Screening Of A PKCζ-specific Kinase Inhibitor PKCzI257.3 Which Inhibits EGF-induced Breast Cancer Cell Chemotaxis

Research Content:Tumor metastasis is the major cause of death of cancer patients.Recent data indicated that chemotaxis,which involves the chemokines expressed at specific organs and their receptors expressed on tumor cells,plays a crucial role in tumor invasion and spreading.Various organs and tissues secrete a gradient of chemotactic cytokines such as epidermal growth factor(EGF) and CXCL12 chemokine,and numerous studies have revealed that chemotaxis of cancer cells were indeed mediated by their receptors i.e.EGF receptor and CXCR4,respectively,and EGF served as a potent chemoattractant for human breast cancer cell lines.Though the molecular mechanisms in cancer cell chemotaxis is complex and far from fully elucidated,and recently the results of previous work suggested that among all the PKC isotypes,PKCζis a key signal transducer shared by both EGFR-and CXCR4-mediated chemotactic signaling pathways in breast and non-small lung cancer cell lines which is always activated or over-expressed.Our previous work has also confirmed that reducing PKCζactivity through siRNA can inhibit migration and metastasis of cancer cells in vivo,which indicates PKCζis a anti-cancer drug target.So,a biochemical screening kit of American Invitrogen corporation was used to screen potential PKCζ-specific kinase inhibitors from a micromolecular library in this study,and our purpose is to evaluate the functional effects of inhibitors in cancer cell chemotaxis,indicate the molecular mechanism of cancer metastasis and further more for more potent cancer metastasis inhibitors.Methods:Part 1:A biochemical screening kit-Z'-LYTE^TM KINASE was used to screen potential PKCζ-specific kinase inhibitors from a micromolecular library comprised of almost two hundred micromolecules.Part 2:MTT assay was used to examine the cytotoxicity of screened micromolecular compound for human breast cancer cell MDA-MB-231;cell chemotaxis and wound healing assay were used to evaluate functional effects of it for cell migration;actin polymerization,cell adhesion and cofilin activation in western blotting assay were used to indicate molecular mechanism of inhibition for cell chemotaxis and migration Results:Part 1:The optimal kinase concentration of PKCζand PKCαscreened were 125,9ng/ml;PKCzI257.3(Molecular Formula:C₁₅H₁₉N₃O) can specificly and significantly inhibit PKCζactivity(IC₅₀=28μM),not for PKCαactivity (IC₅₀=200μM);while commercialized PKC inhibitor G_o|¨6850 can impaire both PKCζand PKCαactivities,with the IC₅₀ at 4.8μM and 0.04μM respectively.Part 2:In breast cancer cell MDA-MB-231,It was indicated that PKCzI257.3 had no significant effects on cell proliferation through MTT assay;chemotaxis and would healing assay had detected that PKCzI257.3 significantly inhibited EGF-induced chemotaxis and migration;under the condition of PKCzI257.3 pre-treatment,cell attachment in general was decreased with statistical significance in adhesion assay; F-actin and western blotting assay had identified PKCzI257.3 can impair the cofilin activation and filamentous actin polymerization.Conclusion:1.Micromolecular compound PKCzI257.3 exhibites a specific inhibition of the PKCζactivity,with neglectable cytotoxicity in MDA-MB-231 cell;a series of cell functional assays have indicated that:PKCzI257.3 effectively inhibites chemotaxis and migration,and even cell attachment,presumably through preventing PKCζdown-stream effector cofilin from activation and actin polymerization.2.Primary analysis has suggested that PKCzI257.3 as a PKCζ-specific inhibitor, might worth to be further characterized or tested as potential therapeutic reagent against cancer metastasis.