Free Radical Research, 2014; Early Online: 1–11 © 2014 Informa UK, Ltd. ISSN 1071-5762 print/ISSN 1029-2470 online DOI: 10.3109/10715762.2014.951838

ORIGINAL ARTICLE

Generation of ROS by CAY10598 leads to inactivation of STAT3 signaling and induction of apoptosis in human colon cancer HCT116 cells I. G. Chae1, D.-H. Kim2, J. Kundu1, C.-H. Jeong1, J. K. Kundu1 & K.-S. Chun1 of Pharmacy, Keimyung University, Daegu, South Korea and  2College of Pharmacy, Seoul National University, Seoul, South Korea

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Abstract Prostaglandin E2 (PGE2) has been reported to play critical roles in cell fate decision by interacting with four types of prostanoid receptors such as EP1, EP2, EP3 and EP4. The present study was aimed at investigating the effect of the EP4-specific agonist CAY10598 in human colon cancer HCT116 cells. Our study revealed that treatment with CAY10598 significantly reduced the cell viability and induced apoptosis in HCT116 cells, as evidenced by the induction of p53 and Bax, release of cytochrome c, cleavage of caspase-9, -7, and -3, and PARP, and the inhibition of Bcl-2, Bcl-xL and survivin expression. Moreover, treatment with CAY10598 diminished the phosphorylation of JAK2, leading to the attenuation of STAT3 activation in HCT116 cells. CAY10598-induced apoptosis in cells which were transiently transfected with EP4 siRNA or treated with an EP4 antagonist prior to incubation with the compound remained unaffected, suggesting an EP4-independent mechanism of apoptosis induction by CAY10598. We found that treatment with CAY10598 generated reactive oxygen species (ROS) and pretreatment of cells with N-acetyl cysteine rescued cells from apoptosis by abrogating the inhibitory effect of CAY10598 on the activation of JAK2/STAT3 signaling. In conclusion, CAY10598 induced apoptosis in HCT116 cells in an EP4-independent manner, but through the generation of ROS and inactivation of JAK2/STAT3 signaling. Keywords: EP4 receptor, CAY10598, ROS, STAT3, colon cancer cells

Introduction Prostaglandins (PGs), one of the products of cyclooxygenase-2 (COX-2), are involved in various physiological functions, such as inflammation, proliferation, apoptosis, and cellular differentiation [1]. PGE2 exerts its biological functions by binding to four types of G protein-coupled receptors (GPCRs) termed EP1, EP2, EP3, and EP4, which are coupled to a distinct downstream second messenger system [2,3]. Both EP1 and EP3 receptors are coupled to Gaq to activate Ca2 signaling and Gai to inhibit adenylyl cyclase, respectively, whereas EP2 and EP4 are linked to Gas to promote the synthesis of adenylyl cyclase [3,4]. Accumulating evidence indicates that PGE2 plays an important role in cancer development through an increase of cell proliferation or the inhibition of apoptosis [5,6]. However, several other investigators have suggested anti-proliferative or apoptotic effects of PGE2 in various cancer cells [7–10]. These conflicting findings may have resulted from the non-selective agonistic property of PGE2 and the differential activation of EP receptors, depending on the cell types, with highly variable patterns of EP expression [9,10]. Among these EP receptors, EP4 has been implicated in the PGE2-mediated increase of cell growth, motility and invasive behavior of human colorectal epithelial cells

[11,12]. Pozzi et  al. suggest that the anti-proliferative effects resulting from COX inhibition by indomethacin or COX-selective inhibitors, are reverted specifically by treatment with PGE2 or the EP4-selective agonist PGE1-OH in vivo [13]. Moreover, azoxymethaneinduced premalignant aberrant crypt foci formation was significantly decreased in EP4-deficient mice as compared with the EP4 wild-type mice [14]. Moreover, treatment with EP4 antagonist ONO-AE2–227 [14] or L-161982 [15,16] reduced colon adenomatous polyp formation or the proliferation of colon cancer cells, respectively. Contrary to these reports, several other studies have demonstrated that the activation of EP4 by its agonists reduce inflammation and induce cytotoxicity under certain circumstances. For instance, treatment with an EP4 agonist, ONO-AE1-329, inhibits lipopolysaccharide (LPS)-induced production of tumor necrosis factor-a (TNF-a) in neutrophils [17]. Others have reported that ONO-AE1-329 induces apoptosis in fibroblasts [18] and inhibits cell proliferation in human airway smooth muscle cells [19] and gastric cancer cells [9], respectively. Moreover, incubation of nonasthmatic eosinophils with 11-deoxy-PGE1, a nonselective agonist of EP4, produced a marked inhibition of cell proliferation, which was recovered by the EP4 antagonist AH23848 [20].

