Several genetic and biochemical studies indicate that the FoxO family is a key downstream target of the PI3K-Akt pathway in development and longevity (Lin et al, Tofacitinib Citrate buy 1997; Brunet et al, 1999). Thus, phosphorylation of FoxO factors in specific serine and/or threonine sites modulates their subcellular localisation (Rena et al, 2002; Barthel et al, 2005; Anton et al, 2007). Once placed in the nucleus, they play tumour suppressor roles through enhanced transcription of proapoptotic genes, such as BCL6, a Bcl-2-interacting mediator of cell death (Bim), and Fas ligand (Dijkers et al, 2000; Yang et al, 2006). Bim is a proapoptotic member of the Bcl-2 family, and is one of the main downstream targets of FoxO3a. After transcription, Bim mRNA undergoes an alternate splicing, giving three isoforms (BimS, BimL and BimEL) with different length (Ewings et al, 2007).

Interestingly, there are in vivo and in vitro evidence demonstrating an essential role of Bim proteins in Bax activation (Ren et al, 2010). Based on this information, we focused this study on the FoxO3a regulation of Bim expression after treatment with pharmacological concentrations of melatonin, in an attempt to gain further mechanistic insights on the molecular pathways leading to melatonin-induced apoptosis in HepG2 liver cancer cells. Materials and methods Cell culture HepG2 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and grown in Dulbecco’s modified Eagle’s medium (DMEM). Primary human hepatocytes were isolated from healthy liver tissue of patients undergoing partial hepatectomy by two-step collagenase perfusion.

Cells were seeded on collagen-coated culture dishes in Williams medium supplemented with 10% fetal bovine serum, 15mmoll?1 HEPES (pH 7.4), 2mmoll?1 glutamine, 100Uml?1 penicillin and 100��gml?1 streptomycin. LY294002 was from Tocris (Bristol, UK). Melatonin and epidermal growth factor (EGF) were obtained from Sigma-Aldrich (St Louis, MO, USA). Viability assays HepG2 cells or primary human hepatocyte were seeded in 96-well plates. Melatonin dissolved in dimethyl sulphoxide (DMSO) was added to the cells at the concentrations as indicated in the figures. Apoptosis was induced in HepG2 cells with 200ngml?1 of the monoclonal antibody (Ab) to human (APO-1/Fas) anti-APO-1, kindly provided by Peter H Krammer.

Cell viability was determined using the CellTiter-Glo (Promega, Fitchburg, WI, USA) and 3-(4,5-Dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays (Sigma-Aldrich). CellTiter-Glo assay was performed according to Batimastat the manufacturer’s instructions (Promega). Luminescence was determined in a Saphire luminometer (Tecan Austria, Gr?dig, Austria). The MTT assay was carried out as described by Denizot and Lang (1986). Briefly, after exposure of cells to melatonin, culture media were changed by serum-free culture media.