Overexpression Of Manganese Superoxide Dismutase Research Articles

Overexpression of manganese superoxide dismutase mitigates ACL injury-induced muscle atrophy, weakness and oxidative damage

Oxidative stress has been implicated in the etiology of skeletal muscle weakness following joint injury. We investigated longitudinal patient muscle samples following knee injury (anterior cruciate ligament tear). Following injury, transcriptomic analysis revealed downregulation of mitochondrial metabolism-related gene networks, which were supported by reduced mitochondrial respiratory flux rates. Additionally, enrichment of reactive oxygen species (ROS)-related pathways were upregulated in muscle following knee injury, and further investigation unveiled marked oxidative damage in a progressive manner following injury and surgical reconstruction. We then investigated whether antioxidant protection is effective in preventing muscle atrophy and weakness after knee injury in mice that overexpress Mn-superoxide dismutase (MnSOD+/−). MnSOD+/− mice showed attenuated oxidative damage, atrophy, and muscle weakness compared to wild type littermate controls following ACL transection surgery. Taken together, our results indicate that ROS-related damage is a causative mechanism of muscle dysfunction after knee injury, and that mitochondrial antioxidant protection may hold promise as a therapeutic target to prevent weakness and development of disability.

Loganic acid regulates the transition between epithelial and mesenchymal-like phenotypes by alleviating MnSOD expression in hepatocellular carcinoma cells

AimsCancer metastasis is the major cause of cancer-related deaths. There are few prior studies reported on molecules targeting C-X-C chemokine receptor (CXCR) family and manganese superoxide dismutase (MnSOD). CXCRs are known to involve in angiogenesis, metastasis, cell survival and MnSOD is reported to be related in Epithelial–mesenchymal transition (EMT). Main methodsCell viability and cell proliferation were measured by MTT and BrdU assay. Protein expression level of CXCR4/7, MMP-2/9, MnSOD, and EMT markers were evaluated by Western blot analysis. mRNA levels of Snail and Occludin were analyzed by Real-time RT-qPCR. Expression of EMT markers in cells was observed by immunocytochemistry. Cell invasion and migrations were evaluated by wound healing assay and boyden chamber assay. Key findingsWe noticed that LGA abolished proliferation, invasive ability, and cellular migration. LGA down-regulated the protein levels of mesenchymal markers such as Twist, Snail, Fibronectin, and Vimentin in CXCL12-treated HCC cells. It also suppressed the gelatinolytic activity of MMP-9/2. The amplification of MnSOD increased EMT-like phenotypes but with LGA treatment, these phenotypes were markedly attenuated. The overexpression of MnSOD increased the ROS levels significantly but ROS levels were decreased upon exposure to LGA and deletion of MnSOD suppressed the levels of various mesenchymal proteins. SignificanceLGA could function as a novel anti-metastatic agent by suppressing metastasis and EMT process via attenuation of MnSOD expression in hepatocellular carcinoma cells.

Potential function of loliolide as a novel blocker of epithelial-mesenchymal transition in colorectal and breast cancer cells

Loliolide (LL), a naturally occurring monoterpenoid lactone isolated from Vicia tenuifolia Roth, can exhibit numerous pharmacological effects such as those related to anti-Parkinson, anti-oxidant, anti-cholinesterase, and anti-depressant. Epithelial-mesenchymal transition (EMT) plays a pivotal role in regulating tumor metastasis. CXCR4 and CXCR7 are G-protein-coupled receptors (GPRs), which can be stimulated by CXCL12. CXCL12/CXCR4/CXCXR7 axis can cause activation of multiple pathways including MAPKs, JAK/STAT pathway, and manganese superoxide dismutase (MnSOD) signaling. These events can initiate EMT process and induce cell invasion and migration. Here, we investigated whether LL can modulate the CXCR4 and CXCR7 and EMT process in colon cancer and breast cancer cells. We found that LL suppressed levels of CXCR4 and CXCR7, and exerted an inhibitory effect on these chemokines even after stimulation by CXCL12. LL suppressed expression of MnSOD and mesenchymal markers, whereas induced epithelial markers. In addition, LL significantly attenuated cellular invasion, migration, and metastasis. We noted that LL inhibited CXCR4/7 and EMT process even after stimulation of CXCL12 and MnSOD overexpression. Therefore, in this study, we provide evidences that targeting CXCR4/7 and MnSOD could inhibit the invasion, migration, and metastasis of cancer cells as well as negatively regulate the EMT process. Overall, our study suggested that LL might act as a potent suppressor of EMT process against colon and breast cancer cells.

