammatory response via NLRP3 inflammasome activation and activates mitophagy to minimize myocardial injury.

Extracellular glutathione recycling by γ-glutamyl transferase in barley root tip exposed to cadmium

A considerable amount of extracellular glutathione (eGSH) is detectable in intact root tips, but even the immersion of roots into water markedly decreased its detectability, suggesting its localization into the rhizosphere. While in water-treated seedlings the amount of eGSH was rapidly restored during the recovery period, in Cd-treated roots its amount was restored much more slowly. Enhanced γ-glutamyl transferase (GGT) activity was detected in 15μM Cd-treated root tips 1h after the end of short-term treatment, reaching its maximum level 2h after the treatment. Thereafter it decreased to the level of control roots. By contrast, higher Cd concentrations inhibited the activity of GGT and more strongly inhibited the restoration of eGSH pool in root tips. Depletion of eGSH was accompanied by inhibition of GGT activity, whereas its elevated level slightly activated it. We showed that elevated eGSH, induced either by exogenously applied GSH or by GGT activity inhibition, triggered enhanced ROS generation and marked root growth inhibition in both control and Cd-treated seedlings. In addition, the toxicity of the Cd–GSH complex was more serious than the toxicity of Cd alone. Our results indicated that just recycling of eGSH or/and Cd/eGSH complex by GGT but not its level in the apoplast plays role in the sensing of toxic compounds and in the activation of defence responses. On the other hand, above a certain threshold concentration both eGSH and Cd/GSH complex due to their high toxicity can impair defence responses, causing detrimental damages in root tissues.

Curcumin protects rat liver from streptozotocin-induced diabetic pathophysiology by counteracting reactive oxygen species and inhibiting the activation of p53 and MAPKs mediated stress response pathways

Curcumin (CUR) is a highly pleiotropic molecule and possesses anti-inflammatory, hypoglycemic, antioxidative, wound-healing and antimicrobial activities. The present study was carried out to investigate whether CUR plays any beneficial role in streptozotocin (STZ) induced hepatic pathophysiology in diabetic rats. STZ exposure increased hepatic damage associated serum markers (ALT, ALP and LDH) as well as NO production in the liver tissue. Moreover, the same exposure enhanced ROS generation and lipid peroxidation; reduced GSH levels and antioxidant enzyme activities. Hyperglycemia induced hepatic pathophysiology also activated stress response pathways (involving phosphorylation of p38, ERK1/2 MAPKs and p53) and reduced mitochondrial membrane potential which in turn led to cellular apoptosis as evidenced from increased hepatic DNA fragmentation as well as FACS analysis. However, treatment with CUR effectively counteracts diabetes-induced, oxidative stress mediated hepatic damage and could act as a therapeutic in lessening liver dysfunction in diabetic subjects.

Study of kinetics of photocatalysis, bacterial inactivation and •OH scavenging activity of electrochemically synthesized Se4+ doped ZnS nanoparticles

Se4+ doped ZnS nanoparticles is synthesized by electrochemical method which is capable of producing flower-like nanoparticles in a size range about 10–30nm, under ambient conditions. UV–VIS spectra discusses the band gap of Se4+ doped ZnS to be 2.1eV which is much lower than that of commercial ZnS (3.68eV). The photocatalytic activity was evaluated on the degradation of indigo carmine dye both under ultraviolet (UV) and sunlight illumination which follows first-order kinetics. It was found that Se doped ZnS photocatalyst showed much higher photodegradation efficiency (6–8 times) than commercial ZnS. A linear correlation is achieved for photo-degradation and inactivation of bacteria as both are ROS dependent phenomena. Hydroxyl radical scavenging ability of these particles was evaluated. The impact of enhanced ROS generation was consistent with higher photocatalytic dye degradation and antibacterial activity of Se4+ doped ZnS nanoparticles.

