Chronic ethanol exposure induces SK-N-SH cell apoptosis by increasing N-methyl-D-aspartic acid receptor expression and intracellular calcium

Wang,Hongbo; Wang,Xiaolong; Li,Yan; Yu,Hao; Wang,Changliang; Feng,Chunmei; Xu,Guohui; Chen,Jiajun; You,Jiabin; Wang,Pengfei; Wu,Xu; Zhao,Rui; Zhang,Guohua

doi:10.3892/etm.2018.5902

Chronic ethanol exposure induces SK-N-SH cell apoptosis by increasing N-methyl-D-aspartic acid receptor expression and intracellular calcium

Authors:
- Hongbo Wang
- Xiaolong Wang
- Yan Li
- Hao Yu
- Changliang Wang
- Chunmei Feng
- Guohui Xu
- Jiajun Chen
- Jiabin You
- Pengfei Wang
- Xu Wu
- Rui Zhao
- Guohua Zhang
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Affiliations: Department of Forensic Pathology, School of Forensic Medicine, China Medical University, Shenyang, Liaoning 110122, P.R. China, No.1 English Department, School of Fundamental Sciences, China Medical University, Shenyang, Liaoning 110122, P.R. China
Published online on: February 28, 2018 https://doi.org/10.3892/etm.2018.5902
Pages: 3791-3800
Copyright: © Wang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

It has been identified that chronic ethanol exposure damages the nervous system, particularly neurons. There is scientific evidence suggesting that neuronal loss caused by chronic ethanol exposure has an association with neuron apoptosis and intracellular calcium oscillation is one of the primary inducers of apoptosis. Therefore, the present study aimed to investigate the inductive effects of intracellular calcium oscillation on apoptosis in SK‑N‑SH human neuroblastoma cells and the protective effects of the N‑methyl‑D‑aspartic acid receptor (NMDAR) antagonist, memantine, on SK‑N‑SH cell apoptosis caused by chronic ethanol exposure. SK‑N‑SH cells were treated with 100 mM ethanol and memantine (4 µM) for 2 days. Protein expression of NR1 was downregulated by RNA interference (RNAi). Apoptosis was detected by Annexin V/propidium iodide (PI) double‑staining and flow cytometry and cell viability was detected using an MTS kit. Fluorescence dual wavelength spectrophotometry was used to determine the intracellular calcium concentration and the levels of NR1 and caspase‑3 were detected using western blotting. NR1 mRNA levels were also detected using qPCR. It was found that chronic ethanol exposure reduced neuronal cell viability and caused apoptosis of SK‑N‑SH cells, and the extent of damage in SK‑N‑SH cells was associated with ethanol exposure concentration and time. In addition, chronic ethanol exposure increased the concentration of intracellular calcium in SK‑N‑SH cells by inducing the expression of NMDAR, resulting in apoptosis, and memantine treatment reduced ethanol‑induced cell apoptosis. The results of the present study indicate that the application of memantine may provide a novel strategy for the treatment of alcoholic dementia.