In Gyeong Chae and Do-Hee Kim contributed equally to this work. Correspondence: Prof. Kyung-Soo Chun, Ph.D., College of Pharmacy, Keimyung University, 1095 Dalgubeoldaero, Dalseo-Gu, Daegu 704-701, South Korea. Tel:  82 53 580 6647. E-mail: [email protected] (Received date: 15 April 2014; Accepted date: 30 July 2014; Published online: 3 September 2014)

2  I. G. Chae et al. Although CAY10598 is a potent and a highly specific agonist of EP4 [21], little is known about the effect of this compound on cell proliferation. Thus, the present study was designed to investigate the role of CAY10598 in cell proliferation and to elucidate its molecular mechanism in human colon cancer HCT116 cells. Materials and methods

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Materials CAY10598, GW627368x and PGE2 were purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). Antibodies against cleaved caspase-9, -3, -7, and PARP, Bcl-2, Bax, cytochrome c, STAT3, p-STAT3 (Y705), p-STAT3 (S727), JAK2, p-JAK2, and survivin were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). The Bcl-xL antibody was purchased from BD-Biosciences (San Jose, CA, USA). Antibodies against each of p53 and horse-raddish peroxidase-conjugated secondary antibodies were purchased from SantaCruz Biotechnology (SantaCruz, CA, USA). The b-Actin antibody was obtained from Sigma Chemical Co. (St Louis, MO, USA). The 2′7′dichlorofluorescin diacetate (DCF-DA) was purchased from Invitrogen (Carlsbad, CA, USA). Hank’s balanced salt solution (HBSS) was purchased from Meditech (Herndon, VA, USA). All other chemicals were of analytical or highest purity grade available.

Western blot analysis The cells were harvested and lysed with an RIPA lysis buffer. The cells were disrupted and extracted at 4°C for 30 min. After centrifugation at 16,000 3 g for 15 min, the supernatant was obtained as the cell lysate. Protein concentrations were measured using a protein kit (Pierce Biotechnology, Rockford, IL, USA). Cellular proteins (30 mg) were resolved in aliquots subjected to 10% SDSpolyacrylamide gel electrophoresis. The resolved proteins were transferred to an Immobilon-P-membrane and allowed to react with a specific antibody. The detection of specific proteins was carried out by a Super-signal pico-chemiluminescent substrate or dura-luminol substrate (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions, and visualized with imagequant™ LAS 4000 (Fujifilm Life Science, Japan). Loading differences were normalized using anti-b-actin antibody. Determination of reactive oxygen species (ROS) production ROS generation was monitored by flow cytometry using the peroxide-sensitive fluorescent probe DCF-DA. Briefly, the cells were plated in 6-well culture plates at a density of 2  105 cells/well were washed twice with HBSS solution and suspended in the complete media, and were then examined under a fluorescence microscope to detect the intracellular accumulation of ROS.

Cell culture and treatment

Apoptosis assay with Annexin-V staining

HCT116 cells were obtained from the American Type Culture Collection (Rockville, MD, USA). Cell types were maintained in RPMI1640 supplemented with 10% fetal bovine serum (FBS) containing 100 U/ml of penicillin and 100 mg/ml of streptomycin and 5% CO2 at 37°C. For all experiments, early passage cells were grown to 80–90% confluence.