Reactive oxygen species generation and antioxidant defense system in hydroponically grown wheat (Triticum aestivum) upon β-pinene exposure: an early time course assessment

We investigated the effect of β-pinene on reactive oxygen species (ROS: lipid peroxidation, membrane integrity, hydrogen peroxide and superoxide ions) generation and activity of antioxidant defense system during early hours of treatment (4, 8, 16 and 24 h) in hydroponically grown Triticum aestivum (wheat). β-Pinene reduced the root and shoot growth of the hydroponically grown wheat. However, the reduction was more pronounced in root length than in shoot length. β-Pinene enhanced ROS generation as indicated by increased levels of malondialdehyde (20–87 %), hydrogen peroxide (9–45 %) and superoxide ion (23–179 %) content, thereby suggesting lipid peroxidation and induction of oxidative stress in a time- and concentration-dependent manner. The oxidative damage was more pronounced at ≥10 µM β-pinene and at ≥8 h after exposure. β-Pinene caused a severe electrolyte leakage from wheat roots indicating membrane disruption and loss of integrity. Enhanced lipid peroxidation and loss of membrane integrity were confirmed by in situ histochemical studies. β-Pinene provoked increase in the activity of lipoxygenase and upregulation in the activities of antioxidant enzymes: catalases, superoxide dismutases, ascorbate peroxidases, guaiacol peroxidases and glutathione reductases. The enhanced activity of lipoxygenases evoked by β-pinene paralleled higher accumulation of MDA, thereby suggesting that antioxidant defense mechanism was not able to prevent β-pinene-induced lipid peroxidation.

Abstract 279: Vascular Aging In Aldosterone Associated Hypertension: Role Of NADPH Oxidase 1

In hypertension, vascular aging is accelerated leading to heart failure, stroke and renal dysfunction. Cellular and molecular mechanisms of age-associated vascular changes are unclear, but enhanced ROS generation by NADPH oxidases (Nox) and aldosterone (aldo) have been suggested. Nox1 is of particular interest due to increased levels in hypertension. Here, we postulated that aldo plays an important role in vascular aging through Nox1-dependent mechanisms. We examined signalling molecules associated with aging in arteries from 2 experimental models: stroke-prone spontaneously hypertensive rats (SHRSP) rats and Nox1 knockout mice infused with aldo (300 ug/Kg/day). Gene expression was assessed by qPCR and protein levels by immunoblotting. Aldo was measured by ELISA. In SHRSP rats, aging-associated mRNA inflammatory markers, such as RANTES (5- fold), MCP-1 (6-fold) and IL-6 (2-fold); as well as aldo levels (6-fold) and H2AX (marker of aging-associated DNA damage - 1.5-fold) were increased compared with control rats, p&lt;0.05. In mice treated with aldo, JNK (pro-inflammatory - 69%) and p66SHC (pro-senescence - 92%) activation were increased in mesenteric arteries (p&lt;0.05); an effect blunted in vessels from Nox1 KO mice. In parallel studies, vascular smooth muscle cells (VSMCs) from adult and aged control mice, as well as, from adult Nox1 transgenic mice (Nox1 overexpression in VSMCs) were extracted and stimulated with H2O2 and aldo. Basal levels of p66SHC (39%) and OGG-1 (48%), markers of senescence, were increased in VSMCs from aged animals, p&lt;0.05. H2O2 (64%) and aldo (84%) increased p66SHC activation in VSMCs from adult mice to similar levels observed in cells from aged animals (p&lt;0.05). However, aldo effects on p66SHC activation were exacerbated in VSMCs from adult Nox1 transgenic mice. In conclusion, aldosterone may play an important role in the aging-like phenotype in vascular injury associated with hypertension. Such processes may involve Nox1 and redox-sensitive p66Shc signalling.

FoxO proteins’ nuclear retention and BH3-only protein Bim induction evoke mitochondrial dysfunction-mediated apoptosis in berberine-treated HepG2 cells

