Rosmarinic Acid Enhances Doxorubicin Activity in MDA-MB-231 Cells: Associations with Intracellular Oxidative Stress, Mitochondrial Dysfunction, and Apoptosis

Figures

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  Figure 1. Concentration-dependent effects of RA and DOX on the viability of MDA-MB-231 and HaCaT cells after 48 h of treatment. (A) Dose–response curve showing the effect of RA on MDA-MB-231 cell viability and the corresponding IC50 value (188.4 ± 4.2 µM). (B) Dose–response curve illustrating the cytotoxic activity of DOX in MDA-MB-231 cells with an IC50 value of 1.2 ± 0.1 µM. (C) Dose-dependent reduction in the viability of HaCaT cells following RA treatment, with an IC50 value of 146.6 ± 6.8 µM. (D) Dose–response curve of DOX in HaCaT cells demonstrating an IC50 value of 2.6 ± 0.1 µM. Cell viability was determined using the MTT assay and expressed as the percentage of untreated control cells. Symbols represent experimental data, while dashed curves indicate the nonlinear regression model used for the determination of IC50. The horizontal dotted line denotes 50% cell viability, and the vertical dashed line indicates the calculated IC50 value. Data are presented as the mean ± SD of three independent biological experiments (n = 3). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple-comparisons post hoc test. Differences were considered significant relative…

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  Figure 2. Pharmacological interaction between RA and DOX determined using the Chou–Talalay method. Drug interactions were evaluated using a fixed RA:DOX molar ratio of 157:1 based on the respective MDA-MB-231 IC50 values. CI values at the specified effect levels and DOX DRI values at the median-effect level were derived from the fitted fixed-ratio concentration–response model. (A) CI values calculated at the IC25, IC50, and IC75 effect levels for MDA-MB-231 and HaCaT cells. CI values < 0.90 indicate synergism, values from 0.90 to 1.10 indicate an additive interaction, and values > 1.10 indicate antagonism. (B) Model-derived DRI values for DOX at Fa = 0.50 in each cell line. DRI values represent descriptive comparisons of the single-agent and combination doses predicted to produce the same effect and do not indicate clinical dose reduction. (C) Fa–CI plot illustrating the relationship between the fraction affected and the calculated pharmacological interaction. (D) Summary table presenting CI values at the specified effect levels and median-effect DOX DRI values for both cell lines. CI data are presented as the mean ± SD of three independent biological experiments (n = 3); DOX DRI values…

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  Figure 3. Intracellular ROS-associated fluorescence assessed using DCFH-DA in MDA-MB-231 cells after RA and DOX treatment. (A) Representative DCFH-DA fluorescence microscopy images of untreated control cells and cells treated with N-acetyl-L-cysteine (NAC; 5 mM), RA (188.4 µM), DOX (1.2 µM), RA + DOX, NAC + RA, NAC + DOX, and NAC + RA + DOX for 48 h. Intracellular ROS-associated fluorescence is indicated by green DCF fluorescence. The RA + DOX combination produced the highest fluorescence intensity, whereas NAC pretreatment markedly reduced intracellular ROS-associated fluorescence in all treatment groups. Scale bar = 50 µm. (B) Quantitative analysis of intracellular ROS-associated DCF fluorescence. Fluorescence values were normalised to the untreated control group and presented as fold change relative to the control. Data are presented as the mean ± SD of three independent biological experiments (n = 3). Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple-comparisons post hoc test. ** p < 0.01 and *** p < 0.001 versus the untreated control group; ### p < 0.001 versus the RA + DOX group.

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  Figure 4. Annexin V-FITC/PI flow-cytometric analysis of apoptosis in MDA-MB-231 cells following 48 h treatment with RA (188.4 µM), DOX (1.2 µM), or their combination (188.4 µM RA + 1.2 µM DOX). (A) Representative Annexin V-FITC/PI dot plots. Cell populations were classified as viable (Annexin V−/PI−), early apoptotic (Annexin V+/PI−), late apoptotic/PI-positive (Annexin V+/PI+), and necrotic (Annexin V−/PI+). (B) Replicate-level quantification of total apoptosis, calculated as the sum of the early- and late-apoptotic populations. Total apoptosis increased from 5.2 ± 0.3% in control cells to 22.4 ± 0.6%, 34.7 ± 0.7%, and 42.8 ± 0.8% following RA, DOX, and RA + DOX treatment, respectively. Bars represent the mean ± SD of three independent biological experiments (n = 3), and individual points represent the values obtained in each experiment. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple-comparisons test. Comparisons are shown relative to the untreated control (*** p < 0.001).

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  Figure 5. Cell-cycle distribution of MDA-MB-231 cells following 48 h treatment with RA (188.4 µM), DOX (1.2 µM), or their combination (RA + DOX; 188.4 µM RA + 1.2 µM DOX). (A) Representative PI flow-cytometry histograms illustrating DNA-content profiles and the distribution of cells in the Sub-G1, G0/G1, S, and G2/M phases. (B) Quantitative analysis of the percentage of cells in each cell-cycle phase. In the RA-treated group, the corrected Sub-G1, G0/G1, S, and G2/M distributions were 12.5%, 52.1%, 18.4%, and 17.0%, respectively, corresponding to a total of 100.0%. Data are presented as the mean ± SD of three independent biological experiments (n = 3). Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple-comparisons post hoc test. Statistical comparisons were made with the untreated control group (* p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant).

