Hong-Duck Um

Link between Mitochondria and NADPH Oxidase 1 Isozyme for the Sustained Production of Reactive Oxygen Species and Cell Death

Although mitochondria and the Nox family of NADPH oxidase are major sources of reactive oxygen species (ROS) induced by external stimuli, there is limited information on their functional relationship. This study has shown that serum withdrawal promotes the production of ROS in human 293T cells by stimulating both the mitochondria and Nox1. An analysis of their relationship revealed that the mitochondria respond to serum withdrawal within a few minutes, and the ROS produced by the mitochondria trigger Nox1 action by stimulating phosphoinositide 3-kinase (PI3K) and Rac1. Activation of the PI3K/Rac1/Nox1 pathway was evident 4-8 h after but not earlier than serum withdrawal initiation, and this time lag was found to be required for an additional activator of the pathway, Lyn, to be expressed. Functional analysis suggested that, although the mitochondria contribute to the early (0-4 h) accumulation of ROS, the maintenance of the induced ROS levels to the later (4-8 h) phase required the action of the PI3K/Rac1/Nox1 pathway. Serum withdrawal-treated cells eventually lost their viability, which was reversed by blocking either the mitochondria-dependent induction of ROS using rotenone or KCN or the PI3K/Rac1/Nox1 pathway using the dominant negative mutants or small interfering RNAs. This suggests that mitochondrial ROS are essential but not enough to promote cell death, which requires the sustained accumulation of ROS by the subsequent action of Nox1. Overall, this study shows a signaling link between the mitochondria and Nox1, which is crucial for the sustained accumulation of ROS and cell death in serum withdrawal-induced signaling.

Bcl-w Promotes Gastric Cancer Cell Invasion by Inducing Matrix Metalloproteinase-2 Expression via Phosphoinositide 3-Kinase, Akt, and Sp1

Given a previous report that Bcl-w is expressed in gastric cancer cells, particularly in those of an infiltrative morphology, we investigated whether Bcl-w expression influences the invasiveness of gastric cancer cells. To accomplish this, Bcl-w was overexpressed in adherent types of gastric adenocarcinoma cell lines, and this was found to result in an increase in their migratory and invasive potentials. These effects were not induced when Bcl-2 was overexpressed in the same cell types. Consistently, Bcl-w, but not Bcl-2, overexpression increased matrix metalloproteinase-2 (MMP-2) expression, and synthetic or natural inhibitors of MMP-2 abolished Bcl-w-induced cell invasion. Bcl-w overexpression also activated phosphoinositide 3-kinase (PI3K), Akt, and Sp1, and the blocking effects of each of these components using pharmacologic inhibitors, dominant-negative mutants, or small interfering RNA abolished the ability of Bcl-w to induce MMP-2 and cell invasion. The inhibition of PI3K/Akt signaling also prevented Sp1 activation. Overall, our data suggest that Bcl-w, which was previously shown to enhance gastric cancer cell survivability, also promotes their invasiveness by inducing MMP-2 expression via the sequential actions of PI3K, Akt, and Sp1.

Phospholipase A2 Activity of Peroxiredoxin 6 Promotes Invasion and Metastasis of Lung Cancer Cells

Peroxiredoxins (PRDX) are a family of thiol-dependent peroxidases. Among the six mammalian members of this family, PRDX6 is the only protein that additionally exhibits phospholipase A2 (PLA2) activity. The physiologic role of this interesting PRDX6 feature is largely unknown at present. In this study, we show that PRDX6 increases the metastatic potential of lung cancer cells. Functional analyses of the enzymatic activities of PRDX6, using specific pharmacologic inhibitors and mutagenesis studies, reveal that both peroxidase and PLA2 activities are required for metastasis. Specifically, peroxidase activity facilitates the growth of cancer cells, and PLA2 activity promotes invasiveness. Further investigation of the latter event discloses that PLA2 activity promotes accumulation of arachidonic acid, which, in turn, induces the invasive pathway involving p38 kinase, phosphoinositide 3-kinase, Akt, and urokinase-type plasminogen activator. This study is the first to define the functions of the enzymatic activities of PRDX6 in metastasis and to show the involvement of arachidonic acid in PRDX6 action in intact cells. These novel findings provide a significant step toward elucidating the role of PRDX6 in cancer and the mechanism of its action. Mol Cancer Ther; 9(4); 825–32. ©2010 AACR.

