Inhibition of Rho-Kinase Abrogates Migration of Human Transitional Cell Carcinoma Cells: Results of an in vitro Study

Introduction: Migration of cells involves a complex signaling network. The aim of the present study was to elucidate the impact of Rho-kinase (ROK) on G protein-coupled receptor-induced migration of human transitional cell carcinoma cells in an in vitro experimental setting. Materials and Methods: Intracellular calcium concentration ([Ca2+](i)) was measured with the indicator dye Fura-2 in response to lysophosphatidic acid, thrombin and sphingosine-1-phosphate. Phospholipase C activity was determined in myo-[H-3]inositol-(0.5 p,mu Ci/ml) labeled cells. Migration was performed using a Boyden chamber. Transient transfection of a dominant-negative mutant of ROK was done with calcium phosphate. For staining of actin filaments, tetramethylrhodamine isothiocyanate-conjugated phalloidin was used. Results: Lysophosphatidic acid, thrombin and sphingosine-1-phosphate cause increases in [Ca2+](i), cellular responses being accompanied by an enhancement of phospholipase C activity and sensitive to the G(i) inhibitor pertussis toxin. Agonists potently stimulated migration of 124 and J82 cells. Inhibition of Rho proteins by Clostridium difficile toxin B abrogated cell migration. Inhibition of ROK using HA1077 and Y-27632 mimicked the properties of toxin B. Expression of a ROK mutant drastically reduced migration. Conclusions:G protein-coupled receptors potently stimulated cell migration in 124 and J82 cells. Rho proteins and ROK play a pivotal role in this signaling cascade. Rho and ROK may be putative targets for new therapy options in bladder cancer. Copyright (c) 2010 S. Karger AG, Basel