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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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Keratin IF are less sensitive to WFA than VIF.Human lung cancer cells, A549, were treated for 3 hrs with DMSO [ctrl] (A), 4.0 μM WFA (B), and 6.0 μM WFA (C), followed by staining with vimentin (A′, B′, C′) and pan-cytokeratin antibodies (A′′, B′′, C′′). Scale bars =10 μm.
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pone-0039065-g008: Keratin IF are less sensitive to WFA than VIF.Human lung cancer cells, A549, were treated for 3 hrs with DMSO [ctrl] (A), 4.0 μM WFA (B), and 6.0 μM WFA (C), followed by staining with vimentin (A′, B′, C′) and pan-cytokeratin antibodies (A′′, B′′, C′′). Scale bars =10 μm.

Mentions: Most cancers arise from epithelial cells, which in their normal differentiated states express keratin (KIF), but not VIF. However, vimentin expression is up-regulated in, and is indeed one of the hallmarks of, the epithelial-mesenchymal transition (EMT) that accompanies the transformation of pre-metastatic to metastatic cells [3]. As a consequence, it is common for cancerous cells to express both VIF and KIF networks during the EMT. Based upon these findings, it has been proposed that specifically disrupting VIF using WFA in cells undergoing the EMT might prove useful in chemotherapy [4]. However, the proposed binding site for WFA in vimentin is highly conserved in all cytoskeletal IF. Therefore, it is important to determine whether WFA specifically disrupts VIF or whether it affects other types of IF as well. Thus, we determined the effects of WFA in human A549 lung cancer cells, which express both KIF and VIF (Fig. 8A). Treatment with a range of concentrations showed that after 3 hrs of exposure to 4 μM WFA, the VIF network reorganizes into the perinuclear region (Fig. 8B′), while KIF retain their normal distribution (Fig. 8B′′). Increasing the concentration of WFA to 6 μM induces the reorganization of both the VIF and KIF networks in 3 hrs (Fig. 8C). To determine whether the difference in sensitivity to WFA is attributable to a difference in the rate of retraction between KIF and VIF we treated the cells with 4 μM WFA for 6 hrs. Under these conditions the extent of reorganization of both IF networks is similar to that seen after 3 hrs (data not shown). Similar differences between VIF and KIF sensitivity to WFA are also observed in HeLa cells (data not shown).


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)

Keratin IF are less sensitive to WFA than VIF.Human lung cancer cells, A549, were treated for 3 hrs with DMSO [ctrl] (A), 4.0 μM WFA (B), and 6.0 μM WFA (C), followed by staining with vimentin (A′, B′, C′) and pan-cytokeratin antibodies (A′′, B′′, C′′). Scale bars =10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0039065-g008: Keratin IF are less sensitive to WFA than VIF.Human lung cancer cells, A549, were treated for 3 hrs with DMSO [ctrl] (A), 4.0 μM WFA (B), and 6.0 μM WFA (C), followed by staining with vimentin (A′, B′, C′) and pan-cytokeratin antibodies (A′′, B′′, C′′). Scale bars =10 μm.
Mentions: Most cancers arise from epithelial cells, which in their normal differentiated states express keratin (KIF), but not VIF. However, vimentin expression is up-regulated in, and is indeed one of the hallmarks of, the epithelial-mesenchymal transition (EMT) that accompanies the transformation of pre-metastatic to metastatic cells [3]. As a consequence, it is common for cancerous cells to express both VIF and KIF networks during the EMT. Based upon these findings, it has been proposed that specifically disrupting VIF using WFA in cells undergoing the EMT might prove useful in chemotherapy [4]. However, the proposed binding site for WFA in vimentin is highly conserved in all cytoskeletal IF. Therefore, it is important to determine whether WFA specifically disrupts VIF or whether it affects other types of IF as well. Thus, we determined the effects of WFA in human A549 lung cancer cells, which express both KIF and VIF (Fig. 8A). Treatment with a range of concentrations showed that after 3 hrs of exposure to 4 μM WFA, the VIF network reorganizes into the perinuclear region (Fig. 8B′), while KIF retain their normal distribution (Fig. 8B′′). Increasing the concentration of WFA to 6 μM induces the reorganization of both the VIF and KIF networks in 3 hrs (Fig. 8C). To determine whether the difference in sensitivity to WFA is attributable to a difference in the rate of retraction between KIF and VIF we treated the cells with 4 μM WFA for 6 hrs. Under these conditions the extent of reorganization of both IF networks is similar to that seen after 3 hrs (data not shown). Similar differences between VIF and KIF sensitivity to WFA are also observed in HeLa cells (data not shown).

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