Abstract
Background
Breast cancer is one of the most common cancers among women worldwide, and metastasis plays an important role in its lethality. Silymarin (SLY) is a natural compound that has exhibited potential anticancer effects. However, its effects on the metastatic properties of different breast cancer cell lines remain unknown. Therefore, this study aimed to investigate the effects of SLY on the metastatic properties of two breast cancer cell lines (MCF-7 and MDA-MB-231) and evaluate differences between two-dimensional (2D) and three-dimensional (3D) cell culture models.
Methods
This study used 2D and 3D cell culture systems to evaluate the effects of SLY on cell proliferation, migration, and epithelial–mesenchymal transition (EMT). The effective dose of SLY was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell viability assays in 2D and 3D cultures. In 2D cultures, wound healing assays, polymerase chain reaction (PCR) to evaluate mRNA levels and immunohistochemical analyses to evaluate protein expression. In 3D cultures, spheroids were formed, and Transwell migration assays, PCR for mRNA levels, and immunohistochemical analyses for protein expression were conducted.
Results
SLY effectively modulated proliferation, migration, and EMT in both cell lines. Notably, the 3D microenvironment exerted differential effects on drug sensitivity: MCF-7 cells showed increased sensitivity to SLY in 3D culture, whereas MDA-MB-231 cells exhibited increased resistance compared to 2D models.
Conclusions
These findings suggest that SLY exhibits promising antimetastatic potential in vitro, warranting further in vivo and clinical investigations to evaluate its efficacy as a supportive therapeutic candidate for breast cancer treatment. This study emphasizes the importance of 3D culture models for accurately evaluating the efficacy of anticancer drugs. Notably, SLY's effect on MMP-9 expression was found to be environment-dependent, showing a significant reduction specifically in the 3D spheroid model, which highlights its potential antimetastatic efficacy in more biomimetic settings.
Supplementary Information
The online version contains supplementary material available at https://doi.org/10.1186/s12906-026-05456-6.
Keywords: Breast cancer, Epithelial–mesenchymal transition, Metastasis, Silymarin, Two-dimensional and three-dimensional cell culture
Background
Cell culture methods are frequently employed in cancer research. In two-dimensional (2D) systems, cells lose their polarization and exhibit limited secretion of the extracellular matrix (ECM). To overcome these limitations, three-dimensional (3D) platforms that more closely mimic in vivo conditions have been developed [1]. 3D systems are crucial for understanding and mimicking cell–cell and cell–matrix interactions, as well as external stimuli [2]. Compared with 2D cultures, 3D cultures more accurately replicate the tumor microenvironment by altering cellular activities, including hypoxia, proliferation, migration, epithelial–mesenchymal transition (EMT), and drug sensitivity [3–5]. Although 2D cultures are useful for rapid and cost-effective drug screening, they are limited by the absence of an ECM and 3D organization [6]. Spheroids mimic in vivo tumors by reflecting cell-to-cell and cell-to-substrate interactions, providing more realistic results in terms of drug response and other factors [7].
Breast cancer (the most common malignant tumor in women) arises from the uncontrolled proliferation of epithelial cells. Approximately 15% of breast cancers are triple-negative breast cancer (TNBC), accounting for over 50% of cancer-related deaths [8]. TNBC is characterized by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expressions [9, 10]. Compared with other breast cancer subtypes, TNBCs exhibit aggressive progression, often presenting larger tumor size and lymph node involvement at diagnosis [9, 11]. MDA-MB-23 is a model of TNBC, whereas MCF-7 represents ER + and PR + breast cancer. Both are widely used cell lines in in vivo studies.
The first step in the metastatic process is the transformation of cancer cells into migratory and invasive phenotypes, a process known as EMT [3]. Cell migration begins with the loss of cell-to-cell adhesion proteins, enabling cells to degrade the ECM and infiltrate surrounding tissues [12]. EMT is characterised by the phenotypic conversion of epithelial cells into a mesenchymal state. This is characterised by the loss of epithelial features, reduced E-cadherin expression and increased N-cadherin expression, as well as enhanced invasive capacity [3, 13, 14]. Consistent with these EMT-associated changes, the expression of cytoskeletal proteins was higher in 2D cultures than in 3D cultures, suggesting that culture dimensionality may influence EMT-related structural organisation. However, this results in an increased cell surface area, thicker filaments, and a loss of flexibility [15–17]. During EMT, mesenchymal cells degrade type IV collagen and laminin while depositing their own collagen and fibronectin matrix. The formation of lamellipodia and filopodia on the cell surface enhances the motility and secretion of matrix metalloproteinases (MMPs), which facilitate invasion by degrading the ECM [18, 19]. In addition, MMP-2 and MMP-9 upregulate genes(e.g., MMP2, MMP9) that cleave type IV collagen [20, 21]. Furthermore, MMPs weaken cell junctions by degrading E-cadherin [22]. Silymarin (SLY), one of the active components of milk thistle extract, exhibits antimetastatic and anti-inflammatory effects, modulating the imbalance between cell death and survival and influencing the expression of cell cycle regulatory proteins (e.g., cyclin-dependent kinases) [23]. According to research, SLY reduces or prevents toxicities induced by chemotherapy and radiation. Furthermore, it can alter the expression of gene products associated with tumor proliferation, invasion, metastasis, and angiogenesis in various cancer types [23, 24]. In the present study, we comparatively evaluated the effects of short-term SLY treatment on EMT-related proteins and migratory behavior in MDA-MB-231 and MCF-7 both 2D and 3D spheroid model.
