ABSTRACT Resveratrol (3,5,4′-trihydroxy-_trans_-stilbene) is a well-known polyphenol that is present in grapes, peanuts, pine seeds, and several other plants. Resveratrol exerts deleterious

effects on various types of human cancer cells. Here, we analyzed the cell death-inducing mechanisms of resveratrol-006 (Res-006), a novel resveratrol derivative in human liver cancer cells

_in vitro_. Res-006 was more effectively suppressed the viability of HepG2 human hepatoma cells than resveratrol (the IC50 values were 67.2 and 354.8 μmol/L, respectively). Co-treatment with

the ER stress regulator 4-phenylbutyrate (0.5 mmol/L) or the ROS inhibitor N-acetyl-_L_-cysteine (NAC, 1 mmol/L) significantly attenuated Res-006-induced HepG2 cell death, suggesting that

events, except for the change in mitochondrial morphology, were prevented by the exposure of the HepG2 cells to the mitochondrial ROS scavenger, Mito-TEMPO (300–1000 μmol/L). The results

suggest that Res-006 may kill HepG2 cells through cell death pathways, including the ER stress initiated by mitochondrial ROS accumulation. The cell death induced by this novel resveratrol

derivative involves crosstalk between the mitochondria and ER stress mechanisms. SIMILAR CONTENT BEING VIEWED BY OTHERS CHALCOMORACIN PROMOTES APOPTOSIS AND ENDOPLASMIC RETICULUM STRESS IN

HEPATOCELLULAR CARCINOMA CELLS Article 09 May 2024 NUPR1 INHIBITOR ZZW-115 INDUCES FERROPTOSIS IN A MITOCHONDRIA-DEPENDENT MANNER Article Open access 01 October 2021 COROSOLIC ACID

SENSITIZES FERROPTOSIS BY UPREGULATING HERPUD1 IN LIVER CANCER CELLS Article Open access 29 August 2022 INTRODUCTION Resveratrol (3,5,4′-trihydroxy-_trans_-stilbene; Res) is a nonflavonoid

polyphenol present in grapes, peanuts, berries, and the constituents of several other plants1. It possesses a wide range of beneficial effects, including anti-cancer, anti-aging,

anti-atherogenic, anti-inflammatory and anti-oxidant activities1,2,3. However, its utilization and development in products have been hampered by its poor solubility, poor chemical stability,

and low bioavailability4,5. Many attempts have sought to improve the bioactivity and bioavailability of Res6,7. For example, trimethylated Res is up to 100-fold more cytotoxic than

unamended Res in cancer cell lines due to the depletion of the intracellular pool of polyamines and altered microtubule polymerization8. Mitochondria are essential in cellular energy

metabolism and are now recognized as being central in apoptotic cell death. Stresses, including growth-factor withdrawal, DNA damage, and exposure to certain chemotherapeutic agents,

activate mitochondria-mediated intrinsic apoptosis pathways9. During intrinsic apoptosis signaling, mitochondrial outer membrane permeabilization (MOMP) leads to the release of pro-apoptotic

proteins (cytochrome _c_, Smac/DIABLO, Omi/HtrA2, AIF and endonuclease G) contained in the intermembrane space9. These pro-apoptotic proteins trigger the execution of cell death by

promoting the caspase activation cascade or by acting as caspase-independent death effectors9. Several recent reports suggested that Res and its derivatives trigger apoptosis through the

intrinsic mitochondrial-dependent pathway, which is associated with mitochondrial dysfunctions, such as mitochondrial membrane potential (MMP) collapse and reactive oxygen species (ROS)

production10,11,12. The endoplasmic reticulum (ER) is a principal cellular compartment for the biosynthesis, folding and modification of membrane and secretory proteins, production of lipids

and sterols, and calcium storage and gated release in eukaryotic cells. The pathological, environmental, or physiological stimuli that interfere with ER functions cause ER stress. To cope

with the ER stress conditions, the ER activates a set of signal transduction pathways, collectively termed the unfolded protein response (UPR), to assist with protein folding and secretion

and to facilitate the degradation of misfolded proteins in the ER lumen13. In mammals, the UPR is mediated by three basic signal transducers: inositol-requiring 1α (IRE1α), PERK

(double-strand RNA-activated protein kinase-like ER kinase), and ATF6α (activating transcription factor 6α)13,14. During ER stress, the cytoplasmic nuclease domain of activated IRE1α

processes the mRNA encoding the XBP-1 (X-box-binding protein 1) transcription factor to generate mature _Xbp1_ mRNA (_Xbp1s_)15. The activated PERK phosphorylates the α subunit of eukaryotic

translation initiation factor 2 (eIF2α) at Ser51 to attenuate global translation16 and increase the translation of mRNAs, such as those encoding the ATF4 transcription factor17. Upon

activation, ATF6α translocates from the ER to the Golgi complex, where it is cleaved by the S1P and S2P proteases to release a cytosolic fragment (ATF6αΔC) that migrates to the nucleus to

activate transcription18,19. For ER stress adaptation, XBP1s and ATF6αΔC by themselves or together induce many UPR genes to enhance ER protein folding, trafficking, secretion, and

ER-associated protein degradation (ERAD)20,21. ATF4 induces the expression of several genes involved in amino acid biosynthesis and transport, anti-oxidative stress, and ER protein folding

and secretion22. However, if the ER stress is too strong and persistent to re-establish ER homeostasis, the ER preferentially elicits several cell-death signaling pathways, including three

