Mitochondrial Superoxide Radical Research Articles

Oxidized guanosines induce mitochondrial dysfunction and loss of viability in β-cells

The production of reactive oxygen species and oxidative stress promote β-cell dysfunction and impair insulin secretion, thereby contributing to the pathogenesis of type 2 diabetes mellitus (T2DM). The nucleobase guanine is highly sensitive to oxidation, which results in the formation of 8-oxoguanosine (8oxoG) and 8-oxodeoxyguanosine (8oxodG). The urinary excretion of 8oxoG is associated with the risk of mortality in people with T2DM, including from diabetic complications such as cardiovascular disease. However, the cellular mechanisms responsible for this association are poorly defined. Therefore, in this study, we examined the effect of 8oxoG, 8oxodG and other oxidized guanosine derivatives, on the INS-1E β-cell line. Exposure of INS-1E cells to 8oxoG and 8oxodG decreased metabolic activity and promoted cell death by apoptosis. The change in cell viability was similar to that induced by treatment of INS-1E cells with the inflammatory cytokines interleukin 1β (Il-1β) and tumour necrosis factor α (TNFα). Changes in mitochondrial membrane permeability and superoxide radical formation were also observed with 8oxoG, but there was no significant change in the oxidation state of mitochondrial cytochromes or hydrogen peroxide levels in the INS-1E cells. Interestingly, exposure to 8oxoG and 8-oxodG also increased the mRNA expression of stress response genes, including NADPH dehydrogenase quinone 1 (NQO1), and thioredoxin-interacting protein (TXNIP). Together, these results support a potential role of oxidized guanosine derivatives in the induction of β-cell dysfunction, which could be relevant to the pathogenesis of T2DM.

Microalgae polysaccharides exert antioxidant and anti-inflammatory protective effects on human intestinal epithelial cells in vitro and dextran sodium sulfate-induced mouse colitis in vivo

Microalgae polysaccharides (MAPS) have emerged as novel prebiotics, but their direct effects on intestinal epithelial barrier are largely unknown. Here, MAPS isolated from Chlorella pyrenoidosa, Spirulina platensis, and Synechococcus sp. PCC 7002 were characterized as mainly branched heteropolysaccharides, and were bioavailable to Caco-2 cells based on fluorescein isothiocyanate labeling and flow cytometry analysis. These MAPS were equally effective to scavenge hydroxyl and superoxide radicals in vitro and to attenuate the H2O2-, dextran sodium sulfate-, tumor necrosis factor α-, and interleukin 1β-induced burst of intracellular reactive oxygen species and mitochondrial superoxide radicals, interleukin-8 production, cyclooxygenase-2 and inducible nitric oxide synthase expression, and/or tight junction disruption in polarized Caco-2 cells. MAPS and a positive drug Mesalazine were intragastrically administered to C57BL/6 mice daily for 7 d during and after 4-d dextran sodium sulfate exposure. Clinical signs and colon histopathology revealed equivalent anti-colitis efficacies of MAPS and Mesalazine, and based on biochemical analysis of colonic tight junction proteins, goblet cells, mucin 2 and trefoil factor 3 transcription, and colonic and peripheral pro-inflammatory cytokines, MAPS alleviated dextran sodium sulfate-induced intestinal epithelial barrier dysfunction, and their activities were even superior than Mesalazine. Overall, MAPS confer direct antioxidant and anti-inflammatory protection to intestinal epithelial barrier function.

Simultaneous two-photon intravital imaging of viscosity and superoxide radical anion by a styrylpyridinium-based fluorescent probe

Simultaneous sensing of cellular microenvironment parameters and redox signaling molecules by a single fluorescent probe allows an efficient access to numerous applications ranging from convenient diagnostic assays to investigation on their pathophysiological roles and cross-link. Focusing on a lack of efficient molecule tools for simultaneous intravital imaging of mitochondrial viscosity and superoxide radical anion (O2•-), we bridged diethylaminobenzene with diphenylphosphinate-modified pyridinium moiety by an olefinic bond to construct a styrylpyridinium-based two-photon fluorescent probe V-OS. V-OS triggered a turn-on response either at 625 nm to viscosity which blocks intramolecular rotation, or at 530 nm to O2•-via nucleophilic attack of O2•- followed by 1,6-elimination. With the aid of the two-photon probe, we not only identified the cross-talk between viscosity and O2•- but successfully mapped the burst of O2•- and the increased viscosity during either cellular ferroptosis process or cerebral ischemia reperfusion injury of living mice.

Association between MnSOD Activity and Cognitive Impairment in Unmedicated First-Episode Schizophrenia: Regulated by MnSOD Ala-9Val Gene Polymorphism.