Methods
Preparation of chemicals
SLY (S2358, Selleckchem, USA) was purchased commercially. The compound was dissolved in prewarmed complete culture medium and serially diluted to create solutions at different concentrations for 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide (MTT) assays.
Cell culture and experimental groups
MDA-MB-231 and MCF-7 cells were cultured in RPMI-1640 medium. For the 2D culture model, the cells were divided into control and SLY-treated groups, with both cell lines represented in each group. Similarly, for the 3D spheroid model, spheroids generated from the two cell lines were assigned to control and SLY-treated groups.
3D culture spheroid formation
3D spheroids were generated using the hanging drop technique. Briefly, cell suspensions were prepared at a concentration of 2 × 104 cells per droplet. Approximately 40 droplets were carefully placed onto the inner surface of the lid of a culture plate. The lid was then inverted and placed over a plate containing sterile PBS to maintain humidity and prevent droplet evaporation. This method was chosen to ensure high uniformity in spheroid size and to provide a cost-effective alternative to ultra-low attachment plates while maintaining rigorous experimental standards. Every five days, 10 µL of fresh medium was added to each droplet to maintain the supply of nutrients. The droplets were incubated for 20 days under standard conditions (37°C, 5% CO₂, and 95% humidity) to allow the formation of spheroids. Spheroid development was monitored periodically, and a final morphological evaluation was performed using phase-contrast microscopy.
MTT assay in 2D and 3D cultures
MTT assay was performed to determine the cytotoxic effect and IC50 value of SLY. Cells were seeded at a density of 5 × 103–4 × 104 cells per well and treated with SLY concentrations (10, 30, 60, 90, and 120 µM) for 24 h. To ensure high statistical power and reproducibility, each concentration was studied in triplicate, and the entire experiment was repeated three times, providing a total of nine independent data points (n = 9) for each concentration. Next, dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazan crystals. The absorbance values at 570 nm (690 nm as reference) were measured using a microplate reader (Shimadzu UV-1601). Cell viability was calculated as a percentage compared with untreated control cells. Raw absorbance data were first converted to percentage cell viability using the formula:
 |
Statistical significance and data distribution were analyzed using SPSS software. Subsequently, the final IC50 values were determined by fitting the normalized viability data to a non-linear regression model (log[inhibitor] vs. normalized response) using GraphPad Prism software. In 2D culture, cells were seeded in 96-well plates. In 3D culture, the spheroids were incubated in hanging droplets for 20 days.
Cell migration and invasion assays
2D wound healing migration assay
The migration potential was evaluated using scratch wound assay. MDA-MB-231 and MCF-7 cells were cultured to 100% confluence in 24-well plates. A standardized wound area was created in a monolayer using a sterile 10-µL pipette tip. Following wound formation, cellular debris was removed by washing with phosphate buffered saline. The cells were then subjected to experimental conditions, and wound closure was monitored for 24 h. Migration was quantified by measuring the percentage of wound area closure at predetermined time intervals using phase-contrast microscopy. Three independent experiments were performed, each with triplicate wells.
Invasion analysis using the 3D transwell method
Each cell line’s invasion potential was evaluated using Transwell invasion assay. Matrigel, which had been stored at − 20 °C, was thawed overnight at + 4 °C, liquefied, and then diluted 1:1 with serum-free media. The upper chambers of Transwell inserts (0.8 µm pore size) were uniformly coated with 100 µL of Matrigel. Next, the coated inserts were placed in 24-well culture plates containing serum-free media in the lower chambers. After incubation for 1 h at 37 °C to allow Matrigel polymerization, 1 × 10⁶ cells were seeded onto the Matrigel-coated inserts in each group. During the 24-h incubation period, cells capable of invading the Matrigel barrier migrated to the serum-free media in the lower chamber. The number of invaded cells was quantified using an automated cell counter (Bio-Rad TC20), which enabled a comparative analysis of the invasion capacity of different experimental groups [25].
Changes in the expression of proteins involved in EMT
Spheroids that formed from cultured cells were collected from each droplet using a micropipette and transferred to poly-L-lysine-coated slides. Changes in the expression of MMP-9, E-cadherin, N-cadherin, fibronectin, and vimentin proteins were evaluated using immunohistochemical staining, and at least 10 samples were used for each antibody.
Immunocytochemical staining in 2D and 3D cultures
For immunohistochemical analysis, the following primary antibodies were employed at the specified dilution ratios: anti-MMP-9 (1:250, sc-393859, Santa Cruz), anti-E-cadherin (1:250, sc-8426, Santa Cruz), anti-N-cadherin (1:200, ab18203, Abcam), anti-fibronectin (1:250, sc-8422, Santa Cruz), and anti-vimentin (1:250, sc-6260, Santa Cruz). All antibodies were diluted in PBS and incubated overnight at 4 °C according to the manufacturers optimized protocols. Immunoreactivity was detected by visualizing the antibodies, avidin–biotin complex, and 3,3′-diaminobenzidine. The semiquantitative assessment of protein expression was performed by calculating H-scores (Histo-scores), as previously described [26–28]. This evaluation integrated both the staining intensity and the proportion of positive cells. Staining intensity was graded on a six-point scale: 0 (negative), 1 (weak), 2 (mild), 3 (moderate), 4 (strong), and 5 (intense). For each antibody and experimental group, the percentage of positive cancer cells was determined by counting a minimum of 500 cells across 10 randomly selected fields. The final H-score was derived by multiplying the intensity grade by the percentage of positive cells, resulting in a potential score range of 0 to 500. This comprehensive quantification approach ensured a robust mathematical representation of the immunoreactivity patterns across the different culture models.
