5-fluorouracil: Mechanisms of action and clinical strategies. of histone H3 lysine 9 acetylation (H3K9ac) and reduced AKT phosphorylation (Ser473). Conclusion: Our data revealed, for the first time, the enhanced inhibitory effect of CUDC-907 against CRC cells when combined with 5-FU, supporting the application of this combination as a potential therapeutic strategy in CRC treatment. experiments and animal model studies have shown that DNA methyltransferase and HDAC inhibitors exhibit anti-tumor activities in CRC. Thus far, seven classes of HDACis have been developed, with four of them, i.e., short-chain fatty acids, cyclic peptides, hydroxamic acids, and benzamides, currently being investigated in the clinic. CUDC-907 is a small-molecule, dual inhibitor of HDAC, and phosphatidylinositide 3-kinase (PI3K) that is currently in Phase 1 clinical testing for the treatment of patients with lymphoma and multiple myeloma. It has been shown to inhibit class I and class II HDAC enzymes as well as suppress the PI3K-AKT-mTOR pathway in solid tumors, in addition to ABT333 inducing apoptosis and inhibiting cancer cell proliferation in xenograft tumors. The aim of the current study is to investigate the effect of CUDC-907 as a single agent and in combination with 5-FU against CRC at the cellular and molecular levels. MATERIALS AND METHODS Cell culture and viability HCT116 human colorectal cell line was obtained from ATCC (Manassas, VA, USA) and was subsequently Mouse monoclonal to MER authenticated by Genetica DNA Laboratories, Inc. (Burlington, NC, USA). The RKO cell line was obtained from ATCC, while the HT-29 and COLO-205 cell lines were obtained from CLS Cell Lines Service (CLS Cell Lines Service GmbH, Eppelheim, Germany). Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 4500 mg/L D-glucose, 4 mM L-glutamine, 110 mg/L sodium pyruvate, 10% fetal bovine serum, 1 penicillinCstreptomycin (PenCStrep), and non-essential amino acids (all purchased from Gibco-Invitrogen, Waltham, MA, USA). For cell viability assays, 1 104 cells were seeded in 96-well flat bottom plates and incubated for 24 h. Thereafter, cells were treated with the indicated dose of 5-FU (Sigma, St. Louis, MO, USA), CUDC-907 (Selleckchem Inc., Houston, TX, USA), or combination of both CUDC-907 and 5-FU at the same concentration. AlamarBlue assay (BUF012B; AbD Serotec, Kidlington, UK) was used to measure cell viability, as we previously described. Briefly, cells under different treatment conditions were incubated with 10 L (10% of total volume) of AlamarBlue substrate in the dark at 37C for 60 min. Subsequently, plate readings were taken using the fluorescent mode (ex 530 nm/em 590 nm) with a BioTek Synergy II microplate reader (BioTek Inc., Winooski, VT, USA). Immunoblotting HCT116 cells were treated with 10 nM CUDC-907, and 48 h later, cells were lysed using RIPA buffer (Norgen-Biotek Corp., Thorold, Canada) supplemented with 1 Halt protease inhibitor cocktail (Pierce Inc., Rockford, IL, USA). Twenty micrograms of total protein were isolated and blotted using the Bio-Rad V3 Western work flow system, as previously described.[10,11] Immunoblotting was performed against acetyl-histone H3 (Lys9, C5B11) rabbit mAb (1:1000 dilution, #9649; Cell Signaling Technology, Danvers, MA, USA) and phospho-Akt (Ser473, D9E) XP rabbit mAb (1:2000 dilution, #4060; Cell Signaling Technology). The membrane was subsequently incubated with anti-rabbit IgG-horseradish peroxidase (HRP) conjugated antibody (1:3000 dilution, #7074p2; Cell Signaling Technology), and probed with mouse anti-human -actin antibody (1:1000 dilution; GenWay Biotech, Inc., San Diego, CA, USA) followed by secondary anti-mouse IgG-HRP conjugated antibody (1:2500 dilution; GE Healthcare, Buckinghamshire, UK). For phospho-Akt (Ser473) detection, cells were starved for 4 h and incubated with insulin ABT333 (20 mg/mL) for 30 min in order to activate the PI3K pathway in the absence or presence of CUDC-907 before cell pellet collection. Imaging was performed using the ChemiDoc?MP imager (Bio-Rad Laboratories, Hercules, CA, USA). Flow cytometry (Cell cycle) HCT116 cells were treated with dimethyl sulfoxide (DMSO) as control, 5-FU, CUDC-907, or combination of 5-FU and CUDC-907. On day 5, cell pellets were collected and washed ABT333 with phosphate-buffered saline (PBS), and then were resuspended in 1 ml of FACS buffer (PBS/0.5% BSA), and then 3 ml of ice-cold 70% ethanol was added ABT333 to fix the cells for 1 h on ice. Cell pellets.