Superoxide Mediates Direct Current Electric Field-Induced Directional Migration of Glioma Cells through the Activation of AKT and ERK

Direct current electric fields (DCEFs) can induce directional migration for many cell types through activation of intracellular signaling pathways. However, the mechanisms that bridge extracellular electrical stimulation with intracellular signaling remain largely unknown. In the current study, we found that a DCEF can induce the directional migration of U87, C6 and U251 glioma cells to the cathode and stimulate the production of hydrogen peroxide and superoxide. Subsequent studies demonstrated that the electrotaxis of glioma cells were abolished by the superoxide inhibitor N-acetyl-l-cysteine (NAC) or overexpression of mitochondrial superoxide dismutase (MnSOD), but was not affected by inhibition of hydrogen peroxide through the overexpression of catalase. Furthermore, we found that the presence of NAC, as well as the overexpression of MnSOD, could almost completely abolish the activation of Akt, extracellular-signal-regulated kinase (Erk)1/2, c-Jun N-terminal kinase (JNK), and p38, although only JNK and p38 were affected by overexpression of catalase. The presenting of specific inhibitors can decrease the activation of Erk1/2 or Akt as well as the directional migration of glioma cells. Collectively, our data demonstrate that superoxide may play a critical role in DCEF-induced directional migration of glioma cells through the regulation of Akt and Erk1/2 activation. This study provides novel evidence that the superoxide is at least one of the “bridges” coupling the extracellular electric stimulation to the intracellular signals during DCEF-mediated cell directional migration.     

Hydrogen peroxide contributes to the manganese superoxide dismutase promotion of migration and invasion in glioma cells

Manganese superoxide dismutase (MnSOD) is over-expressed in most brain tumours, and high MnSOD expression is associated with poor prognosis. The mechanisms still remain largely unknown. In the present study, the elevation of hydrogen peroxide (H(2)O(2)) level and the enhancement of glioma migration/invasion by over-expression of MnSOD were demonstrated. Subsequent studies showed that over-expression of MnSOD significantly increased the activation of mitogen-activated protein kinases (MAPKs) and phosphatidylinositol-3-kinases (PI3Ks), including AKTs, s6-ribosomal protein, ERKs and JNKs. Over-expression of MnSOD was also associated with elevations of matrix metalloproteinases-1(MMP-1) and MMP-9 protein. The promotion of migration/invasion, activation of PI3Ks and MAPKs and up-regulation of MMPs were inhibited by the general reactive oxygen species scavenger N-acetyl-l-cysteine (NAC), over-expression of the H(2)O(2)-detoxifying enzyme mitochondrial catalase (mCat) and specific inhibitors of AKTs or ERKs. Collectively, our study indicated that H(2)O(2) would contribute to the MnSOD-promoted migration/invasion in glioma cells through activation of AKTs and ERKs. This study provided new molecular insights into the understanding of glioma migration and invasion.     

Nitric oxide enhances the manganese superoxide dismutase-dependent suppression of proliferation in HT-1080 fibrosarcoma cells.

The overexpression of manganese superoxide dismutase (MnSOD), an enzyme that catalyzes the removal of superoxide (O2*-) from the mitochondria, has been shown to be closely associated with tumor regression in vivo and loss of the malignant phenotype in vitro. To investigate the mechanism by which MnSOD overexpression mediates this reversal, we have established 29 independent, clonal MnSOD-overexpressing HT-1080 fibrosarcoma cells. MnSOD activity is inversely correlated with cell proliferation in our cell lines. Incubating cells in 3% oxygen can prevent the inhibition of cellular proliferation mediated by MnSOD, suggesting that oxygen is a prerequisite component of the MnSOD-dependent proliferative inhibition. Confocal laser microscopy was used in combination with the oxidant-sensitive fluorescent dyes dihydrorhodamine-123, dihydroethidium, and 2',7'-dichlorodihydrofluorescein diacetate to determine the oxidizing capacity of the MnSOD-overexpressing cells. When compared with parental or control cell lines, there was a significant decrease in the rate of oxidation of the fluorophores in the MnSOD-overexpressing cell lines. Thus, an increase in the oxidizing capacity of the cells does not appear to mediate the inhibition of proliferation associated with MnSOD overexpression. Superoxide dismutase has also been shown to enhance the cytotoxic activity of NO* toward tumor cells. In this study, we have shown that MnSOD overexpression enhances the cytostatic action of the NO* donors, sodium nitroprusside, 3-morpholinosydnonomine, and (Z)-1-[2-aminethyl)-N-(2-ammonioethyl)amino]diazen-1-+ ++ium-1,2-diolate in a dose-dependent manner. In addition, the NO* toxicity is blocked by oxyhemoglobin, a NO* scavenger. Our findings suggest that NO* may play a role in the reversal of tumorigenicity associated with MnSOD overexpression.     

