Mandragora autumnalis: Phytochemical Composition, Antioxidant and Anti-Cancerous Bioactivities on Triple-Negative Breast Cancer Cells

1. Introduction

Breast cancer is one of the many common malignant tumors that affect women[1]. Numerous internal and external factors contribute to the development and occurrence of breast cancer[2]. The milk duct is often the site of emergence; lobules are the site of less serious cases. Cancers that affect the ductal region are referred to as ductal carcinomas, and those that affect the mammary lobules are known as lobular carcinomas[3]. Triple-negative breast cancers, or TNBCs, are considered aggressive forms of breast cancer and result from reduced expression of the human growth factor receptor 2 as well as the progesterone and estrogen receptors[4]. Natural recurrence occurs in TNBCs, which account for 12–17% of all breast cancer cases. Compared to other subtypes of breast cancer, their clinical behavior is comparatively more aggressive. Furthermore, TNBCs account for 24% of newly diagnosed breast cancers, and their incidence has been reported to be steadily rising. These cancers also have poor prognoses and typical metastatic patterns where the average survival rate is approximately 10.2 months; for regional tumors, the 5-year survival rate is 65%, and for tumors that have spread to distant organs, it is 11%[5,6,7]. Globally, the incidence of breast cancer is increasing along with the disease's burden, making breast cancer an important public health concern[8]. Due to population growth and aging, it is predicted that the incidence of breast cancer will increase by more than 40% by 2040, reaching roughly 3 million cases annually. In a similar vein, over 50% more people will die from breast cancer in 2040[9]. Breast cancer has been linked to several established risk factors, such as late menopause, advanced age at first birth, fewer children, decreased breastfeeding, menopausal hormone replacement therapy, oral contraceptives, alcohol consumption, and excess body weight[10].

The use of phytonutrients, also known as nutraceuticals and herbal medicines, is still growing worldwide, with many people turning to these products in various national healthcare settings to treat various health issues. For years, herbal remedies have been used as anticancer medications. They also have anti-inflammatory properties and many anticancer ingredients that have been shown to have direct cytotoxic effects and indirect actions on the tumor microenvironment, cancer immunity, and chemotherapy progression[11,12]. From 200 to 1800 AD, according to Aristotle and Galen's teachings, cancer was thought to be caused by the coagulation of "black bile." Until recently, when the prevalence of biology has led to a 25% decrease in mortality, herbs were crucial in managing cancer symptoms and improving the quality of life and survival of patients with the disease[13]. Compared to conventional chemical drugs, herbal medicine fights cancer in a significantly different way, as it prevents DNA mutation in surviving cells. Another benefit of herbal medicines is that they can create an environment that is unfavorable to the growth of cancer[14].

The Mandragora autumnalis plant has been prized as one of the most significant medicinal plants and has had great cultural significance as an herb since ancient times. It was remarkably used throughout history. Mandragora has a wide range of uses, including medicinal, hallucinogenic, and boosting ovulation. Furthermore, Mandragora autumnalis possesses a narcotic effect, which is most likely caused by the presence of an alternative form of alkaloids[15]. The plant genus Mandragora, a member of the Solanaceae family, is commonly known as "mandrake". Historically, various parts of the Mandragora species, including the root, fruit, and leaves, have been used to treat conditions like ulcers, inflammation, sleeplessness, and eye health issues. The mandrake plant has been used for centuries as a valuable and traditional herbal medicine; however, little research has been conducted on its biological activity and potential phytochemicals [16,17,18].

In this work, we used Liquid Chromatography Mass Spectrometry (LC-MS) analytical examination and qualitative assays to examine the phytochemical composition of Mandragora autumnalis ethanolic leaf extract (MAE). Using the MDA-MB-231 cell line, we also investigated the anticancer potential of MAE by examining its effects on cell proliferation, the induction of apoptosis, and various hallmarks of cancer metastasis on a molecular level, including adhesion, invasion, cell cycle, angiogenesis, migration, and aggregation in TNBC.

2. Results

2.1. Phytochemical Composition, Total Phenolic, and Total Flavonoid Content (TPC and TFC) Assays

The presence of various primary and secondary bioactive metabolites, including terpenoids, tannins, fatty acids, flavonoids, phenols, and steroids, was revealed by a qualitative phytochemical screening of the MAE crude extract (Table 1).