Angiopoietin-Like Protein 4 Is Involved in Manganese Superoxide Dismutase-Mediated Suppression of Breast Cancer Cell Growth.

This study aims to understand the molecular basis of manganese superoxide dismutase (MnSOD) impacts on breast cancer cell growth. Modulation of the level of MnSOD by genetic engineering led significant changes in the expression of angiopoietin-like protein 4 (ANGPTL4) and activity of peroxisome proliferator-activated receptor α (PPARα) in MCF7 cells. PPARα agonist increased ANGPTL4 expression inhibited by MnSOD. Proliferation of MCF7 cells was inhibited by MnSOD, however, ANGPTL4 transduction into MCF7 cells with MnSOD overexpression significantly stimulated cell proliferation. MnSOD induced G0/G1 cell cycle arrest, nevertheless, ANGPTL4 transduction significantly reduced the percentage of cells in G0/G1 phase overexpressing MnSOD. In conclusion, MnSOD suppressed the expression of ANGPTL4 in breast cancer cells via the PPARα signaling pathway, and ANGPTL4 was involved in MnSOD-mediated proliferation inhibition and cell cycle arrest.

Daidzin targets epithelial-to-mesenchymal transition process by attenuating manganese superoxide dismutase expression and PI3K/Akt/mTOR activation in tumor cells

AimsEpithelial–mesenchymal transition (EMT) is a process during which epithelial cells lose their polarity and gain invasive properties to transform into mesenchymal cells. A few recent studies have reported that manganese superoxide dismutase (MnSOD) can effectively modulate EMT phenotype by influencing cellular redox environment via altering the intracellular ratio between O2− and H2O2. Daidzin (DDZ), a naturally occurring isoflavone isolated from Pueraria lobate (Fabaceae), has numerous pharmacologic effects including anti-cholesterol, anti-angiocardiopathy, anti-cancer. However, the potential inhibitory impact of DDZ on cancer metastasis and specifically on the EMT process has not been evaluated. We aimed to evaluate the possible relationship between MnSOD and EMT as well as influence of DDZ on these two processes in colon and prostate carcinoma cells. Main methodsCell viability was measured by MTT and real time cell analysis (RTCA) assay. Protein expression level of EMT markers and Akt/mTOR/PI3K signaling pathway were evaluated by Western blot analysis. Expression of EMT markers in cells was observed by immunocytochemistry. Cell invasion and migrations were evaluated by wound healing assay and Boyden chamber assay. Key findingsDDZ can block EMT accompanied with down-regulation of MnSOD, fibronectin, vimentin, MMP-9, MMP-2, N-cadherin, twist, and Snail, and up-regulation of occludin and E-cadherin in both unstimulated and TGFβ-induced cells. In addition, DDZ exposure also attenuated cell proliferation, invasion, and metastasis by reversing the EMT process in SNU-C2A, DU145, and PC-3 cells. DDZ treatment also modulated activation of PI3K/Akt/mTOR signaling cascades in DU145 cells. Moreover, an overexpression of MnSOD or silencing of MnSOD expression modulated EMT-related proteins, PI3K/Akt/mTOR activation and invasive activity. SignificanceThis is first finding on the DDZ in regulating MnSOD and EMT process by targeting PI3K/Akt/mTOR pathway in both colorectal and prostate cancer cell lines. Our data indicated that DDZ might act as a potent suppressor of EMT by affecting MnSOD expression in tumor cells.

A role for Snail-MnSOD axis in regulating epithelial-to-mesenchymal transition markers expression in RPE cells

Age-related macular degeneration (AMD) is a common cause of vision loss. The epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells, accompanied by oxidative damage, plays a crucial role in AMD. It is well known that manganese superoxide dismutase (MnSOD) encoded by SOD2 is a critical molecule in fighting against oxidative stress, and Snail encoded by SNAI1 is the essential transcription factor for EMT. However, the effect of MnSOD on EMT and the underlying mechanism in RPE cells remains unknown. In this study, we found that MnSOD knockdown triggered the EMT by upregulating Snail, while MnSOD overexpression reversed EMT even with TGFβ treatment in RPE cells, and the anti-oxidative stress activity of MnSOD mediated this observation. In addition, Snail depletion increased both expression and activity of MnSOD while Snail overexpression decreased MnSOD expression and activity, and Dual-luciferase reporter and ChIP assays showed that Snail directly bound to E-box (CACCTG) in the SOD2 promoter. Moreover, MnSOD over-expression and Snail interference co-treatment strengthened the anti-oxidation and EMT reversing. Therefore, our findings demonstrate that MnSOD prevents EMT of RPE cells in AMD through inhibiting oxidative injury to RPE. Moreover, a critical EMT transcription factor, Snail, functions as a new negative transcriptional factor of SOD2. Herein, the Snail-MnSOD axis forms a mutual loop in the development of AMD, which may be a novel systemic treatment target for preventing AMD.