Hydrogen peroxide contributes to the manganese superoxide dismutase promotion of migration and invasion in glioma cells

Abstract

Manganese superoxide dismutase (MnSOD) is over-expressed in most brain tumours, and high MnSOD expression is associated with poor prognosis. The mechanisms still remain largely unknown. In the present study, the elevation of hydrogen peroxide (H₂O₂) level and the enhancement of glioma migration/invasion by over-expression of MnSOD were demonstrated. Subsequent studies showed that over-expression of MnSOD significantly increased the activation of mitogen-activated protein kinases (MAPKs) and phosphatidylinositol-3-kinases (PI3Ks), including AKTs, s6-ribosomal protein, ERKs and JNKs. Over-expression of MnSOD was also associated with elevations of matrix metalloproteinases-1(MMP-1) and MMP-9 protein. The promotion of migration/invasion, activation of PI3Ks and MAPKs and up-regulation of MMPs were inhibited by the general reactive oxygen species scavenger N-acetyl-l-cysteine (NAC), over-expression of the H₂O₂-detoxifying enzyme mitochondrial catalase (mCat) and specific inhibitors of AKTs or ERKs. Collectively, our study indicated that H₂O₂ would contribute to the MnSOD-promoted migration/invasion in glioma cells through activation of AKTs and ERKs. This study provided new molecular insights into the understanding of glioma migration and invasion.     

Mitochondrial or cytosolic catalase reverses the MnSOD-dependent inhibition of proliferation by enhancing respiratory chain activity, net ATP production, and decreasing the steady state levels of H(2)O(2)

Manganese superoxide dismutase (MnSOD) overexpression has been shown to reverse the malignant phenotype in a variety of tumor cell lines. The inhibition of proliferation and reversal of the malignant phenotype has been attributed to an increase in H(2)O(2) production as a result of the dismutation reaction. However, direct evidence in support of this hypothesis has not been forthcoming. To evaluate the contribution of H(2)O(2) in the regulation of cell growth in response to MnSOD overexpression, control and MnSOD-overexpressing HT-1080 fibrosarcoma cells were transfected with constructs that direct catalase to either the mitochondrial or cytosolic compartments. Overexpression of catalase in either compartment reversed the proliferative and clonogenic inhibition associated with MnSOD overexpression, blocked the increase in the steady state levels of H(2)O(2) as measured by flow cytometric analysis of 2', 7'-dichlorofluorescein diacetate, and increased protection from the cytotoxicity of H(2)O(2). In addition, mitochondrial or cytosolic catalase enhances respiration through complex I and II in both control and MnSOD overexpressing cell lines and reverses a MnSOD-dependent decrease in net ATP production. Thus, catalase reverses the proliferative inhibition associated with MnSOD overexpression and may also play an important role in metabolic regulation.     

Cdc42 regulates arsenic-induced NADPH oxidase activation and cell migration through actin filament reorganization

Although arsenic is a human carcinogen, the molecular mechanisms of its action remain to be understood. The present study reports that exposure to arsenic induced actin filament reorganization, resulting in lamellipodia and filopodia structures through the activation of Cdc42 in SVEC4-10 endothelial cells. It was also found that arsenic induced the formation of the superoxide anion (O2*) in SVEC4-10 cells. Immunoprecipitation and Western blotting analysis demonstrated that arsenic stimulation induced serine phosphorylation of p47phox, a key component of NADPH oxidase, indicating that arsenic induces O2* formation through NADPH oxidase activation. Inhibition of arsenic-induced actin filament reorganization by either overexpression of a dominant negative Cdc42 or pretreatment of an actin filament stabilizing regent, jasplakinolide, abrogated arsenic-induced NADPH oxidase activation, showing that the activation of NADPH oxidase was regulated by Cdc42-mediated actin filament reorganization. This study also showed that overexpression of a dominant negative Rac1 was sufficient to abolish arsenic-induced O2*- production, implying that Rac1 activities are required for Cdc42-mediated NADPH oxidase activation in response to arsenic stimulation. Furthermore, arsenic stimulation induced cell migration, which can be inhibited by the inactivation of either Cdc42 or NADPH oxidase. Taken together, the results indicate that arsenic is able to activate NADPH oxidase through Cdc42-mediated actin filament reorganization, leading to the induction of an increase in cell migration in SVEC4-10 endothelial cells.     