The imbalance between pro-oxidants and antioxidants is thought to be responsible for aging and cognitive impairment in many degenerative diseases, including schizophrenia (SZ). As the first antioxidant enzyme to detoxify superoxide radicals in mitochondria, manganese superoxide dismutase (MnSOD) activity and its functional polymorphism of Ala-9Val have been found to be associated with SZ. In this study, we explored the association between MnSOD activity, MnSOD Ala-9Val polymorphism and cognitive dysfunction in unmedicated first-episode (UMFE) SZ patients, which has not been examined. We recruited 234 UMFE SZ patients and 232 healthy controls (HC) and evaluated them with Repeated Battery for the Assessment of Neuropsychological Status (RBANS), plasma MnSOD activity and MnSOD Ala-9Val (rs4880) polymorphism. In addition, we used the Positive and Negative Syndrome Scale (PANSS) to assess the severity of patients’ psychopathological symptoms. Compared with HC, UMFE patients showed extensive cognitive impairment on RBANS, and had higher MnSOD activity. MnSOD Ala-9Val polymorphism was not associated with SZ susceptibility and cognitive impairment, but only affected MnSOD activity in patients. Moreover, only in SZ patients with Val homozygotes, MnSOD activity was significantly correlated with cognitive impairment, especially in RBANS total score, visuospatial/constructional and attention index scores. Our results suggest that cognitive impairment is associated with MnSOD activity in patients with first-episode SZ, which may be regulated by MnSOD Ala-9Val polymorphism.

A Core-Linker-Polyamine (CLP) Strategy Enables Rapid Discovery of Antileishmanial Aminoalkylquinolinecarboxamides That Target Oxidative Stress Mechanism.

A Core-Linker-Polyamine (CLP) strategy has been exploited to develop new antileishmanial agents. It involves the linker-based assembly of alkyl-polyamine side chain as a potential pharmacophore motif with a privileged heterocyclic motif, 4-arylquinoline. A series of aminoalkyl 4-arylquinoline-2-carboxamides and their analogs were synthesized and tested against L. donovani promastigotes. Among all synthesized derivatives, 10 compounds showed significant antipromastigote activities with more efficacy (IC50 : 4.75-8 μM) than an antileishmanial oral drug Miltefosine (IC50 : 8.9±1.55 μM). Most active aminoalkyl-quinoline-carboxamides 9 a and 9 b, displayed negligible cytotoxicity towards human monocytic (THP-1) macrophages. The compounds show antileishmanial activity by generating mitochondrial superoxide radicals. However, they did not show interference with trypanothione reductase, a redox enzyme of Leishmania. Significant change in the morphology of the L. donovani promastigote by the compounds was observed. The Structure-activity relationship analysis suggest the pharmacophoric importance of alkylpolyamine and carboxamide motifs. In silico evaluation indicated that the investigated active molecules 9 a and 9 b possess important drug-likeness, physicochemical and pharmacokinetic-relevant properties.

The Short-Term Exposure to SDHI Fungicides Boscalid and Bixafen Induces a Mitochondrial Dysfunction in Selective Human Cell Lines.

Fungicides are used to suppress the growth of fungi for crop protection. The most widely used fungicides are succinate dehydrogenase inhibitors (SDHIs) that act by blocking succinate dehydrogenase, the complex II of the mitochondrial electron transport chain. As recent reports suggested that SDHI-fungicides could not be selective for their fungi targets, we tested the mitochondrial function of human cells (Peripheral Blood Mononuclear Cells or PBMCs, HepG2 liver cells, and BJ-fibroblasts) after exposure for a short time to Boscalid and Bixafen, the two most used SDHIs. Electron Paramagnetic Resonance (EPR) spectroscopy was used to assess the oxygen consumption rate (OCR) and the level of mitochondrial superoxide radical. The OCR was significantly decreased in the three cell lines after exposure to both SDHIs. The level of mitochondrial superoxide increased in HepG2 after Boscalid and Bixafen exposure. In BJ-fibroblasts, mitochondrial superoxide was increased after Bixafen exposure, but not after Boscalid. No significant increase in mitochondrial superoxide was observed in PBMCs. Flow cytometry revealed an increase in the number of early apoptotic cells in HepG2 exposed to both SDHIs, but not in PBMCs and BJ-fibroblasts, results consistent with the high level of mitochondrial superoxide found in HepG2 cells after exposure. In conclusion, short-term exposure to Boscalid and Bixafen induces a mitochondrial dysfunction in human cells.

