Histone acetylation modifications in cancer biology. (a) The chemical structure of selisistat (EX527) – sirtuin 1 (SIRT1) inhibitor. (b) Cellular localisation of SIRT1. (c) Inhibition of histone deacetylation (DEACETYLATION OFF) (catalyzed by SIRT1 (HDAC)) through EX527. After inhibition, HDACs are unable to remove acetyl groups (Ac) from the N-terminus lysine residues. This leads to an opened state of the chromatin and increases transcriptional activity of RNA polymerase II (RNAPII).

Evaluation of single drug-mediated effects on the viability of the (a) MCF7 and (b) T47D luminal A BC, (c) BJ normal, and (d) MDA-MB-231, (e) BT-549 and (f) MDA-MB-468 BC cells in the MTT assay. BC cells were exposed for 96 h to increasing EX527 concentrations (10–200 µM). The data are presented as the mean ± standard deviation (±SD) of the mean. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA, Tukey’s post-hoc test.

Evaluation of single or combined drug-mediated effects on the proliferation of (a) MCF7, (b) T47D, (c) MDA-MB-231, (d) BT-549, (e) MDA-MB-468 BC cells in the BrdU assay. BC cells were exposed for 48 h to 1/2 and IC₅₀ PAX, EX527 or 1:1 PAX:EX527 (1/2 and IC_{50 mix)}. The data are presented as the mean ± standard deviation (±SD) of the mean. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA, Tukey’s post-hoc test.

Isobolographic analysis of interaction between PAX and EX527 for parallel (a) T47D, (b) BT-549, (c) MDA-MB-468 and non-parallel (d) MCF7, (e) MDA-MB-231 concentration-response effects in BC cells in the MTT assay. The additive interactions between PAX and EX527 are displayed on isobolograms in T47D, BT-549, MDA-MB-468, MCF7 and MDA-MB-231 BC cells. The dotted diagonal line starting from the point (0,0) corresponds to the mixture of PAX and EX527 at the fixed ratio of 1:1. The points: A (on graphs A-C); A’ and A” (on graphs D-E) depicts the theoretically calculated IC_{50 add} values for the isoboles of additivity for parallel and non-parallel concentration-response effects. The points M represents the IC_{50 mix} values for the total concentrations of the experimental mixture of PAX and EX527 that exerted a 50% anti-viability effect (50% isobole) in the MTT assay. The SEM values are illustrated as horizontal and vertical error bars for every IC₅₀ value. The combinations of PAX with EX527 in all analysed BC cell lines were additive in the MTT test because the IC_{50 mix} values for these combinations are placed close to the points A, A’ or A’’. For more detailed information on the isobolographic representation of the data, please refer to²⁹.

The percentage of zebrafish embryos initiated 2 days post-fertilization (dpf) that survived after 1, 2 or 3 days of treatment with indicated concentrations of (a) EX527, (b) PAX and (c) EX527:PAX (IC_{50 mix}). IC₅₀ drug doses refer to the effective therapeutic concentrations were determined earlier by the in vitro MTT assay. Analyses were performed in triplicates, n = 15 per group. Data are presented as mean ± standard deviation of the mean (SD), ***p < 0.001 determined by one-way ANOVA, Tukey’s post-hoc test.

In vivo experiments with zebrafish xenografted model. (a) Drug screening scheme with zebrafish larvae model. (b) The impact of 1/8 IC₅₀ EX527, PAX and the combination of both compounds (1/8 IC_{50 mix}) on T47D xenografted zebrafish model after 3 days of treatment. The representative images of Vybrant^™ DiD-stained tumour cells after treatment were illustrated. (c) Relative expression of human GAPDH versus zebrafish gapdh in zebrafish larvae after 1/8 IC₅₀ EX527, PAX and EX527:PAX (1/8 IC_{50 mix}) treatment. Data are presented as mean ± standard deviation of the mean (SD), ***p < 0.001 determined by one-way ANOVA, Tukey’s post-hoc test.

Schematic representation of molecular interaction pathways between (a) SIRT1 or (b) SIRT3 with tubulin, as indicated by SIGNOR. The diagram showcases the proteins and molecules involved in the interaction network between molecular targets of EX527 and PAX. Different shapes and colours denote the nature of the entities, as explained in the legend panel. (c) The effects of PAX, SIRT and PAX:SIRT (1/8 IC₅₀ or 1/8 IC_{50 mix}) on the gene expressions in zebrafish xenografted BT-549 TNBC in vivo model after 72 treatment and analysed by the qRT-PCR. *p < 0.05, **p < 0.01 by one-way ANOVA, Tukey’s post-hoc test.

Experimentally solved structures of EX527 analogue bound to (a) SIRT1, as revealed by the PDB entry 4I5I. (b) EX527 bound to SIRT3 in the PDB entry 4BVH, (c) Taxol molecule bound to a tubulin unit, as presented by the PDB entry 6WVR. The distinct chemical nature of the ligands, variations in size, and differences in the characteristics of their respective binding pockets render it improbable for them to engage in competition for the same binding site.

Evaluation of single or combined drug-mediated effects on the induction of apoptosis in T47D BC cells. (a) The % of cells with active caspase-3 determined after PAX and EX527 treatment for 48 h using selected ratios of the IC₅₀ determined in the MTT assay. Data are presented as mean ± standard deviation (± SD) of the mean, *** p < 0.001 by one-way ANOVA, Tukey’s post-hoc test. (b) Representative dot plots from the FACS analysis of the T47D luminal-type BC cells after a 48h incubation with a medium PAX, EX527 or PAX:EX527 (1:1). R3-apoptotic cells with active caspase-3. (c) BAX relative gene expression in zebrafish T47D injected xenograft model after 72 h with 1/8 IC₅₀ EX527 or/and PAX treatment determined in qPCR method. *** p < 0.001 determined by one-way ANOVA, Tukey’s post-hoc test. (d) p21 relative expression in zebrafish T47D injected xenograft model after 72 h with 1/8 IC₅₀ EX527 or/and PAX treatment determined in qPCR method. *** p < 0.001 determined by one-way ANOVA, Tukey’s post-hoc test.

Effect of EX527 or/and PAX on the cell cycle progression in the MCF7 luminal BC cells. (a) BC cells were exposed to individual or concomitant EX527 and PAX treatment for 48 h using selected ratios of the IC₅₀ determined in the MTT assay, stained with propidium iodide (PI) and analysed by FACS. The data are presented as the means ± standard deviation (±SD). (b) CCND1 (cyclin D1) relative gene expression in zebrafish MCF7 injected xenograft model after 72 h with 1/8 IC₅₀ EX527 or/and PAX treatment determined in qPCR method. (c) Representative histograms from the FACS analysis of the MCF7 BC cells after a 48h incubation with EX527 or/and PAX. M1-subG1 (pre-G1) phase; M2-G1 phase; M3-S phase; M4-G2 phase.