Fig. 2
PPH has antiproliferative activity in vitro and ex vivo. (a) Time-lapse tubulin polymerization assay showing PTX (5 µM) and PPH (5 µM) induce polymerization of microtubules in vitro and eliminate the nucleation phase. Data represent mean ± SD of three independent experiments. Statistical significance between groups was determined with one-way ANOVA and Tukey’s multiple comparisons test. (b) 3-day PPH treatment dose-dependently inhibits growth of cancer cell cultures with EC50 values in the low micromolar range indicated between brackets. (c) GRinf represents the normalized growth rate inhibition of the drug at infinite concentration and shows a cytotoxic effect (GRinf < 0), cytostatic (GRinf = 0) or partial growth inhibition (GRinf > 0) dependent on cell type. (d) Combining PTX and PPH leads to an additive effect in SKOV3 cells. Heatmap represents mean of 3 biological replicates (N = 3). δ-score < -10: antagonistic effect, -10 < δ-score < 10 additive effect, 10 < δ-score synergistic effect. Dotted square in 2-dimensional heatmap indicates the most synergistic area. (e) PPH reduces colony forming capacity of SKOV3 cells. Cells were treated with PTX (1 nM, 3 nM and 10 nM) or PPH (0.1 µM, 1 µM and 2.5 µM) . Samples were normalized to vehicle control. Bar plot represents mean ± SD of three biologically independent experiments. Statistical significance between groups was determined with one-way ANOVA and Tukey’s multiple comparisons test. (f) EC50 curve showing dose-dependent reduction in metabolic activity of SKOV3 spheroids after 5-day treatment with PTX or PPH. EC50 curve represents mean ± SD of two biologically independent experiments (N = 2) with each 3 technical replicates (n = 3). (g,h) Violin plot indicating the reduction in metabolic activity following 5 days of ex vivo treatment with PTX (0.1 µM) or PPH (50 µM) of 4T1 tumor fragments (g, n = 24) or soft tissue sarcoma (SAR183) (h, n = 16). Statistical significance between groups was determined with a Kruskal–Wallis test and Dunn’s multiple comparisons test. (i) Immunohistochemical Ki67 staining of ex vivo cultured 4T1 tumor fragments after 5 days of ex vivo treatment with PPH (50 µM). Images represent technical replicates. Levels of statistical significance are indicated as * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P < 0.0001. DMSO was used as vehicle control.