Overexpression of MnSOD Protects against Cold Storage-Induced Mitochondrial Injury but Not against OMA1-Dependent OPA1 Proteolytic Processing in Rat Renal Proximal Tubular Cells.

Kidneys from deceased donors undergo cold storage (CS) preservation before transplantation. Although CS is a clinical necessity for extending organ quality preservation, CS causes mitochondrial and renal injury. Specifically, many studies, including our own, have shown that the triggering event of CS-induced renal injury is mitochondrial reactive oxygen species (mROS). Here, we explored the role of OMA1-depedent OPA1 proteolytic processing in rat kidney proximal tubular epithelial (NRK) cells in an in vitro model of renal CS (18 h), followed by rewarming (6 h) (CS + RW). The involvement of mROS was evaluated by stably overexpressing manganese superoxide dismutase (MnSOD), an essential mitochondrial antioxidant enzyme, in NRK cells. Western blots detected rapid OPA1 proteolytic processing and a decrease in ATP-dependent cell viability in NRK cells subjected to CS + RW compared to control cells. Small interfering RNA (siRNA) knockdown of OMA1 reduced proteolytic processing of OPA1, suggesting that OMA1 is responsible for OPA1 proteolytic processing during CS + RW-induced renal injury. Overexpression of MnSOD during CS + RW reduced cell death, mitochondrial respiratory dysfunction, and ATP-dependent cell viability, but it did not prevent OMA1-dependent OPA1 processing. These data show for the first time that OMA1 is responsible for proteolytically cleaving OPA1 in a redox-independent manner during renal cell CS.

Gene Therapy for Systemic or Organ Specific Delivery of Manganese Superoxide Dismutase.

Manganese superoxide dismutase (MnSOD) is a dominant component of the antioxidant defense system in mammalian cells. Since ionizing irradiation induces profound oxidative stress, it was logical to test the effect of overexpression of MnSOD on radioresistance. This task was accomplished by introduction of a transgene for MnSOD into cells in vitro and into organs in vivo, and both paradigms showed clear radioresistance following overexpression. During the course of development and clinical application of using MnSOD as a radioprotector, several prominent observations were made by Larry Oberley, Joel Greenberger, and Michael Epperly which include (1) mitochondrial localization of either manganese superoxide dismutase or copper/zinc SOD was required to provide optimal radiation protection; (2) the time required for optimal expression was 12–18 h, and while acceptable for radiation protection, the time delay was impractical for radiation mitigation; (3) significant increases in intracellular elevation of MnSOD activity were required for effective radioprotection. Lessons learned during the development of MnSOD gene therapy have provided a strategy for delivery of small molecule SOD mimics, which are faster acting and have shown the potential for both radiation protection and mitigation. The purpose of this review is to summarize the current status of using MnSOD-PL and SOD mimetics as radioprotectors and radiomitigators.

Isovitexin Suppresses Stemness of Lung Cancer Stem-Like Cells through Blockage of MnSOD/CaMKII/AMPK Signaling and Glycolysis Inhibition.

