Α-Mangostin Reduced ER Stress-Mediated Tumor Growth Through Autophagy Activation

http://dx.doi.org/10.4110/in.2012.12.6.253 ORIGINAL ARTICLE pISSN 1598-2629 eISSN 2092-6685 α-Mangostin Reduced ER Stress-mediated Tumor Growth through Autophagy Activation Sung-Jin Kim1, Eun-Hye Hong1, Bo-Ra Lee1, Moon-Ho Park1, Ji-Won Kim1, A-Rim Pyun1, Yeon-Jeong Kim2, Sun-Young Chang3, Young-Won Chin4 and Hyun-Jeong Ko1* Laboratory of Immunology and Microbiology, College of Pharmacy, 1Kangwon National University, Chuncheon 200-701, 2Inje University, Gimhae 621-749, 3Ajou University, Suwon 443-749, 4Dongguk University, Goyang 410-773, Korea α-Mangostin is a xanthon derivative contained in the fruit INTRODUCTION hull of mangosteen (Garcinia mangostana L.), and the administration of α-Mangostin inhibited the growth of trans- α-Mangostin is a xanthone derivative included in the fruit planted colon cancer, Her/CT26 cells which expressed hull of mangosteen (Garcinia mangostana L.), and recently α Her-2/neu as tumor antigen. Although -Mangostin was re- it was reported that α-Mangostin induced apoptosis of sev- ported to have inhibitory activity against sarco/endoplasmic 2＋ eral cancer cells via sarcoplasmic/endoplasmic Ca ATPase reticulum Ca2+ ATPase like thapsigargin, it showed different (SAECA) inhibition and autophagy activation (1,2). Although activity for autophagy regulation. In the current study, we α found that α-Mangostin induced autophagy activation in -Mangostin has an inhibitory effect on SAECA like thapsi- mouse intestinal epithelial cells, as GFP-LC3 transgenic mice gargin, a sesquiterpene lactone, initially isolated from a plant, were orally administered with 20 mg/kg of α-Mangostin Thapsia garganica, it was different from thapsigargin in that daily for three days. However, the activation of autophagy by α-Mangostin activated autophagy pathway, whereas thapsi- α-Mangostin did not significantly increase OVA-specific T gargin induced autophagy arrest (3). Disruption of endoplas- cell proliferation. As we assessed ER stress by using XBP-1 mic reticulum (ER) Ca2＋ homeostasis which was mediated by reporter system and phosphorylation of eIF2α, thapsi- the inhibition of SAECA, resulted in Ca2＋ deprivation in ER, gargin-induced ER stress was significantly reduced by α- raised cytosolic Ca2＋ concentration, and consequently apop- Mangostin. However, coadministration of thapsigargin with tosis (4). α-Mangostin completely blocked the antitumor activity of Autophagy, especially macroautophagy, is a lysosome-de- α -Mangostin, suggesting ER stress with autophagy block- pendent degradation pathway for homeostatic regulation of ade accelerated tumor growth in mouse colon cancer model. cells which involved in degradation of cytosolic protein and Thus the antitumor activity of α-Mangostin can be ascrib- organelles (5). Several studies have shown that ER stress in- able to the autophagy activation rather than ER stress induc- tion. duced autophagy flow via JNK activation or PERK/eIF2α [Immune Network 2012;12(6):253-260] phosphorylation (6,7). In the current study, the administration of α-Mangostin efficiently inhibited tumor growth in correla- tion with previous reports showing the anticancer activity of Received on October 23, 2012. Revised on November 1, 2012. Accepted on November 7, 2012. CC This is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribu- tion, and reproduction in any medium, provided the original work is properly cited. *Corresponding Author. Hyun-Jeong Ko, Laboratory of Immunology and Microbiology, College of Pharmacy, Kangwon National University, Chuncheon, Korea. Tel: 82-33-250-6923; Fax: 82-33-255-7865; E-mail: [email protected] Keywords: Autophagy, α-Mangostin, Thapsigargin, ER stress, Antitumor activity, Colon cancer Abbreviations: ER, endoplasmic reticulum; SAECA, sarcoplasmic/endoplasmic Ca2+ ATPase; eIF2α, eukaryotic initiation factor 2 alpha; UPR, unfolded protein response; XBP1, X-box binding protein 1; DC, dendritic cell; PERK, PKR-like ER-res- ident kinase IMMUNE NETWORK Vol. 12, No. 6: 253-260, December, 2012 253 Antitumor Activity of α-Mangostin Sung-Jin Kim, et al. α-Mangostin. Although the role of ER stress in cancer has ng/ml of GM-CSF for 7 days and BM-DC were incubated with been studied in depth (8,9), the effect of ER stress-mediated Ovalbumin (OVA, 100 ng/ml) for 18 hrs. These cells were autophagy under ER stress upon anticancer activity was not further incubated with α-Mangostin (1 uM) for four hours studied well. Thus we tried to find out the effect of ER stress before i.v. injection into mice adoptively transferred with car- with autophagy regulators, including α-Mangostin and thap- boxylfluorescein diacetate succinimidyl ester (CFSE)-labeled sigargin, on antitumor activity. Interestingly, despite the sim- OT-I cells. To prepare CFSE-labeled OT-I cells, OVA-specific ilar inhibitory effect on