Akt is negatively regulated by the MULAN E3 ligase

The serine/threonine kinase Akt functions in multiple cellular processes, including cell survival and tumor development. Studies of the mechanisms that negatively regulate Akt have focused on dephosphorylation-mediated inactivation. In this study, we identified a negative regulator of Akt, MULAN, which possesses both a RING finger domain and E3 ubiquitin ligase activity. Akt was found to directly interact with MULAN and to be ubiquitinated by MULAN in vitro and in vivo. Other molecular assays demonstrated that phosphorylated Akt is a substantive target for both interaction with MULAN and ubiquitination by MULAN. The results of the functional studies suggest that the degradation of Akt by MULAN suppresses cell proliferation and viability. These data provide insight into the Akt ubiquitination signaling network.

Bcl‐XL and STAT3 mediate malignant actions of γ‐irradiation in lung cancer cells

Previous reports suggest that, in addition to its therapeutic effects, ionizing radiation (IR) increases the invasiveness of surviving cancer cells. Here, we demonstrate that this activity of IR in lung cancer cells is mediated by a signaling pathway involving p38 kinase, phosphoinositide 3‐kinase, Akt, and matrix metalloproteinase (MMP‐2). The invasion‐promoting doses of IR also increased and reduced the levels of vimentin and E‐cadherin, respectively, both of which are markers for the epithelial–mesenchymal transition (EMT). Interestingly, all of these malignant actions of IR were mimicked by the overexpression of Bcl‐XL, a pro‐survival member of the Bcl‐2 family, in lung cancer cells. Moreover, both RNA and protein levels of Bcl‐XL were elevated upon irradiation of the cells, and the prevention of this event using small‐interfering RNAs of Bcl‐XL reduced the ability of IR to promote invasion signals and EMT‐associated events. This suggests that Bcl‐XL functions as a signaling mediator of the malignant effects of IR. It was also demonstrated that IR enhances signal transducer and activator of transcription 3 (STAT3) phosphorylation, and the reduction of STAT3 levels via RNA interference prevented IR‐induced Bcl‐XL accumulation, and thus all the tested Bcl‐XL‐dependent events. Overall, the data suggest that IR induces Bcl‐XL accumulation via STAT3, which then promotes cancer cell invasion and EMT‐associated markers. Our findings demonstrate a novel function of Bcl‐XL in cancer, and also advance our understanding of the malignant actions of IR significantly. (Cancer Sci 2010)

Nicotinamide Phosphoribosyltransferase Is Essential for Interleukin-1β-mediated Dedifferentiation of Articular Chondrocytes via SIRT1 and Extracellular Signal-regulated Kinase (ERK) Complex Signaling

Although much is known about interleukin (IL)-1β and its role as a key mediator of cartilage destruction in osteoarthritis, only limited information is available on IL-1β signaling in chondrocyte dedifferentiation. Here, we have characterized the molecular mechanisms leading to the dedifferentiation of primary cultured articular chondrocytes by IL-1β treatment. IL-1β or lipopolysaccharide, but not phorbol 12-myristate 13-acetate, retinoic acid, or epidermal growth factor, induced nicotinamide phosphoribosyltransferase (NAMPT) expression, showing the association of inflammatory cytokines with NAMPT regulation. SIRT1, in turn, was activated NAMPT-dependently, without any alteration in the expression level. Activation or inhibition of SIRT1 oppositevely regulates IL-1β-mediated chondrocyte dedifferentiation, suggesting this protein as a key regulator of chondrocytes phenotype. SIRT1 activation promotes induction of ERK and p38 kinase activities, but not JNK, in response to IL-1β. Subsequently, ERK and p38 kinase activated by SIRT1 also induce SIRT1 activation, forming a positive feedback loop to sustain downstream signaling of these kinases. Moreover, we found that the SIRT1-ERK complex, but not SIRT1-p38, is engaged in IL-1β-induced chondrocyte dedifferentiation via a Sox-9-mediated mechanism. JNK is activated by IL-1β and modulates dedifferentiation of chondrocytes, but this pathway is independent on NAMPT-SIRT1 signaling. Based on these findings, we propose that IL-1β induces dedifferentiation of articular chondrocytes by up-regulation of SIRT1 activity enhanced by both NAMPT and ERK signaling.

Bcl-2 family proteins as regulators of cancer cell invasion and metastasis: a review focusing on mitochondrial respiration and reactive oxygen species

Although Bcl-2 family proteins were originally identified as key regulators of apoptosis, an impressive body of evidence has shown that pro-survival members of the Bcl-2 family, including Bcl-2, Bcl-XL, and Bcl-w, can also promote cell migration, invasion, and cancer metastasis. Interestingly, cell invasion was recently found to be suppressed by multidomain pro-apoptotic members of the Bcl-2 family, such as Bax and Bak. While the mechanisms underlying these new functions of Bcl-2 proteins are just beginning to be studied, reactive oxygen species (ROS) have emerged as inducers of cell invasion and the production of ROS from mitochondrial respiration is known to be promoted and suppressed by the pro-survival and multidomain pro-apoptotic Bcl-2 family members, respectively. Here, I review the evidence supporting the ability of Bcl-2 proteins to regulate cancer cell invasion and metastasis, and discuss our current understanding of their underlying mechanisms, with a particular focus on mitochondrial respiration and ROS, which could have implications for the development of strategies to overcome tumor progression.

