The effects of kahweol, a diterpene present in coffee, on the mitochondria of the human neuroblastoma SH-SY5Y cells exposed to hydrogen peroxide
Introduction
The double-membrane organelles called mitochondria are the most important source of adenosine triphosphate (ATP) in any nucleated mammalian cell (Friedman and Nunnari, 2014). The bioenergetics functions of the mitochondria are dependent on the structure of these organelles. The inner mitochondrial membrane (IMM) presents, among other proteins, the complexes I, II, III, and IV of the electron transfer chain (ETC, also known as respiratory chain) and the complex V (ATP synthase/ATPase), forming the oxidative phosphorylation (OXPHOS) system (Papa et al., 2012). The flux of electrons from the complex I to the complex IV is mediated by electron transfer agents (namely coenzyme Q10/ubiquinone and cytochrome c) and generates an electrochemical gradient across the IMM, which is necessary to the complex V to produce ATP from adenosine diphosphate (ADP) and inorganic phosphate (Pi) (Papa et al., 2012). Oxygen gas (O2) is the final acceptor of electrons in the complex IV, and electron leakage from the ETC leads to the formation of reactive oxygen species (ROS) (Papa et al., 2012). Actually, the mitochondria are a major site of ROS production in different cell types (Dröse and Brandt, 2008; Dröse and Brandt, 2012; Lanciano et al., 2013; Sies et al., 2017). Partial reduction of O2 generates the superoxide anion radical (O2-•), which is converted into hydrogen peroxide (H2O2) by the enzyme manganese-superoxide dismutase (Mn-SOD), located in the mitochondria (Sies et al., 2017). Other antioxidant enzymes, such as catalase (CAT) and glutathione peroxidase (GPx), which may also be found in the mitochondria of some cell types, converts H2O2 into water (Sies et al., 2017). Accumulation of ROS in the mitochondria causes redox impairment of the mitochondrial membranes and inhibition of enzymes involved in the maintenance of the mitochondria-related bioenergetics functions, among other effects, as for instance oxidation of the mitochondrial DNA (Sies et al., 2017; Ott et al., 2007). Increased mitochondrial production of ROS also plays a role in the triggering of cell death by apoptosis through activation of the intrinsic apoptotic pathway, which is dependent on the release of pro-apoptotic factors, such as cytochrome c, from the organelles (Ott et al., 2007; Orrenius et al., 2007). Mitochondrial dysfunction, redox impairment, and increased rates of cell death figure among the molecular and cellular aspects associated with neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease (Brown and Bal-Price, 2003; Papa and De Rasmo, 2013; Zhao et al., 2017). Since there is not a cure for neurodegenerative diseases yet, chemically-induced prevention can be an interesting strategy in order to decrease the incidence of such maladies among humans.
In this context, kahweol (KW; C20H26O3), a diterpene present in coffee, exerts antioxidant, anti-inflammatory, and anti-tumor effects in different experimental models (Lee, 2007; Cárdenas et al., 2011; Cárdenas et al., 2014; Choi et al., 2019; Iwamoto et al., 2019). It has been described that, at least in part, the cytoprotective effects induced by KW can be mediated by the nuclear factor erythroid 2-related factor 2 (Nrf2), a major modulator of the expression of enzymes involved with antioxidant defense and phase II detoxification reactions in virtually any nucleated mammalian cell (Higgins et al., 2008; Kalthoff et al., 2010; Wu et al., 2014). Recently, a growing body of evidences indicates that Nrf2 may also be involved in the modulation of mitochondrial function and dynamics, as demonstrated by different experimental groups (Hayes and Dinkova-Kostova, 2014; Dinkova-Kostova and Abramov, 2015). Hwang and Jeong (2008) have reported that KW activated the phosphoinositide 3-kinase/Akt (PI3K/Akt) and the p38 mitogen-activated protein kinase (MAPK) signaling pathways, leading to Nrf2 upregulation and enhanced heme oxygenase-1 (HO-1) expression in the human neuroblastoma SH-SY5Y cell line (Hwang and Jeong, 2008). The inducible enzyme HO-1 mediates heme degradation, generating free iron, carbon monoxide (CO), and biliverdin, which is converted into bilirubin by biliverdin reductase (BVR) (Jansen and Daiber, 2012; Chung et al., 2013). Bilirubin is a potent antioxidant and also is involved in mitochondrial protection (Muhsain et al., 2015); however, bilirubin may also induce endoplasmic reticulum (ER) stress depending on the circumstances (Müllebner et al., 2015).
Even though there is information showing that KW causes cytoprotection in several experimental models, it was not demonstrated whether and how this diterpene would promote mitochondrial protection in cells facing a pro-oxidant challenge. Therefore, we have decided to evaluate whether a pretreatment with KW would be effective in preventing mitochondrial dysfunction in the human neuroblastoma SH-SY5Y cell line exposed to H2O2. The pro-oxidant agent H2O2 is produced at high rates in brain cells due to the activity of superoxide dismutase (SOD) and monoamine oxidase (MAO) enzymes (Cobley et al., 2018). H2O2 induces redox impairment by directly interacting with lipids present in biomembranes, for example, and by generating hydroxyl radical (•OH) through the Fenton and Haber-Weiss reactions (Chen et al., 2012; Pavlin et al., 2016). We also investigated whether Nrf2 and the protein kinases upstream Nrf2 would be associated with the effects induced by KW in this experimental model. The role of the HO-1 enzyme in the mitochondrial redox biology-related parameters was also examined here.
Section snippets
Materials
We have utilized plastic materials purchased from Corning, Inc. (NY, USA) and Beckton Dickson (NJ, USA) to perform cell culture. The analytical grade reagents necessary to maintain cell culture were obtained from Sigma (MO, USA). Several other specific chemicals and commercial assay kits were acquired as described here.
Cell culture and treatments
We acquired the human dopaminergic neuroblastoma SH-SY5Y cells from the American Type Culture Collection (Manassas, VA, USA) and cultured the cells in Dulbecco's modified Eagle's
KW pretreatment induced cytoprotection in H2O2-treated SH-SY5Y cells
We initially examined the effect of a pretreatment (for 12 h) with different KW concentrations on the viability of SH-SY5Y cells exposed to H2O2 at 300 μM. According to Fig. S1, KW at 0.1–1 μM did not prevent loss of viability in the H2O2-challenged cells. On the other hand, KW at 5 or 10 μM caused cytoprotective effects in this experimental model (p < .05). Therefore, we decided to utilize KW at 10 μM in the other assays we have performed. The effect of a pretreatment with KW at 10 μM on the
Discussion
Here we have shown that KW activated the PI3K/Akt and p38 MAPK/Nrf2/HO-1 signaling pathway, causing mitochondrial protection in the human SH-SY5Y cells exposed to H2O2. It was previously demonstrated that KW is a potent activator of pro-survival signaling pathways involved in the regulation of Nrf2 and HO-1 (Hwang and Jeong, 2008); however, this is the first time that it is demonstrated that KW is able to prevent mitochondrial disruption regarding the redox biology and function of the
Acknowledgement
MRO receives a “Bolsa de Produtividade em Pesquisa 2 - PQ2” fellow from the Conselho Nacional de Pesquisa e Desenvolvimento Tecnológico (CNPq) (protocol number 301273/2018-9). This research was supported by CNPq (protocol numbers 400216/2016-7 and 460903/2014-4).
Declaration of Competing Interests
None.
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