Background Manganese superoxide dismutase (MnSOD) has been reported to promote stemness of lung cancer stem-like cells (LCSLCs) which had higher glycolytic rates compared with non-CSLCs. Isovitexin exhibited an inhibitory effect on the stemness of hepatocellular carcinoma cells. However, whether isovitexin could inhibit the promotion of stemness of LCSLCs mediated by MnSOD through glycolysis remains unclear. Objective Our study was aimed at investigating whether isovitexin inhibits lung cancer stem-like cells (LCSLCs) through MnSOD signaling blockage and glycolysis suppression. Methods Sphere formation and soft agar assays were conducted to determine self-renewal ability. The migration and invasion of LCSLCs were determined by wound healing and transwell assay. The glycolytic activity was assessed by determination of L-lactate metabolism rate. The influences of isovitexin on MnSOD, CaMKII, and AMPK activations as well as the metabolic shift to glycolysis were determined by manipulating MnSOD expression. Results It was found that MnSOD and glycolysis enhanced simultaneously in LCSLCs compared with parental H460 cells. Overexpression of MnSOD activated CaMKII/AMPK signaling and glycolysis in LCSLCs with increased self-renewal, migration, invasion, and expression of stemness-associated markers in vitro and elevated carcinogenicity in vivo. Knockdown of MnSOD induced an inverse effect in LCSLCs. Isovitexin blocked MnSOD/CaMKII/AMPK signaling axis and suppressed glycolysis in LCSLCs, resulting in inhibition of stemness features in LCSLCs. The knockdown of MnSOD significantly augmented isovitexin-associated inhibition of CaMKII/AMPK signaling, glycolysis, and stemness in LCSLCs. However, the overexpression of MnSOD could attenuate the inhibition of isovitexin on LCSLCs. Importantly, isovitexin notably suppressed tumor growth in nude mice bearing LCSLCs by downregulation of MnSOD expression. Conclusion MnSOD promotion of stemness of LCSLCs derived from H460 cell line is involved in the activation of the CaMKII/AMPK pathway and induction of glycolysis. Isovitexin-associated inhibition of stemness in LCSLCs is partly dependent on blockage of the MnSOD/CaMKII/AMPK signaling axis and glycolysis suppression.

Trace element levels in pleural effusions.

Trace element levels in pleural effusions.

The state of antioxidant defense system in young persons with gastroesophageal reflux disease and autoimmune thyrioditis

Aim of research was assessment of the levels of antioxidant biomarkers associated with mitochondrial function in young patients with gastroesophageal reflux disease (GERD) and autoimmune thyroiditis (AIT). This study included 165 patients of them 120 patients with GERD and AIT - the main group, 45 patients with isolated GERD - the comparison group. The examined contingent was presented by students aged 18 to 25 years. The control group consisted of 20 healthy individuals of corresponding gender, age and social status (students). Total antioxidant activity (TAS-TAC) was determined in blood serum of study persons with enzyme immunoassays (ELISA, Elabscience, USA), levels of manganese superoxide dismutase (MnSOD) – with enzyme immunoassays (ELISA, Elabscience, USA) and Klotho protein - with enzyme immunoassays (ELISA, Elabscience, USA). Statistical data processing by the Statistica Basic Academic 13 for Windows En local was made. In examined patients with esophageal and thyroid pathologies the decline of total antioxidant activity has been revealed. The MnSOD level in patients with comorbidity of GERD and AIT and isolated GERD was significantly higher as compare to control group. Significant increasing of Klotho protein in serum of young patients was observed. We assessed imbalance between decline of extracellular antioxidants and activation of mitochondrial antioxidants which is more pronounced in combination of diseases. The increase of biomarkers of mitohondrial antioxidant defense system with non-specific citoprotection mechanism in patients with GERD provides the basis to consider MnSOD and Klotho protein as prognostic indicator of clinical outcome of disease in young age. Under combination of GERD and AIT the tendency to overexpression of MnSOD and depression of total antioxidant activity has been revealed, this may cause the deterioration of mitochondrial function.

Genistein inhibits lung cancer cell stem-like characteristics by modulating MnSOD and FoxM1 expression.

Manganese superoxide dismutase (MnSOD) promotes invasive and migratory activities by upregulating Forkhead box protein M1 (FoxM1) expression. The present study investigated whether modulation of MnSOD and FoxM1 expression was responsible for the antitumor effects of genistein on cancer stem-like cells (CSLCs) derived from non-small cell lung cancer cells (NSCLCs). Spheroids prepared from H460 or A549 cells were defined as lung cancer stem-like cells (LCSLCs) and were treated with genistein. The Cell Counting Kit-8 assay was performed to assess human lung fibroblast IMR-90 cell proliferation, as well as NSCLC H460 and A549 cell proliferation following treatment with genistein. MnSOD, FoxM1, cluster of differentiation (CD)133, CD44, BMI1 proto-oncogene, polycomb ring finger (Bmi1) and Nanog homeobox (Nanog) protein expression levels were examined via western blotting. The sphere formation assay was conducted to evaluate LCSLC self-renewal potential, and LSCLC migratory and invasive activities were analyzed using the wound healing and Transwell invasion assays, respectively. Knockdown and overexpression of MnSOD and FOXM1 via short hairpin-RNA or cDNA transfection were performed. The results indicated that genistein (80 and 100 µM) suppressed H460 and A549 cell viability compared with IMR-90 cells. Sub-cytotoxic concentrations of genistein (20 and 40 µM) inhibited sphere formation activity and decreased the protein expression levels of CD133, CD44, Bmi1 and Nanog in LCSLCs compared with the control group. Genistein also suppressed the migratory and invasive activities of LCSLCs compared with the control group. MnSOD and FoxM1 overexpression antagonized the effects of genistein (40 µM), whereas MnSOD and FoxM1 knockdown enhanced the inhibitory effects of genistein (20 µM) on CSLC characteristics of LCSLCs. Overall, the results suggested that genistein suppressed lung cancer cell CSLC characteristics by modulating MnSOD and FoxM1 expression levels.