NF-κB mediates the adaptation of human U937 cells to hydrogen peroxide

Low doses of oxidative stress can induce cellular resistance to subsequent higher doses of the same stress. By using human U937 leukemia cells, we previously demonstrated that H2O2 can induce such an adaptive response without elevating the cellular capacity to degrade H2O2, and were able to confer the cells a cross-resistance to an H2O2-independent lethal stimulus, C2-ceramide. In this study, it was found that the adaptation is accompanied by the translocation of cytoplasmic NF-κB to the nuclei. This event was promoted or abolished when either IKKα or a dominant negative mutant of IκB, respectively, was overexpressed. The overexpression of IKKα also resulted in the suppression of H2O2-induced cell death and DNA fragmentation, whereas these events were accelerated by the expression of the IκB mutant. The protective effect of IKKα was accompanied neither by an elevation of protein levels of various antioxidant enzymes such as catalase, superoxide dismutase, and glutathione peroxidase, nor by an increase in the cellular capacity to consume H2O2. Moreover, the overexpression of IKKα resulted in an enhancement of H2O2-induced resistance to C2-ceramide. The overall data suggest that NF-κB mediates the H2O2 adaptation induced in a manner independent of H2O2-degrading activity.

Bmal1 suppresses cancer cell invasion by blocking the phosphoinositide 3-kinase-Akt-MMP-2 signaling pathway

Bmal1 is a core factor in the regulation of circadian rhythms. Previous studies have shown that Bmal1 suppresses tumor growth in cell culture and animal models and is down-regulated in certain types of cancer. The aim of the present study was to investigated whether Bmal1 influences the invasiveness of cancer cells. We demonstrated that knockdown of Bmal1 by RNA interference promoted cancer cell invasion, whereas its overexpression reduced cellular invasiveness. These effects were observed in lung cancer and glioma cells, and occurred regardless of p53 status. Therefore, it appears that Bmal1 suppresses the invasion of multiple cancer types in a p53-independent manner. Bmal1 knockdown-induced cancer cell invasion was accompanied by activation of the PI3K-Akt-MMP-2 pathway, and was prevented by inhibitors of PI3K, Akt or MMP-2. This suggests that Bmal1 suppresses cell invasion by blocking the PI3K-Akt-MMP-2 pathway. Since this invasion pathway is activated by the oncogene Bcl-w, we investigated whether Bmal1 affects the activity of Bcl-w. As expected, Bmal1 attenuated the ability of Bcl-w to promote MMP-2 accumulation and cell invasion, supporting the idea that Bmal1 antagonizes Bcl-w activity. Collectively, our data suggest that Bmal1 is a tumor suppressor, capable of suppressing cancer cell growth and invasiveness, and support the recent proposal that there is a tight molecular link between circadian rhythms and tumor formation/progression.

Amplification of the γ-irradiation-induced cell death pathway by reactive oxygen species in human U937 cells

Given the critical involvement of reactive oxygen species (ROS) in cell death, their hierarchical status in the cell pathway has been analyzed by many investigators. However, it has been shown that ROS can act either upstream or downstream of various death mediators depending on experimental settings. To investigate whether the contrasting relationships may exist in a single model system, human U937 cells were irradiated with lethal doses of gamma-rays. This resulted in a promotion of mitochondrial ROS production, which was found to be induced via sequential actions of c-Jun N-terminal kinase (JNK), Bax, and caspase-3. Interestingly, the induced ROS, in turn, re-activated JNK, Bax, and caspase-3 in the same model system. Consistently, the blockade of Bax action by RNA interference or Bcl-2 overexpression abolished the activation of JNK induced after, but not before, the production of ROS. Bcl-2 overexpression also blocked the translocation of Bax from the cytosol to the mitochondria only after the induction of ROS. Functional analyses revealed that the initial ROS-independent activations of JNK, Bax, and caspase-3 are not sufficient for cell death, and thus, should be re-activated by ROS in order to kill the cells. These findings suggest that ROS do not simply mediate the lethal action of gamma-irradiation, but actually amplify it by forming a feedback loop between a downstream effector caspase and the upstream initiation signals leading to the activation of JNK. This role for ROS appears to allow Bcl-2 to block the signaling events, which are initially induced upstream.