Mao

MnSOD upregulation sustains the Warburg effect via mitochondrial ROS and AMPK-dependent signalling in cancer

Manganese superoxide dismutase (MnSOD/SOD2) is a mitochondria-resident enzyme that governs the types of reactive oxygen species egressing from the organelle to affect cellular signaling. Here, we demonstrate that MnSOD upregulation in cancer cells establishes a steady flow of H2O2 originating from mitochondria that sustains AMP-activated kinase (AMPK) activation and the metabolic shift to glycolysis. Restricting MnSOD expression or inhibiting AMPK suppress the metabolic switch and dampens the viability of transformed cells indicating that the MnSOD/AMPK axis is critical in support cancer cell bioenergetics. Recapitulating in vitro findings, clinical and epidemiologic analyses of MnSOD expression and AMPK activation indicated that the MnSOD/AMPK pathway is most active in advanced stage and aggressive breast cancer subtypes. Taken together, our results indicate that MnSOD serves as a biomarker of cancer progression and acts as critical regulator of tumor cell metabolism.

Nitric oxide-dependent Src activation and resultant caveolin-1 phosphorylation promote eNOS/caveolin-1 binding and eNOS inhibition.

The mechanism of caveolin-1–dependent eNOS inactivation is not clear. These studies reveal that NO-mediated Src kinase activation and caveolin-1 phosphorylation promote eNOS binding and inactivation, that is, eNOS negative feedback regulation.

The peroxidase activity of mitochondrial superoxide dismutase

Manganese superoxide dismutase (MnSOD) is an integral mitochondrial protein known as a first-line antioxidant defense against superoxide radical anions produced as by-products of the electron transport chain. Recent studies have shaped the idea that by regulating the mitochondrial redox status and H(2)O(2) outflow, MnSOD acts as a fundamental regulator of cellular proliferation, metabolism, and apoptosis, thereby assuming roles that extend far beyond its proposed antioxidant functions. Accordingly, allelic variations of MnSOD that have been shown to augment levels of MnSOD in mitochondria result in a 10-fold increase in prostate cancer risk. In addition, epidemiologic studies indicate that reduced glutathione peroxidase activity along with increases in H(2)O(2) further increase cancer risk in the face of MnSOD overexpression. These facts led us to hypothesize that, like its Cu,ZnSOD counterpart, MnSOD may work as a peroxidase, utilizing H(2)O(2) to promote mitochondrial damage, a known cancer risk factor. Here we report that MnSOD indeed possesses peroxidase activity that manifests in mitochondria when the enzyme is overexpressed.

Nitrosation-dependent caveolin 1 phosphorylation, ubiquitination, and degradation and its association with idiopathic pulmonary arterial hypertension

In the present study, we tested the hypothesis that chronic inflammation and oxidative/nitrosative stress induce caveolin 1 (Cav-1) degradation, providing an underlying mechanism of endothelial cell activation/dysfunction and pulmonary vascular remodeling in patients with idiopathic pulmonary arterial hypertension (IPAH). We observed reduced Cav-1 protein despite increased Cav-1 messenger RNA expression and also endothelial nitric oxide synthase (eNOS) hyperphosphorylation in human pulmonary artery endothelial cells (PAECs) from patients with IPAH. In control human lung endothelial cell cultures, tumor necrosis factor α–induced nitric oxide (NO) production and S-nitrosation (SNO) of Cav-1 Cys-156 were associated with Src displacement and activation, Cav-1 Tyr-14 phosphorylation, and destabilization of Cav-1 oligomers within 5 minutes that could be blocked by eNOS or Src inhibition. Prolonged stimulation (72 hours) with NO donor DETANONOate reduced oligomerized and total Cav-1 levels by 40%–80%, similar to that observed in IPAH patient–derived PAECs. NO donor stimulation of endothelial cells for >72 hours, which was associated with sustained Src activation and Cav-1 phosphorylation, ubiquitination, and degradation, was blocked by NOS inhibitor L-NAME, Src inhibitor PP2, and proteosomal inhibitor MG132. Thus, chronic inflammation, sustained eNOS and Src signaling, and Cav-1 degradation may be important causal factors in the development of IPAH by promoting PAEC dysfunction/activation via sustained oxidative/nitrosative stress.

