Associated with Inhibition of PI3K/AKT/FOXO Pathway Pancreatic Tumor Suppression by Benzyl Isothiocyanate Is

Abstract Purpose:Ourpreviousstudieshaveshownthatbenzylisothiocyanate(BITC)suppresspancreaticcancergrowthbyinducingapoptosisbutthemolecularmechanismwasunclear.Inthisstudywehypothesizedtheinvolvement of PI3K/AKT/FOXO pathway in BITC-induced apoptosis.ExperimentalDesign:MicewereimplantedBxPC-3tumorxenograftsandorallygavagedwith12mmolBITC. Plasma and tumor BITC concentration was estimated by liquid chromatography/tandem massspectrometry. BxPC-3 and PanC-1 cells were used to elucidate PI3K/AKT/FOXO pathway. Electrophoreticmobility shift assay (EMSA), DNA binding activity, immunofluorescence, and gene transfection were usedto delineate the mechanism.Results: BITC-treated mice showed 43% less tumor growth as compared with control mice andcorrelated well with the therapeutic concentrations of 6.5 mmol/L BITC achieved in plasma and 7.5mmol/gBITCintumortissue.WesternblotanalysesandimmunohistochemistryrevealedthattumorsfromBITC-treated mice showed reduced phosphorylation of PI3K, AKT, PDK1, mTOR, FOXO1, and FOXO3aand increased apoptosis. Complementing our in vivo results, we made similar observations in a dose- andtime-dependent manner in BITC-treated BxPC-3 and Panc-1 cells. Binding of FOXO1 with 14-3-3 proteinswasalsoreduceddrasticallybyBITCtreatmentindicatingnuclearretentionofFOXO1andthisobservationwas further confirmed with EMSA, immunofluorescence, DNA binding, and upregulation of FOXO-responsive proteins Bim, p27, and p21 in BxPC-3 cells. Overexpression of AKT by transient transfectionsignificantly blocked the modulation of FOXO proteins and protected the cells from BITC-mediatedapoptosis and growth suppression.Conclusions:OurresultsprovideconvincingevidenceontheinvolvementofPI3K/AKT/FOXOpathwayin BITC-mediated pancreatic tumor growth suppression.