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Research ArticleCellular and Molecular

Different Antioxidative and Antiapoptotic Effects of Piceatannol and Resveratrol

Ryusuke Hosoda, Hiroki Hamada, Daisuke Uesugi, Naotoshi Iwahara, Iyori Nojima, Yoshiyuki Horio and Atsushi Kuno
Journal of Pharmacology and Experimental Therapeutics March 2021, 376 (3) 385-396; DOI: https://doi.org/10.1124/jpet.120.000096

Abstract

Resveratrol affords protection against reactive oxygen species (ROS)-related diseases via activation of SIRT1, an NAD+-dependent deacetylase. However, the low bioavailability of resveratrol limits its therapeutic applications. Since piceatannol is a hydroxyl analog of resveratrol with higher bioavailability, it could be an alternative to resveratrol. In this study, we compared the cytotoxicity, antioxidative activity, and mechanisms of cytoprotection of piceatannol with those of resveratrol. In C2C12 cells incubated with piceatannol, electrospray ionization mass spectrometry analysis showed that piceatannol was present in the intracellular fraction. A high concentration (50 μM) of piceatannol, but not resveratrol, induced mitochondrial depolarization and apoptosis. However, piceatannol at 10 μM inhibited the increase in mitochondrial ROS level induced by antimycin A, and this ROS reduction was greater than that by resveratrol. The reduction in hydrogen peroxide–induced ROS by piceatannol was also greater than that by resveratrol or vitamin C. Piceatannol reduced antimycin A–induced apoptosis more than did resveratrol. SIRT1 knockdown abolished the antiapoptotic activity of resveratrol, whereas it blocked only half of the antiapoptotic activity of piceatannol. Piceatannol, but not resveratrol, induced heme oxygenase-1 (HO1) expression, which was blocked by knockdown of the transcription factor NRF2, but not by SIRT1 knockdown. HO1 knockdown partially blocked the reduction of ROS by piceatannol. Furthermore, the antiapoptotic action of piceatannol was abolished by HO1 knockdown. Our results suggest that the therapeutic dose of piceatannol protects cells against mitochondrial ROS more than does resveratrol via SIRT1- and NRF2/HO1–dependent mechanisms. The activation of NRF2/HO1 could be an advantage of piceatannol compared with resveratrol for cytoprotection.

Significance Statement This study showed that piceatannol and resveratrol were different in cytotoxicity, oxidant-scavenging activities, and mechanisms of cytoprotection. Protection by piceatannol against apoptosis induced by reactive oxygen species was superior to that by resveratrol. In addition to the sirtuin 1–dependent pathway, piceatannol exerted nuclear factor erythroid 2–related factor 2/heme oxygenase-1–mediated antioxidative and antiapoptotic effects, which could be an advantage of piceatannol compared with resveratrol.

Introduction

Piceatannol (3,3′,4,5′-trans-tetrahydroxystilbene; PIC) and resveratrol (3,4′,5-trans-trihydroxystilbene; RSV) are natural stilbenes found in grape skins and have therapeutic potential against cancers, cardiovascular diseases, inflammation, hypercholesterolemia, and obesity (Piotrowska et al., 2012; Bonkowski and Sinclair, 2016; Seyed et al., 2016; Salehi et al., 2018). PIC has high apoptosis-inducing ability for tumor cells (Piotrowska et al., 2012; Seyed et al., 2016). On the other hand, since PIC possesses one extra hydroxyl residue compared with RSV (Fig. 1A), PIC provides higher antioxidation activity than that of resveratrol (Rhayem et al., 2008; Sato et al., 2014; Sueishi et al., 2017). PIC has been shown to protect cells from toxicities of amyloid-β and the lipid peroxide product 4-hydroxynonenal, both of which increase the production of cellular reactive oxygen species (ROS) (Kim et al., 2007; Jang et al., 2009). In addition to increasing the production of ROS, these compounds also activate ROS-independent mechanisms such as a caspase cascade and calcium influx in cells. Thus, the effects of PIC on cell death induced only by high levels of ROS have not been studied in detail.

Fig. 1.
Fig. 1.

Chemical structures and in vitro antioxidant activity of resveratrol and piceatannol. (A) Chemical structures of resveratrol and piceatannol. (B) The DPPH radical-scavenging activity of PIC and RSV. Data were obtained from three independent experiments. *P < 0.05.

Both PIC and RSV activate SIRT1, an NAD+-dependent protein deacetylase (Howitz et al., 2003). SIRT1 is a mammalian homolog of yeast silent information regulator 2, which prolongs lifespan when overexpressed in yeast (Kaeberlein et al., 1999). Activation of SIRT1 reduces cellular oxidative stress, cell death, and inflammation and promotes mitochondrial biogenesis and lipid metabolism (Horio et al., 2011). Deacetylation of forkhead box O transcription factors (FoxOs) and peroxisome proliferator–activated receptor γ coactivator 1α by SIRT1 leads to their activation to induce the expression of antioxidative enzymes such as superoxide dismutase 2 (SOD2) (Pan and Finkel, 2017). Although RSV itself is an antioxidant, our previous data suggest that induction of antioxidative enzymes mediated via SIRT1 activation is the main mechanism of the antioxidative action of RSV (Tanno et al., 2010; Hori et al., 2013; Hosoda et al., 2013).

RSV and PIC have also been reported to activate nuclear factor erythroid 2–related factor 2 (NRF2) to induce antioxidative action (Lee et al., 2010; Hao et al., 2013; Perez-Leal et al., 2017). NRF2 is a transcription factor that promotes the expression of antioxidative genes such as heme oxygenase-1 (HO1) and NAD(P)H:quinone oxidoreductase 1 (NQO1) (Cuadrado et al., 2019). In cells without stress, activity of NRF2 is repressed by Keap1, which forms a complex with NRF2, leading to ubiquitin-mediated degradation of NRF2. ROS-induced release of NRF2 from Keap1 causes stabilization and nuclear translocation of NRF2, leading to its transcriptional activation. Acetylation/deacetylation of NRF2 also regulates NRF2 activity. It has been reported that acetylation of NRF2 by p300/cAMP response element binding protein-binding protein augments its DNA binding (Sun et al., 2009). There are opposite reports regarding roles of SIRT1 in NRF2 activity. One study showed that SIRT1 attenuates transcription activity of NRF2 via its deacetylation (Kawai et al., 2011), whereas another study showed that SIRT1 increases nuclear NRF2 protein level and its transcription activity (Huang et al., 2013).

