The effect of the phenol compound ellagic acid on Ca2þ homeostasis and cytotoxicity in liver cells

Wei-Zhe Liang a, Chiang-Ting Chou b,c, Jin-Shiung Cheng d, Jue-Long Wang e, Hong-Tai Chang f, I-Shu Chen f, Ti Lu g, Jeng-Hsien Yeh h, Daih-Huang Kuo i, Pochuen Shieh i, Fu-An Chen i, Chun-Chi Kuo j, Chung-Ren Jan a,n

Abstract

Ellagic acid, a natural phenol compound found in numerous fruits and vegetables, causes various physiological effects in different cell models. However, the effect of this compound on Ca2þ homeostasis in liver cells is unknown. This study examined the effect of ellagic acid on intracellular Ca2þ concentration ([Ca2þ]i) and established the relationship between Ca2þ signaling and cytotoxicity in liver cells. The data show that ellagic acid induced concentration-dependent [Ca2þ]i rises in HepG2 human hepatoma cells, but not in HA22T, HA59T human hepatoma cells or AML12 mouse hepatocytes. In HepG2 cells, this Ca2þ signal response was reduced by removing extracellular Ca2þ and was inhibited by store-operated Ca2þ channel blockers (2-APB, econazole or SKF96365) and the protein kinase C (PKC) inhibitor GF109203X. In Ca2þ-free medium, pretreatment with the endoplasmic reticulum Ca2þ pump inhibitor thapsigargin abolished ellagic acid-induced [Ca2þ]i rises. Conversely, incubation with ellagic acid abolished thapsigargin-induced [Ca2þ]i rises. Inhibition of phospholipase C (PLC) with U73122 also abolished ellagic acidinduced [Ca2þ]i rises. Ellagic acid (25–100 μM) concentration-dependently caused cytotoxicity in HepG2, HA22T or HA59T cells, but not in AML12 cells. Furthermore, this cytotoxic effect was partially prevented by prechelating cytosolic Ca2þ with BAPTA-AM only in HepG2 cells. Together, in HepG2 cells, ellagic acid induced [Ca2þ]i rises by inducing PLC-dependent Ca2þ release from the endoplasmic reticulum and Ca2þ entry via PKC-sensitive store-operated Ca2þ channels. Moreover, ellagic acid induced Ca2þ-associated cytotoxicity.