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1	Masaki T, Mochizuki H, Matsushita S, Yokoyama A, Kamakura K and Higuchi S: Association of aldehyde dehydrogenase-2 polymorphism with alcoholic polyneuropathy in humans. Neurosci Lett. 363:288–290. 2004. View Article : Google Scholar : PubMed/NCBI
2	Johansson S, Ekström TJ, Marinova Z, Okvist A, Sheedy D, Garrick T, Harper C, Kuzmin A, Yakovleva T and Bakalkin G: Dysregulation of cell death machinery in the prefrontal cortex of human alcoholics. Int J Neuropsychopharmacol. 12:109–115. 2009. View Article : Google Scholar : PubMed/NCBI
3	McVicker BL, Tuma DJ, Kharbanda KK, Kubik JL and Casey CA: Effect of chronic ethanol administration on the in vitro production of proinflammatory cytokines by rat kupffer cells in the presence of apoptotic cells. Alcohol Clin Exp Res. 31:122–129. 2007. View Article : Google Scholar : PubMed/NCBI
4	Crews F, Nixon K, Kim D, Joseph J, Shukitt-Hale B, Qin L and Zou J: BHT blocks NF-kappaB activation and ethanol-induced brain damage. Alcohol Clin Exp Res. 30:1938–1949. 2006. View Article : Google Scholar : PubMed/NCBI
5	Wang Y, Seitz HK and Wang X: Moderate alcohol consumption aggravates high-fat diet induced steatohepatitis in rats. Alcohol Clin Exp Res. 34:567–573. 2010. View Article : Google Scholar : PubMed/NCBI
6	Akane K, Kojima S, Mak TW, Shiku H and Suzuki H: CD8⁺CD122⁺CD49dlow regulatory T cells maintain T-cell homeostasis by killing activated T cells via Fas/FasL-mediated cytotoxicity. Proc Natl Acad Sci USA. 113:2460–2465. 2016. View Article : Google Scholar : PubMed/NCBI
7	Jeong JB, Choi J, Lou Z, Jiang X and Lee S: Patchouli alcohol, an essential oil of Pogostemoncablin, exhibits anti-tumorigenic activity in human colorectal cancer cells. Int Immunopharmacol. 16:184–190. 2013. View Article : Google Scholar : PubMed/NCBI
8	Zou J and Crews F: Induction of innate immune gene expression cascades in brain slice cultures by ethanol: Key role of NF-κB and proinflammatory cytokines. Alcohol Clin Exp Res. 34:777–789. 2010. View Article : Google Scholar : PubMed/NCBI
9	Baykara B, Micili SC, Tugyan K, Tekmen I, Bagriyanik H, Sonmez U, Sonmez A, Oktay G, Yener N and Ozbal S: The protective effects of carnosine in alcohol-induced hepatic injury in rats. Toxicol Ind Health. 30:25–32. 2014. View Article : Google Scholar : PubMed/NCBI
10	Bolnick JM, Karana R, Chiang PJ, Kilburn BA, Romero R, Diamond MP, Smith SM and Armant DR: Apoptosis of alcohol-exposed human placental cytotrophoblast cells is downstream of intracellular calcium signaling. Alcohol Clin Exp Res. 38:1646–1653. 2014. View Article : Google Scholar : PubMed/NCBI
11	Grynkiewicz G, Poenie M and Tsien RY: A new generation of Ca²⁺ indicators with greatly improved fluorescence properties. J Biol Chem. 260:3440–3450. 1985.PubMed/NCBI
12	La Rovere RM, Roest G, Bultynck G and Parys JB: Intracellular Ca(2+) signaling and Ca(2+) microdomains in the control of cell survival, apoptosis and autophagy. Cell Calcium. 60:74–87. 2016. View Article : Google Scholar : PubMed/NCBI
13	MacDermott AB, Mayer ML, Westbrook GL, Smith SJ and Barker JL: NMDA-receptor activation increases cytoplasmic calcium concentration in cultured spinal cord neurones. Nature. 321:519–522. 1986. View Article : Google Scholar : PubMed/NCBI
14	Finch EA, Turner TJ and Goldin SM: Calcium as a coagonist of inositol 1,4,5-trisphosphate-induced calcium release. Science. 252:443–446. 1991. View Article : Google Scholar : PubMed/NCBI
