Food and Chemical Toxicology 71 (2014) 51–59

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Ilimaquinone induces death receptor expression and sensitizes human colon cancer cells to TRAIL-induced apoptosis through activation of ROS-ERK/p38 MAPK–CHOP signaling pathways Minh Truong Do a,1, MinKyun Na a,1, Hyung Gyun Kim a, Tilak Khanal a, Jae Ho Choi a, Sun Woo Jin a, Seok Hoon Oh a, In Hyun Hwang b, Young Chul Chung c, Hee Suk Kim d, Tae Cheon Jeong e,⇑, Hye Gwang Jeong a,⇑ a

Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon, Republic of Korea Department of Chemistry, University of Iowa, Iowa City, USA c Department of Food and Medicine, International University of Korea, Jinju, Republic of Korea d Department of Food Science and Culinary, International University of Korea, Jinju, Republic of Korea e College of Pharmacy, Yeungnam University, Gyeongsan, Republic of Korea b

a r t i c l e

i n f o

Article history: Received 19 November 2013 Accepted 4 June 2014 Available online 12 June 2014 Keywords: Ilimaquinone CHOP DR4 DR5 TRAIL Apoptosis

a b s t r a c t TRAIL induces apoptosis in a variety of tumor cells. However, development of resistance to TRAIL is a major obstacle to more effective cancer treatment. Therefore, novel pharmacological agents that enhance sensitivity to TRAIL are necessary. In the present study, we investigated the molecular mechanisms by which ilimaquinone isolated from a sea sponge sensitizes human colon cancer cells to TRAIL. Ilimaquinone pretreatment significantly enhanced TRAIL-induced apoptosis in HCT 116 cells and sensitized colon cancer cells to TRAIL-induced apoptosis through increased caspase-8, -3 activation, PARP cleavage, and DNA damage. Ilimaquinone also reduced the cell survival proteins Bcl2 and Bcl-xL, while strongly upregulating death receptor (DR) 4 and DR5 expression. Induction of DR4 and DR5 by ilimaquinone was mediated through up-regulation of CCAAT/enhancer-binding protein homologous protein (CHOP). The up-regulation of CHOP, DR4 and DR5 expression was mediated through activation of extracellular-signal regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) signaling pathways. Finally, the generation of ROS was required for CHOP and DR5 up-regulation by ilimaquinone. These results demonstrate that ilimaquinone enhanced the sensitivity of human colon cancer cells to TRAIL-induced apoptosis through ROS-ERK/p38 MAPK–CHOP-mediated up-regulation of DR4 and DR5 expression, suggesting that ilimaquinone could be developed into an adjuvant chemotherapeutic drug. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Abbreviations: CHOP, CCAAT/enhancer-binding protein homologous protein; DR4, -5, death receptor 4, -5; ERK, extracellular signal-regulated kinase; p38 MAPK, p38 mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; PARP, polyADPribose polymerase; ROS, reactive oxygen species; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling. ⇑ Corresponding authors. Address: College of Pharmacy, Yeungnam University, Gyeongsan 712-749, Republic of Korea Tel.: +82 53 810 2819 (T.C. Jeong). Address: Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon 305-764, Republic of Korea Tel.: +82 42 821 5936 (H.G. Jeong) E-mail addresses: [email protected] (T.C. Jeong), [email protected] (H.G. Jeong). 1 These authors contributed equally. http://dx.doi.org/10.1016/j.fct.2014.06.001 0278-6915/Ó 2014 Elsevier Ltd. All rights reserved.

Colorectal cancer is the third most common malignant neoplasm worldwide (Shike et al., 1990). Recent studies have demonstrated that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL; also known as the Apo2 ligand) is one of the most promising cancer therapeutic drugs. Interest in TRAIL increased following reports that it efficiently induced apoptosis in numerous human tumor cell lines, including colon cancer cells in vitro and in xenograft models, with little toxicity toward normal cells (Koschny et al., 2007; Kim et al., 2000). TRAIL is a membrane-bound TNF-family ligand that interacts with an unusually complex receptor system, which in humans comprises a fully functional TRAIL death receptor 4 (DR4) and DR5, as well as decoy receptor 1 and decoy receptor 2, and osteoprotegerin (Kelley and Ashkenazi, 2004). Interestingly, the TRAIL signaling pathway is blocked in normal cells due to low levels