Annexin-V staining was performed using Annexin-Vfluorescein isothiocyanate (FITC) staining kit (BDBiosciences) following the manufacturer’s instructions. HCT116 cells (2  105 cells/well) were incubated with or without CAY10598, for 24 h after seeding. At 24 h after treatment, the cells were detached using trypsin-EDTA. Briefly, sample treated cells were washed with PBS and resuspended in a binding buffer containing Annexin-VFITC and propidium iodide (P.I.). After 15 min incubation in the dark at room temperature, fluorescence intensity was measured using flow cytometry.

Cell viability assay The anti-proliferative effect of CAY10598 against HCT116 cells was measured by using a solution of tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy methoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium, inner salt] (MTS) (Promega, Madison, WI, USA). Briefly, the cells were plated in 96-well culture plates at a density of 2  103 cells/well in RPMI1640 culture medium and allowed to attach for 24 h. After incubation, the medium was discarded and replaced with 100 ml of new medium containing various concentrations of CAY10598. After 24 h of culture, 20 ml MTS reagent was added to each well followed by 1 h incubation. The optical density was measured at 490 nm in a microplate reader (Tecan Trading AG, Switzerland). Cell viability was described as a percentage relative to the control group. All determinations were confirmed using at least three independent replicate experiments.

Electrophoretic mobility shift assay (EMSA) Cells treated with CAY10598 were gently washed twice with ice-cold PBS and centrifuged at 5,000  g for 8 min at 4°C. The pellets were suspended in 100 ml of hypotonic buffer (10 mM HEPES, pH 7.9; 10 mM KCl, 2 mM MgCl2, 1 mM DTT, 0.1 mM EDTA and 0.1 mM PMSF) for 15 min on ice, and 1 ml of 10% Nonidet P-40 solution was added for 5 min. The mixture was then centrifuged at 14,000  g for 15 min. The supernatant (i.e., the cytosolic fraction) was kept for Western blot analysis. The nuclei were suspended in 80 ml of buffer (50 mM HEPES, pH 7.9; 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.1 mM PMSF, and 10% glycerol) for 30 min on ice and centrifuged at 12,000  g for 15 min. The supernatant

CAY10598 induces apoptosis in HCT116 cells   3

Real-time quantitative polymerase chain reaction Real-time polymerase chain reaction (PCR) was performed on the complementary DNAs (cDNAs) using the selective primers for EP1 (sense; 5′-GATGGTGGGCCAGCTT GTC-3′, antisense; 5′-GCCACCAACACCAGCATTG-3′), EP2 (sense; 5′-GTGCTGACAAGGGACTTCATGT-3′, antisense; 5′-TGTTCCTCGAAAGGCCAAGTAC-3′), EP3 (sense; 5′-AAGGCCACGGGATCTCAGT-3′, antisense; 5′-TGATCCCCATAAGCTGAATGG-3′), EP4 (sense; 5′-CTT GGAGGCAGGAATTTGCTT-;3′, antisense; 5′-AAAGT CCTCAGTGAGGTGGTGTCT-3′), and GAPDH (sense; 5′-TGCACCACCAACTGCTTAGC;3′, antisense; 5′-GGCA TGGACTGTGGTCATGA-3′). PCRs were performed in a Light Cycler 480 (Roche Diagnostics, Mannheim, Germany) using the Light Cycler DNA Master SYBR Green I kit (Roche Diagnostics) according to the manufacturer’s instruction. The PCR thermal profile was 95°C for 10 min, and 45 cycles of 95°C for 10 s, 60°C for 40 s followed by a cooling step at 40°C for 30 s. For relative quantification, the crossing point (Cp) value of EP receptors was norma­ lized by keeping the Cp value of GAPDH as a control. Statistical analyses When necessary, data were expressed as mean  SD of at least three independent experiments, and statistical analysis for single comparison was performed using the Student’s t-test. The criterion for statistical significance was * and ** as p  0.01 and 0.001, respectively, compared to the corresponding control. Results The EP4 agonist CAY10598 induces apoptosis in HCT116 cells Since previous studies have demonstrated conflicting results on the proliferative effect of EP4 on different cancer cell lines, we sought to examine whether the EP4 agonist CAY10598 influenced the growth of HCT116

cells. Figure 1A shows the chemical structure of CAY10598. In accordance with paper of Shoji et al.[22], we found that HCT116 cells express endogenous EP1, EP2, and EP4 mRNA expression but not EP3 (Figure 1B). Next, we examined whether EP4 agonist CAY10598 could regulate the growth of HCT116 cells. As shown in Figure  1C, the cell growth was inhibited in a timeand concentration-dependent manner in CAY10598-

