Necrostatin-1 protects C2C12 myotubes from CoCl2-induced hypoxia

Chen,Rui; Xu,Jiehua; She,Yanling; Jiang,Ting; Zhou,Shanyao; Shi,Huacai; Li,Cheng

doi:10.3892/ijmm.2018.3466

Necrostatin-1 protects C2C12 myotubes from CoCl2-induced hypoxia

Authors:
- Rui Chen
- Jiehua Xu
- Yanling She
- Ting Jiang
- Shanyao Zhou
- Huacai Shi
- Cheng Li
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Affiliations: Guangdong Traditional Medical and Sports Injury Rehabilitation Research Institute, Guangdong Second Provincial General Hospital, Guangzhou, Guangdong 510317, P.R. China, Department of Nuclear Medicine, The Third Affiliated Hospital, Sun Yat‑sen University, Guangzhou, Guangdong 510630, P.R. China, Department of Radiology, The Third Affiliated Hospital, Sun Yat‑sen University, Guangzhou, Guangdong 510630, P.R. China
Published online on: February 6, 2018 https://doi.org/10.3892/ijmm.2018.3466
Pages: 2565-2572
Copyright: © Chen et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Necrostatin-1 (Nec-1) is a selective and potent allosteric inhibitor of necroptosis by specifically inhibiting the activity of receptor‑interacting protein (RIP) 1 kinase. The aim of the present study was to determine the effect of Nec‑1 on an anoxia model comprising mouse skeletal C2C12 myotubes. In the present study, a hypoxic mimetic reagent, cobalt chloride (CoCl2), was used to induce hypoxia in C2C12 myotubes. The cytotoxic effects of CoCl2‑induced hypoxia were determined by a Cell Counting kit‑8 assay and flow cytometry. Transmission electron microscopy (TEM) was used to characterize the morphological characteristics of dead cells at the ultrastructural level. To clarify the signaling pathways in CoCl2‑mediated cell death, the expression levels of RIP1, RIP3, extracellular signal‑regulated kinase (ERK)1/2, hypoxia‑inducible factor (HIF)‑1α and B cell lymphoma‑2 adenovirus E1B 19‑kDa interacting protein 3 (BNIP3) were investigated by western blotting. Oxidative stress was determined using 2',7'‑dichlorofluorescin diacetate to measure intracellular reactive oxygen species (ROS) and the fluorescent dye JC‑1 was used to measure mitochondrial membrane potential (Δψm). The results showed that the ratios of apoptotic and necrotic C2C12 cells were increased following CoCl2 treatment, typical necroptotic morphological characteristics were able to observe by TEM, whereas Nec‑1 exhibited a protective effect against CoCl2‑induced oxidative stress. Treatment with Nec‑1 significantly decreased the levels of RIP1, p‑ERK1/2, HIF‑1α, BNIP3 and ROS induced by CoCl2, and promoted C2C12 differentiation. Nec‑1 reversed the CoCl2‑induced decrease in mitochondrial membrane potential. Together, these findings suggested that Nec‑1 protected C2C12 myotubes under conditions of CoCl2-induced hypoxia.

