Cell Viability Tests Using Cancer Cell Lines. The colorectal
adenocarcinoma cell line (CACO2, ATTC HTB-37) and breast cancer cell line (MCF-7, ATTC HTB-22) were cultured in Dulbeccoâ€™s modified Eagle medium (DMEM; Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco), penicillin (100 UÂ·mLâˆ’1) (Sigma-Aldrich), and streptomycin (100 mMÂ·mLâˆ’1) (SigmaAldrich) and maintained at 37 Â°C in an atmosphere of 5% CO2. Cell cultures were maintained as described previously.48 After reaching 80% confluence, the cells were removed with the aid of a plastic carrier(TPP). The cell concentration was adjusted to 1 Ã— 105 cellsÂ·mLâˆ’1
.Cells were then incubated in a 96-well (TPP) plate along with cyclotides at concentrations of 0.33, 0.65, 1.3, 2.6, 5.25, and 10.5 Î¼M.The fibroblast cell line L929 was used as control and grown under the same conditions as the cancer cell lines. For each concentration of the peptide, three replicates were performed, and the procedure was repeated three times. The MTT assay (Sigma-Aldrich) was used for cell viability analysis, with readings taken at 48 h. For this assay, 155Î¼L of culture medium was removed and added to 10 Î¼L of MTT; this was then incubated for 3 h at 37 Â°C in the dark. Following incubation,60 Î¼L of DMSO (J.T. Baker) was added to each well to dilute the formazan crystals. The absorbance was determined using a microplate reader at 575 nm. Cell viability was expressed as a percentage compared to the untreated negative control cells and the positive control cells, which were treated with the lysis solution [10 mM TrisHCl, pH 7.4; 1 mM EDTA, 0.1% Triton X-100 (J.T. Baker)].