Raekil Park

Roles of NADPH Oxidases in Cisplatin-Induced Reactive Oxygen Species Generation and Ototoxicity

In our previous study, we clearly demonstrated the roles of pro-inflammatory cytokines, including tumor necrosis factor-α, interleukin-1β (IL-1β), and IL-6, and subsequent reactive oxygen species (ROS) generation on the pathogenesis of cisplatin ototoxicity in vitro and in vivo. ROS generation in cisplatin-treated HEI-OC1 auditory cells was also correlated with changing mitochondrial membrane potential. However, the roles of NADPH oxidase in cisplatin-induced ROS generation and ototoxicity have not been fully elucidated. Herein, immunohistochemical studies demonstrated that treatment of cisplatin induced the expression of NADPH oxidase isoforms NOX-1 and NOX-4 in HEI-OC1 auditory cells. Expression of mRNA for NOX-1, NOX-4, NOXO1, NOXA1, p47phox, and p67phox was also increased. Inhibition of NADPH oxidase with diphenyleniodonium chloride or apocynin abolished ROS production and the subsequent apoptotic cell death in cisplatin-treated cells. Furthermore, suppression of NOX1 and NOX4 expression by small interfering RNA transfection markedly abolished the cytotoxicity and ROS generation by cisplatin. Together, our data suggest that ROS generated, in part, through the activation of NADPH oxidase plays an essential role in cisplatin ototoxicity.

Cisplatin Cytotoxicity of Auditory Cells Requires Secretions of Proinflammatory Cytokines via Activation of ERK and NF-κB

The ototoxicity of cisplatin, a widely used chemotherapeutic agent, involves a number of mechanisms, including perturbation of redox status, increase in lipid peroxidation, and formation of DNA adducts. In this study, we demonstrate that cisplatin increased the early immediate release and de novo synthesis of proinflammatory cytokines, including TNF-α, IL-1β, and IL-6, through the activation of ERK and NF-κB in HEI-OC1 cells, which are conditionally immortalized cochlear cells that express hair cell markers. Both neutralization of proinflammatory cytokines and pharmacologic inhibition of ERK significantly attenuated the death of HEI-OC1 auditory cells caused by cisplatin and proinflammatory cytokines. We also observed a significant increase in the protein and mRNA levels of proinflammatory cytokines in both serum and cochleae of cisplatin-injected rats, which was suppressed by intraperitoneal injection of etanercept, an inhibitor of TNF-α. Immunohistochemical studies revealed that TNF-α expression was mainly located in the spiral ligament, spiral limbus, and the organ of Corti in the cochleae of cisplatin-injected rats. NF-κB protein expression, which overlapped with terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling-positive signal, was very strong in specific regions of the cochleae, including the organ of Corti, spiral ligament, and stria vascularis. These results indicate that proinflammatory cytokines, especially TNF-α, play a central role in the pathophysiology of sensory hair cell damage caused by cisplatin.

Quercetin protects the hydrogen peroxide-induced apoptosis via inhibition of mitochondrial dysfuntion in H9c2 cardiomyoblast cells

Quercetin possesses a broad range of pharmacological properties, including protection of LDL from oxidation. However, little is known about the mechanism by which quercetin rescues cardiomyoblasts from oxidative damage. This study was designed to investigate the protective mechanism of quercetin on H(2)O(2)-induced toxicity of H9c2 cardiomyoblasts. Oxidative stress, such as H(2)O(2), ZnCl(2), and menadione, significantly decreased the viability of H9c2 cells, which was accompanied with apparent apoptotic features, including fragmentation of genomic DNA as well as activation of caspase protease. However, quercetin markedly inhibited the apoptotic characteristics via reduction of intracellular reactive oxygen species generation. Also, it prevented the H(2)O(2)-mediated mitochondrial dysfunction, including disruption of mitochondria membrane permeability transition as well as an increase in expression of apoptogenic Bcl-2 proteins, Bcl-2 and Bcl-X(L). Furthermore, pretreatment of quercetin inhibited the activation of caspase-3, thereby both cleavage of poly(ADP-ribose) polymerase and degradation of inhibitor of caspase-activated DNase/DNA fragmentation factor by H(2)O(2) were completely abolished. Taken together, these data suggest that protective effects of quercetin against oxidative injuries of H9c2 cardiomyoblasts may be achieved via modulation of mitochondrial dysfunction and inhibition of caspase activity.