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of TRAIL death receptors on the cell surface and a high expression of decoy receptors and FLICE-like inhibitory protein, which inhibits caspase 8, caspase 10, and apoptosis (Koschny et al., 2007; Kim et al., 2000). In cancer cells, binding of a TRAIL trimer to DR4 and DR5 leads to receptor trimerization and recruitment of Fas-associated protein with death domain (FADD) followed by procaspase 8 and procaspase 10. This recruitment results in the cleavage and activation of procaspase 8 and procaspase 10, which activates effector caspases leading to apoptosis (Wu and Lippman, 2011). Cancer cells resist TRAIL-induced apoptosis via several mechanisms. Dysfunction of DR4 and DR5 due to mutations (Kim et al., 2000) or decreased protein levels at the cell surface can lead to TRAIL resistance (Kurbanov et al., 2007; van Geelen et al., 2011). Moreover, up-regulation of antagonistic decoy receptors bind to TRAIL but do not have the functional domains necessary to transduce apoptosis signals. The defects in FADD and caspase-8, overexpression of survival proteins such as Bcl-2 or Bcl-xL, or loss of Bax function also can lead to development of TRAIL resistance in cancer cells (Song et al., 2007; Wu and Lippman, 2011). Metabolic substances from marine organisms exhibit a variety of anti-cancer (Jimeno et al., 2004; Lu et al., 2007) anti-angiogenesis (Senthilkumar et al., 2013), and anti-inflammation (Kim and Kim, 2013) pharmacological activities. Ilimaquinone was originally isolated from the Hawaiian sponge Hippiospongia metachromia in 1979. The chemical structure of ilimaquinone contains a benzoquinone moiety, combined with trans-decalin ring system bearing an exocyclic double bond as a rearranged drimane-type sesquiterpene unit (Capon and MacLeod, 1987). Since the initial isolation of ilimaquinone, more than 170 sesquiterpene quinone analogs have been identified, forming a characteristic class of secondary metabolites with abundant structural variants with promising biological activities (Marcos et al., 2010). The first complete synthesis of ilimaquinone and related analogs was accomplished in 1995, and a number of attempts with different approaches have been reported, which reflects the biological significance of this class of compounds. Ilimaquinone has a variety of biological properties as an antiHIV (Loya and Hizi, 1993), anti-microbial (Rangel et al., 1997), anti-inflammatory (Bourguet-Kondracki et al., 1991) and anti-cancer compound (Lu et al., 2007). Mechanistic studies have proposed that ilimaquinone induces breakdown of the Golgi apparatus in a microtubule-independent manner to interfere with vesicular protein transport (Takizawa et al., 1993; Veit et al., 1993). However, as an anti-cancer agent ilimaquinone may inhibit cell growth without affecting the Golgi apparatus (Lu et al., 2007). A previous study reported that ilimaquinone inhibited cell proliferation through G1 cell cycle arrest and up-regulation and nuclear translocation of the transcription factor CCAAT/enhancer-binding protein (C/EBP)-homologous protein/growth arrest and DNA damage-inducible protein 153 (CHOP/GADD153) in cancer cells (Lu et al., 2007). Colon cancer cells are resistant to TRAIL, and the actions of TRAIL can be enhanced through pretreatment with other chemotherapeutic drugs. Therefore, in this study we examined whether ilimaquinone can enhance the sensitivity TRAIL-induced apoptosis in colon cancer cells. Our findings indicate that ilimaquinone upregulates DR4 and DR5 expression through activation ROS-ERK/ p38 MAPK–CHOP signaling pathways, leading to sensitization of colon cancer cells to TRAIL-induced apoptosis.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from USB Corp. (Cleveland, OH). A live/dead viability/cytotoxicity kit was purchased from Invitrogen (Carlsbad, CA, USA). A protein assay kit was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA). The enhanced chemiluminescence (ECL) system and polyvinylidene difluoride (PVDF) membranes were purchased from Amersham Pharmacia Biotech (Uppsala, Sweden). Oligonucleotide polymerase chain reaction (PCR) primers were custom synthesized by Bioneer Co. (Seoul, South Korea). Antibodies against p44/42 MAPK (ERK/2), p-p44/42 MAPK (T202/Y204) (p-ERK1/2), p38 MAPK (p38), p-p38 MAPK (T180/Y182) (p-p38), SAPK/JNK, p-SAPK/JNK (T183/Y185) (p-JNK), Bcl-2, Bcl-xL, poly(ADP-ribose) polymerase (PARP), and cleaved PARP (Asp214), as well as secondary antibodies (HRP-linked anti-rabbit and anti-mouse IgG), were purchased from Cell Signaling Technologies (Beverly, MA). Antibodies against DR4, DR5, caspase-8, caspase-3 and b-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Alexa Fluor 488-conjugated anti-mouse IgG antibody was purchased from Invitrogen (Carlsbad, CA, USA). All other chemicals and reagents were of analytical grade. 2.2. Extraction and isolation of ilimaquinone Ilimaquinone (purity 98%) was obtained from the previous chemical investigation of three sponges Smenospongia aurea, S. cerebriformis, and Verongula rigida (Hwang et al., 2013). Briefly, dried ethanol extract (3.6 kg) of the sponges was fractionated (Fr. 1 to 13) by silica gel vacuum liquid chromatography (VLC), eluting with a stepwise gradient of hexane/acetone/methanol/water. Fr. 10 (39.3 g) was further divided into nine fractions (Fr. 10-1 to 10-9) using the same four solvents on silica gel VLC (21  17.5 cm). Fr. 10-7 (3.7 g) was subjected to C18 MPLC (15.5  4 cm) with an isocratic solvent system of methanol–water (85:15) to yield six subfractions (Fr. 10-7-1 to 10-7-6). Predominant nuclear magnetic resonance (NMR) signals for ilimaquinone were detected in Fr. 10-7-2. Further purification of Fr. 10-7-2 by C18 HPLC (250  21.2 mm, 10 lm) eluting with an isocratic solvent system of methanol–water (78:22) over 200 min resulted in the isolation of ilimaquinone (tR = 136 min). Ilimaquinone: 1H NMR (CDCl3, 600 MHz): 2.08, 1.42 (each 1H, m, H-1), 1.84, 1.16 (each 1H, m, H-2), 2.29, 2.05 (each 1H, ddd, J = 13.7, 8.6, 5.4, H-3), 1.49, 1.32 (each 1H, m, H-6), 1.37 (2H, m, H-7), 1.14 (1H, m, H-8), 0.74 (1H, d, J = 12.0, H10), 4.43, 4.41 (each 1H, s, H-11), 1.02 (3H, s, H-12), 0.96 (3H, d, J = 6.4, H-13), 0.82 (3H, s, H-14), 2.51, 2.45 (each 1H, d, J = 13.7, H-15), 5.83 (1H, s, H-19), 3.84 (3H, s, H-22); 13C NMR (CDCl3, 125 MHz): 23.34 (C-1), 28.11 (C-2), 33.13 (C-3), 160.69 (C-4), 40.63 (C-5), 36.82 (C-6), 28.80 (C-7), 38.25 (C-8), 43.50 (C-9), 50.30 (C-10), 102.66 (C-11), 20.73 (C-12), 18.01 (C-13), 17.52 (C-14), 32.52 (C-15), 117.49 (C-16), 153.49 (C-17), 182.51 (C-18), 102.17 (C-19), 161.90 (C-20), 182.20 (C-21), 57.01 (C-22). 2.3. Cell culture and treatment HCT 116 and HT-29 human colon cancer cell lines were obtained from the American Type Culture Collection (Rockville, MD). Cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; Invitrogen). Cells were cultured in a humidified 5% CO2 incubator at 37 °C in complete medium supplemented with 10% FBS to 80% confluence for use in all assays. Ilimaquinone, PD98059, SB202190 and SP600125 were dissolved in dimethyl sulfoxide (DMSO), TRAIL was dissolved in phosphate-buffered saline containing 0.1% bovine serum albumin, and working concentrations were added directly to serum-free culture media. Cells were treated with DMSO as a vehicle control. The final concentration of DMSO did not exceed 0.1% (v/v) and did not affect cell viability. 2.4. Measurement of cell viability Cells were cultured at 37 °C in medium containing 10% FBS at a density of 4  104/500 lL in 48-well plates. After incubation for 24 h, the growth medium was replaced with serum-free medium and the cells were pretreated with different concentrations of ilimaquinone (0.5–10 lM) or an equal volume of DMSO for 8 h, followed by treatment with TRAIL 10 ng/mL for an additional 24 h at 37 °C. After treatment, cells were treated with MTT solution (final concentration, 0.5 mg/mL) for 1 h. The dark blue formazan crystals formed in intact cells were solubilized with DMSO, and the absorbance at 570 nm was measured with a microplate reader (Varioskan; Thermo Electron, Waltham, MA). Cell viability was calculated based on the absorbance of the ilimaquinone and/or TRAIL-treated cells relative to that of vehicle-treated control cells. 2.5. Live/dead assay

