Cell culture and viability analysis

The study used two types of breast cancer cell lines: hormone-sensitive (MCF-7) and hormone-insensitive (MDA-MB-231). The MCF-7 and MDA-MB-231 cell lines were grown in DMEM (Dulbecco's Modified Eagle Medium) with 2 mM L-glutamine, 50 IU/mL penicillin, and 50 mg/mL streptomycin. The optimal cytotoxic concentration or concentrations for alone GEN and the non-cytotoxic optimal concentration for alone Poly I:C and GEN + Poly I:C combination treatment were assessed using the WST-1 viability assay. For this study, MCF-7 and MDA-MB-231 cells were seeded into 96-well cell culture plates at a density of 2 × 104 cells per well. The cells were then treated with Poly I:C (at 5 and 10 µM) [14] and GEN (at 5, 10, 25, 50, and 100 µM) [22] and incubated for 24, 48, and 72 h. Following this, 10 µL of WST-1 dye was added to each well and incubated for 1 h at 37 °C. Measurements were then taken using an Elisa Reader in the 460–620 nm wavelength range. The viability of control cells was considered 100%, and the viability rates of the experimental cells were expressed as percentages.

Annexin V analysis

To assess the apoptotic effect of GEN, Poly I:C, and GEN + Poly I:C combination on MCF-7 and MDA-MB-231 cells, 1 × 105 cells were seeded into a 6-well plate. After treatment with GEN, Poly I:C, and GEN + Poly I:C combinations, the cells were incubated at 37 °C with 5% CO2 for 24 h. Following the incubation, the cells were treated with trypsin at 37 °C with 5% CO2 for 5 min, centrifuged at 1200 rpm for 5 min, and washed twice with Phosphate Buffer Saline (PBS), each wash lasting 5 min. Annexin V dye was added to each tube containing the suspended cell groups and incubated in the dark for 30 min at room temperature. After incubation, each tube was analyzed using the Muse™ Cell Analyzer (Merck Millipore, Germany), and all experiments were repeated three times.

Cell cycle analysis

To study the impact of GEN, Poly I:C, and the combination of GEN and Poly I:C on cell cycle arrest in MCF-7 and MDA-MB-231 cells, 1 × 105 cells were seeded into a 6-well plate. After treatment with GEN, Poly I:C, and GEN + Poly I:C combinations, the cells were then incubated at 37 °C with 5% CO2 for 24 h. Subsequently, the cells were removed, fixed with 70% cold ethanol at −20 °C for at least 3 h, and then stained with 200 μL of Muse® Cell Cycle Reagent for 30 min following the protocol of the Muse® Cell Cycle Kit (Merck Millipore, Germany). The stained cell pellets were incubated for 30 min at room temperature in the dark and then analyzed using the Muse® Cell Analyzer (Merck Millipore, Germany). All experiments were repeated three times.

Acridine orange (AO) staining

To observe the apoptotic effect of GEN, Poly I:C, and GEN + Poly I:C combination on MCF-7 and MDA-MB-231 cells, 1 × 105 cells were seeded into a 6-well plate. After treatment with GEN, Poly I:C, and GEN + Poly I:C combinations, the cells were then incubated at 37 °C with 5% CO2 for 24 h. Following the incubation, the cells were washed twice with PBS each for 5 min, fixed with 4% Paraformaldehyde (PFA) for 30 min, and the wells were washed twice with PBS each for 5 min. Subsequently, the staining procedure was performed using Acridine Orange (Sigma-Aldrich, USA) solution for 30 min at room temperature in the dark. The stained wells were washed twice with PBS each for 5 min and then imaged using the EVOS™FLoid (Life Technologies, USA) cell imaging system.

Immunofluorescence (IF) analysis

To investigate the impact of GEN, Poly I:C, and the combination of GEN and Poly I:C on the cellular localization of TLR3, TRIF, IRF3, p-NF-kB, and AP1 proteins in MCF-7 and MDA-MB-231 cells, 1 × 105 cells were seeded into 6-well plates. Following treatment with GEN, Poly I:C, and GEN + Poly I:C combinations, the cells were then incubated at 37 °C and 5% CO2 for 24 h. After incubation, the cells were fixed with 4% PFA for 30 min, permeabilized using PBS containing 0.1% Triton X-100, and blocked with PBS containing 1% BSA and 5% goat serum for 30 min. Subsequently, the fixed cells were incubated with primary antibodies for 3 h, then washed twice with PBS each for 5 min, and incubated with Alexa Fluor 488-conjugated secondary antibody for 1 h. Finally, the nucleus was stained with DAPI and visualized using the EVOS™FLoid (Life Technologies, USA) cell imaging system.

