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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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Jasplakinolide inhibits cell motility and causes actin aggregates to form.(A) MDCK cells were plated at low density and imaged as in Figure 5A but in the presence of 200 nM jasplakinolide. The cells cease to move and actin aggregates begin forming within 1 h of compound addition. (B) B16-F1 cells were imaged as in Figure 5B but in the presence of 200 nM jasplakinolide. Cells were spread and moving before addition of jasplakinolide but ceased to move and accumulated actin aggregates within 1 h of treatment with compound. The cells also showed fragmentation of the lamellipodium, which was not observed in cells treated with cucurbitacin I.
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pone-0014039-g008: Jasplakinolide inhibits cell motility and causes actin aggregates to form.(A) MDCK cells were plated at low density and imaged as in Figure 5A but in the presence of 200 nM jasplakinolide. The cells cease to move and actin aggregates begin forming within 1 h of compound addition. (B) B16-F1 cells were imaged as in Figure 5B but in the presence of 200 nM jasplakinolide. Cells were spread and moving before addition of jasplakinolide but ceased to move and accumulated actin aggregates within 1 h of treatment with compound. The cells also showed fragmentation of the lamellipodium, which was not observed in cells treated with cucurbitacin I.

Mentions: The formation of actin aggregates in cucurbitacin I-treated cells indicates that the compound is having an effect on actin depolymerization. In an attempt to learn more about the mechanism by which cucurbitacin I causes actin aggregation, we compared its effects to that of jasplakinolide, a compound that directly binds F-actin and stabilizes the filaments. Treatment of migrating B16-F1 cells with jasplakinolide resulted in cessation of movement within 5 min (Movie S6), as it does in fibroblasts [29]. Within 2 h of compound addition, cells had retracted all processes and became rounded. The cells recovered after compound removal, and after 4 h, they had begun to move again (Movie S6). Jasplakinolide also caused motility to cease and the formation of actin aggregates in MDCK cells (Figure 8A). In B16-F1 cells, jasplakinolide caused the formation of small actin aggregates, but the more dramatic effect was the collapse and fragmentation of the lamellipodium (Figure 8B and Movie S6). This was not observed with treatment with cucurbitacin I, indicating that the effect of the compounds on the actin cytoskeleton is likely to be mechanistically different.


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)

Jasplakinolide inhibits cell motility and causes actin aggregates to form.(A) MDCK cells were plated at low density and imaged as in Figure 5A but in the presence of 200 nM jasplakinolide. The cells cease to move and actin aggregates begin forming within 1 h of compound addition. (B) B16-F1 cells were imaged as in Figure 5B but in the presence of 200 nM jasplakinolide. Cells were spread and moving before addition of jasplakinolide but ceased to move and accumulated actin aggregates within 1 h of treatment with compound. The cells also showed fragmentation of the lamellipodium, which was not observed in cells treated with cucurbitacin I.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0014039-g008: Jasplakinolide inhibits cell motility and causes actin aggregates to form.(A) MDCK cells were plated at low density and imaged as in Figure 5A but in the presence of 200 nM jasplakinolide. The cells cease to move and actin aggregates begin forming within 1 h of compound addition. (B) B16-F1 cells were imaged as in Figure 5B but in the presence of 200 nM jasplakinolide. Cells were spread and moving before addition of jasplakinolide but ceased to move and accumulated actin aggregates within 1 h of treatment with compound. The cells also showed fragmentation of the lamellipodium, which was not observed in cells treated with cucurbitacin I.
Mentions: The formation of actin aggregates in cucurbitacin I-treated cells indicates that the compound is having an effect on actin depolymerization. In an attempt to learn more about the mechanism by which cucurbitacin I causes actin aggregation, we compared its effects to that of jasplakinolide, a compound that directly binds F-actin and stabilizes the filaments. Treatment of migrating B16-F1 cells with jasplakinolide resulted in cessation of movement within 5 min (Movie S6), as it does in fibroblasts [29]. Within 2 h of compound addition, cells had retracted all processes and became rounded. The cells recovered after compound removal, and after 4 h, they had begun to move again (Movie S6). Jasplakinolide also caused motility to cease and the formation of actin aggregates in MDCK cells (Figure 8A). In B16-F1 cells, jasplakinolide caused the formation of small actin aggregates, but the more dramatic effect was the collapse and fragmentation of the lamellipodium (Figure 8B and Movie S6). This was not observed with treatment with cucurbitacin I, indicating that the effect of the compounds on the actin cytoskeleton is likely to be mechanistically different.

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