7-Chloro-6-piperidin-1-yl-quinoline-5,8-dione (PT-262), a novel ROCK inhibitor blocks cytoskeleton function and cell migration Chih-Chien Tsai a,b , Huei-Fang Liu a , Kai-Cheng Hsu a,c , Jinn-Moon Yang a,c , Chinpiao Chen d , Kuang-Kai Liu a , Tzu-Sheng Hsu b , Jui-I. Chao a,e, * a Department of Biological Science and Technology, National Chiao Tung University, Hsinchu 30068, Taiwan b Institute of Pharmacology and Toxicology, Tzu Chi University, Hualien 970, Taiwan c Institute of Bioinformatics and Systems Biology, National Chiao Tung University, Hsinchu 30068, Taiwan d Department of Chemistry, National Dong Hwa University, Hualien 970, Taiwan e Institute of Molecular Medicine and Bioengineering, National Chiao Tung University, Hsinchu 30068, Taiwan 1. Introduction The reorganization of cytoskeleton plays important roles in the regulation of cancer cell migration and metastasis. The RhoGT- Pases are key regulators of cytoskeleton dynamics [1,2]. The RhoGTPase family members include RhoA, Rac1, and Cdc42 in mammalian cells. RhoA is involved in the regulation of cytoskele- ton reorganization and focal adhesion, whereas Rac1 and Cdc42 work together at the regulation of cell leading edges to form lamellipodia and filopodia [3,4]. RhoA is a key family of RhoGTPases that participates in cancer migration and metastasis [5,6]. Overexpression of RhoA has been found in a variety of cancers including, lung, bladder, testicular, ovarian, colon, and breast [7]. ROCK (Rho-associated coiled-coil forming protein kinase) is a RhoA downstream protein [5]. There are two isoforms of ROCK, known as ROCK1 and ROCK2 [8]. Both ROCK1 and ROCK2 can regulate the activity of myosin light chain (MLC) proteins by direct MLC phosphorylation [9,10]. Cell migration of actomyosin contractility is mediated by the phosphorylation of MLC for cell movement [1]. Moreover, ROCK has been shown to induce the stress fiber formation and cancer cell migration and metastasis [5,11,12]. Thus, the development of strategies or drugs to block the RhoA–ROCK pathway is highly desirable for cancer therapy. Y-27632, [(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclo- hexane carboxamide dihydrochloride], is a specific ROCK inhibitor [10,13]. The inhibiting ROCK mechanism of Y-27632 is through binding to the catalytic site of ROCK by competing with the ATP binding site [14]. Inhibition of ROCK by Y-27632 blocks cancer cell Biochemical Pharmacology 81 (2011) 856–865 ARTICLE INFO Article history: Received 30 November 2010 Accepted 13 January 2011 Available online 26 January 2011 Keywords: PT-262 ROCK Cell migration Cytoskeleton Lung cancer ABSTRACT The 5,8-quinolinediones are precursors for producing multiple types of bioactive products. In this study, we investigated a new compound derived from 5,8-quinolinediones, 7-chloro-6-piperidin-1-yl- quinoline-5,8-dione (designated as PT-262), which markedly induced cytoskeleton remodeling and migration inhibition in lung carcinoma cells. Comparison with various cytoskeleton inhibitors, including paclitaxel, colchicine and phallacidin, the cell morphology following treatment with PT-262 was similar to phallacidin on the cell elongation and abnormal actin polymerization. However, PT-262 did not directly bind to actin filaments. ROCK (Rho-associated coiled-coil forming protein kinase) is a downstream effector of RhoA to mediate the phosphorylation of myosin light chain (MLC) and cytoskeleton reorganization. The RhoA–ROCK–MLC pathway has been shown to promote cancer cell migration and metastasis. Interestingly, PT-262 was more effective on inhibiting ROCK kinase activities than specific ROCK inhibitors Y-27632 and H-1152. PT-262 induced cytoskeleton remodeling and migration inhibition in A549 lung carcinoma cells. The total MLC and phosphorylated MLC proteins and stress fibers were blocked after treatment with PT-262. Nonetheless, the RhoA protein and GTPase activity were not altered by PT-262. A computational model suggests that PT-262 interacts with the ATP- binding site of ROCK protein. Together, these findings demonstrate that PT-262 is a new ROCK inhibitor. ß 2011 Elsevier Inc. All rights reserved. Abbreviations: ROCK, Rho-associated coiled-coil forming protein kinase; MLC, myosin light chain; MBS, myosin binding subunit; DMSO, dimethyl sulfoxide; PI, propidium iodide; MTT, 3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyl tetrazolium bromide; FBS, fetal bovine serum; ERK, extracellular signal-regulated kinase; PBS, phosphate-buffered saline; F-actin, actin filament. * Corresponding author at: Department of Biological Science and Technology, National Chiao Tung University, 75, Bo-Ai Street, Hsinchu 30068, Taiwan. Tel.: +886 3 5712121x56965; fax: +886 3 5131309. E-mail address: jichao@faculty.nctu.edu.tw (J.-I. Chao). Contents lists available at ScienceDirect Biochemical Pharmacology journal homepage: www.elsevier.com/locate/biochempharm 0006-2952/$ – see front matter ß 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bcp.2011.01.009