Cj9111 On The Mechanism Of The Inhibitory Protein Kinase C And Protein Kinase C As A Target For Anti-transfer Experiments

Protein kinase C (PKC) plays an important role in cell—signal transduction during cellular growth and differentiation as well as in tumor promotion,oncogene activation and carcinogenesis. So, Using PKC as the target enzyme,we have isolated a PKC inhibitor CJ9111, a natural product,from traditional Chinese medical herb Caulis Spatholobi by column chromatography with gradient elution.In the further studies,CJ9111 was isolated and purified as a single component by preparative HPLC. Determined by HLPC analysis the purity of CJ9111, thus obtained,is over 99%.The mechanism of PKC inhibition by CJ9111 was thoroughly studied. We tested the effect of CJ9111 on the metastasis of murine melanoma B16 cells, and demonstrated preliminarily that there were some relation between PKC activity and the expression of adhesive receptors on cell membraneand tne expression of both oncogenes and anti—oncogenes.The effects of CJ9111 on protein kinase C and cAMP—dependent kinase activities were estimated by enzyme preparation, It was shown that CJ9111 inhibited both the activities of PKC and PKA,and the inhibitions of PKC and PKA were dose—dependent. and the IC50 were 3.54ug/ml(P<0.05) and 20.69ug/ml(P<0.05) respectively.The well known inhibitors, Staurosporine and H-7 ,have 50% inhibitory doseat 2.8ng/ml,0.12mg/ml for PKC and 4.4ng/ml,0.30mg/ml for PKA respectively.This indicates that CJ9111 is a potent and relatively selective inhibitor of PKC. The inhibition of PKC was not overcome by adding an excess amount of phospholipid or Ca²⁺, These meant that CJ9111 do not interact with binding site of phospholipid or Ca²⁺in the regulatory domain. Furthermore, CJ9111 was found to inhibit the activation of PKC by PdBu, and the inhibition is dose—dependent, indicating that CJ9111 competed with Phorbol ester (also DAG) for the binding site in the regulatory domain of the enzyme.Treatment of B16 murine melanoma cells with CJ9111 for 24h in culture and subsequent i.v. injection of the tumor cells into C57BL / 6...