A large class of secondary metabolites known as polyphenolic compounds is responsible for most plants' antioxidant qualities. Our results showed that the TPC of MAE is 58.98±7.40 mg GAE/g and TFC is 36.47±0.87 mg QE/g, as shown in Table 2.

2.2. LC-MS Analysis of MAE Extract

The mass-to-charge ratios (m/z) and retention times (RT) of 34 compounds were determined by LC-MS analysis of MAE in both positive and negative ionization. The LC-MS analysis in positive ionization mode (Table 3A, Figure 1) revealed a chemically diverse profile consisting of multiple classes of bioactive compounds. The most abundant group was alkaloids, represented by hyoscyamine, tropine, methylisopelletierine, tropinone, and solacaproine. The phenolic compounds group was marked by the presence of chlorogenic acid, and flavonoids were identified through the detection of quercetin and chrysin. Fatty acid derivatives were another major group, including hexadecanamide (palmitic amide), ethyl palmitate, ethyl oleate, linoleic acid, and ethyl linolenate. Esters such as ethyl 3-hydroxybutanoate, 3-(methylthio)propyl acetate, and ethyl hydrocinnamate were also detected in notable quantities. Additionally, furan derivatives like 5-hydroxymethyl-2-furancarboxaldehyde and ketones and aldehydes such as 1-hydroxy-2-propanone acetate and β-ionone were present. A unique compound, simulanoquinoline, representing the quinoline alkaloids, was also observed. On the other hand, among the principal compounds detected under negative ionization mode (Table 3B, Figure 1) were polyphenolic acids and their derivatives, including chlorogenic acid with a high intensity, and caffeic acid. Also, Linolenic acid, a polyunsaturated fatty acid, and coumarins with their derivatives, including scopoletin, and 4-Methylumbelliferyl acetate, were present. Furthermore, flavonoids and glycosides, including rutin and Hyperoside, are identified. All things considered, these results show MAE has a rich phytochemical profile, bolstering its potential as a source of bioactive compounds for additional pharmacological research.

2.3. MAE Extract Has a Potent Radical Scavenging Potential

The results of this investigation demonstrated concentration-dependent free-radical scavenging activity for MAE extract (Figure 2). MAE demonstrated a high reducing power, where the DPPH free radical scavenging of MAE at a concentration of 600 µg/mL was 54.94 ± 0.89%. The TPC, TFC, and LC-MS data are consistent with these results, indicating that the extract's antioxidant capacity increases with phenolic and flavonoid concentration.

2.4. MAE Significantly Inhibits the Proliferation of MDA-MB-231 Cells

To examine the potential anti-proliferative impact of MAE extract on breast cancer, different concentrations of each extract (0, 50, 100, 200, 400, and 600 μg/mL) were examined at 24, 48, and 72 hours of treatment. Findings indicate that MAE treatment reduced the viability of cells in a concentration- and time-dependent manner. After 48 hr of treatment with 50, 100, 200, 400, and 600 µg/mL of MAE, the proliferation of MDAMB-231 cells was 92.18±7.32, 66.66±8.82, 22.67±0.17, 17.19± 0.92,18.00± 1.42 % respectively (Figure 3A). Using the MTT assay, the IC₅₀ of MAE was 467.28, 132.33, and 109.4 µg/mL at 24, 48, and 72 h. Based on that and the IC₅₀ values, 100 and 200 µg/mL of MAE were used in further experiments. Crucially, the viability assay against MDA-MB-231 cells revealed that DOXO was more harmful to MDA-MB-231 cells with a CC₅₀ of 0.0036 µg/mL after 72 hours (Figure 3B).

To examine the potential correlation between the antiproliferative effects of the extracts and the generation of ROS induced by MAE, MDAMB-231 cells were pretreated with N-acetyl cysteine (NAC), a ROS scavenger[19]. The findings indicated that NAC significantly reduced NAC-mediated cell death (Figure 3C). Specifically, the viability of cells treated with 100 μg/mL MAE for 24 hours was 64.33% ± 0.007% in the absence of NAC and was significantly increased to 165.36% ± 0.001% when cells were pre-treated with NAC. Similarly, the viability of cells treated with 200 μg/mL MAE was 36.25% ± 0.002% in the absence of NAC, and NAC pretreatment significantly elevated cell viability to 281.47% ± 0.01%. These results suggest that MAE exerts its anti-proliferative effect in TNBC cells through a ROS-dependent mechanism.

