such as benzamides including MS 275, polypeptides including depsipeptide and the hydroxamates which include vorinostat, MK-4827 panabinostat, and belinostat, all of which are currently being evaluated in multiple phase II and III clinical trials . Hydroxamates are so called pan HDAC inhibitors as they inhibit the three classes of zinc dependent HDAC enzymes. Despite intense research in the field of HDAC inhibitors over the past decade, their exact molecular mechanism of action still remains relatively unsolved. HDAC inhibitor treatment induces a global hyperactylation state of the histone proteins and affects a downstream regulation of gene expression of up to 20% of the genome,dependent on cell line and HDAC inhibitor molecule used .
A powerful tool to profile altered cellular factors in response to drug treatment at the protein level is represented by 2 D gel electrophoresis in combination with Rivaroxaban molecular weight MS . In this study, we used this technology to analyse the differentially expressed proteome of the human colon cancer cell line HCT116 induced by treatment with the HDAC inhibitor belinostat. We observed a doseresponse effect of belinostat on cell survival of the HCT116 cell line, and identified 45 differentially expressed proteins by MS many of which are involved in apoptoic and anti apoptoic processes. 2 Materials and methods 2.1 Cell culture and drug treatment The human colon cancer cell line HCT116 was propagated in RPMI 1640 medium supplemented with glutamine, penicillin, streptomycin and 10% fetal bovine serum in a humidified atmosphere with 5% CO2 at 371C.
Belinostat was synthesized as described in patent application , dissolved in sterile water, aliquoted and stored at 201C until use. Increasing concentrations of belinostat were added to the cell cultures DNA-PK hemmer when they approached confluence in 175 cm2 Nunc cell culture easy flasks and incubated for 24 h prior to protein extraction. 2.2 Clonogenic assay In vitro colony forming assays were essentially performed as described previously . Briefly, HCT116 cells were cultured with belinostat for the indicated concentrations and seeded onto 35mm dishes in 3% w/v agar containing a sheep erythrocyte feeder layer. Agar plates were cultured for 1421 days at 371C and colonies counted using a digital colony counter and Sorcerer image analysis software . Data were analysed using GraphPad Prism software . 2.
3 Immunoblotting Bortezomib ic50 Cellular protein was extracted by a cell lysis buffer were separated by SDS PAGE in NuPAGE TM 12% gels and visualized by Coomassie Blue staining or transferred to a nitrocellulose membrane. Membranes were molecule blocked with skimmed milk overnight at 41C, washed thoroughly, and incubated with polyclonal antibodies raised against acetylated lysine residues , gelsolin , nucleolin , and nucleophosmin followed by incubation with biotinylated goat antirabbit IgG or biotinylated rabbit antimouse IgG and avidinhorseradish peroxidase . Detection of acetylated proteins was performed by the horseradish peroxidase catalysed oxidation of 3,30,5,50 tetramethylbenzidine. 2.4 2 D gel electrophoresis Protein samples from untreated HCT116 cells, and cells treated with 1 and 10 mM belinostat were analysed in triplicates by 2 D PAGE 17 cm pH 58 and pH 36 linear IPG strips . Protein samples were chloroform/methanol .