Pathologic ROS are derived from various sources, including the electron transport chain in mitochondria, especially in organs rich in mitochondria (Balaban et al., 2005), cytochrome P450 in the liver (Veith and Moorthy, 2018), and NADPH oxidases in the cardiovascular systems (Zhang et al., 2020). Among them, mitochondrial ROS are liberated into the cytoplasm and the nucleus and are implicated in various pathologic conditions, such as muscular diseases and neurodegenerative diseases (Balaban et al., 2005; Bock and Tait, 2020). Besides SOD2 induction, SIRT1 activation by RSV induces autophagy/mitophagy to attenuate mitochondrial oxidative stress (Morselli et al., 2011; Lee et al., 2012; Sebori et al., 2018). In a model of culture cells, it has been shown that RSV decreases cellular ROS and apoptosis induced by antimycin A, a product of Streptomyces that inhibits mitochondrial electron transport chain complex III, via SIRT1 (Tanno et al., 2010; Hori et al., 2013). In contrast, it is not known whether PIC protects cells from cytotoxicity induced by ROS generated from mitochondria.

We have shown that RSV has the potential for treatment of ROS-related diseases, including Duchenne muscular dystrophy and heart failure (Tanno et al., 2010; Kuno et al., 2018; Sebori et al., 2018). However, it has been pointed out that a weak point of RSV is its low bioavailability when orally administered (Almeida et al., 2009). Since PIC is an analog of RSV and has been reported to have higher bioavailability than that of RSV (Setoguchi et al., 2014), PIC could be an alternative candidate for treatment of ROS-related diseases. However, it remains unclear whether the cytotoxicity and antioxidative properties of PIC are different from those of RSV.

In the present study, we compared the cytotoxicity, antimitochondrial ROS activity, and involvement of SIRT1 and NRF2 in the antiapoptotic function of PIC with those of RSV in C2C12 myoblasts.

Materials and Methods

Reagents.

PIC was obtained from Tokyo Kasei (Tokyo, Japan). RSV, antimycin A, hydrogen peroxide (H2O2), and N-acetyl-l-cysteine (NAC) were purchased from FUJIFILM Wako Pure Chemicals (Osaka, Japan). Vitamin C was obtained from Kanto Chemical (Tokyo, Japan). MitoSOX Red was purchased from Thermo Fisher Scientific (Waltham, MA). Hoechst33342 was obtained from Dojindo (Kumamoto, Japan).

Mass Spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) was performed with a Q Exactive Plus (Thermo Fischer Scientific) in negative ionization mode (ESI−) with a methanol carrier solution at a flow rate of 0.5 µl/min and with an injection volume of 2 μl.

Measurement of 2,2-Diphenyl-1-Picrylhydrazyl Radical-Scavenging Activity.

The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity was measured as previously reported (Morales and Jimenez-Perez, 2001) with a slight modification. Briefly, 500 μl of a test sample in water was mixed with the same volume of 0.15 mM DPPH dissolved in 99.5% ethanol. The mixture was shaken for 30 minutes at room temperature, and then absorbance of the mixture was measured at 517 nm. Data are shown as percent inhibition according to the formula [Abs (control) – Abs (sample)/Abs (control)] × 100. The IC50 was calculated by 10(Log(A/B) × (50 − C)/(D − C) + Log(B), where A is the concentration of the test compound directly above 50% inhibition, B is the concentration of the test compound directly below 50% inhibition, C is the percent inhibition at B, and D is the percent inhibition at A.

Cell Culture.

C2C12 myoblasts were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Wako Pure Chemical) with 4.5 g/l d-glucose supplemented with 1% antibiotic-antimycotic mixed stock solution (Nacalai Tesque, Kyoto, Japan) and 10% fetal bovine serum. The culture medium was switched to serum-free DMEM to eliminate cytoprotective factors in serum and to obtain reproducible cytotoxicity in experiments for cell death, mitochondrial membrane potential, mitochondrial ROS, and gene expression analyses.

Detection of Intracellular and Extracellular Piceatannol.

C2C12 cells in a 10-cm dish were incubated with or without the culture medium containing 100 μM PIC for 1 hour. After removal of the culture medium, the cells were washed twice with PBS and lysed with 0.5 ml of CelLytic M Cell Lysis Reagent (Sigma-Aldrich). The cell lysates were then sonicated and centrifuged. The supernatant fraction was mixed with an equal volume of ethyl acetate and shaken vigorously for 30 minutes at room temperature. The ethyl acetate layer, which contained PIC, was isolated and then evaporated. PIC in the culture medium (10 ml) was also extracted with ethyl acetate. Dried samples were stored at −80°C. The samples were dissolved in 100 μl of methanol and examined by ESI-MS. Experiments were independently carried out at least three times.

Immunoblot Analysis.

C2C12 cells were lysed with CelLytic M Cell Lysis Reagent with 1% protease inhibitor cocktail (Nacalai Tesque) and centrifuged at 10,000g for 10 minutes at 4°C. The supernatant fractions were analyzed by immunoblot analysis as described previously (Hosoda et al., 2013). Protein concentration was measured with the Protein Quantification Kit-Rapid (Dojindo), and equal protein amounts per lane (7 μg) were analyzed using the following antibodies: rabbit polyclonal anti-SIRT1 (Sakamoto et al., 2004) (1:1000) and mouse monoclonal anti-tubulin (T5168, 1:10,000; Sigma-Aldrich) antibodies.

Apoptosis-Inducing Effects of PIC and RSV.

