Abstract
Lasonolide A, a novel polyketide-derived macrolide, was previously identified from an extract of the marine sponge Forcepia sp. in an assay for protein kinase C (PKC) inhibitors. Cytotoxicity testing and profiling of lasonolide A in the National Cancer Institute (NCI) 60 cell panel screen revealed that it was potent toward a broad range of cell lines and also suggested a unique mechanism of action. Contrary to expected results, we found lasonolide A to be a strong activator of PKC in Panc-1 pancreatic carcinoma cells. Downstream mitogen-activated protein kinases, ERK 1/2 and p38 were also rapidly phosphorylated in response to lasonolide A, as was Akt. Microscopy studies revealed that lasonolide A induced blebbing and contraction of the cells within minutes of exposure, and the eventual loss of adherence. However, membrane integrity was maintained and the effects were reversible if lasonolide A was washed from the cells after their loss of adherence. Pretreatment of cells with a myosin II inhibitor, blebbistatin, slowed the early onset, but did not prevent the morphological effects of lasonolide A. Cells stained for actin filaments showed some reduction in stress fiber structure after lasonolide A exposure; however, it did not affect the polymerization of purified actin in vitro. Bisindolemaleimide, a PKC inhibitor, and wortmannin, a phosphoinositide 3-kinase; inhibitor, did not reduce lasonolide A-induced contraction or blebbing or the activation of mitogen-activated protein kinases, although Akt phosphorylation was prevented by wortmannin pretreatment. Our results indicate that lasonolide A activates multiple signal transduction pathways and suggest that the origin is upstream of PKC.
- BIM-1, bisindolemaleimide
- PKC, protein kinase C
- MARCKS, myristoylated alanine-rich protein kinase C substrate
- ERK1/2, p44 and p42 extracellular signal-regulated protein kinases 1 and 2
- FITC, fluorescein isothiocyanate
- PMA, phorbol 12-myristate 13-acetate
- MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
- LDH, lactate dehydrogenase
- PI3K, phosphoinositide 3-kinase
- DPBS, Dulbecco's phosphate-buffered saline
- MAP, mitogen-activated protein.
Footnotes
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This work was supported by the National Institutes of Health National Cancer Institute [Grant CA-93455].
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This is Harbor Branch Oceanographic Institute of Florida Atlantic University contribution number 1785.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.109.155531
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↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
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ABBREVIATIONS:
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↵1 Current affiliation: Health Canada, Center for Biologics Research, Ottawa, Ontario, Canada.
- Received May 4, 2009.
- Accepted August 18, 2009.
- © 2009 by The American Society for Pharmacology and Experimental Therapeutics
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