Cycloviolacin O2 showed antitumor effects are minor or absent at tolerable (sublethal) doses, and cycloviolacin O2 has a
very abrupt in vivo toxicity profile, with lethality after
single injection at 2 mg/kg, but no signs of discomfort to
the animals at 1.5 mg/kg. Repeated dosing of 1 mg/kg gave a local-inflammatory reaction at the site of injection after 2â€“3 days; lower doses were without complications.
Human Cancer Cell-Line Assay In Vitro
The cyclotide was initially tested in vitro using the FMCA.20 Cycloviolacin O2 was dissolved in 10% ethanol (equal to a final concentration of ethanol of 1% in the assay) and tested at series of concentrations obtained by twofold dilutions. Microtiter plates (Nunc, Roskilde, Denmark) were prepared with test solution in triplicates for each concentration. Also, six solvent-control wells (10% ethanol), six blank wells (medium), and six negativecontrol wells (phosphate-buffered saline solution) were prepared
on each microtiter plate. FMCA experiments were performed
Toxicity In Vivo
Eight animals (male NMRI) received a single intravenous (i.v.) injection of different doses of cycloviolacin O2 (0.5â€“2.0 mg/kg).
Hollow Fiber Assay In Vitro and In Vivo
Cycloviolacin O2 was dissolved in 10% ethanol (equal to a final concentration of ethanol of 0.1% in the assay) and tested at five concentrations obtained by 10-fold dilutions using 15 lM as the maximum concentration in the assay. The hollow fibers, encapsulating the cell lines, were exposed to cycloviolacin O2 in six-well plates containing 3 ml supplemented medium cyclotide solution at 378C and 5% CO2 for 72 h.
Animals receiving repeated injections of 0.5 mg/kg of cycloviolacin O2 showed no signs of discomfort, gained
weight like the controls (Table II), and presented with normal hematology (Hb, RBC, Hct, WBC, and Trc) at the end of
the study. However, there was a nonsignificant trend for the
suppression of red blood cell counts (8.2Â±0.6 X 10^12/l in
controls vs. 7.6Â±0.7 in low dose vs. 7.2Â±0.4 in high-dosegroup), hemoglobin (137Â±9 vs. 131Â±9 vs. 127Â±4 g/l),hematocrite (0.40Â±0.03 vs. 0.39Â±0.03 vs. 0.37Â±0.02),and thrombocytes (967Â±173 vs. 917Â±111 vs. 856Â±234 X 10^9/l) in animals treated with cycloviolacin O2. No trend was observed for white blood cell counts (5.0Â±1.8 vs. 4.9Â±2.5 vs. 5.9Â±0.7 X 10^9/l).
Cycloviolacin O2 exerted a concentration-dependent toxicity toward the lymphoma cell line U-937 GTB, the leukemia cell-line CCRF-CEM, the small cell lung cancer cell-line NCI-H69, and the colon carcinoma cell-line HT29.
Cycloviolacin O2 exhibited a concentration-dependent cytotoxicity in CCRF-CEM, U-937 GTB and NCI-H69 cell lines.
Cancer Cell Toxicity