UPR pathway-mediated apoptosis pathways, over time23,24. Under chronic ER stress, IRE1α recruits the tumor necrosis factor receptor-associated factor 2 (TRAF2) and apoptosis

signal-regulating kinase 1 (ASK1) and then causes the activation of c-Jun N-terminal kinase (JNK), which is implicated in apoptosis25,26. In addition, prolonged IRE1α-mediated activation of

the regulated IRE1-dependent decay (RIDD) pathway may promote apoptosis by degrading mRNAs encoding essential ER-translocating proteins27 and microRNAs repressing the translation of

pro-apoptotic _caspase-2_ mRNA28. Although the PERK-eIF2α phosphorylation-ATF4 and ATF6α pathways conduct important adaptive mechanisms to relieve the ER stress, ATF4 and ATF6αΔC converge on

the promoter of the gene encoding C/EBP homologous protein (CHOP)21,29, an important pro-apoptotic transcription factor of ER stress-mediated cell death23, which controls the expression of

the pro-apoptotic (_Bim_)30 and anti-apoptotic (_Bcl-2_) genes31. Furthermore, CHOP expression increases the expression of several pro-apoptotic genes, such as ER oxidoreductase 1-α (ERO1α),

growth arrest and DNA damage 34 (GADD34), tribbles-related protein3 (Trb3) and death receptor 5 (Dr5)23. A growing body of data indicates that Res and Res derivatives can induce

pro-apoptotic ER stress in cancer cells by disrupting the N-linked glycosylation of proteins or by increasing intracellular calcium levels32,33,34. Here, we sought to detect the cell death

effects of a new Res derivative in a human liver cancer cell line and to determine the underlying mechanism of its activity, which involves apoptosis associated with mitochondrial

dysfunctions and ER stress. MATERIALS AND METHODS REAGENTS AND ANTIBODIES Resveratrol (Res), N-acetyl-_L_-cysteine (NAC), 4-phenylbutyric acid (4-PBA), BAPTA-AM, TEMPOL, Mito-TEMPO, and

Hoechst 33258 were purchased from Sigma-Aldrich (St Louis, MO, USA). Tunicamycin and thapsigargin were purchased from EMD Millipore (Billerica, MA, USA). MitoTracker Red and JC-1 were

purchased from Molecular Probes (Eugene, OR, USA). Resveratrol-005 (Res-005) and resveratrol-006 (Res-006, Korea Patent #1016328390000) were synthesized by Prof Hyoungsu KIM (see

Supplementary information for details). A Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technology (Rockville, MD, USA). The antibodies, including anti-BAK, anti-BAX,

anti-Bcl-2, anti-Bcl-xL, anti-cleaved caspase-3, anti-CHOP, anti-IRE1α, anti-JNK, anti-p-JNK, anti-PARP, and anti-PERK, were purchased from Cell Signaling Technology (Danvers, MA, USA).

Anti-β-actin and horseradish peroxidase-conjugated anti-Flag were purchased from Sigma-Aldrich (St Louis, MO, USA). In addition, the following antibodies were used: anti-eIF2α from Santa

Cruz Biotechnology (Dallas, TX, USA), anti-BiP from BD Bioscience (San Jose, CA, USA), anti-KDEL from Assay Designs (Farmingdale, NY, USA), anti-p-IRE1α from Novus Biologicals (Littleton,

CO, USA), anti-MTCO1 and anti-SDHA from Abcam (Cambridge, UK), and anti-p-eIF2α from Invitrogen (Carlsbad, CA, USA). The secondary peroxidase-conjugated antibodies were purchased from Thermo

Fisher Scientific (Waltham, MA, USA) or Jackson ImmunoResearch (West Grove, PA, USA). CELL CULTURE HepG2 human hepatocellular carcinoma cancer cells were obtained from the American Type

Culture Collection (ATCC, Manassas, VA, USA). The Huh-7 cells were provided by Dr Sung Key JANG (Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Korea)35.

Both the HepG2 and Huh-7 cells were cultured in DMEM (Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (ATCC) and 5% penicillin-streptomycin (Gibco). The THLE-2 cells

(ATCC) originated from human primary normal liver cells and were provided by Dr Jong-heon KIM (Cancer Cell and Molecular Biology Branch, National Cancer Center, Korea). THLE-2 cells were

plated on culture plates precoated with a solution containing 0.01 mg/mL fibronectin (Thermo Scientific, Waltham, MA, USA), 0.03 mg/mL bovine collagen type I (Thermo Scientific), and 0.01

mg/mL bovine serum albumin (Sigma-Aldrich) dissolved in bronchial epithelial basal medium (BEBM, Lonza, Basel, Switzerland). The THLE-2 cells were cultured in BEBM supplemented with 5 ng/mL

EGF (R&D Systems, Minneapolis, MN, USA), 70 ng/mL phosphoethanolamine (Sigma-Aldrich), 10% fetal bovine serum (ATCC, Manassas, VA, USA), and BEGM SingleQuots (Lonza) with no

gentamicin/amphotericin (GA) and epinephrine. The cells were grown in a 5% CO2 incubator at 37 °C. CELL VIABILITY ASSAYS The cell viability assay was performed using a CCK-8 kit (Dojindo