15	Bezprozvanny I, Watras J and Ehrlich BE: Bell-shaped calcium-response curves of Ins(1,4,5)P3- and calcium-gated channels from endoplasmic reticulum of cerebellum. Nature. 351:751–754. 1991. View Article : Google Scholar : PubMed/NCBI
16	Flatscher-Bader T and Wilce PA: Impact of alcohol abuse on protein expression of midkineand excitatory amino acid transporter 1 in the human prefrontal cortex. Alcohol Clin Exp Res. 32:1849–1858. 2008. View Article : Google Scholar : PubMed/NCBI
17	Sattler R and Tymianski M: Molecular mechanisms of calcium-dependent excitotoxicity. J Mol Med (Berl). 78:3–13. 2000. View Article : Google Scholar : PubMed/NCBI
18	Pérez-Otaño I and Ehlers MD: Homeostatic plasticity and NMDA receptor trafficking. Trends Neurosci. 28:229–238. 2005. View Article : Google Scholar : PubMed/NCBI
19	Lau CG and Zukin RS: NMDA receptor trafficking in synaptic plasticity and neuropsychiatric disorders. Nat Rev Neurosci. 8:413–426. 2007. View Article : Google Scholar : PubMed/NCBI
20	Glue P and Nutt D: Overexcitement and disinhibition. Dynamic neurotransmitter interactions in alcohol withdrawal. Br J Psychiatry. 157:491–499. 1990. View Article : Google Scholar : PubMed/NCBI
21	Addolorato G, Mirijello A, Leggio L, Ferrulli A and Landolfi R: Management of alcohol dependence in patients with liver disease. CNS Drugs. 27:287–299. 2013. View Article : Google Scholar : PubMed/NCBI
22	Petralia RS, Wang YX, Hua F, Yi Z, Zhou A, Ge L, Stephenson FA and Wenthold RJ: Corrigendum to organization of NMDA receptors at extrasynaptic locations. Neuroscience. 167:68–87. 2010. View Article : Google Scholar : PubMed/NCBI
23	Vicini S, Wang JF, Li JH, Zhu WJ, Wang YH, Luo JH, Wolfe BB and Grayson DR: Functional and pharmacological differences between recombinant N-methyl-D-aspartate receptors. J Neurophysiol. 79:555–566. 1998. View Article : Google Scholar : PubMed/NCBI
24	Chiu J, Brien JF, Wu P, Eubanks JH, Zhang L and Reynolds JN: Chronic ethanol exposure alters MK-801 binding sites in the cerebral cortex of the near-term fetal guinea pig. Alcohol. 17:215–221. 1999. View Article : Google Scholar : PubMed/NCBI
25	Devaud LL, Morrow AL and Nguyen UT: Ovariectomy has minimal effects on neuroadaptations associated with ethanol dependence in female rats. Neurochem Int. 37:433–442. 2000. View Article : Google Scholar : PubMed/NCBI
26	Zhang G, Dong Y, Zhang B, Ichinose F, Wu X, Culley DJ, Crosby G, Tanzi RE and Xie Z: Isoflurane-induced caspase-3 activation is dependent on cytosolic calcium and can be attenuated by memantine. J Neurosci. 28:4551–4560. 2008. View Article : Google Scholar : PubMed/NCBI
27	Wasfi IA, Al-Awadhi AH, Al-Hatali ZN, Al-Rayami FJ and Al Katheeri NA: Rapid and sensitive static headspace gas chromatography-mass spectrometry method for the analysis of ethanol and abused inhalants in blood. J Chromatogr B Analyt Technol Biomed Life Sci. 799:331–336. 2004. View Article : Google Scholar : PubMed/NCBI
28	Joshi R, Tawfik A, Edeh N, McCloud V, Looney S, Lewis J, Hsu S and Ogbureke KU: Dentin sialophosphoprotein (DSPP) gene-silencing inhibits key tumorigenic activities in human oral cancer cell line, OSC2. PLoS One. 5:e139742010. View Article : Google Scholar : PubMed/NCBI
29	Wang P, Wang Q, Yang L, Qin QL and Wu YJ: Characterization of lysophosphatidylcholine-induced changes of intracellular calcium in Drosophila S2 cells. Life Sci. 131:57–62. 2015. View Article : Google Scholar : PubMed/NCBI