ATN-161 Ameliorates Ischemia/Reperfusion-induced Oxidative Stress, Fibro-inflammation, Mitochondrial damage, and Apoptosis-mediated Tight Junction Disruption in bEnd.3 Cells.

—We have previously demonstrated the significance of endothelial cell-expressed α5β1 integrin in ischemic stroke, having shown that α5β1 integrin endothelial cell-selective knockout mice are significantly resistance to ischemic stroke injury via preservation of the tight junction protein claudin-5 and subsequent stabilization of the blood–brain barrier (BBB). In addition, inhibition of α5β1 by the small peptide noncompetitive integrin α5 inhibitor, ATN-161, is beneficial in a mouse model of ischemic stroke through reduction of infarct volume, edema, stabilization of the BBB, and reduced inflammation and immune cell infiltration into the brain. In continuation with our previous findings, we have further evaluated the mechanistic role of ATN-161 in vitro and found that oxygen and glucose deprivation and reperfusion (OGD/R)-induced inflammation, oxidative stress, apoptosis, mitochondrial depolarization, and fibrosis attenuate tight junction integrity via induction of α5, NLRP3, p-FAK, and p-AKT signaling in mouse brain endothelial cells. ATN-161 treatment (10 µM) effectively inhibited OGD/R-induced extracellular matrix (ECM) deposition by reducing integrin α5, MMP-9, and fibronectin expression, as well as reducing oxidative stress by reducing mitochondrial superoxide radicals, intracellular ROS, inflammation by reducing NLRP3 inflammasome, tight junction loss by reducing claudin-5 and ZO-1 expression levels, mitochondrial damage by inhibiting mitochondrial depolarization, and apoptosis via regulation of p-FAK and p-AKT levels. Taken together, our results further support therapeutically targeting α5 integrin with ATN-161, a safe, well-tolerated, and clinically validated peptide, in ischemic stroke.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10753-021-01509-9.

Progesterone Modulates Mitochondrial Functions in Human Glioblastoma Cells.

A substantial literature supports the notion that cancer is a metabolic disease. Mitochondria are sexually dimorphic, and progesterone (P4) plays a key regulatory role in mitochondrial functions. We investigated the effect of P4 on mitochondrial functions in three human glioblastoma multiforme (GBM) cell lines. In dose-response and time-response studies, GBM cells were exposed to different concentrations of P4 followed by mitochondrial stress-testing with a Seahorse analyzer. Data were analyzed for oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and spare respiratory capacity (SRC) to determine the effects of P4 exposure on mitochondrial respiration and rate of glycolysis. We also examined the effect of P4 on mitochondrial superoxide radical generation by confocal microscopy. As early as 1h post-P4 exposure, we found a substantial dose-dependent inhibitory effect of P4 on OCR, ECAR, and SRC in all GBM cell lines. P4 treatment altered the levels of basal respiration, maximum respiration, nonmitochondrial oxygen consumption, ATP production, and proton leak. P4 given at 80-μM concentration showed the maximum inhibitory effect compared to controls. Live imaging data showed an 11-22% increase in superoxide radical generation in all three GBM cell lines following 6h exposure to a high concentration of P4. Our data show that high-dose P4 exerts an inhibitory effect on both mitochondrial respiration and glycolysis in GBM cells. These effects would lead to decreased tumor size and rate of growth, representing a potential treatment to control the spread of GBM.

The program of renal fibrogenesis is controlled by microRNAs regulating oxidative metabolism

Excessive accumulation of extracellular matrix (ECM) is the hallmark of fibrotic diseases. In the kidney, it is the final common pathway of prevalent diseases, leading to chronic renal failure. While cytokines such as TGF-β play a fundamental role in myofibroblast transformation, recent work has shown that mitochondrial dysfunction and defective fatty acid oxidation (FAO), which compromise the main source of energy for renal tubular epithelial cells, have been proposed to be fundamental contributors to the development and progression of kidney fibrosis. MicroRNAs (miRNAs), which regulate gene expression post-transcriptionally, have been reported to control renal fibrogenesis. To identify miRNAs involved in the metabolic derangement of renal fibrosis, we performed a miRNA array screen in the mouse model of unilateral ureteral obstruction (UUO). MiR-150-5p and miR-495-3p were selected for their link to human pathology, their role in mitochondrial metabolism and their targeting of the fatty acid shuttling enzyme CPT1A. We found a 2- and 4-fold upregulation of miR-150-5p and miR-495-5p, respectively, in both the UUO and the folic acid induced nephropathy (FAN) models, while TGF-β1 upregulated their expressions in the human renal tubular epithelial cell line HKC-8. These miRNAs synergized with TGF-β regarding its pro-fibrotic effect by enhancing the fibrosis-associated markers Acta2, Col1α1 and Fn1. Bioenergetics studies showed a reduction of FAO-associated oxygen consumption rate (OCR) in HKC-8 cells in the presence of both miRNAs. Consistently, expression levels of their mitochondrial-related target genes CPT1A, PGC1α and the mitochondrial transcription factor A (TFAM), were reduced by half in renal epithelial cells exposed to these miRNAs. By contrast, we did not detect changes in mitochondrial mass and transmembrane potential (ΔѰm) or mitochondrial superoxide radical anion production. Our data support that miR-150 and miR-495 may contribute to renal fibrogenesis by aggravating the metabolic failure critically involved in tubular epithelial cells, ultimately leading to fibrosis.