Mammalian forkhead-box family members belonging to the ‘O’ category (FoxO) manipulate a plethora of genes modulating a wide array of cellular functions including cell cycle regulation, apoptosis, DNA damage repair, and energy metabolism. FoxO overexpression and nuclear accumulation have been reported to show correlation with hindered tumor growth in vitro and size in vivo, while FoxO’s downregulation via phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway has been linked with tumor promotion. In this study, we have explored for the first time intervention of berberine, a plant-derived isoquinoline alkaloid, with FoxO family proteins in hepatoma cells. We observed that berberine significantly upregulated the mRNA expression of both FoxO1 and FoxO3a. Their phosphorylation-mediated cytoplasmic sequestration followed by degradation was prevented by berberine-induced downmodulation of the PI3K/Akt/mTOR pathway which promoted FoxO nuclear retention. PTEN, a tumor suppressor gene and negative regulator of the PI3K/Akt axis, was upregulated while phosphorylation of its Ser380 residue (possible mechanism of PTEN degradation) was significantly decreased in treated HepG2 cells. Exposure to berberine induced a significant increase in transcriptional activity of FoxO, as shown by GFP reporter assay. FoxO transcription factors effectively heightened BH3-only protein Bim expression, which in turn, being a direct activator of proapoptotic protein Bax, altered Bax/Bcl-2 ratio, culminating into mitochondrial dysfunction, caspases activation, and DNA fragmentation. The pivotal role of Bim in berberine-mediated cytotoxicity was further corroborated by knockdown experiments where Bim-silencing partially restored HepG2 cell viability during berberine exposure. In addition, a correlation between oxidative overload and FoxO’s nuclear accumulation via JNK activation was evident as berberine treatment led to a pronounced increase in JNK phosphorylation together with enhanced ROS generation, lipid peroxidation, decreased activities of superoxide dismutase and catalase, and diminished glutathione levels. Thus, our findings suggest that the antiproliferative effect of berberine may in part be due to mitochondria-mediated apoptosis with Bim acting as a pivotal downstream factor of FoxO-induced transcriptional activation.

Additivity, antagonism, and synergy in arsenic trioxide-induced growth inhibition of C6 glioma cells: Effects of genistein, quercetin and buthionine-sulfoximine

Arsenic trioxide (ATO) induces clinical remission in acute promyelocytic leukemia and growth inhibition in various cancer cell lines in vitro. Recently, genistein and quercetin were reported to potentiate ATO-provoked apoptosis in leukemia and hepatocellular carcinoma cells. Genistein acted via enhanced ROS generation and quercetin via glutathione depletion. Searching for potential strategies for the treatment of malignant gliomas in this study the capacity of these flavonoids to sensitize rat C6 astroglioma cells for the cytotoxic action of ATO was investigated. ATO inhibited cell growth in a concentration- and time-dependent manner. This effect was accompanied neither by enhanced radical generation nor lipid peroxidation and was not attributed to apoptosis. ATO treatment concentration-dependently increased glutathione levels. Genistein enhanced radical generation. Combined with ATO it inhibited cell growth additively. Additivity was also obtained after cotreatment with ATO and H2O2. Quercetin acted antagonistically on ATO-induced growth inhibition. Quercetin increased glutathione levels. In contrast, buthionine-sulfoximine (BSO) depleted cellular glutathione and acted synergistically with ATO. In conclusion, in C6 cells neither genistein nor quercetin are suited as sensitizing agent, in contrast to BSO. Depletion of cellular glutathione content rather than an increase of ROS generation plays a central role in the enhancement of ATO-toxicity in C6 cells.

Cinnamaldehyde-Induced Apoptosis in Human Hepatoma PLC/PRF/5 Cells Involves the Mitochondrial Death Pathway and is Sensitive to Inhibition by Cyclosporin A and z-VAD-fmk

Cinnamaldehyde (CIN) has been shown to exert chemopreventive activity against several types of human cancer cells. We previously reported that CIN induced apoptosis of human hepatoma PLC/PRF/5 cells and this effect was associated with activation of the pro-apoptotic Bcl-2 family of proteins and the MAPK cascade. To further clarify the underlying mechanism of CIN-induced apoptosis, we examined in this study its relationship with the mitochondrial death pathway using the mitochondrial permeability transition (MPT) inhibitor, cyclosporin A (CsA), and the general caspase inhibitor, z-VAD-fmk. Results indicated that CIN-induced apoptosis involved enhanced ROS generation, disruption of mitochondrial potential, and the mitochondrial release of cytochrome c and Smac/DIABLO into the cytosol, which in turn promoted caspase-3 to its active form and the subsequent cleavage of PARP. Treatment with CIN also downregulated protein levels of the anti-apoptotic factors XIAP and Bcl-2 with concomitant accumulation of the pro-apoptotic Bax in a timedependent manner. These mitochondria-related apoptotic effects induced by CIN were however blocked by CsA and z-VAD-fmk pretreatments, which prevented cells from undergoing programmed cell death triggered by CIN. Furthermore, the increase of Bax and decrease of Bcl-2 and XIAP protein expression due to CIN treatment were also reversely modulated by the two inhibitors. Taken together, these results suggested that CIN is an apoptotic inducer that acts on the mitochondrial death pathway in PLC/PRF/5 cells and its effect could be blocked by CsA and z-VAD-fmk.