Molecular Technologies) according to the manufacturer's instructions. Briefly, the cells were plated in 96-well plates and grown overnight. The next day, the cells were treated with the

indicated chemicals for 24 h, and then, the cells were treated with the CCK-8 solution for 3 h. Absorbance was measured at 450 nm using a microplate reader (Molecular Devices, Sunnyvale,

CA, USA). TRANSFECTION HepG2 cells (1×105) were plated in 60-mm dishes or collagen-coated 35-mm coverglass bottom dishes and cultured overnight. The next day, the flag-tagged

ATF6α-expressing plasmid (pCMV-Flag3x-ATF6α, a kind gift from Ron Prywes, Addgene plasmid #11975) or mitochondria-targeted EYFP-expressing plasmid (pEYFP-Mito, Clontech, Mountain View, CA,

USA) was transfected into HepG2 cells using the Fugene6 transfection reagent (Promega, Madison, WI, USA) for 48 h. The cells were treated with the chemicals (Tm, Tg, or Res-006) for the

indicated time. WESTERN BLOT ANALYSIS Cells were lysed with NP40 lysis buffer (1% Nonidet P-40, 0.05% sodium dodecyl sulfate, 50 mmol/L Tris-Cl pH 7.5, 150 mmol/L NaCl, 0.5 mmol/L sodium

vanadate, 100 mmol/L sodium fluoride, and 50 mmol/L β-glycerophosphate) supplemented with Halt protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). The homogenates were

centrifuged at 12 000×_g_ for 15 min at 4 °C, and the supernatants were collected. The protein concentration was determined using a BCA protein assay kit (Bio-Rad, Hercules, CA, USA).

Protein samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride or nitrocellulose membranes (GE Healthcare Life

Sciences, Marlborough, MA, USA). The membranes were blocked for 3 h at room temperature with 5% skim milk in Tris-buffered saline Tween buffer (0.1% Tween 20, 20 mmol/L Tris-HCl, pH 7.5, and

150 mmol/L NaCl). The membranes were then incubated with the indicated primary antibodies overnight at 4 °C and then with the horseradish peroxidase-conjugated secondary antibody.

Membrane-bound antibodies were detected by enhanced chemiluminescence (ECL) (Thermo Scientific). FLOW CYTOMETRY ANALYSIS OF APOPTOSIS OR THE CELLULAR ROS LEVEL HepG2 cells (2×105) were

plated in 6-well plates and cultured overnight. The cells were treated with the indicated chemicals. After treatment, the cells were harvested and then washed twice with cold-phosphate

buffered saline (PBS). Next, the cells were double-stained with annexin V and 7-aminoactinomycin D (7AAD) (BD Pharmingen, Franklin Lakes, NJ, USA) in binding buffer for 15 min. Finally, the

cells were analyzed by flow cytometry using a FACSCanto II apparatus (BD Biosciences, Franklin Lakes, NJ, USA). FlowJo software (Ashland, OR, USA) was used for the analysis. HepG2 cells

(2×105) were plated in 6-well plates and cultured overnight. The next day, the cells were treated with the indicated chemicals. After treatment, the cells were stained with dihydroethidium

(15 μmol/L, Sigma-Aldrich) in culture medium for 30 min. The cells were harvested and analyzed by flow cytometry as described above. IMMUNOFLUORESCENCE HepG2 cells (2×105) were plated in

6-well plates coated with collagen 0.01% in PBS and cultured overnight. The next day, the cells were treated with the indicated chemicals, fixed with 4% paraformaldehyde in PBS for 15 min,

and permeabilized with 0.2% Triton X-100 in PBS for 2 min. The cells were blocked with 1% bovine serum albumin in PBS for 30 min and incubated with the indicated primary antibody overnight

at 4 °C. The cells were further incubated with a fluorescein isothiocyanate-conjugated secondary antibodies at room temperature for 1 h. The nuclei were stained with Hoechst 33258

(Sigma-Aldrich). Finally, the cells were observed by confocal laser microscopy using an FV1200-OSR microscope (Olympus, Tokyo, Japan). MITOCHONDRIAL MEMBRANE POTENTIAL (MMP) ANALYSIS THLE-2

and HepG2 cells (2×105) were plated in collagen-coated 35 mm coverglass bottom dishes (SPL, Pocheon-si, Gyeonggi-do, Korea) and cultured overnight. The next day, the cells were treated with

the indicated chemicals. After treatment, the cells were stained with MitoTracker Red (200 nmol/L) or JC-1 (2.5 μmol/L) and Hoechst 33258 (4 μg/mL) in culture medium for 1 h. Fluorescence

images of living cells were obtained using an FV1200-OSR confocal laser microscope. TIME-LAPSE CONFOCAL MICROSCOPY Mitochondria-targeted EYFP-expressing HepG2 cells were treated with mock or

Res-006 for 20 min, and then the mitochondria were imaged for 4 min and 30 s using an FV1200-OSR confocal laser microscope (Olympus). Frames were taken every 30 s. The microscopic field was

63.4 μm×63.4 μm. SEMI-QUANTITATIVE PCR AND QRT-PCR Total RNA was prepared from the HepG2 cells treated with the indicated chemicals using an RNeasy Plus Mini Kit (Qiagen, Venlo,