Mitochondrial Superoxide Dismutase: What the Established, the Intriguing, and the Novel Reveal About a Key Cellular Redox Switch.

Significance: Reactive oxygen species (ROS) are now widely recognized as central mediators of cell signaling. Mitochondria are major sources of ROS. Recent Advances: It is now clear that mitochondrial ROS are essential to activate responses to cellular microenvironmental stressors. Mediators of these responses reside in large part in the cytosol. Critical Issues: The primary form of ROS produced by mitochondria is the superoxide radical anion. As a charged radical anion, superoxide is restricted in its capacity to diffuse and convey redox messages outside of mitochondria. In addition, superoxide is a reductant and not particularly efficient at oxidizing targets. Because there are many opportunities for superoxide to be neutralized in mitochondria, it is not completely clear how redox cues generated in mitochondria are converted into diffusible signals that produce transient oxidative modifications in the cytosol or nucleus. Future Directions: To efficiently intervene at the level of cellular redox signaling, it seems that understanding how the generation of superoxide radicals in mitochondria is coupled with the propagation of redox messages is essential. We propose that mitochondrial superoxide dismutase (SOD2) is a major system converting diffusion-restricted superoxide radicals derived from the electron transport chain into highly diffusible hydrogen peroxide (H2O2). This enables the coupling of metabolic changes resulting in increased superoxide to the production of H2O2, a diffusible secondary messenger. As such, to determine whether there are other systems coupling metabolic changes to redox messaging in mitochondria as well as how these systems are regulated is essential.

KU-596 decreases mitochondrial superoxide and improves bioenergetics following downregulation of manganese superoxide dismutase in diabetic sensory neurons

Neuronal mitochondrial dysfunction and oxidative stress are key pathophysiologic mechanisms of diabetic peripheral neuropathy (DPN). KU-596 is a small molecule modulator of heat shock protein 90 (Hsp90) that can reverse clinically relevant measures of DPN in diabetic animal models. Mechanistically, drug efficacy requires Hsp70 and correlates with improving mitochondrial maximal respiratory capacity (MRC) and decreasing oxidative stress in diabetic sensory neurons. The goal of this study was to determine if ex vivo treatment of diabetic neurons with KU-596 improves MRC by decreasing glucose-induced oxidative stress in an Hsp70-dependent manner. Sensory neurons were isolated from non-diabetic or diabetic mice wild type (WT) or Hsp70 knockout (Hsp70 KO) mice and treated with KU-596 in the presence of low or high glucose concentrations. In diabetic WT and Hsp70 KO neurons, hyperglycemia significantly increased superoxide levels, but KU-596 only decreased superoxide in WT neurons. Similarly, KU-596 significantly improved MRC in diabetic WT neurons maintained in high glucose but did not improve MRC in diabetic Hsp70 KO neurons under the same conditions. Since manganese superoxide dismutase (MnSOD) is the main mechanism to detoxify mitochondrial superoxide radicals, the cause and effect relationship between improved respiration and decreased oxidative stress was examined after knocking down MnSOD. Downregulating MnSOD in diabetic WT neurons increased hyperglycemia-induced superoxide levels, which was still significantly decreased by KU-596. However, KU-596 did not improve MRC following MnSOD knockdown. These data suggest that the ability of KU-596 to improve MRC is not necessarily dependent on decreasing mitochondrial superoxide in a MnSOD-dependent manner.