To further validate MAE's anti-proliferative properties, protein lysates from MAE-treated cells were immunoblotted with an anti-Ki67 antibody, which is strongly linked to the proliferation of cancer cells. Ki67 is highly expressed in TNBC and is a known predictive and prognostic marker for many cancers[20]. The treatment of MDA-MB-231 cells with MAE at 200 µg/mL significantly reduced Ki67 protein levels in a concentration-dependent manner by 0.47±0.07 fold, in comparison to the vehicle-treated control cells, as shown in Figure 3D. These findings demonstrate that MAE inhibits MDA-MB-231 cells' ability to proliferate by interfering with their growth.

2.5. MAE Induces Intrinsic apoptosis in MDA-MB-231 Cells

After subjecting MDA-MB-231 cells to a 24-hour MAE treatment, the cells were viewed under an inverted phase-contrast microscope. The morphological observation of MAE-treated cells showed a concentration-dependent decrease in the total number of cells per microscopic field. Furthermore, MAE-treated cells displayed characteristics of apoptosis. At higher magnifications, apoptotic bodies were visible along with cytoplasmic shrinkage, loss of epithelial morphology, membrane blebbing, and rounded shape in the cells (Figure 4A). Additional examination of MAE-treated cells using DAPI staining demonstrated the development of apoptotic bodies, chromatin lysis, and nuclear condensation (Figure 4B). Next, we aimed to confirm that cells treated with MAE undergo activated apoptosis. To achieve this, we first assessed the levels of procaspase-3, a protein that is essential to apoptosis[21]. Western blotting was used to analyze the protein levels of the apoptosis effector enzyme pro-Caspase 3 and its cleavage products in order to confirm that MAE activated apoptosis. The results showed reduced levels of the Pro-Caspase 3 protein in a concentration-dependent manner.

The levels of procaspase-3 in cells treated with MAE at 200 μg/mL decreased significantly, reaching 0.54 ± 0.12-fold (Figure 4C). The levels of cleaved caspase 3 fragments were also markedly elevated by MAE treatment concurrently. The increase in Caspase3 cleavage products was significant, with a 1.27±0.02 increase at 200 μg/mL of MAE. This implies that the intrinsic cascade of apoptosis was initiated by MAE extracts, which caused the proteolytic cleavage of pro-Caspase 3 into its active form, caspase 3.

Another anti-apoptotic protein known to be important for the intrinsic apoptosis pathway is B-cell lymphoma 2 (Bcl-2)[22]. According to our findings, MAE significantly reduced Bcl-2 protein levels to reach 0.70±0.05 decrease at 200 µg/mL of MAE compared to the control (Figure 4C). Bcl-2-associated X-protein (Bax), a pro-apoptotic protein, is another apoptosis regulator[23]. In our investigation, cells treated with 200 µg/mL MAE showed significantly higher Bax levels with 1.64±0.02 fold (Figure 4C). These findings suggest that MAE targets apoptotic pathways to mediate its anticancer effects.

2.6. MAE Enhances the Aggregation of MDA-MB231 Cells

Epithelial-mesenchymal transition (EMT), a process where epithelial cells acquire a mesenchymal phenotype characterized by a decrease in cell-cell adhesion and enhanced migratory and invasive capabilities, which is one of the hallmarks of cancer progression towards metastasis[24]. An aggregation assay was used to assess how MAE affected the MDA-MB-231 cells' cell-cell adhesion characteristics. As demonstrated in Figure 5, there was a concentration-dependent rise in cell-cell aggregates following the treatment of cells with 100 and 200 µg/mL MAE, with significant increases of 73.39 ±12.35 and 80.80±8.86 %, respectively. One of the main constituents of adherens junctions, which are specialized structures that facilitate robust cell-cell adhesion in epithelial tissues, is E-cadherin, also known as epithelial cadherin[25]. Particularly in TNBC, E-cadherin downregulation facilitates the migration of breast cancer cells through the extracellular matrix and their invasion of blood and lymphatic vessels, and encourages the spread of disease to other organs. E-cadherin protein levels were raised in MDA-MB-231 cells treated with 100 and 200 µg/mL MAE in a dose-dependent manner, and significantly at 200 µg/mL, there was a discernible difference from the control, with a 1.67 ± 0.22-fold change.