Netherlands). The cDNA was prepared with a High Capacity cDNA RT Kit (Ambion, Life Technologies, Waltham, MA, USA) for semi-quantitative PCR using standard methods or for qRT-PCR normalized

to the levels of β-actin as previously described36. The primers for the semi-quantitative PCR analysis were as follows: forward primer for _Xbp1_ mRNA splicing analysis,

5′-CCGCAGCAGGTGCAGG-3′ and reverse primer 5′-GGGGCTTGGTATATATGTGG-3′; forward primer for _Gapdh_ mRNA, 5′-ACATCAAGAAGGTGGTGAAG-3′ and reverse primer 5′-CTGTTGCTGTAGCCAAATTC-3′. The primers

for the qRT-PCR analysis are as follows: _β-actin_ forward primer 5′-TCCCCCAACTTGAGATGTATGAAG-3′ and _β-actin_ reverse primer 5′-AACTGGTCTCAAGTCAGTGTACAGG-3′; _Xbp1s_ forward primer

5′-CCGCAGCAGGTGCAGG-3′ and _Xbp1s_ reverse primer 5′-GAGTCAATACCGCCAGAATCCA-3′; _Xbp1t_ forward primer 5′- GCAAGCGACAGCGCCT-3′ and _Xbp1t_ reverse primer 5′- TTTTCAGTTTCCTCCTCAGCG-3′;

_ERdj4_ forward primer 5′- GGAAGGAGGAGCGCTAGGTC-3′ and _ERdj4_ reverse primer 5′-ATCCTGCACCCTCCGACTAC-3′; _Chop_ forward primer 5′-ATGGCAGCTGAGTCATTGCCTTTC-3′ and _Chop_ reverse primer

5′-AGAAGCAGGGTCAAGAGTGGTGAA-3′; and _Grp78_ (_BiP_) forward primer 5′-GCCTGTATTTCTAGACCTGCC-3′ and _Grp78_ (_BiP_) reverse primer 5′-TTCATCTTGCCAGCCAGTTG-3′. STATISTICAL ANALYSIS All data

are represented as the mean±SEM of three or four independent experiments. The data were analyzed using GraphPad Prism 5 (GraphPad Software, Inc, La Jolla, CA, USA). Unpaired 2-tailed

Student's _t_-tests were performed to determine the statistical significance for paired samples. _P_<0.05 was considered significant. RESULTS RES DERIVATIVE

3,5-DIETHOXY-3′,4′-DIHYDROXY-TRANS-STILBENE DISPLAYS ER STRESS AND/OR OXIDATIVE STRESS-MEDIATED CYTOTOXICITY IN HEPG2 CELLS To investigate the cytotoxic effect of Res and its derivatives

(Figure 1A), one normal human liver cell line, THLE-2, and two human hepatoma cell lines, Huh-7 and HepG2, were treated with various concentrations of the compounds (Figure 1B-1H). The

dehydrogenase-based cell viability assay revealed that the Res derivative designated Res-006 had stronger cellular toxicity than Res and Res-005 in all cell lines used (Figure 1B, 1D, 1F,

and 1G). The IC50 value (67.2 μmol/L) of Res-006 in HepG2 cells was approximately 2-fold lower than that (139.2 μmol/L) in THLE-2 cells (Figure 1C, 1E, and 1G). The cytotoxic effect of 65

μmol/L Res-006 against HepG2 cells was higher than that against THLE-2 and Huh-7 cells (Figure 1H). In addition, the time-dependent expression of apoptotic marker proteins (cleaved caspase-3

and PARP) in lysates of Res-006-treated HepG2 cells was higher compared with that of Res-006-treated THLE-2 and Huh-7 cells (Figure 1I). Therefore, subsequent experiments were focused on

the cytotoxic effects of Res-6 against HepG2 cells. To investigate whether Res-006 can induce apoptosis in HepG2 cells, we examined the apoptotic effect of Res-006 by flow cytometry and

Western blot analyses. In the mock-treated group, <6.0% of cells underwent apoptosis (Q2+Q4 in Figure 2A). In contrast, in cells treated with 65 μmol/L Res-006, 26.1% of cells underwent

apoptosis (Figure 2A). Moreover, both caspase-3 and PARP were significantly cleaved and activated by Res-006 (Figure 2B). The results indicated that Res-006 can significantly induce

apoptosis in HepG2 cells. Several recent reports suggested that Res and its derivatives can elicit ER stress-mediated cell death32 by increasing the level of cellular calcium by inhibiting

sarco/endoplasmic reticulum Ca2+ATPase (SERCA)34 or disrupting the _N_-linked glycosylation of proteins33. In addition, oxidative stress induced by the drugs may be responsible for cell

death12,37. To verify whether ER stress and/or oxidative stress are associated with Res-006-mediated HepG2 cell death, the ER stress regulator 4-phenylbutyrate (PBA) and/or the ROS inhibitor