Effect of spermine treatment on the functioning of Thellungiella salsuginea antioxidant system

The effect of spermine (Spm) treatment on the content of polyamines (PAs) and activities of antioxidant enzymes in the roots and leaves of Thellungiella salsuginea (Pall.) O.E. Schulz plants grown under optimal conditions were studied. The genes encoding three forms of ascorbate peroxidase (APX; APX1, APX2, and APX4) and genes of key enzymes of proline metabolism (Pro, P5CS1, 1P5CD) were identified, and their expression intensity was measured. Six-day-old plants were treated with Spm (1 and 2 mM) and with the inhibitor of polyamine oxidase (PAO) activity, N,N-(2-hydroxyethyl)hydrazine (HEH, 1 and 2 mM) separately or in combination by adding these compounds to nutrient medium. Roots and leaves responded differently to Spm treatment. In the leaves, the content of PAs reduced due to a decreased in the spermidine (Spd) content, whereas in the roots the total pool of PAs increased due to putrescine (Put) and Spd accumulation. Treatment with Spm activated PAO in the roots but not in the leaves; HEH removed this increase, but the intercellular Spm concentration was not substantially changed. It was suggested that treatment with Spm suppressed the biosynthesis of intracellular Spm and, on the other hand, stimulated the reverse conversion of Spm into Spd and further into Put due to the activation of one of the PAO isoforms. Plant treatment with Spm was not accompanied by a noticeable activation enzymes degrading hydrogen peroxide in the roots: APX, (except of peroxidase II), and catalase. However, the activity of Cu/Zn-SOD doubled and the activity of Mn-SOD reduced. In the leaves, slight activation of peroxidases I and III, the inhibition of Cu/Zn- and Mn-SOD, differential changes in the time-coursed of gene expression of three APX isoforms, and activated gene expression of key enzymes of Pro metabolism were observed. At the same time, the level of MDA did not increase either in the leaves or in the roots. This indicates that treatment of Th. salsuginea plants with Spm under optimal growing conditions did not enhance ROS generation and did not manifest prooxidant properties.

Curcumin induces radiosensitivity of in vitro and in vivo cancer models by modulating pre-mRNA processing factor 4 (Prp4)

Radiation therapy plays a central role in adjuvant strategies for the treatment of both pre- and post-operative human cancers. However, radiation therapy has low efficacy against cancer cells displaying radio-resistant phenotypes. Ionizing radiation has been shown to enhance ROS generation, which mediates apoptotic cell death. Further, concomitant use of sensitizers with radiation improves the efficiency of radiotherapy against a variety of human cancers. Here, the radio-sensitizing effect of curcumin (a derivative of turmeric) was investigated against growth of HCT-15 cells and tumor induction in C57BL/6J mice. Ionizing radiation induced apoptosis through ROS generation and down-regulation of Prp4K, which was further potentiated by curcumin treatment. Flow cytometry revealed a dose-dependent response for radiation-induced cell death, which was remarkably reversed by transfection of cells with Prp4K clone. Over-expression of Prp4K resulted in a significant decrease in ROS production possibly through activation of an anti-oxidant enzyme system. To elucidate an integrated mechanism, Prp4K knockdown by siRNA ultimately restored radiation-induced ROS generation. Furthermore, B16F10 xenografts in C57BL/6J mice were established in order to investigate the radio-sensitizing effect of curcumin in vivo. Curcumin significantly enhanced the efficacy of radiation therapy and reduced tumor growth as compared to control or radiation alone. Collectively, these results suggest a novel mechanism for curcumin-mediated radio-sensitization of cancer based on ROS generation and down-regulation of Prp4K.