Keywords:
Ca2þ
Cytotoxicity
Ellagic acid
Liver cells
Phenolic compound

1. Introduction

Phenolic compounds have a wide range of pharmacological actions including anticarcinogenic properties in different models (Abliz et al., 2015; Gossé et al., 2005; Weisburg et al., 2013). Ellagic acid, a natural phenolic antioxidant, is found in many fruits, nut galls and plant extracts in the forms of hydrolysable tannins called ellagitannins such as raspberries, strawberries, grapes, pomegranate, black currants, camu-camu, mango, guava, walnuts, almonds, longan seeds and green tea (García-Niño and Zazueta, 2015; Rasool et al., 2015; Williner et al., 2003). Structurally, ellagic acid presents four rings representing the lipophilic domain, four phenolic groups and two lactones, which form hydrogen-bonds sides and act as electron acceptors respectively, and that represent the hydrophilic domain (García-Niño and Zazueta, 2015).
Ellagic acid was shown to have antioxidant, antihepatotoxic, antisteatosic, anticholestatic, antifibrogenic, antihepatocarcinogenic and antiviral properties (García-Niño and Zazueta, 2015; Rasool et al., 2015; Williner et al., 2003). In recent years, ellagic acid has gained attention due to its multiple biological activities and several molecular targets. Previous studies have shown that ellagic acid may evoke a spectrum of cell signaling pathways to attenuate or slow down the development of neurodegenerative disorders (Ahmed et al., 2016). Ellagic acid possesses potent neuroprotective effects through its free radical scavenging properties, iron chelation, and mitigation of mitochondrial dysfunction (Ahmed et al., 2016). In liver research, ellagic acid has been shown to improve hepatic steatosis and serum lipid composition in diabetic mice (Yoshimura et al., 2013). Additionally, ellagic acid reduces the liver oxidative stress induced by ischemiareperfusion injury and protects hepatocytes from damage by inhibiting mitochondrial production of reactive oxygen species (Hwang et al., 2010). Furthermore, a lot of ellagic acid derivatives such as 3,3′-di-O-methyl ellagic acid-4′-O-β-D-xylopyranoside (JNE2) (Zhang et al., 2014a, 2014b) or urolithin A (Wang et al., 2015) has been shown to cause cytotoxicity in human hepatoma cell lines. Although ellagic acid has various physiological effects on liver, the effect of ellagic acid on Ca2þ homeostasis in liver cells is unknown.
Unlike other cations, Ca2þ is a pivotal second messenger in almost all cells (Clapham, 1995). Failure to maintain regulated intracellular Ca2þ concentration ([Ca2þ]i) levels may cause many diseases (Berridge, 2012). A transient rise in [Ca2þ]i is called a Ca2þ signal (Bootman, 1994). This Ca2þ signal can control various cellular responses such as growth, death, gene expression, secretion, contraction, fertilization, etc. (Clapham, 1995; Berridge, 2006). [Ca2þ]i is controlled by extracellular Ca2þ entry or intracellular Ca2þ release, or both (Clapham, 1995; Berridge, 2006). Ca2þ may enter cells through various channels. In non-excitable cells such as human hepatoma cells, the main Ca2þ channel is the store-operated Ca2þ channel which is triggered by depletion of the endoplasmic reticulum Ca2þ store (Putney, 1986). In excitable cells such as neurons, the voltage-gated Ca2þ channels play a crucial role (Clapham, 1995; Berridge, 2012). The main Ca2þ store in most cell types is the endoplasmic reticulum. Thus it is important to explore the mechanisms of different compounds-induced Ca2þ entry and Ca2þ release, in order to understand the impact of these compounds on physiology of the cells. Because the effect of ellagic acid on [Ca2þ]i in liver cells has never been explored, the aim of this study was to explore whether ellagic acid induced [Ca2þ]i rises in liver cells.
HepG2 human hepatoma cells can be applied for evaluating different compounds-induced physiological effects including Ca2þ signaling (Huang et al., 2015; Zhang et al., 2015). The effect of ellagic acid on [Ca2þ]i was also examined in HA22T, HA59T human hepatoma cells or AML12 mouse hepatocytes. AML12 cells were established from the normal liver of a 3-month-old male mouse. This cell is often used as a normal control in liver studies (Wu et al., 1994; Kuete et al., 2014). The [Ca2þ]i rises were characterized and the pathways underlying ellagic acid-evoked Ca2þ influx and Ca2þ release were explored. The cytotoxic effect of ellagic acid was also investigated in HepG2, HA22T, HA59T or AML12 cells.

2. Materials and methods

2.1. Chemicals

The reagents for cell culture were from Gibcos (Gaithersburg, MD, USA). Aminopolycarboxylic acid-acetoxy methyl (Fura-2-AM), 2-aminoethoxydiphenyl borate (2-APB) and 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxy methyl (BAPTAAM) were from Molecular Probess (Eugene, OR, USA). Ellagic acid and all the other compounds were from Sigma-Aldrichs (St. Louis, MO, USA). The purity of ellagic acid (Z98%) was isolated from Phyllanthus amarus extract and determined by HPTLC densitometry (Dhalwal et al., 2006).

2.2. Cell culture

HepG2, HA22T and HA59T human hepatoma cells, and AML12 mouse hepatocytes were purchased from Bioresource Collection and Research Center (Taiwan). HepG2 cells were cultured in minimal essential medium (MEM), HA22T and HA59T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), and AML12 cells were cultured in 90% 1:1 mixture of DMEM and Ham’s F12 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin. The cells were cultured at 37 °C in a humidified 5% CO2 atmosphere.

2.3. Experimental solutions

Ca2þ-containing medium (pH 7.4) contained 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES), and 5 mM glucose. Ca2þ-free medium (pH 7.4) contained 140 mM NaCl, 5 mM KCl, 3 mM MgCl2, 0.3 mM ethylene glycol tetraacetic acid (EGTA), 10 mM HEPES, and 5 mM glucose. Phosphate buffer saline (PBS, pH 7.4) contained 137 mM NaCl, 10 mM phosphate and 2.7 mM KCl. Ellagic acid was dissolved in ethanol as a 0.1 M stock solution. The other reagents are dissolved in water, ethanol or dimethyl sulfoxide (DMSO) as concentrated stocks. The concentration of organic solvents in the experimental solution was less than 0.1% and did not alter viability or basal [Ca2þ]i.