C2C12 cells were treated with a vehicle (dimethylsulfoxide) and various concentrations of RSV or PIC in serum-free DMEM for 4 hours. Apoptotic cell death was analyzed by nuclear condensation and by flow cytometry. Cell nuclei were stained with Hoechst33342 (1:2000) and observed by fluorescence microscopy. The percentage of cells with condensed nuclei was averaged from five fields of each treatment. For analysis by flow cytometry, cells were stained using the Muse Annexin V and Dead Cell Kit (Millipore). The cells were fractionated into four fractions according to the manufacturer’s protocol: nonapoptotic cells, annexin V (−), and 7-amino-actinomycin D (7-AAD) (−); early apoptotic cells, annexin V (+), and 7-AAD (−); late-stage apoptotic and dead cells, annexin V (+), and 7-AAD (+); and necrotic cells, annexin V (−), and 7-AAD (+). Three independent experiments were carried out.

Assay for Mitochondrial Membrane Depolarization.

C2C12 cells were treated with dimethylsulfoxide as a vehicle and various concentrations of RSV or PIC in serum-free DMEM for 4 hours. Mitochondrial membrane potential and dead cells were analyzed by flow cytometry with the Muse MitoPotential Kit (Millipore). Cells were fractionated into four fractions according to the manufacturer’s protocol: live cells, MitoPotential Dye (+), and 7-AAD (−); depolarized live cells, MitoPotential Dye (−), and 7-AAD (−); depolarized and dead cells, MitoPotential Dye (−), and 7-AAD (+); and dead cells, MitoPotential Dye (+), and 7-AAD (+). Three independent experiments were carried out.

RNA Analysis.

Total RNA isolation from cultured cells and reverse transcriptase reaction were performed as described previously (Hori et al., 2013; Hosoda et al., 2013). Quantitative polymerase chain reaction (PCR) was performed in StepOne (Applied Biosystems, Foster City, CA) using TaqMan Universal Master Mix II with UNG (Applied Biosystems) and TaqMan Gene Expression Assays for SOD2 (Mm00449726_m1), myogenin (Mm00446194_m1), myoblast determination protein 1 (MyoD) (Mm00440387_m1), myosin heavy chain 4 (MYH4) (Mm01332541_m1), and GAPDH (Mm99999915_g1). For analyses of mouse SIRT1, catalase, HO1, NQO1, GCLM, and β-actin, GoTaq qPCR Master Mix (Promega) with the following oligonucleotide primers was used: 5′-GAC GCT GTG GCA GAT TGT TA-3′ and 5′-GGA ATC CCA CAG GAG ACA GA-3′ for SIRT1, 5′-GGA GGC GGG AAC CCA ATA G-3′ and 5′-GTG TGC CAT CTC GTC AGT GAA-3′ for mouse catalase, 5′-GTC AAG CAC AGG GTG ACA GA-3′ and 5′-CTG CAG CTC CTC AAA CAG CT-3′ for mouse HO1, 5′-ACA TGA ACG TCA TTC TCT GG-3′ and 5′-ACC AGT TGA GGT TCT AAG AC-3′ for mouse NQO1, 5′-TGT GTG ATG CCA CCA GAT TG-3′ and 5′-ATG CTT TCT TGA AGA GCT TCC T-3′ for mouse GCLM, and 5′-CTG GCT CCT AGC ACC ATG AAG AT-3′ and 5′-GGT GGA CAG TGA GGC CAG GAT-3′ for mouse β-actin. All assays were performed in duplicate and by the standard curve method using serial cDNA dilution. β-Actin or GAPDH was used as an internal control. Experiments were independently carried out at least three times.

Effects of PIC and RSV on the Level of Mitochondrial ROS Induced by Antimycin A or Hydrogen Peroxide.

C2C12 cells were treated with the vehicle, 30 μM RSV, or 30 μM PIC in serum-free DMEM for 4 hours and were further incubated with 45 μM antimycin A for 4 hours to generate mitochondrial ROS. The cells were stained with MitoSOX Red (Thermo Fischer Scientific) to analyze the level of mitochondrial ROS according to the manufacturer’s protocol, and red fluorescence was monitored using fluorescence microscopy. We also examined the effect of RSV, PIC, vitamin C, or NAC on MitoSOX Red fluorescence in cells treated with 250 μM H2O2 for 1 hour. Cells were pretreated with the vehicle (DMSO), 10 μM RSV, 10 μM PIC, 10 μM vitamin C, or 10 μM NAC for 24 hours and were then coincubated with the vehicle or H2O2. All images were captured under the same conditions, and the fluorescence intensity was quantified by ImageJ software (National Institutes of Health ). The average fluorescence intensity was obtained from five to six fields for each experiment, and then the data from three independent experiments were analyzed.

Effects of PIC and RSV on Necrotic Cell Death Induced by Mitochondrial Oxidative Stress.

C2C12 cells were treated with the vehicle, 30 μM RSV, or 30 μM PIC in serum-free DMEM for 4 hours, and then 100 μM antimycin A was added to the medium to induce necrosis. After 45 minutes, cells that had died from necrosis were analyzed by the Muse Cell Analyzer with Muse Count and Viability Kit (Millipore) according to the manufacturer’s protocol.

Transfection of Small Interfering RNA.

C2C12 cells were transfected with small interfering RNAs (siRNAs, 50 nM) for SIRT1 (SASI_Mm01_00105675, Sigma-Aldrich), NRF2 (SASI_mM01_00013-0750), HO1 (SASI_Mm01_0015-9368), or control siRNA (SIC-001; Sigma-Aldrich) by using LipofectAMINE RNAiMAX Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. Cell death, MitoSOX Red, and mRNA levels were examined at 48 hours after siRNA transfection.

Antiapoptotic Effects of PIC and RSV.

At 24 hours after siRNA transfection, the cells were treated with the vehicle, 10 μM RSV, or 10 μM PIC in serum-free DMEM for 24 hours and were then treated with 30 μM antimycin A for 4 hours to induce apoptosis in the presence of the vehicle, RSV, or PIC. The cells were fixed with 4% paraformaldehyde, and cell nuclei were stained by Hoechst33342 (1:2000 dilution). Apoptotic cells were determined by nuclear condensation. The percentage of cells with condensed nuclei was determined from five fields in each experiment, and the data from three independent experiments were analyzed.