Hemodynamic Regulation of Reactive Oxygen Species: Implications for Vascular Diseases

Arterial blood vessels functionally and structurally adapt to altering hemodynamic forces in order to accommodate changing needs and to provide stress homeostasis. This ability is achieved at the cellular level by converting mechanical stimulation into biochemical signals (i.e., mechanotransduction). Physiological mechanical stress helps maintain vascular structure and function, whereas pathologic or aberrant stress may impair cellular mechano-signaling, and initiate or augment cellular processes that drive disease. Reactive oxygen species (ROS) may represent an intriguing class of mechanically regulated second messengers. Chronically enhanced ROS generation may be induced by adverse mechanical stresses, and is associated with a multitude of vascular diseases. Although a causal relationship has clearly been demonstrated in large numbers of animal studies, an effective ROS-modulating therapy still remains to be established by clinical studies. This review article focuses on the role of various mechanical forces (in the form of laminar shear stress, oscillatory shear stress, or cyclic stretch) as modulators of ROS-driven signaling, and their subsequent effects on vascular biology and homeostasis, as well as on specific diseases such as arteriosclerosis, hypertension, and abdominal aortic aneurysms. Specifically, it highlights the significance of the various NADPH oxidase (NOX) isoforms as critical ROS generators in the vasculature. Directed targeting of defined components in the complex network of ROS (mechano-)signaling may represent a key for successful translation of experimental findings into clinical practice.

Resolvin E1 enhances phagocytosis‐induced neutrophil apoptosis

Resolvin E1 (RvE1) is an endogenous lipid mediator derived from the ω‐3 polyunsaturated fatty acid eicosapentaenoic acid that displays potent pro‐resolving actions in experimental models of inflammation. However, little is known about the impact of RvE1 on neutrophil apoptosis, a critical control point in the resolution of inflammation. Culture of human neutrophils with RvE1 did not affect neutrophil longevity, RvE1 accelerated apoptosis evoked by phagocytosis of opsonized Escherichia coli or yeast. RvE1 through the leukotriene B4 receptor BLT1 enhanced ROS generation by NADPH oxidase, leading to subsequent activation of caspase‐8 and caspase‐3. Moreover, RvE1 mitigated phagocytosis‐induced ERK activation. RvE1 also attenuated myeloperoxidase (through Mac‐1), serum amyloid A (through the formyl peptide receptor 2) and bacterial DNA (through TLR9)‐evoked concurrent ERK and phosphatidylinositol 3‐kinase/Akt‐mediated apoptosis‐suppressing signals. These led to reductions in the expression of the antiapoptotic protein Mcl‐1, collapse of mitochondrial transmembrane potential and activation of caspase‐3. Our results identify a novel BLT1 receptor‐mediated mechanism, promotion of phagocytosis‐induced neutrophil apoptosis by which RvE1 could enhance neutrophil apoptosis and subsequently promote inflammation resolution. (Grant support: CIHR MOP‐97742).

Methylglyoxal induces DNA crosslinks in ECV304 cells via a reactive oxygen species-independent protein carbonylation pathway

Methylglyoxal (MG) is a reactive dicarbonyl compound that is produced endogenously from glycolytic intermediates and via gluconeogenesis. Elevated MG levels in diabetes patients are believed to cause diabetic complications. MG-induced crosslinking products from the covalent binding of DNA or protein alone or together could be relevant to carcinogenesis and multiple complications in diabetes. However, the mechanisms governing DNA crosslink formation by MG are unclear. We investigated whether MG could induce DNA crosslinks in human ECV304 cells and the possible mechanism of this action. The level of DNA crosslinks and reactive oxygen species production were assayed by a modified alkaline Comet assay and a 2′,7′ dichlorofluorescin diacetate (DCHF-DA) assay, respectively. MG caused a time- and dose-dependent increase in DNA crosslinks and a dose-dependent increase in protein carbonylation in ECV304 cells. Addition of 2mM MG resulted in a transient increase in protein carbonylation, and this increase peaked within 2h and then rapidly decreased. Most notably, MG did not cause significantly enhanced ROS generation in ECV304 cells. Co-treatment with carbonyl-scavenging drugs, such as aminoguanidine, N-acetyl-l-cysteine, and glutathione, significantly inhibited the formation of DNA crosslinks by MG, whereas co-treatment with the antioxidant ascorbic acid did not. In conclusion, our results imply that MG induces DNA crosslink formation in ECV304 cells via a reactive oxygen species-independent protein carbonylation pathway. Our findings also suggest that non-toxic aminothiol antioxidants with carbonyl scavenging capabilities are potential therapeutic agent for MG-related diseases, such as diabetes and neurodegeneration. Furthermore, our findings also imply that DNA nonbinding proteins, bovine serum albumin might be able to crosslink calf thymus DNA in the presence of MG.