Real-Time Polymerase Chain Reaction (RT-PCR) analysis in 2D and 3D cultures
Total RNA was extracted from MCF-7 and MDA-MB-231 using TRIzol Reagent for genetic analysis. The RNA samples were converted into cDNA. The mRNA expression levels of CDH1 (E-cadherin), CDH2 (N-cadherin), VIM, and MMP9 mRNA was analyzed using the StepOnePlus Real-Time PCR System. Amplifications were performed using cDNA, site-specific primers (Oligomer Biotechnology, Ankara), SYBR Green, and nuclease-free water. To ensure internal control, GAPDH was utilized. The primer sequences of CDH1, CDH2, VIM, and MMP9 were designed based on the FASTA format from NCBI (https://www.ncbi.nlm.nih.gov/) (Table 1).
Table 1.
Primer sequences used for RT-PCR analysis of CDH1, CDH2, VIM, MMP9, and GAPDH genes
Statistical analysis
The data obtained from experimental studies were analyzed using SPSS. Quantitative data were evaluated using the Kruskal–Wallis test, whereas qualitative data were analyzed using the Chi-square test. Statistical significance was considered at p < 0.05. Genetic analysis was performed using REST2009 V2.0.13, where p < 0.05 represented a statistically significant result.
Results
Cell viability analyses
The cytotoxic effects of SLY on MCF-7 and MDA-MB-231 breast cancer cells were quantitatively assessed using the MTT assay across both 2D monolayer and 3D spheroid culture models. Our findings demonstrate that SLY exerts a dose-dependent reduction in cell viability, with its efficacy significantly modulated by the culture architecture. The IC50 values were determined by fitting the normalized viability data to a non-linear regression model using GraphPad Prism software. For 2D cultures, the IC50 values were 60 µM for MDA-MB-231 cells and 90 µM for MCF-7 cells. In 3D spheroid cultures, the IC50 values were 120 µM for MDA-MB-231 and 60 µM for MCF-7 cells, respectively (Fig. 1). While a general dose-dependent trend was observed, some variability in cell viability across concentrations was noted. This observed variability, particularly in 3D spheroid models, is consistent with the inherent complexity of spheroid architectures. Such fluctuations often arise from heterogeneous drug penetration gradients, the presence of distinct cellular metabolic zones within the 3D structure, and biological variations in spheroid size and density, which can modulate the cytotoxic response compared to 2D monolayers [29, 30].
Fig. 1.
Dose-dependent cytotoxic effects and IC50 values of SLY. The cells were treated with 10–120 µM for 24 h. Thereafter, cell viability was assessed using MTT assay. The results are expressed as a percentage of the viability relative to the control (100%). A Viability of MCF-7 and MDA-MB-231 cells in 2D monolayer cultures. The IC50 values were then calculated from the dose–response data, 90 µM for MCF-7 and 60 µM for MDA-MB-231 cells. B Viability of MCF-7 and MDA-MB-231 cells in 3D spheroid cultures. The IC50 values were determined as 60 µM for MCF-7 and 120 µM for MDA-MB-231 spheroids. Results are expressed as a percentage of viability relative to the untreated control (100%). Data represent the mean ± standard deviation (SD) of triplicate measurements from three independent experiments. Statistical significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 compared to the control group
Migration and invasion tendencies of cells following SLY treatment
Effects of SLY on MDA-MB-231 and MCF-7 cells in 2D wound healing assay
The migratory tendencies of MDA-MB-231 and MCF-7 cells were evaluated using 2D wound healing assay after SLY treatment. Once cells reached confluency, a scratch was made, and cell migration toward the wound area was assessed by measuring the remaining wound gap at hourly intervals. Wound areas were measured under a ZEISS axiovert A1 invert microscope using the ZEN 3.3 (Blue edition) imaging system. Complete wound closure was observed in the control groups of MDA-MB-231 and MCF-7 cells at the end of 24 h. By contrast, in SLY-treated cells, the wound area remained approximately half the size of the initial gap. Compared with the control, the migration rate of SLY-treated cells in 2D was approximately twice as slow, which was statistically significant (p < 0.05) (Figs. 2 and 3A-B).
Fig. 2.
Wound area images of MCF-7 and MDA-MB-231 breast cancer cells
Fig. 3.
Effect of SLY on the migration capacity of MCF-7 and MDA-MB-231 cells in 2D wound healing assay. A Quantitative analysis of wound area closure over 24 h in MCF-7 cells treated with SLY compared to the control group. B Quantitative analysis of wound area closure over 24 h in MDA-MB-231 cells treated with SLY compared to the control group. The wound area was measured in pixels at 0, 6, 12, 18, and 24 h post-scratch. A larger wound area in the SLY-treated groups compared to the control (particularly evident in MDA-MB-231 at 12 h and 18 h) indicates the inhibitory effect of SLY on cell migration. Data are presented as mean ± standard deviation (SD) from three independent experiments. Statistical significance between the control and SLY-treated groups is indicated by asterisks (**p < 0.01, ***p < 0.001, ****p < 0.0001)
Effects of SLY on the invasion and migration of MDA-MB-231 and MCF-7 cells in 3D transwell invasion assay
The invasive and migratory capacities of MDA-MB-231 and MCF-7 cells in 3D spheroid model were compared between SLY-treated and control groups using Transwell invasion assay.
