Induced ROS Production Research Articles

Mitochonic acid 5 activates the MAPK\u2013ERK\u2013yap signaling pathways to protect mouse microglial BV-2 cells against TNF\u03b1-induced apoptosis via increased Bnip3-related mitophagy

BackgroundThe regulation of microglial function via mitochondrial homeostasis is important in the development of neuroinflammation. The underlying mechanism for this regulatory function remains unclear. In this study, we investigated the protective role of mitochonic acid 5 (MA-5) in microglial mitochondrial apoptosis following TNFα-induced inflammatory injury.MethodsTNFα was used to induce inflammatory injury in mouse microglial BV-2 cells with and without prior MA-5 treatment. Cellular apoptosis was assessed using the MTT and TUNEL assays. Mitochondrial functions were evaluated via mitochondrial membrane potential JC-1 staining, ROS flow cytometry analysis, mPTP opening assessment, and immunofluorescence of cyt-c. Mitophagy was examined using western blots and immunofluorescence. The pathways analysis was carried out using western blots and immunofluorescence with a pathway blocker.ResultsOur results demonstrated that TNFα induced apoptosis in the microglial BV-2 cell line by activating the caspase-9-dependent mitochondrial apoptotic pathway. Mechanistically, inflammation reduced mitochondrial potential, induced ROS production, and contributed to the leakage of mitochondrial pro-apoptotic factors into the cytoplasm. The inflammatory response reduced cellular energy metabolism and increased oxidative stress. By contrast, treatment with MA-5 reduced mitochondrial apoptosis via upregulation of mitophagy. Increased mitophagy degraded damaged mitochondria, disrupting mitochondrial apoptosis, neutralizing ROS overproduction, and improving cellular energy production. We also identified that MA-5 regulated mitophagy via Bnip3 through the MAPK–ERK–Yap signaling pathway. Inhibiting this signaling pathway or knocking down Bnip3 expression prevented MA-5 from having beneficial effects on mitochondrial homeostasis and increased microglial apoptosis.ConclusionsAfter TNFα-induced inflammatory injury, MA-5 affects microglial mitochondrial homeostasis in a manner mediated via the amplification of protective, Bnip3-related mitophagy, which is mediated via the MAPK–ERK–Yap signaling pathway.

Effects of local Interleukin‐6 on Skeletal Muscle Mitochondrial Physiology

Skeletal muscle is vital to metabolic health and physical function. Skeletal muscle mitochondria have been extensively interrogated as organelles whose function and dysfunction are closely tied with chronic diseases such as diabetes and sarcopenia. There is accumulating evidence suggesting that chronic inflammation that is prevalent with aging and obesity is associated with skeletal muscle abnormalities such as functional impairments in mitochondria. Despite compelling links between inflammation, oxidative stress, and skeletal muscle dysfunction, the local effects of inflammatory cytokines on skeletal muscle mitochondrial physiology are currently not well‐understood. The purpose of this study was to determine how the inflammatory cytokine interleukin‐6 (IL‐6) influences skeletal muscle mitochondrial function. To determine the acute effects of IL‐6, mitochondria were isolated from skeletal muscle of healthy male mice (C57BL6) and were exposed to IL‐6 (IL6+,50 ng/ml) or vehicle (CTRL) for one hour. Multiplexed measurements of mitochondrial respiration (high‐resolution respirometry) and reactive oxygen species production (ROS, fluorometry) revealed that IL‐6 significantly increased ROS production as a percent of oxygen (4.14%±0.40) compared to CTRL (2.74%±0.34). Similarly, IL‐6 increased ROS production in differentiated C2C12 myotubes that were incubated for 18 hrs with 50 ng/mL IL‐6 (4.71%±.57) compared to CTRL (2.21%±.34). To evaluate the possibility that IL‐6 triggers ROS production by stimulating the mitochondrial transition pore (mPTP), isolated mitochondria were ale treated with cyclosporin A (CsA), a mPTP inhibitor in the presence or absence of IL‐6. Inhibition of mPTP attenuated IL‐6 induced ROS production (IL6+ (4.14%±0.40) vs. IL6+/CsA (2.78%±0.58). These results suggest that IL‐6 triggers an increase in mitochondrial ROS production that appears to be mediated by opening of the mPTP. Prolonged opening of the mPTP caused by chronically elevated IL‐6 may contribute to increased oxidative stress that is evident in skeletal muscle with aging and obesity.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Microparticles in sickle cell disease.

