MOLECULAR CARCINOGENESIS 55:1096–1110 (2016)

Carnosic Acid Inhibits STAT3 Signaling and Induces Apoptosis Through Generation of ROS in Human Colon Cancer HCT116 Cells Do-Hee Kim,1 Ki-Woong Park,2 In Gyeong Chae,2 Juthika Kundu,2 Eun-Hee Kim,3 Joydeb Kumar Kundu,2 and Kyung-Soo Chun2* 1

College of Pharmacy, Seoul National University, Seoul, South Korea College of Pharmacy, Keimyung University, Daegu, South Korea 3 CHA Cancer Institute, CHA University, Seoul, South Korea 2

Carnosic acid (CA), the main antioxidant compound of Rosmarinus officinalis L., has been reported to possess anticancer activity. However, the molecular mechanisms underlying the anticancer effects of CA remain poorly understood. Our study revealed that CA treatment significantly reduced the viability of human colon cancer HCT116, SW480, and HT-29 cells. Treatment with CA induced apoptosis, which was associated with the induction of p53 and Bax, inhibition of Mdm2, Bcl-2, and Bcl-xl expression, activation of caspase-9, and -3, and the cleavage of PARP in HCT116 cells. CA inhibited the constitutive phosphorylation, the DNA binding and the reporter gene activity of STAT3 in HCT116 cells by blocking the phosphorylation of upstream JAK2 and Src kinases. Moreover, CA attenuated the expression of STAT3 target gene products, such as survivin, cyclin D1, D2, and D3. In STAT3-overexpressed HCT116 cells, CA inhibited cell viability and the expression of cyclin D1 and survivin. Furthermore, CA treatment induced the generation of ROS in these colon cancer cells. Pretreatment of cells with ROS scavenger N-acetyl cysteine abrogated the inhibitory effect of CA on the JAK2-STAT3/Src-STAT3 signaling and rescued cells from CA-induced apoptosis by blocking the induction of p53 and the cleavage of caspase-3 and PARP in HCT116 cells. However, L-buthionine-sulfoximine, a pharmacological inhibitor of GSH synthesis, increased CA-induced ROS production, thereby potentiating apoptotic effect of CA. In conclusion, our study provides the first report that CA induced apoptosis in HCT116 cells via generation of ROS, induction of p53, activation of caspases, and inhibition of STAT3 signaling pathway. © 2015 Wiley Periodicals, Inc. Key words: carnosic acid; apoptosis; reactive oxygen species; signal transducer and activator of transcription-3; colon cancer

INTRODUCTION Colorectal cancer (CRC) is the third most frequent cancer worldwide. The incidence of CRC is now rapidly increasing in previously known low risk areas, such as Latin America, Asia, and Africa. More than one million new cases of CRC are diagnosed every year [1]. Since chemotherapy contributes to overall patient’s survival with compromised quality of life, a wide variety of naturally occurring substances, derived from plant-based diet have been extensively used to prevent colon carcinogenesis [2]. Evidences from epidemiological studies suggest that the risk of cancer is twofold higher in persons having a low intake, as compared to those with high intake, of fruits and vegetables [3]. The Mediterranean diet has long been known to improve human health. Herbs like rosemary (Rosmarinus officinalis L.) and sage (Silvia officinalis L.) are commonly consumed in diet and can prevent various human diseases. Carnosic acid (CA, chemical structure shown in Figure 1A), largely present in sage and rosemary, has been shown to possess anti-inflammatory as well as antiproliferative activity in human leukemia HL-60 and U937 cells [4,5]. It has also been reported to inhibit the ß 2015 WILEY PERIODICALS, INC.

proliferation of various CRC cell lines such as Caco-2, HT-29, and LoVo cells in culture [6]. However, the molecular mechanisms underlying the antiproliferative and apoptosis inducing effects of CA in CRC cells are yet to be fully elucidated. We, therefore, attempted to investigate the anticancer effects of CA on human colon cancer HCT116 cells and to elucidate its underlying mechanisms. One of the hallmarks of cancer is the evasion of tumor cells from apoptosis [7], which is characterized by morphological changes such as cell membrane

Do-Hee Kim, Ki-Woong Park and In Gyeong Chae contributed equally to this work. Conflict of interest: None. Grant sponsor: Ministry of Science, ICT and Future Planning; Grant number: 2014R1A2A2A01004698 *Correspondence to: College of Pharmacy, Keimyung University, 1095 Dalgubeoldaero, Dalseo-Gu, Daegu 704-701, South Korea. Received 12 July 2014; Revised 4 May 2015; Accepted 28 May 2015 DOI 10.1002/mc.22353 Published online 8 July 2015 in Wiley Online Library (wileyonlinelibrary.com).