N-acetyl-_L_-cysteine (NAC) were applied along with Res-006. Inhibition of either ER stress or ROS antagonized the cell death activity of Res-006 toward HepG2 cells (Figure 2C). Moreover,

compared with the treatment with drug alone, co-treatment with 4-PBA and NAC further increased cell viability (Figure 2C), indicating that pro-apoptotic ER stress and/or ROS may govern the

cell death induced by Res-006 in HepG2 cells. However, treatment with the Ca2+ chelator BAPTA/AM did not restore the viability of Res-006-treated HepG2 cells (Figure 2D), suggesting that an

increase in intracellular Ca2+ levels may not relate with Res-006-mediated cell death. Next, to determine the effect of Res-006 on the N-linked glycosylation of the proteins, we monitored

the N-linked glycosylation of ATF6α, an N-linked glycosylated ER membrane protein18 in HepG2 cells. Tunicamycin (Tm), an established inhibitor of N-linked glycosylation38, was used to

generate a positive control. The 3xFlag-tagged human ATF6α proteins were transiently overexpressed in HepG2 cells. The deglycosylation-induced mobility change of 3xFlag-ATF6α was examined by

Western blot analysis with tunicamycin, the SERCA inhibitor thapsigargin (Tg), or Res-006-treated HepG2 cells. As expected, the 3xFlag-ATF6α in the Tm-treated cell lysates migrated faster

compared with the mock or Tg-treated cell lysates (Figure 2E), indicating that the protein was deglycosylated by tunicamycin treatment. However, there were no fast migrating bands observed

in Res-006-treated lysates until 24 h (Figure 2E), suggesting that the Res-006 may not elicit ER stress through the disruption of the N-linked glycosylation of the proteins. Taken together,

these results suggest that Res-006 induces ER stress- and/or ROS-mediated cell death, which is not associated with acute calcium mobilization or N-linked glycosylation inhibition. RES-006

DYSREGULATES MITOCHONDRIAL DYNAMICS AND DISRUPTS MMP IN HEPG2 CELLS It is possible that mitochondrial dysfunction can induce ER stress37,39 and vice versa24,40,41. To explore this, we first

used mitochondria-targeted enhanced yellow fluorescent protein (Mito-EYFP) to monitor the morphological changes of mitochondria during Res-006 treatment in HepG2 cells (Figure 3A). Following

the addition of Res-006, the mitochondrial morphology changed from interconnected filaments to large spheres within 60 min, whereas the morphology was barely changed in the mock-treated

cells (Figure 3A). Because mitochondrial morphology is determined by a dynamic equilibrium between organelle fusion and fission, we imaged mitochondria for 4.5 min in mock-treated and

Res-006-treated cells (Figure 3B and Supplementary Movie S1 and S2). In the mock-treated cells, the mitochondria were highly mobile, and several fusion and fission events were observed

during the recordings (Supplementary Movie S1). However, in the Res-006-treated cells, many mitochondria had already become ovoid or spherical, possibly due to fusion before the time-lapse

experiments. Later on, the fusion or fission events were rarely observed. One fusion event was observed, which generated a large spherical mitochondrion (arrows and ovals in Figure 3B and

Supplementary Movie S2). In addition to reduced fusion and fission, the time-lapse videos revealed striking defects in the mobility of the mitochondria in Res-006-treated HepG2 cells. These

results suggest that Res-006 alters mitochondrial dynamics, which can lead to a mitochondrial morphology change. Next, we tested whether Res-006 treatment could disrupt MMP, which is an

important parameter of mitochondrial function. MitoTracker Red, a lipophilic cationic dye that is sensitive to the MMP, was used to stain Res-006-treated HepG2 cells. The number of stained

mitochondria and their fluorescence intensities were gradually reduced in Res-006-treated cells until 24 h; however, they were not changed in mock-treated cells (Figure 3C and 3E) and

Res-006-treated THLE-2 cells (Supplementary Figure S1). However, the levels of the mitochondrial DNA-encoded protein MTCO1 and a nuclear-encoded protein SDHA remained unchanged in

Res-006-treated HepG2 cells until 24 h (Figure 3D). In addition, immunofluorescence analysis of MTCO1 showed that the fluorescence intensity of mitochondria labeled with the anti-MTCO1

antibody did not decrease in Res-006-treated cells for 24 h compared with the mock-treated cells; whereas the fluorescence intensity of MMP-dependent MitoTracker Red was significantly

decreased by the drug treatment (Figure 3E). The observations suggested that Res-006 treatment disrupts mitochondrial function but does not cause the removal of malfunctioning mitochondria

from the cells. RES-006-INDUCED CELL DEATH IS PREVENTED BY SUPEROXIDE SCAVENGER Mitochondria are an important source of ROS within most mammalian cells42,43. In addition, cellular redox

homeostasis is interconnected with MMP42,44,45. The level of accumulated ROS was measured by flow cytometry following staining of the cells with the superoxide indicator dihydroethidium

(DHE). The ROS levels in cells treated for 2 h with Res-006 was significantly higher (approximately 2.3 fold) than in the mock-treated cells (Figure 4A). From these results, we hypothesized

that in Res-006 treated cells, the ROS accumulation leads to mitochondrial dysfunction (Figure 3A-3E) and cell death (Figure 2B), which can be ameliorated by its modulation. To verify this

hypothesis, HepG2 cells were co-treated with the superoxide dismutase mimetic TEMPOL (Tem) and Res-006. The TEMPOL dose-dependently increased the viability of Res-006-treated cell

populations up to approximately 80% (Figure 4B, 4D, and 4E). Given that Res-006 led to mitochondrial dysfunctions (Figure 3A-3C and 3E) associated with mitochondrial ROS generation, we next

used a mitochondria-targeted TEMPOL (Mito-TEMPO, Mito) to specifically reduce the mitochondrial ROS levels. Intriguingly, the Mito-TEMPO treatments completely prevented cell death induced by