Several pathophysiological pathways in sickle cell disease (SCD), the most prevalent hemoglobinopathy worldwide, result in activation of circulating blood cells and the release of submicron vesicles, so-called microparticles (MPs). MPs are candidate biomarkers in vascular disease that exhibit functional biological properties. Compared to healthy individuals, higher level of plasma MPs, mostly derived from platelets and red blood cells (RBC), has been repeatedly observed in SCD patients in their steady-state condition. In contrast, conflicting results have been obtained on the impact of SCD complications and hydroxyurea treatment on circulating MP concentrations, largely due to non-standardized pre- and analytical procedures. Several factors responsible for the increased release of MPs by RBC have been identified in SCD such as sickling/unsickling, oxidative stress and abnormal activity of RBC acid sphingomyelinase. Besides their well-known pro-coagulant effect, sickle RBC-derived MPs produced ex vivo can induce ROS production by endothelial cells and promote a pro-inflammatory and pro-adhesive phenotype that may lead to renal occlusion in SCD mice. However, the functional properties of circulating MPs in human sickle cell disease remain to be studied and fully characterized.

EKSTRAK BUAH PEPAYA (Carica papaya L.) MENINGKATKAN KADAR CATALASE DAN GLUTATHIONE HATI TIKUS YANG TERPAPARLEAD ACETATE

Pollution is an entry or inclusion living things, subtances, energy, and or other components into environment and or change of environment compositions because of human activity or natural process with the result that decrease of quality to certain level that causes doesn’t work environment. Pollution can be caused many subtances that is heavy metal, i.e. lead acetate (Pb). Lead acetate affect physiological aberration, biochemical, and behavior. Heavy metal induction induce ROS production act as destructive oxidants. Body capability to oxidant neutralize serviced by superoxyde dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), glutathione (GSH), vitamin C, vitamin E, and other antioxidants in the cell. Antioxidants synthesized by plants, which is papaya (Carica papaya L.). Papaya (Carica papaya L.) effect on CAT and GSH levels was performed on male mice wistar strain. Mice induced by lead acetate 20 gr / Kg weight dose for 6 weeks, given papaya extract 100, 200, and 400 mg/Kg weight dose for 6 weeks. Further glutathione and catalase levels are measured on mice liver. This study proves, papaya extract increase catalase levels (p = 0,000) and glutathione levels (p = 0,000) on mice liver induced lead acetate. Papaya extract 400 mg/Kg/day most effective dose to increase catalase and glutathione levels on mice liver induced lead acetate.

Gamma irradiation of aloe-emodin induced structural modification and apoptosis through a ROS- and caspase-dependent mitochondrial pathway in stomach tumor cells

Purpose: The changes in molecular structure and the physiological properties of a gamma-irradiated aloe-emodin were examined.Materials and methods: Aloe-emodin was gamma-irradiated at doses ranging from 0 to 150 kGy, and the molecular structure was then analyzed using high-performance liquid chromatography (HPLC). AGS cells were cultured in RPMI medium and treated gamma irradiated aloe-emodin. Cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Apoptosis efficiency was investigated by cell cycle arrest, cell morphology, and signaling pathway. The structure of new radiolytic peak was identified by the hydrogen-nuclear magnetic resonance (1H NMR).Results: HPLC results showed that gamma irradiation induced new radiolytic peaks that were distinguishable from the aloe-emodin standard, and the area of new peaks was increased as the radiation dose increased. Gamma-irradiated aloe-emodin treatment significantly increased the cytotoxicity in AGS tumor cells. We also found that 150 kGy aloe-emodin increased the expression of Bax, cytosolic cytochrome c, PARP cleavage, and the activation of caspases-8, -9, -3, Bid, and Bcl-2. Treatment of 150 kGy aloe-emodin induced ROS production, DNA fragmentation, alterations of cell morphology, and the migration in AGS cells. Gamma-irradiated aloe-emodin induced an increase of sub-G1 phase and depolarization of mitochondrial membrane potential in AGS cells. We also confirmed that fractionated AEF1 (new radiolytic peak) induce the cell death, migration, an increase of sub-G1 phase and cytochrome c in a ROS-dependent manner.Conclusions: The radiolysis product (AEF1) of aloe-emodin transformed by gamma-irradiation strongly induced apoptotic cell death in AGS cells, indicating AEF1 is a potential candidate drug for use in anti-cancer drug.