Interplay between steroid receptors and neoplastic progression in sarcoma tumors

Steroid hormones are expressed at low levels in mesenchymal cells and are highly expressed in soft tissue sarcoma. In human soft tissue fibrosarcoma cell line (HT-1080), the epidermal growth factor (EGF) stimulates the express of matrix metal (MMPs) expression through a Src-dependent mechanism. In human fibrosarcomas, increased expression of MMPs correlates with the metastatic progression. Our recent data in human breast cancer cell line MCF-7, demonstrates that EGF stimulates estradiol receptor (ER) phosphorylation on tyrosine at position 537 thereby promoting the association of a complex among EGF receptor (EGFR), androgen receptor (AR), ER, and Src that activates EGF-dependent signaling pathway. In the present study, we demonstrate that, in HT-1080 cells, the Src kinase activity is involved in EGFR phosphorylation and this activity is regulated by an interplay between Src, steroid receptors, and EGFR. In these cells, estradiol (E(2) )/ER and synthetic androgen (R1881)/AR trans-activate EGFR leading to the downstream signaling and to ERK activation. Indeed, the association between ER/AR and EGFR enhances metastatic progression of fibrosarcoma tumors. A population pilot study performed on 16 patients with soft tissue neoplasias highlights that MMPs expression correlates with progression of anaplastic sarcoma as well as overexpression of EGFR. These findings suggest that there is a crosstalk among AR, ER, and EGFR that lead to src activation also in fibrosarcoma cells.     

A Quantitative Comparison of Human HT-1080 Fibrosarcoma Cells and Primary Human Dermal Fibroblasts Identifies a 3D Migration Mechanism with Properties Unique to the Transformed Phenotype

Here, we describe an engineering approach to quantitatively compare migration, morphologies, and adhesion for tumorigenic human fibrosarcoma cells (HT-1080s) and primary human dermal fibroblasts (hDFs) with the aim of identifying distinguishing properties of the transformed phenotype. Relative adhesiveness was quantified using self-assembled monolayer (SAM) arrays and proteolytic 3-dimensional (3D) migration was investigated using matrix metalloproteinase (MMP)-degradable poly(ethylene glycol) (PEG) hydrogels (“synthetic extracellular matrix” or “synthetic ECM”). In synthetic ECM, hDFs were characterized by vinculin-containing features on the tips of protrusions, multipolar morphologies, and organized actomyosin filaments. In contrast, HT-1080s were characterized by diffuse vinculin expression, pronounced β1-integrin on the tips of protrusions, a cortically-organized F-actin cytoskeleton, and quantitatively more rounded morphologies, decreased adhesiveness, and increased directional motility compared to hDFs. Further, HT-1080s were characterized by contractility-dependent motility, pronounced blebbing, and cortical contraction waves or constriction rings, while quantified 3D motility was similar in matrices with a wide range of biochemical and biophysical properties (including collagen) despite substantial morphological changes. While HT-1080s were distinct from hDFs for each of the 2D and 3D properties investigated, several features were similar to WM239a melanoma cells, including rounded, proteolytic migration modes, cortical F-actin organization, and prominent uropod-like structures enriched with β1-integrin, F-actin, and melanoma cell adhesion molecule (MCAM/CD146/MUC18). Importantly, many of the features observed for HT-1080s were analogous to cellular changes induced by transformation, including cell rounding, a disorganized F-actin cytoskeleton, altered organization of focal adhesion proteins, and a weakly adherent phenotype. Based on our results, we propose that HT-1080s migrate in synthetic ECM with functional properties that are a direct consequence of their transformed phenotype.     

Induction of superoxide dismutase and cytotoxicity by manganese in human breast cancer cells.

MnCl2 induced manganese-containing superoxide dismutase (MnSOD) expression (mRNA, immunoreactive protein, and enzyme activity) in human breast cancer Hs578T cells. The induction of MnSOD immunoreactive protein in Hs578T cells was inhibited by tiron (a metal chelator and superoxide scavenger), pyruvate (a hydrogen peroxide scavenger), or 2-deoxy-d-glucose (DG, an inhibitor of glycolysis and the hexose monophosphate shunt), but not by 5,5-dimethyl-1-pyrroline-1-oxide (a superoxide scavenger), N-acetyl cysteine (a scavenger for reactive oxygen species and precursor of glutathione), diphenylene iodonium (an inhibitor of flavoproteins such as NADPH oxidase and nitric oxide synthase), or SOD (a superoxide scavenger). Northern blotting demonstrated that tiron or DG affected at the mRNA level, while pyruvate affected Mn-induced MnSOD expression at both the mRNA and protein levels. These results demonstrate that Mn can induce MnSOD expression in cultured human breast cancer cells. Mn also induced apoptosis and necrosis in these cells. Since inhibitors of Mn-induced MnSOD induction did not affect cell viability, MnSOD induction is probably not the cause of the Mn-induced cell killing.     