Endothelial barrier protection by local anesthetics: Ropivacaine and lidocaine block tumor necrosis factor-α-induced endothelial cell Src activation

Background:Pulmonary endothelial barrier dysfunction mediated in part by Src-kinase activation plays a crucial role in acute inflammatory disease. Proinflammatory cytokines, such as tumor necrosis factor-&agr; (TNF&agr;), activate Src via phosphatidylinositide 3-kinase/Akt-dependent nitric oxide generation, a process initiated by recruitment of phosphatidylinositide 3-kinase regulatory subunit p85 to TNF-receptor-1. Because amide-linked local anesthetics have well-established anti-inflammatory effects, the authors hypothesized that ropivacaine and lidocaine attenuate inflammatory Src signaling by disrupting the phosphatidylinositide 3-kinase–Akt–nitric oxide pathway, thus blocking Src-dependent neutrophil adhesion and endothelial hyperpermeability. Methods:Human lung microvascular endothelial cells, incubated with TNF&agr; in the absence or presence of clinically relevant concentrations of ropivacaine and lidocaine, were analyzed by Western blot, probing for phosphorylated/activated Src, endothelial nitric oxide synthase, Akt, intercellular adhesion molecule-1, and caveolin-1. The effect of ropivacaine on TNF&agr;-induced nitric oxide generation, co-immunoprecipitation of TNF-receptor-1 with p85, neutrophil adhesion, and endothelial barrier disruption were assessed. Results:Ropivacaine and lidocaine attenuated TNF&agr;-induced Src activation (half-maximal inhibitory concentration [IC50] = 8.611 × 10−10 M for ropivacaine; IC50 = 5.864 × 10−10 M for lidocaine) and endothelial nitric oxide synthase phosphorylation (IC50 = 7.572 × 10−10 M for ropivacaine; IC50 = 6.377 × 10−10 M for lidocaine). Akt activation (n = 7; P = 0.006) and stimulus-dependent binding of TNF-receptor-1 and p85 (n = 6; P = 0.043) were blocked by 1 nM of ropivacaine. TNF&agr;-induced neutrophil adhesion and disruption of endothelial monolayers via Src-dependent intercellular adhesion molecule-1- and caveolin-1-phosphorylation, respectively, were also attenuated. Conclusions:Ropivacaine and lidocaine effectively blocked inflammatory TNF&agr; signaling in endothelial cells by attenuating p85 recruitment to TNF-receptor-1. The resultant decrease in Akt, endothelial nitric oxide synthase, and Src phosphorylation reduced neutrophil adhesion and endothelial hyperpermeability. This novel anti-inflammatory “side-effect” of ropivacaine and lidocaine may provide therapeutic benefit in acute inflammatory disease.

Bioluminescent detection of peroxynitrite with a boronic acid-caged luciferin.

Peroxynitrite, a highly reactive biological oxidant, is formed under pathophysiologic conditions from the diffusion-limited reaction of nitric oxide and superoxide radical anion. Peroxynitrite has been implicated as the mediator of nitric oxide toxicity in many diseases and as an important signaling disrupting molecule (L. Liaudet et al., Front. Biosci.14, 4809-4814, 2009) [1]. Biosensors effective at capturing peroxynitrite in a specific and fast enough manner for detection, along with readouts compatible with in vivo studies, are lacking. Here we report that the boronic acid-based bioluminescent system PCL-1 (peroxy-caged luciferin-1), previously reported as a chemoselective sensor for hydrogen peroxide (G.C. Van de Bittner et al., Proc. Natl. Acad. Sci. USA107, 21316-21321, 2010) [2], reacts with peroxynitrite stoichiometrically with a rate constant of 9.8±0.3×10(5)M(-1)s(-1) and a bioluminescence detection limit of 16nM, compared to values of 1.2±0.3M(-1)s(-1) and 231nM for hydrogen peroxide. Further, we demonstrate bioluminescent detection of peroxynitrite in the presence of physiological competitors: carbon dioxide, glutathione, albumin, and catalase. We also demonstrate the utility of this method to assess peroxynitrite formation in mammalian cells by measuring peroxynitrite generated under normal culture conditions after stimulation of macrophages with bacterial endotoxin lipopolysaccharide. Thus, the PCL-1 method for measuring peroxynitrite generation shows superior selectivity over other oxidants under in vivo conditions.

Interaction of a Specific Population of Human Embryonic Stem Cell-Derived Progenitor Cells with CD11b Cells Ameliorates Sepsis-Induced Lung Inflammatory Injury

n n Human embryonic stem cells differentiated under mesoderm-inducing conditions have important therapeutic properties in sepsis-induced lung injury in mice. Single cell suspensions obtained from day 7 human embryoid bodies (d7EBs) injected i.v. 1 hour after cecal ligation and puncture significantly reduced lung inflammation and edema as well as production of tumor necrosis factor-α and interferon-γ in lungs compared with controls, whereas interleukin-10 production remained elevated. d7EB cell transplantation also reduced mortality to 50% from 90% in the control group. The protection was ascribed to d7EB cell interaction with lung resident CD11b+ cells, and was correlated with the ability of d7EB cells to reduce it also reduced production of proinflammatory cytokines by CD11+ cells, and to endothelial NO synthase–derived NO by d7EB cells, leading to inhibition of inducible macrophage-type NO synthase activation in CD11b+ cells. The protective progenitor cells were positive for the endothelial and hematopoietic lineage marker angiotensin converting enzyme (ACE). Only the ACE+ fraction modulated the proinflammatory profile of CD11b+ cells and reduced mortality in septic mice. In contrast to the nonprotective ACE-cell fraction, the ACE+ cell fraction also produced NO. These findings suggest that an ACE+ subset of human embryonic stem cell–derived progenitor cells has a highly specialized anti-inflammatory function that ameliorates sepsis-induced lung inflammation and reduces mortality.n n

