Abstract
Discovery of novel bioactive compounds is important not only for therapeutic purposes but also for understanding the mechanisms of biological processes. To screen bioactive compounds that affect nuclear morphology in marine organism extracts, we employed a microscopy-based assay using DNA staining of human cancer cells. A crude extract from a marine sponge Mycale aff. nullarosette, collected from the east coast of Japan, induced cellular binucleation. Fractionation of the extract led to the isolation of mycalolides A and B, and 38-hydroxymycalolide B as the active components. Mycalolides have been identified as marine toxins that induce depolymerization of the actin filament. Live cell imaging revealed that low concentrations of mycalolide A produce binucleated cells by inhibiting the completion of cytokinesis. At higher concentrations, however, mycalolide A causes immediate disruption of actin filaments and changes in cell morphology, yielding rounded cells. These results suggest that the completion of cytokinesis is a process requiring high actin polymerization activity. Furthermore, luciferase reporter assays with mycalolide A treatments support the view that the level of globular actin can affect transcription of a serum response gene.
Highlights
Marine invertebrates, including marine sponges, are a good source of natural products with potent bioactivities
Liquid chromatography followed by mass spectrometry (LC-MS) and NMR analyses revealed that the fraction 11-3(1) contained mycalolide A (82% purity) (Supplementary Fig. S1B,C)
As the purity of mycalolide A in 11-3(1) fraction was only 82% measured using LC-MS, we prepared a more purified mycalolide A (92% purity) from another Mycale sample collected at Kagoshima (Supplementary Fig. S2), by which similar cell phenotypes were observed as for 11-3(1) (Supplementary Fig. S3), and used this for quantitative comparisons with other actin polymerization inhibitors (Figs 7 and 8)
Summary
Marine invertebrates, including marine sponges, are a good source of natural products with potent bioactivities. Actin is involved in the transcriptional regulation of a subset of genes[3,4] Many of these processes are controlled through the balance between actin polymerization (to form filamentous actin, or F-actin) and depolymerization (to dissociate into monomeric globular actin, or G-actin)[5,6]. Mycalolides that are composed of a macrocyclic ring containing three adjacent oxazoles were identified as marine toxins that induce actin depolymerization by binding to G-actin[9,10,11]. Three fractions that produced binucleation and actin depolymerization in cells were found to contain mycalolides A, B, and 38-hydroxymycalolide B. Actin fibers were immediately disrupted, causing the loss of cellular tension These results are consistent with the biochemical property observed in vitro for mycalolides that bind to F-actin to promote depolymerization. The luciferase reporter assay suggests that mycalolide A can bind to nuclear actin and affect transcription from a serum response gene
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