Synergistic Effects of Doxorubicin and Quercetin on ROS-Associated Apoptosis and EGFR/FOXP3 Modulation in OVCAR3 Cells

Figures

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  Figure 1. Effects of DOX and Q on cell viability in OVCAR3 and HaCaT cells. Cells were treated with increasing concentrations of DOX (0–25 µM) and Q (0–200 µM) for 24 and 48 h. Cell viability was assessed by MTT assay and expressed as percentage relative to untreated control cells (mean ± SD, n = 3). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. (A) DOX treatment resulted in a dose- and time-dependent decrease in cell viability in both OVCAR3 and HaCaT cells. IC50 values were calculated as 4.38 µM (24 h) and 1.42 µM (48 h) in OVCAR3 cells, and 12.60 µM (24 h) and 7.03 µM (48 h) in HaCaT cells. (B) Q treatment showed a time-dependent reduction in cell viability. In OVCAR3 cells, IC50 values were not reached at 24 h and were calculated as 35.94 µM at 48 h. In HaCaT cells, IC50 values were not reached at 24 h and were calculated as 183.92 µM at 48 h.

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  Figure 2. Effects of NAC pretreatment on cell viability in OVCAR3 cells. OVCAR3 cells were pretreated with 5 mM NAC for 2 h prior to treatment with DOX (DOX; 1.42 µM), Q (Q; 35.94 µM), or their combination for 48 h. Cell viability was assessed using the MTT assay and expressed as a percentage relative to untreated control cells (mean ± SD, n = 3). NAC pretreatment increased cell viability in DOX-, Q-, and DOX+Q-treated groups compared with the corresponding treatments without NAC. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. Statistical significance is indicated as ** p < 0.01 and *** p < 0.001 versus the corresponding treatment without NAC.

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  Figure 3. CI analysis of DOX and Q in OVCAR3 cells. Cells were treated with DOX and Q at fixed concentration ratios for 48 h. Combination ratios (1:10, 1:20, and 1:50) were selected based on relative IC50 values and fixed-ratio combination design principles commonly used in CI-based synergy analyses. CI values were calculated using the Chou–Talalay method based on the median-effect principle. Data were derived from three independent biological replicates (n = 3). (A) CI values plotted as a function of fraction affected (Fa). CI values ranged from 0.68 to 0.29 across Fa levels of 0.25 to 0.90. (B) CI values calculated at fixed concentration ratios (1:10, 1:20, and 1:50) across multiple Fa levels (0.25, 0.50, 0.75, and 0.90). Values below 1 indicate synergistic interaction.

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  Figure 4. Isobologram analysis of DOX and Q combination in OVCAR3 cells. Cells were treated with DOX and Q alone or in combination at fixed concentration ratios for 48 h. The isobologram illustrates the dose combinations required to achieve fraction affected (Fa) levels of 0.5, 0.75, and 0.9. The diagonal lines represent the theoretical additive effect at each Fa level. Experimental combination points (closed symbols) located below the additive lines indicate synergistic interaction, whereas points above the lines would indicate antagonism. The green arrow indicates the direction toward the synergy region, where experimental combination points located below the theoretical additivity line reflect synergistic interactions between DOX and Q. In this analysis, combination data points were predominantly located below the additive lines across all tested Fa levels, consistent with a synergistic interaction between DOX and Q in OVCAR3 cells. Data are representative of three independent biological replicates (n = 3).

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  Figure 5. Apoptosis analysis of DOX, Q, and their combination in OVCAR3 cells by flow cytometry. OVCAR3 cells were treated with DOX, Q, or their combination at concentrations corresponding to their 48 h IC50 values. Apoptosis was assessed by Annexin V-FITC/PI staining followed by flow cytometry. Representative dot plots show the distribution of viable (Annexin V−/PI−), early apoptotic (Annexin V+/PI−), late apoptotic/necrotic (Annexin V+/PI+), and necrotic (Annexin V−/PI+) cell populations. The combination treatment was associated with an increased proportion of total apoptotic cells (early + late apoptosis) compared with single-agent treatments. Data are presented as mean ± SD from three independent biological replicates (≥20,000 events per sample, n = 3). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. Flow cytometric acquisition and compensation parameters are provided in Supplementary Table S1.