2. Materials and methods 2.1. Reagents and antibodies TRAIL and N-acetyl-L-cysteine (NAC) were purchased from Sigma Chemical (St. Louis, MO). PD98059 (MEK/ERK inhibitor), SB202190 (p38 MAPK inhibitor) and SP600125 (JNK inhibitor) were purchased from Calbiochem (San Diego, CA, USA).

To measure cell viability we also used a two-color fluorescence cell viability assay based on the simultaneous determination of live and dead cells using two probes that measure recognized parameters of cell viability-intracellular esterase activity and plasma membrane integrity. A non-fluorescent polyanionic dye (calcein AM) is retained by live cells, in which it generates green fluorescence by esterase conversion. In contrast, the ethidium homodimer (EthD-1) enters cells through

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TC-30 ; DR5 forward, 50 -GCC CCA CAA CAA AAG AGG TC-30 ; DR5 reverse, 50 -GGA GGT CAT TCC AGT GAG TG-30 ; 18S rRNA forward, 50 -GCT GGA ATT ACC GCG GCT30 ; and 18S rRNA reverse, 50 -CGG CTA CCA CAT CCA AGG AA-30 . The quantity of each transcript was calculated as described in the instrument manual and normalized relative to the mRNA level of the 18S rRNA housekeeping gene.

damaged membranes and binds to nucleic acids, thereby generating a red fluorescence in dead cells. Briefly, HCT 116 cells were pretreated with ilimaquinone 5 lM for 8 h, and then treated with 10 ng/mL TRAIL for an additional 24 h. The cells were stained with a 2 lM calcein AM and 4 lM EthD-1 working solution, and incubated at 37 °C for 30 min. The cells were then analyzed under a fluorescence microscope (Nikon).