Myotube Differentiation of C2C12 Myoblasts.

For the differentiation, C2C12 cells were cultured in DMEM with 1.0 g/l d-glucose (FUJIFILM Wako Pure Chemicals) containing 2% horse serum (Thermo Fisher Scientific) for 1 week as previously reported (Fujiwara et al., 2019). During differentiation, cells were coincubated with the vehicle, 10 μM RSV, or 10 μM PIC.

Statistical Analysis.

The data are expressed as means ± S.D. Comparisons between multiple groups were performed by one-way and two-way analyses of variance followed by a post hoc Student-Newman-Keuls test. Differences were considered significant if the P value was less than 0.05. All analyses were carried out using SigmaStat (Systat).

Results

Higher Reducing Activity of PIC In Vitro.

PIC, a hydroxylated analog of RSV, is expected to have a higher reducing ability than that of RSV. We compared the capacities of PIC and RSV to scavenge DPPH, a hydrogen acceptor. The DPPH-scavenging capacity of PIC was greater than that of RSV (Fig. 1B). The concentrations of PIC and RSV required for 50% of the maximal DPPH-scavenging activity were approximately 18 and 81 μM, respectively. Thus, the DPPH-scavenging activity of PIC was 4.5-fold higher than that of RSV.

Entry of PIC into Cells.

We previously reported that resveratrol-4′-O-β-d-glucoside (4′G-RSV), a glycosyl RSV, exerts a cytoprotective function against ROS, although 4′G-RSV itself does not enter cells. In fact, 4′G-RSV is firstly deglycosylated and is converted to RSV in the extracellular space, and then RSV enters cells and affords cytoprotection (Hosoda et al., 2013). We examined whether PIC directly enters cells. ESI-MS analysis showed a peak of PIC m/z 243] in a sample from a medium containing 100 μM PIC (Supplemental Fig. 1A). When cells were treated with a medium without PIC, extracellular (medium) and intracellular fractions did not show such peaks (Supplemental Fig. 1B). After C2C12 cells had been treated with 100 μM PIC for 1 hour, extracellular and intracellular fractions were analyzed. PIC (m/z 243) was present in the extracellular fraction as well as in the intracellular fraction (Supplemental Fig. 1C). Additional peaks did not appear in the extracellular and intracellular fractions (Supplemental Fig. 1C). The peak for PIC was not detected in the intracellular fraction isolated from cells harvested after incubation with PIC for 1 minute (Supplemental Fig. 1D).

Mitochondrial Depolarization by PIC but not by RSV.

Both PIC and RSV have apoptosis-inducing activity for tumor cells (Piotrowska et al., 2012; Seyed et al., 2016; Salehi et al., 2018). It has been shown that PIC induced depolarization of the mitochondrial membrane potential and apoptosis of a lymphoma cell line in a concentration-dependent manner with a half-maximum concentration of 25 μM (Wieder et al., 2001). We compared the effects of PIC and RSV on mitochondrial membrane potential and cell death in nontumor C2C12 cells. After incubation with various concentrations of PIC or RSV under a serum-free condition, the cells were stained with MitoPotential Dye and 7-AAD (Fig. 2). Live cells exclude 7-AAD, a dye with high DNA binding affinity, whereas dead cells with ruptured plasma membranes are stained with 7-AAD. Cells were fractionated into four groups, and live cells with depolarized mitochondrial membranes could be distinguished from those with intact mitochondrial membrane potential (Fig. 2A). Treatment with 50 μM PIC increased the percentage of live cells with depolarized mitochondrial membranes (Fig. 2, A and B) and reduced live cells with intact mitochondrial membrane potential (Fig. 2, A and C). Cells with depolarized mitochondria were also increased by 30 μM PIC, whereas RSV at concentrations up to 50 μM had no effect on the mitochondrial membrane potential (Fig. 2, A–C). Thus, PIC, but not RSV, at a high concentration affected mitochondrial membrane potential in C2C12 cells under our experimental conditions.

Fig. 2.
Fig. 2.

Effects of piceatannol and resveratrol on mitochondrial depolarization in C2C12 cells. (A) C2C12 cells were treated with RSV or PIC at 0, 10, 30, and 50 µM for 4 hours; stained with MitoPotential Dye and 7-AAD; and analyzed by flow cytometry. Cells were fractionated into four groups according to the manufacturer’s protocol: live cells with intact mitochondrial membrane potential, MitoPotential Dye (+), and 7-AAD (−); depolarized live cells, MitoPotential Dye (−), and 7-AAD (−); depolarized dead cells, MitoPotential Dye (−), and 7-AAD (+); and dead cells, MitoPotential Dye (+), and 7-AAD (+). The percentages of live cells with depolarized mitochondria (B) and live cells with intact mitochondrial membrane potential (C) are shown. One-way ANOVA followed by a Student-Newman-Keuls test was performed to analyze the statistical difference. n = 6, *P < 0.05. MT, mitochondria.

Induction of Apoptosis by PIC, but not by RSV, at High Concentrations.

Depolarization of mitochondrial membrane potential leads to mitochondrial dysfunction and triggers cell death. We compared the effects of PIC and RSV on two types of cell death: apoptosis and necrosis. In this experiment, C2C12 cells were incubated with various concentrations of RSV or PIC for 4 hours in serum-free DMEM. Apoptotic cell death was determined by condensed nuclei after nuclear staining (Fig. 3A). Under the experimental conditions, RSV at concentrations up to 50 μM showed no cytotoxicity, whereas 30 and 50 μM PIC increased the number of cells with nuclear condensation (Fig. 3, A and B). Cytotoxicity of PIC and RSV was further analyzed by flow cytometry using annexin V and 7-AAD, which detect apoptotic and necrotic cell death, respectively. The flow cytometric analysis fractionated cells into four fractions: live, early apoptotic, late apoptotic, and necrotic cells (Fig. 3C). RSV at concentrations up to 50 μM did not increase apoptosis (Fig. 3, C–E). On the other hand, 50 µM PIC increased both early apoptosis and late apoptosis (Fig. 3, C–E). Necrosis was not increased by RSV or PIC (Fig. 3F).