2.4. [Ca2þ]i measurements

[Ca2þ]i was measured as previously described (Hsu et al., 2011). Briefly, trypsinized cells (106 cells/ml) were loaded with 2 μM fura-2-AM for 30 min at 25 °C in medium. Cells were subsequently washed with Ca2þ-containing medium twice and was made into a suspension in Ca2þ-containing medium at a concentration of 107 cells/ml. Fura-2 fluorescence measurements were performed in a water-jacketed cuvette (25 °C) with continuous stirring which had 1 ml of medium and 0.5 million cells. Fluorescence was monitored with a Shimadzu RF-5301PC spectrofluorophotometer immediately after 0.1 ml cell suspension was added to 0.9 ml Ca2þ-containing or Ca2þ-free medium, by recording excitation signals at 340 nm and 380 nm and emission signal at 510 nm at 1-s intervals. For calibration of [Ca2þ]i, maximum and minimum fluorescence values were obtained by adding the detergent Triton X-100 (0.1%) and the Ca2þ chelator EGTA (10 mM) sequentially at the end of each experiment. Cell viability was routinely greater than 95% after the treatment as assayed by trypan blue exclusion. Control experiments showed that cells bathed in a cuvette with 100 μM ellagic acid had a viability of 95% after 20 min of fluorescence measurements. [Ca2þ]i was calculated as described previously assuming a Kd of 155 nM (Grynkiewicz et al., 1985). Mn2þ quenching of fura-2 fluorescence was performed in Ca2þ-containing medium containing 50 μM MnCl2. MnCl2 was added to cell suspension in the cuvette 1 min before starting the fluorescence recording. Data were recorded at excitation signal at 360 nm (Ca2þ-insensitive) and emission signal at 510 nm at 1-s intervals as previously described (Merritt et al., 1989).

2.5. Cell viability assay

Viability was assessed as previously described (Hsu et al., 2011). The assay was based on cleavage of the tetrazolium salt WST-1 (4[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) by active mitochondria to produce a colored formazan salt. The intensity of color correlated with the percentage of live cells. Measurements were conducted following manufacturer’s instructions (Roche Molecular Biochemical, Indianapolis, IN, USA). Cells were seeded in 96-well plates (104 cells/well) in medium for 24 h in the presence of ellagic acid. The cell viability detecting reagent WST-1 (10 μl pure solution) was added to samples after treatment with ellagic acid, and cells were incubated for 2 h at 37 °C in a humidified atmosphere with 5% CO2. In experiments using BAPTA-AM to chelate cytosolic Ca2þ, cells were treated with 5 μM BAPTA-AM for 1 h before addition of ellagic acid. The cells were washed once with Ca2þ-containing medium and incubated with/without ellagic acid for 24 h. The absorbance of samples (A450) was determined using a microtiter reader (model MRX II, Dynex Technologies, Chantilly, VA, USA). Absolute optical density was normalized to the absorbance of unstimulated cells in each plate and expressed as a percentage of the control value, which was treated with vehicle only (0.1% DMSO), taken as 100% growth.

2.6. Statistics

Data are reported as mean7S.E.M. of three separate experiments. Data were analyzed by one-way analysis of variances (ANOVA) using the Statistical Analysis System (SASs, SAS Institute Inc., Cary, NC, USA). Multiple comparisons between group means were performed by post hoc analysis using the Tukey’s HSD (honestly significantly difference) procedure. A P-value less than 0.05 was considered significant.

