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Nitric Oxide Prevents H2O2-Induced Apoptosis in SK-N-MC Human Neuroblastoma Cells Yeong-Min Yoo, Eui-Man Jung, Changhwan Ahn, Eui-Bae Jeung

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Nitric Oxide Prevents H2O2-Induced Apoptosis in SK-N-MC Human Neuroblastoma Cells Yeong-Min Yoo, Eui-Man Jung, Changhwan Ahn, Eui-Bae Jeung Int. J. Biol. Sci. 2018, Vol. 14 1974 Ivyspring International Publisher International Journal of Biological Sciences 2018; 14(14): 1974-1984. doi: 10.7150/ijbs.28050 Research Paper Nitric oxide prevents H2O2-induced apoptosis in SK-N-MC human neuroblastoma cells Yeong-Min Yoo, Eui-Man Jung, Changhwan Ahn, Eui-Bae Jeung Laboratory of Veterinary Biochemistry and Molecular Biology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk 28644, Republic of Korea Corresponding author: Eui-Bae Jeung, PhD. Laboratory of Veterinary Biochemistry and Molecular Biology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk 28644, Republic of Korea. E-mail: [email protected] © Ivyspring International Publisher. This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) license (https://creativecommons.org/licenses/by-nc/4.0/). See http://ivyspring.com/terms for full terms and conditions. Received: 2018.06.22; Accepted: 2018.09.04; Published: 2018.11.02 Abstract Nitric oxide (NO) is a cellular signaling molecule in many physiological and pathological processes including neuroprotector. Here we examined the antiapoptotic effect of NO in SK-N-MC cells. H2O2 treatment (10-200 μM) induced cell death in a dose-dependent manner and pretreatment of cells with 100 μM S-nitroso-N-acetylpenicillamine (SNAP), an NO donor, attenuated the occurrence of H2O2-induced cell death. DAPI staining showed H2O2-induced nuclear fragmentation and NO treatment suppressed it. NO inhibited the proteolytic activation of caspase-3 and mitochondrial cytochrome c release. Treatment of soluble guanylyl cyclase inhibitor ODQ decreased the protective effect of SNAP on H2O2-treated cells and increased caspase 3-like enzyme activity and activation, cytochrome c release, PARP cleavage, and DNA fragmentation, indicating that cGMP is a key mediator in NO-mediated antiapoptosis. The cGMP analog 8-Br-cGMP blocked H2O2-induced apoptotic cell death; reduction of caspase-3 enzyme, cytochrome c release, and caspase-8 and -9. These preventive effects of SNAP and 8-Br-cGMP were suppressed by PKG inhibitor KT5823. Levels of PKGI, PKGII, and p-VASP proteins were increased by SNAP and 8-Br-cGMP and suppressed by KT5823 treatment. These results indicate that PKG is a downstream signal mediator in the suppression of apoptosis by NO and cGMP. Akt activation was inhibited the PI3K inhibitors LY294002 and Wortmannin, resulting in the inhibition of cell viability and increase of cytochrome c release. SNAP induced phosphorylation of Akt and Bad and then increased the interactions between 14-3-3β and p-Bad. These data suggest that the NO suppresses H2O2-induced SK-N-MC cell apoptosis by suppressing apoptosis signal mediating the interaction between 14‐3‐3β and Bad phosphorylation via PKG/PI3K/Akt. Key words: Nitric oxide; hydrogen peroxide; PKG; PI3K; Akt Introduction Hydrogen peroxide (H2O2) has been known as a produced by the conversion of L-arginine to signaling molecule in various biological processes L-citrulline, a reaction catalyzed by one of three NO including cell proliferation, differentiation, migration, synthases (NOS): neuronal NOS (nNOS), endothelial autophagy, and apoptosis [1-4]. These molecules can NOS (eNOS), and inducible NOS (iNOS) [8, 9]. NO decompose into a hydroxyl radical through Fenton has been shown as a tissue-protection through its reaction or Haber-Weiss reaction [5]. Hydroxyl physiologic regulation of vascular tone, inhibition of radicals can react with and damage vital cellular platelet aggregation, attenuation of leukocyte components, especially the mitochondria. At last cells adherence to the endothelium, scavenging of or tissues induces cell death, or accelerates aging and oxygen-derived free radicals, maintenance of normal cancer [6, 7]. vascular permeability, inhibition of smooth muscle Nitric oxide (NO) has been known to be proliferation, immune defenses, and stimulation of http://www.ijbs.com Int. J. Biol. Sci. 2018, Vol. 14 1975 endothelial cell regeneration [9]. Furthermore, NO is established in 1971 from the supraorbital metastatic to be a mediator/protector of ischemia and site in a 14-year-old Caucasian female with an Askin’s reperfusion tissue-mediated injury [9] and tumor related to Ewing's sarcoma. The SK-N-MC cells neuroprotector in neurodegenerative diseases show little or no dopamine-beta-hydroxylase activity including Parkinson’s disease (PD), Alzheimer’s but increase choline acetyltransferase activity disease (AD), Huntington’s disease (HD), and compared to other neuroblastoma cell lines such as amyotrophic lateral sclerosis (ALS) [8]. the SK-N-SH, SK-N-BE, and SH-SY5Y. Therefore, in In PC12 cells, NO protects against serum the present study, the NO suppresses H2O2-induced