Fig 1.
The anti-proliferative effects of HDIs and CDDP in breast cancer cell lines.
(A) The anti-proliferative effects of VPA, SAHA, and CDDP on T47D, MCF7, MDA-MB-231 breast cancer cell lines after 96 h treatment with various concentration (0.1–1000 μg/ml) of active substances. The cell viability was measured by the MTT assay. (B) The anti-proliferative effects of VPA, SAHA, and CDDP on T47D breast cancer cell line. The cell viability was measured by the MTT assay. T47D cells were exposed to concomitant or sequential VPA and CDDP treatment using different ratios of the IC50 (1/2 IC50, IC50, 3/2 IC50). VPA+CDDP—valproic acid and cisplatin concomitant, CDDP→VPA—cisplatin (1h) followed by valproic acid, VPA→CDDP—valproic acid (1h) followed by cisplatin. Statistical analysis was performed using one-way ANOVA test (*** p<0.001). The results are means ± standard deviation (SD). (C) Examination of cell proliferation by MTT assay. T47D cells were exposed to concomitant or sequential SAHA and CDDP treatment using different ratios of the IC50 (1/2 IC50, IC50, 3/2 IC50). SAHA+CDDP—vorinostat and cisplatin concomitant, CDDP→SAHA cisplatin (1h) followed by vorinostat, SAHA→CDDP—vorinostat (1h) followed by cisplatin. The results are presented as mean ± standard deviation (SD). Statistical analysis was performed using one-way ANOVA test (*** p<0.001).
Fig 2.
(A-F) Log-probit dose-response relationship curves (DRRCs) for HDIs and CDDP.
DRRCs for cisplatin (CDDP), suberoylanilide hydroxamic acid (SAHA) and valproic acid (VPA) administered alone, and in combinations at the fixed-ratio of 1:1 (in red), illustrating the anti-proliferative effects of the drugs in three cancer cell lines (MCF7, T47D and MDA-MB-231) measured in vitro by the MTT assay. Doses of CDDP, SAHA and VPA administered separately and the mixture of the drugs at the fixed-ratio combination of 1:1 (in red) were transformed into logarithms, whereas the anti-proliferative effects produced by the drugs in three cancer cell lines (MCF7, T47D and MDA-MB-231) measured in vitro by the MTT assay were transformed into probits according to Litchfield and Wilcoxon (1949). Linear regression equations of DRRCs are presented on the graph; where y—is the probit of response, and x—is the logarithm (to the base 10) of a drug dose, R2 –coefficient of determination. Test for parallelism revealed that the experimentally determined DRRCs for CDDP, SAHA and VPA (administered alone) are not parallel to one another (for more details see Table 1.).
Table 1.
Anti-proliferative effects of CDDP, SAHA and VPA administered singly in three breast cancer cell lines (MCF7, T47D and MDA-MB-231) measured in vitro by the MTT assay.
Table 2.
Type I isobolographic analysis of interactions (for non-parallel DRRCs) between CDDP and SAHA or VPA at the fixed-ratio combination of 1:1 in three cancer cell lines (MCF7, T47D and MDA-MB-231) measured in vitro by the MTT assay.
Fig 3.
(A-F) Isobolographic analysis of interactions between HDIs and CDDP.
Isobolograms showing additive interactions between cisplatin (CDDP), suberoylanilide hydroxamic acid (SAHA) and valproic acid (VPA) with respect to their anti-proliferative effects on three cancer cell lines (MCF7, T47D and MDA-MB-231) measured in vitro by the MTT assay. The median inhibitory concentrations (IC50) for CDDP, SAHA and VPA are plotted graphically on the X- and Y-axes, respectively. The solid lines on the X and Y axes represent the S.E.M. for the IC50 values for the studied drugs administered alone. The lower and upper isoboles of additivity represent the curves connecting the IC50 values for CDDP and SAHA or VPA administered alone. The dotted line starting from the point (0, 0) corresponds to the fixed-ratio of 1:1 for the combination of CDDP with SAHA or VPA. The diagonal dashed line connects the IC50 for CDDP and SAHA or VPA on the X- and Y-axes. The points A’ and A” depict the theoretically calculated IC50 add values for both, lower and upper isoboles of additivity. The point M represents the experimentally-derived IC50 mix value for total dose of the mixture expressed as proportions of CDDP and SAHA or VPA that produced a 50% anti-proliferative effect (50% isobole) in three cancer cell lines (MCF7, T47D and MDA-MB-231) measured in vitro by the MTT assay. On the graph, the S.E.M. values are presented as horizontal and vertical error bars for every IC50 value. The experimentally-derived IC50 mix value is placed close to the point A”, indicating additive interaction between CDDP and SAHA or VPA in three cancer cell lines (MCF7, T47D and MDA-MB-231) measured in vitro by the MTT assay.
Fig 4.
(A-F) Effects of HDIs and CDDP on caspase-3 activation in breast cancer cells.
Apoptotic changes induced by VPA or SAHA and CDDP alone or in combinations. Three breast cancer cell lines (T47D, MCF7, MDA-MB-231) were cultivated for 48 h with different doses of active drugs and their mixtures (2.0 = 100% IC50, 1.4 = 70% IC50, 1.0 = 50% IC50,) and analyzed by flow cytometry. The values present the percentage of the cells with active caspase-3. The results are depicted as means ± SD, n = 5 from 5 separate experiments. Statistical analysis was performed using one-way ANOVA test (*** p<0.001, ** p<0,01, * p<0,05).
Fig 5.
(A-F) Effects of CDDP and HDIs on cell cycle progression in breast cancer cells.
Effect of HDIs alone and in combination with CDDP on cell cycle in T47D, MCF7 and MDA-MB-231 cells. The cell lines were cultivated for 48 h with VPA or SAHA and CDDP (2.0 = 100% IC50) and analyzed by flow cytometry. The results are presented as mean + SD, n = 10 from five separate experiments.