Role of OATP2A1 in PGE 2 secretion from human colorectal cancer cells via exocytosis in response to oxidative stress Taku Kasai 1 , Takeo Nakanishi 1 , Yasuhiro Ohno, Hiroaki Shimada, Yoshinobu Nakamura, Hiroshi Arakawa, Ikumi Tamai n Faculty of Pharmaceutical Science, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan article info Article history: Received 2 July 2015 Received in revised form 29 January 2016 Accepted 1 February 2016 Available online 2 February 2016 Keywords: Colorectal cancer Transporter OATP2A1 Prostaglandin E 2 Exocytosis abstract Chronic inflammation induced by reactive oxygen species is associated with increased risk of developing colorectal cancer (CRC), and prostaglandin E 2 (PGE 2 ), which serves as a key mediator of inflammatory responses, plays an important role in CRC initiation and progression. Therefore, in the present study, we aimed to investigate the role of prostaglandin transporter OATP2A1/SLCO2A1 in the changes of PGE 2 disposition in CRC cells in response to oxidative stress. H 2 O 2 induced translocation of cytoplasmic OATP2A1 to plasma membranes in LoVo and COLO 320DM cells, but not in Caco-2 cells. The shift of subcellular OATP2A1 was abolished in the presence of anti-oxidant N-acetyl-L-cysteine or an inhibitor of protein kinase C, which evokes exocytosis. Exposure of LoVo cells to H 2 O 2 caused an increase in the amount of extracellular PGE 2 without changing the sum of intra- and extracellular PGE 2 . OATP2A1 knockdown decreased extracellular PGE 2 in LoVo cells. In addition, extracellular PGE 2 was significantly reduced by exocytosis inhibitor cytochalasin D, suggesting that H 2 O 2 -induced PGE 2 release occurs in an exocytotic manner. Furthermore, mRNA expression of vascular endothelial growth factor (VEGF) was significantly reduced in LoVo cells by knockdown of OATP2A1. These results suggest that cytoplasmic OATP2A1 likely facilitates PGE 2 loading into suitable intracellular compartment(s) for efficient exocytotic PGE 2 release from CRC cells exposed to oxidative stress. & 2016 Elsevier Inc. All rights reserved. 1. Introduction Chronic inflammation is a risk factor for increased incidence of colorectal cancer (CRC). At sites of inflammation, a number of nonspecific cytokines produced in and released from activated inflammatory cells induce inflammation-associated genes, in- cluding cyclooxygenase (COX)-2. COX-2 expression is upregulated in human CRC patients [1], and it has been detected in their me- tastases in clinical studies [2]. Serum and mucosal levels of PGE 2 are elevated in human CRC and familial adenomatous polyposis patients [3,4]. There is considerable evidence that PGE 2 is involved in the aggressive phenotype of CRCs by enhancing tumor cell growth and motility [5,6]. Further, COX-2-selective nonsteroidal anti-inflammatory drugs are anti-tumorigenic in various animal models for CRC [7,8]. Therefore, COX-2-derived PGE 2 is thought to play a key role in tumor progression of CRC. Oxidative stress associated with chronic inflammation is linked to PGE 2 production in colorectal tumors. Previous studies have shown that oxidative stress markers are upregulated in human CRCs [9–11]. Clinically, levels of oxygen radicals in whole blood are higher in patients with a history of sporadic CRC than in controls [12]. Since overproduction of reactive oxygen species (ROS) con- tributes to increased PGE 2 production through arachidonic acid release [13], as well as upregulating cytoplasmic phospholipase [14] and inducing COX-2 expression [14,15], oxidative stress-in- duced PGE 2 release could facilitate CRC initiation and cell pro- liferation. Although molecular mechanisms underlying increased production of PGE 2 have been well studied, little is known about how PGE 2 is secreted from cells to afford a sufficient extracellular concentration for receptor-mediated activation of the biological action of PGE 2 . PGs cannot permeate through lipid bilayers [16], and therefore would have to be actively transported across the plasma Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/yexcr Experimental Cell Research http://dx.doi.org/10.1016/j.yexcr.2016.02.002 0014-4827/& 2016 Elsevier Inc. All rights reserved. Abbreviations: BCG, bromocresol green; BIM, bisindolylmaleimide; BSP, bromo- sulfophthalein; COX, cyclooxygenase; CRC, colorectal cancer; EGTA, ethylene glycol tetraacetic acid; EP, prostaglandin E receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; 15-PGDH, 15-hydroxyprostaglandin dehydrogenase; HPRT1, hy- poxanthine guaninephosphoribosyl transferase; MAPK, mitogen-activated protein kinase; NAC, N-acetyl-L-cysteine; NS, non-specific; OATP, organic anion trans- porting polypeptide; PGE 2 , prostaglandin E 2 ; ROS, reactive oxygen species; TGBz, TGBz T34; VEGF, vascular endothelial growth factor n Corresponding author. E-mail address: tamai@p.kanazawa-u.ac.jp (I. Tamai). 1 These authors contributed equally. Experimental Cell Research 341 (2016) 123–131