targeting sphingosine kinase 1 in carcinoma cells decreases proliferation and survival by compromising pkc activity and cytokinesis针对鞘氨醇激酶1在癌细胞减少损害pkc活性和胞质分裂增殖和生存.pdf

Targeting Sphingosine Kinase 1 in Carcinoma Cells Decreases Proliferation and Survival by Compromising PKC Activity and Cytokinesis 1 2 1,3 1,4 Nataliya Kotelevets , Doriano Fabbro , Andrea Huwiler , Uwe Zangemeister-Wittke * 1 Institute of Pharmacology, University of Bern, Bern, Switzerland, 2 Novartis Pharma AG, NIBR Basel, Switzerland, 3 Pharmazentrum Frankfurt/ZAFES, University Hospital ¨ ¨ Frankfurt, Frankfurt am Main, Germany, 4 Institute of Biochemistry, University of Zurich, Zurich, Switzerland Abstract Sphingosine kinases (SK) catalyze the phosphorylation of proapoptotic sphingosine to the prosurvival factor sphingosine 1- phosphate (S1P), thereby promoting oncogenic processes. Breast (MDA-MB-231), lung (NCI-H358), and colon (HCT 116) carcinoma cells were transduced with shRNA to downregulate SK-1 expression or treated with a pharmacologic SK-1 inhibitor. The effects of SK-1 targeting were investigated by measuring the level of intracellular sphingosine, the activity of protein kinase C (PKC) and cell cycle regulators, and the mitotic index. Functional assays included measurement of cell proliferation, colony formation, apoptosis, and cell cycle analysis. Downregulation of SK-1 or its pharmacologic inhibition increased intracellular sphingosine and decreased PKC activity as shown by reduced phosphorylation of PKC substrates. In MDA-MB-231 cells this effect was most pronounced and reduced cell proliferation and colony formation, which could be mimicked using exogenous sphingosine or the PKC inhibitor RO 31-8220. SK-1 downregulation in MDA-MB-231 cells increa