Metformin attenuates renal medullary hypoxia in diabetic nephropathy through inhibition uncoupling protein‐2

The purpose of the study is to examine the effect of metformin on oxygen metabolism and mitochondrial function in the kidney of an animal model of insulinopenic diabetes in order to isolate any renoprotective effect from any concomitant effect on blood glucose homeostasis. Sprague-Dawley rats were injected with streptozotocin (STZ) (50mgkg-1 ) and when stable started on metformin treatment (250mgkg-1 ) in the drinking water. Rats were prepared for in vivo measurements 25 to 30days after STZ injection, where renal function, including glomerular filtration rate and sodium transport, was estimated in anesthetized rats. Intrarenal oxygen tension was measured using oxygen sensors. Furthermore, mitochondrial function was assessed in mitochondria isolated from kidney cortex and medulla analysed by high-resolution respirometry, and superoxide production was evaluated using electron paramagnetic resonance. Insulinopenic rats chronically treated with metformin for 4weeks displayed improved medullary tissue oxygen tension despite of no effect of metformin on blood glucose homeostasis. Metformin reduced UCP2-dependent LEAK and differentially affected medullary mitochondrial superoxide radical production in control and diabetic rats. Metformin attenuates diabetes-induced renal medullary tissue hypoxia in an animal model of insulinopenic type 1 diabetes. The results suggest that the mechanistic pathway to attenuate the diabetes-induced medullary hypoxia is independent of blood glucose homeostasis and includes reduced UCP2-mediated mitochondrial proton LEAK.

Abstract 17190: Dual Hit of HIV-1 and Opioids Promote Oxidative Stress Mediated Pulmonary Endothelial Dysfunction

Introduction: HIV related pulmonary hypertension is one of the serious non-infectious cardiovascular complications in HIV infected individuals, especially among intravenous drug users. We previously demonstrated that opioids in combination with viral protein(s) result in reactive oxygen species (ROS) mediated induction of autophagy in pulmonary endothelial cells leading to shift in the cells from apoptosis to apoptosis resistant proliferative status associated with angio-proliferative remodeling observed in PAH. Here, we delineate the major sources of HIV-1 and morphine induced oxidative burst and its role in development of PAH using in-vivo model. Methods: Human pulmonary microvascular endothelial cells (HPMECs) were treated with morphine and HIV-Tat in the presence or absence of ROS scavengers followed by measurement of ROS. Mitochondrial specific superoxide radicals were measured with MitoSOX dye and ratio of mitochondrial DNA to nuclear DNA was determined by real-time PCR. To assess the in-vivo effect of HIV-1 and morphine on pulmonary vascular dysfunction, male HIV-Tg/WT Fischer rats (n=7-13per group) were subcutaneously administered 10 mg/kg body wt. morphine or saline daily IP for 21 days. Right ventricle systolic pressure (RVSP) and fulton Index were measured along with immunohistochemistry analysis of lungs. Result: Increased oxidative stress in HPMECs in response to combined treatment of morphine and tat (M+T) was noted to be contributed by mitochondria, NADPH oxidase and xanthine oxidase as observed by the decrease in ROS production of M+T treated cells on pre-treatment with ROS scavenger viz., Mitotempo (mitochondria), VAS3947 (NADPH oxidase) and allopurinol (Xanthine oxidase). However, M+T mediated early apoptosis and late hyperproliferation of HPMECs was prevented in presence of NADPH oxidase inhibitor. In parallel we observed elevated mitochondrial specific ROS at 2 hr and increased mitophagy at 3h followed by increase in total mitochondrial mass at 4-6h of MT treatment. Further, we observed significant increase in RVSP and RV hypertrophy in HIV-Tg rats exposed to morphine compared to WT rats. Immunohistochemical analysis also showed significant pulmonary vascular remodelling including endothelial proliferation in lung sections from HIV+morphine rats. Conclusion: Our findings suggest NADPH oxidase specific ROS role in autophagy-dependent increased proliferation of M+T treated HPMECs. Further our in-vivo findings provide evidence of potentiation of PAH development with the dual hit HIV-1 and opioids.

Selenotriapine – An isostere of the most studied thiosemicarbazone with pronounced pro-apoptotic activity, low toxicity and ability to challenge phenotype reprogramming of 3-D mammary adenocarcinoma tumors

Triapine, the most studied α-N-heterocyclic thiosemicarbazone, revealed potent activity against advanced leukemia, but was ineffective against a variety of solid tumors. Moreover, methemoglobinemia, which is a side effect of triapine administration, may limits all clinical application. To enhance anticancer activity and reduce side effects, we applied an isosteric replacement of sulfur to selenium atom was performed by synthesis and characterization of selenium triapine analog, 3-aminopyridine-2-carboxaldehyde selenosemicarbazone (selenotriapine). Compared to triapine, selenotriapine revealed superior pro-apoptotic activity with activation of intrinsic apoptotic pathway in both human monocytic leukemia (THP-1) and mammary adenocarcinoma (MCF-7) cell lines. For MCF-7 2-D cultures, selenotriapine induced notable increase in mitochondrial superoxide radical generation and dissipation of mitochondrial transmembrane potential. A significant delay in growth of MCF-7 spheroids (3-D culture) was accompanied by phenotypic stem cell reprogramming (Oct-4 expression). Additionally, selenotriapine demonstrated a very low toxicity profile as compared to triapine, confirmed over alleviated extent of methemoglobin formation and higher IC50 value in brine shrimp cytotoxicity assay.