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Figure 1. Cytotoxic effect of CA in HCT116 cells. (A) Chemical structure of CA. (B) HCT116 cells were treated with indicated concentrations of CA for 24, 48, or 72 h. Cell viability was determined by the MTS assay. Values are expressed as means  SD.  P < 0.001, compared to control. (C) Cells were treated with indicated concentrations of CA for 24 h and cell morphology was analyzed by

fluorescence microscopy (200). (D) The apoptotic index (%) was determined by flow cytometry upon treatment of cells with CA (20, 50, or 100 mM) for 24 h and staining with Annexin V and P.I. Right panel shows statistical analysis of apoptosis. Data are representative of three independent experiments.

blebbing, cell shrinkage, nuclear condensation, and formation of apoptotic bodies [8]. Numerous naturally occurring polyphenols inhibit the proliferation and induce apoptosis in various cancer cells [9]. Apoptosis is induced by two cellular mechanisms: intrinsic (mitochondria-dependent) and extrinsic (death receptor-mediated) signaling [10]. The intrinsic pathway of apoptosis is mediated through the depolarization of mitochondrial membrane and the mitochondrial release of cytochrome c, which activates pro-caspase-9 resulting in subsequent cleavage

of procaspase-7 and-3 and the poly-(ADP-ribose) polymerase (PARP). Apoptotic signals originated through the activation of death receptor, on the other hand, leads to cleavage of procaspase-8 and -10, thereby activating caspase-3 either directly or through mitochondria-mediated pathway. The mitochondrial membrane integrity is maintained partly by the relative mitochondrial membrane localization of B-cell lymphoma (Bcl) family proteins. While the anti-apoptotic proteins Bcl-2 and Bcl-xl maintain the mitochondrial membrane integrity, Bax localization

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to mitochondrial membrane causes membrane depolarization and subsequent activation of caspase cascades. Cancer cells are characterized by constitutively elevated expression of anti-apoptotic proteins Bcl-2 and Bcl-xl, and the diminished expression of pro-apoptotic protein Bax [11]. The balance between the expression of pro- versus anti-apoptotic proteins determines the cell fate. A series of cell cycle regulatory and cell proliferation markers, such as cyclins, cyclin-dependent kinases (Cdk), and survivin are overexpressed in cancer. A redox-sensitive transcription factor, signal transducer and activator of transcription-3 (STAT3), regulates the transcriptional activation of these anti-apoptotic and proliferative gene products. STAT3 is consitutively active in cancer cells and the aberrant activation of STAT3-mediated signaling has been implicated in colon carcinogenesis [12–14]. The activation of STAT3 involves the phosphorylation of its tyrosine-705 (Y705) and serine-727 (S727) residues by various upstream kinases including Janus-activated kinases (JAKs), Src family kinases, and mitogen-activated protein (MAP) kinases. The phosphorylated STAT3 dimerizes and then translocates to the nucleus, where it binds to specific DNA response elements in the promoter regions of its target genes [15,16]. The blockade of STAT3 activation inhibits cell proliferation and induces apoptosis. Thus, STAT3 is a prime target of many anticancer agents [17]. In the present study, we report that CA induces anti-proliferative and apoptotic effects in HCT116 cells through interference with STAT3 signaling pathway via generation of ROS. MATERIALS AND METHODS Materials CA (purity 99%) and N-acetyl cysteine (NAC) were purchased from Sigma–Aldrich (St. Louis, MO). Antibodies against cleaved caspase-9, -3, PARP, Bcl-2, Bclxl, Bax, STAT3, p-STAT3 (Y705), JAK2, p-JAK2, Src, pSrc, cyclin D1, D2, and D3, and survivin were obtained from Cell Signaling Technology Inc. (Beverly, MA). Primary antibodies against each of p53, murine double minute-2 (Mdm2), Lamin A, and horseraddish peroxidase-conjugated secondary antibodies were purchased from SantaCruz Biotechnology (SantaCruz, CA). b-Actin antibody was obtained from Sigma–Adrich. The 20 -70 dichlorofluorescin diacetate (DCF-DA) was procured from Invitrogen (Carlsbad, CA). Hank’s balanced salt solution (HBSS) was purchased from Meditech (Herndon, VA). L-buthionine-sulfoximine (BSO) was purchased from Sigma– Adrich. All other chemicals were of analytical or highest purity grade available. Cell Culture and Treatment HCT116, SW480, and HT-29 cells were obtained from American Type Culture Collections and Molecular Carcinogenesis