Res-006 at concentrations over 300 μmol/L (Figures 4C-4E). The flow cytometry of cells stained with annexin V and 7AAD clearly showed that the Mito-TEMPO treatment nearly abrogated

Res-006-induced cell death (Figure 4F). In addition, the Mito-TEMPO treatment markedly inhibited caspase-3 activation and PARP cleavage induced by Res-006, although TEMPOL also showed

significant inhibitory effects against the events (Figure 4G). Taken together, these data indicate that Res-006 induces cell death, which can be prevented by the removal of mitochondrial

ROS. MITOCHONDRIA-TARGETED SUPEROXIDE SCAVENGER PREVENTS RES-006-MEDIATED MMP DISRUPTION To explore the efficiency of the inhibition of ROS accumulation by two superoxide scavengers in

Res-006-treated HepG2 cells, flow cytometry was used to examine dihydroethidium (DHE)-stained cells. Both superoxide scavengers significantly inhibited ROS accumulation induced by Res-006

treatment. Notably, the ROS levels in the Mito-TEMPO treated cells were as low as those in the mock-treated cells (Figure 5A). Next, to verify whether the increased viability due to ROS

scavenging could be correlated with the recovery of MMP in Res-006-treated HepG2 cells, we used JC-1, a cationic MMP probe that accumulates in energized mitochondria. The cells that were

exposed to Res-006 for 2 h displayed greatly reduced red J-aggregate fluorescence and significantly increased cytoplasmic green monomer fluorescence compared with the mock-treated cells

(Figure 5B), indicating that Res-006 treatment causes the rapid collapse of MMP, which may have preceded apoptotic cell death (Figure 2B). However, both superoxide scavengers significantly

restored red J-aggregate fluorescence, suggesting that ROS scavenging can prevent the MMP loss in Res-006-treated HepG2 cells (Figure 5B). In addition, the TEMPOL and Mito-TEMPO treatments

greatly restored both the number and fluorescence intensities of the MitoTracker Red-stained mitochondria in Res-006-treated cells (Figure 5C); however, they did not prevent Res-006-mediated

mitochondrial fragmentation and swelling, indicating that the mitochondrial morphological changes may not be related with the Res-006-mediated ROS accumulation and cell death. RES-006

INDUCES ER STRESS RESPONSES Treatment with 4-phenylbutyric acid, an ER stress inhibitor, ameliorated Res-006-mediated cellular toxicity (Figure 2C). Thus, we investigated whether Res-006

could operate as an ER stress inducer. ROS-006 treatment induced the quick activation of all three UPR sensors (IRE1α, PERK, and ATF6α) in HepG2 cells13,14 (Figure 6A-6D). First, IRE1α

phosphorylation46 and its mediated downstream events, including increases in JNK phosphorylation25, _Xbp1_ mRNA splicing47, and XBP1s-dependent _Erdj4_ mRNA expression48, occurred in

Res-006-treated cells (Figure 6A and 6D). Second, Res-006 treatment induced the activation of the PERK/eIF2α-dependent pathway, including marked and persistent PERK phosphorylation, eIF2α

phosphorylation at 3 h (Figure 6B), and increases in _Chop_ transcripts (Figure 6D)29,49 and CHOP proteins (Figure 6B)50. Third, the activation of the last UPR sensor ATF6α was determined by

the observation of the S1P and S2P protease-mediated cleavage fragment (3XFlag-ATF6αΔC) generated19 from Flag-tagged ATF6α protein exogenously expressed in Res-006-treated HepG2 cells

(Figure 6C). As expected, the level of the cleavage product (3XFlag-ATF6αΔC) was increased in the HepG2 cells treated with the ER stress inducer, dithiothreitol (DTT) (Figure 6C)18,19.

Similarly, the Flag-tagged cleavage products increased over time in the Res-006-treated HepG2 cells. Furthermore, consistent with the ATF6α activation, the Res-006 treatment significantly

increased the expression of an ATF6α downstream target gene _Grp78_ (Figure 6C and 6D)20,21. Taken together, these results clearly suggest that Res-006 is a strong ER stress inducer that can

activate all three UPR pathways in HepG2 cells. RES-006 INDUCES MITOCHONDRIAL ROS-MEDIATED ER STRESS Since the removal of mitochondrial ROS prevented Res-006-mediated cell death, we

questioned whether mitochondrial ROS scavenging can reduce ER stress responses in Res-006-treated HepG2 cells. Mitochondria-targeted Mito-TEMPO treatment significantly prevented IRE1α and

JNK phosphorylation (Figure 7A). It strongly inhibited IRE1α-mediated _Xbp1_ mRNA splicing (Figure 7B) and subsequent expression of _Erdj4_, an XBP1s target gene (Figure 7C) in Res-006

treated cells. In addition, the Mito-TEMPO treatment robustly inhibited PERK phosphorylation (Figure 7B) and strongly suppressed pro-apoptotic CHOP expression (Figure 7A and 7C). Lastly, it

significantly reduced the expression of _Grp78_, an ATF6α downstream target gene (Figure 7C), suggesting that ATF6α activation was blocked by ROS scavenger treatment in Res-006-treated HepG2

cells. Although non-mitochondria-targeted TEMPOL could substantially suppress ER stress responses induced by Res-006 treatment, it was not as strong as Mito-TEMPO (Figure 7A-7C).