Expression of genes belonging to the interacting TLR cascades, NADPH-oxidase and mitochondrial oxidative phosphorylation in septic patients.

Background and objectivesSepsis is a complex disease that is characterized by activation and inhibition of different cell signaling pathways according to the disease stage. Here, we evaluated genes involved in the TLR signaling pathway, oxidative phosphorylation and oxidative metabolism, aiming to assess their interactions and resulting cell functions and pathways that are disturbed in septic patients.Materials and methodsBlood samples were obtained from 16 patients with sepsis secondary to community acquired pneumonia at admission (D0), and after 7 days (D7, N = 10) of therapy. Samples were also collected from 8 healthy volunteers who were matched according to age and gender. Gene expression of 84 genes was performed by real-time polymerase chain reactions. Their expression was considered up- or down-regulated when the fold change was greater than 1.5 compared to the healthy volunteers. A p-value of ≤ 0.05 was considered significant.ResultsTwenty-two genes were differently expressed in D0 samples; most of them were down-regulated. When gene expression was analyzed according to the outcomes, higher number of altered genes and a higher intensity in the disturbance was observed in non-survivor than in survivor patients. The canonical pathways altered in D0 samples included interferon and iNOS signaling; the role of JAK1, JAK2 and TYK2 in interferon signaling; mitochondrial dysfunction; and superoxide radical degradation pathways. When analyzed according to outcomes, different pathways were disturbed in surviving and non-surviving patients. Mitochondrial dysfunction, oxidative phosphorylation and superoxide radical degradation pathway were among the most altered in non-surviving patients.ConclusionOur data show changes in the expression of genes belonging to the interacting TLR cascades, NADPH-oxidase and oxidative phosphorylation. Importantly, distinct patterns are clearly observed in surviving and non-surviving patients. Interferon signaling, marked by changes in JAK-STAT modulation, had prominent changes in both survivors and non-survivors, whereas the redox imbalance (iNOS signaling, oxidative phosphorylation and superoxide radical degradation) affecting mitochondrial functions was prominent in non-surviving patients.

Sigma-2 receptor agonist derivatives of 1-Cyclohexyl-4-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propyl]piperazine (PB28) induce cell death via mitochondrial superoxide production and caspase activation in pancreatic cancer

BackgroundDespite considerable efforts by scientific research, pancreatic cancer is the fourth leading cause of cancer related mortalities. Sigma-2 receptors, which are overexpressed in several tumors, represent promising targets for triggering selective pancreatic cancer cells death.MethodsWe selected five differently structured high-affinity sigma-2 ligands (PB28, PB183, PB221, F281 and PB282) to study how they affect the viability of diverse pancreatic cancer cells (human cell lines BxPC3, AsPC1, Mia PaCa-2, and Panc1 and mouse Panc-02, KCKO and KP-02) and how this is reflected in vivo in a tumor model.ResultsImportant cytotoxicity was shown by the compounds in the aggressive Panc02 cells, where cytotoxic activity was caspase-3 independent for four of the five compounds. However, both cytotoxicity and caspase-3 activation involved generation of Reactive Oxygen Species (ROS), which could be partially reverted by the lipid antioxidant α-tocopherol, but not by the hydrophilic N-acetylcysteine (NAC) indicating crucial differences in the intracellular sites exposed to oxidative stress induced by sigma-2 receptor ligands. Importantly, all the compounds strongly increased the production of mitochondrial superoxide radicals except for PB282. Despite a poor match between in vitro and the in vivo efficacy, daily treatment of C57BL/6 mice bearing Panc02 tumors resulted in promising effects with PB28 and PB282 which were similar compared to the current standard-of-care chemotherapeutic gemcitabine without showing signs of systemic toxicities.ConclusionsOverall, this study identified differential sensitivities of pancreatic cancer cells to structurally diverse sigma-2 receptor ligands. Of note, we identified the mitochondrial superoxide pathway as a previously unrecognized sigma-2 receptor-activated process, which encourages further studies on sigma-2 ligand-mediated cancer cell death for the targeted treatment of pancreatic tumors.