Modulation of MnSOD and FoxM1 Is Involved in Invasion and EMT Suppression by Isovitexin in Hepatocellular Carcinoma Cells.

BackgroundManganese superoxide dismutase (MnSOD) induces FoxM1 expression, subsequently contributing to migration in several cancer cells. Isovitexin (ISOV) was recently found to downregulate MnSOD and FoxM1, decreasing stemness in hepatocellular carcinoma (HCC) stem-like cells (HCSLCs). The current study aimed to determine whether inhibition of migration, invasion and EMT in HCSLCs by ISOV results from MnSOD/FoxM1 signaling blockade and subsequent Twist1, Slug, ZEB1 and MMP-2 downregulation.Materials and MethodsWe examined the migratory and invasive capabilities and EMT phenotype in HCC cells and their HCSLCs, respectively, by wound-healing assay, transwell invasion assay and Western blot after treatment with non-cytotoxic concentrations of ISOV, and explored the mechanism by which ISOV affects migration, invasion and EMT by MnSOD or FoxM1 knockdown and/or overexpression in HCSLCs or HCC cells.ResultsThe results showed that ISOV not only downregulated MnSOD and FoxM1 but also suppressed the migratory and invasive capabilities and reversed the EMT phenotype in HCSLCs, which was reflected by elevated E-cadherin protein amounts, and reduced N-cadherin, Twist1, Slug, ZEB1 and MMP-2 protein levels. The suppressive effects of ISOV on the migratory and invasive capabilities and EMT phenotype could be potentiated by MnSOD or FoxM1 knockdown in HCSLCs, and attenuated by MnSOD or FoxM1 overexpression in HCC cells. Importantly, FoxM1 overexpression reversed MnSOD knockdown combined with ISOV suppression on the migratory and invasive capabilities and EMT phenotype in HCSLCs, while having little effects on MnSOD expression.ConclusionCollectively, the above findings demonstrated that ISOV suppresses migration, invasion and EMT in HCSLCs by blocking MnSOD/FoxM1 signaling subsequently inhibiting the expression of EMT-related transcription factors and MMP-2.

Withaferin A inhibits expression of ataxia telangiectasia and Rad3-related kinase and enhances sensitivity of human breast cancer cells to cisplatin.

Ataxia telangiectasia and Rad3-related (ATR) is a serine/threonine-specific kinase that plays an important role in the maintenance of genomic integrity. In this study, we investigated the role of ATR in cell-cycle arrest by withaferin A (WA), a cancer preventative steroidal lactone derived from Withania somnifera plant abundant in India and surrounding countries. The WA treatment decreased the viability of MCF-7, MDA-MB-231, and SUM159 cells. Exposure of breast cancer cells to WA also resulted in suppression of protein level as well as phosphorylation of ATR and its downstream effector kinase (checkpoint kinase 1; CHK1). Both transcriptional and posttranscriptional mechanisms were involved in the WA-mediated downregulation of ATR protein. Downregulation of ATR protein expression resulting from WA exposure was not attenuated by overexpression of manganese superoxide dismutase. In contrast, the overexpression of CHK1 attenuated WA-mediated G2 /M arrest and augmented S10 phosphorylation of histone H3, a marker of mitotic arrest. The protein level of ATR was lowered by about 50% in breast tumors of WA-treated mouse mammary tumor virus-neu mice when compared with vehicle-treated controls but the difference was not significant due to small sample size. WA treatment sensitized MDA-MB-231 and SUM159 cells to growth inhibition and apoptosis induction by cisplatin. Cisplatin treatment resulted in increased autophosphorylation of ATR (T1989) and CHK1 (S345) phosphorylation that was markedly suppressed in the presence of WA. These results indicate that WA is an inhibitor of ATR in human breast cancer cells.