Collectively, these results strongly support the view that mitochondrial ROS that accumulate during Res-006 treatment induces ER stress responses that can induce cell death. DISCUSSION

Currently, the chemical derivatization of Res produced two novel cytotoxic drugs with improved cell death activity compared with Res. Res-006 produced mitochondrial dysfunction and ER

stress, which triggers the death of HepG2 cells. However, partial restoration of the mitochondrial dysfunctions via ROS scavengers, especially a mitochondria-targeted ROS scavenger

(Mito-TEMPO) robustly prevented ER stress and cell death. We conclude that the pro-oxidant activity of Res-006 is critical in inducing ER stress and cell death. The Res-006 treatment quickly

induced ROS accumulation in advance of cell death (compare Figure 4A with Figure 2B), suggesting that ROS accumulation triggered cell death. If so, what is the source of ROS in

Res-006-treated HepG2 cells? Treatment with the mitochondria-targeted ROS scavenger, Mito-TEMPO, robustly blocked ROS accumulation and restored MMP in Res-006-treated cells (Figure 5A and

5B), indicating that mitochondria are the main source of ROS. In addition, Res-006 treatment immediately caused a change in mitochondrial morphology, which led to large spherical

mitochondria (Figure 3A and 3B and Supplementary Movie S1 and S2). The changes may have been caused by changes in mitochondrial fusion and/or fission and its movement, suggesting that the

chemical alters mitochondrial dynamics. Mitochondrial morphology and dynamics are interlinked with cellular and mitochondrial redox homeostasis51. Cells deficient in mitochondrial fusion

proteins (Mfn1, Mfn2, or Opa1) display a fragmented mitochondrial morphology and increased ROS levels and then die. Conversely, chemical or genetic inhibitions of mitochondrial fission

proteins (Drp1 or Fis) induce mitochondrial elongation and reduce ROS production51,52. Thus, mitochondrial fragmentation allows increases in ROS production. However, in our experimental

conditions, treatment with the mitochondrial fission inhibitor, Mdivi-1, did not prevent Res-006-induced cell death, but rather inversely increased it (data not shown), suggesting that

Res-006-mediated ROS production and cell death may not be related with increased mitochondrial fission. The time-lapse imaging experiments provided evidence that Res-006 can induce

mitochondrial fusion at early time points, which rendered the mitochondria as large and spherical in HepG2 cells (Figure 3A and 3B, and Supplementary Movie S1 and S2). Therefore, the

morphological changes of mitochondria may not be triggered by the inhibition of mitochondrial fusion proteins (Mfn1, Mfn2, or Opa1) in Res-006-treated HepG2 cells. In addition, the removal

of ROS could not prevent the morphological changes of the mitochondria induced by the Res-006 treatment (Figure 5C). Thus, these data strongly suggest that ROS accumulation is not

responsible for the morphological change. Inversely, it is also possible that the morphological change of mitochondria is not related with ROS accumulation and cell death in Res-006-treated

HepG2 cells. The exact mechanism and role of the morphological change remain to be clarified. Although several reports suggested that Res and its derivatives target multiple intracellular

components (such as tumor suppressors p53 and Rb and apoptosis and survival regulators, Bax, Bak, AKT, Bcl-2, and Bcl-xL; see reference review3), including mitochondrial proteins11,53, which

can induce mitochondrial ROS and mitochondria-mediated apoptosis pathways (Figure 7D), there is increasing evidence that Res and Res derivatives can induce ER stress and mediate cell death

in several cancer cell types by disrupting the N-linked glycosylation of proteins or by increasing the level of intracellular Ca2+54. In this study, the novel Res derivative, Res-006, also

elicited ER stress. However, the ER stress inhibitor, 4-phenylbutyric acid, partially blocked Res-006-mediated ER stress (data not shown) and cell death (Figure 2C), whereas a

mitochondria-targeted ROS scavenger robustly inhibited both ER stress and cell death (Figures 4C-4G and 7A-7C), indicating that the ER stress partially contributes to Res-006-mediated cell

death and occurs downstream of mitochondria-ROS accumulation. Furthermore, Res-006 may not be a direct ER stress inducer inhibiting ER-mediated N-linked glycosylation or increasing

intracellular calcium level because the results in Figure 2D and 2E showed that the drug-mediated cell death was not linked with the general conditions that ER stress induced. Until now,

there have been no reports that ROS accumulation caused by Res or Res derivatives can elicit ER stress, but the role of other drug-mediated mitochondrial ROS in ER stress induction has been

demonstrated in a variety of cell types39,55. Drug-mediated ROS generation precedes UPR induction and is efficiently blocked by ROS scavengers55. In terms of mechanisms, the drugs trigger

mitochondrial ROS production through the regulation of gene expression or enzyme activity of mitochondrial ROS-producing proteins, such as NADPH oxidase 4 or cytochrome _c_ reductase