FPRL1-mediated induction of superoxide in LL-37-stimulated IMR90 human fibroblast

Molecular mechanisms underlying the generation of reactive oxygen species in LL-37-stimulated cells are poorly understood. Previously, we demonstrated that in human fibroblasts the exposure to WKYMVm induced p47^phox phosphorylation and translocation and, in turn, NADPH oxidase activation. These effects were mediated by the activation of the Formyl-peptide receptor-like 1 (FPRL1) and the downstream signaling involved ERKs phosphorylation and PKCα- and PKCδ-activation. Since LL-37 uses FPRL1 as a receptor to mediate its action on several cell types, we investigated in LL-37-stimulated IMR90 cells molecular mechanisms involved in NADPH-dependent superoxide generation. The exposure to LL-37, which is expressed in fibroblasts, induced ERKs activation, p47^phox phosphorylation and translocation as well as NADPH oxidase activation. These effects were prevented by pertussis toxin, PD098059 and WRWWWW, a FPRL1-selective antagonist. Furthermore, the stimulation with LL-37 of HEK293 cells, transfected to stably express FPRL1, induced a rapid activation of ERKs and p47^phox phosphorylation.     

Functional and biochemical studies of CD9 in fibrosarcoma cell line

CD9, a member of the tetraspanin family, plays important roles in a variety of cell activities. Fibrosarcoma is a malignant tumor that arises from fibroblasts. Low CD9 expression is found in fibrosarcoma tumor, but function of CD9 in fibrosarcoma has been rarely studied. In this study, stable cell lines for CD9 overexpression and vector were generated in HT1080, a human fibroscarcoma cell line, and cellular functions were widely investigated. In CD9-HT1080 cells, CD9 mainly localized in the membrane and co-localized with F-actin in the filopodia of cell surface. In functional assays, we demonstrated that CD9 could up-regulate total and active caspase-3 expression and induce cell apoptosis, but cell proliferation remained unchanged. CD9 overexpression inhibited HT1080 cell adhesion to FN but promoted cell spreading on FN. We also observed CD9 reduced cell migration using FN a chemoattractant and inhibited cell colony formation in soft agar medium. To explore the biochemical mechanism for functional changes, we investigated the effects of CD9 overexpression on cellular pathways and protein association. CD9 overexpression induced Akt phosphorylation on FN but did not change total Akt expression. Phosphorylation of p38 but not ERK was increased by CD9 overexpression, total p38 and ERK were not affected. CD9 overexpression did not affect the expression of TGFα, EGFR, β1, and EWI-2, but EWI-F expression was up-regulated. Moreover, CD9 could associate with TGFα, EGFR, β1, EWI-2, and EWI-F in HT1080 cell line. Take together, CD9 overexpression had promoting effects on cell apoptosis and cell spreading, but had inhibitory effects on cell adhesion, migration, and cell colony formation. These effects might be ascribed to CD9 associations with EWI-2/EWI-F/β1 complex and EGFR pathway, and the activation of Akt and p38 signalings as well.     

Notoginsenoside R1 inhibits TNF-alpha-induced fibronectin production in smooth muscle cells via the ROS/ERK pathway

The matrix fibronectin protein plays an important role in vascular remodeling. Notoginsenoside R1 is the main ingredient with cardiovascular activity in Panax notoginseng; however, its molecular mechanisms are poorly understood. We report that notoginsenoside R1 significantly decreased TNF-alpha-induced activation of fibronectin mRNA, protein levels, and secretion in human arterial smooth muscle cells (HASMCs) in a dose-dependent manner. Notoginsenoside R1 scavenged hydrogen peroxide (H2O2) in a dose-dependent manner in the test tube. TNF-alpha significantly increased intracellular ROS generation and then ERK activation, which was blocked by notoginsenoside R1 or DPI and apocynin, inhibitors of NADPH oxidase, or the antioxidant NAC. Our data demonstrated that TNF-alpha-induced upregulation of fibronectin mRNA and protein levels occurs via activation of ROS/ERK, which was prevented by treatment with notoginsenoside R1, DPI, apocynin, NAC, or MAPK/ERK inhibitors PD098059 and U0126. Notoginsenoside R1 significantly inhibited H2O2-induced upregulation of fibronectin mRNA and protein levels and secretion; it also significantly inhibited TNF-alpha and H2O2-induced migration. These results suggest that notoginsenoside R1 inhibits TNF-alpha-induced ERK activation and subsequent fibronectin overexpression and migration in HASMCs by suppressing NADPH oxidase-mediated ROS generation and directly scavenging ROS.     

