Withaferin a alters intermediate filament organization, cell shape and behavior

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.

Figure 1. WFA treatment alters the subcellular organization of VIF.

BJ-5ta cells were treated for 3 hrs (A–C and F) with DMSO alone (A), 0.5 μM WFA (B), 1 μM WFA (C), and 2 μM WFA (F). In addition, cells treated with 2 μM WFA are depicted after 1 hr (D) and 2 hrs (E) which show that the changes in VIF organization take place gradually. Cells were fixed and processed for immunofluorescence with vimentin antibodies. Arrow: a region depicted at higher magnification in the inset, showing non-filamentous vimentin particles and short IF or squiggles. Scale bars =10 μm.

Figure 2. 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.

Figure 3. Cysteine-328 is not required for the effects of WFA on VIF.

SW13-1HF5 cells which are null 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.

Figure 4. WFA treatment induces an increase in the phosphorylation of vimentin serine-38.

BJ-5ta cells were treated for 3 hrs with DMSO (A) or 2 μM (B) WFA, then fixed and double labeled with vimentin (A′ and B′) and pSer38 vimentin (A′′ and B′′) antibodies. Scale bars =10 μm. Arrow: a region depicted at higher magnification in the inset showing vimentin particles stained with pSer38 vimentin antibody. (C) Whole cell lysates of cells treated with DMSO (ctrl) or 2 μM WFA for 60 min, 120 min, and 180 min, were separated by SDS-PAGE and stained with anti-vimentin and anti-vimentin pSer38 vimentin antibodies.

Figure 5. WFA does not affect the sedimentation velocity of vimentin.

Sedimentation velocity profile of vimentin (0.15 mg/ml) reconstituted in 5 mM Tris-HCl, pH 8.4 (dashed lines) or 2 mM NaCl, pH 7.5 buffer (solid lines) alone (red lines), with DMSO (green lines), or with 25 μM WFA dissolved in DMSO (blue lines). Note that the curves of the two groups of runs are practically superimposable indicating that DMSO and DMSO plus WFA do not have any effect on the complex formation of vimentin oligomers.

Figure 6. 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).

Figure 7. WFA affects the organization of keratin and neuron specific IF.

MCF-7 cells, which express only keratin IF, were treated for 3 hrs with DMSO (A) and 4.0 μM WFA (B; the lowest effective concentration in these cells) and stained with pan-cytokeratin antibodies. Differentiated PC12 cells were treated for 3 hrs with DMSO (C) or 1.0 μM WFA (D, the lowest effective concentration for these cells), then stained with neurofilament-M (C′ and D′) and peripherin antibodies (C′′ and D′′). Scale bars =10 μm.

Figure 8. 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.

Figure 9. 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.

Figure 10. WFA also alters the organization of microtubules and microfilaments.

BJ-5ta cell were treated with DMSO (A and C) and 2 μM WFA (B and D) for 3 hrs and stained with vimentin antibodies (A′, B′, C′, D′), tubulin antibodies (A′′ and B′′), and phalloidin to visualize to actin (C′′ and D′′). Scale bars =10 μm.