2.8. Western blotting

2.6. TUNEL assay

After treatment, cells were harvested and resuspended in lysis buffer (50 mmol/ L Tris–HCl, pH 7.4, 1% NP40, 0.25% sodium deoxycholate, 150 mmol/L NaCl, 1 mmol/ L EDTA). The lysates were boiled for 5 min, resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) in a 10% polyacrylamide gel, and blotted onto PVDF membranes. The membranes were blocked with 5% skim milk and then incubated with the appropriate primary antibodies, followed by horseradish peroxidase-conjugated secondary antibody. The blots were visualized using an ECL Western blot kit according to the manufacturer’s protocol. Equal sample loading was confirmed by immunoblotting for b-actin.

Apoptosis was detected by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling (TUNEL) assay using the DeadEnd™ Fluorometric TUNEL System (Promega) according to the manufacturer’s instructions. HCT 116 cells were seeded on sterile coverslips in 12-well plates at a density of 3  104 cells/well overnight and pretreated with 5 lM ilimaquinone for 16 h, and then treated with 10 ng/mL TRAIL for an additional 8 h. Cells were then washed twice with PBS, fixed with 4% paraformaldehyde for 25 min, washed twice with PBS, and permeabilized with 0.2% Triton X-100 for 5 min. After two more washes, each glass slide was covered with equilibration buffer for 10 min. Then the buffer was aspirated, and the glass slides were incubated with rTdT buffer at 37 °C for 1 h in the dark. Chromosomal DNA was stained with DAPI; stained cells were mounted on glass slides, and examined using an EVOSÒ FL imaging system (Life technologies, Carlsbad, CA, USA).

2.9. Immunofluorescence analysis of DR4 and DR5 membrane localization HCT 116 cells were seeded on sterile coverslips in 12-well plates overnight and treated with 10 lM ilimaquinone for 24 h. Cells were fixed with 4% paraformaldehyde, incubated with mouse monoclonal antibodies recognizing DR4 and DR5 (1:200 in PBS containing 1% BSA and 0.1% Tween 20 (PBST/BSA)) followed by incubation with Alexa Fluor 488-conjugated anti-mouse IgG (1:200 in PBST/BSA). Image was analyzed by an EVOSÒ FL imaging system (Life technologies, Carlsbad, CA, USA).

2.7. Quantitative real-time RT-PCR (qRT-PCR) After 24 h of treatment with 1–10 lM ilimaquinone, total RNA was isolated from untreated and ilimaquinone-treated HCT 116 cells using RNAiso plus Reagent (Takara, Tokyo, Japan) according to the manufacturer’s protocol. The concentration and purity of the extracted RNA were measured using a Nanodrop instrument. After RNA isolation, cDNA was synthesized using a reverse transcription kit (Promega). Product formation during PCR was monitored continuously using Sequence Detection System software (ver. 1.7; Applied Biosystems, Foster City, CA). PCR products were detected directly by monitoring increases in reporter dye (SYBRÒ) signals. The levels of CHOP, DR4 and DR5 mRNA levels in exposed cells were compared with those in control cells at each time point using the comparative cycle threshold (Ct) method. The following primers were used: CHOP forward, 50 -CAA CTG CAG AGA TGG CAG CT-30 ; CHOP reverse, 50 -CTG ATG CTC CCA ATT GTT CA-30 ; DR4 forward, 50 -CAC AGC AAT GGG AAC ATA GC-30 ; DR4 reverse, 50 -CAG GGA CTT CTC TCT TCT

2.10. Measurement of intracellular ROS We used the fluorescent probe H2DCFDA to measure ilimaquinone-induced intracellular ROS generation. The assay was conducted according to a protocol published previously (Wang and Joseph, 1999). Briefly, the confluent HCT 116 cells in the 48-well plates were pre-incubated with 20 lM H2DCFDA for 30 min at 37 °C, followed by treating the cells with ilimaquinone for 1 h. The fluorescence intensity (relative fluorescence units) was measured at excitation and emission wavelengths of 485 and 530 nm, respectively, using a fluorescence spectrophotometer.

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Fig. 1. Ilimaquinone sensitizes colon cancer cells to TRAIL-induced death. (A) Chemical structure of ilimaquinone. (B) Effect of ilimaquinone on TRAIL-induced death in HCT 116 cells measured using a live/dead cell viability assay. Green staining highlights live cells and red staining highlights dead cells. The effect of ilimaquinone on TRAILinduced survival inhibition in HCT 116 cells, (C) and HT-29 cells, (D) measured by MTT assay. All experiments were performed in triplicate. Bars represent SD. P < 0.05 vs. control. #P < 0.05 vs. cells treated with TRAIL and ilimaquinone alone. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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2.11. siRNA transfection Commercial small interfering RNA (siRNA) targeting ERK2 and p38 were obtained from Bioneer Co. (Seoul, South Korea). HCT 116 cells, grown to 50% confluence, were transfected with ERK2 or p38 siRNA at 50 nM or a non-specific control siRNA according to the manufacturer’s instructions. The subsequent experiments were performed after 48 h of transfection.