Reactive oxygen species produced by NADPH oxidase, xanthine oxidase, and mitochondrial electron transport system mediate heat shock-induced MMP-1 and MMP-9 expression

In addition to ultraviolet radiation, human skin is also exposed to infrared radiation (IR) from natural sunlight. IR typically increases the skin temperature. This study examined whether or not heat shock-induced ROS stimulates MMPs in keratinocyte HaCaT cells. In HaCaT cells, heat shock was found to increase the intracellular ROS levels, including hydrogen peroxide and superoxide. The heat shock treatment induced MMP-1 and MMP-9, but not MMP-2, at the mRNA and protein levels. Moreover, heat shock caused the rapid activation of the three distinct MAPKs, ERK, JNK, and p38 kinase. The heat shock-induced expression of MMP-1 and MMP-9 was significantly suppressed by a pretreatment with the antioxidant NAC or catalase. On the other hand, SOD inhibited heat shock-induced activity of MMP-9 induction, but not MMP-1. A pretreatment with NAC or catalase, but not SOD, attenuated the phosphorylation of ERK, JNK, and p38 kinase by heat shock. The potential sites of ROS generation by heat shock along with its role in the heat shock-induced expression of MMP-1 and MMP-9 were next analyzed. These results indicate that heat shock-induced ROS is promoted via NADPH oxidase, xanthine oxidase, and mitochondria. Indeed, the NADPH oxidase and xanthine oxidase activities were increased by heat shock. Overall, the ROS produced by heat shock may play an important role in the heat shock-induced activation of MAPKs, which can induce MMP-1 and-9 expressions.     

Mechanisms of H2O2-induced oxidative stress in endothelial cells

Hydrogen peroxide, produced by inflammatory and vascular cells, induces oxidative stress that may contribute to endothelial dysfunction. In smooth muscle cells, H₂O₂ induces production of O₂⁻ by activating NADPH oxidase. However, the mechanisms whereby H₂O₂ induces oxidative stress in endothelial cells are poorly understood. We examined the effects of H₂O₂ on O₂⁻ levels on porcine aortic endothelial cells (PAEC). Treatment with 60 μmol/L H₂O₂ markedly increased intracellular O₂⁻ levels (determined by conversion of dihydroethidium to hydroxyethidium) and produced cytotoxicity (determined by propidium iodide staining) in PAEC. Overexpression of human manganese superoxide dismutase in PAEC reduced O₂⁻ levels and attenuated cytotoxicity resulting from treatment with H₂O₂. L-NAME, an inhibitor of nitric oxide synthase (NOS), and apocynin, an inhibitor of NADPH oxidase, reduced O₂⁻ levels in PAEC treated with H₂O₂, suggesting that both NOS and NADPH oxidase contribute to H₂O₂-induced O₂⁻ in PAEC. Inhibition of NADPH oxidase using apocynin and NOS rescue with L-sepiapterin together reduced O₂⁻ levels in PAEC treated with H₂O₂ to control levels. This suggests interaction-distinct NOS and NADPH oxidase pathways to superoxide. We conclude that H₂O₂ produces oxidative stress in endothelial cells by increasing intracellular O₂⁻ levels through NOS and NADPH oxidase. These findings suggest a complex interaction between H₂O₂ and oxidant-generating enzymes that may contribute to endothelial dysfunction.     

Catalase, but not MnSOD, inhibits glucose deprivation-activated ASK1-MEK-MAPK signal transduction pathway and prevents relocalization of Daxx: hydrogen peroxide as a major second messenger of metabolic oxidative stress

Overexpression of catalase, but not manganese superoxide dismutase (MnSOD), inhibited glucose deprivation-induced cytotoxicity and c-Jun N-terminal kinase 1 (JNK1) activation in human prostate adenocarcinoma DU-145 cells. Suppression of JNK1 activation by catalase overexpression resulted from inhibition of apoptosis signal-regulating kinase 1 (ASK1) activation by preventing dissociation of thioredoxin (TRX) from ASK1. Overexpression of catalase also inhibited relocalization of Daxx from the nucleus to the cytoplasm as well as association of Daxx with ASK1 during glucose deprivation. Taken together, hydrogen peroxide (H(2)O(2)) rather than superoxide anion (O(2) (*-)) acts as a second messenger of metabolic oxidative stress to activate the ASK1-MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)-mitogen-activated protein kinase (MAPK) signal transduction pathway.