Protective role of morin, a flavonoid, against high glucose induced oxidative stress mediated apoptosis in primary rat hepatocytes.

Apoptosis is an early event of liver damage in diabetes and oxidative stress has been linked to accelerate the apoptosis in hepatocytes. Therefore, the compounds that can scavenge ROS may confer regulatory effects on high-glucose induced apoptosis. In the present study, primary rat hepatocytes were exposed to high concentration (40 mM) of glucose. At this concentration decreased cell viability and enhanced ROS generation was observed. Depleted antioxidant status of hepatocytes under high glucose stress was also observed as evident from transcriptional level and activities of antioxidant enzymes. Further, mitochondrial depolarisation was accompanied by the loss of mitochondrial integrity and altered expression of Bax and Bcl-2. Increased translocation of apoptotic proteins like AIF (Apoptosis inducing factor) & Endo-G (endonuclease-G) from its resident place mitochondria to nucleus was also observed. Cyt-c residing in the inter-membrane space of mitochondria also translocated to cytoplasm. These apoptotic proteins initiated caspase activation, DNA fragmentation, chromatin condensation, increased apoptotic DNA content in glucose treated hepatocytes, suggesting mitochondria mediated apoptotic mode of cell death. Morin, a dietary flavonoid from Psidium guajava was effective in increasing the cell viability and decreasing the ROS level. It maintained mitochondrial integrity, inhibited release of apoptotic proteins from mitochondria, prevented DNA fragmentation, chromatin condensation and hypodiploid DNA upon exposure to high glucose. This study confirms the capacity of dietary flavonoid Morin in regulating apoptosis induced by high glucose via mitochondrial mediated pathway through intervention of oxidative stress.

Abstract 2071: Docosahexaenoic acid-induced apoptosis is directly linked to mitochondria-mediated reactive oxygen species production in human cervical cancer cells

Abstract Reactive oxygen species (ROS) produced by docosahexaenoic acid (DHA) have an important function in cancer cell death. However, the exact mechanism of ROS production, after DHA stimulation, is not clearly understood. Here, we determined that elevated levels of ROS generated by mitochondrial respiration is directly associated with DHA-induced cervical cancer cell death. The levels of caspase 3 activity, TUNEL-positive staining cells and Sub-G1 portion were markedly increased in DHA-treated cancer cells, suggesting that apoptosis is responsible for the DHA-induced cervical cancer cell death. Furthermore, DHA was able to induce both mitochondrial complex I substrate- and complex II substrate-supported mitochondrial ROS production in isolated mitochondria from rodent liver. Meanwhile, a reduction in oxygen consumption rate and an increase in mitochondrial ROS production as measured by MitoSOX, were also observed in DHA-treated cancer cells, indicating that DHA can directly act on mitochondrial respiration and enhance ROS generation. The role of DHA-induced mitochondrial ROS production in apoptosis was further identified by the findings that DHA reduced the mitochondrial membrane potential, resulting in cardiolipin oxidation and cytochrome c release from mitochondria, and that N-acetylcysteine, an antioxidant almost completely blocked these processes as well as ROS production occurred in mitochondria and remarkably reversed the apoptotic cell death triggered by DHA. From the results presented here, we conclude that mitochondria actively participate in the DHA-induced apoptotic cell death by the generation of mitochondrial ROS. (This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Ministry of Education, Science and Technology (2011-0006232 and 2011-0003060)). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2071. doi:1538-7445.AM2012-2071