2.12. Statistical analysis All experiments were repeated at least three times. Results were expressed as means ± SD. Statistical significance was determined using a one-way analysis of variance (ANOVA) test. A value of P < 0.05 was taken to indicate statistical significance.

3. Results 3.1. Structure determination of ilimaquinone The 1H NMR spectrum of the purified compound from Fr. 10-7-2 showed signals for an exomethylene at dH 4.43 and 4.41 (each 1H, s, H-11), two methyl singlets at dH 1.02 (3H, s, H-12) and 0.82 (3H, s, H-14), and a methyl doublet at dH 0.96 (3H, d, J = 6.4, H-13), typical of rearranged drimane type sesquiterpenoids. In addition, 13C NMR signals for distinctive two carbonyl carbons at dC 182.51 (C-18) and 182.20 (C-21), and four olefinic carbons at dC 117.49 (C-16), 153.49 (C-17), 102.17 (C-19), and 161.90 (C-20), excluding two olefinic carbons for exomethylene unit, indicated an oxygenated benzoquinone moiety. This compound was identified as ilimaquinone by comparing the 1H and 13C NMR data with values in the literature (Capon and MacLeod, 1987) (Fig. 1A).

HCT 116

A

3.2. Ilimaquinone enhances TRAIL-mediated survival inhibition in colon cancer cells To investigate the effects of ilimaquinone on TRAIL-mediated cell death, we used a cell viability live/dead assay. HCT 116 cells were pretreated with 5 lM ilimaquinone for 8 h, and then with 10 ng/mL TRAIL for an additional 24 h. Ilimaquinone clearly potentiated TRAIL-mediated cell death in HCT 116 colon cancer cells (Fig. 1B). A previous study reported that HT-29 cells, compared with HCT 116, are relatively resistant to TRAIL treatment (Galligan et al., 2005). We performed a MTT assay in both TRAILsensitive HCT 116 and TRAIL-resistant HT-29 colon cancer cells. Treatment with 10 ng/mL TRAIL for 24 h slightly, albeit significantly, inhibited cell viability in HCT 116 cells but not in HT-29 cells. HCT 116 and HT-29 cells were pretreated with ilimaquinone for 8 h, and then with 10 ng/mL TRAIL for an additional 24 h. Interestingly, pretreatment with ilimaquinone significantly enhanced TRAIL-mediated survival inhibition in both HCT 116 and HT-29 cells in a dose-dependent manner (Fig. 1C and D). These results indicate that ilimaquinone potentiates TRAIL-mediated colon cancer cell death.

3.3. Ilimaquinone potentiates TRAIL-mediated apoptosis in colon cancer cells We found that ilimaquinone significantly enhanced TRAIL-mediated cell death. To investigate whether the death caused by ilimaquinone and TRAIL treatment was related to apoptosis, we analyzed caspase-3 and PARP protein expression as markers of

HT-29

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Fig. 2. Ilimaquinone enhances TRAIL-induced apoptosis in colon cancer cells. The effect of ilimaquinone and TRAIL on caspase-3 and PARP protein expression in HCT 116 cells (A) and HT-29 cells (B) as determined by Western blotting. Results are representative of three independent experiments. (C) Ilimaquinone enhances TRAIL-induced DNA damage in HCT 116 cells. Ilimaquinone and/or TRAIL-treated cells were analyzed with a TUNEL assay. DAPI, 40 ,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate.

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an additional 24 h. Caspase-3 and PARP protein levels in cell lysates were analyzed by Western blotting. Pretreatment with ilimaquinone significantly enhanced TRAIL-induced activated caspase-3 and cleaved PARP protein levels in both HCT 116 and HT-29 colon cancer cells (Fig. 2A and B). Additionally, DNA damage-induced apoptosis was also observed in a terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling (TUNEL) assay in which pretreatment with ilimaquinone enhanced the apoptosis-inducing potential of TRAIL in HCT 116 cells (Fig. 2C). These results indicate that ilimaquinone potentiated TRAILmediated cell death by inducing apoptosis through activation of the caspase pathway in colon cancer cells.

ProCaspase-8 Active Caspase-8 ProCaspase-3 Active Caspase-3 β-actin Full-length PARP

3.4. Ilimaquinone down-regulates expression of survival proteins in colon cancer cells

Cleaved PARP β-actin Bcl-xL Bcl-2 β-actin -

1

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Fig. 3. Ilimaquinone down-regulates expression of survival proteins in HCT 116 colon cancer cells. Bcl2, Bcl-xL, PARP, caspase-8, caspase-3 and b-actin protein levels in cell lysates were determined by Western blotting. Results are representative of three independent experiments.

apoptosis because the activation of caspase-3 leads to PARP protein degradation. HCT 116 and HT-29 cells were pretreated with 5 lM ilimaquinone for 8 h and then treated with 10 ng/mL TRAIL for

Relative DR4 or DR5 mRNA to 18S rRNA (fold of control)

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3.5. Ilimaquinone up-regulates the death receptor expression in colon cancer cells