Fig. 3.
Fig. 3.

Effects of piceatannol and resveratrol on cell death in C2C12 cells. C2C12 cells were treated with RSV or PIC at 0, 10, 30, and 50 µM for 4 hours, and then cells were stained with Hoechst33342 (A and B) or analyzed by flow cytometry (C–F). (A) Representative images of nuclear staining with Hoechst33342 in C2C12 cells. An arrow indicates a condensed nucleus, an index of apoptosis. Scale bar, 50 µm. (B) Summary data of the percentages of cells with nuclear condensation. The mean percentage of apoptotic cells was obtained from five fields for each treatment, and data from three independent experiments were analyzed. One-way ANOVA followed by a Student-Newman-Keuls test was performed to analyze the statistical difference. *P < 0.05. (C) Representative profiles of cells stained with annexin V and 7-AAD. Cells were fractionated into four fractions: live cells, annexin V (−), and 7-AAD (−); early apoptotic cells, annexin V (+), and 7-AAD (−); late apoptotic cells, annexin V (+), and 7-AAD (+); and necrotic cells, annexin V (−), and 7-AAD (+). The percentages of cells with early apoptosis (D), late apoptosis (E), and necrosis (F) are shown. One-way ANOVA followed by a Student-Newman-Keuls test was performed to analyze the statistical difference. n = 4. *P < 0.05. Apop,, apoptosis.

Effects of PIC on Levels of Mitochondrial ROS Induced by Antimycin A or H2O2.

Although 50 μM PIC itself was cytotoxic (Figs. 2 and 3), PIC had higher oxidant-scavenging ability than that of RSV (Fig. 1), indicating the possibility that PIC at a low concentration has beneficial effects in cells with pathologically high levels of ROS. Since mitochondria are organelles that are a pathologic source of ROS (Balaban et al., 2005; Bock and Tait, 2020), we compared the effects of PIC and RSV on levels of ROS directly generated from mitochondria by using antimycin A, an inhibitor of mitochondrial complex III. Treatment of C2C12 cells with 30 μM RSV or PIC alone slightly increased the levels of MitoSOX Red fluorescence, an indicator of mitochondrial superoxide anion (Fig. 4, A and B). Treatment with antimycin A increased the fluorescence level of MitoSOX Red, which was suppressed by pretreatment with PIC or RSV (Fig. 4, A and B). PIC at 30 μM decreased MitoSOX Red fluorescence levels to approximately one-fourth of those in antimycin A–treated cells; however, 30 μM RSV reduced the levels of ROS only to three-quarters (Fig. 4, A and B). Thus, PIC achieved an approximately 3-fold decrease in the mitochondrial level of ROS compared with that achieved by RSV in cells treated with antimycin A.

Fig. 4.
Fig. 4.

Effects of piceatannol and resveratrol on mitochondrial levels of ROS induced by antimycin A or H2O2. (A) Representative images of MitoSOX Red fluorescence in C2C12 cells treated with a vehicle, 30 μM RSV, or 30 μM PIC for 4 hours and then stimulated with a vehicle or 45 μM antimycin A (AA) for 4 hours under a serum-free condition. (B) Mean level of MitoSOX Red fluorescence intensity in each treatment. Scale bar, 100 µm. The average fluorescence intensity was obtained from six fields for each experiment, and the data from three independent experiments were analyzed. (C) Representative images of MitoSOX Red fluorescence in cells pretreated with a vehicle, 10 μM RSV, 10 μM PIC, 10 μM vitamin C (VC), or 10 μM NAC for 24 hours followed by coincubation of a vehicle or 250 μM H2O2 for 1 hour. (D) Summary data of MitoSOX Red fluorescence. The average of MitoSOX florescence per cell was obtained from randomly selected five fields for each experiment, and the data from three independent experiments were analyzed. One-way ANOVA followed by a Student-Newman-Keuls test was performed to analyze the statistical difference among groups. *P < 0.05. a.u., arbitrary unit.

The level of MitoSOX Red fluorescence was also increased by H2O2 (Fig. 4, C and D). RSV pretreatment reduced MitoSOX Red fluorescence level to approximately one-third of that in H2O2-treated cells. On the other hand, the H2O2-induced increase in MitoSOX Red fluorescence was almost completely suppressed by PIC (Fig. 4, C and D). Pretreatment with the same concentration of the antioxidative molecule vitamin C decreased the level of MitoSOX Red fluorescence to a level similar to that with RSV pretreatment. The other antioxidant NAC at 10 μM did not influence H2O2-induced MitoSOX Red fluorescence (Fig. 4, C and D).

Equal Antinecrotic Activities of PIC and RSV.

Mitochondrial ROS can induce necrotic cell death. Next, we examined the effects of 30 μM PIC and RSV on antimycin A–induced necrosis. To induce necrotic cell death, C2C12 cells were treated with a high concentration of antimycin A for 45 minutes, and necrosis was monitored by flow cytometry after staining with 7-AAD. Neither PIC nor RSV alone affected necrosis (Supplemental Fig. 2, A and B). Antimycin A treatment increased the percentage of necrotic cells to 28.6% under the experimental conditions. Pretreatment with PIC and pretreatment with RSV decreased necrotic cell death induced by antimycin A to 23.2% and 23.4%, respectively (Supplemental Fig. 2, A and B). Thus, the potency of the antinecrotic activity of PIC was similar to that of RSV.

Higher Antiapoptotic Ability of PIC via SIRT1-Dependent and -Independent Mechanisms.