Doxorubicin in myocardial lysophosphatidic acid signaling

Generation of reactive oxygen species has been implicated in the toxicity of nearly 50% of the current FDA approved cancer therapeutic drugs. Mitochondrial respiration is a major source of reactive oxygen species production and that the presence of the mitochondrial antioxidant enzyme, manganese superoxide dismutase, is essential for the survival of all aerobic life. Our studies of animals using the prototype redox‐active anticancer drug, doxorubicin, indicate that overexpression of manganese superoxide dismutase protects the heart against doxorubicin‐induced cardiac injury and reduction of manganese superoxide dismutase by heterozygous knockout mice increases cardiac injury after exposure to doxorubicin. While the bioactive glycerophospholipid lysophosphatidic acid rises by seven‐fold over control subjects, suggesting lysophosphatidic acid may contribute to the doxorubicin‐induced cardiac toxicity. Lysophosphatidic acid production involves hydrolysis of lysophosphatidylcholine by the secreted enzyme autotaxin, whereas lipid phosphate phosphatase‐3 (LPP3) catalyzes lysophosphatidic acid dephosphorylation to generate lipid products that are not receptor active. In this application, we present the first evidence that doxorubicin‐induced cardiac injury enhances myocardial autotaxin levels and decreases myocardial LPP3 expression, and this is associated with increased serum lysophosphatidic acid levels. Reactive oxygen species production arises as a burst of superoxide from mitochondria following doxorubicin‐induced cardiac injury. The redox‐sensitive transcription factor NFAT (a nuclear factor of activated T‐cells) has been shown to bind to the autotaxin promoter and induce its expression. Similarly, oxidant stress may deplete LPP3 levels in the context of cardiac injury through reduced LPP3 expression or enhanced LPP3 degradation. Thus, we hypothesize that doxorubicin‐induced cardiac injury alters autotaxin and LPP3 expression through mitochondrial superoxide production to drive lysophosphatidic acid signaling and cardiomyocyte dysfunction. The following interrelated specific aims are designed to provide step‐wise and in‐depth studies in vitro, in vivo, and in experimental therapeutics settings. Specific aim 1 will assess the role of myocardial superoxide production in autotaxin expression and lysophosphatidic acid production in doxorubicin‐induced cardiac injury. Specific aim 2 will determine the role of mitochondrial superoxide production in LPP3 depletion and lysophosphatidic acid regulation in doxorubicin‐induced cardiac injury. We could identify whether modulation of cellular versus mitochondrial antioxidant status confers a differential protective effect in doxorubicin‐induced cardiac injury models.Support or Funding InformationAAA‐ FGAP Fellow grant funded by the American Association of Anatomists.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

P20 - 2018 SFRBM LIFETIME ACHIEVEMENT AWARD LECTURE MnSOD at the Crossroads of Life and Death: We've Only Just Begun

Manganese superoxide dismutase (MnSOD), a mitochondria-localized primary antioxidant enzyme, is essenstial for the survival of all areobic life. Decades of research on MnSOD have extended our understanding of the role of superoxide radicals and their associated reactive oxygen derived species (ROS) in mitochondria well beyond the once prevalent view of their being the toxic byproducts of cellular energy production to include their being important in cell signaling. Our studies on the regulation of MnSOD expression during cancer development and progression have contributed to the knowledge that cancer cells are usually under higher oxidative stress conditions than normal cells are and that different redox potentials can trigger different responses. A low redox potential triggers survival pathways, whereas a high redox potential triggers cell death mediators. This results in a therapeutic window in which redox-active antioxidants can manipulate intracellular redox stress levels that induce normal cells to produce prosurvival responses while cancer cells produce, on the other hand, lethal responses. These antioxidant/prooxidant properties are best represented by MnSOD, which cycles active site manganese through varying states of oxidation. Thus, overexpression of MnSOD in normal tissues protects tissues against oxidative stress-induced injury, which includes protection of cardiac function from the influences of the anticancer drug doxorubicin, lung fibrosis from ionizing radiation, and brain damage from beta amyloid. In contrast, overexpression of MnSOD in malignant cells results in reduced cancer cell survival. Identification of these paradoxical properties of MnSOD has led to the development of potential therapeutics that are capable of acting as redox active oxidants or of mimicking endogenous enzyme functions. This presentation introduces the prospect of a novel avenue to therapeutic control using redox active antioxidants. It also introduces a novel redox-based liquid biopsy approach that uses oxidatively modified extracellular vesicles (EVs) as mediators of therapy-induced normal tissue injury and prognosis markers of cancer responses to therapy. Our expectation is that the results from extensive proof-of-concept studies, coupled with an in-depth understanding of the mechanisms mediating this exciting concept, will lead to further developments of novel redox- based therapeutic agents for clinical applications.

