Angiopoietin-Like Protein 4 Potentiates DATS-Induced Inhibition Of

FOOD & NUTRITION RESEARCH, 2017 VOL. 61, 1338918 https://doi.org/10.1080/16546628.2017.1338918 ORIGINAL ARTICLE Angiopoietin-like protein 4 potentiates DATS-induced inhibition of proliferation, migration, and invasion of bladder cancer EJ cells; involvement of G2/M-phase cell cycle arrest, signaling pathways, and transcription factors-mediated MMP-9 expression Seung-Shick Shina*, Jun-Hui Songb*, Byungdoo Hwangb, Sung Lyea Parkb, Won Tae Kimc, Sung-Soo Parka, Wun-Jae Kimc and Sung-Kwon Moonb aDepartment of Food Science and Nutrition, Jeju National University, Jeju, South Korea; bDepartment of Food and Nutrition, Chung-Ang University, Anseong, South Korea; cDepartment of Urology, Chungbuk National University, Cheongju, South Korea ABSTRACT ARTICLE HISTORY Background: Diallyl trisulfide (DATS), a bioactive sulfur compound in garlic, has been highlighted Received 31 January 2017 due to its strong anti-carcinogenic activity. Accepted 27 May 2017 Objective: The current study investigated the molecular mechanism of garlic-derived DATS in KEYWORDS cancer cells. Additionally, we explored possible molecular markers to monitoring clinical ANGPTL4; bladder cancer; responses to DATS-based chemotherapy. diallyl trisulfide; migration; Design: EJ bladder carcinoma cells were treated with different concentration of DATS. Molecular invasion; microarray changes including differentially expressed genes in EJ cells were examined using immunoblot, FACS cell cycle analysis, migration and invasion assays, electrophoresis mobility shift assay (EMSA), microarray, and bioinformatics analysis. Results: DATS inhibited EJ cell growth via G2/M-phase cell cycle arrest. ATM-CHK2-Cdc25c- p21WAF1-Cdc2 signaling cascade, MAPKs, and AKT were associated with the DATS-mediated growth inhibition of EJ cells. DATS-induced inhibition of migration and invasion was correlated with down-regulated MMP-9 via reduced activation of AP-1, Sp-1, and NF-κB. Through microarray gene expression analysis, ANGPTL4, PLCXD1, and MMP3 were identified as candidates of molecular targets of DATS. Introduction of each gene to EJ cells revealed that ANGPTL4 was associated with the DATS-induced inhibition of cell growth, migration, and invasion. Conclusions: ANGPTL4 regulates DATS-mediated inhibition of proliferation, migration, and invasion of EJ cells, and thus, has potential as a prognostic marker for bladder cancer patients. Introduction into allicin, which is responsible for the aroma of fresh garlic [4]. Allicin is very unstable and is readily converted Bladder cancer is one of the most common cancers into sulfur-containing compounds such as diallyl sulfide of the human genitourinary system worldwide. (DAS), diallyl disulfide (DADS), diallyl trisulfide (DATS), According to the American Cancer Society, bladder and other allyl polysulfides [3,5]. Recently, these organic cancer accounts for approximately 5% of new cancer sulfur compounds have attracted great attention as a cases, and in 2016 an estimated 77,000 cases were novel pool of cancer preventive agents [3,5]. For example, diagnosed, among which 16,390 patients died due to a growing body of evidence indicates that DATS inhibits the disease in the USA [1]. Thus, the development of prostate, lung, gastric, and breast cancer progression by new therapeutic options is crucial to long-term sur- inducing apoptosis [6,7]. vival of bladder cancer patients. Cell-cycle progression is regulated by cell-cycle Garlic (Allium sativum) is renowned as a medicinal checkpoints at the G1, S, and G /M phases [8]. The food in traditional and alternative medicine. 2 G /M checkpoint, which prevents DNA-damaged Epidemiological studies support a positive association 2 cells from entering mitosis, is regulated by cyclin- between intake of Allium vegetables and low incidence dependent kinase 1 (CDK1), also known as Cdc2, of cancers [2,3]. When fresh garlic is crushed or chopped, and its activating partner cyclin B1 [9]. Activation the sulfoxide constituent alliin is enzymatically converted CONTACT Sung-Kwon Moon [email protected] Department of Food and Nutrition, Chung-Ang University, 4726 Seodong-Daero, Daedeok- Myeon, 17546 Anseong, Republic of Korea *These authors contributed equally to this work. © 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 2 S.