Propofol increases morbidity and mortality in a rat model of sepsis

IntroductionSevere sepsis is associated with approximately 50% mortality and accounts for tremendous healthcare costs. Most patients require ventilatory support and propofol is commonly used to sedate mechanically ventilated patients. Volatile anesthetics have been shown to attenuate inflammation in a variety of different settings. We therefore hypothesized that volatile anesthetic agents may offer beneficial immunomodulatory effects during the course of long-term intra-abdominal sepsis in rats under continuous sedation and ventilation for up to 24 hours.MethodsSham operation or cecal ligation and puncture (CLP) was performed in adult male Wistar rats followed by mechanical ventilation. Animals were sedated for 24 hours with propofol (7 to 20 mg/kg/h), sevoflurane, desflurane or isoflurane (0.7 minimal alveolar concentration each).ResultsSeptic animals sedated with propofol showed a mean survival time of 12 hours, whereas >56% of all animals in the volatile groups survived 24 hours (P <0.001). After 18 hours, base excess in propofolu2009+u2009CLP animals (−20.6u2009±u20092.0) was lower than in the volatile groups (isofluraneu2009+u2009CLP: -11.7u2009±u20094.2, sevofluraneu2009+u2009CLP: -11.8u2009±u20093.5, desfluraneu2009+u2009CLP -14.2u2009±u20093.7; all P <0.03). Plasma endotoxin levels reached 2-fold higher levels in propofolu2009+u2009CLP compared to isofluraneu2009+u2009CLP animals at 12 hours (P <0.001). Also blood levels of inflammatory mediators (tumor necrosis factor-α, interleukin-1β, interleukin-10, CXCL-2, interferon-γ and high mobility group protein-1) were accentuated in propofolu2009+u2009CLP rats compared to the isofluraneu2009+u2009CLP group at the same time point (P <0.04).ConclusionsThis is the first study to assess prolonged effects of sepsis and long-term application of volatile sedatives compared to propofol on survival, cardiovascular, inflammatory and end organ parameters. Results indicate that volatile anesthetics dramatically improved survival and attenuate systemic inflammation as compared to propofol. The main mechanism responsible for adverse propofol effects could be an enhanced plasma endotoxin concentration, leading to profound hypotension, which was unresponsive to fluid resuscitation.

Nitroglycerin drives endothelial nitric oxide synthase activation via the phosphatidylinositol 3-kinase/protein kinase B pathway

Nitroglycerin (GTN) has been clinically used to treat angina pectoris and acute heart episodes for over 100 years. The effects of GTN have long been recognized and active research has contributed to the unraveling of numerous metabolic routes capable of converting GTN to the potent vasoactive messenger nitric oxide. Recently, the mechanism by which minute doses of GTN elicit robust pharmacological responses was revisited and eNOS activation was implicated as an important route mediating vasodilation induced by low GTN doses (1-50nM). Here, we demonstrate that at such concentrations the pharmacologic effects of nitroglycerin are largely dependent on the phosphatidylinositol 3-kinase, Akt/PKB, and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) signal transduction axis. Furthermore, we demonstrate that nitroglycerin-dependent accumulation of 3,4,5-InsP(3), probably because of inhibition of PTEN, is important for eNOS activation, conferring a mechanistic basis for GTN pharmacological action at pharmacologically relevant doses.

Natural Allelic Variations in Glutathione Peroxidase-1 Affect Its Subcellular Localization and Function

Glutathione peroxidase 1 (GPx-1) has been implicated in the etiology of several common diseases due to the association between specific allelic variations and cancer risk. The most common among these variations are the codon 198 polymorphism that results in either a leucine or proline and the number of alanine repeat codons in the coding sequence. The molecular and biologic consequences of these variations remain to be characterized. Toward achieving this goal, we have examined the cellular location of GPx-1 encoded by allelic variants by ectopically expressing these genes in MCF-7 human breast carcinoma cells that produce undetectable levels of GPx-1, thus achieving exclusive expression in the same cellular environment. A differential distribution between the cytoplasm and mitochondria was observed, with the allele expressing the leucine-198 polymorphism and 7 alanine repeats being more cytoplasmically located than the other alleles examined. To assess whether the distribution of GPx-1 between the cytoplasm and mitochondria had a biologic consequence, we engineered derivative GPx-1 proteins that were targeted to the mitochondria by the addition of a mitochondria targeting sequence and expressed these proteins in MCF-7 cells. These cells were examined for their response to oxidative stress, energy metabolism, and impact on cancer-associated signaling molecules. The results obtained indicated that both primary GPx-1 sequence and cellular location have a profound impact on cellular biology and offer feasible hypotheses about how expression of distinct GPx-1 alleles can affect cancer risk. Cancer Res; 74(18); 5118-26. ©2014 AACR.