Development of a functionalized UV-emitting nanocomposite for the treatment of cancer using indirect photodynamic therapy

BackgroundPhotodynamic therapy is a promising cancer therapy modality but its application for deep-seated tumor is mainly hindered by the shallow penetration of visible light. X-ray-mediated photodynamic therapy (PDT) has gained a major attention owing to the limitless penetration of X-rays. However, substantial outcomes have still not been achieved due to the low luminescence efficiency of scintillating nanoparticles and weak energy transfer to the photosensitizer. The present work describes the development of Y2.99Pr0.01Al5O12-based (YP) mesoporous silica coated nanoparticles, multifunctionalized with protoporphyrin IX (PpIX) and folic acid (YPMS@PpIX@FA) for potential application in targeted deep PDT.ResultsA YP nanophosphor core was synthesized using the sol–gel method to be used as X-ray energy transducer and was then covered with a mesoporous silica layer. The luminescence analysis indicated a good spectral overlap between the PpIX and nanoscintillator at the Soret as well as Q-band region. The comparison of the emission spectra with or without PpIX showed signs of energy transfer, a prerequisite for deep PDT. In vitro studies showed the preferential uptake of the nanocomposite in cancer cells expressing the folate receptorFolr1, validating the targeting efficiency. Direct activation of conjugated PpIX with UVA in vitro induced ROS production causing breast and prostate cancer cell death indicating that the PpIX retained its activity after conjugation to the nanocomposite. The in vivo toxicity analysis showed the good biocompatibility and non-immunogenic response of YPMS@PpIX@FA.ConclusionOur results indicate that YPMS@PpIX@FA nanocomposites are promising candidates for X-ray-mediated PDT of deep-seated tumors. The design of these nanoparticles allows the functionalization with exchangeable targeting ligands thus offering versatility, in order to target various cancer cells, expressing different molecular targets on their surface.

Thymol Elicits HCT-116 Colorectal Carcinoma Cell Death Through Induction of Oxidative Stress.

Colon cancer is one of the most deadly and common carcinomas occurring worldwide and there have been many attempts to treat this cancer. The present work was designed in order to evaluate thymol as a potent drug against colon cancer. Cytotoxicity of thymol at different concentrations was evaluated against a human colon carcinoma cell line (HCT-116 cells). Fluorescent staining was carried out to evaluate the level of ROS as well as mitochondrial and DNA fragmentation and immunoblot analysis were performed to confirm apoptosis and mitoptosis. Results of the study demonstrated that thymol efficiently created an oxidative stress environment inside HCT-116 cells, a colorectal carcinoma cell line, through induction of ROS production along with intense damage to DNA and mitochondria, as observed through Hoechst and rhodamine 123 staining, respectively. Moreover, expression of PARP-1, p-JNK, cytochrome-C and caspase-3 proteins was up-regulated, suggesting HCT-116 cells underwent mitoptotic cell death. Therefore, thymol could be used as a potent drug against colon cancer due to its lower toxicity and prevalence in natural medicinal plants.

Classical Swine Fever Virus Infection and Its NS4A Protein Expression Induce IL-8 Production through MAVS Signaling Pathway in Swine Umbilical Vein Endothelial Cells.

