Persistent inhibition of pore-based cell migration by sub-toxic doses of miuraenamide, an actin filament stabilizer

Abstract

Opposed to tubulin-binding agents, actin-binding small molecules have not yet become part of clinical tumor treatment, most likely due to the fear of general cytotoxicity. Addressing this problem, we investigated the long-term efficacy of sub-toxic doses of miuraenamide, an actin filament stabilizing natural compound, on tumor cell (SKOV3) migration. No cytotoxic effects or persistent morphological changes occurred at a concentration of miuraenamide of 20 nM. After 72 h treatment with this concentration, nuclear stiffness was increased, causing reduced migration through pores in a Boyden chamber, while cell migration and chemotaxis per se were unaltered. A concomitant time-resolved proteomic approach showed down regulation of a protein cluster after 56 h treatment. This cluster correlated best with the Wnt signaling pathway. A further analysis of the actin associated MRTF/SRF signaling showed a surprising reduction of SRF-regulated proteins. In contrast to acute effects of actin-binding compounds on actin at high concentrations, long-term low-dose treatment elicits much more subtle but still functionally relevant changes beyond simple destruction of the cytoskeleton. These range from biophysical parameters to regulation of protein expression, and may help to better understand the complex biology of actin, as well as to initiate alternative regimes for the testing of actin-targeting drugs.

Figure 1

Low dose treatment of SKOV3 with miuraenamide A (Miu) showed no effects on cell viability, proliferation and actin cytoskeleton morphology. (a) Cell viability after treatment for 72 h of SKOV3. (b) PI exclusion assay after treatment for 72 h with 20 nM Miu. (c) Cell cycle analysis. (d) Proliferation after treatment with increasing concentrations of Miu. (e) Actin staining of SKOV3 cells treated with 20 nM Miu for the indicated time points (rhodamine-phalloidin, red, and nucleus, Hoechst 33342, blue). White bars: 10 µm. ns, not significant. One-Way ANOVA/Bonferroni’s Multiple Comparison Test, *p < 0.05. n = 3.

Figure 2

Analysis of SKOV3 cell migration. SKOV3 cells were pre-treated with 20 nM Miu over 72 h, during the migration process no compound was present. (a) Boyden chamber migration assay over 24 h migration through a 8 µm pore membrane. Paired two tailed t-test, *p < 0.05. (b) Wound healing assay, neg Ctrl: negative control, migration in FCS free medium. One representative image per treatment is shown. (c) Chemotaxis assay over 24 h along an FCS gradient. FMI, forward migration index, +/+ Ctrl: positive control, medium with FCS on both sides, no gradient. One-Way ANOVA/Bonferroni’s Multiple Comparison Test, *p < 0.05. n = 3.

Figure 3

Analysis of migration associated parameters. (a) Adhesion and (b) Spreading of SKOV3 cells pre-treated with 20 nM Miu over 72 h were quantified after 90 min by counting adhered or spread cells. (c) HRP secretion assay with HeLa-ssHRP. Brefeldin A (BFA) was used as positive control, fresh Miu was added ( + 4 h) or not (−4 h) during secretion, n = 4. One-Way ANOVA/Bonferroni’s Multiple Comparison Test, *p < 0.05.

Figure 4

Atomic force measurements of Miu treated SKOV3 cells and Rat tail collagen I 3D gel migration of SKOV3 cells. Cells were treated for 72 h with 20 nM Miu. (a) Total cell stiffness was measured with a force map of 80 × 80 µm. (b) Nuclear stiffness measurement with single force curves in contact mode, 10–20 cells per experiment were measured. (c) Cells were pre-treated for 72 h with 20 nM miuraenamide (Miu) and migrated over 20 h in rat tail collagen I gels (2 mg/ml). Left panel: Quantification of cellular phenotypes normalized to total counted cell number (total no.). Right panel: Example images of cellular phenotypes recorded by life cell imaging with transmitted light microscopy (upper row) or fluorescence microscopy (rhodamine-phalloidin, red, and nucleus, Hoechst 33342, blue, lower row). Bars indicate 10 µm. Arrows indicate the nucleus deformed due to restriction. Paired two tailed t-test, *p < 0.05. ns: not significant, n = 4.

Figure 5

Time course of proteome changes after treatment with Miu. SKOV3 cells were treated with Miu 20 nM for indicated time points. (a) Heat plot with ten defined row clusters, red areas indicate up- and green areas indicate down-regulation, log2 scale. (b) Defined cluster up- and down-regulated proteins after 56 h treatment depicted in lanes as time series, log2 scale. (c) Dot plot of enriched pathways with a threshold of FDR < 0.5 showed down-regulated proteins after 56 h treatment, orange dots: FDR < 0.2, red dots: FDR < 0.2 and enrichment value > 2.

Figure 6

Proteome analysis of MRTF-associated genes and expression of p300 transcription co-activator. SKOV3 cells were treated with 20 nM miuraenamide (Miu) for 56 h. (a) MRTF-associated genes were selected by GeneCards® Keyword Search “MRTF”. Miu treated cells (black bars) or harvested at stimulation start point without treatment (Ctrl, white bars) were depicted, log2 scale. b: Protein level of p300 in the Western blot (upper panel with stain free gel as loading control) and quantification (lower panel). One representative image per treatment is shown, n = 4. Paired two tailed t-test, *p < 0.05. The Western blot is cropped; the complete blot is displayed in the Supplementary materials.