Cytotoxicity analysis

In this study, we investigated the inhibitory effects of CIS and EA on cancer cell proliferation in cisplatin-sensitive and -resistant MDA-MB-231 breast cancer cell lines. The cells were treated with increasing concentrations of CIS and EA. Both EA and CIS decreased the growth of sensitive and resistant cell lines, according to cell viability studies. Cell growth curves were used to calculate IC₅₀ values. The IC₅₀ values in the cisplatin-sensitive MDA-MB-231 cell line were 29 µM for EA and 38.2 µM for CIS (Fig. 1A). The IC₅₀ values in the cisplatin-resistant MDA-MB-231 cells were determined to be 49.5 µM for EA and 80.2 µM for CIS, respectively (Fig. 1B). Co-administration of CIS and EA in combinatorial treatments effectively (p < 0.05) suppressed cell growth in cisplatin-sensitive MDA-MB-231 cell lines (Fig. 1A). Even though the combined therapy in the cisplatin-resistant MDA-MB-231 cell lines led to a statistically significant decrease in cell viability when compared to the control group, the level of inhibition was similar to that seen with EA treatment alone (Fig. 1B).

Fig. 1

(A) Cytotoxicity analysis results of cisplatin-sensitive MDA-MB-231 breast cancer cells (48 h). (B) Cytotoxicity analysis results of cisplatin-resistant MDA-MB-231 breast cancer cells (48 h)

Alterations in ABCB1 gene and protein expression in cisplatin-sensitive and resistant MDA-MB-231 cells

After 24 h of treatment, the EA-treated group significantly reduced the expression of the ABCB1 gene in cisplatin-sensitive MDA-MB-231 breast cancer cells as compared to the dimethyl sulfoxide (DMSO) control group (p = 0.018). When the DMSO group was compared to the CIS and CIS + EA groups after 48 h of treatment, the ABCB1 gene expression in both treatment groups was significantly lower (p = 0.036 and p = 0.045, respectively) (Fig. 2A).

In cisplatin-resistant MDA-MB-231 breast cancer cells, a significant decrease in ABCB1 gene expression was detected in the EA-treated group compared to the DMSO group after 48 h of treatment (p = 0.013) (Fig. 2B). Immunocytochemical analysis showed that the levels of ABCB1 protein expression were consistent with the mRNA expression results (Fig. 2C and D).

In the cisplatin-sensitive group, ABCB1 immunopositivity percentages were 12.83 ± 3.06, 18.33 ± 2.16, and 17.17 ± 2.86 in the DMSO group (at 12, 24, and 48 h, respectively), 10.67 ± 2.16, 3.00 ± 1.79, and 10.67 ± 2.16 in the EA group (at 12, 24, and 48 h, respectively), 6.83 ± 1.47, 8.17 ± 1.47, and 5.83 ± 2.32 in the CIS group (at 12, 24, and 48 h, respectively), and 10.50 ± 1.87, 11.33 ± 1.75, and 3.17 ± 1.72 in the CIS + EA group (at 12, 24, and 48 h, respectively).

In the cisplatin-resistant group, ABCB1 immunopositivity percentages were 11.50 ± 1.87, 13.50 ± 1.87, and 11.67 ± 2.16 in the DMSO group (at 12, 24, and 48 h, respectively), 14.17 ± 2.32, 13.50 ± 2.43, and 8.17 ± 1.47 in the EA group (at 12, 24, and 48 h, respectively), 7.83 ± 2.32, 15.17 ± 1.72, and 11.17 ± 2.14 in the CIS group (at 12, 24, and 48 h, respectively), and 8.83 ± 2.32, 12.67 ± 2.16 and 11.83 ± 2.48 in the CIS + EA group (at 12, 24, and 48 h, respectively).