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1	Kawamura I, Takemura G, Kanamori H, Takeyama T, Kawaguchi T, Tsujimoto A, Goto K, Maruyama R, Watanabe T, Shiraki T, et al: Repeated phlebotomy augments angiogenesis to improve blood flow in murine ischemic legs. Am J Physiol Heart Circ Physiol. 299:H372–H378. 2010. View Article : Google Scholar : PubMed/NCBI
2	Masschelein E, Van Thienen R, D'Hulst G, Hespel P, Thomis M and Deldicque L: Acute environmental hypoxia induces LC3 lipidation in a genotype-dependent manner. FASEB J. 28:1022–1034. 2014. View Article : Google Scholar
3	Samaras N, Samaras D, Chambellan A, Pichard C and Thibault R: Pulmonary rehabilitation: The reference therapy for undernourished patients with chronic obstructive pulmonary disease. Biomed Res Int. 2014:2484202014. View Article : Google Scholar : PubMed/NCBI
4	Saxena S, Shukla D, Saxena S, Khan YA, Singh M, Bansal A, Sairam M and Jain SK: Hypoxia preconditioning by cobalt chloride enhances endurance performance and protects skeletal muscles from exercise-induced oxidative damage in rats. Acta Physiol (Oxf). 200:249–263. 2010. View Article : Google Scholar
5	Rovetta F, Stacchiotti A, Faggi F, Catalani S, Apostoli P, Fanzani A and Aleo MF: Cobalt triggers necrotic cell death and atrophy in skeletal C2C12 myotubes. Toxicol Appl Pharm. 271:196–205. 2013. View Article : Google Scholar
6	Cervellati F, Cervellati C, Romani A, Cremonini E, Sticozzi C, Belmonte G, Pessina F and Valacchi G: Hypoxia induces cell damage via oxidative stress in retinal epithelial cells. Free Radical Res. 48:303–312. 2014. View Article : Google Scholar
7	Yuan Y, Hilliard G, Ferguson T and Millhorn DE: Cobalt inhibits the interaction between hypoxia-inducible factor-alpha and von Hippel-Lindau protein by direct binding to hypoxia-inducible factor-alpha. J Biol Chem. 278:15911–15916. 2003. View Article : Google Scholar : PubMed/NCBI
8	Linkermann A and Green DR: Necroptosis. New Engl J Med. 370:455–465. 2014. View Article : Google Scholar : PubMed/NCBI
9	Laster SM, Wood JG and Gooding LR: Tumor necrosis factor can induce both apoptic and necrotic forms of cell lysis. J Immunol. 141:2629–2634. 1988.PubMed/NCBI
10	Degterev A, Huang Z, Boyce M, Li Y, Jagtap P, Mizushima N, Cuny GD, Mitchison TJ, Moskowitz MA and Yuan J: Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat Chem Biol. 1:112–119. 2005. View Article : Google Scholar
11	Han W, Xie J, Fang Y, Wang Z and Pan H: Nec-1 enhances shikonin-induced apoptosis in leukemia cells by inhibition of RIP-1 and ERK1/2. Int J Mol Sci. 13:7212–7225. 2012. View Article : Google Scholar : PubMed/NCBI
12	Cho YS, Challa S, Moquin D, Genga R, Ray TD, Guildford M and Chan FK: Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation. Cell. 137:1112–1123. 2009. View Article : Google Scholar : PubMed/NCBI
13	Chen JK, Zhan YJ, Yang CS and Tzeng SF: Oxidative stress-induced attenuation of thrombospondin-1 expression in primary rat astrocytes. J Cell Biochem. 112:59–70. 2011. View Article : Google Scholar
14	Locatelli SL, Cleris L, Stirparo GG, Tartari S, Saba E, Pierdominici M, Malorni W, Carbone A, Anichini A and Carlo-Stella C: BIM upregulation and ROS-dependent necroptosis mediate the antitumor effects of the HDACi Givinostat and Sorafenib in Hodgkin lymphoma cell line xenografts. Leukemia. 28:1861–1871. 2014. View Article : Google Scholar : PubMed/NCBI
15	Obitsu S, Sakata K, Teshima R and Kondo K: Eleostearic acid induces RIP1-mediated atypical apoptosis in a kinase-independent manner via ERK phosphorylation, ROS generation and mitochondrial dysfunction. Cell Death Dis. 4:e6742013. View Article : Google Scholar : PubMed/NCBI
16	Choi H, Merceron C, Mangiavini L, Seifert EL, Schipani E, Shapiro IM and Risbud MV: Hypoxia promotes noncanonical autophagy in nucleus pulposus cells independent of MTOR and HIF1A signaling. Autophagy. 12:1631–1646. 2016. View Article : Google Scholar : PubMed/NCBI
