Inhibition Of Phospholipase C And Protein Kinase C By Alkylphosphocholines

Hexadecylphosphocholine and a series of analogs were investigated with regard to their antiproliferative activity, their interference with phosphatidylinositol-specific phospholipase C (PI-PLC) and their interaction with protein kinase C (PKC). The compounds investigated included hexadecylphosphocholine, octadecyl-2-(N-methyl-piperidino)-ethyl-phosphate (D-20133), octadecyl-(1,1-dimethyl-piperidino-4-yl)-phosphate (D-21266), octadecyl-2-(trimethyl-arsonio)-ethyl-phosphate (D-21805), hexadecylphospho-L-serine and hexadecyl-phosphono-L-serine. The agents inhibit (3)H-thymidine incorporation into DNA of NIH3T3 cells with IC(50) values ranging from 3-6 mu M. All compounds reduce PI-PLC catalyzed generation of inositol-1,4, 5-trisphosphate (IP(3)) with IC(50) values between 2 and 4 mu M. PI-PLC beta(1) and PI-PLC gamma(1) catalyzed IP(3) formation was found to be equally affected. The reduction of IF, formation results in a concomitant depression of the growth factor-induced increase in cytosolic free Ca(2+). Since PI-PLC in addition to IP(3) generates diacylglycerol (DAG), the functional consequences of an inhibition of DAG formation were addressed. DAG is known to act as a cofactor for c-and n-type PKC isoforms. An inhibition of DAG formation should, therefore, interfere with the mitogen-induced activation of DAG-responsive PKC isozymes. Evidence for an inhibition of thrombin-induced intracellular activation of a DAG-responsive PKC is presented. The intracellular biochemical function of BAG-responsive PKC isoforms and the biological consequences of their inhibition were unknown. The NIH3T3 cells express cPKC alpha and nPKC epsilon. By employing dominant negative and constitutively active mutants of these PKC isoforms, it could be demonstrated that the alpha and epsilon isoforms phosphorylate the myristoylated alanine rich C kinase substrate (MARCKS) in vivo. As the nonphosphorylated MARCKS complexes calmodulin, an inhibitor of PKC alpha and epsilon should reduce the levels of free cytosolic calmodulin and thereby enhance effects due to the depression of the Ca(2+) signal. PKC epsilon was found to be required for the Ras-mediated transcriptional activation of the fos promoter. Thus, an interference with the activation of PKC epsilon should also contribute to the growth-inhibitory effect of alkylphosphocholines. Many alkylphosphocholines were found to inhibit PKC in cell-free extracts and to interfere with phorbol eater-mediated cellular functions. This inhibition was found to be due to a competition with phosphatidylserine, a biological cofactor of PKC. However, hexadecylphosphoserine and hexadecylphosphonoserine, which do not act as PKC inhibitors, are equally efficient with regard to their antiproliferative activity as alkylphosphocholines which exert a PKC inhibition. It was concluded, therefore, that a direct inhibition of PKC is not required for the antiproliferative activity of these compounds. All growth-inhibitory alkylphosphocholines and analogs studied so Car, however, inhibit PI-PLC at growth-inhibitory concentrations. These findings strongly suggest that the antitumor activity of these agents is correlated to their effect on PI-PLC and the resulting blockade in mitogenic signaling. (C) 1998 Prous Science. All rights reserved.