3. Results

3.1. Ellagic acid concentration-dependently induced [Ca2þ]i rises in Ca2þ-containing medium or Ca2þ-free medium in HepG2 cells but not in HA22T, HA59T or AML12 cells

The effect of ellagic acid on basal [Ca2þ]i was examined. In HepG2 cells, Fig. 1A shows that the basal [Ca2þ]i was 5072 nM. At concentrations between 25 μM and 75 μM, ellagic acid induced [Ca2þ]i rises in a concentration-dependent manner in Ca2þ-containing medium. At a concentration of 10 μM, ellagic acid did not alter [Ca2þ]i. At a concentration of 75 μM, ellagic acid induced [Ca2þ]i rises that attained to net (baseline subtracted) increases of 37073 nM (n¼3) followed by a decay. The Ca2þ response saturated at 75 μM ellagic acid because 100 μM ellagic acid evoked similar responses as that induced by 75 μM ellagic acid (not shown). Fig. 1B shows that in Ca2þ-free medium, 75 μM ellagic acid induced [Ca2þ]i rises of 14573 nM. Ellagic acid between 25 μM and 75 μM also concentration-dependently induced [Ca2þ]i rises in Ca2þ-free medium. Fig. 1C shows the concentration-response plots of ellagic acid-induced responses. The EC50 value was 3573 μM or 2773 μM in Ca2þ-containing medium or Ca2þ-free medium, respectively, fitting to a Hill equation. Ellagic acid between 10 μM and 75 μM did not induce [Ca2þ]i rises in other liver cell types including HA22T (Fig. 1D), HA59T cells (Fig. 1E) or AML12 cells (Fig. 1F).

3.2. Ellagic acid-induced [Ca2þ]i rises involved Ca2þ influx as assayed by measuring Mn2þ influx in HepG2 cells

Experiments were performed to confirm that ellagic acid-induced [Ca2þ]i rises involved Ca2þ influx in HepG2 cells. Fig. 1 shows that ellagic acid-induced Ca2þ response saturated at 75 μM, therefore the response induced by 75 μM ellagic acid was used as the condition studied in the following experiments. Because Mn2þ enters cells through similar pathways as Ca2þ but quenches fura-2 fluorescence at all excitation wavelengths (Merritt et al., 1989), quenching of fura-2 fluorescence excited at the Ca2þ-insensitive excitation wavelength of 360 nm by Mn2þ implies Ca2þ influx. Fig. 2 shows that 75 μM ellagic acid induced an immediate decrease in the 360 nm excitation signal by 3273 (n¼3) arbitrary units (trace b compared to trace a, time point of 50 s). The data suggest that ellagic acid-induced [Ca2þ]i rises involved Ca2þ influx.

3.3. Store-operated Ca2þ channel blockers or a PKC inhibitor inhibited ellagic acid-induced [Ca2þ]i rises in HepG2 cells

The pathway of ellagic acid-induced Ca2þ influx was explored in HepG2 cells. The store-operated Ca2þ channel blockers 2-APB (50 μM) (Sun et al., 2013), econazole (0.5 μM) (Jiang et al., 2006) or SKF96365 (5 μM) (Xie et al., 2014); or the protein kinase C (PKC) inhibitor GF109203X (2 μM) (Hah and Lee, 2003) was applied 1 min before 75 μM ellagic acid in Ca2þ-containing medium. Addition of 2-APB, econazole, SKF96365, or GF109203X alone did not alter baseline [Ca2þ]i (data not shown). In Ca2þ-containing medium, Fig. 3A shows that 2-APB, econazole, SKF96365, or GF109203X inhibited ellagic acid-induced [Ca2þ]i rises in Ca2þ-containing medium by 5075%, 4975%, 5075%, or 4874%, respectively (Po0.05) (n¼3). Fig. 3B shows the original tracings of 2-APB-, econazole-, SKF96365-or GF109203X-inhibited ellagic acid-induced [Ca2þ]i rises.

3.4. The endoplasmic reticulum was the dominant Ca2þ store in ellagic acid-induced Ca2þ release in HepG2 cells

Efforts were made to explore the intracellular Ca2þ store involved in ellagic acid-induced release in HepG2 cells. Fig. 4A shows that in Ca2þ-free medium, thapsigargin (1 μM), an inhibitor of the endoplasmic reticulum Ca2þ pumps in human hepatoma cells (Thastrup et al., 1990; Sun et al., 2013), induced [Ca2þ]i rises of 12573 nM, and subsequently added 75 μM ellagic acid failed to induce [Ca2þ]i rises. Fig. 4B shows that, addition of 75 μM ellagic acid prevented the [Ca2þ]i rises induced by thapsigargin. The data suggest that thapsigargin-sensitive endoplasmic reticulum stores appeared to be dominant in ellagic acid-induced Ca2þ release in HepG2 cells.

