Combination of immunoprecipitation (IP)-ATP_Glo kinase assay and melanogenesis for the assessment of potent and safe PAK1-blockers in cell culture.

摘要: Cucurbitacin I (CBI) is a triterpene from bitter melon called Goya grown in Okinawa, Japan, and directly inhibits both the Tyr-kinase JAK2 G protein RAC, leading to inactivation of PAK1 (RAC/CDC42-activated kinase 1). Bio 30, propolis produced New Zealand, contains CAPE (caffeic acid phenethyl ester) as major anti-cancer ingredient which down-regulates PAK1. Since essential for growth RAS cancer cells such A549 cell line carry an oncogenic K-RAS mutant, melanogenesis skin cells, here using these PAK1-blockers model compounds, we introduce new approach quick assessment culture. First, combining immuno-precipitation (IP) lysate vitro ATP_Glo assay kit (called "Macaroni-Western" assay), confirmed that CBI 30 inactivate lung 24 h, inhibit their PAK1-dependent 72 h. Furthermore, verified PAK1/PAK4-dependent melanoma by far more than 50%, while only with merginal effect on per se. are rather simple quick, combination two culture assays would be highly useful selecting "potent" (highly cell-permeable) "safe" (non-toxic) natural or synthetic PAK1-blockers.