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  Figure 6. JC-1 flow-cytometric analysis of mitochondrial membrane potential (ΔΨm) in MDA-MB-231 cells following 48 h treatment with RA (188.4 µM), DOX (1.2 µM), or their combination (188.4 µM RA + 1.2 µM DOX). (A) Representative JC-1 flow-cytometry plots generated from 10,000 acquired events per experimental group. JC-1 red fluorescence represents mitochondrial JC-1 aggregates associated with preserved membrane potential, whereas green fluorescence represents JC-1 monomers associated with mitochondrial membrane depolarisation. (B) Replicate-level quantification of the JC-1 red/green fluorescence ratio. The ratio decreased from 3.82 ± 0.09 in control cells to 1.81 ± 0.05, 1.22 ± 0.04, and 0.94 ± 0.04 following RA, DOX, and RA + DOX treatment, respectively. Bars represent the mean ± SD of three independent biological experiments (n = 3), and individual points represent values obtained in each experiment. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple-comparisons test. Comparisons are shown relative to the untreated control (*** p < 0.001).

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  Figure 7. Immunocytochemical evaluation of cleaved Caspase-9-associated immunoreactivity in MDA-MB-231 cells after 48 h of treatment with RA (188.4 µM), DOX (1.2 µM), and their combination (RA + DOX; 188.4 µM RA + 1.2 µM DOX). (A) Representative immunocytochemical images showing cleaved Caspase-9-associated immunoreactivity in untreated control cells and treated groups. Immunoreactive cells were identified by brown cytoplasmic DAB staining, whereas the nuclei were counterstained with hematoxylin. Weak immunoreactivity was observed in the control group, while RA and DOX treatments increased cytoplasmic immunoreactivity. The strongest immunoreactivity was detected in the RA + DOX combination group. These findings were interpreted as treatment-associated differences in cleaved Caspase-9-associated immunoreactivity rather than definitive evidence of Caspase-9 activation. The images were acquired using a light microscope at 100× magnification. Scale bar = 100 µm. (B) Quantitative analysis of cleaved Caspase-9-associated immunoreactivity using the H-score method. H-score values increased from 22.3 ± 8.7 in the control group to 124.5 ± 12.4 after RA treatment, 186.2 ± 15.8 after DOX treatment,…

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  Figure 8. Nuclear morphological assessment of MDA-MB-231 cells following 48 h treatment with RA (188.4 µM), DOX (1.2 µM), and their combination (RA + DOX; 188.4 µM RA + 1.2 µM DOX) (A) Representative NucBlue fluorescence images showing nuclear morphology in untreated control cells and treated groups. Cell nuclei are visualized by blue fluorescence. Untreated control cells exhibited uniformly stained nuclei with regular morphology and high cellular density. Treatment with RA and DOX reduced cell density and increased nuclear condensation, while the combination of RA + DOX produced the most pronounced nuclear condensation and nuclear fragmentation, consistent with apoptotic cell death. The images were acquired using a fluorescence microscope at 100× magnification. Scale bar = 100 µm. (B) Quantitative analysis of apoptotic nuclear morphology. The percentage of nuclei exhibiting apoptotic features, including chromatin condensation and nuclear fragmentation, was determined following NucBlue staining. Data are presented as the mean ± SD of three independent biological experiments (n = 3). Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple-comparisons post hoc…

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  Figure 9. Relative expression of apoptosis-related genes in MDA-MB-231 cells after 48 h of treatment with RA (188.4 µM), DOX (1.2 µM), and their combination (RA + DOX; 188.4 µM RA + 1.2 µM DOX). Gene expression levels were normalised to the untreated control group and are presented as a fold change relative to the control. The combination of RA + DOX significantly increased the expression of BAX, CASP9, CASP3, and TP53, while the expression of CDKN1A (p21) showed a modest and non-significant increase. Data are presented as the mean ± SD of three independent biological experiments (n = 3). The dashed horizontal line indicates the normalised control expression level (fold change = 1.0). *** p < 0.001 versus the untreated control group; ns, not significant.

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  Figure 10. Relative expression of anti-apoptotic and proliferation-associated genes mRNA in MDA-MB-231 cells after 48 h of treatment with RA (188.4 µM), DOX (1.2 µM), and their combination (RA + DOX; 188.4 µM RA + 1.2 µM DOX). Gene expression is presented as the percentage decrease relative to the untreated control group. The combination of RA + DOX significantly reduced the expression of BCL2, MKI67, and PCNA. Data are presented as the mean ± SD of three independent biological experiments (n = 3). *** p < 0.001 versus the untreated control group.

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  Figure 11. Bioinformatic analysis of apoptosis-related genes associated with RA + DOX treatment. (A) PPI network generated using STRING based on the experimentally assessed genes TP53, BAX, BCL2, CASP9, and CASP3, together with CYCS as a pathway-associated contextual node. CYCS expression and cytochrome-c release were not experimentally evaluated. (B) GO enrichment analysis showing the principal biological processes associated with the selected gene set. (C) KEGG enrichment analysis identifying the significantly enriched signalling pathways. (D) Conceptual representation of the predicted TP53–BAX/BCL2–CYCS–CASP9–CASP3 pathway. The bioinformatic findings are hypothesis-generating and do not constitute experimental validation of CYCS expression, cytochrome-c release, or pathway activation. The red dashed lines in panels B and C indicate the significance threshold [−log10(p) = 3]. The white arrows in panel D indicate the predicted directional relationships among pathway components.