Isolation and Characterization of Inhibitor against PDGF-Induced Phosphatidylinositol Turnover from Streptomyces sp. MT 1882-5

Abstract

Signal transduction pathways are the attractive targets for the discovery of anticancer agents because the investigations can focus on the sites of the fundamental impediment leading to uncontrolled cell growth. Phosphatidylinositol(PI) turnover is a major mechanism of signal transduction in mammalian cells and is considered to be implicated in the regulation of cell proliferation. So, inhibitors of PDGF-induced PI turnover in fibroblast NIH 3T3γ1 cell were screened from metabolites of actinomycetes. As a result, isolate MT 1882-5 was selected as the producer of an inhibitor because of its strong and reproducible inhibitory activity. To identify the isolate, the morphological characteristics, the analysis of components in cell wall hydrolysate, and the electron microscopic observation were examined. The color of mature aerial mycelium was in the gray series and spore morphology was round, spiny and spiral. The compositions of diaminopimelic acid(DAP) isomers and amino acids in cell wall hydrolysate were LL-DAP, alanine, glutamic acid and glycine. Therefore the strain was identified as Streptomyces sp. and designated Streptomyces sp. MT 1882-5. An inhibitor of PI turnover from a culture broth of Streptomyces sp. MT 1882-5 was purified as yellowish amorphous powder through ethylacetate extraction and several chromatographic methods such as Amberlite XAD-2, silica gel, Sephadex LH-20 and Lobar RP-18 column chromatography, and designated MT 1882-Ⅱ. From the spectroscopic data of UV, NMR and FAB mass, the purified inhibitor, MT 1882-Ⅱ, was identified as glucopiericidin A which substituted a glucose moiety in the C-10 position of piericidin A1. MT 1882-Ⅱ inhibited PDGF-induced PI turnover in NIH 3T3γ1 cell with an IC50 value, 5㎍/㎖, but did not inhibit EGF-induced PI turnover in A431 cell. It did not inhibit in vitro phospholipase Cγ1 (PLCγ1) activity involved in PI turnover. Inhibition of MT 1882-Ⅱ on PDGF-induced PI turnover in NIH 3T3γ1 cell results from reducin...