Classical swine fever virus (CSFV) infection causes a severe disease of pigs, which is characterized by hemorrhage, disseminated intravascular coagulation, and leucopenia. IL-8, a main chemokine and activator of neutrophils, regulates the permeability of endothelium, which may be related to the hemorrhage upon CSFV infection. Until now, the molecular mechanisms of IL-8 regulation during CSFV infection are poorly defined. Here, we showed that CSFV infection induced IL-8 production and the upregulation of IL-8 required virus replication in swine umbilical vein endothelial cells (SUVECs). Additionally, MAVS expression was increased and was required for IL-8 production upon CSFV infection. Moreover, ROS was involved in CSFV-induced IL-8 production. Subsequent studies demonstrated that ROS was involved in MAVS-induced IL-8 production and CSFV induced ROS production through MAVS pathway. These results indicate that CSFV induces IL-8 production through MAVS pathway and production of ROS. The role of NS4A in the pathogenesis of CSFV is not well-understood. In this study, we further demonstrated that CSFV NS4A induced IL-8 production through enhancing MAVS pathway and promoted CSFV replication. In addition, we discovered that CSFV NS4A was localized in the cell nucleus and cytoplasm, including endoplasmic reticulum (ER) and mitochondria. Taken together, these results provide insights into the mechanisms of IL-8 regulation and NS4A functions during CSFV infection.

Type 2 Diabetes Promotes Cell Centrosome Amplification via AKT-ROS-Dependent Signalling of ROCK1 and 14-3-3σ

Background/Aims: Type 2 diabetes is associated with oxidative stress and DNA damage which can cause centrosome amplification. Thus, the study investigated centrosome amplification in type 2 diabetes and the underlying mechanisms. Methods: Centrosome numbers in human peripheral blood mononuclear blood cells (PBMC) from healthy subjects and patients with type 2 diabetes were compared to access the association between type 2 diabetes and centrosome amplification. Colon cancer cells were used to investigate the molecular mechanisms underlying the centrosome amplification triggered by high glucose, insulin and palmitic acid. Western blot analysis was used to quantify the level of protein and protein phosphorylation. Immunofluorescent staining was performed to detect centrosomes. ROS was quantified using flow cytometry technique. Transcriptpmic profiling was performed using Illumina HiSeq^TM500 platform. Results: We found that centrosome amplification was increased PBMC from the type 2 diabetic patients, which correlated with the levels of fasting blood glucose and HbA1c. High glucose, insulin and palmitic acid, alone or in combinations, induced ROS production and centrosome amplification. Together, they increased AKT activation as well as the expression, binding and centrosome translation of ROCK1 and 14-3-3σ. Results from further analyses showed that AKT-ROS-dependent upregulations of expression, binding and centrosome translocation of ROCK1 and 14-3-3σ was the molecular pathway underlying the centrosome amplification in vitro triggered by high glucose, insulin and palmitic acid. Moreover, the key in vitro molecular signalling events activated by high glucose, insulin and palmitic acid were verified in PBMC from the patients with type 2 diabetes. Conclusion: Our results show that type 2 diabetes promotes cell centrosome amplification, and suggest that the diabetic pathophysiological factors-activated AKT-ROS-dependent signalling of ROCK1 and 14-3-3σ is the underlying molecular mechanism.

Phosphorylation of the Cytoskeletal Protein CAP1 Regulates Non-Small Cell Lung Cancer Survival and Proliferation by GSK3β

Adenylate cyclase-associated protein 1 (CAP1) is an evolutionarily conserved protein that regulates actin dynamics. Our previous study indicates that CAP1 is overexpressed in NSCLC tissues and correlated with poor clinical outcomes. Further establishing the role and dissecting underlying mechanisms are imperative before targeting CAP1 can become a possibility for cancer treatment. Here we report our findings that knockdown of CAP1 inhibited cell proliferation and induced apoptosis in vitro and in vivo. Moreover, phosphor mutants of CAP1 at the S307/S309 regulatory site had compromised rescue effects for both the invasiveness and the proliferation in CAP1-knockdown cells and GSK3β kinase inhibitor LiCl inhibited cell phosphorylation site S307/S309 by up-regulating the expression of p53, BAK, BAD and cleaved PARP induced ROS production, decreased lung cancer cell viability, adhesion, proliferation, migration and invasion, and induction of apoptosis. These novel mechanistic insights may ultimately open up avenues for strategies targeting CAP1 in the treatment of lung cancer, tailored for specific types of the highly diverse disease.

Graphene and graphene oxide induce ROS production in human HaCaT skin keratinocytes: the role of xanthine oxidase and NADH dehydrogenase.