3.5. Ellagic acid induced [Ca2þ]i rises in a phospholipase C (PLC)dependent manner in HepG2 cells

The role of PLC stimulation plays an important role in releasing Ca2þ from the endoplasmic reticulum (Bootman et al., 1994; Clapham, 1995). Because ellagic acid released Ca2þ from the endoplasmic reticulum, the role of PLC in this process was examined in HepG2 cells. U73122, a PLC inhibitor in most cells including human hepatoma cells (Banfi et al., 1999), was used to see whether the activation of this enzyme was required for ellagic acid-induced Ca2þ release. First, effort was exerted to assure the effectiveness of U73122 as a PLC inhibitor under our experimental condition. ATP is necessary for PLC activation in [Ca2þ]i increases in human hepatoma cells (Xie et al., 2014). Fig. 5A shows that ATP (10 μM) induced [Ca2þ]i rises of 9574 nM. Fig. 5B shows that incubation with 2 μM U73122 did not change basal [Ca2þ]i but abolished ATPinduced [Ca2þ]i rises. This suggests that U73122 effectively suppressed PLC activity. Fig. 5B shows that 75 μM ellagic acid-induced [Ca2þ]i rises were set as 100% (control). Incubation with U73122 inhibited ellagic acid-induced [Ca2þ]i rises and the combination of U73122 and ATP had the same effect.

3.6. Ellagic acid decreased cell viability that was triggered by preceding [Ca2þ]i rises in HepG2 cells but not in HA22T or HA59T cells

The next experiments were performed to examine whether ellagic acid-induced cytotoxicity was triggered by preceding [Ca2þ]i rises in HepG2, HA22T or HA59T cells. The intracellular Ca2þ chelator BAPTA-AM (Tsien, 1980; Zhang et al., 2015) was used to prevent [Ca2þ]i rises during ellagic acid treatment. Fig. 6A shows that 5 μM BAPTA-AM loading abolished 10–100 μM ellagic acid-induced [Ca2þ]i rises in Ca2þ-containing medium. This suggests that BAPTA-AM effectively prevented [Ca2þ]i rises during ellagic acid treatment. In the presence of 25–100 μM ellagic acid, cell viability decreased in a concentration-dependent manner in HepG2 (Fig. 6B), HA22T (Fig. 6C) or HA59T (Fig. 6D) cells, but not in AML 12 cells (Fig. 6E). In addition, in HepG2 cells, BAPTA-AM loading partially inhibited ellagic acid (50 or 75 μM)-induced cell death by 12.270.5% or 1570.5% (Po0.05), respectively. However, BAPTA-AM loading did not prevent ellagic acid (25–100 μM)-induced cell death in HA22T or HA59T cells.