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(B) Relative mRNA expression (Fold induction relative to EP1)

containing nuclear proteins was collected and stored at  70°C after determination of the protein concentration. The STAT3 oligonucleotide probe (5′-AGC TTC ATT TCC CGT AAA TCC CTA-3′) (Bioneer, Korea) was labeled with [g-32P]ATP by T4 polynucleotide kinase and purified on a Nick column. The binding reaction was performed in 25 ml of the mixture containing 5 ml of incubation buffer (10 mM Tris-HCl, pH 7.5; 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 4% (v/v) glycerol, and 0.1 mg/ ml sonicated salmon sperm DNA), 10 mg of nuclear extracts, and the labeled probe at 100,000 cpm. After 50 min incubation at room temperature, 2 ml of 0.1% bromophenol blue was added, and samples were electrophoresed on a 6% nondenaturing polyacrylamide gel at 150 V for 1.5 h. Finally, the gel was dried and exposed to an X-ray film.

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Figure 1. Cytotoxic effect of CAY10598 in HCT116 cells. (A) Chemical structure of CAY10598. (B) Real-time RT-PCR analysis was conducted on HCT116 cells with primers specific for endogenous EP1, EP2, EP3, and EP4 mRNAs. Values were normalized to GAPDH levels. We calculated the fold induction relative to EP1 level. The results presented are mean  S.D. (n  3). (C) Cells were treated with CAY10598 (1, 10, 25 or 50 mM) for indicated time, and cell viability was determined by the MTS assay. The results are presented as means  S.D. (n  3). *p  0.01 and **p  0.001 as compared to control. (D) Cells were treated with indicated concentrations of CAY10598 for 24 h and cell morphology was analyzed by fluorescence microscopy ( 200). (E) The apoptotic index (%) was determined by flow cytometry upon treatment of cells with CAY10598 (1, 10 or 25 mM) for 24 h and staining with Annexin-V and P.I. Data are representative of three independent experiments. Bar graphs represent percentage of apoptotic cells in CAY10598-treated HCT116 cells. Data is presented as mean  SD. (*p  0.01).

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treated HCT116 cells. In another experiment, the growth inhibitory effect of CAY10598 was verified by the morphological changes observed under the phase contrast microscope. Analysis of cell morphology showed that the number of viable cells was obviously decreased by CAY10598 treatment, as evidenced by the appearance of the cytoplasmic bubbles and segments of cell bodies (Figure 1D). To further assess apoptosis, Annexin-VFITC dye was utilized to detect the translocation of phosphatidylserine from the inner (cytoplasmic) leaflet of the plasma membrane to the cell surface. Apoptotic cells were significantly induced in a concentration-dependent manner by CAY10598 treatment for 24 h (Figure 1E). Effects of CAY10598 on the expression of apoptotic markers To elucidate the mechanisms underlying the growth inhibitory effects of CAY10598, the expression of several major apoptosis regulating proteins was measured by the Western blot analysis (Figure 2). When HCT116 cells were treated with 1, 10, and 25 mM CAY10598 for 24 h, the expression of p53 was markedly increased

