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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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Cucurbitacin I does not directly affect actin polymerization, depolymerization or steady-state levels of F-actin in vitro.(A) Pyrene-labeled rabbit muscle G-actin polymerization was measured in the presence or absence of 50 µM cucurbitacin I as described in Materials and Methods. Values represent the mean with standard deviation (SD) for n = 4. The compound had no effect on actin polymerization. (B) Pyrene-G-actin was polymerized to pyrene-F-actin and then diluted to induce actin filament disassembly, as described in Materials and Methods, in the presence or absence of cucurbitacin or phallicidin at the indicated concentrations. Phallacidin inhibited actin depolymerization, but cucurbitacin I only weakly delayed the start of depolymerization and only at relatively high concentrations, while not affecting the rate of depolymerization (note similar slopes once depolymerization starts). Values represent the mean with SD for n = 9. (C) Cucurbitacin I does not directly stabilize F-actin. Purified human non-muscle G-actin was polymerized to F-actin and then pelleted immediately (lane 1) or after dilution to initiate depolymerization and overnight incubation in the absence (lane 2) or presence of 200 nM jasplakinolide (lane 3) or 200 nM cucurbitacin I (lane 4). Pellets were solubilized in SDS sample buffer, and samples were then electrophoresed on an SDS-polyacrylamide gel and stained with Coomassie blue. Jasplakinolide prevented filament depolymerization, but cucurbitacin I had no effect.
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pone-0014039-g010: Cucurbitacin I does not directly affect actin polymerization, depolymerization or steady-state levels of F-actin in vitro.(A) Pyrene-labeled rabbit muscle G-actin polymerization was measured in the presence or absence of 50 µM cucurbitacin I as described in Materials and Methods. Values represent the mean with standard deviation (SD) for n = 4. The compound had no effect on actin polymerization. (B) Pyrene-G-actin was polymerized to pyrene-F-actin and then diluted to induce actin filament disassembly, as described in Materials and Methods, in the presence or absence of cucurbitacin or phallicidin at the indicated concentrations. Phallacidin inhibited actin depolymerization, but cucurbitacin I only weakly delayed the start of depolymerization and only at relatively high concentrations, while not affecting the rate of depolymerization (note similar slopes once depolymerization starts). Values represent the mean with SD for n = 9. (C) Cucurbitacin I does not directly stabilize F-actin. Purified human non-muscle G-actin was polymerized to F-actin and then pelleted immediately (lane 1) or after dilution to initiate depolymerization and overnight incubation in the absence (lane 2) or presence of 200 nM jasplakinolide (lane 3) or 200 nM cucurbitacin I (lane 4). Pellets were solubilized in SDS sample buffer, and samples were then electrophoresed on an SDS-polyacrylamide gel and stained with Coomassie blue. Jasplakinolide prevented filament depolymerization, but cucurbitacin I had no effect.

Mentions: If cucurbitacin I stabilizes F-actin, then this might lead to a change in the ratio of F- to G-actin in cells. In cells treated for 2 h with cucurbitacin I, there was a shift in the F-/G-actin ratio from 0.2 to 2.5 (Figure 9). In order to determine whether cucurbitacin I acts directly on actin filaments or acts indirectly through other actin-binding proteins, the effect of the compound on purified actin was measured. The rate of actin polymerization from pyrene-G-actin in the presence or absence of cucurbitacin I in vitro was indistinguishable (Figure 10A). To test the effect of the compounds on actin filament disassembly, pyrene-G-actin was polymerized to pyrene-F-actin and then diluted to induce depolymerization in the presence or absence of compound. Cucurbitacin I at high concentrations had a weak inhibitory effect on actin depolymerization, but this was much weaker than the known direct actin-stabilizing compound phallacidin (Figure 10B). The effect was also qualitatively different from that of phallacidin. High concentrations of cucurbitacin I seemed to delay the onset of depolymerization, but once the filaments began to disassemble, the rates of depolymerization were similar to the DMSO control (note similar slopes in the presence of cucurbitacin I as in its absence in Figure 10B). Furthermore, in an in vitro actin depolymerization assay based instead on pelleting of F-actin, 200 nM cucurbitacin I had no effect on actin depolymerization, whereas 200 nM jasplakinolide prevented depolymerization (Figure 10C).