Abstract 399: Inhibition of NFE2L2-ARE Pathway by mtROS Contributes to Development of Cardiomyopathy and Left Ventricular Dysfunction in Chagas Disease

Aims: We investigated the effects of mitochondrial reactive oxygen species (mtROS) on nuclear factor (erythroid 2)-like 2 (NFE2L2) transcription factor activity during Trypanosoma cruzi (Tc) infection and determined whether enhancing the mtROS scavenging capacity preserved the heart function in Chagas disease. Results: C57BL/6 wild type (WT, female) mice infected with Tc exhibited myocardial loss of mitochondrial membrane potential, complex II (CII)-driven coupled respiration, and ninefold increase in mtROS production. In vitro and in vivo studies showed that Tc infection resulted in an ROS-dependent decline in the expression, nuclear translocation, antioxidant response element (ARE) binding, and activity of NFE2L2, and 35-99% decline in antioxidants’ (gamma-glutamyl cysteine synthase [γGCS], heme oxygenase-1 [HO1], glutamate-cysteine ligase modifier subunit [GCLM], thioredoxin (Trx), glutathione S transferase [GST], and NAD(P)H dehydrogenase, quinone 1 [NQO1]) expression. An increase in myocardial and mitochondrial oxidative adducts, myocardial interventricular septum thickness, and left ventricle (LV) mass, a decline in LV posterior wall thickness, and disproportionate synthesis of collagens (COLI/COLIII), αSMA, and SM22α were noted in WT.Tc mice. Overexpression of manganese superoxide dismutase (MnSOD) in cultured cells (HeLa or cardiomyocytes) and MnSODtg mice preserved the NFE2L2 transcriptional activity and antioxidant/oxidant balance, and cardiac oxidative and fibrotic pathology were significantly decreased in MnSODtg.Tc mice. Importantly, echocardiography finding of a decline in LV systolic (stroke volume, cardiac output, ejection fraction) and diastolic (early/late peak filling ratio, myocardial performance index) function in WT.Tc mice was abolished in MnSODtg.Tc mice. Innovation and Conclusion: The mtROS inhibition of NFE2L2/ARE pathway constitutes a key mechanism in signaling the fibrotic gene expression and evolution of chronic cardiomyopathy. Preserving the NFE2L2 activity arrested the mitochondrial and cardiac oxidative stress, cardiac fibrosis, and heart failure in Chagas disease.

Exercise characteristics in manganese superoxide dismutase overexpressing mice

Oxidative stress, the production of free radicals and reactive oxygen species, can be a result of prolonged and high intensity exercise and may lead to skeletal muscle fatigue and dysfunction, and decreased exercise performance. Manganese superoxide dismutase (MnSOD), a naturally existing mitochondrial antioxidant enzyme, provides the first line of defense against oxidative stress caused by superoxide anions. Transgenic mice overexpressing MnSOD have been studied within the context of disease pathology, but skeletal muscle function and exercise capacity in these mice have not been characterized. Therefore, the objective of this study was to investigate if grip strength, maximal exercise capacity, and exercise endurance differ between transgenic mice that overexpress MnSOD in the mitochondrial matrix (SOD2) and their wild‐type (WT) littermates. Female SOD2 and WT mice (8–10 mo old) performed 3 consecutive forelimb grip strength tests, with 1‐min rest between trials. Next, these mice performed a maximal exercise capacity test, which consisted of a 10‐min warm up at 10–12.5 m•min−1 and 5–10° incline, followed by 2‐min stages of increasing treadmill speed by 3 m•min−1 every stage; treadmill remained at 10° incline and mice ran to exhaustion. Then, 48 hours after the completion of the maximal exercise capacity test, all mice performed an exercise endurance test, consisting of a 10‐min warm up at 10–12.5 m•min−1 and 5–10° incline, followed by a run to exhaustion at a constant treadmill speed of 22 m•min−1 and incline of 10°. Grip strength, maximal exercise capacity, and exercise endurance were not different between SOD2 and WT mice, respectively (p>0.05, all). These findings suggest that overexpression of MnSOD in the mitochondrial matrix does not alter grip strength, treadmill exercise capacity, or endurance in mice.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Upregulation of FoxM1 by MnSOD Overexpression Contributes to Cancer Stem-Like Cell Characteristics in the Lung Cancer H460 Cell Line.

