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Cucurbitacin I inhibits cell motility by indirectly interfering with actin dynamics.

Knecht DA, LaFleur RA, Kahsai AW, Argueta CE, Beshir AB, Fenteany G - PLoS ONE (2010)

Bottom Line: 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.Cucurbitacin I results in accumulation of actin filaments in cells by a unique indirect mechanism.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut, USA. david.knecht@uconn.edu

ABSTRACT

Background: 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 also known to cause changes in the organization of the actin cytoskeleton.

Methodology/principal findings: We 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/significance: Cucurbitacin 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.

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Related in: MedlinePlus

Cucurbitacin I inhibits motility of B16-F1 melanoma cells.Phase-contrast images of migrating B16-F1 cells were captured every 30 s on a laminin-coated dish. 200 nM cucurbitacin I was then added to the chamber. The panels show representative time points before and after compound addition with “0 h” being when compound was added.
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pone-0014039-g004: Cucurbitacin I inhibits motility of B16-F1 melanoma cells.Phase-contrast images of migrating B16-F1 cells were captured every 30 s on a laminin-coated dish. 200 nM cucurbitacin I was then added to the chamber. The panels show representative time points before and after compound addition with “0 h” being when compound was added.

Mentions: We next looked at the effect of cucurbitacin I on motile B16-F1 melanoma cells and Dictyostelium discoideum amoebae. When 200 nM cucurbitacin I was applied to migrating B16-F1 cells, translocation rapidly ceased (Movies S2 and S3). After 45 min, the compound was washed out, but it took about an hour before the first cells began to regain movement. Full recovery of the population occurred gradually over the next 5 h. When imaged at higher temporal and spatial resolution, lamellipodial ruffling and protrusion stopped within 30 s of treatment with cucurbitacin I, and within 1 min the lamellipodia began to retract (Figure 4 and Movie S3). Within 10 min, the cells had become rounded and small blebs appeared around the periphery. We then tested the effect of cucurbitacin I on the motility of Dictyostelium amoebae. No significant change in average speed or cell morphology were seen at concentrations as high as 2 µM cucurbitacin I (Figure 5 and Movie S4).


Cucurbitacin I inhibits cell motility by indirectly interfering with actin dynamics.

Knecht DA, LaFleur RA, Kahsai AW, Argueta CE, Beshir AB, Fenteany G - PLoS ONE (2010)

Cucurbitacin I inhibits motility of B16-F1 melanoma cells.Phase-contrast images of migrating B16-F1 cells were captured every 30 s on a laminin-coated dish. 200 nM cucurbitacin I was then added to the chamber. The panels show representative time points before and after compound addition with “0 h” being when compound was added.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2991314&req=5

pone-0014039-g004: Cucurbitacin I inhibits motility of B16-F1 melanoma cells.Phase-contrast images of migrating B16-F1 cells were captured every 30 s on a laminin-coated dish. 200 nM cucurbitacin I was then added to the chamber. The panels show representative time points before and after compound addition with “0 h” being when compound was added.
Mentions: We next looked at the effect of cucurbitacin I on motile B16-F1 melanoma cells and Dictyostelium discoideum amoebae. When 200 nM cucurbitacin I was applied to migrating B16-F1 cells, translocation rapidly ceased (Movies S2 and S3). After 45 min, the compound was washed out, but it took about an hour before the first cells began to regain movement. Full recovery of the population occurred gradually over the next 5 h. When imaged at higher temporal and spatial resolution, lamellipodial ruffling and protrusion stopped within 30 s of treatment with cucurbitacin I, and within 1 min the lamellipodia began to retract (Figure 4 and Movie S3). Within 10 min, the cells had become rounded and small blebs appeared around the periphery. We then tested the effect of cucurbitacin I on the motility of Dictyostelium amoebae. No significant change in average speed or cell morphology were seen at concentrations as high as 2 µM cucurbitacin I (Figure 5 and Movie S4).

Bottom Line: 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.Cucurbitacin I results in accumulation of actin filaments in cells by a unique indirect mechanism.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut, USA. david.knecht@uconn.edu

ABSTRACT

Background: 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 also known to cause changes in the organization of the actin cytoskeleton.

Methodology/principal findings: We 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/significance: Cucurbitacin 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.

Show MeSH
Related in: MedlinePlus