The human cancer cell lines used were RPMI-8226/s (myeloma) and U-937 GTB (lymphoma), procured and maintained as described earlier. Cell-growth medium was prepared from RPMI-1640 stock, supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mM glutamine, 100 ug/mL streptomycin, and 100 U/mL penicillin. Fractions of V. tricolor and purified cyclotides were dissolved in 10% EtOH (equal to a final concentration of EtOH of 1% in the assay). V-shaped 96-well microtiter plates (Nunc, Roskilde, Denmark) were prepared with 20 uL per well of test solution in triplicate for each concentration. Also, six solvent-control wells (20 uL per well of 10% EtOH), six blank wells (200 uL per well of cell-growth medium), six positive-control wells (20 uL per well of 10% Triton X-100), and six negative-control wells (20 uL per well of phosphate-buffered saline solution, PBS) were prepared on each microtiter plate. All experiments were repeated once. Initial cell viability was assessed by the trypan blue dye exclusion test. The tumor cells suspended in cell-growth medium were dispensed on the prepared microtiter plates (20 000 cells/180 uL per well) and incubated at 37 C and 5% CO2. After 72 h of incubation, the cells were washed with PBS, and 100 uL of fluorescein diacetate (10 g/mL in a physiological buffer) was added to each well. The plates were incubated at 37 C and 5% CO2 for 40 min, and the generated fluorescence was measured in a 96-well scanning fluorometer, exciting fluorescence at 485 nm and sequentially reading at 538 nm.10 The fluorescence (as measured) is proportional to the number of living cells, and thus cell survival can be quantified as a survival index (SI), defined (in units of percent) as the fluorescence of the test wells relative to the average fluorescence for control wells, with average for the blank wells subtracted. IC50 values, which correspond to the 50% survival index, were calculated using nonlinear regression in GraphPad Prism (GraphPad Software, Inc., San Diego). Quality-assessment criteria for a successful experiment included a fluorescence signal in control wells of more than 10 times the average value for blank wells, with an average value for the coefficient of variation in blank and control wells being less than 30%.
The crude fraction of V. tricolor was subjected to fractionation guided by bioactivity, using the fluorometric microculture cytotoxicity assay (FMCA)11 for the human lymphoma cell line U-937 GTB and the human myeloma cell line RPMI-8226/s. The most potent cytotoxic compounds isolated were the three cyclotides described above. After 72 h of treatment, all three showed cytotoxicity in a dose-dependent manner, and their IC50 values were in the micromolar range. The potencies of the three cyclotides are in the range of the clinically used anticancer drug doxorubicin. In the myeloma cell line, vitri A was approximately 3 and 4 times more potent than varv A and varv E, respectively, and in the lymphoma cell line, vitri A turned out to be approximately 10 and 7 times more potent than varv A and varv E, respectively.