Fig. 2

(A) Relative ABCB1 gene expression in cisplatin-sensitive MDA-MB-231 cells at 12, 24, and 48 h. Data (2–ΔΔCt ratio to beta-actin) are shown as the mean relative to the DMSO control group (mean = 1).*p < 0.05;Kruskal-Wallis test with Bonferroni correction.(B) Relative ABCB1 gene expression in cisplatin-resistant MDA-MB-231 cells at 12, 24, and 48 h. Data (2–ΔΔCt ratio to beta-actin) are shown as the mean relative to the DMSO control group (mean = 1). *p < 0.05; One-Way ANOVA with Bonferroni correction. (C) ABCB1 immunoexpression in MDA-MB-231 cisplatin-sensitive and (D) MDA-MB-231 cisplatin-resistant cells after 12, 24, and 48 h of treatment, stained with AEC and hematoxylin

Alterations in VEGF gene and protein expression in cisplatin-sensitive and resistant MDA-MB-231 cells

After 24 h of treatment, there was a significant decrease in VEGF gene expression in the CIS-treated group relative to the DMSO control group in cisplatin-sensitive MDA-MB-231 breast cancer cells (p = 0.005). When the DMSO and EA groups were compared at 48 h, the EA-treated group’s VEGF gene expression was significantly lower (p = 0.045) (Fig. 3A).

In cisplatin-resistant MDA-MB-231 breast cancer cells, VEGF gene expression was significantly reduced in the EA and CIS + EA treatment groups compared to the DMSO control group after 24 h of treatment (p = 0.045 and p = 0.023, respectively) (Fig. 3B). VEGF protein expression levels were found by immunocytochemical staining to be in agreement with the mRNA expression findings. Notably, the DMSO groups showed elevated VEGF protein expression. Additionally, EA treatment was associated with a marked reduction in VEGF staining (Fig. 3C and D).

In the cisplatin-sensitive group, VEGF immunopositivity percentages were 70.67 ± 2.16, 73.83 ± 3.49 and 72.83 ± 3.31 in the DMSO group (at 12, 24, and 48 h, respectively), 50.00 ± 2.61, 40.50 ± 1.87 and 21.00 ± 2.37 in the EA group (at 12, 24, and 48 h, respectively), 52.17 ± 2.32, 25.67 ± 2.34 and 38.33 ± 2.80 in the CIS group (at 12, 24, and 48 h, respectively), and 65.17 ± 2.32, 39.50 ± 4.23 and 55.50 ± 2.74 in the CIS + EA group (at 12, 24, and 48 h, respectively).

In the cisplatin-resistant group, VEGF immunopositivity percentages were 40.50 ± 1.87, 38.17 ± 2.32 and 50.00 ± 3.29 in the DMSO group (at 12, 24, and 48 h, respectively), 18.67 ± 2.16, 32.50 ± 2.74 and 6.50 ± 1.87 in the EA group (at 12, 24, and 48 h, respectively), 19.83 ± 2.32, 28.33 ± 2.16 and 9.33 ± 2.16 in the CIS group (at 12, 24, and 48 h, respectively), and 16.33 ± 2.80, 7.50 ± 1.87 and 8.83 ± 2.32 in the CIS + EA group (at 12, 24, and 48 h, respectively).

Fig. 3

(A) Relative VEGF gene expression levels in cisplatin-sensitive MDA-MB-231 cells, and (B) in cisplatin-resistant MDA-MB-231 cells at 12, 24 and 48 h time points. Data (2–ΔΔCt ratio to beta-actin mRNA) are expressed as the mean relative to the DMSO group (mean: 1). *: p < 0.05; Kruskal-Wallis test with Bonferroni correction pos-hoc test. All experiments were conducted four times. (C) VEGF immunoexpression in MDA-MB-231 cisplatin-sensitive and (D) MDA-MB-231 cisplatin-resistant cells after 12, 24, and 48 h of treatment, stained with AEC and hematoxylin

Alterations in MMP2 and MMP9 gene and protein expression in cisplatin-sensitive and resistant MDA-MB-231 cells

In cisplatin-sensitive MDA-MB-231 breast cancer cells, after 12 h of treatment, a significant decrease in MMP2 and MMP9 gene expression was observed in the EA-treated group compared to the DMSO control group (p = 0.002 and p = 0.007, respectively). MMP2 gene expression was still considerably lower in the EA group after 24 h (p = 0.018). MMP2 and MMP9 gene expression was significantly lower in the CIS-treated group than in the DMSO-treated group at 48 h (p = 0.007 and p = 0.002, respectively) (Fig. 4A and C).

