MTT cell viability assay and Lactate dehydrogenase leakage assay

Each of the five cancer cell lines — non-small cell lung cancer H157, melanocyte MDA-MB-435S, human prostate carcinoma PC-3, human glioblastoma astrocytoma U251MG, human breast cancer MCF-7 — as well as the cell line for normal human microvessel endothelial cells HMEC-1 were seeded into a 96-well plate at densities of 5000 cells/well. After incubation for 24 h at 37 °C with 5% CO₂, the cells were starved for 6 h by replacing the medium with serum-free medium. Thereafter, synthesised peptides (in 10-fold concentrations from 10⁻⁴ to 10⁻⁹ M in serum-free medium) were incubated with the cells for 24 h after which 10 μl of MTT solution (5 mg/ml) was added to each well under dark conditions. Following a further 4 h of incubation, 100 μl of DMSO superseded medium was replaced to each well to dissolve the formazan crystals. The OD value of each well was read by the Synergy HT plate reader at 550 nm.

The cell (PC-3 cells) loss of intracellular LDH was measured via LDH Cytotoxicity Assay Kit (Thermo Fisher Scientific). Equivalent cells treated with sterile water or lysis buffer (supplied with the kit) for 45 min served as the spontaneous and maximum LDH activity controls, respectively. Then, 50 μl of supernatant was collected from each well and mixed with equal volume of supplied reaction mixture and incubated at room temperature for 30 min. Fifty microlitre of supplied stop solution was added to each well. The absorbance at 680 nm was measured using Synergy HT plate reader and subtracted from the 490 nm absorbance. Similarly, to obtain the kinetics of cytoplasmic LDH release, 50 μl of supernatant from equivalent PC-3 cells treated with 100 μM, 10 μM and 1 μM of R2PLx peptide, respectively, at different time points (6, 12, 24 and 36 h), was collected and the changes in the release of cytoplasmic LDH were monitored.