The nutraceutical treatment induced death by apoptosis, upregulation of p53 and downregulation of c-myc, pAkt, and Bcl-2. Given the central role of these molecular targets in cell proliferation and death, the potential preventive benefits of CF in human cancers are self-evident. Methods Cell culture Breast (SKRB3), colorectal (HCT116), lung (H1650, H1975), melanoma (M14), mesothelioma (MSTO-211H, NCI-2452, Ist-Mes1, MPP89, Ist-Mes2) cancer cell lines, and fibroblast (HFF) and mesothelio (MeT5A) cell lines were gradually conditioned in DMEM/F12 + Glutamax (Invitrogen

Life Technologies, Paisley, UK) supplemented with 10% FBS and antibiotics and maintained selleck compound at 37°C and 5% CO2. Cellfood CF (liquid) was kindly provided by Eurodream srl (La Spezia, Italy) and stored at room temperature. CF was diluted in phosphate buffered saline (PBS) and sterilized using a 0.45 μm syringe-filter before use. Cell growth assays For cell growth experiments, cells were plated in quintuplicates in 96-well culture plates (Nunc, Milan, Italy) at a density of 3 × 103 cells/well. 24 h later, the medium was replaced with fresh SC79 solubility dmso growth medium containing 1:200, 1:400, 1:800, 1:1600 dilutions of CF. At 24 and 48 h of treatment, XTT labelling reagent (final concentration 0.5 mg/ml) was added to each well, and the samples were incubated for an additional 4 h at

37°C. The XTT assay (Cell proliferation Kit (XTT), Roche Molecular Biochemicals, Indianapolis, IN) is based on the cleavage of the click here yellow tetrazolium salt XTT to form an orange formazan dye by metabolic active Forskolin cells. Absorbance was measured at 492 nm with a reference wavelength at 650 nm and the absorbance values of treated cells were presented as a percentage of the absorbance versus non treated cells (CNTRL). All experiments were repeated three times. The anti-proliferative CF activity was assessed in monolayer cell culture conditions by plating

cell lines in a T25 flask. After 24 h, CF (5 μl per ml of medium corresponding to a 1:200 dilution) was added for the time indicated in the experiments. Nothing else was added in CNTRL. The expansion of cell culture proliferation was quantified by manual cell counting. Experiments were repeated in triplicate and media values were calculated. Clonogenic assay Five hundred viable cells per well (treated with CF and CNTRL) were plated in a 35 mm dish and allowed to grow in normal medium for 10-14 days and then stained for 30 min at room temperature with a 6% glutaraldehyde, 0.5% crystal violet solution. Pictures were captured digitally. All experiments were repeated at a minimum twice for each cell line. Flow cytometry For cell cycle analyses, cells were fixed in 70% ethanol and stored at -20°C over night. Fixed cells were treated with 1 mg/ml RNase A (cat. 12091021, Invitrogen Life Technologies, Paisley, UK) for 1 h at 37°C and DNA was stained with Propidium Iodide (Sigma, St. Louis, MO, USA).