maintained in RPMI supplemented with 10% fetal bovine serum and antibiotics (100 U/ml penicillin G and 100 mg/ml streptomycin) at 378C in a humidified incubator containing 5% CO2 and 95% air. In all the experiments, cells were seeded at 2  105 cells/ml and incubated with CA at 50–60% confluence. All chemicals were dissolved in ethanol and the final ethanol concentration was less than 0.1%. Cell Viability Assay The anti-proliferative effect of CA against HCT116 cells was measured by using a solution of tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) (Promega, Madison, WI). Briefly, cells (2  103) were incubated in triplicate in a 96-well plate in presence or absence of CA in a final volume of 0.1 ml for different time intervals at 378C. Thereafter, 20 ml of MTS solution was added to each well and incubated for 60 min. The number of viable cells was measured in a 96-well plate at an optical density of 492 nm on a microplate reader (Tecan Trading AG, Switzerland). Cell viability was described as the relative percentage of control. The IC50 was calculated using GraphPad Prism 4.0 software. Annexin V Staining Annexin V staining was performed using FITCAnnexin V staining kit (BD Biosciences, San Jose, CA) following the manufacturer’s instructions. Briefly, CA treated cells were washed with PBS and resuspended in binding buffer containing Annexin V and propidium iodide (P.I.). Flourescence intensity was measured using flow cytometry (BD Biosciences). Western Blot Analysis Cells were harvested and lysed with RIPA buffer and collected protein samples were quantified by using bicinchoninic acid protein assay kit (Pierce Biotechnology, Rockford, IL). The protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis was done according to the protocol described earlier [18]. Immunoblot membranes were incubated with Super-signal picochemiluminescent substrate or dura-luminol substrate (Thermo Scientific, Waltham, MA) according to manufacturer’s instruction and visualized with imageTM quant LAS 4000 (Fujifilm Life Science, Japan). The STAT3-Luciferase Reporter Gene Assay Cells were seeded into 12-well plates at a density of 5  104 cells per well prior to transfection. Cells were transfected with p-STAT3-TA-luc (BD Biosciences) or control vector using Genefectin transfection reagent (GenetroneBiotech,Korea).After24 htransfection,cells weretreatedwithCAforadditional24 handcelllysiswas carriedoutwith1reporterlysisbuffer.Aftermixingthe cell lysates with luciferase substrate (Promega, WI), the luciferase activity was measured by using luminometer.

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The b-galactosidase assay was done according to the supplier’s instruction (Promega Enzyme Assay System) for normalizing the luciferase activity and the results were expressed as fold transactivation. Preparation of Cytosolic and Nuclear Extracts The cytosolic and nuclear extracts were prepared by using NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit (Thermo Scientific). Cells were washed with ice cold PBS, collected and centrifuged at 1,600g for 15 min at 48C. Pellets were suspended in 50 ml of Cytoplasmic Extraction Reagent (CER) I for 15 min, added CER II for additional 2 min. The mixture was centrifuged for 10 min at 16,000g. Supernatant was collected as cytosolic extract. The pellets were washed with Nuclear Extraction Reagent and incubated on ice for 1 h and centrifuged at 16,000g for 15 min. The supernatant containing nuclear proteins was collected and stored at 708C after determination of protein concentration by using Bradford Reagent (Bio-Rad Laboratories, Hercules, CA). Electrophoretic Mobility Gel Shift Assay (EMSA) The EMSA for STAT3 DNA binding was performed using a DNA–protein binding detection kit according to the manufacturer’s protocol (GIBCO BRL, Grand Island, NY). The nuclear extract was prepared from cells incubated with or without CA. The STAT3 oligonucleotide probe 50 -AGC TTC ATT TCC CGT AAA TCC CTA-30 (Bionics, Seoul, Korea) was labeled with [g-32P] ATP and the EMSA was performed according to the protocol described earlier [19]. Transient Transfection The pCMV-STAT3-Ha plasmid for expression of STAT3-HA tagged proteins was transfected into HCT116 cells. The pCMV-STAT3-HA plasmid was kindly provided by Prof. Young-Joon Surh (Seoul National University). Transient transfections were performed using the Lipofectamine transfection reagents according to the instructions supplied by the manufacturer (Invitrogen). After 48 h transfection, cells were treated with CA of indicated concentration and the cell lysis was performed with indicated lysis buffer. Measurement of Reactive Oxygen Species (ROS) Accumulation Cells were treated with CA in the presence or absence of NAC for 2 h and then loaded with 25 mM of DCF-DA. After incubation for 30 min at 378C in a 5% CO2 incubator, cells were washed twice with HBSS solution, suspended in the complete media and were examined under a fluorescence microscope to detect the intracellular accumulation of ROS. Statistical Analysis When necessary, data were expressed as mean  SD of at least three independent experiments and Molecular Carcinogenesis

statistical analysis was done by using SigmaPlot 10.0. A P-value

Carnosic acid inhibits STAT3 signaling and induces apoptosis through generation of ROS in human colon cancer HCT116 cells.

Carnosic acid (CA), the main antioxidant compound of Rosmarinus officinalis L., has been reported to possess anticancer activity. However, the molecul...
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