Manganese superoxide dismutase promotes migration and invasion in lung cancer cells via upregulation of the transcription factor forkhead box M1. Here, we assessed whether upregulation of forkhead box M1 by manganese superoxide dismutase overexpression mediates the acquisition of cancer stem-like cell characteristics in non-small cell lung cancer H460 cells. The second-generation spheroids from H460 cells were used as lung cancer stem-like cells. The levels of manganese superoxide dismutase, forkhead box M1, stemness markers (CD133, CD44, and ALDH1), and transcription factors (Bmi1, Nanog, and Sox2) were analyzed by Western blot. Sphere formation in vitro and carcinogenicity of lung cancer stem-like cells were evaluated by spheroid formation assay and limited dilution xenograft assays. Knockdown or overexpression of manganese superoxide dismutase or/and forkhead box M1 by transduction with short hairpin RNA(shRNA) or complementary DNA were performed for mechanistic studies. We showed that manganese superoxide dismutase and forkhead box M1 amounts as well as the expression levels of stemness markers and transcription factors sphere formation in vitro, and carcinogenicity of lung cancer stem-like cells were higher than in monolayer cells. Lung cancer stem-like cells transduced with manganese superoxide dismutase shRNA or FoxM1 shRNA exhibited decreased sphere formation and lower amounts of stemness markers and transcription factors. Overexpression of manganese superoxide dismutase or FoxM1 in H460 cells resulted in elevated sphere formation rates and protein levels of stemness markers and transcription factors. Meanwhile, manganese superoxide dismutase knockdown or overexpression accordingly altered forkhead box M1 levels. However, forkhead box M1 knockdown or overexpression had no effect on manganese superoxide dismutase levels but inhibited or promoted lung cancer stem-like cell functions. Interestingly, forkhead box M1 overexpression alleviated the inhibitory effects of manganese superoxide dismutase knockdown in lung cancer stem-like cells. In a panel of non-small cell lung cancer cells, including H441, H1299, and H358 cells, compared to the respective monolayer counterparts, the expression levels of manganese superoxide dismutase and forkhead box M1 were elevated in the corresponding spheroids. These findings revealed the role of forkhead box M1 upregulation by manganese superoxide dismutase overexpression in maintaining lung cancer stem-like cell properties. Therefore, inhibition of forkhead box M1 upregulation by manganese superoxide dismutase overexpression may represent an effective therapeutic strategy for non-small cell lung cancer.

BRAF-Oncogene-Induced Senescence and the Role of Thyroid-Stimulating Hormone Signaling in the Progression of Papillary Thyroid Carcinoma.

Oncogene-induced senescence (OIS) explains the phenomenon of cellular senescence triggered by the action of oncogenes. It is a mechanism adopted by a cell to inhibit progression of benign tumors into malignancy, occurs in premalignant lesions, and is almost never present in malignant lesions. BRAF mutations occur in about 40-45% of all papillary thyroid carcinomas (PTCs) and of which 99.7% is the BRAFV600E mutation. A unique phenotype of the BRAFV600E mutation is the upregulation of the thyroid-stimulating hormone receptor (TSHR) on thyrocyte membranes. Despite the overexpression of the receptor, BRAFV600E cells undergo cell cycle arrest leading to OIS via a negative feedback signaling mechanism. A simultaneous increase in serum thyroid-stimulating hormone (TSH) in response to hypothyroidism (common in autoimmune diseases such as Hashimoto's thyroiditis) would cause senescent tumor cells to overcome OIS and proceed towards malignancy, hence showing the importance of TSH/TSHR signaling in the development of PTCs. Increase in TSH/TSHR signaling triggers an increase in levels of downstream enzymes such as manganese superoxide dismutase (MnSOD) and dual-specific phosphatase 6 (DUSP6) which eventually results in the production of oncogenic proteins such as c-Myc. Therefore, the detection of these genetic alterations as effective biomarkers for premalignant lesions of PTC is important in clinical settings and techniques such as polymerase chain reaction-mediated restriction fragment length polymorphism (PCR-RFLP) and real-time PCR can be used to detect the BRAFV600E point mutation and overexpression of TSHR, MnSOD, and DUSP6, respectively.