title: Combination of immunoprecipitation (IP)-ATP_Glo kinase assay and melanogenesis for the assessment of potent and safe PAK1-blockers in cell culture.
abstract: Cucurbitacin I (CBI) is a triterpene from a bitter melon called Goya grown in Okinawa, Japan, and directly inhibits both the Tyr-kinase JAK2 and the G protein RAC, leading to the inactivation of PAK1 (RAC/CDC42-activated kinase 1). Bio 30, a propolis produced in New Zealand, contains CAPE (caffeic acid phenethyl ester) as the major anti-cancer ingredient which directly down-regulates RAC, leading to the inactivation of PAK1. Since PAK1 is essential for the growth of RAS cancer cells such as A549 cell line which carry an oncogenic K-RAS mutant, and the melanogenesis in skin cells, here using these PAK1-blockers as model compounds, we introduce a new approach to the quick assessment of PAK1-blockers in cell culture. First, combining the immuno-precipitation (IP) of PAK1 from cell lysate and the in vitro ATP_Glo kinase assay kit (called "Macaroni-Western" assay), we confirmed that both CBI and Bio 30 inactivate PAK1 in A549 lung cancer cells in 24 h, and inhibit their PAK1-dependent growth in 72 h. Furthermore, we verified that CBI inhibits the PAK1/PAK4-dependent melanogenesis in melanoma cells by far more than 50%, while Bio 30 inhibits the melanogenesis only by 50%, with only a merginal effect on their growth per se. Since the "Macaroni-Western" kinase assay and melanogenesis are both rather simple and quick, the combination of these two cell culture assays would be highly useful for selecting both "potent" (highly cell-permeable) and "safe" (non-toxic) natural or synthetic PAK1-blockers.
authors.affiliation: PAK Research Center.
authors.forename: Binh Cao Quan
authors.initials: BC
authors.lastname: Nguyen
authors.forename: Pham Thi
authors.initials: PT
authors.lastname: Be Tu
authors.forename: Shinkichi
authors.initials: S
authors.lastname: Tawata
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Maruta
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002109:Caffeic Acids / Q000494:pharmacology; D045744:Cell Line, Tumor; D006801:Humans; D047468:Immunoprecipitation; D008543:Melanins / Q000096:biosynthesis*; D051379:Mice; D010626:Phenylethyl Alcohol / Q000031:analogs & derivatives / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011933:Reagent Kits, Diagnostic; D014315:Triterpenes / Q000494:pharmacology; D054462:p21-Activated Kinases / Q000037:antagonists & inhibitors*
chemical_list: D000970:Antineoplastic Agents; D002109:Caffeic Acids; D008543:Melanins; D047428:Protein Kinase Inhibitors; D011933:Reagent Kits, Diagnostic; D014315:Triterpenes; C515903:PAK1 protein, human; D054462:p21-Activated Kinases; C055494:caffeic acid phenethyl ester; D010626:Phenylethyl Alcohol; C038106:cucurbitacin I