(Figure 2A). The ratio of pro-apoptotic Bax and the anti-apoptotic Bcl-2 is considered as a molecular rheostat determining cell survival/death [23]. As shown in Figure 2A, CAY10598 treatment led to a concomitant decrease in the level of Bcl-2, whereas the expression of Bax was upregulated in a concentration-dependent manner. In addition, we measured the level of Bcl-xL, which was reduced by CAY10598 treatment in HCT116 cells (Figure 2A). Moreover, treatment of cells with CAY10598 for 24 h decreased the expression of survivin, a cell survival protein known to inhibit apoptosis [24]. Many pro-apoptotic signals have been known to trigger mitochondrial cytochrome c release [25]. The growth inhibitory effect in HCT116 cells treated with CAY10598 was accompanied by the release of cytochrome c, which subsequently led to the activation of caspases (Figure 2). Caspases are important mediators of apoptosis and contribute to the overall apoptotic morphology by cleavage of various cellular substrates [25]. Incubation with CAY10598 activated caspase-9, -7, and -3, and induced the cleavage of PARP in HCT116 cells (Figure 2C). PARP, a known caspase substrate, is a 116 kDa nuclear protein that is specifically cleaved by active caspase-3 into an 85 kDa apoptosis fragment [26].

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Figure 2. Effects of CAY10598 on the cellular markers of apoptosis in HCT116 cells. HCT116 cells were treated with CAY10598 (1, 10 and 25 mM) for 24 h. The levels of (A) p53, Bax, Bcl-2, Bcl-xL, survivin, and cytochrome c, and (B) cleaved PARP, cleaved caspase-3, -7, and -9 were determined by Western blot analysis. Data are representative of three different experiments.

Apoptosis induced by CAY10598 is independent of EP4 receptor We have next examined whether CAY10598-induced apoptosis in HCT116 cells is causally linked to the EP4 receptor. HCT116 cells were treated with CAY10598 in the absence or presence of the EP4 siRNA or EP4 antagonist GW627368x. As illustrated in Figure 3A, GW62­ 7368x failed to attenuate CAY10598-induced cytotoxicity in HCT116 cells. Cells were transiently transfected with EP4 siRNA or scrambled siRNA (Figure 3B). When EP4 siRNA transfected cells were treated with CAY10598, the cell viability remained unchanged compared with that of control siRNA transfected cells (Figure 3C). CAY10598-induced apoptosis is mediated through inhibition of the JAK2/STAT3 pathway To further elucidate the molecular basis for the CAY10598induced apoptosis, we have examined the effect of the

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Figure 3. EP4-independent growth inhibitory effect of CAY10598 in HCT116 cells. (A) HCT116 cells were treated with or without EP4specific antagonist GW627368x (10 mM) for 1 h prior to incubation with CAY10598 (25 mM) for 24 and then cell viability was measured by MTS assay as described in the section Materials and methods. (B–C) HCT116 cells were transiently transfected with scrambled or EP4 siRNA for 24 h prior to incubation with CAY10598 (10, 15 or 25 mM) for 24 h. (B) The mRNA levels of EP4 and b-actin were determined by RT-PCR. (C) The cell viability was measured by the MTS assay.

compound on the activation of JAK2 and STAT3, which promotes cell survival by transcriptional activation of various proliferative genes. Treatment of HCT116 cells with CAY10598 for 24 h resulted in the significant inhibition of constitutive phosphorylation of JAK2 and that of STAT3 at both tyrosine 705 and serine 727 residues in a concentration-dependent manner (Figure 4A). The expression of total JAK2 was also decreased by CAY10598 treatment. In addition, CAY10598 (25 mM) exerted a significant inhibitory effect on STAT3 DNA binding activity in HCT116 cells (Figure 4B). Involvement of ROS in CAY10598-induced apoptosis in HCT116 cells Since ROS is a universal entity mediating apoptosis [27], we examined the effect of CAY10598 on ROS generation. The intracellular accumulation of ROS induced by CAY10598 was measured through fluorescent microscopy and fluorescence-activated cell sorting (FACS) by using

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Figure 4. Effects of CAY10598 on JAK2/STAT3 pathway in HCT116 cells. (A) HCT116 cells were treated with indicated concentrations of CAY10598, and then the phosphorylation levels of JAK2 and STAT3 were examined by Western blot analysis. (B) Nuclear extract prepared from cells treated with CAY10598 (1 or 25 mM) was assessed for the DNA-binding of STAT3.

