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Withaferin a alters intermediate filament organization, cell shape and behavior.

Grin B, Mahammad S, Wedig T, Cleland MM, Tsai L, Herrmann H, Goldman RD - PLoS ONE (2012)

Bottom Line: In cells co-expressing keratin IF and VIF, the former are significantly less sensitive to WFA with respect to inducing perinuclear aggregates.Lower doses of the drug do not kill cells but cause them to senesce.In light of our findings that WFA affects multiple IF systems, which are expressed in many tissues of the body, caution is warranted in its use as an anti-cancer agent, since it may have debilitating organism-wide effects.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Feinberg School of Medicine at Northwestern University, Chicago, Illinois, United States of America. r-goldman@northwestern.edu

ABSTRACT
Withaferin A (WFA) is a steroidal lactone present in Withania somnifera which has been shown in vitro to bind to the intermediate filament protein, vimentin. Based upon its affinity for vimentin, it has been proposed that WFA can be used as an anti-tumor agent to target metastatic cells which up-regulate vimentin expression. We show that WFA treatment of human fibroblasts rapidly reorganizes vimentin intermediate filaments (VIF) into a perinuclear aggregate. This reorganization is dose dependent and is accompanied by a change in cell shape, decreased motility and an increase in vimentin phosphorylation at serine-38. Furthermore, vimentin lacking cysteine-328, the proposed WFA binding site, remains sensitive to WFA demonstrating that this site is not required for its cellular effects. Using analytical ultracentrifugation, viscometry, electron microscopy and sedimentation assays we show that WFA has no effect on VIF assembly in vitro. Furthermore, WFA is not specific for vimentin as it disrupts the cellular organization and induces perinuclear aggregates of several other IF networks comprised of peripherin, neurofilament-triplet protein, and keratin. In cells co-expressing keratin IF and VIF, the former are significantly less sensitive to WFA with respect to inducing perinuclear aggregates. The organization of microtubules and actin/microfilaments is also affected by WFA. Microtubules become wavier and sparser and the number of stress fibers appears to increase. Following 24 hrs of exposure to doses of WFA that alter VIF organization and motility, cells undergo apoptosis. Lower doses of the drug do not kill cells but cause them to senesce. In light of our findings that WFA affects multiple IF systems, which are expressed in many tissues of the body, caution is warranted in its use as an anti-cancer agent, since it may have debilitating organism-wide effects.

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WFA also alters the organization of microtubules and microfilaments.BJ-5ta cell were treated with DMSO (A and C) and 2 μM WFA (B and D) for 3 hrs and stained with vimentin antibodies (A′, B′, C′, D′), tubulin antibodies (A′′ and B′′), and phalloidin to visualize to actin (C′′ and D′′). Scale bars =10 μm.
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pone-0039065-g010: WFA also alters the organization of microtubules and microfilaments.BJ-5ta cell were treated with DMSO (A and C) and 2 μM WFA (B and D) for 3 hrs and stained with vimentin antibodies (A′, B′, C′, D′), tubulin antibodies (A′′ and B′′), and phalloidin to visualize to actin (C′′ and D′′). Scale bars =10 μm.

Mentions: Since IF interact extensively with microtubules (MT), and actin/microfilaments (MF) [19], we determined whether WFA alters these cytoskeletal systems. In control BJ-5ta cells, normal arrays of MTs are seen radiating from a centrally located microtubule-organizing center (MTOC) near the nucleus (Fig. 10A′′). Following 3 hrs of exposure to 2 μM WFA, VIF are reorganized into a perinuclear aggregate (Fig. 10B′). There appear to be fewer MTs at the cell center compared to controls and these are distributed mainly toward the cell periphery. In addition, the MTOC is not apparent in 89.2%±3% (n=615) of the cells compared to 2%±1% (n=600) in controls (compare Figs 10A′′ and B′′). Following WFA treatment many MTs appear wavy, rather than straight as seen in controls (compare Fig. 10A′′ and B′′). Under the same conditions of WFA treatment there is an apparent increase in the number of actin stress fibers and the appearance of extensive “sheets” of actin not observed in control cells (compare Figs 10 C′′ and D′′).