After 24 h, 28% of MCF-7 cells in the control migrated through the Matrigel to the lower chamber. By contrast, only 4% of cells in the SLY-treated group reached the lower chamber. These results suggest that 24 h of SLY treatment reduced MCF-7 migration in the 3D spheroid model by approximately 85% (Fig. 4).
Fig. 4.
Effects of SLY on the invasive and migratory capacities of MCF-7 and MDA-MB-231 cells. After 24 h, 28% of MCF-7 cells in the control group migrated through the Matrigel to the lower chamber, whereas only 4% of the SLY-treated group migrated, indicating an approximately 85% reduction in migration. In MDA-MB-231 spheroids, 55% of control cells migrated compared with 32% in the SLY-treated group. This finding corresponds to a 42% decrease in migration following SLY treatment. The results are presented as the percentage of migrated cells relative to the total cell population
In MDA-MB-231 cells, 55% migrated to the lower chamber in the control group after 24 h compared with 32% in the SLY-treated group. Consequently, SLY treatment for 24 h decreased the migration of MDA-MB-231 cells in 3D spheroid model by 42% (Fig. 4).
Immunohistochemical evaluations
Immunohistochemical assessment of adhesion, cytoskeletal, and invasion marker proteins following 2D and 3D cultures
To evaluate the impact of SLY on the epithelial-mesenchymal transition (EMT) profile, we quantified the expression of key biomarkers using H-score analysis in both 2D and 3D culture models (Figs. 5 and 6) (Tables 2 and 3).
Fig. 5.
Quantitative assessment of EMT and invasion markers in MCF-7 cells across 2D and 3D cultures. The protein expression levels of E-cadherin, N-cadherin, Vimentin, Fibronectin, and MMP-9 were evaluated using the semi-quantitative H-score method. The bar graphs represent the comparative H-scores between the Control and SLY-treated groups in both 2D monolayer and 3D spheroid model. Data are expressed as Mean ± Standard Deviation (SD), derived from counting at least 500 cells in 10 random fields per group. Statistical significance was analyzed using independent samples t-tests, with exact p-values and significance levels (p < 0.05, **p < 0.001, ns: non-significant) indicated above the bars. The results highlight the preservation of the epithelial phenotype and the significant reduction of MMP-9 in MCF-7 spheroids following SLY treatment
Fig. 6.
Quantitative assessment of EMT and invasion markers in MDA-MB-231 cells across 2D and 3D cultures. The protein expression levels of E-cadherin, N-cadherin, Vimentin, Fibronectin, and MMP-9 were quantified using the H-score method to evaluate the antimetastatic potential of SLY. The graphs compare the H-scores of Control and SLY-treated groups in 2D and 3D environments. Data represent the Mean ± Standard Deviation (SD) from a minimum of 500 cells across 10 fields. Exact p-values are provided for each comparison, with significance denoted as p < 0.05, **p < 0.001, and ns for non-significant results. Notably, the 3D spheroid model reveals a complex modulation of mesenchymal markers (Fibronectin and Vimentin) and a significant suppression of MMP-9, reflecting the context-dependent impact of SLY on this aggressive triple-negative breast cancer cell line
Table 2.
H-score comparison in 2D culture
| Cell Line | Antibody | Control (Mean ± SD) |
SLY (Mean ± SD) |
p-value |
|---|---|---|---|---|
| MCF-7 | E-cadherin | 242.38 ± 13.54 | 239.67 ± 2.90 | 0.631 |
| N-cadherin | 281.97 ± 8.77 | 330.92 ± 2.99 | < 0.05 | |
| Fibronectin | 172.77 ± 4.79 | 197.07 ± 2.79 | < 0.05 | |
| Vimentin | 307.73 ± 3.76 | 281.55 ± 3.93 | < 0.05 | |
| MMP-9 | 259.22 ± 4.08 | 209.00 ± 2.39 | < 0.05 | |
| MDA-MB-231 | E-cadherin | 250.90 ± 3.20 | 265.42 ± 2.33 | < 0.05 |
| N-cadherin | 266.83 ± 2.70 | 276.53 ± 2.50 | < 0.05 | |
| Fibronectin | 278.60 ± 3.00 | 278.80 ± 1.96 | 0.903 | |
| Vimentin | 306.23 ± 6.82 | 173.00 ± 4.22 | < 0.05 | |
| MMP-9 | 148.42 ± 2.16 | 204.03 ± 2.86 | < 0.05 |
Table 3.
