Cyclotide-containing fractions from SPE(eluted at 20 and 30% acetonitrile in 1% formic acid) and purified cyclotides (Rivi1âˆ’4) from R. virgata were used to determine cytotoxicity against MDA-MB-231 (breast cancer), MM96L (melanoma), HT-29 (colon cancer), and HFF-1 (human foreskin fibroblast) cells using an MTT assay (Sigma). Purified cyclotides Rivi1âˆ’4 were also tested against human umbilical vein endothelia cells (HUVECs).All cells were plated in 96-well plates at 3 Ã— 103 cells/well (100 Î¼L) in different media, including 10% FBS/EBM-2 media supplemented with SingleQuots (which includes growth factors, cytokines, and antibiotics; Lonza) for HUVECs, 15% FBS/DMEM (Dulbeccoâ€™s modified Eagle medium; Gibco) for HFF-1 cells,10% FBS/DMEM for HT-29 and MDA-MB-231 cells, and 10% FBS/RPMI with 2 mM L-glutamine and 1 mM sodium pyruvate (Gibco) for MM96L cells. Cells were incubated at 37 Â°C in 5% CO2 for 24 h
prior to the assay. Medium was first removed and replaced with fresh serum-free EBM-2 and DMEM media (100 Î¼L wellâˆ’1
) followed by the addition of 10 Î¼L of fraction from SPE at concentrations ranging from 0.75 to 100 Î¼g/mL with the exception of HT-29, which was used from 0.35 to 50 Î¼g/mL, and Rivi 1âˆ’4, which was used from 0.075 to 10 Î¼M. The plate was incubated for 2 h before a 10 Î¼L aliquot of MTT (5 mg mLâˆ’1 in phosphate buffered saline [PBS]) was added to each well and incubated for a further 3 h. The supernatant
was removed, and the formazan crystals were dissolved in 100 Î¼L of dimethyl sulfoxide (DMSO). The plate was measured at 600 nm using a BioTek PowerWave XS spectrophotometer. Paclitaxel (Sigma) was used as a positive control at concentrations ranging from 0.00075 to 10 Î¼M. Paclitaxel was incubated with cells for either 24 or 48 h.Peptides and paclitaxel were tested in triplicate, and the data were analyzed using the statistical software package GraphPad Prism to obtain IC50 values from the sigmoidal concentrationâˆ’response curves.
Rivi1âˆ’3 were prepared at concentrations ranging from 0.003 to 10 Î¼M. Melittin (0.15âˆ’20 Î¼M; Sigma) was included as a positive control to determine toxicity to human red blood cells according to the method described by Chan et al.
IC50 of Rivi1 against HUVEC, HFF-1, MM96L, MDA-MB-231, HT-29 and RBC cells is >>3Î¼M, >>3Î¼M, >>3Î¼M, >>10Î¼M, >>10Î¼M, >>10Î¼M.
IC50 of Rivi2 against HUVEC, HFF-1, MM96L, MDA-MB-231, HT-29 and RBC cells is 1.6Â±0.02Î¼M, 2.1Â±0.02Î¼M, 1.3Â±0.01Î¼M, 0.95Â±0.10Î¼M, 5.9Â±0.04Î¼M,>>10Î¼M.
IC50 of Rivi3 against HUVEC, HFF-1, MM96L, MDA-MB-231, HT-29 and RBC cells is 2.6Â±0.06Î¼M, 1.9Â±0.02Î¼M, 1.1Â±0.05Î¼M, 2.3Â±0.01Î¼M, ND, >>10Î¼M.
IC50 of Rivi4 against HUVEC, HFF-1, MM96L, MDA-MB-231, HT-29 and RBC cells is 2.8Â±0.02Î¼M, 1.7Â±0.01Î¼M, 0.93Â±0.02Î¼M, 4.4Â±0.04Î¼M, ND, ND.