Limits...
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.

Show MeSH

Related in: MedlinePlus

WFA treatment inhibits cell motility.(A) The average speed of BJ-5ta fibroblasts was calculated before treatment (white bar), during incubation with 2 μM WFA for 4 hrs (black bars) and after the cells were allowed to recover in fresh medium (gray bars). (B and C) Cells were treated with 2 μM WFA for 3 hrs and then placed into fresh medium followed by fixation and processing for immunofluorescence with vimentin antibodies after 6h rs (B) and 9 hrs (C). Scale bars =10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3376126&req=5

pone-0039065-g002: WFA treatment inhibits cell motility.(A) The average speed of BJ-5ta fibroblasts was calculated before treatment (white bar), during incubation with 2 μM WFA for 4 hrs (black bars) and after the cells were allowed to recover in fresh medium (gray bars). (B and C) Cells were treated with 2 μM WFA for 3 hrs and then placed into fresh medium followed by fixation and processing for immunofluorescence with vimentin antibodies after 6h rs (B) and 9 hrs (C). Scale bars =10 μm.

Mentions: The effects of withaferin A (WFA) on vimentin IF (VIF) networks were determined in human fibroblasts (BJ-5ta) by immunofluorescence. In controls, VIF networks extend throughout the cytoplasm, from the nucleus to peripheral regions (Fig. 1A). After a three-hour exposure to WFA, VIF networks retract from the cell periphery towards the nucleus. The extent of retraction varies with the concentration of WFA. After 3 hr in 0.5 μM WFA, the retraction is less extensive (Fig. 1B) when compared to 1.0 μM WFA, which causes more extensive retraction (Fig. 1C). Eighty-two percent (82±1.04%, n=315) of cells treated with 2 μM WFA show an extensive reorganization of VIF into the perinuclear region after 3 hr. In addition, a significant number of non-filamentous vimentin particles and short IF or squiggles, both precursors to long IF, are observed between the juxtanuclear aggregates and the cell surface [12]. These latter structures which represent normal intermediate steps in the disassembly/assembly of mature VIF are significantly more visible following WFA treatment. In addition, the reorganization of VIF involves several intermediate stages of retraction seen at one and two hours following exposure to 2 μM WFA, prior to the formation of the large perinuclear aggregates (Fig. 1D–F). The effects of WFA treatment are reversible following 3 hr of exposure. The normal organization of the VIF network is gradually re-established in the majority of cells within 6 to 9 hrs following removal of WFA from the cell culture medium (Fig. 2 B, C). We use 2 μM WFA in BJ-5ta cells throughout the rest of this study, unless otherwise noted, as it has a maximal reversible impact on VIF organization over short time periods.


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 treatment inhibits cell motility.(A) The average speed of BJ-5ta fibroblasts was calculated before treatment (white bar), during incubation with 2 μM WFA for 4 hrs (black bars) and after the cells were allowed to recover in fresh medium (gray bars). (B and C) Cells were treated with 2 μM WFA for 3 hrs and then placed into fresh medium followed by fixation and processing for immunofluorescence with vimentin antibodies after 6h rs (B) and 9 hrs (C). Scale bars =10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0039065-g002: WFA treatment inhibits cell motility.(A) The average speed of BJ-5ta fibroblasts was calculated before treatment (white bar), during incubation with 2 μM WFA for 4 hrs (black bars) and after the cells were allowed to recover in fresh medium (gray bars). (B and C) Cells were treated with 2 μM WFA for 3 hrs and then placed into fresh medium followed by fixation and processing for immunofluorescence with vimentin antibodies after 6h rs (B) and 9 hrs (C). Scale bars =10 μm.
Mentions: The effects of withaferin A (WFA) on vimentin IF (VIF) networks were determined in human fibroblasts (BJ-5ta) by immunofluorescence. In controls, VIF networks extend throughout the cytoplasm, from the nucleus to peripheral regions (Fig. 1A). After a three-hour exposure to WFA, VIF networks retract from the cell periphery towards the nucleus. The extent of retraction varies with the concentration of WFA. After 3 hr in 0.5 μM WFA, the retraction is less extensive (Fig. 1B) when compared to 1.0 μM WFA, which causes more extensive retraction (Fig. 1C). Eighty-two percent (82±1.04%, n=315) of cells treated with 2 μM WFA show an extensive reorganization of VIF into the perinuclear region after 3 hr. In addition, a significant number of non-filamentous vimentin particles and short IF or squiggles, both precursors to long IF, are observed between the juxtanuclear aggregates and the cell surface [12]. These latter structures which represent normal intermediate steps in the disassembly/assembly of mature VIF are significantly more visible following WFA treatment. In addition, the reorganization of VIF involves several intermediate stages of retraction seen at one and two hours following exposure to 2 μM WFA, prior to the formation of the large perinuclear aggregates (Fig. 1D–F). The effects of WFA treatment are reversible following 3 hr of exposure. The normal organization of the VIF network is gradually re-established in the majority of cells within 6 to 9 hrs following removal of WFA from the cell culture medium (Fig. 2 B, C). We use 2 μM WFA in BJ-5ta cells throughout the rest of this study, unless otherwise noted, as it has a maximal reversible impact on VIF organization over short time periods.

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