The extraordinary physicochemical properties of graphene-based nanomaterials (GBNs) make them promising tools in nanotechnology and biomedicine. Considering the skin contact as one of the most feasible exposure routes to GBNs, the mechanism of toxicity of two GBNs (few-layer-graphene, FLG, and graphene oxide, GO) towards human HaCaT skin keratinocytes was investigated. Both materials induced a significant mitochondrial membrane depolarization: 72 h cell exposure to 100 μg mL-1 FLG or GO increased mitochondrial depolarization by 44% and 56%, respectively, while the positive control valinomycin (0.1 μg mL-1) increased mitochondrial depolarization by 48%. Since the effect was not prevented by cyclosporine-A, it appears to be unrelated to mitochondrial transition pore opening. By contrast, it seems to be mediated by reactive oxygen species (ROS) production: FLG and GO induced time- and concentration-dependent cellular ROS production, significant already at the concentration of 0.4 μg mL-1 after 24 h exposure. Among a panel of specific inhibitors of the major ROS-producing enzymes, diphenyliodonium, rotenone and allopurinol significantly reverted or even abolished FLG- or GO-induced ROS production. Intriguingly, the same inhibitors also significantly reduced FLG- or GO-induced mitochondrial depolarization and cytotoxicity. This study shows that FLG and GO induce a cytotoxic effect due to a sustained mitochondrial depolarization. This seems to be mediated by a significant cellular ROS production, caused by the activation of flavoprotein-based oxidative enzymes, such as NADH dehydrogenase and xanthine oxidase.

Targeting flavin-containing enzymes eliminates cancer stem cells (CSCs), by inhibiting mitochondrial respiration: Vitamin B2 (Riboflavin) in cancer therapy.

Here, we performed high-throughput drug-screening to identify new non-toxic mitochondrial inhibitors. This screening platform was specifically designed to detect compounds that selectively deplete cellular ATP levels, but have little or no toxic side effects on cell viability. Using this approach, we identified DPI (Diphenyleneiodonium chloride) as a new potential therapeutic agent. Mechanistically, DPI potently blocks mitochondrial respiration by inhibiting flavin-containing enzymes (FMN and FAD-dependent), which form part of Complex I and II. Interestingly, DPI induced a chemo-quiescence phenotype that potently inhibited the propagation of CSCs, with an IC-50 of 3.2 nano-molar. Virtually identical results were obtained using CSC markers, such as CD44 and CD24. We further validated the effects of DPI on cellular metabolism. At 10 nM, DPI inhibited oxidative mitochondrial metabolism (OXPHOS), reducing mitochondrial driven ATP production by >90%. This resulted in a purely glycolytic phenotype, with elevated L-lactate production. We show that this metabolic inflexibility could be rapidly-induced, after only 1 hour of DPI treatment. Remarkably, the mitochondrial inhibitory effects of DPI were reversible, and DPI did not induce ROS production. Cells maintained in DPI for 1 month showed little or no mitochondrial activity, but remained viable. Thus, it appears that DPI behaves as a new type of mitochondrial inhibitor, which maintains cells in a state of metabolic-quiescence or “suspended animation”. In conclusion, DPI treatment can be used to acutely confer a mitochondrial-deficient phenotype, which we show effectively depletes CSCs from the heterogeneous cancer cell population. These findings have significant therapeutic implications for potently targeting CSCs, while minimizing toxic side effects. We also discuss the possible implications of DPI for the aging process. Interestingly, previous studies in C. elegans have shown that DPI prevents the accumulation of lipofuscin (an aging-associated hallmark), during the response to oxidative stress. Our current results are consistent with data showing that flavins (FAD, FMN and/or Riboflavin) are auto-fluorescent markers of i) increased mitochondrial “power” (OXPHOS) and ii) elevated CSC activity. Finally, we believe that DPI is one of the most potent and highly selective CSC inhibitors discovered to date. Therefore, our current findings suggest a new impetus to create novel analogues of i) DPI (Diphenyleneiodonium chloride) and ii) DPI-related compounds (Diphenyliodonium chloride), using medicinal chemistry, to optimize this very promising and potent anti-CSC activity. We propose to call these new molecules “Mitoflavoscins”. For example, DPI is ∼30 times more potent than Palbociclib (IC-50 = 100 nM), which is an FDA-approved CDK4/6 inhibitor, that broadly targets proliferation in any cell type, including CSCs.