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The expression of caspase family proteins, such as caspase-8 and caspase-3, as well as pro-apoptotic proteins such as Bcl-2 and Bcl-xL are required for TRAIL-induced apoptosis in cancer cells (Bodmer et al., 2000; Seol et al., 2001; Wu and Lippman, 2011). HCT 116 cells were treated with 1–10 lM ilimaquinone for 24 h and the levels of these proteins in cell lysates determined by Western blotting. Results indicated that treatment with ilimaquinone alone increased active caspase-8 and -3, followed by increased cleaved PARP levels. Moreover, ilimaquinone significantly reduced Bcl-2 and Bcl-xL protein levels (Fig. 3). These results suggest that ilimaquinone potentiates TRAIL-induced apoptosis through regulation of these survival proteins in colon cancer cells.

18 16 14 12 10 8 6 4 2 0

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Fig. 4. Ilimaquinone up-regulates DR4, DR5 and CHOP expression in HCT 116 colon cancer cells. (A) Effect of ilimaquinone on DR4 and DR5 mRNA levels. Total RNA was extracted and the levels of DR4 and DR5 mRNA determined by qRT-PCR. All experiments were performed in triplicate. Bars represent the SD. P < 0.05 vs. control. (B) Effect of ilimaquinone on DR4 and DR5 protein expression. DR4, DR5 and b-actin protein levels in cell lysates were determined by Western blotting. Results are representative of three independent experiments. (C) Fluorescence microscopy analysis of DR4 and DR5 membrane localization in HCT 116 colon cancer cells. Cells were treated ilimaquinone 10 lM for 24 h. Localization of DR4 and DR5 on cell surface was evaluated by immunofluorescence and analyzed by fluorescence microscopy. Green staining highlights DR4 and DR5 expression on cell surface. (D) Effect of ilimaquinone on CHOP mRNA expression. Total RNA was extracted and CHOP mRNA levels were determined by qRT-PCR. All experiments were performed in triplicate. Bars represent SD. P < 0.05 vs. control.

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and Dixit, 1998; Wu and Lippman, 2011). Low levels of DR4 and DR5 expression on the cell surface cause TRAIL resistance (Kurbanov et al., 2007; van Geelen et al., 2011). Thus, we investigated the effects of ilimaquinone on DR4 and DR5 expression in colon cancer cells. Treatment with ilimaquinone alone significantly induced DR4 and DR5 mRNA levels in a dose-dependent manner (Fig. 4A). Ilimaquinone also markedly induced DR4 and DR5 protein levels in HCT 116 cells (Fig. 4B). To confirm DR4 and DR5 membrane expression are actually elevated, we used fluorescence microscopy analysis. Immunofluorescence staining showed that ilimaquinone treatment induced expression of DR4 and DR5 on the plasma membrane of HCT 116 cells (Fig. 4C). Furthermore, we found that the mRNA level of the transcription factor CHOP, which regulates DR4 and DR5 expression (Yamaguchi and Wang, 2004), was significantly enhanced by ilimaquinone treatment (Fig. 4D). These results suggest that the up-regulation of DR4 and DR5 expression by ilimaquinone potentiates TRAIL-induced apoptosis. 3.6. Activation of the ERK and p38 MAPK signaling pathways are required for ilimaquinone-mediated up-regulation of death receptors Previous studies demonstrated that MAPK activation plays an important role in death receptor up-regulation (Oh et al., 2010; Ichijo, 1999). To further investigate the upstream signaling pathways that mediate up-regulation of CHOP and DR5 expression by ilimaquinone, HCT 116 cells were treated with 1–10 lM ilimaquinone for 2 h and the expression of MAPK proteins analyzed by Western blotting. Ilimaquinone markedly induced p-JNK, p-p38

A

3.7. Ilimaquinone up-regulates CHOP, DR4 and DR5 through ROS generation To determine the role of ROS generation in ilimaquinone-mediated CHOP and DR5 up-regulation, we assessed intracellular ROS

B Relative CHOP, DR4 and DR5 mRNA to 18S rRNA (fold of control)

p-JNK JNK p-p38 p38 p-ERK ERK

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and p-ERK in HCT 116 cells (Fig. 5A). To clarify which MAPK mediated CHOP, DR4 and DR5 expression induced by ilimaquinone, HCT 116 cells were pretreated with 10 lM SP600125 (JNK inhibitor), 10 lM SB202190 (p38 inhibitor) and 20 lM PD98059 (ERK inhibitor) prior to treatment with 10 lM ilimaquinone. SB202190 and PD98059, but not SP600125, significantly inhibited ilimaquinone-induced CHOP, DR4 and DR5 mRNA levels (Fig. 5B). Furthermore, SB202190 and PD98059 markedly suppressed ilimaquinone-induced DR4 and DR5 protein levels (Fig. 5C). To confirm the activation of ERK and p38 MAPK signaling and subsequent upregulation of death receptor expression play an important role for enhancing susceptibility of cancer cells to TRAIL by ilimaquinone, silencing of ERK2 and p38 was performed using siRNA. Knockdown of ERK2 by siRNA was reported to significantly attenuate TRAIL sensitivity caused by azapirone (Gupta et al., 2013). Western blot results indicated that ERK2 and p38 were knocked out successfully in HCT 116 cancer cells (Supplementary Fig. S1). Knockdown of ERK2 and p38 significantly restored the reduction of cell viability caused by combination of ilimaquinone and TRAIL in HCT 116 cells (Fig. 5D). These results demonstrate that activation of ERK and p38 MAPK by ilimaquinone are required for up-regulation of CHOP, DR5 expression and TRAIL sensitivity in colon cancer cells.