We compared the effects of PIC and RSV on apoptosis induced by antimycin A. To examine the involvement of SIRT1 in the antiapoptotic function, SIRT1 was knocked down via siRNA. Treatment of cells with SIRT1 siRNA substantially reduced the SIRT1 protein level (Fig. 5A) and SIRT1 knockdown alone did not affect apoptosis determined by nuclear condensation under this experimental condition (Fig. 5, B and C). Antimycin A increased the percentage of apoptotic cells to 44.3%. Pretreatment with either PIC or RSV suppressed antimycin A–induced apoptosis, and the reduction of apoptosis by PIC was greater than that by RSV. RSV at 10 μM reduced the percentage of apoptosis to 20.2% under the condition of antimycin A treatment (Fig. 5, B and C). On the other hand, in the presence of 10 μM PIC, the percentage of apoptotic cells was reduced to 10.4%. The antiapoptotic effect of RSV was substantially suppressed by siRNA-mediated SIRT1 knockdown. In contrast, SIRT1 knockdown increased the percentage of apoptosis to 22.0% in cells treated with PIC (Fig. 5, B and C), suggesting that PIC protected the remaining approximately 20% of cells via mechanisms unrelated to SIRT1. The results indicated that PIC affords protection against mitochondrial ROS-induced cell death through SIRT1-dependent and -independent mechanisms.

Fig. 5.
Fig. 5.

The role of SIRT1 in antiapoptotic effects of piceatannol and resveratrol. (A) (Upper) Representative immunoblots for SIRT1 and α-tubulin in C2C12 cells transfected with control siRNA or SIRT1 siRNA. (Lower) Relative expression levels of SIRT1 protein normalized by α-tubulin level. n = 3. *P < 0.05 by two-tailed unpaired t test. (B) C2C12 cells transfected with either control siRNA or SIRT1 siRNA were pretreated with a vehicle, 10 μM PIC, or 10 μM RSV for 24 hours and then treated with 30 μM antimycin A (AA) for 4 hours to induce apoptosis in a serum-free condition. Cells were stained by Hoechst33342, and nuclear condensation was examined. Representative images of nuclear staining with Hoechst33342. An arrow indicates a condensed nucleus, a marker of apoptosis. Scale bar, 100 µm. (C) Percentage of apoptotic cells in each treatment. The average of the percentage of apoptotic cells was obtained from five fields for each treatment, and the data from three independent experiments were analyzed. Two-way ANOVA followed by a Student-Newman-Keuls test was performed to analyze the statistical difference among groups. *P < 0.05. a.u., arbitrary unit

Effects of SIRT1 and NRF2 on HO1 Upregulation by PIC.

PIC exerted more potent antioxidative and antiapoptotic activities than those of RSV (Figs. 4 and 5), and PIC decreased apoptotic cells via SIRT1-dependent and -independent mechanisms (Fig. 5). We compared the effects of PIC and RSV on induction of genes related to the antioxidative reaction regulated by SIRT1 and NRF2. The involvement of SIRT1 was also examined. Quantitative reverse transcription-polymerase chain reaction analysis confirmed SIRT1 knockdown by siRNA against SIRT1. Neither RSV nor PIC changed SIRT1 expression (Fig. 6A). Among target genes of NRF2 examined in the present study, HO1 mRNA was upregulated by PIC but not by RSV. This upregulation was not affected by SIRT1 knockdown (Fig. 6A). Levels of NQO1 and GCLM, the other targets of NRF2, were not changed by PIC or RSV. Although previous studies showed that RSV at a higher concentration upregulated SOD2, a target gene of FoxO transcription factors (Tanno et al., 2010; Hori et al., 2013), neither 10 μM RSV nor 10 μM PIC increased mRNA levels of SOD2 and catalase, another target of FoxOs (Fig. 6A).

Fig. 6.
Fig. 6.

Roles of SIRT1 and NRF2 in effects of piceatannol and resveratrol on changes in expression of genes related to antioxidative reaction. (A) C2C12 cells were treated with a vehicle (control), 10 μM RSV, or 10 μM PIC for 24 hours under a serum-free condition after transfection of control or SIRT1 siRNA. SIRT1, HO1, NQO1, GCLM, SOD2, and catalase mRNA levels were measured by a quantitative PCR method. N = 4. (B) Cells were treated with the vehicle, RSV, or PIC as in (A) after transfection of control or NRF2 siRNA. Levels of NRF2, HO1, NQO1, and GCLM mRNA were analyzed. N = 3. Two-way ANOVA followed by a Student-Newman-Keuls test was performed to analyze the statistical difference among groups. *P < 0.05.

We further examined whether upregulation of HO1 by PIC is dependent on NRF2. Transfection of NRF2 siRNA reduced NRF2 mRNA levels by 83% of that in cells transfected with control siRNA, and neither RSV nor PIC changed the NRF2 mRNA level (Fig. 6B). HO1 upregulation by PIC was blocked by NRF2 knockdown (Fig. 6B). PIC slightly suppressed NQO1 and GCLM expression, and NRF2 knockdown further decreased the expression levels of these genes (Fig. 6B). These results suggested that basal expression levels of these genes partly depend on NRF2.

Roles of Heme Oxygenase-1 in Reductions in Mitochondrial ROS Level and Apoptosis Afforded by Piceatannol.

We examined whether HO1 is involved in the antioxidative and antiapoptotic actions of PIC. HO1 level was effectively reduced by siRNA against HO1 (Fig. 7A). PIC decreased the fluorescence level of MitoSOX Red induced by antimycin A to 24% of that in cells transfected with control siRNA without PIC. This ROS reduction by PIC was greater than that by RSV (from 100% to 56%), which is consistent with results shown in Fig. 4. In the absence of antimycin A, HO1 knockdown alone did not change MitoSOX Red fluorescence. Compared with control siRNA cells, HO1 knockdown augmented antimycin A–induced increase in fluorescence level by 52% (Fig. 7, B and C). MitoSOX Red fluorescence level of cells treated with control siRNA was not affected by HO1 knockdown alone, but it was potentiated under the condition of antimycin A treatment (Fig. 7, B and C). HO1 siRNA treatment decreased the antioxidative function of PIC and RSV (Fig. 7, B and C). Although PIC decreased the fluorescence level of MitoSOX Red more than did RSV in cells treated with control siRNA, HO1 siRNA treatment cancelled the antioxidative function of PIC more than did RSV. Under the condition of HO1 knockdown, MitoSOX Red fluorescence level in PIC-treated cells was the same as that in cells treated with RSV (Fig. 7, B and C).