In cisplatin-resistant MDA-MB-231 breast cancer cells, a significant decrease in MMP2 gene expression was observed in the CIS-treated group compared to the DMSO control group after 12 h of treatment (p = 0.002). At 24 and 48 h, MMP2 and MMP9 gene expressions were significantly reduced in the CIS + EA treatment groups compared to DMSO (for 24 h: p = 0.011 and p = 0.002, respectively; for 48 h: p = 0.004 and p = 0.007, respectively) (Fig. 4B and D).

Immunocytochemical staining of MMP2 and MMP9 expression levels was consistent with the mRNA expression results. Notably, high levels of MMP2 and MMP9 expression were observed in the DMSO control groups. Furthermore, staining intensity was reduced following EA treatment (Fig. 4E, F, G and H).

In the cisplatin-sensitive group, MMP2 immunopositivity percentages were 77.67 ± 3.08, 75.83 ± 2.32 and 70.17 ± 3.06 in the DMSO group (at 12, 24, and 48 h, respectively), 51.17 ± 3.06, 40.67 ± 2.80 and 53.50 ± 2.88 in the EA group (at 12, 24, and 48 h, respectively), 62.00 ± 2.90, 55.67 ± 2.80 and 58,33 ± 2.58 in the CIS group (at 12, 24, and 48 h, respectively), and 66.00 ± 3.22, 45.50 ± 4.32 and 59.17 ± 3.76 in the CIS + EA group (at 12, 24, and 48 h, respectively). In the cisplatin-resistant group, MMP2 immunopositivity percentages were 41.17 ± 2.86, 50.67 ± 3.14 and 48.67 ± 2.16 in the DMSO group (at 12, 24, and 48 h, respectively), 32.67 ± 2.16, 26.83 ± 2.32 and 20.33 ± 3.39 in the EA group (at 12, 24, and 48 h, respectively), 30.50 ± 1.87, 26.50 ± 1.87 and 23.00 ± 2.37 in the CIS group (at 12, 24, and 48 h, respectively), and 30.50 ± 3.83, 24.00 ± 2.61 and 17.17 ± 2.48 in the CIS + EA group (at 12, 24, and 48 h, respectively).

In the cisplatin-sensitive group, MMP9 immunopositivity percentages were 78.50 ± 3.27, 78.17 ± 3.06 and 79.00 ± 2.61 in the DMSO group (at 12, 24, and 48 h, respectively), 33.17 ± 2.64, 11.50 ± 1.87 and 21.83 ± 2.48 in the EA group (at 12, 24, and 48 h, respectively), 38.33 ± 2.16, 19.00 ± 2.61 and 20.67 ± 2.16 in the CIS group (at 12, 24, and 48 h, respectively), and 32.33 ± 1,86, 14,83 ± 3,19 and 26,50 ± 1,87 in the CIS + EA group (at 12, 24, and 48 h, respectively). In the cisplatin-resistant group, MMP9 immunopositivity percentages were 60.17 ± 1.47, 58.33 ± 2.16 and 46.50 ± 3.39 in the DMSO group (at 12, 24, and 48 h, respectively), 51.50 ± 2.43, 30.83 ± 2.32 and 40.17 ± 2.86 in the EA group (at 12, 24, and 48 h, respectively), 49.67 ± 2.16, 34.17 ± 2.64 and 43.83 ± 2.32 in the CIS group (at 12, 24, and 48 h, respectively), and 22.00 ± 2.90, 22.00 ± 2.61 and 24.00 ± 2.61 in the CIS + EA group (at 12, 24, and 48 h, respectively).

