Abstract
BackgroundCucurbitacins are plant natural products that inhibit activation of the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway by an unknown mechanism. They are also known to cause changes in the organization of the actin cytoskeleton.Methodology/Principal FindingsWe show that cucurbitacin I potently inhibits the migration of Madin-Darby canine kidney (MDCK) cell sheets during wound closure, as well as the random motility of B16-F1 mouse melanoma cells, but has no effect on movement of Dictyostelium discoideum amoebae. Upon treatment of MDCK or B16-F1 cells with cucurbitacin I, there is a very rapid cessation of motility and gradual accumulation of filamentous actin aggregates. The cellular effect of the compound is similar to that observed when cells are treated with the actin filament-stabilizing agent jasplakinolide. However, we found that, unlike jasplakinolide or phallacidin, cucurbitacin I does not directly stabilize actin filaments. In in vitro actin depolymerization experiments, cucurbitacin I had no effect on the rate of actin filament disassembly at the nanomolar concentrations that inhibit cell migration. At elevated concentrations, the depolymerization rate was also unaffected, although there was a delay in the initiation of depolymerization. Therefore, cucurbitacin I targets some factor involved in cellular actin dynamics other than actin itself. Two candidate proteins that play roles in actin depolymerization are the actin-severing proteins cofilin and gelsolin. Cucurbitacin I possesses electrophilic reactivity that may lead to chemical modification of its target protein, as suggested by structure-activity relationship data. However, mass spectrometry revealed no evidence for modification of purified cofilin or gelsolin by cucurbitacin I.Conclusions/SignificanceCucurbitacin I results in accumulation of actin filaments in cells by a unique indirect mechanism. Furthermore, the proximal target of cucurbitacin I relevant to cell migration is unlikely to be the same one involved in activation of the JAK2/STAT3 pathway.
Highlights
Cucurbitacins are plant triterpenoids with a number of interesting biological properties
We show that cucurbitacin I inhibits the migration of Madin-Darby canine kidney (MDCK) cells and B16-F1 mouse melanoma cells, it has no effect on Dictyostelium discoideum cells
A high throughput wound closure assay with MDCK cells was used to screen the National Cancer Institute (NCI) Diversity Set for inhibitors that slowed the rate of cell migration [28]
Summary
Cucurbitacins are plant triterpenoids with a number of interesting biological properties. And disassembly of actin filaments is required for cell migration, and compounds that affect polymerization of actin or the stability of actin filaments inhibit the motility of cells (for reviews, see [26,27]) This may be the mechanism by which cucurbitacin I inhibits the movement of cells, but it is as yet unknown how cucurbitacin I mediates its effects on the cytoskeleton. Cucurbitacins are plant natural products that inhibit activation of the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway by an unknown mechanism. They are known to cause changes in the organization of the actin cytoskeleton
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