Carbon Monoxide Induces Heme Oxygenase-1 via Activation of Protein Kinase R–Like Endoplasmic Reticulum Kinase and Inhibits Endothelial Cell Apoptosis Triggered by Endoplasmic Reticulum Stress

Carbon monoxide (CO), a reaction product of the cytoprotective heme oxygenase (HO)-1, is antiapoptotic in a variety of models of cellular injury, but the precise mechanisms remain to be established. In human umbilical vein endothelial cells, exogenous CO activated Nrf2 through the phosphorylation of protein kinase R–like endoplasmic reticulum kinase (PERK), resulting in HO-1 expression. CO-induced activation of PERK was followed by the phosphorylation of eukaryotic translation initiation factor 2&agr; and the expression of activating transcription factor 4. However, CO fails to induce X-box binding protein-1 expression and activating transcription factor 6 cleavage. CO had no significant effect on synthesis of endoplasmic reticulum (ER) chaperone proteins such as the 78-kDa glucose-regulated proteins 78 and 94. Instead, CO prevented X-box binding protein 1 expression and activating transcription factor 6 cleavage induced by ER-stress inducers such as thapsigargin, tunicamycin and homocysteine. CO also prevented endothelial apoptosis triggered by these ER inducers through suppression of C/EBP homologous protein expression, which was associated with its activation of p38 mitogen-activated protein kinase. Similarly, endogenous CO produced from endothelial HO-1 induced by either exogenous CO or a pharmacological inducer was also cytoprotective against ER stress through C/EBP homologous protein suppression. Our findings suggest that CO renders endothelial cells resistant to ER stress not only by downregulating C/EBP homologous protein expression via p38 mitogen-activated protein kinase activation but also by upregulating Nrf2-dependent HO-1 expression via PERK activation. Thus, the HO-1/CO system might be potential therapeutics in vascular diseases associated with ER stress.

Sulforaphane protects against cytokine- and streptozotocin-induced β-cell damage by suppressing the NF-κB pathway

Sulforaphane (SFN) is an indirect antioxidant that protects animal tissues from chemical or biological insults by stimulating the expression of several NF-E2-related factor-2 (Nrf2)-regulated phase 2 enzymes. Treatment of RINm5F insulinoma cells with SFN increases Nrf2 nuclear translocation and expression of phase 2 enzymes. In this study, we investigated whether the activation of Nrf2 by SFN treatment or ectopic overexpression of Nrf2 inhibited cytokine-induced beta-cell damage. Treatment of RIN cells with IL-1beta and IFN-gamma induced beta-cell damage through a NF-kappaB-dependent signaling pathway. Activation of Nrf2 by treatment with SFN and induction of Nrf2 overexpression by transfection with Nrf2 prevented cytokine toxicity. The mechanism by which Nrf2 activation inhibited NF-kappaB-dependent cell death signals appeared to involve the reduction of oxidative stress, as demonstrated by the inhibition of cytokine-induced H(2)O(2) production. The protective effect of SFN was further demonstrated by the restoration of normal insulin secreting responses to glucose in cytokine-treated rat pancreatic islets. Furthermore, pretreatment with SFN blocked the development of type 1 diabetes in streptozotocin-treated mice.

Microphthalmia transcription factor and PU.1 synergistically induce the leukocyte receptor osteoclast-associated receptor gene expression.

We have recently reported the identification of a novel member of the leukocyte receptor family, osteoclast-associated receptor (OSCAR), which has two Ig-like domains and functions as a bone-specific regulator of osteoclast differentiation. Here, we have cloned the OSCAR promoter region to examine its regulation by transcription factors. The 1.7-kb promoter region of the mouse OSCAR gene contains two potential E-box elements for microphthalmia transcription factor (MITF) and three putative PU.1 sites. MITF or PU.1 alone activates the OSCAR reporter construct 5–6-fold, and the combination of MITF and PU.1 synergistically activates the OSCAR reporter activity up to 110-fold. The mRNA expression patterns of MITF, PU.1, and OSCAR in TRANCE-treated (RAW 264.7) or TRANCE/M-CSF-treated cells (primary osteoclasts) reveal that MITF mRNA expression is induced at a much earlier time point than OSCAR gene expression. In contrast to MITF, PU.1 mRNA levels remain relatively constant at all time points, suggesting that TRANCE-induced MITF, not PU.1 expression, is one of the critical regulatory mechanisms for optimal OSCAR expression during osteoclastogenesis. In addition, we have shown that the combination of MITF and constitutively active MKK6-expressing plasmids synergistically activates OSCAR reporter activity. Taken together, our results strongly suggest that PU.1 and MITF transcription factors synergistically activate OSCAR gene expression. Moreover, the activation of OSCAR gene expression by PU.1/MITF is further enhanced by the TRANCE-induced MKK6/p38 signaling cascade.

Nitric oxide is a regulator of bone remodelling.

Nitric oxide (NO) is known to be implicated in the metabolism of bone, especially as a mediator of cytokine effects on the remodelling of bone tissue. In this study we examine whether NO affects the osteoblast activation or the osteoclast differentiation of primary mouse osteoblast‐like and osteosarcoma ROS 17/2.8 cell lines.

Protective effects of morphine in peroxynitrite-induced apoptosis of primary rat neonatal astrocytes: potential involvement of G protein and phosphatidylinositol 3-kinase (PI3 kinase).