Fig. 4

(A) Relative MMP2 gene expression levels in cisplatin-sensitive MDA-MB-231 cells, and (B) in cisplatin-resistant MDA-MB-231 cells at 12, 24 and 48 h time points. (C) Relative MMP9 gene expression levels in cisplatin-sensitive MDA-MB-231 cells, and (D) in cisplatin-resistant MDA-MB-231 cells at 12, 24 and 48 h time points. Data (2–ΔΔCt ratio to beta-actin mRNA) are expressed as the mean relative to the DMSO group (mean: 1). *: p < 0.05; Kruskal-Wallis test with Bonferroni correction pos-hoc test. All experiments were conducted four times. (E) MMP2 immunoexpression in MDA-MB-231 cisplatin-sensitive and (F) MDA-MB-231 cisplatin-resistant cells after 12, 24, and 48 h of treatment, stained with AEC and hematoxylin. (G) MMP9 immunoexpression in MDA-MB-231 cisplatin-sensitive and (H) MDA-MB-231 cisplatin-resistant cells after 12, 24, and 48 h of treatment, stained with AEC and hematoxylin

Alterations in Bcl-2 and Bax gene and protein expression in cisplatin-sensitive and resistant MDA-MB-231 cells

In cisplatin-sensitive MDA-MB-231 breast cancer cells, Bcl-2 gene expression was significantly decreased in the EA-treated group compared to the DMSO control group at 12 and 48 h of treatment (p < 0.001 and p = 0.027, respectively). At 12 h, a significant reduction in Bcl-2 gene expression was also observed in the CIS + EA group compared to DMSO (p = 0.002). Furthermore, the CIS + EA group exhibited significantly higher Bax gene expression at 12 and 48 h in comparison to DMSO group (p = 0.002 and p = 0.007, respectively). At 24 h, Bcl-2 gene expression was significantly lower in all treatment groups (p < 0.001) when DMSO group was compared to EA, CIS, and CIS + EA groups (Fig. 5A and C).

After 12 h of treatment, the Bcl-2 gene expression in cisplatin-resistant MDA-MB-231 breast cancer cells was significantly lower in the CIS and CIS + EA treatment groups than in the DMSO group (p < 0.001). At 24 h, the CIS group’s Bax gene expression was considerably higher compared to that of the DMSO group (p = 0.018). In comparison to the DMSO group, the EA, CIS, and CIS + EA groups showed significantly lower Bcl-2 gene expression after 48 h (p = 0.001, p = 0.012, and p = 0.003, respectively) (Fig. 5B and D).

Fig. 5

(A) Relative Bcl-2 gene expression levels in cisplatin-sensitive MDA-MB-231 cells, and (B) in cisplatin-resistant MDA-MB-231 cells at 12, 24 and 48 h time points. Data (2–ΔΔCt ratio to beta-actin mRNA) are expressed as the mean relative to the DMSO group (mean: 1). *: p < 0.05; One-Way ANOVA with Bonferroni correction test. (C) Relative Bax gene expression levels in cisplatin-sensitive MDA-MB-231 cells, and (D) in cisplatin-resistant MDA-MB-231 cells at 12, 24 and 48 h time points. Data (2–ΔΔCt ratio to beta-actin mRNA) are expressed as the mean relative to the DMSO group (mean: 1). *: p < 0.05; Kruskal-Wallis test with Bonferroni correction pos-hoc test. All experiments were conducted four times

Molecular docking analysis results

EA demonstrates a strong affinity for the chosen target proteins, according to molecular docking analysis (Table 2). Notably, 16 hydrogen bonds were observed in the interaction with the Bax protein, with a binding energy of −7.7 kcal/mol. Furthermore, it was found that the binding energy between EA and the MMP9 protein was − 8.4 kcal/mol. EA may significantly suppress genes that are crucial to the progression of cancer, especially when combined with medicines like CIS, which are used in both our experimental and clinical settings.

Table 2 Binding position, energy value and number of hydrogen bonds of EA to proteins