Di-O-Demethylcurcumin Protects SK-N-SH.Pdf
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Neurochemistry International 80 (2015) 110–119 Contents lists available at ScienceDirect Neurochemistry International journal homepage: www.elsevier.com/locate/nci Di-O-demethylcurcumin protects SK-N-SH cells against mitochondrial and endoplasmic reticulum-mediated apoptotic cell death induced by Aβ25-35 Decha Pinkaew a, Chatchawan Changtam b, Chainarong Tocharus c, Sarinthorn Thummayot c, Apichart Suksamrarn d, Jiraporn Tocharus a,* a Department of Physiology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand b Division of Physical Science, Faculty of Science and Technology, Huachiew Chalermprakiet University, Samutprakarn 10540, Thailand c Department of Anatomy, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand d Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Ramkhamhaeng University, Bangkok 10240, Thailand ARTICLE INFO ABSTRACT Article history: Alzheimer’s disease (AD) is a neurodegenerative and progressive disorder. The hallmark of pathological Received 2 July 2014 AD is amyloid plaque which is the accumulation of amyloid β (Aβ) in extracellular neuronal cells and Received in revised form 20 October 2014 neurofibrillary tangles (NFT) in neuronal cells, which lead to neurotoxicity via reactive oxygen species Accepted 21 October 2014 (ROS) generation related apoptosis. Loss of synapses and synaptic damage are the best correlates of cog- Available online 24 October 2014 nitive decline in AD. Neuronal cell death is the main cause of brain dysfunction and cognitive impairment. Aβ activates neuronal death via endoplasmic reticulum (ER) stress and mitochondria apoptosis pathway. Keywords: This study investigated the underlying mechanisms and effects of di-O-demethylcurcumin in prevent- Alzheimer’s disease Amyloid beta ing Aβ-induced apoptosis. Pretreatment with di-O-demethylcurcumin for 2 h, which was followed by Aβ25- Apoptosis 35 (10 μM) in human neuroblastoma SK-N-SH cells improved cell viability by using MTS assay and decreased Di-O-demethylcurcumin neuronal cell apoptosis. Pretreatment with di-O-demethylcurcumin attenuated the number of nuclear Endoplasmic reticulum stress condensations and number of apoptotic cells in Aβ25-35-induced group in a concentration-dependent manner Mitochondrial death pathway by using transmission electron microscope (TEM) and flow cytometry, respectively. Di-O-demethylcurcumin also increased the ratio of Bcl-XL/Bax protein, and reduced intracellular ROS level, cytochrome c protein expression, cleaved caspase-9 protein expression, and cleaved caspase-3 protein expression. Addition- ally, di-O-demethylcurcumin treatment also reduced the expression of ER stress protein markers, including protein kinase RNA like endoplasmic reticulum kinase (PERK) phosphorylation, eukaryotic translation initiation factor 2 alpha (eIF2α) phosphorylation, inositol-requiring enzyme 1 (IRE1) phosphorylation, X-box-binding protein-1 (XBP-1), activating transcription factor (ATF6), C/EBP homologous protein (CHOP), and cleaved caspase-12 protein. CHOP and cleaved caspase-12 protein are the key mediators of apop- tosis. Our data suggest that di-O-demethylcurcumin is a candidate protectant against neuronal death through its suppression of the apoptosis mediated by mitochondrial death and ER stress pathway. © 2014 Elsevier Ltd. All rights reserved. 1. Introduction 2002; Selkoe, 2001) that cause loss of neurons and synapses in the brain. Amyloid β (Aβ) is derived from the proteolytic cleavage of the Alzheimer’s disease (AD) is a progressive neurodegenerative amyloid precursor protein (APP) by β- and γ-secretase enzymes, and disease of the central nervous system (CNS), affecting, generally, accumulate in the extracellular neuronal cells (Sisodia et al., 2001). elderly people. AD is one of the most common forms of dementia, NFT is accumulated within the neurons as a result of abnormal phos- which is determined clinically by diagnosing for multiple cogni- phorylation of the microtubules-associated tau-protein. Recent tive deficits including memory loss, emotional disturbance, etc. studies suggest that Aβ accumulation has been causatively impli- (Hauptmann et al., 2006). The pathological hallmarks of AD are cated in the neuronal dysfunction and neuronal loss that underlie amyloid plaques and neurofibrillary tangles (NFT) (Hardy and Selkoe, the clinical manifestations of AD (Dante et al., 2003). Further- more, several lines of research have suggested that Aβ exerts neuronal toxicity through the production of reactive oxygen species (ROS) (Behl et al., 1994; Butterfield et al., 2007), which leads to neu- ronal apoptosis (Ramalingam and Kim, 2012) due to the involvement * Corresponding author. Department of