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Fig. 5. Ilimaquinone induces CHOP, DR4 and DR5 expression through the ERK and p38 MAPK signaling pathways in HCT 116 colon cancer cells. (A) The effect of ilimaquinone on the phosphorylation of the MAPK signaling proteins. P-JNK, JNK, p-p38, p38, p-ERK, ERK, and b-actin protein levels in cell lysates were determined by Western blotting. (B) Effect of SP600125 (JNK inhibitor), SB202190 (p38 inhibitor) and PD98059 (ERK inhibitor) on ilimaquinone-mediated up-regulation of CHOP, DR4 and DR5 mRNA expression. Total RNA was extracted and CHOP, DR4 and DR5 mRNA levels determined by qRT-PCR. All experiments were performed in triplicate. Bars represent the SD. #P < 0.05 vs. control. P < 0.05 vs. cells treated with ilimaquinone alone. (C) Effect of SP600125, SB202190 and PD98059 on ilimaquinone-mediated up-regulation of DR4 and DR5 protein levels. DR4, DR5 and b-actin protein levels in cell lysates were determined by Western blotting. Results are representative of three independent experiments. (D) Knockdown of ERK2 and p38 by siRNAs significantly restored the reduction of cell viability caused by combination of ilimaquinone and TRAIL in HCT 116 cells. HCT 116 cells were transfected with ERK2 siRNA, p38 siRNA or a non-specific control siRNA for 48 h. The siRNA-transfected cells were pretreated with ilimaquinone for 8 h, and then with TRAIL for an additional 24 h. Cell viability was measured by MTT assay. All experiments were performed in quadruplicate. Bars represent SD. P < 0.05 vs. control. #P < 0.05 vs. cells treated with both TRAIL and ilimaquinone.

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Fig. 6. Ilimaquinone up-regulates CHOP, DR4 and DR5 expression through ROS generation in HCT 116 colon cancer cells. (A) Ilimaquinone induces intracellular ROS production. Intracellular ROS production was determined using the fluorescent probe H2DCFDA. All experiments were performed in triplicate. Bars represent SD. P < 0.05 vs. control. NAC suppresses ilimaquinone-induced CHOP (B), DR4 (C) and DR5 (D) mRNA levels. Total RNA was extracted and CHOP, DR4 and DR5 mRNA levels were determined by qRT-PCR. All experiments were performed in triplicate. Bars represent SD. #P < 0.05 vs. control. P < 0.05 vs. cells treated with ilimaquinone alone.

production in colon cancer cells treated with ilimaquinone. As showed in Fig. 6A, ilimaquinone significantly induced intracellular ROS generation in HCT 116 cells. When HCT 116 cells were pretreated with 2 mM NAC for 1 h, followed by 10 lM ilimaquinone for an additional 24 h, NAC significantly inhibited ilimaquinoneinduced CHOP, DR4 and DR5 mRNA expression (Fig. 6B–D). These observations suggest that ilimaquinone induces intracellular ROS generation resulted in up-regulation CHOP, DR4 and DR5 expression in colon cancer cells.

4. Discussion Cancer is a leading cause of death worldwide (Jemal et al., 2011). Solid cancer is initially treated with surgery to remove the bulk of the tumor, followed by chemotherapy and/or radiotherapy to kill residual cancer cells. However, this treatment regimen often causes substantial death of healthy cells. Thus, agents that specifically and strongly enhance cell death-related signaling pathways in cancer cells are ideal for cancer therapies. TRAIL has attracted the most interest in cancer therapy as it induces apoptosis in cancer cells, but not in normal cells, and lacks toxicity in animal models (Koschny et al., 2007; Kim et al., 2000). Although many types of cancer cells are sensitive to TRAIL-induced apoptosis, others are resistant. Therefore, current studies are focused on finding strategies that optimize the therapeutic efficacy or sensitize cancer cells to TRAIL (Stuckey and Shah, 2013). In the present study, we demonstrated that pretreatment with ilimaquinone enhanced TRAIL-induced apoptosis in human colon cancer cells. Several mechanisms mediating ilimaquinone sensitization of human cancer cells to TRAIL were also clarified. Previous study demonstrated that HCT 116 cancer cells are TRAIL-sensitive and HT-29 cancer cells are TRAIL-resistant (Galligan et al., 2005). We found that ilimaquinone potentiated TRAIL-mediated survival inhibition in both TRAIL-sensitive HCT 116 and TRAIL-resistant