DCF-DA as a probe capable of detecting peroxides such as H2O2. Treatment with CAY10598 for 16 h induced intracellular accumulation of ROS (Figure 5A–B), which was abrogated by pretreatment with a prototypic ROS scavenger NAC (Figure C–D). Moreover, pretreatment with NAC rescued cells from CAY10598-induced apoptosis, as evidenced by Annexin-V-FITC staining (Figure 5E) and MTT assay (Figure 5F). Pretreatment of NAC also averted CAY10598-induced increase in p53 level, the inhibition of Bcl-2 expression and the cleavage of caspase-3 and PARP (Figure 5G). Furthermore, the inhibition of JAK2, phosphorylated JAK2 and STAT3 by CAY10598 was recovered by NAC treatment in HCT116 cells (Figure 5H). Effect of CAY10598 on cell growth in various cancer cells In order to determine whether CAY10598 can also inhibit the growth of other cell lines, we examined the effect of the compound on the viability of breast cancer (MCF-7), renal cancer (Caki) and other colon cancers (SW480) cells. Similar to its effects on HCT116 cells, CAY10598 significantly reduced the viability of MCF-7 (Figure 6A), Caki (Figure 6B) and SW480 (Figure 6C) in a concentration-dependent manner. In addition, real-time PCR was conducted to measure endogenous mRNA levels of EP receptors in each cells (Supplementary Figure 1 available online at http://informahealthcare.com/doi/abs/10.3109/ 10715762.2014.951838). EP1, EP2, and EP4 were detected in Caki, SW480, and MCF-7 cells, but not EP3. We found a very strong expression of EP1 in Caki and SW480 cells, whereas MCF-7 cells showed high EP2 and EP4 expression. Treatment of Caki cells with CAY10598 for 24 h resulted in the significant inhibition of constitutive

phosphorylation of JAK2 and STAT3 in a concentrationdependent manner (Supplementary Figure 2 available online at http://informahealthcare.com/doi/abs/10.3109/ 10715762.2014.951838). Interestingly, in accordance with HCT116 cells, the expression of total JAK2 was also decreased by CAY10598 treatment. In addition, CAY10598 of 25 mM exerted a significant inhibitory effect on the extent of phosphorylation at tyrosine 705 and serine 727 of STAT3 in SW480 and MCF-7 cells (Figure 4B). However, we could not detect constitutive phosphorylated JAK2 in these cells. These data suggest that CAY10598 induces the apoptosis in various cancer cells through the attenuation of STAT3 signaling, but the inhibition of STAT3 could be dependent on or independent of the JAK2 pathway according to cell types. Discussion PGE2 is known to play multifaceted roles in carcinogenesis [28]. While numerous studies have demonstrated that PGE2 enhances tumor cell proliferation, several others have reported its anti-proliferative effects in cancer cells [6–8]. The diverging roles of PGE2 in cell fate decisions may result from the cell type-specific and variable expression of four types of EP receptors, which have differential roles in tumorigenesis [9,10]. Whereas the activation of EP1, EP2 and EP4 are known to promote cancer cell proliferation, that of EP3 exerts anti-proliferative effects [28]. It is, therefore, plausible that simultaneous non-specific binding of PGE2 to different EP receptors may account for the variable effects of PGE2 on tumor cell proliferation. Moreover, the delineation of the mechanisms and roles of a particular EP receptor in cells stimulated with PGE2 as

CAY10598 induces apoptosis in HCT116 cells   7

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Figure 5. Role of intracellular ROS in CAY10598-induced apoptosis in HCT116 cells. (A–B) Cells were treated with CAY10598 (1, 10 or 25 mM) for 16 h and then examined for the intracellular accumulation of ROS measured by flow cytometry (A) and under the fluorescence microscope using DCF-DA fluorescence staining method ( 200) (B). (C–D) Cells were treated with NAC (5 mM) for 30 min before treatment with CAY10598 (25 mM) for 16 h. ROS levels were measured either by flow cytometry (C) or fluorescence microscopy (D). (E–F) Cells were treated with CAY10598 for 16 h in the absence or presence NAC, and the cell viability was determined by the FACS analysis (E) and MTS assay (F). In Figure 5E, bar graphs represent percentage of apoptotic cells in CAY10598 (CAY) and/or NAC-treated HCT116 cells. The results presented are mean  S.D. (n  3). ** Significantly different from the control (p  0.001). (G–H) Under the same experimental condition, the expression apoptosis markers (G) and phosphorylation of JAK2 and STAT3 (H) were determined by Western blot analysis.