-S. SHIN ET AL. of the cyclin B1-CDK1 complex is controlled by VA, USA). Detailed information on cells and materials either inhibitory phosphorylation of CDK1 by is available in the Supporting Information. WEE1 and MYT1 kinases or activation of Cdc25c phosphatase by ATM/CHK2 [9]. In addition, che- DATS treatment and cell counting motherapeutic reagents modulate mitogen-activated protein kinase (MAPK) and AKT cascades, which EJ cells were seeded in 6-well plates and treated with are key signaling pathways associated with cell DATS (0, 50, 100, and 150 µM) for 24 h. The cells were death and growth inhibition of bladder cancer cells detached from the plates by treatment with 0.25% trypsin [10,11]. Furthermore, expression of MMP-9 (gelati- containing 0.2% EDTA (Corning, NY, USA). Fifty micro- nase B, a 92-kDa gelatinase) is closely related with liters of detached cells were mixed with 50 μLof0.4% the migration and invasion ability of bladder tumor trypan blue (Sigma-Aldrich) by gentle pipetting, after cells via the activation of transcription factors, which 20 μL of the mixture was loaded into each chamber including AP-1, Sp-1, and NF-κB[12,13]. Thus, tar- of a hemocytometer, and the cells were counted. geting of cell cycle regulation, signaling pathways, and transcription factor-associated MMP-9 modula- MTT assay tion might prevent tumor proliferation and metasta- sis, consequently reducing mortality. Cellular proliferation was measured by the MTT (3-(4,5- Angiopoietin-like protein 4 (ANGPTL4) is an dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) endogenous inhibitor of lipoprotein lipase that is assay as described previously with some modification regulated by fatty acids through PPAR regulatory [19]. In brief, EJ cells were treated with different con- pathways [14]. Although the major function of centrations of DATS (0, 50, 100, and 150 μM) for 24 hr. ANGPTL4 is to regulate adipogenesis, recent studies Then, the medium was removed and the cells were have suggested diverse roles in various cancers incubated with 0.5 mg/mL of MTT solution. After incu- including colorectal cancer [15], hepatocellular carci- bation for 2 hr at 37°C in a 5% CO2 incubator, the noma (HCC) [16], breast cancer [17], and prostate supernatant was removed and 100 uL of DMSO was cancer [18]. added. After incubation for 1 hr, cell proliferation was Although the inhibitory effects of DATS on cancer determined by measuring the absorbance at 540 nm on cell proliferation have been well demonstrated, the a microplate reader. Cell morphology was analyzed underlying molecular mechanisms remain largely using phase-contrast microscopy. unclear. In this study, we investigated the mechanism of DATS-mediated inhibition of proliferation, migra- Cell-cycle analysis tion, and invasion of EJ bladder cancer cells through comprehensive analysis of signaling pathways, cell After treatment with DATS (0, 50, 100, and 150 μM) cycle regulation, and transcription factor-associated for 24 hr, the cells were collected and washed twice MMP-9 regulation. Microarray analysis identified with 1 × PBS. To determine cell cycle distribution, ANGPTL4 as a crucial factor associated with the 5 mL of ice-cold ethanol (70% (v/v)) was added drop- DATS-mediated anti-tumor effect in EJ cells. wise and resuspended the cell pellets and stored at 4°C overnight. The cells were then pelleted and resuspended in 1 ml of 1 × PBS containing RNase A Methods and materials (100 μg/mL) and propidium iodide (100 μg/mL), fol- lowed by incubation at 37°C for 30 min. Cell-cycle Cells and materials distribution was analyzed by a flow cytometer DATS (SMB00289) was purchased from Sigma-Aldrich (Facstar, Becton Dickinson, Franklin Lakes, NJ, USA) (St. Louis, MO, USA). Antibodies were purchased from supplied with BD Cell FIT software. Santa Cruz Biotechnology (Santa Cruz, CA, USA) or Cell Signaling Technology (Danvers, MA, USA). The Wound-healing migration assay nuclear extract kit and electrophoretic mobility shift assay (EMSA) gel shift kit were obtained from Cells (3 × 105) were plated in 2 mL of medium in each Panomics (Fremont, CA, USA). cDNAs of ANGPTL4, well of a 6-well plate and grown to 90% confluence. The PLCXD1, MMP3, and vectors pOTB7 and pCNS were surface area of the cells was scratched with a 2-mm-wide obtained from the Korean Human Gene Bank. The tip. After washing with PBS three times, the plate was human bladder carcinoma cell line EJ was purchased incubated with culture media in the presence or absence from the American Type Culture Collection (Manassas, of DATS for 24 h. The rate of cells migrating into the FOOD & NUTRITION RESEARCH 3 scratched area was measured and photographed using RNA extraction an inverted microscope at a magnification of 40 ×. Total RNA was isolated from EJ cells treated with or without DATS using TRIzol reagent (Thermo Fisher Boyden chamber invasion assay Scientific). RNA quality was validated by 1% agarose gel electrophoresis. Invasion

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