(136) Assay Card

General Information
Summary Parigidin-br3 showed cytotoxic activity against MCF-7 (breast cancer) and CACO2 (colorectal adenocarcinoma) cells, with IC50 values of ∼2.5 μM and less than 10% hemolytic activity.
Condition 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 Dulbecco’s modified Eagle medium (DMEM; Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco), penicillin (100 U·mL−1) (Sigma-Aldrich), and streptomycin (100 mM·mL−1) (SigmaAldrich) and maintained at 37 °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 × 105 cells·mL−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 μM.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, 155μL of culture medium was removed and added to 10 μL of MTT; this was then incubated for 3 h at 37 °C in the dark. Following incubation,60 μL 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)].
Result Activity assays of Parigidin-br3 against CACO2 and MCF-7 cellas evidenced by a 66% loss of CACO2 cell viability at 10 μM and an IC50 value of 2.5 μM, and a 57% loss of MCF-7 cell viability at the same concentration (10 μM) and an IC50 value of 2.5 μM. By comparison, the fibroblast cell line L929 was less affected by Parigidin-br3, with an IC50 > 10 μM.
AssayType Cancer Cell Toxicity

Pinto, M.F., Silva, O.N., Viana, J.C., Porto, W.F., Migliolo, L., B. da Cunha, N., Gomes Jr, N., Fensterseifer, I.C., Colgrave, M.L., Craik, D.J. and Dias, S.C (2016) Characterization of a Bioactive Acyclotide from Palicourea rigida Journal of natural products 79:2767-2773

Proteins Assayed Parigidin-br3