Tumor suppressor NPRL2 induces ROS production and DNA damage response

The SEA/GATOR complex is an essential regulator of the mTORC1 pathway. In mammals the GATOR1 complex is composed of the proteins DEPDC5, NPRL2 and NPRL3. GATOR1 serves as an mTORC1 inhibitor and activates the mTORC1-modulating RagA GTPase. However, several GATOR members have mTORC1 independent functions. Here we characterize mammalian cells overexpressing the GATOR1 component NPRL2. We demonstrate that, in the cells with active p53, ectopic expression of NPRL2 induces NOX2-dependent production of reactive oxygen species and DNA damage. Overexpressed NPRL2 accumulates in the nucleus, together with apoptosis-inducing factor (AIF). These events are accompanied by phosphorylation of p53, activation of a DNA-damage response and cell cycle arrest in G1 phase, followed by apoptosis. In the cells negative for active p53, NPRL2 ectopic expression leads to activation of CHK1 or CHK2 kinases and cell cycle arrest in S or G2/M phases. Combined, these results demonstrate a new role for the NPRL2, distinct from its function in mTORC1 regulation.

Cepharanthine exhibits a potent anticancer activity in p53-mutated colorectal cancer cells through upregulation of p21Waf1/Cip1.

Cepharanthine(CEP), a biscoclurine alkaloid isolated from StephaniacepharanthaHayata, has demonstrated anticancer activity in several different types of cancer cells. Colorectal cancer(CRC) is one of the most common cancers in both men and women. Mutated p53 in CRC was reported to be associated with resistance to commonly used chemotherapeutic agents including, 5‑fluorouracil, oxaliplatin and irinotecan. Many studies reported that mutation of p53 induced chemoresistance through several mechanisms, including induction of drug efflux, disruption of cell cycle regulation, evasion of apoptosis and upregulation of DNA repair. This study aimed to evaluate the anticancer activity of CEP in p53 mutant versus p53 wild-type colorectal cancer cells and determine its underlying mechanisms of action. Our results showed that CEP induced colorectal cancer cell death in a concentration-dependent manner. Remarkably, CEP was more effective in controlling the growth of the p53 mutant colorectal cancer cell lines, HT‑29 and SW-620, than the p53 wild-type colorectal cancer cell lines, COLO‑205 and HCT-116. Further studies on the underlying mechanisms revealed that CEP could induce cell cycle arrest and apoptosis in both HT‑29 and COLO‑205 cells. Treatment with CEP dramatically increased p21Waf1/Cip1 expression levels of the p53 mutant cell line HT‑29 and to a lesser extent, the p53 wild-type cell line COLO‑205. In addition, cyclinA and Bcl‑2 expression levels of both cell lines were significantly downregulated following treatment with CEP. CEP also induced ROS formation in colorectal cancer cells. Taken together, we concluded that CEP effectively induced cell cycle arrest and apoptosis which may be mediated through upregulation of p21Waf1/Cip1, downregulation of cyclinA and Bcl‑2 and induction of ROS production in colorectal cancer cells. These findings suggested that CEP could potentially be a novel anticancer agent for p53 mutant colorectal cancer cells which are often resistant to current chemotherapeutic agents.

Thioredoxin-albumin fusion protein prevents copper enhanced zinc-induced neurotoxicity via its antioxidative activity