Western blotting analysis

To determine the levels of TLR3 (sc-32232, Santa Cruz), TRIF (sc-514384, Santa Cruz), IRF3 (sc-33641, Santa Cruz), p-NF-kB (3033, Cell signaling), and AP1 (MA5-15,172; Thermo Scientific) proteins in MCF-7 and MDA-MB-231 cells with the combination of GEN, Poly I:C, and GEN + Poly I:C, 1 × 105 cells were seeded into T75 flasks. After treatment with GEN, Poly I:C, and GEN + Poly I:C combinations, the cells were incubated at 37 °C and 5% CO2 for 24 h. For protein isolation, the cells were collected by scraping from the flask and centrifuged at 12,000 rpm for 15 min. The obtained pellet was resuspended with a cold lysis buffer for 15 min; the total protein amount was determined using a protein amount determination kit (Thermo Scientific). β-Actin protein was used as the housekeeping gene. From the obtained protein stock, an appropriate amount of protein was diluted for loading, mixed with sample loading buffer, and separated by SDS-PAGE (4% stacking gel; 10% separating gel). The proteins in the gel were transferred to the membrane by electro-transfer (dry blotting system, Bio-Rad). Following the western blotting method, the membrane was shaken in blocking solution (5% milk powder, 0.05% Tween 20, 1 × TBS) for 30 min and washed three times with washing solution (0.1% Tween 20, 1 × TBS) each for 5 min. After this process, the primary antibody incubated for overnight at 4 °C and then the secondary antibody [Goat anti-Mouse (31,430) and Rabbit IgG (31,460) (H + L) Secondary Antibody, HRP (Thermo Scientific)] was incubated for 2 h at room temperature specifically for the proteins. The signal was detected with the imaging device (Syngene GBox) using the ECL (enhanced chemiluminescence) kit (Thermo Scientific).

Enzyme-linked immunosorbent Assay (ELISA)

To determine the effect of GEN, Poly I:C, and GEN + Poly I:C combination on INF-β and TNF-α levels in MCF-7 and MDA-MB-231 cells, 1 × 105 cells were seeded into 6-well plates after treatment with GEN, Poly I:C, and GEN + Poly I:C combination the cells were incubated at 37 °C and 5% CO2 for 24 h. After incubation, the medium was taken, and INF-β (E-EL-H0085) and TNF-α (E-EL-H0109) were determined with the ELISA kit according to the procedure recommended by the company (Elabscience).

Statistical analysis

Statistical analyses were performed using GraphPad (V9.0) software package programs. P values ​​less than 0.05 will be considered statistically significant. The intensities of the bands obtained from Western analysis will be determined using the Image J program. The normality of the data distribution was assessed using the Shapiro–Wilk test. Following confirmation of normal distribution, a two-way analysis of variance (ANOVA) was conducted to evaluate the effects of two independent variables. Post hoc comparisons between groups were performed using Tukey’s multiple comparison test.

Effect of GEN, Poly I:C, and GEN + Poly I:C combination on cell growth

The concentrations of GEN and Poly I:C, as well as the treatment durations (24, 48, and 72 h), were evaluated to identify conditions that affect cell viability in MCF-7 and MDA-MB-231 cells using WST-1 assays (Fig. 1). Poly I:C alone (5 and 10 µM) resulted in modest reductions in cell viability, suggesting limited cytotoxicity at these concentrations (Fig. 1A). Based on these results, 5 µM Poly I:C was selected for combination treatments to minimize toxicity while allowing potential TLR3 activation. GEN treatment led to a concentration- and time-dependent reduction in cell viability (Fig. 1B). GEN and 5 µM Poly I:C combination resulted in a slightly greater reduction in viability at higher GEN concentrations (especially 100 µM), particularly after 24 h (Fig. 1C). Although the observed effects of the combination were more pronounced than GEN alone, the differences were relatively modest. Thus, while the GEN + Poly I:C combination demonstrated enhanced cytotoxicity compared to individual treatments, the magnitude of this enhancement was limited. Thus, further studies are needed to confirm the biological significance and underlying mechanisms (Fig. 1D).