4. Discussion

Phenolic compounds that are made up of various groups of metabolites commonly found in the human diet and environment have been extensively studied recently. Particularly, some studies have shown that ellagic acid (a naturally occurring polyphenolic compound) possesses exceptional pharmacological properties against disease (Ahmed et al., 2016; García-Niño and Zazueta, 2015; Rasool et al., 2015; Williner et al., 2003). Ellagic acid was shown to elicit anticarcinogenic effects by inhibiting tumor cell proliferation, inducing apoptosis, breaking DNA binding to carcinogens, blocking virus infection, and disturbing inflammation, angiogenesis, and drug-resistance processes required for tumor growth and metastasis (Zhang et al., 2014a, 2014b). Furthermore, the molecular mechanisms that ellagic acid activates include the scavenging of free radicals, regulation of phase I and II enzymes, modulation of proinflammatory and profibrotic cytokines synthesis, the regulation of biochemical pathways involved in the synthesis and degradation of lipids as well as the maintenance of essential trace elements levels (García-Niño and Zazueta, 2015; Rasool et al., 2015; Williner et al., 2003). Therefore, this compound has potential to be evaluated in medical treatments to prevent or reduce toxicity in liver.
Previous studies have shown that phenolic compounds such as octyl gallate (Guo et al., 2010), artocarpol A (Kuan et al., 2005) or bisphenol A (Deutschmann et al., 2013) affected Ca2þ homeostasis in different cell models. The effect of ellagic acid on Ca2þ signaling has also been studied. In rat ventricular myocytes, ellagic acid reduced L-type Ca2þ current (Olgar et al., 2014). However, whether ellagic acid affected physiological responses via Ca2þ signaling in liver cells is unknown. This study shows that ellagic acid induced concentration-dependent [Ca2þ]i rises in HepG2 cells. The Ca2þ signal was composed of Ca2þ entry and Ca2þ release because it was reduced by approximately 55% by removing extracellular Ca2þ. The Mn2þ quenching data also suggest that Ca2þ influx occurred during ellagic acid incubation.
The [Ca2þ]i rises induced by ellagic acid in HepG2 cells were not presented in other types of liver cells including HA22T, HA59T or AML12 cells. The record of Bioresource Collection and Research Center (Taiwan) shows that the characteristics of HepG2 cells are different from that of HA22T, HA59T or AML12 cells. The major difference is the status of 3-hydroxy-3-methylglutaryl-CoA reductase expression. HA22T, HA59T or AML12 cells do not have this enzyme. Our data suggest that ellagic acid-induced [Ca2þ]i rises were dependent on the 3-hydroxy-3-methylglutaryl-CoA reductase status in these cells. It has been shown that 3-hydroxy-3-methylglutaryl-CoA reductase activity regulated Ca2þ mobilization in isolated rat hepatocytes (Roitelman et al., 1991; Zammit and Caldwell, 1991). Therefore, the mechanisms underlying ellagic acid-induced [Ca2þ]i rises appears to vary among different liver cell types.
Store-operated Ca2þ entry was shown to be the dominant Ca2þ entry pathway in HepG2 cells (Cui et al., 2015; Putney, 1986; Sun et al., 2013). Ellagic acid-induced [Ca2þ]i rises were significantly inhibited by 50–55% by 2-APB, econazole, or SKF96365, which is also similar to the magnitude of ellagic acid-induced Ca2þ influx. These three compounds have been used to inhibit store-operated Ca2þ entry in liver cells (Jiang et al., 2006; Sun et al., 2013; Xie et al., 2014). Therefore, it appears that store-operated Ca2þ entry was involved in ellagic acid-induced Ca2þ influx in HepG2 cells. Furthermore, the activity of PKC is known to associate with Ca2þ homeostasis (Tepperman et al., 2005). A normally maintained PKC level is needed to induce a full Ca2þ response (Doolan et al., 1998). Our data show that ellagic acid-evoked [Ca2þ]i rises were inhibited by approximately 50% by PKC inhibition. Therefore, ellagic acid-induced [Ca2þ]i rises were contributed by PKC-regulated store-operated Ca2þ entry in HepG2 cells.
Because ellagic acid induced [Ca2þ]i rises in the absence of extracellular Ca2þ, the role of Ca2þ stores in these rises was explored. The thapsigargin-sensitive endoplasmic reticulum stores might be the dominant one because thapsigargin pretreatment abolished ellagic acid-induced [Ca2þ]i rises. Thapsigargin has been shown to induce [Ca2þ]i rises by blocking the ability of the endoplasmic reticulum Ca2þ-ATPase to sequester Ca2þ into the endoplasmic reticulum, leading to depletion of the Ca2þ store (Thastrup et al., 1990; Sun et al., 2013). Although previous studies have shown that ellagic acid activated sarco-endoplasmic reticulum Ca2þ-ATPase in isolated murine ventricular myocardia (Namekata et al., 2013), our data show that ellagic acid pretreatment abolished thapsigargin-induced Ca2þ release in HepG2 cells, which is consistent with the hypothesis that thapsigargin inhibits the endoplasmic reticulum Ca2þ-ATPase. Therefore, cell types derived from different origins may have different mechanisms of Ca2þ signaling, depending on the physiological function of this particular cell.