Zinc (Zn) is a co-factor for a vast number of enzymes, and functions as a regulator for immune mechanism and protein synthesis. However, excessive Zn release induced in pathological situations such as stroke or transient global ischemia is toxic. Previously, we demonstrated that the interaction of Zn and copper (Cu) is involved in the pathogenesis of Alzheimer’s disease and vascular dementia. Furthermore, oxidative stress has been shown to play a significant role in the pathogenesis of various metal ions induced neuronal death. Thioredoxin-Albumin fusion (HSA-Trx) is a derivative of thioredoxin (Trx), an antioxidative protein, with improved plasma retention and stability of Trx. In this study, we examined the effect of HSA-Trx on Cu2+/Zn2+-induced neurotoxicity. Firstly, HSA-Trx was found to clearly suppress Cu2+/Zn2+-induced neuronal cell death in mouse hypothalamic neuronal cells (GT1-7 cells). Moreover, HSA-Trx markedly suppressed Cu2+/Zn2+-induced ROS production and the expression of oxidative stress related genes, such as heme oxygenase-1. In contrast, HSA-Trx did not affect the intracellular levels of both Cu2+ and Zn2+ after Cu2+/Zn2+ treatment. Finally, HSA-Trx was found to significantly suppress endoplasmic reticulum (ER) stress response induced by Cu2+/Zn2+ treatment in a dose dependent manner. These results suggest that HSA-Trx counteracted Cu2+/Zn2+-induced neurotoxicity by suppressing the production of ROS via interfering the related gene expressions, in addition to the highly possible radical scavenging activity of the fusion protein. Based on these findings, HSA-Trx has great potential as a promising therapeutic agent for the treatment of refractory neurological diseases.

Leptin induces ROS via NOX5 in healthy and neoplastic mammary epithelial cells.

NADPH oxidase (NOX) complexes (a family of seven isoforms) drive cellular ROS production in patho-logical processes such as cancer. NOX-driven ROS production is involved in cell mechanisms from signalling to oxidative stress. Leptin, an adipokine overexpressed in obese patients, has been investigated in studies on breast carcinogenesis, but its effects on oxidative stress remain largely unexplored, especially in breast cancer. The study used three human mammary epithelial cell models presenting different neoplastic status (healthy primary HMECs, neoplastic MCF-7 cells and neoplastic MDA-MB-231 cells) to determine the effects of leptin on short-term ROS production and to characterize the enzymes involved. All three cell models significantly expressed NADPH oxidase isoform5 (NOX5) in our culture conditions. All models showed induced ROS production regardless of leptin concentration (10ng/ml mimicking good health, 100ng/ml mimicking obesity). Cell treatment with either siRNA against NOX5, NOX inhibitor DPI or a calcium channel blocker (verapamil) confirmed the putative involvement of the NOX5 isoenzyme in ROS production. Moreover, cell treatments suppressed ROS production under leptin at both concentrations. Neoplastic cells appeared unable to downregulate NOX5 mRNA expression under leptin. Leptin emerged as a potential activator of ROS production in human epithelial mammary cells, where the ROS production was apparently linked to NOX5 activation. This novel finding could shed light on the potential role of obesity-associated hyperleptinemia in mammary cells via the activation of NOXenzymes.

Atorvastatin but Not Pravastatin Impairs Mitochondrial Function in Human Pancreatic Islets and Rat \u03b2-Cells. Direct Effect of Oxidative Stress

Statins are a class of drugs widely prescribed as frontline therapy for lowering plasma LDL-cholesterol in cardiovascular risk prevention. Several clinical reports have recently suggested an increased risk of type 2 diabetes associated with chronic use of these drugs. The pathophysiology of this effect remains to be fully elucidated but impaired β-cell function constitutes a potential mechanism. The aim of this study was to explore the effect of a chronic treatment with lipophilic and hydrophilic statins on β-cell function, using human pancreatic islets and rat insulin-secreting INS-1 cells; we particularly focused on the role of mitochondria and oxidative stress. The present study demonstrates, for the first time, that atorvastatin (lipophilic) but not pravastatin (hydrophilic) affected insulin release and mitochondrial metabolism due to the suppression of antioxidant defense system and induction of ROS production in pancreatic β-cell models. Mevalonate addition and treatment with a specific antioxidant (N-AcetylCysteine) effectively reversed the observed defects. These data demonstrate that mitochondrial oxidative stress is a key element in the pathogenesis of statin-related diabetes and may have clinical relevance to design strategies for prevention or reduction of statin induced β-cell dysfunction and diabetes in patients treated with lipophilic statins.

High Boron-loaded DNA-Oligomers as Potential Boron Neutron Capture Therapy and Antisense Oligonucleotide Dual-Action Anticancer Agents.