Fig. 1

Effect of Poly I:C and GEN on the viability of MCF-7 and MDA-MB-231 breast cancer cells. (a) Cell viability of MCF-7 and MDA-MB-231 cells treated with Poly I:C (5 and 10 µM) for 24, 48, and 72 h, (b) Effect of GEN treatment (5, 10, 25, 50, and 100 µM) on the viability of MCF-7 and MDA-MB-231 cells at different time points (24, 48, and 72 h), (c) Combined effect of GEN (5, 10, 25, 50, and 100 µM) and 100 µM Poly I:C on cell viability at 24, 48, and 72 h, (d) Comparison of cell viability between 100 µM GEN treatment alone and 100 µM GEN + 5 µM Poly I:C combination in both cell lines. Data are presented as mean ± standard deviation (n = 3). Statistical significance is indicated by *p < 0.01, **p < 0.05, ***p < 0.001, and ****p < 0.0001 compared to the control group

Genistein exhibits inducible effect on apoptotic cell death

Annexin V analysis was conducted to assess the apoptotic effects of GEN, Poly I:C, and their combination in MCF-7 and MDA-MB-231 cells (Fig. 2). In MCF-7 cells, the total apoptotic rate was 32.96 ± 0.09% following 100 µM GEN treatment, 12.61 ± 0.36% with 5 µM Poly I:C, and 35.53 ± 0.38% with the GEN + Poly I:C combination, compared to 10.43 ± 0.31% in the control group (p < 0.01). Similarly, in MDA-MB-231 cells, total apoptosis was 32.51 ± 0.36% upon GEN, 18.51 ± 0.41% upon Poly I:C, and 36.55 ± 0.46% upon the combination treatment, compared with the control (13.19 ± 0.26%) (p < 0.01). These results indicate that GEN induces apoptotic response in both cell lines, and the apoptotic effects of combination were more pronounced following combination treatment (Fig. 2).

Fig. 2

Induction of apoptosis in MCF-7 and MDA-MB-231 breast cancer cells following treatment with Poly I: C and GEN. (a) Representative flow cytometry dot plots showing apoptosis profiles of MCF-7 and MDA-MB-231 cells after treatment with control (untreated), Poly I:C, GEN, or GEN + Poly I:C. Apoptotic cells were assessed using Annexin V/propidium iodide (PI) staining. The lower-left quadrant represents live cells, the lower-right quadrant shows early apoptotic cells (Annexin V-positive/PI-negative), the upper-right quadrant represents late apoptotic or dead cells (Annexin V-positive/PI-positive), and the upper-left quadrant shows necrotic cells (PI-positive/Annexin V-negative). (b) Quantification of total apoptotic cells (early + late apoptosis) in MCF-7 and MDA-MB-231 cells under different treatment conditions. Data are presented as mean ± standard deviation (n = 3), and statistical significance is indicated by **p < 0.05, ***p < 0.001, and ****p < 0.0001 compared to the control group

Acridine orange staining reveals morphological features of apoptosis

To qualitatively assess the apoptotic effects of GEN, Poly I:C, and their combination, acridine orange (AO) staining was performed on MCF-7 and MDA-MB-231 cells (Fig. 3). Cells treated with 100 µM GEN exhibited characteristic apoptotic features such as membrane blebbing and nuclear condensation. These morphological changes appeared slightly more pronounced in the combination treatment group (GEN + Poly I:C), particularly in MCF-7 cells. AO/PI staining indicated that GEN alone and GEN + Poly I:C treatments induced apoptotic death in two different breast cancer cells.

