(95) Assay Card

General Information
Summary Cell viability and morphology studies on the human lymphoma cell line U-937 GTB showed that cells exposed to cycloviolacin O2 displayed disintegrated cell membranes within 5 min.
Condition The human lymphoma cell line U-937 GTB was used. Cytotoxicity was measured using the FMCA. Initial cell viability was assessed using the trypan blue dye exclusion test. Cancer cells suspended in cell-growth medium were incubated at several time points and then washed with PBS and fluorescein diacetate was added. The plates were incubated for 40 min, and fluorescence was then measured at 538 nm, following excitation at 485 nm. For microscopy work, slides were stained according to the May-Gr├╝nwald-Giemsa (MGG) method and then air-dried. A quantitative measure of the gross morphology was obtained by classifying the cells as necrotic or apoptotic.
Result Cycloviolacin achieves its full effect within 4 h, but 4 h is the shortest possible end-point in the FMCA. From microscopy studies, cycloviolacin O2 rapidly induced cell death, with disrupted membranes observed already after 5 min and more than 60% of the cells were dead after 1 h. Both gross morphology and the rapid onset indicated that necrosis rather than apoptosis is the mechanism of action.
AssayType Cancer Cell Toxicity

References
Svangard E, Burman R, Gunasekera S, Lovborg H, Gullbo J, Goransson U (2007) Mechanism of action of cytotoxic cyclotides: cycloviolacin O2 disrupts lipid membranes. J Nat Prod 70:643-647

Cross-references
Proteins Assayed cycloviolacin O2