Inflammatory Niche Signalling Drives Genotoxic Stress in Hematopoietic Stem Cells and Predicts Leukemic Evolution in Human Leukemia Predisposition Syndromes

Primary alterations of the mesenchymal niche can induce myelodysplasia and acute myeloid leukemia in mouse models, introducing a concept of niche-driven leukemogenesis (Raaijmakers et al, Nature 2010). The molecular mechanisms and human relevance of this concept, however, have remained elusive.We addressed these key questions by modelling Shwachman-Diamond-Syndrome (SDS), a human monogenic congenital disorder caused by loss-of function mutation in the SBDS gene and characterized by skeletal defects, bone marrow failure and a striking propensity for leukemic evolution.Targeted Sbds deletion from mesenchymal progenitor cells (MPCs) in mice (OsxCre/+Sbdsf/f; OCSf/f) resulted in bone abnormalities faithfully recapitulating human disease, including short stature and early-onset osteoporosis. Skeletal defects were associated with genotoxic stress in hematopoietic stem and progenitor cells (HSPCs) as demonstrated by mitochondrial membrane hyperpolarization, oxidative stress, DNA damage and cell cycle checkpoint activation (transcriptional modulation of DNA damage response/repair pathways and G0-G1 cell cycle arrest). DNA damage could be partially rescued by in vivo administration of the ROS scavenger N-acetylcysteine supporting the notion of niche induced DNA damage in HSPCs induced by mitochondria-derived superoxide radicals.Mechanistically, Sbds deficiency caused activation of the p53 tumor suppressorpathway in MPCs (upregulation of P53 and transcriptional activation of downstream targets (GSEA). Genetic deletion of Trp53 from MPCs (Osxcre/+Sbdsf/fTrp53f/f mice) rescued the skeletal phenotype and genotoxic stress in HSPCs. Comparison of the transcriptome of MPCs from OCSf/f mice to their highly FACS-purified mesenchymal (CD45-CD235-7AAD-CD31-CD271+CD105+) human equivalents from SDS patients (RNAseq; n=5) demonstrated a striking overlap in disrupted gene programs (GSEA), including ribosome biogenesis and significant overexpression of the proinflammatory molecules such as S100A8 and S100A9, bona fide p53 downstream targets. Activation of p53 and inflammatory molecules was an MPC-autonomous consequence of Sbds depletion as demonstrated by ex vivo knockdown of the gene in OP9 cells. S100A8/A9 overexpression and secretion from MPCs from OCSf/f mice was confirmed by FCM and serum ELISA. Exposure of HSPCs to recombinant murine S100A8/9 resulted in increased DNA damage and apoptosis associated with transcriptional activation of TLR4 downstream signaling, a bona fide S100A8A9 receptor. In vivo TLR4 blockade by neutralizing antibodies resulted in reduced γH2AX foci in HSPCs from OCSf/f mice, in support of the existence of a Tpr53-S100A8/A9-TLR4 axis driving genotoxic stress. Formal demonstration that niche-derived S100A8/9 is sufficient to drive genotoxic stress in HSPCs was provided by transplantation of wild-type hematopoietic cells into recipient S100A8/A9 transgenic mice (Cheng et al., 2008) resulting in accumulation of mitochondrial superoxide radicals and DNA-damage in wild-type HSPCs.Finally, to further define the clinical relevance of this inflammatory MPC-HSPC axis to human disease, we performed massive parallel RNA-sequencing of FACS purified mesenchymal cells from homogeneously treated low-risk MDS patients (n=45). Overexpression of S100A8 and S100A9 in MPCs(confirmed by IHC) was found in a considerable subset of patients (17/45; 38%). S100A8/9+ mesenchymal cells displayed transcriptional activation of p53 and TLR programs, in line with findings in the mouse model. Strikingly, patients in the niche-S100A8/9+ group displayed a higher frequency of leukemia evolution (29.4% vs. 14.2%) with significantly shorter evolution time (average 3.4 (1-7.5) vs 18.5 (7-40); p=.03) and progression-free survival (median 11.5 vs. 53 months, p=.03), independent of established prognostic factors and risk classification systems.Collectively, the data define niche-HSPC inflammatory signaling through the p53-S100A8/A9-TLR axis as an actionable determinant of genotoxic stress and disease outcome in human preleukemia, opening the way to niche-instructed, therapeutic targeting to attenuate leukemic evolution. DisclosuresNo relevant conflicts of interest to declare.

SOD2 and Sirt3 Control Osteoclastogenesis by Regulating Mitochondrial ROS.