H-score comparison in 3D culture
| Cell Line | Antibody | Control (Mean ± SD) |
SLY (Mean ± SD) |
p-value |
|---|---|---|---|---|
| MCF-7 | E-cadherin | 258.72 ± 1.76 | 248.44 ± 2.48 | < 0.05 |
| N-cadherin | 225.65 ± 8,98 | 256.75 ± 2.78 | 0.522 | |
| Fibronectin | 277.30 ± 3.02 | 260.77 ± 4.15 | < 0.05 | |
| Vimentin | 289.03 ± 1.88 | 265.40 ± 3.70 | < 0.05 | |
| MMP-9 | 266.87 ± 3.58 | 228.33 ± 1.67 | < 0.05 | |
| MDA-MB-231 | E-cadherin | 268.68 ± 3.14 | 236.24 ± 3.76 | < 0.05 |
| N-cadherin | 247.25 ± 2.90 | 268.25 ± 2.32 | < 0.05 | |
| Fibronectin | 197.28 ± 2.44 | 239.35 ± 3.54 | < 0.05 | |
| Vimentin | 310.42 ± 2.23 | 267.63 ± 3.34 | < 0.05 | |
| MMP-9 | 198.70 ± 1.97 | 236.65 ± 2.84 | < 0.05 |
In the 2D culture model of MCF-7 cells, the expression of the adhesion molecule N-cadherin significantly increased in the SLY-treated group (p < 0.05). However, the decrease in E-cadherin expression was not statistically significant. In MDA-MB-231 cells, E-cadherin and N-cadherin levels showed increases following SLY treatment (p < 0.05). Among cytoskeletal proteins, vimentin expression was downregulated in MCF-7 cells after SLY treatment (p < 0.05), whereas fibronectin expression was upregulated (p < 0.05). By contrast, fibronectin levels remained unchanged in MDA-MB-231 cells, whereas vimentin expression decreased in response to SLY treatment (p < 0.05). Regarding invasion potential, MMP-9 expression decreased in SLY-treated MCF-7 cells (p < 0.05), whereas an increase was observed in SLY-treated MDA-MB-231 cells (p < 0.05) (Table 2 and Fig. 7).
Fig. 7.
Immunohistochemical staining images of EMT markers in 2D MCF-7 and MDA-MB-231 cells
In 3D spheroids, SLY treatment led to a decrease in E-cadherin protein expression in MCF-7 cells compared with the control (p < 0.05). Although an increase in N-cadherin protein levels was observed, the change was not statistically significant (p = 0.522). In addition, significant reductions in fibronectin and vimentin protein levels were detected (p < 0.05). MMP-9 protein expression was significantly reduced in the SLY-treated group (p < 0.05). In MDA-MB-231 cells, SLY treatment resulted in a significant decrease in E-cadherin protein levels and an increase in N-cadherin protein expression. Furthermore, the cells exhibited an increase in fibronectin protein expression and a significant decrease in vimentin protein levels. MMP-9 protein immunoreactivity was also significantly elevated following SLY treatment (p < 0.05). These results suggest that SLY has a cell-type-dependent impact on EMT-related markers in 3D cultures (Table 3 and Fig. 8).
Fig. 8.
Immunohistochemical staining images of EMT markers in 3D MCF-7 and MDA-MB-231 spheroids
Assessment of gene expression levels
CDH1, CDH2, VIM, and MMP9 mRNA levels in 2D and 3D MCF-7 cells were analyzed against the respective control. In 2D cells, CDH1 mRNA levels increased by 1.2-fold, CDH2 mRNA levels increased by 2.14-fold (p < 0.001), VIM mRNA levels increased by 1.42-fold, and MMP9 mRNA levels increased by 4.11-fold (p < 0.001) compared with the control. In 3D cells, CDH1 mRNA levels increased by 6.5-fold (p < 0.001), CDH2 mRNA levels increased by 5.81-fold (p < 0.001), VIM mRNA levels decreased by 0.013-fold (p < 0.001), and MMP9 mRNA levels decreased by 0.48-fold compared with the control (Fig. 9).
Fig. 9.
Fold changes in mRNA levels in MCF-7 and MDA-MB-231 cells compared with related controls. Relative mRNA expression analysis of CDH1, CDH2, VIM, and MMP9 in MCF-7 and MDA-MB-231 cells grown in 2D and 3D culture systems. Quantitative RT-PCR data were normalized to the respective control and expressed as fold change relative to control (dashed line at 1.0). *p < 0.001. GAPDH was used as the reference gene for normalization
CDH1, CDH2, VIM, and MMP9 mRNA levels in 2D and 3D MDA-MB-231 cells were analyzed against the respective control. In 2D cells, CDH1 mRNA levels increased by 8-fold (p < 0.001), CDH2 mRNA levels increased by 1.36-fold, VIM mRNA levels increased by 1.59-fold, and MMP9 mRNA levels increased by 6.41-fold (p < 0.001) compared with the control. In 3D cells, CDH1 mRNA levels increased by 1.36-fold, CDH2 mRNA levels increased by 3.92-fold (p < 0.001), VIM mRNA levels increased by 4.9-fold (p < 0.001), and MMP9 mRNA levels decreased by 0.043-fold (p < 0.001) compared with the control (p < 0.001) (Fig. 9).
