Determining the Effect of a CBC-based Oxime on Cell Viability and Apoptosis in Sea Urchin Embryos

Natural products are an important source of existing and potential new anti-cancer drugs. We have previously shown that cannabichromene (CBC) oxime and several CBC oxime esters have potent anti-mitotic activity when used in bioassays with sea urchin embryos. In this study we explored the effect of the CBC oxime on cell viability and apoptosis in sea urchin embryos. Newly fertilized or gastrula stage embryos were combined with 10µM or 50uM CBC oxime as well as a DMSO only control. At various timepoints, samples of the embryos were removed from the cultures and stained with three different fluorescent dyes. NucBlue (ThermoFisher) was used to determine cell number, apoptotic effects were observed with CellEvent caspace3/7 (ThermoFisher), and cell viability was determined with Live-or-Dye (Biotium). As expected, there was a clear effect of the CBC oxime on the rate of cell division. The embryos exposed to both 10uM and 50uM CBC oxime showed a reduction in cell division compared to the controls, with an almost total inhibition in the higher concentration. Cell viability assays using the vital stain Live-or-Dye showed a concentration dependent effect of the CBC oxime on cell viability, with levels of cell death reaching almost 80% in the 50uM concentration by 3 hours after drug addition. Finally, staining with CellEvent caspace3/7, which indicates the presence of active caspase activity, demonstrated concentration dependent induction of apoptosis following exposure to the CBC oxime drug. Therefore, the loss of cell viability observed was due to apoptosis rather than necrosis. There are clear toxic effects seen when this drug is exposed to developing sea urchin embryos, suggesting that it might have potential use as a chemotherapy agent capable of inducing apoptosis in cancer cells. We plan to test this hypothesis on human cancer cell lines in the near future