Fig. 3
Tc52 blocks cells in G2 and alters tubulin network. Treatment of HeLa tumor cells with toxins targeting different steps in M-phase in combination with Tc52. M-phase drugs were administered in increasing concentrations either alone or together with 6 μM Tc52 (EC50) for 24 h. a: Viability assays from treatments with cytochalasin B (CytB), colcemid (Col) or docetaxel (Doc) alone and in combination with 6 μM Tc52. Tc52 itself reduced viability to 77.1 % +/− 1.78 SEM. Tc52 together with CytB displays additive toxicity to CytB alone (parallel curves), whereas Tc52 acts protective in combination with high doses of Col or Doc, as evident from the Combination Index calculated by the algorithm of Chou and Talalay [38] (last panel, Col +/− 0.023 SEM, Doc +/− 0.032 SEM, compared to hypothetical value 1). b: Cell-cycle analysis of single and combinatory treatments after 24 h application of 4 μM cytochalasin B (CytB), 27 nM colcemid (Col) and 50 nM docetaxel (Doc) alone or in combination with 6 μM Tc52. Single treatments were compared to control or to the respective combination. Significance was calculated by two-way ANOVA with Dunnett's Multiple Comparison Test p < 0.05 = *, p < 0.01 = **, p < 0.001 = *** for comparison of treatments to control, and with two-tailed T-test between single and double treatments. Tc52 has no impact on cell-cycle profile, Col increases number of cells in G2/M (1.5-fold), accompanied by a drop in G1 (1.5-fold). This is more pronounced with CytB (1.8-fold and 28.8-fold, respectively) or Doc (2.3-fold and 23.3-fold, respectively). Cells in S-phase are significantly reduced, 6-fold with CytB and 3.6-fold with Doc. SubG1 is increased for Col (12.4-fold), CytB (6.8-fold) and Doc (17-fold). Tc52 addition does not change cell-cycle profile of Col treatment, but reduces number of cells with >4 N content in CytB and Doc treated samples 1.8-fold and 1.3-fold compared to single treatments, respectively. G2/M content and SubG1 are increased only compared to CytB treatment 1.4-fold and 1.3-fold, respectively. Filled bars: G1; Open bars: S; Hatched bars: G2/M; Pointed bars >4 N; Vertical line bars: subG1. c: Cells were treated for 24 h with toxins as in 3b, fixed with formaldehyde and analyzed by DAPI staining for the presence of mitotic figures. 100 cells/experiment were evaluated (in docetaxel-treated samples only 50 cells/experiment) in independent triplicates. The percentage of mitotic cells (Mitotic Index, MI) was calculated and values compared to control (*). In addition, double treated samples were compared to single treatments (+). Significance was calculated by two-tailed T-test, p < 0.01 = **/++, p < 0.001 = ***/+++. CytB reduced MI 3.6-fold, Col and Doc enhanced MI 2.1-fold and 11.4-fold, respectively. Tc52 severely reduces MI in all cases. d: Immunofluorescence analysis of actin and tubulin in drug-treated HeLa cells. Cells were treated and fixed as described in 3c. Tubulin was detected by indirect immunofluorescence whereas f-actin was directly detected by Atto488-coupled phalloidin. Control cells (Ctr) show normal mitosis (filled arrow) and proper actin and tubulin network. Tc52-treated samples display changes in cell morphology such as tubulin bundles at the cell periphery (open arrow) and alterations of f-actin pattern. Cytochalasin B (CytB) changes actin distribution, inducing either aggregation or dim f-actin staining as well as binuclear cells (asterisk). Tubulin is unaffected. Double treated samples (Tc52/CytB) show a combination of changes in f-actin as well as tubulin networks (open arrow), but lack bi-nuclear cells. Colcemid-treated cells (Col) display improper mitotic spindle formation and cells blocked in metaphase (filled arrow). Double treated samples (Tc52/Col) are free of mitotic figures and display altered tubulin network (open arrow). Docetaxel (Doc) induces high levels of mitotic cells (filled arrow) and lobed nuclei, accompanied by strong staining of short tubulin fibers and mitotic spindles. As cells are blocked in mitosis, it was not possible to evaluated changes in the actin network. Double treated samples (Tc52/Doc) completely lack mitoses, and arrangement of tubulin fibers is improved and cells display proper actin network