Physiology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand. Tel.: +66 53 945362; fax: +66 53 945365. of the mitochondrial death pathway and the endoplasmic reticu- E-mail address: [email protected] (J. Tocharus). lum (ER) stress (Takuma et al., 2005). ER is an intracellular organelle http://dx.doi.org/10.1016/j.neuint.2014.10.008 0197-0186/© 2014 Elsevier Ltd. All rights reserved. D. Pinkaew et al./Neurochemistry International 80 (2015) 110–119 111 related to protein folding, intracellular calcium homeostasis, lipid medium (MEM), fetal bovine serum (FBS), penicillin, and strepto- synthesis, steroids and cholesterol. Cellular conditions in ER, such mycin were purchased from GIBCO-BRL (Gaithersburg, MD, USA). as depletion of calcium, alter glycosylation, and the oxidative stress The following antibodies were used for the western blot analysis: leads to unfolded protein. Unfolded protein accumulation in ER anti-Bcl-XL, anti-Bax, anti-cytochrome c, anti-cleaved caspase-3, lumen is termed as ER stress (Nakagawa et al., 2000); this accu- anti-cleaved caspase-9, anti-β-actin, anti-mouse IgG peroxidase- mulation activates unfolded protein response (UPR), thereby signaling conjugated secondary antibody, anti-rabbit IgG peroxidase- responses for ER homeostasis by decreasing protein synthesis, and conjugated secondary antibody (Millipore, Bedford, MA, USA), increasing chaperone and degradation (Schroder and Kaufman, 2005; anti-Grp78, anti-CHOP, anti-PERK, anti-phospho-PERK, anti-phospho- Vembar and Brodsky, 2008). However, prolonged ER stress is as- eIF2α (Cell Signaling Technology, MA, USA), anti-ATF6, anti-XBP-1 sociated with apoptotic cell death (Boyce and Yuan, 2006), which (Santa Cruz Biotechnology, CA, USA), anti-phospho-IRE1α and anti- is caused by activating three signal transducers that are protein caspase-12 (Abcam, Cambridge, UK) and anti-β-actin. kinase RNA like endoplasmic reticulum kinase (PERK), activating transcription factor (ATF6) and inositol-requiring enzyme 1 (IRE1); then, upregulates the expression of the C/EBP homologous protein 2.2. Methods (CHOP) (Chen et al., 2012), caspase-12 protein (Nakagawa et al., 2000), and glucose related protein 78 (Grp78) (Kim et al., 2008). Aβ25-35 was dissolved in deionized distilled water at concentra- Moreover, apoptosis mainly involves excessive ROS-induced mito- tion of 1 mM and storage at −80 °C. Before it was used in each of chondrial dysfunction upon the increase in the permeability of the experiments, it was aggregated at 37 °C for a week, after which mitochondria membrane (Chen and Yan, 2007). These results in the it was diluted to the required concentration with sterile water. inducing of proapoptotic protein such as Bax translocate from the cytoplasm to the mitochondria membrane and a decrease in the anti- 2.2.1. Cell culture apoptotic proteins (Bcl-2, Bcl-XL). Thereafter, cytochrome c gets The SK-N-SH cells were cultured in MEM supplemented with 10% released to the cytoplasm and combines with apoptotic protease heat inactivated FBS, 100 units/ml penicillin, and 100 μg/ml strep- activating factor 1 (APAF-1) and procaspase-9 to become the active tomycin in humidified 95% air, at 37 °C and 5% CO in an incubator. form of caspase-9 protein which stimulates the caspase-3 protein, 2 The cells were passaged by trypsinization every 2–3 days. and this leads to cell death (Li et al., 1997; Slee et al., 1999). There- fore, evidences suggest that suppression of Aβ-mediated neuronal apoptosis would be a target to attenuate progressive neuronal 2.2.2. Preparation of di-O-demethylcurcumin damages and provide a strategy for the given approach to the de- Curcumin (850 mg, 2.30 mmol), obtained from Curcuma longa velopment and treatment of AD. as described previously (Changtam et al., 2010), was dissolved in Curcumin is the major constituent of curcuminoids isolated from dry CH2Cl2 (90 ml); the mixture was stirred at 0 °C or 5 min and BBr3 turmeric (Curcuma longa L.), with demethoxycurcumin and (1 ml) was slowly added. The reaction mixture was stirred at 0 °C bisdemethoxycurcumin being the minor constituents. Previous for 30 min and more BBr3 (1 ml) was slowly added. After stirring studies have shown that curcumin has a wide range of beneficial at 0 °C for 1 h, water (200 ml) was added and the mixture was ex- properties, including antioxidant activity, anti-inflammatory activ- tracted with EtOAc. The combined organic phase was washed with ity, anticancer activity, neuroprotective effects, and antiviral activity water, dried over anhydrous Na2SO4 and the solvent was removed (Bandgar et al., 2014; Belviranli et al., 2013; Bhullar et al., 2013; under vacuum. The crude products were separated by column Morsy and El-Moselhy, 2013; Rath et al., 2013). Several groups of chromatography