HT-29 colon cancer cells. Further data indicated that cancer cell death caused by the combined treatment of ilimaquinone and TRAIL was related to apoptosis because ilimaquinone enhanced TRAIL-induced activated caspase-3, PARP cleavage, and DNA damage in colon cancer cells. Ilimaquinone treatment alone robustly down-regulated expression of survival proteins, such as Bcl-2 and Bcl-xL, in HCT 116 cells. Previous studies demonstrated that the down-regulation of Bcl-2 and Bcl-xL proteins sensitized resistant cancer cells to TRAIL-induced apoptosis (Taniai et al., 2004; Chawla-Sarkar et al., 2004). Moreover, ilimaquinone significantly reduced pro-caspase-8 and pro-caspase 3, which were reported to play an important role in TRAIL-induced apoptosis in cancer cells (Bodmer et al., 2000; Seol et al., 2001). Previous studies indicated that DR4 and DR5 receptors possess crucial functions in transduction of TRAIL activity to activate cell death-related signaling pathways in cancer cells (Ashkenazi and Dixit, 1998). However, the decreased expression or loss of DR4 and DR5 function in cancer cells leads to resistance to TRAIL (Wu and Lippman, 2011; Kurbanov et al., 2007; van Geelen et al., 2011). Our results indicated that ilimaquinone markedly up-regulated DR4 and DR5 expression in colon cancer cells. The ability of ilimaquinone to induce death receptor expression identified a molecular mechanism for ilimaquinone sensitized TRAIL-induced apoptosis in colon cancer cells. This result agrees with previous reports that potent sensitizers, such as gossypol (Sung et al., 2010), dibenzylideneacetone (Prasad et al., 2011), 2-methoxy-5amino-N-hydroxybenzamide (Stolfi et al., 2011), azapirone (Gupta et al., 2013), and tocotrienols (Park et al., 2010), enhanced TRAIL-induced apoptosis in cancer cells by up-regulating death receptors. The pro-apoptotic transcription factor CHOP/GADD153 is induced by various cellular stresses such as endoplasmic reticulum stress and several pharmacological agents (van der Sanden et al., 2004). Endoplasmic-reticulum-stress-induced apoptosis enhances DR5 expression through up-regulation of CHOP expression in

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human cancer cells (Yamaguchi and Wang, 2004). The crucial role of the CHOP transcription factor in DR5 up-regulation by sensitizers has been demonstrated previously (Sung et al., 2010; Prasad et al., 2011; Stolfi et al., 2011; Gupta et al., 2013; Park et al., 2010; Yoon et al., 2013). We found that treatment with ilimaquinone robustly induced CHOP expression in HCT 116 cells. A previous study showed that ilimaquinone significantly up-regulated CHOP/GADD153 expression and nuclear translocation in cancer cells (Lu et al., 2007). Moreover, oxidative stress plays a crucial role as a mediator of cell death (Jacobson, 1996). ROS generation has been proposed to be involved in the up-regulation of DR4 and DR5 by numerous cancer chemopreventive agents (Sung et al., 2010; Prasad et al., 2011; Gupta et al., 2013; Park et al., 2010; Yoon et al., 2013). Our results indicated that ilimaquinone significantly induced intracellular ROS production in HCT 116 cells. The antioxidant NAC significantly abolished the up-regulation of CHOP, DR4 and DR5 expression induced by ilimaquinone. Therefore, ROS generation is required for ilimaquinone-induced CHOP, DR4 and DR5 expression in colon cancer cells. Finally, sensitizers that upregulated CHOP, DR4 and DR5 expression mediated through activation of ERK and p38 MAPK signaling were reported in previous studies (Sung et al., 2010; Stolfi et al., 2011; Gupta et al., 2013; Park et al., 2010). With the use of specific inhibitors or siRNAs, we determined that the activation of the ERK and p38 MAPK signaling pathways was required for ilimaquinone-mediated up-regulation of CHOP and DR5 expression and TRAIL sensitivity in colon cancer cells. In conclusion, the results of the present study indicated that ilimaquinone significantly potentiated TRAIL-induced apoptosis in colon cancer cells and ilimaquinone markedly induced DR4, DR5 expression and TRAIL sensitivity through activation of ROSERK/p38 MAPK–CHOP signaling pathways. Our results suggest that ilimaquinone could be developed as an adjuvant chemotherapeutic drug. Conflict of Interest The authors have declared no conflict of interest. Transparency Document The Transparency document associated with this article can be found in the online version.

Acknowledgments This study was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and future Planning (NRF2013R1A2A2A01067892) and the National Research Foundation of Korea Grant funded by the Korean Government (MEST) (NRF2010-0020484). Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.fct.2014.06.001. References Ashkenazi, A., Dixit, V.M., 1998. Death receptors: signaling and modulation. Science 281, 1305–1308. Bodmer, J.L., Holler, N., Reynard, S., Vinciguerra, P., Schneider, P., Juo, P., Blenis, J., Tschopp, J., 2000. TRAIL receptor-2 signals apoptosis through FADD and caspase-8. Nat. Cell Biol. 2, 241–243.

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