Multiwavelength videomicrofluorometric study of cytotoxic properties of a marine peptide, didemnin B, using...

Didemnin B (DB), a marine natural product, has very encouraging biological activity in vitro (Antineoplastic, immunosuppressive, antiviral). To learn more about its intracellular effects and targets, videomicrofluorometry on single living cells and a protocol of multiple labeling: Hoechst 342 for nuclear DNA, Rhodamine 123 for mitochondria and Nile Red for plasma membrane, have been used. DB behaves differently from Adriamycin, inducing at its IC50 dose of (20 nM) an accumulation of the CEM-WT lymphoblasts in the S phase of the cell cycle while we observed a 50% decrease of the mitochondrial labeling by R123, showing a decrease of the mitochondrial energetic state. Cytostatic dose of DB (250 nM) confirms these observations. However the treatment with a dose reported as apoptotic (1000 nM) induces a much faster effect (corresponding to that of 72 hours at the IC50 dose), 24 hours incubation induced a drastic decrease of nuclear DNA content as well as of the mitochondria energetic state. The evolution of NAD(P)H cellular content exhibited an increase that seems to indicate that the decrease of mitochondrial energetic state was dependent on inhibition of the mitochondrial activity due to an effect of DB at the mitochondrial level, either direct or mediated. Furthermore, the decrease of mitochondrial labeling appears as a very early event in the mechanisms leading to apoptosis.