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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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Related in: MedlinePlus

WFA does not affect the sedimentation velocity of vimentin.Sedimentation velocity profile of vimentin (0.15 mg/ml) reconstituted in 5 mM Tris-HCl, pH 8.4 (dashed lines) or 2 mM NaCl, pH 7.5 buffer (solid lines) alone (red lines), with DMSO (green lines), or with 25 μM WFA dissolved in DMSO (blue lines). Note that the curves of the two groups of runs are practically superimposable indicating that DMSO and DMSO plus WFA do not have any effect on the complex formation of vimentin oligomers.
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pone-0039065-g005: WFA does not affect the sedimentation velocity of vimentin.Sedimentation velocity profile of vimentin (0.15 mg/ml) reconstituted in 5 mM Tris-HCl, pH 8.4 (dashed lines) or 2 mM NaCl, pH 7.5 buffer (solid lines) alone (red lines), with DMSO (green lines), or with 25 μM WFA dissolved in DMSO (blue lines). Note that the curves of the two groups of runs are practically superimposable indicating that DMSO and DMSO plus WFA do not have any effect on the complex formation of vimentin oligomers.

Mentions: Our results show that cysteine-328 is not required for the WFA induced reorganization of VIF in cells. However, in vitro studies clearly show that WFA binds to vimentin [2]. Based upon these findings we tested whether WFA alters any parameter of the polymerization of wild-type vimentin into IF in vitro. First, we analyzed the steps in assembly by sedimentation velocity ultracentrifugation in the presence and the absence of WFA (Fig. 5). We did this under two conditions previously demonstrated to provide optimal starter units for subsequent filament assembly: 5 mM Tris-HCl, pH 8.4 and 2 mM Na3PO4, pH 7.5 [28]. In both cases, neither DMSO alone nor WFA with DMSO had any influence on the sedimentation behavior of vimentin, which exhibited an s-value of 5.6 S (Tris-buffer) and 6.2 S (Na3PO4-buffer).


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 does not affect the sedimentation velocity of vimentin.Sedimentation velocity profile of vimentin (0.15 mg/ml) reconstituted in 5 mM Tris-HCl, pH 8.4 (dashed lines) or 2 mM NaCl, pH 7.5 buffer (solid lines) alone (red lines), with DMSO (green lines), or with 25 μM WFA dissolved in DMSO (blue lines). Note that the curves of the two groups of runs are practically superimposable indicating that DMSO and DMSO plus WFA do not have any effect on the complex formation of vimentin oligomers.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0039065-g005: WFA does not affect the sedimentation velocity of vimentin.Sedimentation velocity profile of vimentin (0.15 mg/ml) reconstituted in 5 mM Tris-HCl, pH 8.4 (dashed lines) or 2 mM NaCl, pH 7.5 buffer (solid lines) alone (red lines), with DMSO (green lines), or with 25 μM WFA dissolved in DMSO (blue lines). Note that the curves of the two groups of runs are practically superimposable indicating that DMSO and DMSO plus WFA do not have any effect on the complex formation of vimentin oligomers.
Mentions: Our results show that cysteine-328 is not required for the WFA induced reorganization of VIF in cells. However, in vitro studies clearly show that WFA binds to vimentin [2]. Based upon these findings we tested whether WFA alters any parameter of the polymerization of wild-type vimentin into IF in vitro. First, we analyzed the steps in assembly by sedimentation velocity ultracentrifugation in the presence and the absence of WFA (Fig. 5). We did this under two conditions previously demonstrated to provide optimal starter units for subsequent filament assembly: 5 mM Tris-HCl, pH 8.4 and 2 mM Na3PO4, pH 7.5 [28]. In both cases, neither DMSO alone nor WFA with DMSO had any influence on the sedimentation behavior of vimentin, which exhibited an s-value of 5.6 S (Tris-buffer) and 6.2 S (Na3PO4-buffer).

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