Reactive oxygen species (ROS) are an indispensable element of cellular signal transduction in various cell types, including bone cells. In particular, osteoclasts (OCs), cells specialized for bone resorption, utilize ROS as second messengers during receptor activator of NF-κB ligand (RANKL)-induced differentiation and activation. In addition, because of the high energy demands of bone-resorbing activity, OCs contain large amounts of mitochondria, the source of the majority of total ROS. In this study, we focused on the regulation of ROS generated from mitochondria during osteoclastogenesis. We observed that the level of mitochondrial superoxide dismutase 2 (SOD2), an enzyme responsible for reducing superoxide radicals in mitochondria, was increased by RANKL. siRNA-mediated knockdown (KD) of SOD2 increased ROS levels and enhanced OC differentiation. Conversely, overexpression of SOD2 reduced osteoclastogenesis by decreasing ROS levels. Moreover, we found that NAD-dependent deacetylase sirtuin 3 (Sirt3), an activator of SOD2 in mitochondria, was induced by RANKL. Sirt3-targeted siRNA decreased SOD2 activity by reducing deacetylation of lysine 68 of SOD2, leading to increased osteoclastogenesis. Furthermore, in vivo KD of SOD2 or Sirt3 in ICR mouse calvariae decreased bone volume and increased OC surface, supporting the results of in vitro experiments. Taken together, our findings demonstrate for the first time to our knowledge that the regulation of mitochondrial ROS by SOD2 and Sirt3 plays an important role in fine-tuning the OC differentiation program. © 2016 American Society for Bone and Mineral Research.

Diffusible Factors Secreted by Glioblastoma and Medulloblastoma Cells Induce Oxidative Stress in Bystander Neural Stem Progenitors.

Harmful effects that alter the homeostasis of neural stem or progenitor cells (NSPs) can affect regenerative processes in the central nervous system. We investigated the effect of soluble factors secreted by control or 137Cs-γ-irradiated glioblastoma or medulloblastoma cells on redox-modulated endpoints in recipient human NSPs. Growth medium harvested from the nonirradiated brain tumor cells, following 24 h of growth, induced prominent oxidative stress in recipient NSPs as judged by overall increases in mitochondrial superoxide radical levels (p < .001), activation of c-jun N-terminal kinase, and decrease in the active form of FoxO3a. The induced oxidative stress was associated with phosphorylation of p53 on serine 15, a marker of DNA damage, induction of the cyclin-cyclin dependent kinase inhibitors p21Waf1 and p27Kip1, and perturbations in cell cycle progression (p < .001). These changes were also associated with increased apoptosis as determined by enhanced annexin V staining (p < .001) and caspase 8 activation (p < .05) and altered expression of critical regulators of self-renewal, proliferation, and differentiation. Exposure of the tumor cells to radiation only slightly altered the induced oxidative changes in the bystander NSPs, except for medium from irradiated medulloblastoma cells that was more potent at inducing apoptosis in the NSPs than medium from nonirradiated cells (p < .001). The elucidation of such stressful bystander effects provides avenues to understand the biochemical events underlying the development or exacerbation of degenerative outcomes associated with brain cancers. It is also relevant to tissue culture protocols whereby growth medium conditioned by tumor cells is often used to support the growth of stem cells.

Lipotoxic brain microvascular injury is mediated by activating transcription factor 3-dependent inflammatory and oxidative stress pathways

Dysfunction of the cerebrovasculature plays an important role in vascular cognitive impairment (VCI). Lipotoxic injury of the systemic endothelium in response to hydrolyzed triglyceride-rich lipoproteins (TGRLs; TGRL lipolysis products) or a high-fat Western diet (WD) suggests similar mechanisms may be present in brain microvascular endothelium. We investigated the hypothesis that TGRL lipolysis products cause lipotoxic injury to brain microvascular endothelium by generating increased mitochondrial superoxide radical generation, upregulation of activating transcription factor 3 (ATF3)-dependent inflammatory pathways, and activation of cellular oxidative stress and apoptotic pathways. Human brain microvascular endothelial cells were treated with human TGRL lipolysis products that induced intracellular lipid droplet formation, mitochondrial superoxide generation, ATF3-dependent transcription of proinflammatory, stress response, and oxidative stress genes, as well as activation of proapoptotic cascades. Male apoE knockout mice were fed a high-fat/high-cholesterol WD for 2 months, and brain microvessels were isolated by laser capture microdissection. ATF3 gene transcription was elevated 8-fold in the hippocampus and cerebellar brain region of the WD-fed animals compared with chow-fed control animals. The microvascular injury phenotypes observed in vitro and in vivo were similar. ATF3 plays an important role in mediating brain microvascular responses to acute and chronic lipotoxic injury and may be an important preventative and therapeutic target for endothelial dysfunction in VCI.