Discussion
Recently, SLY attracted considerable attention as a natural anticancer agent because of its low toxicity and reduced impact on healthy cells [22, 23, 25, 31]. The present study evaluated the cytotoxic and antimetastatic effects of SLY in MCF-7 and MDA-MB-231 cells using 2D and 3D systems. Although 2D models are predominantly used in existing literature, 3D spheroid model more accurately mimic the tumor microenvironment and therefore provide more physiological results [32, 33]. In the present study, the IC50 value of SLY was determined to be 60 and 120 µM in 2D and 3D MDA-MB-231 cells and 90 and 60 µM in 2D and 3D MCF-7 cells, respectively. These findings suggest that MDA-MB-231 exhibits greater resistance to SLY in a 3D environment, which is consistent with the findings of Abdollahi et al. (2014) [34]. Boo (2017) and Yazdi Rouholamini et al. (2018) reported higher IC50 values in 3D settings [35, 36]. SLY significantly inhibited cell migration at concentrations of 60 µM for MDA-MB-231 and at 90 µM for MCF-7 in 2D settings. Interestingly, our results revealed that MCF-7 cells exhibited a lower IC50 value in the 3D spheroid model compared to the 2D monolayer culture, indicating an increased sensitivity to Silymarin in the 3D environment. While 3D cultures typically demonstrate higher drug resistance due to limited diffusion and altered gene expression [37, 38], certain compounds can exhibit enhanced efficacy in spheroid model depending on their mechanism of action. This suggests that the 3D architecture and cell–cell interactions can either sensitize or protect cancer cells depending on their specific molecular subtype and baseline metastatic potential. The increased resistance in MDA-MB-231 spheroids may be linked to their more aggressive, mesenchymal phenotype, which potentially utilizes the 3D matrix to activate survival signals, whereas the epithelial-like MCF-7 cells may become more vulnerable when organized into spheroids. This increased sensitivity in MCF-7 spheroids may be attributed to the spatial reorganization of estrogen receptors and the modulation of survival signaling pathways, such as PI3K/Akt, which are known molecular targets of Silymarin [39, 40]. Furthermore, the 3D architecture may facilitate a more physiologically relevant cellular response, where the induction of apoptosis by Silymarin is amplified through enhanced cell-to-cell communication and metabolic stress within the spheroid core [41, 42]. These findings suggest that the 3D microenvironment can uniquely alter the pharmacodynamics of Silymarin, highlighting the importance of using biomimetic models to evaluate its therapeutic potential [43] (Fig. 10).
Fig. 10.
Schematic overview of the effects of SLY on metastatic properties of breast cancer cells in 2D and 3D culture models. MCF-7 and MDA-MB-231 cells were treated with SLY and evaluated in both 2D and spheroid-based (3D) systems. In the 2D model, proliferation, migration (scratch assay), and EMT were assessed using molecular and immunohistochemical analyses. In the 3D spheroid model, spheroids were analyzed for migration using Transwell assays alongside PCR and immunohistochemistry. Overall, SLY modulated proliferation, migration, and EMT in both cell lines, with the 3D spheroid model providing a more physiologically relevant representation of its anticancer effects
In the 3D Transwell assay, the proportion of migrating cells was 55% for MDA-MB-231 and only 4% for MCF-7. The administered dose of SLY inhibited migration by 42% in MDA-MB-231 and by 85% in MCF-7. These results reveal a more pronounced antimetastatic effects of SLY on MCF-7. According to previous studies, SLY appears to inhibit invasion and migration in a dose- and cell-type-dependent manner, with a stronger effect on adhesion proteins in epithelial-like cells, particularly in 3D cells [39, 44, 45]. In line with previous studies, silibinin B has been shown to be more effective at lower doses in aggressive cancer cells under 2D conditions, whereas higher doses are required for epithelial-like cancer cells. This difference is thought to arise from the potential impact of silibinin B on adhesion proteins. The significant reduction in cell migration at low doses using 3D provides strong evidence that silibinin B exerts antimetastatic effects by modulating adhesion proteins.
EMT characterized by the loss of epithelial features and the acquisition of a mesenchymal phenotype involves the disruption of cell polarity, increased N-cadherin expression, and decreased E-cadherin expression, resulting in enhanced invasive capacity [3, 13, 14, 46]. Adhesion molecules mediate physical cell–cell contacts and play crucial roles in intracellular signaling [47]. Notably, MCF-7 fails to form spheroids when E-cadherin is silenced, whereas the overexpression of E-cadherin in cells that do not normally form spheroids (e.g., SKBR3) supports their formation [48, 49]. Conversely, N-cadherin contributes to spheroid formation in MDA-MB-231 [35]. In the present study, we observed increases in E-cadherin and N-cadherin levels in the 3D control, which is consistent with previous literature and suggests that adhesion molecule expression is enhanced during spheroid formation. However, CDH1 significantly decreased in 3D MDA-MB-231 cells following SLY treatment, implying that SLY may suppress epithelial characteristics in these cells. This decrease was not observed in MCF-7 cells, possibly because of basal differences in E-cadherin levels specific to these cells.
Vimentin expression significantly decreased at the protein level among cytoskeletal proteins in 2D settings. By contrast, an increase in mRNA levels alongside a decrease in protein levels was observed in 3D MDA-MB-231 cells. This discrepancy may be because of posttranscriptional regulation or insufficient time for protein translation. In MCF-7 spheroids, the mRNA and protein levels of vimentin were reduced, which supports the idea that SLY preserves the epithelial phenotype in this cell line. According to research, silibinin modulates EMT by increasing E-cadherin levels while reducing N-cadherin and vimentin levels [50–52]. Furthermore, SLY has been demonstrated to modulate fibronectin expression via the STAT3 signaling pathway in MDA-MB-231 and MCF-7 cells [53]. In the present study, fibronectin expression decreased in 2D MCF-7 cells and remained unchanged in MDA-MB-231 cells. However, in 3D settings, fibronectin expression increased in both cell lines. This increase may be associated with the development of a mesenchymal phenotype alongside spheroid formation.