SAECA, α-Mangostin did not increase CD8＋ T cells (＞90% of which were Vα2-positive) were iso- ER stress in vitro, whereas α-Mangostin reduced ER stress lated from OT-I mice using magnetic beads. These cells were induced by thapsigargin treatment. Collectively, these results labeled with 5 uM CFSE and i.v. transferred into their syn- suggested that autophagy activation by α-Mangostin could genic C57BL/6 mice. Two days after DC vaccination, lym- overcome ER stress mediated by Ca2＋ ATPase blockade by phoid cells from the lymph nodes or spleen of the recipient itself. However, autophagy arrest by thapsigargin completely mice were stained with phycoerythrin (PE)-conjugated anti-V inhibited the antitumor effect by α-Mangostin, suggesting α2 and anti-CD8 antibodies and then analyzed by flow cyto- that autophagy activation in cancer might be an important metry. strategy to achieve successful antitumor activity in the presence of ER stress. Antibodies All Abs for flow cytometry were from BD Biosciences (San MATERIALS AND METHODS Jose, CA) including anti-CD8-APC and anti-Vα2-PE Ab. For the western blotting, anti-phospho-eIF2α, anti-β-actin and Mice anti-rabbit/mouse HRP antibodies (Cell Signaling Technology) BALB/c mice and C57BL/6 mice (Orient-Bio Ltd, Charles River were used. Laboratories, Seoul, Korea) were purchased at 6 weeks of age and used in all of the experiments. GFP-LC3 mice were from Confocal microscopy for autophagy activation Riken with consent on their use from Dr. Mizushima, Noboru GFP-LC3 mice were sacrificed, and the small intestine was in Tokyo Medical and Dental University (10). Mice were fed dissected and promptly washed with PBS. The tissue was with standard lab. chow and water ad libitum. The animals fixed in formalin for 18 hours. Fixed tissue was embedded were maintained in the KNU animal facility at 20∼22oC under in OCT and sectioned on a cryotome into 8μm sections. 40∼60% relative humidity and a 12 h/12 h (light/dark) cycle Slides were washed with PBS and directly applied to attach for at least 7 days prior to the experiment. All experiments coverglass. Images of the sections were collected using Zeiss were approved by the Institutional Animal Care and Use confocal system (13). Committee of Kangwon National University. In addition, the ethical guidelines described in the KFDA Guide for the Care Endoplasmic reticulum assay and Use of Laboratory Animals was followed throughout the To assess cellular ER stress, we checked the splicing of XBP1, experiments. the target of ER stress sensor kinase IRE1α, which was well-known to be activated by ER stress (14). We used the Tumor cell lines and tumor models CT26 cells, and transfected them with XBP1-venus DNA Mouse colon carcinoma CT26 (American Type Culture Collec- (kindly provided by Masayuki Miura, RIKEN, Japan) by lip- tion, Manassas, VA), and human Her-2/neu expressing CT26 ofectamine 2000 (Invitrogen) following the manufacturer’s in- (Her-2/CT26) were used (11). To establish subcutaneous structions (14). To induce the ER stress in the CT26 cells, (s.c.) tumors, 2×105 Her-2/CT26 tumor cells were injected DMSO as negative control or thapsigargin (200 nM; Sigma) on the left flank of BALB/c mice. The tumor growth was were added after plating cells in 96-well plate (2×104 cells/ measured by caliper 3 times a week. 200μl/well) and serially diluted α-Mangostin or DMSO as negative control were added, respectively. After 24 hrs in- o DC vaccine and OT-I proliferation assay cubation at 37 C, 5% CO2, cells were lysed in digitonin buffer Bone marrow-derived DC was prepared as described pre- (50 mM Tris-HCl (pH7.5), 1 mM EDTA, 10 mM EGTA and viously (12). Bone marrow cells were incubated with 10 10μM digitonin) at room temperature for 30 min. We used 254 IMMUNE NETWORK Vol. 12, No. 6: 253-260, December, 2012 Antitumor Activity of α-Mangostin Sung-Jin Kim, et al. the GEMINI EM (Molecular Device) to measure fluorescence of the spliced XBP1 (emission at 520 nm; excitation at 485 nm). Western blot CT26 cells were cultured and treated with thapsigargin (200 nM) in the presence of DMSO or α-Mangostin. Western blot analysis was performed using mAbs to phosphor-eIF2α or β-actin as previously reported (14). Figure 1. Administration of α-Mangostin induced antitumor effect. BALB/c mice were s.c. injected with of Her2/CT26 cells (2×105 cells/mouse). One day after tumor challenge, mice were orally Preparation of α-Mangostin administrated with 20 mg/kg of α-Mangostin daily. The growth of solid tumor was measured by caliper three times a week. *p＜0.05 The pericarps of G. mangostana L. were collected in Indone- and **p＜0.01 (Student’s t-test; n=5/group). Tumor growth was sia. The separation and identification of α-Mangostin was monitored and mean values for tumor volume±SE are shown. conducted as described previously (15).

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