a natural endogenous ligand requires annulment of the contribution by other EP receptors.. In this respect, specific agonists and antagonists of different EP receptors are used to verify the exact roles of a particular EP receptor in carcinogenesis. Although the activation of EP4 has been implicated in the development and progression of cancer [11,12] and the anti-proliferative effects of EP4specific antagonists have been reported [15], there have been studies demonstrating that a certain EP4-specific agonist also reduces the growth of gastric cancer cells by decreasing their DNA synthesis [9]. This perplexing role of the EP4 receptor in cancer has led us to examine the

effect of CAY10598, a specific EP4 agonist, on the growth of human colorectal cancer HCT116 cells and to elucidate its underlying molecular mechanisms. There have been limited studies with CAY10598 since its development as a specific agonist of the EP4 receptor. We have, therefore, first examined the effect of CAY10598 on the viability of HCT116 cells. Treatment with CAY10598 significantly reduced the cell viability in a time- and concentration-dependent manner. This decreased cell viability by CAY10598 has been correlated with the induction of apoptosis, as revealed by Annexin-V staining. Our findings are in good agreement with several previous studies. For example, treatment of PGE2 decreased the proliferation of HCT116 cells lacking cyclooxygenase-2 (COX-2), a key enzyme involved in the production of PGE2, albeit its tumorigenic potential in other human colorectal cancer (HCA7) cells [29]. Moreover, another EP4 agonist, PGE1-OH, enhanced B cell receptor-mediated apoptosis of immature B lymphoma cells [30]. We further confirmed the anti-proliferative effect of CAY10598 in other cancer cells, such as MCF-7, SW480 and Caki cells. We next attempted to delineate the molecular mechanisms underlying the anti-proliferative effect of CAY10598 in HCT116 cells. Our findings that treatment with CAY10598 activated casapse-9, -7 and -3, and increased the cleavage of DNA repair enzyme PARP, suggest that CAY10598 induced apoptosis in HCT116 cells by following the intrinsic mechanisms of cell death. The cytotoxic effect of CAY10598 in HCT116 cells was further supported by the increased expression of proapoptotic proteins Bax and p53, and the reduced expression of anti-apoptotic proteins Bcl-2 and survivin. While the pro-apoptotic Bax gene is a direct target of p53 [31], the overexpression of p53 in p53-null cells attenuates Bcl-2 expression [32]. Thus, CAY10598-induced p53 activation may result in cell death through downregulation of Bcl-2 and upregulation of Bax, thereby initiating caspase activation. Our findings that co-treatment of HCT116 cells with an EP antagonist GW627368x failed to rescue cells from CAY10598-induced cell death and that the cytotoxic effect of CAY10598 remained unaltered in cells transfected with EP4 siRNA suggest that the compound exhibited cytotoxicity independent of EP4 activation. We have, therefore, extended our focus on other cell signaling pathways beyond EP4, to elucidate the mechanistic basis of CAY10598-induced apoptosis in HCT116 cells. Since CAY10598 diminished the expression of cell survival proteins Bcl-2 and survivin, which are the target gene products of STAT3, we have examined the effects of CAY10598 on the activation of STAT3 and its upstream kinase JAK2. Our study revealed that treatment with CAY10598 markedly reduced the constitutive phosphorylation of JAK2 and that of STAT3 at both tyrosine 705 and serine 727 residues, and negated the DNA binding of STAT3 in HCT116 cells. We have found that CAY10598 not only inhibited JAK2 phosphorylation but also reduced the level of total JAK2. It is still unclear whether the

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p-STAT3 (Tyr705)

Bcl-2

p-STAT3 (Ser727)