Depletion of Ca2þ from the endoplasmic reticulum is activated by stimulation of PLC, which results in increases in cytosolic inositol trisphosphate (IP3) levels (Bootman, 1994; Clapham, 1995). Once IP3 binds to its receptors on internal stores such as the endoplasmic reticulum, it opens the receptors and causes Ca2þ release (Bootman, 1994; Clapham, 1995). Our data show that Ca2þ release induced by ellagic acid was abolished when PLC activity was inhibited by U73122. Therefore, it seems that ellagic acid-induced Ca2þ release was through a PLC-dependent pathway in HepG2 cells.
In previous studies, ellagic acid was shown to cause cytotoxicity in different kinds of cancer cells such as HSC-2 human oral carcinoma cells (Weisburg et al., 2013) and PC3 human prostate cancer cells (Vicinanza et al., 2013). Furthermore, ellagic acid derivatives such as 3,3′-di-O-methyl ellagic acid-4′-O-β-D-xylopyranoside (JNE2) (Zhang et al., 2014a, 2014b) or urolithin A (Wang et al., 2015) at concentrations of 20-100 μM induced cytotoxicity in HepG2 cells. Consistently, our data show that ellagic acid (25–100 μM) concentration-dependently killed HepG2, HA22T or HA59T cells. Notably, at this concentration range, ellagic acid did not cause cytotoxicity in normal liver cells (AML12 mouse hepatocytes). Cell viability could be altered in a Ca2þ-dependent or -independent manner (Nicotera et al., 2009; Sun et al., 2013). Our study shows that ellagic acid-induced cytotoxicity was partially prevented under the condition that cytosolic Ca2þ was chelated by BAPTA-AM in HepG2 cells, but not in HA22T or HA59T cells. This implies that the cytotoxicity induced by ellagic acid was associated with [Ca2þ]i rises in HepG2 cells, but not in HA22T and HA59T cells.
In addition to the cytotoxic effects induced by ellagic acid in hepatoma cells, previous studies have shown that ellagic acid had a protective role in liver. Ellagic acid suppressed resistin secretion in vivo and improved obesity-induced dyslipidemia and hepatic steatosis in KK-A(y) mice (Yoshimura et al., 2013). Additionally, antioxidant and cytoprotective properties of ellagic acid prevented liver damage induced by ischemia-reperfusion injury in in vivo explained by the low bioavailability of ellagic acid. The main ellagic acid metabolites circulating in plasma are known as urolithins. Urolithins are present in plasma as glucuronide or sulphate conjugates, at concentrations in the nM range (Grossi et al., 2014; Larrosa et al., 2010).
Although poor absorption from the gut, rapid metabolism, and lack of transport to the target organs may limit the bioavailability and clinical usefulness of ellagic acid and urolithins upon oral administration, many studies have developed drug delivery systems, such as chitosan-glycerol phosphate in situ gelling system for the sustained subcutaneous delivery of ellagic acid (Sharma et al., 2007), ellagic acid-loaded poly (D,L-lactide-co-glycolide) nanoparticles for oral administration (Bala et al., 2006), and using a new pH-sensitive polymer [Eudragit P-4135F (P-4135F)] to deliver ellagic acid to the lower small intestine in rats (Jeong et al., 2001). The results indicate that the bioavailability of ellagic acid has improved. An increasing number of nanoparticles, liposomes, microemulsions, and polymeric implantable devices are emerging as viable alternatives for delivering therapeutic concentrations of natural compounds into the systemic circulation (Bansal et al., 2011). Furthermore, chemical modifications or more formulations that can bypass their poor oral bioavailability have been developed. Because ellagic acid and its metabolites have preventive and therapeutic potential against human liver cancers, advanced drug delivery systems for enhanced bioavailability and mechanisms to maintain effective therapeutic concentrations in the blood should also be considered.

5. Conclusions

In sum, in HepG2 but not HA22T, HA59T or AML12 cells, the natural phenolic compound ellagic acid caused PKC-regulated Ca2þ influx via 2-APB, econazole SKF96365-sensitive store-operated Ca2þ entry and induced Ca2þ release from the endoplasmic reticulum. Ellagic acid caused cytotoxicity in a Ca2þ-associated manner in HepG2 cells. Our findings that ellagic acid was able to kill three types of human hepatoma cells while leaving mouse hepatocytes unharmed validate the potential of using ellagic acid clinically to treat hepatoma. Our study might contribute to the unveiling of pharmacology of ellagic acid in Ca2þ signaling in human hepatoma cells and the data can serve as a basis for future pharmaceutical development of seeking new drugs to treat liver cancer. To this end, it is important to extend the knowledge about the action mechanisms of this compound and to perform additional preclinical studies using in vitro and in vivo models.

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