Although previous studies have demonstrated a modulation in fibronectin expression following SLY treatment in 2D settings, there are limited data on 3D settings, which warrants further investigation. To the best of our knowledge, the present study is the first to demonstrate the effects of SLY on vimentin and fibronectin expression levels in 3D settings. We acknowledge that MDA-MB-231 spheroids exhibited significant structural fragility compared to MCF-7 spheroids, a phenomenon primarily attributed to their aggressive mesenchymal phenotype and inherently reduced cell–cell adhesion. In this study, spheroids were intentionally generated using a scaffold-free approach, without the addition of an exogenous extracellular matrix (ECM) or the use of co-culture systems. This methodological choice was made to isolate and evaluate the direct effects of SLY on breast cancer cell populations, thereby understanding the fundamental mechanisms of the drug independently of the complex influences exerted by the ECM or interactions with other cell types. While this approach provides critical insights into intrinsic cellular responses and direct cell–cell associations, it likely contributed to the loose and fragile nature of the MDA-MB-231 structures, making them highly susceptible to fragmentation during fixation and immunohistochemical processing. We recognize this as a study limitation and aim to incorporate ECM scaffolds or advanced 3D culture platforms in future investigations to better preserve the structural architecture of such aggressive cell lines while further delineating these complex interactions [54, 55].Consequently, the IHC results for MDA-MB-231 in the 3D model should be interpreted with caution. Overall, our findings suggest that SLY modulates the EMT process differently depending on the cell type and culture environment. The simultaneous upregulation of both epithelial–mesenchymal markers in 3D cultures suggests that SLY may influence partial EMT. An imbalance between mesenchymal and epithelial markers during this process could be a potential strategy for slowing down or halting EMT progression.
Notably, this increase in Fibronectin expression occurred despite a significant reduction in cell migration and invasion in 3D MDA-MB-231 cultures. While Fibronectin is traditionally associated with promoting motility, high levels can significantly enhance cell-to-matrix adhesion strength, effectively 'anchoring' the cells to the extracellular matrix and thereby impeding their migratory capacity [56, 57]. Furthermore, the potent inhibition of matrix metalloproteinases, particularly MMP-9, by SLY likely prevents the proteolytic degradation of the matrix required for invasion, regardless of the increased Fibronectin levels [58]. Previous studies have also suggested that Silibinin can modulate integrin signaling and stabilize the cellular interface, which may shift the functional role of Fibronectin from a pro-migratory factor to a stabilizing adhesive molecule [52, 59]. Thus, the observed increase in Fibronectin may represent a compensatory but functionally ineffective response, or a shift towards a more stationary phenotype induced by SLY. Similarly, In our 3D MDA-MB-231 model, we observed a notable discrepancy between the downregulation of MMP9 mRNA and the increased protein immunoreactivity. Such discordance between transcript and protein levels is frequently reported in 3D spheroid model and may stem from post-transcriptional regulatory mechanisms or enhanced protein stability within the dense extracellular matrix of the spheroid [60, 61]. Furthermore, it is important to note that increased MMP-9 expression does not inherently reflect increased enzymatic activity. The significant impairment of cellular invasion observed in our functional assays following SLY treatment suggests that SLY effectively reduces the proteolytic capacity of these cells, likely by modulating both the expression and the functional activation of matrix metalloproteinases. Overall, these results reveal that SLY does not suppress EMT via a single mechanism involving E-cadherin, N-cadherin, vimentin, and fibronectin but rather exerts context- and cell-type-dependent effects. Further mechanistic studies are warranted to delineate the precise signaling pathways through which SLY exerts its antimetastatic effects.
EMT signaling pathways have been shown to induce the expression of genes that encode MMP2 and MMP9. These enzymes degrade type IV collagen, which is present in the basement membrane. This promotes the invasion of underlying tissues following EMT [20, 21]. MMPs can cleave E-cadherin in the cell membrane, disrupting adherens junctions and weakening the epithelial layer. This facilitates the acquisition of a mesenchymal phenotype and migration into the extracellular matrix [22]. In their study investigating the effects of silibinin on EMT expression in MDA-MB-231 and MCF-7 cells, Si et al. (2020) observed a modulation in MMP-9 after treatment compared with controls [52]. Another study reported that increased soluble E-cadherin interferes with adherens junctions and modulates ADAMs and MMPs, thereby affecting cell migration, proliferation, and survival [62]. Silibinin has also been reported to inhibit COX-2 in breast cancer, thereby reducing 12-O-tetradecanoylphorbol-13-acetate-induced MMP-9 [63]. Byun et al. (2017) further demonstrated that silibinin downregulates MMP9 via the MEK/ERK signaling pathways, leading to decreased MMP-9 levels [60].
In the present study, RT-PCR analysis revealed an increase in MMP9 mRNA levels in both cell lines under 2D settings, whereas a significant decrease in MMP9 expression was observed in 3D settings. This reduction is thought to be related to SLY’s tendency to increase soluble E-cadherin levels in 2D settings. However, when applied in 3D environments, the increase in soluble E-cadherin was markedly lower, which correlates with the observed decrease in MMP9.
SLY exhibited stronger antimetastatic properties against MDA-MB-231 cells compared with MCF-7 cells. In 2D settings, SLY significantly reduced the migration speed (p < 0.05). Meanwhile, in 3D settings, it decreased MDA-MB-231 invasion by 42%. Notably, SLY altered EMT markers. PCR and immunohistochemistry revealed reduced E-cadherin and increased N-cadherin expression, particularly in 3D-cultured MDA-MB-231 cells. This finding suggests a shift toward a mesenchymal phenotype. Furthermore, SLY suppressed MMP-9 in 3D settings, which could inhibit Matrigel invasion.