β-Actin

-

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+ +

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Figure 5. (Continued).

inhibition of JAK2 phosphorylation by CAY10598 resulted from its downregulation of total JAK2 expression. Marubayashi et al. have reported that JAK2 is mutated in leukemia cells and that mutated JAK2 may be degraded by the proteasomal pathway [33]. However, according to

the study by Herreros-Villanueva et al. JAK2 mutation has not been observed in colorectal cancer cells [34]. Thus, it would be worthwhile to examine the mechanisms by which CAY10598 decreases total JAK2 expression in HCT116 cells. Since persistent activation of STAT3 increases the

CAY10598 induces apoptosis in HCT116 cells   9

(A) Cell viability(% of control)

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** ****

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(C)

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Apoptosis

Caki

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Figure 7. Putative mechanism of inhibition of cell growth in CAY10598-treated HCT116 cells.

*

80 60

1 µM 15 µM 25 µM 50 µM

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p-JAK2

p53

(B) 120

ROS

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(h)

CAY10598

Figure 6. The effects of CAY10598 on the cell viability of various cancer cells. MCF-7, Caki, and SW480 cells were treated with CAY10598 for indicated time, and cell viability was determined by the MTS assay.

proliferation of colon cancer cells in culture and enhances the growth of colon cancer cell xenograft tumors in nude mice [35], and the inhibition of STAT3 signaling induces apoptosis and cell cycle arrest in colon carcinoma cells [36], the inhibitory effects of CAY10598 on JAK2 and STAT3 activation would be considered as the potential mechanisms of its cytotoxic effects in HCT116 cells. The generation of ROS has been accounted for in triggering tumor cell death [37]. There are conflicting reports that certain pro-apoptotic agents inhibit the JAK/STAT activation differentially by blocking ROS production [38] or by generating ROS [39,40]. Recent literature suggests

that ROS may inhibit JAK2 phosphorylation via posttranslational modification of its critical cysteine residues [41]. The apoptosis-inducing protein p53 is also activated in response to oxidative stress [42]. Since CAY10598 diminished activation of JAK2/STAT3 and induced the expression of p53, we examined if CAY10598 generates ROS in HCT116 cells. We found that CAY10598 induced ROS generation, which was diminished by the ROS scavenger NAC. Pretreatment of cells with NAC attenuated CAY10598-induced cleavage of caspase-3 and PARP, and p53 expression. Moreover, pretreatment with NAC abrogated CAY10598-induced inhibition of Bcl-2 expression, and the phosphorylation of JAK2 and STAT3. Thus, CAY10598-induced ROS generation may lead to activation of p53 and inhibition of JAK2/STAT3, thereby resulting in the downregulation of Bcl-2 and the induction of Bax expression and caspase cleavage. Thus, our study revealed for the first time that in spite of being an EP4 agonist, CAY10598 induces apoptosis in HCT116 cells independently of EP4 activation but through induction of p53 and inhibition of JAK2/STAT3 activation via generation of ROS. While these findings ratify the potential therapeutic benefit of CAY10598 in colon cancer, we extended our study to verify whether the compound induces apoptosis selectively in cancer cells. Our study

10  I. G. Chae et al.

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revealed that CAY10598 also induces cytotoxicity in normal human keratinocytes (data not shown). Based on this observation, the significance of the present study may be reflected in identifying CAY10598 as a potentially cytotoxic agent that induced both normal and cancer cell death independent of EP4 activation. Thus, caution should be exercised in utilizing CAY10598 as a specific EP4 agonist in interpreting the role of EP4-mediated signaling in cancer. Moreover, the observed effect of CAY10598 on the modulation of JAK2/STAT3 signaling and ROS generation in HCT116 cells suggest that further structural modification of the compound to enhance its cancer cellspecific cytotoxicity may lead to the development of novel anticancer therapeutics.­­­­­­­ Acknowledgement This research 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 (2014R1A2A2A01004698).

[12] [13]

[14] [15]

[16] [17]

[18]

Declaration of interest The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.

[19]

[20]

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Supplementary material available online Supplementary Figures 1–2.

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