Withaferin a alters intermediate filament organization, cell shape and behavior.

Grin B, Mahammad S, Wedig T, Cleland MM, Tsai L, Herrmann H, Goldman RD - PLoS ONE (2012)

WFA also alters the organization of microtubules and microfilaments.BJ-5ta cell were treated with DMSO (A and C) and 2 μM WFA (B and D) for 3 hrs and stained with vimentin antibodies (A′, B′, C′, D′), tubulin antibodies (A′′ and B′′), and phalloidin to visualize to actin (C′′ and D′′). Scale bars =10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3376126&req=5

pone-0039065-g010: WFA also alters the organization of microtubules and microfilaments.BJ-5ta cell were treated with DMSO (A and C) and 2 μM WFA (B and D) for 3 hrs and stained with vimentin antibodies (A′, B′, C′, D′), tubulin antibodies (A′′ and B′′), and phalloidin to visualize to actin (C′′ and D′′). Scale bars =10 μm.
Mentions: Since IF interact extensively with microtubules (MT), and actin/microfilaments (MF) [19], we determined whether WFA alters these cytoskeletal systems. In control BJ-5ta cells, normal arrays of MTs are seen radiating from a centrally located microtubule-organizing center (MTOC) near the nucleus (Fig. 10A′′). Following 3 hrs of exposure to 2 μM WFA, VIF are reorganized into a perinuclear aggregate (Fig. 10B′). There appear to be fewer MTs at the cell center compared to controls and these are distributed mainly toward the cell periphery. In addition, the MTOC is not apparent in 89.2%±3% (n=615) of the cells compared to 2%±1% (n=600) in controls (compare Figs 10A′′ and B′′). Following WFA treatment many MTs appear wavy, rather than straight as seen in controls (compare Fig. 10A′′ and B′′). Under the same conditions of WFA treatment there is an apparent increase in the number of actin stress fibers and the appearance of extensive “sheets” of actin not observed in control cells (compare Figs 10 C′′ and D′′).

Bottom Line: In cells co-expressing keratin IF and VIF, the former are significantly less sensitive to WFA with respect to inducing perinuclear aggregates.Lower doses of the drug do not kill cells but cause them to senesce.In light of our findings that WFA affects multiple IF systems, which are expressed in many tissues of the body, caution is warranted in its use as an anti-cancer agent, since it may have debilitating organism-wide effects.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Feinberg School of Medicine at Northwestern University, Chicago, Illinois, United States of America. r-goldman@northwestern.edu

ABSTRACT
Withaferin A (WFA) is a steroidal lactone present in Withania somnifera which has been shown in vitro to bind to the intermediate filament protein, vimentin. Based upon its affinity for vimentin, it has been proposed that WFA can be used as an anti-tumor agent to target metastatic cells which up-regulate vimentin expression. We show that WFA treatment of human fibroblasts rapidly reorganizes vimentin intermediate filaments (VIF) into a perinuclear aggregate. This reorganization is dose dependent and is accompanied by a change in cell shape, decreased motility and an increase in vimentin phosphorylation at serine-38. Furthermore, vimentin lacking cysteine-328, the proposed WFA binding site, remains sensitive to WFA demonstrating that this site is not required for its cellular effects. Using analytical ultracentrifugation, viscometry, electron microscopy and sedimentation assays we show that WFA has no effect on VIF assembly in vitro. Furthermore, WFA is not specific for vimentin as it disrupts the cellular organization and induces perinuclear aggregates of several other IF networks comprised of peripherin, neurofilament-triplet protein, and keratin. In cells co-expressing keratin IF and VIF, the former are significantly less sensitive to WFA with respect to inducing perinuclear aggregates. The organization of microtubules and actin/microfilaments is also affected by WFA. Microtubules become wavier and sparser and the number of stress fibers appears to increase. Following 24 hrs of exposure to doses of WFA that alter VIF organization and motility, cells undergo apoptosis. Lower doses of the drug do not kill cells but cause them to senesce. In light of our findings that WFA affects multiple IF systems, which are expressed in many tissues of the body, caution is warranted in its use as an anti-cancer agent, since it may have debilitating organism-wide effects.

Show MeSH
Related in: MedlinePlus