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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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Longer exposure to WFA induces apoptosis or senescence.(A) BJ-5ta cells were treated for 24 hrs with DMSO and 2 μM WFA followed by staining with annexin V and assayed by FACS. (B) Whole cell lysates were separated by SDS-PAGE and blotted with anti-vimentin. BJ-5ta cells treated for 24 hrs with0.5 μM WFA (C) and1 μM WFA (D) were stained with vimentin antibodies. BJ-5ta fibroblasts were incubated with DMSO alone (E) or 1 μM WFA (F) continuously for 5 days and stained for senescence-associated ß-galactosidase. (H) Cell proliferation of BJ-5ta cells was monitored by counting cells every 3 days during continuous incubation with DMSO alone (black line) or 1.0 μM WFA (red line). Scale bars =10 μm.
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pone-0039065-g009: Longer exposure to WFA induces apoptosis or senescence.(A) BJ-5ta cells were treated for 24 hrs with DMSO and 2 μM WFA followed by staining with annexin V and assayed by FACS. (B) Whole cell lysates were separated by SDS-PAGE and blotted with anti-vimentin. BJ-5ta cells treated for 24 hrs with0.5 μM WFA (C) and1 μM WFA (D) were stained with vimentin antibodies. BJ-5ta fibroblasts were incubated with DMSO alone (E) or 1 μM WFA (F) continuously for 5 days and stained for senescence-associated ß-galactosidase. (H) Cell proliferation of BJ-5ta cells was monitored by counting cells every 3 days during continuous incubation with DMSO alone (black line) or 1.0 μM WFA (red line). Scale bars =10 μm.

Mentions: We also examined the effects of long-term treatment of cells with WFA as this would be required in cancer treatment. To this end, BJ-5ta cells were treated with 2 μM WFA for 24 hrs. Following this period of exposure, 76.1%±3.2% of the cells die as determined by Trypan blue exclusion. We also determined that apoptosis is the most likely cause of cell death as 69.0±2.2% of the cells treated with WFA are positive for annexin V [32]; compared to 25.8±2.4% in control cells (Fig. 9A). Induction of apoptosis by WFA is confirmed by immunoblotting for the caspase-dependent breakdown of vimentin to a 48 kDa fragment (Fig. 9B) [33].


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)

Longer exposure to WFA induces apoptosis or senescence.(A) BJ-5ta cells were treated for 24 hrs with DMSO and 2 μM WFA followed by staining with annexin V and assayed by FACS. (B) Whole cell lysates were separated by SDS-PAGE and blotted with anti-vimentin. BJ-5ta cells treated for 24 hrs with0.5 μM WFA (C) and1 μM WFA (D) were stained with vimentin antibodies. BJ-5ta fibroblasts were incubated with DMSO alone (E) or 1 μM WFA (F) continuously for 5 days and stained for senescence-associated ß-galactosidase. (H) Cell proliferation of BJ-5ta cells was monitored by counting cells every 3 days during continuous incubation with DMSO alone (black line) or 1.0 μM WFA (red line). Scale bars =10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0039065-g009: Longer exposure to WFA induces apoptosis or senescence.(A) BJ-5ta cells were treated for 24 hrs with DMSO and 2 μM WFA followed by staining with annexin V and assayed by FACS. (B) Whole cell lysates were separated by SDS-PAGE and blotted with anti-vimentin. BJ-5ta cells treated for 24 hrs with0.5 μM WFA (C) and1 μM WFA (D) were stained with vimentin antibodies. BJ-5ta fibroblasts were incubated with DMSO alone (E) or 1 μM WFA (F) continuously for 5 days and stained for senescence-associated ß-galactosidase. (H) Cell proliferation of BJ-5ta cells was monitored by counting cells every 3 days during continuous incubation with DMSO alone (black line) or 1.0 μM WFA (red line). Scale bars =10 μm.
Mentions: We also examined the effects of long-term treatment of cells with WFA as this would be required in cancer treatment. To this end, BJ-5ta cells were treated with 2 μM WFA for 24 hrs. Following this period of exposure, 76.1%±3.2% of the cells die as determined by Trypan blue exclusion. We also determined that apoptosis is the most likely cause of cell death as 69.0±2.2% of the cells treated with WFA are positive for annexin V [32]; compared to 25.8±2.4% in control cells (Fig. 9A). Induction of apoptosis by WFA is confirmed by immunoblotting for the caspase-dependent breakdown of vimentin to a 48 kDa fragment (Fig. 9B) [33].

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