withdrawal-cell death by inhibiting the activation of human neuroblastoma SK-N-MC cell apoptosis by caspase through cGMP production and activation of suppressing apoptosis signal through activating protein kinase G (PKG) [10]. Also, this molecule PKG/PI3K/Akt and result in the increase of the suppresses 6-hydroxydopamine (6-OHDA)-induced interaction between 14‐3‐3β and Bad phosphorylation. apoptosis by activating PKG via phosphatidylinositol-3 kinase (PI3K)/Akt-dependent Results Bad phosphorylation [11]. In the primary neurons, The protection of NO against H2O2-induced NO-activated survival signaling involves nerve apoptosis in SK-N-MC cells growth factor (NGF)-like effects, including enhanced We investigated the protection of NO against phosphorylation of TrkA and activation of PI3K/Akt H2O2-induced apoptosis in human neuroblastoma and MAPK pathways [12]. Andoh et al. [13] reported SK-N-MC cells. H2O2 treatment (10-200 μM) induced that the cytoprotective effect of NO in human cell death in a dose-dependent manner and neuroblastoma SH-SY5Y cells appears to proceed via pretreatment of cells with 100 μM SNAP, an NO the PKG-mediated pathway and the expression of donor, attenuated the occurrence of H2O2-induced cell thioredoxin and thioredoxin peroxidase-1. Ciani et al. death; viabilities of cells treated with SNAP were [14] showed that NO protection in serum about 90, 83, 75, 70, and 52%, respectively, of the starved-SK-N-BE neuroblastoma cell apoptosis is control value (100%) (Figure 1A). DAPI staining associated with cAMP-response element-binding showed H2O2-induced nuclear fragmentation and NO protein (CREB) activation. The SK-N-MC cells are a treatment suppressed it (Figure 1B). human neuroblastoma cell line. This cell line was Figure 1. Protective effect of SNAP against the H2O2-induced death of SK-N-MC cells. The SK-N-MC cells were cultured in DMEM supplemented with 10% heat-inactivated FBS at 37°C in 5% CO2, 95% air in a humidified cell incubator. Cells were treated with H2O2 for 24 hr, and SNAP was added 10 hr prior to H2O2 treatment. Cell viability assay was performed by Cell Counting Kit-8 (A). DAPI staining was analyzed by fluorescence microscopy (B). Values were represented as mean ± SD from three independent experiments. Scale bar, 50 μm. **P < 0.01, ***P < 0.01 versus no treatment of SNAP. #p < 0.01, ##p < 0.01, ###p < 0.001 versus 100 μM SNAP treatment. +++p < 0.001, no treatment of 100 μM SNAP versus 100 μM SNAP treatment. http://www.ijbs.com Int. J. Biol. Sci. 2018, Vol. 14 1976 Figure 2. Protective effect of SNAP and caspase inhibitors on the survival of SK-N-MC cells. The SK-N-MC cells were cultured in DMEM supplemented with 10% heat-inactivated FBS at 37°C in 5% CO2, 95% air in a humidified cell incubator. Cells were treated with H2O2 for 24 hr, and SNAP was added 10 hr prior to H2O2 treatment. Cells were treated with SNAP (100 μM), Ac-YVAD-cho (200 μM), or Ac-DEVD-cho (200 μM) in media. At 24 hr, cell viability was determined by Cell Counting Kit-8 (A). SK-N-MC cells were collected, washed with ice-cold PBS, and lysed in 100 mM HEPES buffer, pH 7.4, containing protease inhibitors. Caspase-3 enzyme activity was determined by measuring the absorbance of the fraction at 405 nm, with 200 μM Ac-DEVD-pNA as a substrate for caspase-3 enzyme (B). Western blot analyses of caspase-3 activation and cytochrome c (C) were determined as described in Materials and methods. Values were represented as mean ± SD from three independent experiments. ***p < 0.01 versus nontreatment of SNAP. ###p < 0.001, 100 μM H2O2 treatment versus 100 μM SNAP/H2O2 or Ac-DEVD-cho/H2O2 treatment. The effect of SNAP and inhibitors of caspase Ac-DEVD-cho, respectively, but not by the addition of on SK-N-MC cell apoptosis Ac-YVAD-cho (Figure 2C). These results indicate that the NO on H2O2-treated SK-N-MC cells may inhibit We examined the effect of SNAP and inhibitors by an inhibition of caspase 3-like enzyme activation of caspase on SK-N-MC cell apoptosis and caspase and/or activity. 3-like activity. The caspase-3-like protease inhibitor Ac-DEVD-cho prevented H2O2-induced cell death, The role of cGMP in NO-mediated protection whereas the caspase-1-like protease inhibitor To determine the role of cGMP in NO-mediated Ac-YVAD-cho did not (Figure 2A). The protective protection, the effects of SNAP on SK-N-MC cells effect of Ac-DEVD-cho was comparable with the were examined in the presence of an inhibitor of effect of SNAP (100 μM) treatment. Caspase-3-like soluble guanylyl cyclase ODQ (Figure 3). ODQ did enzyme activity was measured in the cytosol from not affect the cell viability of cells but inhibited the H2O2-treated cells by a colorimetric assay using protective effect of SNAP on H2O2-treated cells substrate-specific tetrapeptides (Figure 2B). (Figure 3A). Also, treatment with ODQ prevented the Caspase-3-like enzyme activity in H2O2-treated cell capacity of SNAP to decrease caspase-3-like enzyme extracts was about 6-fold higher than that of untreated activity (Figure 3B) and to identify caspase-3 control cells.
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