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  Figure 6. Caspase-3 and caspase-9 activities in OVCAR3 cells following treatment with DOX, Q, and their combination. OVCAR3 cells were treated with DOX, Q, or their combination at concentrations corresponding to their 48 h IC50 values. Data are presented as mean ± SD from three independent biological replicates (n = 3). (A) Caspase-3 activity expressed as fold change relative to control. DOX and Q treatments increased caspase-3 activity to 2.85-fold and 1.65-fold, respectively, while the combination treatment resulted in a 4.72-fold increase. (B) Caspase-9 activity expressed as fold change relative to control. DOX and Q treatments increased caspase-9 activity to 3.12-fold and 1.82-fold, respectively, whereas the combination treatment resulted in a 5.48-fold increase. Statistical significance is indicated as * p < 0.05, ** p < 0.01, and *** p < 0.001 versus control (one-way ANOVA followed by Tukey’s post hoc test).

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  Figure 7. Intracellular ROS levels in OVCAR3 cells following treatment with DOX, Q, and their combination. OVCAR3 cells were treated with DOX, Q, or their combination for 48 h. Intracellular ROS levels were assessed using the DCFH-DA fluorescent probe followed by flow cytometric analysis. Fluorescence intensity was measured in the FITC channel (excitation: 488 nm, emission: 525 nm), and ROS levels were expressed as fold change relative to control (mean ± SD, n = 3). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. Statistical significance is indicated as ** p < 0.01, and *** p < 0.001 versus control. Flow cytometric acquisition and compensation parameters are provided in Supplementary Table S1.

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  Figure 8. Effects of NAC pretreatment on intracellular ROS levels in OVCAR3 cells following DOX and Q treatment. (A) Intracellular ROS production in OVCAR3 cells treated with DOX, Q, and their combination (DOX+Q) at concentrations corresponding to their 48 h IC50 values. ROS levels were measured using the DCFH-DA fluorescence assay. The DOX+Q combination markedly increased ROS levels (6.82-fold) compared with control (1.00), while DOX and Q alone induced moderate increases (3.45-fold and 2.18-fold, respectively). (B) NAC rescue experiment. Cells were pretreated with 5 mM NAC for 2 h prior to DOX+Q co-treatment. NAC pretreatment reduced ROS levels from 6.82-fold (DOX+Q) to 1.52-fold, consistent with attenuation of oxidative stress. Data are presented as mean ± SD (n = 3). Statistical significance is indicated as ** p < 0.01 and *** p < 0.001 versus control (one-way ANOVA followed by Tukey’s post hoc test). ns, not significant versus control.

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  Figure 9. Immunocytochemical assessment of caspase-9 immunoreactivity in OVCAR3 cells following treatment with DOX, Q, and their combination. OVCAR3 cells were treated with DOX, Q, or their combination at concentrations corresponding to their 48 h IC50 values for 48 h. (A–D) Representative light microscopy images (200× magnification) showing caspase-9 immunostaining in control (A), Q-treated (B), DOX-treated (C), and DOX+Q-treated (D) cells. Scale bar = 20 µm. (E) Semi-quantitative analysis of caspase-9 immunoreactivity based on H-score evaluation. Data are presented as mean ± SD (n = 3). Statistical significance is indicated as * p < 0.05, ** p < 0.01, and *** p < 0.001 versus control (one-way ANOVA followed by Tukey’s post hoc test).

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  Figure 10. Effects of DOX, Q, and their combination on mRNA expression profiles in OVCAR3 cells. OVCAR3 cells were treated with DOX, Q, or their combination at concentrations corresponding to their 48 h IC50 values for 48 h. Relative mRNA expression levels were determined by RT-qPCR and normalized to GAPDH. Data are presented as fold change relative to control (mean ± SD, n = 3). (A) BAX, (B) BCL-2, (C) BAX/BCL-2 ratio, (D) NF-κB p65, (E) EGFR, and (F) FOXP3. Statistical significance is indicated as ns, non-significant; * p < 0.05, ** p < 0.01, and *** p < 0.001 versus control (one-way ANOVA followed by Tukey’s post hoc test).

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  Figure 11. Schematic representation of cellular responses associated with combined DOX and Q treatment in OVCAR3 cells. The diagram illustrates the proposed interactions between oxidative stress, mitochondrial signaling, and regulatory pathways following combination treatment. Increased intracellular ROS levels are linked to alterations in mitochondrial apoptotic balance, including modulation of BAX and BCL-2 and signaling events associated with caspase-9 and caspase-3 activation. In addition, changes in EGFR- and NF-κB-related signaling are depicted, reflecting potential effects on proliferation and survival pathways. The modulatory effect of NAC on ROS levels is also indicated, consistent with a modulatory role in treatment-associated cellular responses. Red double upward arrows (↑↑) indicate a marked increase or enhancement. Red downward arrows (↓) indicate a decrease or reduction. Black solid arrows indicate activation, induction, or progression of a biological process, whereas black dashed arrows indicate an indirect or putative association.