17	Feng CC, Lin CC, Lai YP, Chen TS, Marthandam Asokan S, Lin JY, Lin KH, Viswanadha VP, Kuo WW and Huang CY: Hypoxia suppresses myocardial survival pathway through HIF-1α-IGFBP-3-dependent signaling and enhances cardiomyocyte autophagic and apoptotic effects mainly via FoxO3a-induced BNIP3 expression. Growth Factors. 34:73–86. 2016. View Article : Google Scholar : PubMed/NCBI
18	Mammucari C, Milan G, Romanello V, Masiero E, Rudolf R, Del Piccolo P, Burden SJ, Di Lisi R, Sandri C, Zhao J, et al: FoxO3 controls autophagy in skeletal muscle in vivo. Cell Metab. 6:458–471. 2007. View Article : Google Scholar : PubMed/NCBI
19	Kim JY, Kim YJ, Lee S and Park JH: BNip3 is a mediator of TNF-induced necrotic cell death. Apoptosis. 16:114–126. 2011. View Article : Google Scholar
20	Wang H and Zhang B: Cobalt chloride induces necroptosis in human colon cancer HT-29 cells. Asian Pac J Cancer Prev. 16:2569–2574. 2015. View Article : Google Scholar : PubMed/NCBI
21	Huang YC, Tsai MS, Hsieh PC, Shih JH, Wang TS, Wang YC, Lin TH and Wang SH: Galangin ameliorates cisplatin-induced nephrotoxicity by attenuating oxidative stress, inflammation and cell death in mice through inhibition of ERK and NF-kappaB signaling. Toxicol Appl Pharmacol. 329:128–139. 2017. View Article : Google Scholar : PubMed/NCBI
22	Liu S, Ai Q, Feng K, Li Y and Liu X: The cardioprotective effect of dihydromyricetin prevents ischemia–reperfusion-induced apoptosis in vivo and in vitro via the PI3K/Akt and HIF-1α signaling pathways. Apoptosis. 21:1366–1385. 2016. View Article : Google Scholar : PubMed/NCBI
23	Zhang M, Li J, Geng R, Ge W, Zhou Y, Zhang C, Cheng Y and Geng D: The inhibition of ERK activation mediates the protection of necrostatin-1 on glutamate toxicity in HT-22 cells. Neurotox Res. 24:64–70. 2013. View Article : Google Scholar : PubMed/NCBI
24	Gao S, Andreeva K and Cooper NG: Ischemia-reperfusion injury of the retina is linked to necroptosis via the ERK1/2-RIP3 pathway. Mol Vis. 20:1374–1387. 2014.PubMed/NCBI
25	Zhao M, Lu L, Lei S, Chai H, Wu S, Tang X, Bao Q, Chen L, Wu W and Liu X: Inhibition of receptor interacting protein kinases attenuates cardiomyocyte hypertrophy induced by palmitic acid. Oxid Med Cell Longev. 2016:14516762016. View Article : Google Scholar : PubMed/NCBI
26	Chen R, Jiang T, She Y, Xu J, Li C, Zhou S, Shen H, Shi H and Liu S: Effects of cobalt chloride, a hypoxia-mimetic agent, on autophagy and atrophy in skeletal C2C12 myotubes. Biomed Res Int. 2017:70975802017.PubMed/NCBI
27	Chen G, Cheng X, Zhao M, Lin S, Lu J, Kang J and Yu X: RIP1-dependent Bid cleavage mediates TNFα-induced but Caspase-3-independent cell death in L929 fibroblastoma cells. Apoptosis. 20:92–109. 2015. View Article : Google Scholar
28	Das A, McDonald DG, Dixon-Mah YN, Jacqmin DJ, Samant VN, Vandergrift WA III, Lindhorst SM, Cachia D, Varma AK, Vanek KN, et al: RIP1 and RIP3 complex regulates radiation-induced programmed necrosis in glioblastoma. Tumor Biol. 37:7525–7534. 2016. View Article : Google Scholar
29	Chavez-Valdez R, Martin LJ, Flock DL and Northington FJ: Necrostatin-1 attenuates mitochondrial dysfunction in neurons and astrocytes following neonatal hypoxia-ischemia. Neuroscience. 219:192–203. 2012. View Article : Google Scholar : PubMed/NCBI
30	Festjens N, Vanden Berghe T, Cornelis S and Vandenabeele P: RIP1, a kinase on the crossroads of a cell's decision to live or die. Cell Death Differ. 14:400–410. 2007. View Article : Google Scholar : PubMed/NCBI
31	Locatelli SL, Careddu G, Stirparo GG, Castagna L, Santoro A and Carlo-Stella C: Dual PI3K/ERK inhibition induces necroptotic cell death of Hodgkin Lymphoma cells through IER3 downregulation. Sci Rep. 6:357452016. View Article : Google Scholar : PubMed/NCBI
32	Xie X, Zhao Y, Ma CY, Xu XM, Zhang YQ, Wang CG, Jin J, Shen X, Gao JL, Li N, et al: Dimethyl fumarate induces necroptosis in colon cancer cells through GSH depletion/ROS increase/MAPKs activation pathway. Brit J Pharmacol. 172:3929–3943. 2015. View Article : Google Scholar
33	Zhao W, Feng H, Sun W, Liu K, Lu JJ and Chen X: Tert-butyl hydroperoxide (t-BHP) induced apoptosis and necroptosis in endothelial cells: Roles of NOX4 and mitochondrion. Redox Biol. 11:524–534. 2017. View Article : Google Scholar : PubMed/NCBI