Opiates, such as morphine, have been used extensively in the clinical management of pain due to their potent analgesic effect. Astrocytes, representing a major non-neuronal cell population in the CNS, contain opioid receptors that are actively involved in several brain functions. This study was designed to evaluate the effects by which morphine, a preferential mu-opioid receptor agonist, contributes to cytotoxicity of nitric oxide (NO) species, including NO and peroxynitrite (ONOO-), in primary rat neonatal astrocytes. Primary astrocytes isolated from the cerebral cortex of 1- to 2-day-old Sprague-Dawley rats were treated with morphine, naloxone, and 3-morpholinosydnonimine (SIN-1), a donor of peroxynitrite. Morphine significantly protected primary rat astrocytes from apoptosis mediated by sodium nitroprusside, an NO donor, and SIN-1 in a dose-dependent manner, whereas it did not in other types of cells including C6 glioma, RAW 264.7, and HL-60 cells. Moreover, naloxone antagonized the protective effects of morphine on SIN-1-induced apoptosis. Morphine also inhibited the nuclear condensation and fragmentation of SIN-1-treated cells that was antagonized by naloxone pretreatment. The protective role of morphine in SIN-1-induced apoptosis was dependent on an intracellular antioxidant system such as GSH. Furthermore, the effects of morphine on SIN-1-induced cytotoxicity were prohibited by pretreatment with the G(i) protein inhibitor, pertussis toxin, and the phosphatidylinositol 3-kinase (PI3 kinase) inhibitors, wortmannin and LY294002. Taken together, these results suggest that morphine may protect primary rat astrocytes from apoptosis by NO species via the signaling cascades that involve both G protein and PI3 kinase.

Genistein protects pancreatic β cells against cytokine-mediated toxicity

In the past few decades, the use of genistein as an anti-inflammatory agent has gained much attention. Our current study focuses on the preventive effects of genistein on cytokine-induced pancreatic beta-cell damage. Treatment of RINm5F (RIN) rat insulinoma cells with interleukin (IL)-1beta and interferon (IFN)-gamma induced cell damage, which was correlated with nitric oxide (NO) production. Genistein completely prevented cytokine-mediated cytotoxicity and NO production, a finding that correlated well with reduced levels of the inducible form of NO synthase (iNOS) mRNA and protein. The molecular mechanism of genistein inhibition of iNOS gene expression appeared to involve the inhibition of NFkappaB activation. The cytokine induced increases in NFkappaB binding activity, nuclear p50 and p65 subunit levels, and IkappaBalpha degradation in cytosol compared to unstimulated cells; genistein abolished all of these parameters. The cytoprotective effects of genistein are also mediated through the suppression of ERK-1/2 and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathways. In a second set of experiments, rat islets were used. The findings on beta-cell protective effects of genistein were essentially the same as for the RIN cell data, namely genistein prevented cytokine-induced NO production, iNOS expression, ERK-1/2 activation, JAK/STAT activation, and impairment of glucose-stimulated insulin secretion. Collectively, these results suggest that genistein might be used to preserve functional beta-cell mass.

Evidence that Cisplatin-induced Auditory Damage is Attenuated by Downregulation of Pro-inflammatory Cytokines Via Nrf2/HO-1

Recently, we demonstrated that pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 played a critical role in cisplatin-induced cochlear injury and that flunarizine, known as a T-type Ca2+ channel antagonist, induced a cytoprotective effect against cisplatin cytotoxicity in HEI-OC1 cells by the activation of NF-E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) cascade through PI3K-Akt signaling but calcium-independent pathway. We report here that flunarizine markedly attenuates cisplatin-induced pro-inflammatory cytokine secretion and their messenger RNA transcription as well as cisplatin cytotoxicity through the activation of Nrf2/HO-1 and downregulation of NF-κB. In HEI-OC1 cells, overexpression of Nrf2/HO-1 by gene transfer or pharmacological approaches attenuated cisplatin-induced cytotoxicity and pro-inflammatory cytokine production. On the contrary, inhibition of Nrf2/HO-1 signaling by pharmacological inhibitors or specific small interfering RNAs significantly abolished the beneficial effects of flunarizine. Flunarizine also attenuated cisplatin-mediated MAPK activation and pharmacological inhibition of MAPKs, especially MEK1/ERK, blocked cisplatin-induced NF-κB activation in HEI-OC1 cells. Furthermore, WT-Nrf2 overexpression effectively blocked MAPK activation after cisplatin exposure. Finally, orally administrated Sibelium™, the trade name of flunarizine, suppressed the increase of pro-inflammatory cytokines by cisplatin in both serum and cochleas of mice, whereas it increased HO-1 expression in cochleas. These results indicate that flunarizine induces a protective effect against cisplatin ototoxicity through the downregulation of NF-κB by Nrf2/HO-1 activation and the resulting inhibition of pro-inflammatory cytokine production in vitro and in vivo.