HDAC1, 2, 3 and XPO1 highly express in BC cells
Western blotting was used to detect the protein expressions of HDAC1, 2, 3, the target protein of tucidinostat, and XPO1, the target protein of selinexor, in human normal breast cells MCF 10A and BC cells MCF-7, MDA-MB-175, MDA-MB-134, T47D. As shown in Fig. 1, compared with MCF 10A, the protein expressions of HDAC1, 2, 3 and XPO1 in MCF-7, MDA-MB-175, MDA-MB-134, and T47D cells were significantly up-regulated (p < 0.05).
Effects of tucidinostat and selinexor on the BC cells viability
The MTT assay was used to detect the cells viability of MCF-7, MDA-MB-175, MDA-MB-134, and T47D at different concentrations of tucidinostat and selinexor, and the corresponding half maximal inhibitory concentration (IC50) values were calculated and showed in Fig. 2. The IC50 values of tucidinostat on MCF-7, MDA-MB-175, MDA-MB-134, and T47D were 13.6, 9.2, 24.4 and 19.3 µM, respectively. The IC50 values of selinexor on MCF- 7, MDA-MB-175, MDA-MB-134 and T47D were 2.6, 6.8, 9.7 and 12.0 µM, respectively. It can be seen that the IC50 values of tucidinosta or selinexor on MCF-7 and MDA-MB-175 cells were lower than those on MDA-MB-134 and T47D cells, suggesting that the cytotoxic effects of tucidinosta and selinexor on MCF-7 and MDA-MB-175 cells were better than those on MDA-MB-134 and T47D cells. In addition, selinexor obviously had lower IC50 values for all types of BC cells compared with tucidinostat.
The MTT assay was also used to observe the effect of combined intervention of tucidinostat and selinexor on the cells viability of MCF-7, MDA-MB-175, MDA-MB-134, and T47D. As shown in Fig. 3, for MCF-7 and MDA-MB-175 cells, the inhibitory effects of tucidinostat combined with selinexor on cells viability were significantly better than that of tucidinostat or selinexor alone (p < 0.05). Their corresponding CI plot analysis showed synergistically inhibition at the majority of concentrations. However, for MDA-MB-134 and T47D cells, the inhibitory effects of tucidinostat combined with selinexor on cells viability did not show better than that of single-agent intervention significantly (p > 0.05). Their corresponding CI plot analysis also showed additively inhibition at the majority of concentrations. The combined drug treatment of 20 µM tucidinostat and 10 µM Selinexor, which combination has lowest CI values, marked a transition from drug concentrations that prevented the growth of MCF-7 and MDA-MB-175 only to a concentration that effectively prevented the growth of cancer cells. Therefore, we studied the combined administration of this corresponding concentration later.
Effects of tucidinostat combined with selinexor on the proliferations of wt-TP53 BC cells
To observe the effects of continuous intervention of tucidinostat combined with selinexor for 5 days on the proliferations of wt-TP53 BC cells. As shown in Fig. 4, tucidinostat or selinexor alone could significantly inhibit the number of MCF-7 and MDA-MB-175 cells after 5 days of intervention (p < 0.05 vs. Control). Among them, the inhibitory effects of selinexor on the proliferations of MCF-7 and MDA-MB-175 cells was significantly better than that of tucidinostat (p < 0.05 vs. Tucidinostat). The intervention of tucidinostat combined with selinexor could significantly enhance the proliferation inhibitory effects on MCF-7 and MDA-MB-175 cells (p < 0.05 vs. Selinexor).
Effects of tucidinostat combined with selinexor on the invasion of wt-TP53 BC cells
Transwell was used to observe the effects of tucidinostat combined with selinexor on the invasion of MCF-7 and MDA-MB-175 cells after 24 h intervention. As shown in Fig. 5, tucidinostat or selinexor alone could significantly inhibit the invasion of MCF-7 and MDA-MB-175 cells after 24 h interventions (p < 0.05 vs. Control). Among them, the inhibitory effects of selinexor on the invasion of MCF-7 and MDA-MB-175 cells were significantly better than that of tucidinostat (p < 0.05 vs. Tucidinostat). The combination of selinexor could significantly enhance the cell invasion inhibitory effects of tucidinostat on MCF-7 and MDA-MB-175 cells (p < 0.05).
Effects of tucidinostat combined with selinexor on the apoptosis of wt-TP53 BC cells
Flow cytometry was used to observe the effects of tucidinostat combined with selinexor on the apoptosis of MCF-7 and MDA-MB-175 cells after 5 days of intervention. As shown in Fig. 6, tucidinostat or selinexor alone could significantly promote the apoptosis of MCF-7 and MDA-MB-175 cells (p < 0.05 vs. Control). Among them, the promoting effects of selinexor on the apoptosis of MCF-7 and MDA-MB-175 cells were significantly better than that of tucidinostat (p < 0.05 vs. Tucidinostat). The combination of selinexor could significantly enhance the apoptosis promotion effects of tucidinostat on MCF-7 and MDA-MB-175 cells (p < 0.05).
Effects of tucidinostat combined with selinexor on the protein expressions in wt-TP53 BC cells
Western blotting was used to detect the expression levels of acetylated p53, nuclear p53, total p53, p21, Cyclin D1, Bcl-2 and Bax in MCF-7 and MDA-MB-175 cells after 5 days of intervention by tucidinostat and selinexor. As shown in Fig. 7, tucidinostat intervention could significantly up-regulate the expression levels of acetylated p53, nuclear p53 and total p53 in MCF-7 and MDA-MB-175 cells (p < 0.05 vs. Control), and significantly promote the expressions of p21 and Bax, while the expressions of Cyclin D1 and Bcl-2 protein were suppressed (p < 0.05 vs. Control). Selinexor had no significant effect on the expression of acetylated p53 (p > 0.05 vs. Control), but its effect on up-regulating the expression of nuclear P53 protein was stronger than that of tucidinostat (p < 0.05 vs. Tucidinostat). Selinexor could also significantly up-regulate the expressions of P21 and Bax, and down-regulate the expressions of Cyclin D1 and Bcl-2 (p < 0.05 vs. Control). The combination of tucidinostat and selinexor could further affect the expressions of above protein accordingly (p < 0.05 vs. the groups of Tucidinostat or Selinexor).