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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

Cysteine-328 is not required for the effects of WFA on VIF.SW13-1HF5 cells which are  for cytoplasmic IF, were transfected with wild-type vimentin (A) and vimentin C328N (C). The cells were then treated for 3 hrs with DMSO (A and C) or 9 μM WFA (B and D). Scale bars =10 μm.
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pone-0039065-g003: Cysteine-328 is not required for the effects of WFA on VIF.SW13-1HF5 cells which are for cytoplasmic IF, were transfected with wild-type vimentin (A) and vimentin C328N (C). The cells were then treated for 3 hrs with DMSO (A and C) or 9 μM WFA (B and D). Scale bars =10 μm.

Mentions: As described above, there is evidence that WFA binds covalently to vimentin's only cysteine residue (cys328), which is located in the 2B region of the central rod domain [2]. Therefore, we reasoned that an excellent control for our experiments would be the expression of a cysteine- vimentin. Cysteine-328 was converted to asparagine (vimC328N) in the vimentin cDNA. This mutant construct was transfected into SW13-1HF5 cells which are for vimentin and do not express other types of cytoskeletal IF. Within 24 hrs following transfection, the mutant protein assembles into IF networks that are indistinguishable from those assembled from wild-type vimentin (Fig. 3A, C). Upon incubation of these transfected cells with 9 μM WFA (the lowest effective concentration required for control cells), the VIF network formed by vimC328N retracts into the perinuclear region in a fashion indistinguishable from controls, which express wild-type vimentin (Fig. 3B, D). Unfortunately, vimC328N does not work as a control, because it shows that the cysteine residue is not required for the WFA-induced reorganization of VIF networks.


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)

Cysteine-328 is not required for the effects of WFA on VIF.SW13-1HF5 cells which are  for cytoplasmic IF, were transfected with wild-type vimentin (A) and vimentin C328N (C). The cells were then treated for 3 hrs with DMSO (A and C) or 9 μM WFA (B and D). Scale bars =10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0039065-g003: Cysteine-328 is not required for the effects of WFA on VIF.SW13-1HF5 cells which are for cytoplasmic IF, were transfected with wild-type vimentin (A) and vimentin C328N (C). The cells were then treated for 3 hrs with DMSO (A and C) or 9 μM WFA (B and D). Scale bars =10 μm.
Mentions: As described above, there is evidence that WFA binds covalently to vimentin's only cysteine residue (cys328), which is located in the 2B region of the central rod domain [2]. Therefore, we reasoned that an excellent control for our experiments would be the expression of a cysteine- vimentin. Cysteine-328 was converted to asparagine (vimC328N) in the vimentin cDNA. This mutant construct was transfected into SW13-1HF5 cells which are for vimentin and do not express other types of cytoskeletal IF. Within 24 hrs following transfection, the mutant protein assembles into IF networks that are indistinguishable from those assembled from wild-type vimentin (Fig. 3A, C). Upon incubation of these transfected cells with 9 μM WFA (the lowest effective concentration required for control cells), the VIF network formed by vimC328N retracts into the perinuclear region in a fashion indistinguishable from controls, which express wild-type vimentin (Fig. 3B, D). Unfortunately, vimC328N does not work as a control, because it shows that the cysteine residue is not required for the WFA-induced reorganization of VIF networks.

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