Fig. 7.
Fig. 7.

The involvement of heme oxygenase-1 in reductions in mitochondrial ROS and apoptosis afforded by PIC. (A) HO1 mRNA levels normalized to GAPDH in C2C12 cells transfected with control or HO1 siRNA. (B) Representative images of MitoSOX Red fluorescence. Scale bar, 100 µm. Cells were transfected with control- or HO1-siRNA and were then pretreated with a vehicle, 10 μM RSV, or 10 μM PIC for 24 hours under a serum-free condition. Cells were stimulated with 30 μM antimycin A (AA) for 1 hour to produce mitochondrial ROS, which was analyzed using MitoSOX Red. (C) Average of intensity of MitoSOX Red fluorescence per cell. Randomly selected 15 fields from independent three experiments were analyzed. (D) Representative images of nuclear staining with Hoechst33342. An arrow indicates a condensed nucleus, a marker of apoptosis. Scale bar, 100 µm. (E) The average of the percentage of cells with condensed nuclei. Randomly selected 15 fields from three independent experiments were analyzed. Two-way ANOVA followed by a Student-Newman-Keuls test was performed to analyze the statistical difference among groups in (C and E). *P < 0.05.

Apoptosis of cells transfected with control siRNA was not affected by HO1 knockdown in the absence of antimycin A (Fig. 7, D and E). HO1 siRNA did not affect the antiapoptotic function of RSV, and RSV reduced the number of apoptotic cells in the presence of HO1 siRNA. In contrast to the effect of HO1 siRNA on RSV-treated cells, HO1 knockdown increased apoptosis in PIC-treated cells, and it completely abolished the antiapoptotic function of PIC (Fig. 7, D and E).

Effects of RSV and PIC on Myotube Differentiation of C2C12 Myoblasts.

Finally, we examined the effects of RSV and PIC on the differentiation of C2C12 cells because RSV has been reported to promote differentiation of C2C12 myoblast cells (Montesano et al., 2013). Treatment of cells with a differentiation medium containing 2% horse serum for 7 days changed the shape of cells, and myotubes were formed (Supplemental Fig. 3A). Levels of myogenin and MYH4 mRNAs were increased by the differentiation medium, although the expression of MyoD, a master transcription factor of myogenesis, was not changed (Supplemental Fig. 3B). The addition of RSV or PIC to the differentiation medium did not affect the appearance of cells (Supplemental Fig. 3A). RSV and PIC did not affect myogenin and MyoD mRNA levels, but RSV or PIC treatment enhanced the expression levels of MYH4 (Supplemental Fig. 3B). The magnitude of the increase in MYH4 expression induced by RSV was greater than that induced by PIC (Supplemental Fig. 3B).

Discussion

Under the condition of treatment of cells with antimycin A or H2O2, PIC reduced mitochondrial ROS and apoptotic cells more than did RSV (Figs. 4 and 5). These findings indicate that cytoprotection by PIC is more potent than that by RSV against mitochondrial ROS. The reduction in apoptosis by RSV was mostly dependent on SIRT1, and the involvement of SIRT1 in PIC’s antiapoptotic action was partial (Fig. 5). In contrast, PIC, but not RSV, upregulated HO1, which was blocked by NRF2 knockdown but not by SIRT1 knockdown (Fig. 6). HO1 knockdown abolished the effects of PIC on mitochondrial ROS and apoptosis but had no influence on the effects of RSV (Fig. 7). These findings suggest that the cytoprotection by PIC is mainly attributed to HO1 induction via NRF2 activation.

PIC was detected in the intracellular fraction of cells treated with PIC (Supplemental Fig. 1), and an additional peak was not detected. These suggested that PIC entered cells without metabolic modifications. It also means that PIC can reach intracellular targets including NRF2 and SIRT1 to exert its functions. In addition, it is unlikely that we would detect PIC bound to the outer side of the plasma membrane because PIC was not detected in the intracellular fraction from cells harvested after PIC incubation for 1 minute (Supplemental Fig. 1).

Oxidation or covalent modification of thiol groups of Keap1 disrupt its repression of NRF2, resulting in NRF2 translocation to the nucleus (Yamamoto et al., 2018). Lee et al. (2010) hypothesized that cysteine residues of Keap1 are targets of PIC for its NRF2 activation and showed that HO1 induction by PIC was blocked by thiol reducing agents. The authors speculated that PIC directly interacts with a critical thiol(s) of Keap1 because the catechol structure of PIC is likely to be oxidized to form quinone, which targets cysteine thiols. Another compound with a catechol structure had also been reported to be oxidized to quinone, which binds to thiol groups of Keap1 (Sumi et al., 2009). Since RSV does not have a catechol structure, such a structural difference may explain why RSV did not activate NRF2.

In cells treated with H2O2, the reduction in MitoSOX Red fluorescence levels by PIC was greater than that by RSV or vitamin C (Fig. 4, C and D). Our data suggest that this powerful ROS-reducing function of PIC is derived from both its radical-scavenging property (Fig. 1) and HO1 induction (Fig. 7). However, the radical-scavenging activity of piceatannol itself seems to play a minor role in cytoprotection by PIC because knockdown of HO1 almost completely abolished the antiapoptotic effect of PIC.

HO1 is tethered to the membrane of the endoplasmic reticulum (Dunn et al., 2014). Free heme promotes the formation of a cytotoxic hydroxy radical from H2O2 through the Fenton reaction. HO1 catabolizes free heme into Fe2+, carbon monoxide, and biliverdin (Gozzelino et al., 2010). Bilirubin converted from biliverdin by biliverdin reductase exerts antioxidant activity (Gozzelino et al., 2010). Interestingly, it has been reported that HO1 and biliverdin reductase localize in the mitochondria of the rat liver, where HO1 reduces mitochondrial heme content (Converso et al., 2006). Furthermore, HO1 translocates to mitochondria in response to mitochondrial oxidative stress in gastric mucosal cells, resulting in protection from mitochondrial damage and apoptosis (Bindu et al., 2011). Therefore, upregulated HO1 by PIC might have located in the mitochondria and reduced mitochondrial ROS and apoptosis.