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 does not directly affect actin polymerization, depolymerization or steady-state levels of F-actin in vitro.(A) Pyrene-labeled rabbit muscle G-actin polymerization was measured in the presence or absence of 50 µM cucurbitacin I as described in Materials and Methods. Values represent the mean with standard deviation (SD) for n = 4. The compound had no effect on actin polymerization. (B) Pyrene-G-actin was polymerized to pyrene-F-actin and then diluted to induce actin filament disassembly, as described in Materials and Methods, in the presence or absence of cucurbitacin or phallicidin at the indicated concentrations. Phallacidin inhibited actin depolymerization, but cucurbitacin I only weakly delayed the start of depolymerization and only at relatively high concentrations, while not affecting the rate of depolymerization (note similar slopes once depolymerization starts). Values represent the mean with SD for n = 9. (C) Cucurbitacin I does not directly stabilize F-actin. Purified human non-muscle G-actin was polymerized to F-actin and then pelleted immediately (lane 1) or after dilution to initiate depolymerization and overnight incubation in the absence (lane 2) or presence of 200 nM jasplakinolide (lane 3) or 200 nM cucurbitacin I (lane 4). Pellets were solubilized in SDS sample buffer, and samples were then electrophoresed on an SDS-polyacrylamide gel and stained with Coomassie blue. Jasplakinolide prevented filament depolymerization, but cucurbitacin I had no effect.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2991314&req=5

pone-0014039-g010: Cucurbitacin I does not directly affect actin polymerization, depolymerization or steady-state levels of F-actin in vitro.(A) Pyrene-labeled rabbit muscle G-actin polymerization was measured in the presence or absence of 50 µM cucurbitacin I as described in Materials and Methods. Values represent the mean with standard deviation (SD) for n = 4. The compound had no effect on actin polymerization. (B) Pyrene-G-actin was polymerized to pyrene-F-actin and then diluted to induce actin filament disassembly, as described in Materials and Methods, in the presence or absence of cucurbitacin or phallicidin at the indicated concentrations. Phallacidin inhibited actin depolymerization, but cucurbitacin I only weakly delayed the start of depolymerization and only at relatively high concentrations, while not affecting the rate of depolymerization (note similar slopes once depolymerization starts). Values represent the mean with SD for n = 9. (C) Cucurbitacin I does not directly stabilize F-actin. Purified human non-muscle G-actin was polymerized to F-actin and then pelleted immediately (lane 1) or after dilution to initiate depolymerization and overnight incubation in the absence (lane 2) or presence of 200 nM jasplakinolide (lane 3) or 200 nM cucurbitacin I (lane 4). Pellets were solubilized in SDS sample buffer, and samples were then electrophoresed on an SDS-polyacrylamide gel and stained with Coomassie blue. Jasplakinolide prevented filament depolymerization, but cucurbitacin I had no effect.
Mentions: If cucurbitacin I stabilizes F-actin, then this might lead to a change in the ratio of F- to G-actin in cells. In cells treated for 2 h with cucurbitacin I, there was a shift in the F-/G-actin ratio from 0.2 to 2.5 (Figure 9). In order to determine whether cucurbitacin I acts directly on actin filaments or acts indirectly through other actin-binding proteins, the effect of the compound on purified actin was measured. The rate of actin polymerization from pyrene-G-actin in the presence or absence of cucurbitacin I in vitro was indistinguishable (Figure 10A). To test the effect of the compounds on actin filament disassembly, pyrene-G-actin was polymerized to pyrene-F-actin and then diluted to induce depolymerization in the presence or absence of compound. Cucurbitacin I at high concentrations had a weak inhibitory effect on actin depolymerization, but this was much weaker than the known direct actin-stabilizing compound phallacidin (Figure 10B). The effect was also qualitatively different from that of phallacidin. High concentrations of cucurbitacin I seemed to delay the onset of depolymerization, but once the filaments began to disassemble, the rates of depolymerization were similar to the DMSO control (note similar slopes in the presence of cucurbitacin I as in its absence in Figure 10B). Furthermore, in an in vitro actin depolymerization assay based instead on pelleting of F-actin, 200 nM cucurbitacin I had no effect on actin depolymerization, whereas 200 nM jasplakinolide prevented depolymerization (Figure 10C).

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