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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 has no effect on the in vitro assembly of human recombinant vimentin.(A) Recombinant human vimentin (0.2 mg/ml) was assembled for 10 min at 37°C in (i) 50 mM NaCl; (ii) with 0.25% DMSO; (iii) with 50 μM WFA; and (iv) for 30 min with 50 μM WFA at a protein concentration of 0.5 mg/ml. The filaments were fixed with glutaraldehyde and visualized by negative stain electron microscopy. The arrows in (ii) indicate lateral annealing and apparent fusion of individual filaments. (scale bars, 0.2 μm). (B) Viscometric analysis of vimentin assembly in the absence (ctrl) and presence of 50 μM WFA at 37°C in 50 mM NaCl. (C) Centrifugation assay of vimentin assembled in the absence (c) and presence of WFA (w). VIF were assembled for the indicated times (5 to 15 min) in 160 mM NaCl then centrifuged for 5 min at 10 psi in an Airfuge. Samples were separated by SDS-PAGE and stained with Coomassie. The position of vimentin is indicated (55 kDa).
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pone-0039065-g006: WFA has no effect on the in vitro assembly of human recombinant vimentin.(A) Recombinant human vimentin (0.2 mg/ml) was assembled for 10 min at 37°C in (i) 50 mM NaCl; (ii) with 0.25% DMSO; (iii) with 50 μM WFA; and (iv) for 30 min with 50 μM WFA at a protein concentration of 0.5 mg/ml. The filaments were fixed with glutaraldehyde and visualized by negative stain electron microscopy. The arrows in (ii) indicate lateral annealing and apparent fusion of individual filaments. (scale bars, 0.2 μm). (B) Viscometric analysis of vimentin assembly in the absence (ctrl) and presence of 50 μM WFA at 37°C in 50 mM NaCl. (C) Centrifugation assay of vimentin assembled in the absence (c) and presence of WFA (w). VIF were assembled for the indicated times (5 to 15 min) in 160 mM NaCl then centrifuged for 5 min at 10 psi in an Airfuge. Samples were separated by SDS-PAGE and stained with Coomassie. The position of vimentin is indicated (55 kDa).

Mentions: The rate and extent of the assembly of VIF in the presence of WFA was observed by electron microscopy of negatively stained samples and viscometry as previously described [28]. In the presence of up to 50 μM WFA the structure of fully assembled VIF is not significantly altered compared to controls (compare Fig. 6A-i and Fig. 6A-ii to Fig. 6A-iii and Fig. 6A-iv). VIF appear uniform in length and width as observed by negative stain electron microscopy. Some VIF occasionally show lateral annealing, however, this effect is also seen in control samples (DMSO alone; Fig. 6A-ii, arrows). Furthermore, this DMSO-induced change is more obvious at higher protein concentrations. At 0.5 mg/ml protein (Fig. 6A-iv) and 1% DMSO (data not shown) VIF more often terminate in clump-like structures. Correspondingly, the behavior of VIF during viscometry and the rate of VIF assembly is only slightly altered by the addition of WFA (Fig. 6B), compatible with the electron microscopic data. Furthermore, VIF assembly also appears to be normal as determined by centrifugation/pelleting assays. Both with and without WFA, ∼100% of the vimentin is found in the pellet by 15 minutes following the initiation of assembly (Fig. 6C).


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 has no effect on the in vitro assembly of human recombinant vimentin.(A) Recombinant human vimentin (0.2 mg/ml) was assembled for 10 min at 37°C in (i) 50 mM NaCl; (ii) with 0.25% DMSO; (iii) with 50 μM WFA; and (iv) for 30 min with 50 μM WFA at a protein concentration of 0.5 mg/ml. The filaments were fixed with glutaraldehyde and visualized by negative stain electron microscopy. The arrows in (ii) indicate lateral annealing and apparent fusion of individual filaments. (scale bars, 0.2 μm). (B) Viscometric analysis of vimentin assembly in the absence (ctrl) and presence of 50 μM WFA at 37°C in 50 mM NaCl. (C) Centrifugation assay of vimentin assembled in the absence (c) and presence of WFA (w). VIF were assembled for the indicated times (5 to 15 min) in 160 mM NaCl then centrifuged for 5 min at 10 psi in an Airfuge. Samples were separated by SDS-PAGE and stained with Coomassie. The position of vimentin is indicated (55 kDa).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3376126&req=5

pone-0039065-g006: WFA has no effect on the in vitro assembly of human recombinant vimentin.(A) Recombinant human vimentin (0.2 mg/ml) was assembled for 10 min at 37°C in (i) 50 mM NaCl; (ii) with 0.25% DMSO; (iii) with 50 μM WFA; and (iv) for 30 min with 50 μM WFA at a protein concentration of 0.5 mg/ml. The filaments were fixed with glutaraldehyde and visualized by negative stain electron microscopy. The arrows in (ii) indicate lateral annealing and apparent fusion of individual filaments. (scale bars, 0.2 μm). (B) Viscometric analysis of vimentin assembly in the absence (ctrl) and presence of 50 μM WFA at 37°C in 50 mM NaCl. (C) Centrifugation assay of vimentin assembled in the absence (c) and presence of WFA (w). VIF were assembled for the indicated times (5 to 15 min) in 160 mM NaCl then centrifuged for 5 min at 10 psi in an Airfuge. Samples were separated by SDS-PAGE and stained with Coomassie. The position of vimentin is indicated (55 kDa).
Mentions: The rate and extent of the assembly of VIF in the presence of WFA was observed by electron microscopy of negatively stained samples and viscometry as previously described [28]. In the presence of up to 50 μM WFA the structure of fully assembled VIF is not significantly altered compared to controls (compare Fig. 6A-i and Fig. 6A-ii to Fig. 6A-iii and Fig. 6A-iv). VIF appear uniform in length and width as observed by negative stain electron microscopy. Some VIF occasionally show lateral annealing, however, this effect is also seen in control samples (DMSO alone; Fig. 6A-ii, arrows). Furthermore, this DMSO-induced change is more obvious at higher protein concentrations. At 0.5 mg/ml protein (Fig. 6A-iv) and 1% DMSO (data not shown) VIF more often terminate in clump-like structures. Correspondingly, the behavior of VIF during viscometry and the rate of VIF assembly is only slightly altered by the addition of WFA (Fig. 6B), compatible with the electron microscopic data. Furthermore, VIF assembly also appears to be normal as determined by centrifugation/pelleting assays. Both with and without WFA, ∼100% of the vimentin is found in the pellet by 15 minutes following the initiation of assembly (Fig. 6C).

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