30	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
31	Qi SH, Liu Y, Hao LY, Guan QH, Gu YH, Zhang J, Yan H, Wang M and Zhang GY: Neuroprotection of ethanol against ischemia/reperfusion-induced brain injury through decreasing c-Jun N-terminal kinase 3 (JNK3) activation by enhancing GABA release. Neuroscience. 167:1125–1137. 2010. View Article : Google Scholar : PubMed/NCBI
32	Nazam Ansari M, Bhandari U, Islam F and Tripathi CD: Evaluation of antioxidant and neuroprotective effect of ethanolic extract of Embelia ribes Burm in focal cerebral ischemia/reperfusion-induced oxidative stress in rats. Fundam Clin Pharmacol. 22:305–314. 2008. View Article : Google Scholar : PubMed/NCBI
33	Hwang DW, Givens B and Nishijima I: Ethanol-induced developmental neurodegeneration in secretin receptor-deficient mice. Neuroreport. 20:698–701. 2009. View Article : Google Scholar : PubMed/NCBI
34	Oliveira-da-Silva A, Vieira FB, Cristina-Rodrigues F, Filgueiras CC, Manhães AC and Abreu-Villaça Y: Increased apoptosis and reduced neuronal and glial densities in the hippocampus due to nicotine and ethanol exposure in adolescent mice. Int J Dev Neurosci. 27:539–548. 2009. View Article : Google Scholar : PubMed/NCBI
35	Harper C: The neuropathology of alcohol-related brain damage. Alcohol Alcohol. 44:136–140. 2009. View Article : Google Scholar : PubMed/NCBI
36	Mooney SM and Miller MW: Effects of prenatal exposure to ethanol on the expression of bcl-2, bax and caspase 3 in the developing rat cerebral cortex and thalamus. Brain Res. 911:71–81. 2001. View Article : Google Scholar : PubMed/NCBI
37	Brooks PJ: Brain atrophy and neuronal loss in alcoholism: A role for DNA damage? Neurochem Int. 37:403–412. 2000. View Article : Google Scholar : PubMed/NCBI
38	Kumral A, Tugyan K, Gonenc S, Genc K, Genc S, Sonmez U, Yilmaz O, Duman N, Uysal N and Ozkan H: Protective effects of erythropoietin against ethanol-induced apoptotic neurodegenaration and oxidative stress in the developing C57BL/6 mouse brain. Brain Res Dev Brain Res. 160:146–156. 2005. View Article : Google Scholar : PubMed/NCBI
39	Mooney SM and Miller MW: Nerve growth factor neuroprotection of ethanol-induced neuronal death in rat cerebral cortex is age dependent. Neuroscience. 149:372–381. 2007. View Article : Google Scholar : PubMed/NCBI
40	Li Y, Li R, Zhu S, Zhou R, Wang L, DU J, Wang Y, Zhou B and Mai L: Cordycepin induces apoptosis and autophagy in human neuroblastoma SK-N-SH and BE(2)-M17 cells. Oncol Lett. 9:2541–2547. 2015. View Article : Google Scholar : PubMed/NCBI
41	Hurley MM, Martin D and Raisz LG: Changes in ethanol concentration during incubation in multiwell tissue culture trays. Proc Soc Exp Biol Med. 186:139–141. 1987. View Article : Google Scholar : PubMed/NCBI
42	Borgs P, Way DL, Witte MH and Witte CL: Effective stabilization of ethanol levels in multiple-well tissue culture plates. Alcohol. 10:31–35. 1993. View Article : Google Scholar : PubMed/NCBI
43	Hanson CJ, Bootman MD and Roderick HL: Cell signalling: IP3 receptors channel calcium into cell death. Curr Biol. 14:R933–R935. 2004. View Article : Google Scholar : PubMed/NCBI
44	MacLennan DH, Rice WJ and Green NM: The mechanism of Ca²⁺ transport by sarco(endo)plasmic reticulum Ca²⁺-ATPases. J Biol Chem. 272:28815–28818. 1997. View Article : Google Scholar : PubMed/NCBI