When comparing the efficacy of SLY between the two cell lines, a distinction must be made between its cytotoxic and antimetastatic effects. Our results indicate that while MCF-7 cells are more susceptible to SLY-induced growth inhibition (lower IC50), MDA-MB-231 cells demonstrate a higher degree of metabolic resistance, particularly in 3D spheroid model. However, from an antimetastatic perspective, SLY appears to exert a more profound impact on the aggressive phenotype of MDA-MB-231. Despite their higher IC50, the significant reduction in invasion and the modulation of EMT markers in MDA-MB-231 spheroids suggest that SLY effectively targets the metastatic machinery of these triple-negative cells. In contrast, the antimetastatic effect in MCF-7, while present, is less dramatic due to the inherently lower baseline invasiveness of this epithelial cell line. Thus, SLY’s therapeutic potential is characterized by its ability to facilitate cell death in luminal-like cells while simultaneously suppressing the invasive drive of more aggressive, mesenchymal-like breast cancer cells.
Molecular mechanisms underlying the antimetastatic effects of SLY
The antimetastatic potential of SLY observed in our 2D and 3D spheroid model can be attributed to its multi-targeted modulation of key signaling pathways involved in EMT. Previous studies have demonstrated that silibinin, the primary active component of SLY, suppresses the TGF-β/Smad signaling pathway, which is a master regulator of EMT. Specifically, SLY has been shown to downregulate TGF-β2 expression and inhibit the phosphorylation of Smad2/3, thereby preventing the transcriptional activation of mesenchymal genes such as MMP9 and vimentin ([30, 62]). Furthermore, SLY exerts its inhibitory effects via the Wnt/β-catenin pathway by promoting the proteasomal degradation of β-catenin and reducing its nuclear translocation, which is crucial for maintaining the epithelial phenotype and E-cadherin expression [39]. Additionally, the suppression of the MAPK/ERK and PI3K/Akt pathways by SLY has been linked to reduced cellular proliferation and impaired migratory capacity in aggressive breast cancer models [63]. Our findings of significantly reduced MMP-9 expression and enhanced E-cadherin levels in 3D spheroids are consistent with these mechanistic axes, suggesting that SLY acts as a potent inhibitor of the TGF-β and Wnt-mediated EMT programs.
Conclusions
SLY was found to have an effect on MDA-MB-231 cells, inducing significant changes, particularly with regard to invasion, migration, and EMT. The effects were more pronounced in 3D, suggesting that the 3D environment more accurately reflects the impact of SLY. Our findings suggest that SLY may modulate certain metastatic processes by influencing E-cadherin and N-cadherin levels and reducing MMP-9 expression, although these effects appear to be context-dependent. Although the findings suggest that SLY holds potential as an antimetastatic agent, further investigations and supportive therapies are needed to fully validate its efficacy.
Future research should focus on elucidating the specific molecular pathways, such as the detailed crosstalk between PI3K/Akt and other EMT-related signaling cascades, that drive the differential response of MCF-7 and MDA-MB-231 cells in 3D environments. Additionally, incorporating more complex co-culture models involving stromal cells or immune components would further enhance the biomimetic accuracy of the 3D system. Finally, in vivo validation of Silymarin’s antimetastatic potential in patient-derived xenograft (PDX) models remains a crucial next step to translate these in vitro findings into clinical therapeutic strategies.
Supplementary Information
Acknowledgements
I would like to thank Prof. Dr. İsmet Doğan, Head of the Biostatistics Department at the Faculty of Medicine at Afyonkarahisar Health Sciences University, for his support.
Abbreviations
- 2D
Two-dimensional cell culture
- 3D
Three-dimensional cell culture
- CDH1
E-cadherin gene
- CDH2
N-cadherin gene
- DMSO
Dimethyl sulfoxide
- DNA
Deoxyribonucleic acid
- ECM
Extracellular matrix
- EMT
Epithelial–mesenchymal transition
- ER
Estrogen receptor
- MCF-7
Michigan Cancer Foundation-7
- MDA-MB-231
M.D. Anderson—Metastatic Breast—231
- MMP
Matrix metalloproteinase
- MTT
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
- PR
Progesterone receptor
- RT-PCR
Real-time polymerase chain reaction
- SLY
Silymarin
- TNBC
Triple-negative breast cancer
- VIM
Vimentin protein
Authors’ contributions
Conceptualization, TA, FF, and HC; Methodology, TA, ESAS, and FF; Validation, TA, FF, and HC; Formal Analysis, TA and FF; Investigation, TA; Resources, TA; Data Curation, TA and FF; Writing–Original Draft Preparation, TA and FF; Writing–Review & Editing, TA; Visualization, TA, FF, ESAS, FF, and HC; Supervision, TA and HC; Project Administration, TA, FF, and HC; Funding Acquisition, TA and HC. All authors read and approved the final manuscript.
Funding
This study was supported by Pamukkale University’s Scientific Research Projects Coordination (BAP) Department as part of a doctoral thesis project.
Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.Artificial intelligence tools were used to assist with language editing and graphic design. The authors developed and verified all scientific content, interpretation and conclusions.
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Supplementary Materials
Data Availability Statement
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.Artificial intelligence tools were used to assist with language editing and graphic design. The authors developed and verified all scientific content, interpretation and conclusions.