Fig.3

Effect of GEN and Poly I:C on MCF-7 and MDA-MB-231 breast cancer cell viability. Representative fluorescence microscopy images showed the morphology of MCF-7 (left) and MDA-MB-231 (right) cells under different treatment conditions. Scale bars were shown in each image

Genistein induced cell cycle arrest at G2/M phase

The cell cycle analysis was performed to assess the effects of GEN, Poly I:C, and their combination on cell cycle distribution in MCF-7 and MDA-MB-231 cells (Fig. 4). A modest but statistically significant increase in G2/M phase accumulation was observed following GEN treatment in both cell lines. In MCF-7 cells, GEN treatment led to an increase in the G2/M population from 33.9% (control) to 58.9%, and 64.4% with the GEN + Poly I:C combination (p < 0.01). Similarly, in MDA-MB-231 cells, the G2/M phase population increased from 28.1% (control) to 44.0% with GEN, and to 45.4% with the combination treatment (p < 0.01). These results suggest that GEN, alone or in combination with Poly I:C, may contribute to G2/M phase arrest, particularly in MCF-7 cells.

Fig. 4

Cell cycle analysis of MCF-7 and MDA-MB-231 cells following treatment with Poly I:C, GEN or GEN + Poly I:C. (a) Representative DNA content histograms obtained by flow cytometry, showing cell cycle distribution (G0/G1, S, and G2/M phases), (b) Quantification of the G2/M phase percentage for MCF-7 and MDA-MB-231 cells under each treatment condition. Data are presented as the mean ± SD (n = 3). Statistical significance is indicated by *p < 0.01, and **p < 0.001 compared to the control group

Genistein changes cellular localization of TLR3 pathway-related proteins

Immunofluorescence analysis was performed to evaluate the effects of GEN, Poly I:C, and their combination on the cellular localization of TLR3 pathway-related proteins (TLR3, TRIF, IRF3, and p-NF-kB) in MCF-7 and MDA-MB-231 cells (Fig. 5). Compared to the control group, treatment with Poly I:C appeared to enhance cytoplasmic localization of TLR3 and p-NF-kB, along with a noticeable nuclear translocation of TRIF and IRF3 in both cell lines. Treatment with GEN alone showed relatively modest changes in localization of these proteins. However, the combination of GEN and Poly I:C seemed to further increase cytoplasmic expression of TLR3 and p-NF-kB and enhance nuclear translocation of TRIF and IRF3, particularly in MCF-7 cells. While these observations may suggest enhanced TLR3 pathway engagement under combination treatment, the extent and functional consequences of these localization changes require further validation through quantitative analysis and complementary assays.

Fig.5

IF detection of TLR-3, TRIF, IRF3, and p-NF-kB in breast cancer cells treated with Poly I:C, GEN or GEN + Poly I:C (a) Representative images of MCF-7 and (b) MDA-MB-231 cells under each indicated treatment. TLR-3, TRIF, IRF3, and p-NF-kB are stained in green, while nuclei are counterstained with DAPI (blue). Scale bars are shown in each panel

TLR3 pathway activation and cytokine induction by Poly I:C alone and in combination with GEN

The expression levels of TLR3 pathway-associated proteins (TLR3, TRIF, IRF3, AP-1, and p-NF-kB) were analyzed by Western blotting in MCF-7 and MDA-MB-231 cells following treatment with GEN, Poly I:C, and their combination (Fig. 6A). Compared to the control group, Poly I:C treatment was associated with increased expression of TLR3, IRF3, AP-1, and p-NF-kB proteins in both cell lines. The combination of GEN and Poly I:C resulted in higher expression levels than GEN treatment alone. This effect appeared more prominent in MCF-7 cells. In parallel, total levels of TNF-α and IFN-β proteins, measured by ELISA, were elevated in both cell lines following Poly I:C treatment, with a further increase observed upon GEN + Poly I:C treatment (Fig. 6B). The increases were generally more pronounced in MCF-7 cells compared to MDA-MB-231 cells. These findings suggest that GEN could modulate the expression of TLR3-related proteins and cytokines when used alone or in combination with Poly I:C.

Fig.6

Western blot and ELISA analysis results. (a) Western blot analysis of TLR3, TICAM, IRF-3, AP-1, and p-NF-kB protein levels in MCF-7 and MDA-MB-231 breast cancer cells following treatment with Poly I:C (P), GEN (G) or GEN + Poly I:C (G + P). β-Actin served as an internal control. (b) Quantification of TNF-α and IFN-β secretion by MCF-7 and MDA-MB-231 cells following treatment with Poly I:C, GEN or GEN + Poly I:C, measured by ELISA. Data are presented as the mean ± SD (n = 3). Statistical significance is indicated by *p < 0.01, **p < 0.05, ***p < 0.001, and ****p < 0.0001 compared to the control group