Antiapoptotic actions of RSV and PIC were attenuated by SIRT1 knockdown (Fig. 5). However, RSV and PIC failed to induce SOD2 and catalase, which are target genes of FoxOs activated by SIRT1, probably because of a lower dose of RSV than the doses used in previous studies (Tanno et al., 2010; Hori et al., 2013). As a possible SIRT1-dependent protective mechanism, we previously reported that knockdown of the transcription factor p53, a deacetylation target of SIRT1, reduced antimycin A–induced apoptosis in C2C12 cells and that treatment of cells with RSV reduced the level of acetyl-p53 (Hori et al., 2013). Since deacetylation of p53 by SIRT1 negatively regulates p53 activity (Luo et al., 2001), inhibition of p53 may be involved in the SIRT1-mediated antiapoptotic actions of RSV and PIC. In addition, we reported that activation of autophagy/mitophagy is involved in the decrease in mitochondrial ROS by RSV in C2C12 cells (Sebori et al., 2018). Elimination of damaged mitochondria via mitophagy may also play a role in the reduction of ROS by RSV and the antiapoptotic action of RSV.

RSV at a higher concentration did not affect mitochondrial membrane potential and apoptosis in C2C12 cells (Figs. 2 and 3), although administration of RSV at more than 2 g per day in humans often causes gastrointestinal symptoms such as diarrhea and abdominal pain (Patel et al., 2011). On the other hand, PIC promoted mitochondrial membrane depolarization and induced apoptosis at higher concentrations (Figs. 2 and 3). PIC has high anticancer activity for various tumors (Piotrowska et al., 2012). Numerous mechanisms of the anticancer activity of PIC have been reported. The reported mechanisms include activation of caspases, inhibition of Janus kinase, downregulation of cell cycle inducers, activation of poly(ADP-ribose) polymerase, decrease of B-cell lymphoma 2 and B-cell lymphoma-extra large, upregulation of Tumor Necrosis Factor Receptor Superfamily Member 6 and its ligand, inhibition of mitochondrial F1-ATPase, and inhibition of ribonuclease reductase (Wieder et al., 2001; Piotrowska et al., 2012; Seyed et al., 2016). Experimental conditions including cell types, concentration of PIC, and treatment duration were different in previous studies. However, a relatively high concentration of PIC (i.e., 50 μM) was used in most studies for analyses of antitumor effects. Nevertheless, it is important to pay attention to the optimum therapeutic dose of PIC when we apply PIC to cytoprotection against cellular insult such as oxidative stress.

Although there has been no study on the pharmacokinetics of PIC in humans, a study using rats showed that PIC possesses higher metabolic stability than that of RSV (Setoguchi et al., 2014). The elimination half-life of PIC after intravenous administration was 313 minutes, and PIC was still detected 12 hours after administration (Roupe et al., 2004). On the other hand, RSV is subjected to rapid and extensive metabolism after oral administration (Walle et al., 2004), and the elimination half-life of RSV was quite rapid and less than 3 hours in humans (Almeida et al., 2009; Kawamura et al., 2020). Although PIC and RSV are nonspecific compounds and target various molecules, both of them have been shown to have therapeutic potential (Piotrowska et al., 2012; Bonkowski and Sinclair, 2016; Seyed et al., 2016; Salehi et al., 2018). The results of those studies and the present study suggest that PIC is an alternative and promising agent for treatment of various diseases in which ROS are strongly involved, although further studies regarding the safety and pharmacokinetics of PIC in humans are required.

Authorship Contributions

Participated in research design: Hosoda, Hamada, Horio, Kuno.

Conducted experiments: Hosoda, Uesugi, Iwahara, Nojima.

Performed data analysis: Hosoda, Hamada, Iwahara, Nojima, Horio, Kuno.

Wrote or contributed to the writing of the manuscript: Hosoda, Horio, Kuno.

Footnotes

    • Received May 11, 2020.
    • Accepted December 10, 2020.

  • This work was supported in part by research grants from the Chugoku Regional Innovation Research Center (H.H., Y.H.), the Hiroshige Kondo Foundation (Y.H.), and Grants-in-Aid for Regional R&D Proposal-Based Program from Northern Advancement Center for Science and Technology of Hokkaido Japan.

  • All authors have no conflicts of interest to declare.

  • https://doi.org/10.1124/jpet.120.000096.

  • ↵Embedded ImageThis article has supplemental material available at jpet.aspetjournals.org.

Abbreviations

7-AAD
7-amino-actinomycin D
DMEM
Dulbecco’s modified Eagle’s medium
DPPH
2,2′-diphenyl-1-picrylhydrazyl
ESI-MS
electrospray ionization mass spectrometry
FoxO
forkhead box O transcription factor
GAPDH
glyceraldehyde-3-phosphate dehydrogenase
GCLM
glutamate-cysteine ligase modifier subunit
4′G-RSV
resveratrol-4′-O-β-d-glucoside
HO1
heme oxygenase-1
H2O2
hydrogen peroxide
Keap1
Kelch-like ECH-associated protein 1
MYH4
myosin heavy chain 4, MyoD myoblast determination protein 1
NAC
N-acetyl-l-cysteine
NQO1
NAD(P)H:quinone oxidoreductase 1
NRF2
nuclear factor erythroid 2–related factor 2
PCR polymerase chain reaction PIC
piceatannol
ROS
reactive oxygen species
RSV
resveratrol
siRNA
small interfering RNA
SIRT1
sirtuin 1
SOD2
superoxide dismutase 2

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Research ArticleCellular and Molecular

Comparison of Piceatannol with Resveratrol

Ryusuke Hosoda, Hiroki Hamada, Daisuke Uesugi, Naotoshi Iwahara, Iyori Nojima, Yoshiyuki Horio and Atsushi Kuno
Journal of Pharmacology and Experimental Therapeutics March 1, 2021, 376 (3) 385-396; DOI: https://doi.org/10.1124/jpet.120.000096
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