Cell cytotoxicity of cyclotides where studied using the MTT assay perfrmed on two cancer cell lines MCF-7 and MCF-7/ADR. Cells (1 x 104 per 100 μμDMEM media per well) were seeded in 96-well flat-bottom microtiter plates and incubated (37C, 5.0 CO2) for 4 h prior to treatments (Cy02, psyle A, C, E and doxorubicin) with the following concentrations: 10, 5, 2, 1, 0.5, 0.2 μM in 100 μl of media. After 72 h incubation, 100 μl of media was removed from each well, 10 μl of MTT (5 mg/ml in PBS) was added to each well in the absence of light, and formazen crystals were produced over a 4 h incubation period. To dissolve crystals, 150 μl of 0.04N HCl in iso-propanol was added to each well and optical density measured using a Maltiskan Plus Spectrophotometer reading at 540 nm. For coexposure experiments, the same protocol was performed but concentrations modified to 50:50 ratios of each cyclotide (CyO2 and psyle A, C, E) and doxorubicin at the following concentrations: 5, 2.5, 1, 0.5, 0.25 and 0.1 μM. Each test was performed with five replicates and repeated in triplicate. Replicates were averaged and the blank subtracted from control and test concentrations. Optical density of treated wells was compared with controls (100% survival) and percent cell survival calculated and plotted.
SYTOX Green assays were performed to test membrane permeabilization.
The MCF-7 and MCF-7/ADR cells (1 3 105 per 200 μμDMEM media per well) were seeded into 96-well flat-bottom microtiter plates and treated with 5 μM of cyclotide, doxorubicin, or the positive control (mellitin) and 0.04 μM SYTOX in PBS for 30 min. Fluorescence measurements (485 nm excitation, 530 nm emission wavelengths and medium shaking every 0.2 s) were taken at 1 min intervals on a MicroPlate Reader using the Tungsten light source and recorded in the Gen5 System Software. Each test was performed in triplicate and repeated three times. The fluorescence (average fluorescence of test wells relative to control wells minus the blank) was normalized to mellitin which displays 100% membrane permeabilization, and then percent leakage was calculated.