(complex III), respectively39. Although the precise mechanism of how mitochondrial ROS induce ER stress requires further investigation, it is proposed that oxidative protein damage and/or

its mediated ER calcium release may trigger ER stress39,55. However, Res-006-mediated mitochondrial ROS may not induce ER stress through the disruption of ER calcium homeostasis because

treatment with the intracellular calcium chelator BAPTA/AM could not inhibit Res-006-mediated cell death (Figure 2D). This issue requires further studies. In conclusion, our results

demonstrate that cell death in human hepatoma HepG2 cells induced by the novel Res derivative, Res-006, is mediated by the activation of ER stress and the dysfunction of mitochondria that

require ROS generation. The proposed cell death pathway induced by Res-006 is depicted in Figure 7D. The death involves cross-talk between the mitochondria and ER stress mechanisms. Our

study provides a rationale for the development of a new resveratrol derivative as chemotherapeutic agent targeting both mitochondria and the ER. AUTHOR CONTRIBUTION Jae-woo PARK, Woo-gyun

CHOI, Hyoungsu KIM, Hong-pyo KIM, and Sung-hoon BACK designed the research; Jae-woo PARK, Woo-gyun CHOI, Su-wol CHUNG, and Phil-jun LEE performed the experiments; Byung-sam KIM, Hun-taeg

CHUNG, Sungchan CHO, Jong-heon KIM and Byoung-heon KANG contributed to the acquisition of the data; Jae-woo PARK, Woo-gyun CHOI, Phil-jun LEE, Hyoungsu KIM, Hong-pyo KIM, and Sung-hoon BACK

analyzed and interpreted the data; Sung-hoon BACK wrote the draft manuscript, which was subsequently edited by all the authors, all of whom have read and approved the final manuscript.

SUPPLEMENTARY INFORMATION The supplementary information on the website of Acta Pharmacologica Sinica provides the confocal time-lapse movies of EYFP-labeled mitochondria in Mock- or

Res-006-treated HepG2 cells, the MitoTracker Red-labeled mitochondria images of Res-006-treated THLE-2 and HepG2 cells, and the synthesis methods for Res-005 and Res-006. REFERENCES * Baur

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references ACKNOWLEDGEMENTS This work was supported by the Basic Science Research Program (2011-0011433 and 2014R1A1A4A01004329), the Bio & Medical Technology Development Program

(2012M3A9C3050632), and the Priority Research Centers Program (2014R1A6A1030318) of the National Research Foundation of Korea (NRF) funded by the Korean government. AUTHOR INFORMATION Author

notes * Jae-woo Park and Woo-gyun Choi: These authors contributed equally to this work. AUTHORS AND AFFILIATIONS * School of Biological Sciences, University of Ulsan, Ulsan, 44610, Korea

Jae-woo Park, Woo-gyun Choi, Su-wol Chung, Byung-sam Kim, Hun-taeg Chung & Sung-hoon Back * School of Pharmacy, Ajou University, Suwon, 16499, Korea Phil-jun Lee, Hyoungsu Kim & 

Hong-pyo Kim * Targeted Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology, Chungbuk, 28116, Cheongwon Sungchan Cho * Department of Biomolecular Science,

University of Science and Technology, Daejeon, 34554, Korea Sungchan Cho * and Department of System Cancer Science, Cancer Cell and Molecular Biology Branch, Research Institute, Graduate

School of Cancer Science and Policy, National Cancer Center, Goyang, 10408, Korea Jong-heon Kim * School of Biological Sciences, Ulsan National Institute of Science and Technology (UNIST),

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ADDITIONAL INFORMATION Supplementary information is available on the website of Acta Pharmacologica Sinica. SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURE S1 Mitochondria labeling with

MMP-dependent MitoTracker Red in Res-006-treated THLE-2 and HepG2 cells (DOC 1679 kb) SUPPLEMENTARY METHODS Synthesis of Resveratrol-005 (DOC 253 kb) SUPPLEMENTARY MOVIE S1 a mock-treated

HepG2 cell. (AVI 3471 kb) SUPPLEMENTARY MOVIE S2 a Res-006-treated HepG2 cell (AVI 679 kb) POWERPOINT SLIDES POWERPOINT SLIDE FOR FIG. 1 POWERPOINT SLIDE FOR FIG. 2 POWERPOINT SLIDE FOR FIG.

3 POWERPOINT SLIDE FOR FIG. 4 POWERPOINT SLIDE FOR FIG. 5 POWERPOINT SLIDE FOR FIG. 6 POWERPOINT SLIDE FOR FIG. 7 POWERPOINT SLIDE FOR FIG. 8 RIGHTS AND PERMISSIONS Reprints and permissions

ABOUT THIS ARTICLE CITE THIS ARTICLE Park, Jw., Choi, Wg., Lee, Pj. _et al._ The novel resveratrol derivative 3,5-diethoxy-3′,4′-dihydroxy-_trans_-stilbene induces mitochondrial

ROS-mediated ER stress and cell death in human hepatoma cells _in vitro_. _Acta Pharmacol Sin_ 38, 1486–1500 (2017). https://doi.org/10.1038/aps.2017.106 Download citation * Received: 17

January 2017 * Accepted: 19 May 2017 * Published: 10 August 2017 * Issue Date: November 2017 * DOI: https://doi.org/10.1038/aps.2017.106 SHARE THIS ARTICLE Anyone you share the following

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SharedIt content-sharing initiative KEYWORDS * resveratrol * resveratrol-006 * HepG2 human hepatoma cells * mitochondria * ROS * endoplasmic reticulum stress * 4-phenylbutyrate * NAC *

Mito-TEMPO * apoptosis