Delphinidin-3-O-galactoside protects mouse hepatocytes from (−)-epigallocatechin-3-gallate–induced cytotoxicity via up-regulation of heme oxygenase-1 and heat shock protein 70
Introduction
Green tea contains characteristic polyphenolic constituents, generally known as green tea polyphenols, which include (−)-epigallocatechin-3-gallate (EGCG), (−)-epicatechin gallate, (−)-epigallocatechin, and (−)-epicatechin. (−)-Epigallocatechin-3-gallate, the most abundant polyphenol, can have versatile preventive effects on several chronic diseases [1], [2]. The mechanisms of action underlying these effects may be antioxidation against reactive oxygen species (ROS) as well as induction of antioxidant proteins such as heme oxygenase-1 (HO-1) [3], [4]. However, recent studies show that high-dose EGCG down-regulated the expression of HO-1, superoxide dismutase (SOD), and catalase [5], [6]. To support these observations, EGCG o-quinones were reported to rapidly react with the thiol group of glutathione, leading to prooxidation, cytotoxicity, and genotoxicity [7], [8], [9]. Moreover, several groups reported that high-dose EGCG caused hepatotoxicity, as demonstrated by increased formation of malonyldialdehyde and 4-hydroxynonenal. In addition, EGCG enhanced the endoplasmic reticulum (ER) stress in vivo and in vitro, which is relevant to the pathogenesis of Alzheimer disease via neuronal cell death [10], [11], [12]. Therefore, it is significant to identify the compounds that mask or suppress the adverse effects of EGCG.
Anthocyanins are natural colorants belonging to the flavonoid family and the largest group of water-soluble pigments in the plant kingdom. They are widely distributed in fruits, vegetables, and red wines [13], from which we intake significant amounts of anthocyanins, but anthocyanin pigments, in general, are chemically unstable and rapidly broken down under neutral pH conditions [14]. Nevertheless, recently, these anthocyanins have been shown to possess numerous biological functions including ROS scavenging [15]. In addition, these compounds were shown to have antioxidative, anti-inflammatory, and antidiabetic activity in animal models [16], [17], [18]. In fact, cyanidin-3-O-glucoside, one of the most abundant anthocyanins, induced expression of HO-1, reduced cyclooxygenase-2 and inducible nitric oxide synthase expressions, and protected against ROS-induced DNA damage in endothelial cells, smooth muscle, hepatoma cells, and tumor promotion in mouse JB6 cells [19], [20], [21]. In addition, delphinidin (aglycon form) also possesses a strong antioxidant property and other potentially beneficial traits such as anti-inflammation [16]. To our knowledge, there are no known reports on the prooxidative property of anthocyanins (glycoside form), which differ from EGCG.
It is well known that self-protective proteins such as heat shock proteins (HSPs) are a class of stress-inducible proteins that act as molecular chaperones and protect cells against proteotoxic damages from a variety of physiological and environmental stimulus, including heat shock and oxidative stresses [22], [23], [24]. Interestingly, Renis et al [25] reported that anthocyanins protected CaCo2 human colonic adenocarcinoma cells from DNA damage via induction of HSP70. Hence, we hypothesized that anthocyanins are capable of protecting themselves from the EGCG-induced adverse effects by inducing self-protective proteins and antioxidant enzymes. To test this hypothesis, we investigated whether delphinidin-3-O-galactoside (D3G) treatment had a protective effect against EGCG-induced adverse effects such as cytotoxicity and ER stress. In this study, we demonstrated that D3G not only increased HO-1 and HSP70 messenger RNA (mRNA) expressions but also decreased the EGCG-mediated cytotoxicity and ER stress responses. These findings may provide new insights into the molecular mechanisms underlying the potential masking effects of D3G on EGCG-induced cytotoxicity for hepatocytes cells.
Section snippets
Cell culture and reagent
Hepa1c1c-7 mouse hepatocytes cells (passages 15-24) were cultured in Dulbecco modified Eagle medium (DMEM) and supplemented with 10% fetal bovine serum at 37°C in 5% CO2. Both DMEM and fetal bovine serum were purchased from Invitrogen (Carlsbad, California). Delphinidin-3-O-galactoside was purified by Asahi Breweries Ltd (Ibaraki, Japan). (−)-Epigallocatechin-3-gallate was purchased from Wako Pure Chemicals (Osaka, Japan), dissolved in dimethyl sulfoxide (DMSO) (Wako Pure Chemicals) and stored
Effects of D3G and EGCG on cell viability
Cytotoxic effects of EGCG and D3G were determined in heapa1c1c-7 cells by using WST-8 assay. Incubation of the cells with EGCG for 6-hour concentration dependently decreased the cell viability as compared with the vehicle control (Fig. 1A) and exerted more pronounced cytotoxicity at 24 hours (Fig. 1B). In contrast, D3G had no cytotoxicity at 6 hours (Fig. 1C) but decreased cell viability by 40% and 48% at 300 and 500 μM at 24 hours, respectively (Fig. 1D).
Effects of D3G on EGCG-induced cytotoxicity
Subsequently, we tested whether D3G
Discussion
Several independent groups have recently reported that high-dose green tea extracts, or EGCG, caused hepatotoxicity in mice and human [10], [29], [30]. It is worth noting that at high doses, EGCG is readily autooxidized in a cell culture medium to produce the superoxide H2O2 and, perhaps, other ROS that can lead to prooxidation-mediated adverse effects such as genotoxicity and ER stress [7], [8], [9], [10], [11], [31]. Therefore, it is significant to identify the compounds that mask or suppress
Acknowledgment
This work was supported by Research and Development Projects for Application in Promoting New Policy of Agriculture Forestry and Fisheries, from the Ministry of Agriculture, Forestry and Fisheries, Japan (Grant No. 23005).
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