Boron cluster-modified therapeutic nucleic acids with improved properties are of interest in gene therapy and in cancer boron neutron capture therapy (BNCT). High metallacarborane-loaded antisense oligonucleotides (ASOs) targeting epidermal growth factor receptor (EGFR) were synthesized through post-synthetic Cu (I)-assisted “click” conjugation of alkyne-modified DNA-oligonucleotides with a boron cluster alkyl azide component. The obtained oligomers exhibited increased lipophilicity compared to their non-modified precursors, while their binding affinity to complementary DNA and RNA strands was slightly decreased. Multiple metallacarborane residues present in the oligonucleotide chain, each containing 18 B-H groups, enabled the use of IR spectroscopy as a convenient analytical method for these oligomers based on the diagnostic B-H signal at 2400–2650 cm−1. The silencing activity of boron cluster-modified ASOs used at higher concentrations was similar to that of unmodified oligonucleotides. The screened ASOs, when used in low concentrations (up to 50 μM), exhibited pro-oxidative properties by inducing ROS production and an increase in mitochondrial activities in HeLa cells. In contrast, when used at higher concentrations, the ASOs exhibited anti-oxidative properties by lowering ROS species levels. In the HeLa cells (tested in the MTT assay) treated (without lipofectamine) or transfected with the screened compounds, the mitochondrial activity remained equal to the control level or only slightly changed (±30%). These findings may be useful in the design of dual-action boron cluster-modified therapeutic nucleic acids with combined antisense and anti-oxidant properties.

Tussilagonone-induced Nrf2 pathway activation protects HepG2 cells from oxidative injury

Tussilagonone is a compound derived from the medicinal plant Tussilago farfara L., which is used as a traditional medicine for respiratory diseases, including asthma and pneumonia. Recent reports suggest that tussilagonone exhibits anti-inflammatory effects; however, the scope of protective functions has not been elucidated yet. In this study, we demonstrate that tussilagonone enhances cellular detoxification by increasing quinone reductase activity in Hepa1c1c7 cells. In addition, tussilagonone decreased tert-butyl hydroperoxide(t-BHP)-induced ROS production and cell death, suggesting that it also acts as a potent antioxidant. To verify the molecular mechanism underlying tussilagonone activity, we examined the expression of nuclear factor erythroid 2-related factor 2(Nrf2)—a transcription factor that regulates antioxidant protein expression—in HepG2 cells. Significantly, these results showed that tussilagonone induces Nrf2 activation and nuclear accumulation, resulting in the upregulation of the detoxifying enzymes NAD(P)H quinone dehydrogenase 1(NQO1) and heme oxygenase-1(HO-1) that protect cells from oxidative stress. Further molecular analyses revealed that tussilagonone-induced Nrf2 activation was mediated by ERK1/2 in HepG2 cells. Collectively, these data indicate that tussilagonone attenuates t-BHP-induced ROS and activates quinone reductase activity via Nrf2 pathway activation and target gene expression, and thereby acts as an antioxidant that protects HepG2 cells from oxidative stress and associated damage.

Arjunolic Acid Improves the Serum Level of Vitamin B12 and Folate in the Process of the Attenuation of Arsenic Induced Uterine Oxidative Stress.

Continuation of prolonged treatment against arsenicosis with conventional chelating therapy is a global challenge. The present study was intended to evaluate the defensive effect of arjunolic acid against arsenic-induced oxidative stress and female reproductive dysfunction. Wistar strain adult female rats were given sodium arsenite (10mg/kg body weight) in combination with arjunolic acid (10mg/kg body weight) orally for two estrous cycles. Electrozymographic analysis explored that arjunolic acid co-treatment counteracted As3+-induced ROS production in uterine tissue by stimulating the activities of endogenous enzymatic antioxidants. Arjunolic acid was able to enhance the protection against mutagenic uterine DNA breakage, necrosis, and ovarian-uterine tissue damages in arsenicated rats by improving the ovarian steroidogenesis. The mechanisms might be coupled with the augmentation of antioxidant defense system, partly through the elimination of arsenic with the involvement of S-adenosyl methionine pool where circulating levels of vitamin B12, folic acid, and homocysteine play critical roles as evidenced from our present investigation.