2.7. MAE Lowers the Adhesion Potential of MDA Cells

Integrins, among other members of the adhesion molecule family, are the primary mediators of cell-ECM interaction. Elevated integrin β1 expression confers survival benefits to cancer cells and is linked to their capacity for migration and metastasis[26]. As seen in Figure 6A, the treatment of cells with 100 and 200 µg/mL MAE significantly reduced their adhesive abilities to 53.14103 ± 0.08237 and 48.45218 ± 0.031702%, respectively, compared to the control cells. Here, the application of 100 and 200 µg/mL MAE to MDA-MB-231 cells resulted in a significant decrease in the concentration-dependent increase in integrin β1 protein levels by 0.68 and 0.60-fold, contrasted, in turn, with the control cells that were subjected to the vehicle (Figure 6B). All of these findings support the hypothesis that MAE disrupts the integrin-ECM axis, which may limit MDA cells' ability to spread.

2.8. MAE Decreases MDA Cells' Ability to Migrate

Cell migration is crucial during physiological processes like immune responses, wound healing, and embryonic morphogenesis. It is also a crucial process that plays a major role in the early stages of cancer metastasis and is a primary feature of a malignant phenotype[27]. Using the wound-healing assay, the impact of MAE extract on MDA cell migration was investigated. MAE reduced the MDA cells' ability to migrate and fill the wound in a concentration- and time-dependent manner, as shown in Figure 7A. For instance, treating MDA cells with 100 and 200 μg/mL of MAE significantly decreased their migration ability by 0.59 ± 0.13 and 0.40±0.07 fold, respectively, compared to the control cells. These results were further confirmed by the transwell migration assay. MAE treatment significantly reduced MDA-MB-231 cells' capacity to migrate from the upper to the lower chamber Figure 7B.

2.9. MAE Inhibits the Invasive Properties of MDA Cells

An important stage in the initial phases of the cancer metastatic cascade, in which cancer cells spread from the primary tumor site and invade secondary sites, is the invasion of other tissues[28]. When compared to the control, MAE treatment significantly decreased the invasive potential of MDA-MB-231 cells in a dose-dependent manner using the Matrigel-coated trans-well chambers by 0.45±0.13 and 0.26±0.03 fold for 100 and 200 µg/mL, respectively (Figure 8).

STAT3 is a well-known and important participant in the development of tumors. Thus, focusing on the STAT3 pathway is a viable approach for creating new cancer medications because it stimulates the migration and invasion of cancer cells, prevents apoptosis, and promotes cell proliferation[29]. We measured the amount of the active phosphorylated form of STAT3 in MAE-treated MDA-MB-231 cells to see if Mandragora autumnalis mediates its anticancer activity by targeting the STAT3 pathway. Our findings demonstrated that, in comparison to the control, MAE treatment at 100 and 200 µg/mL significantly decreased the levels of STAT3 by 0.65 ± 0.08 and 0.48 ± 0.11 fold, respectively (Figure 8). This finding implies that the downregulation of the STAT3 signaling pathway is a necessary component of the MAE-mediated impact of TNBC progression and metastasis. Also, one widely known mechanism that encourages the migration and invasion of cancer cells is the breakdown of the extracellular matrix (ECM) by matrix metalloproteinases (MMPs)[30]. MMP-9 activity levels were significantly reduced by 200μg/mL MAE with 0.57 fold change in comparison to vehicle-treated cells. These findings suggest that MAE may inhibit MMPs to lessen the invasive potential of MDA-MB-231 cells.

2.10. MAE Triggers MDA-MB-231 Cells to Arrest in the G0/G1 Phase of the Cell Cycle

MDA-MB-231 cells were treated with 200 µg/mL of MAE for 24 hours, and the cell cycle distribution was investigated using flow cytometry. As seen in Figure 9A, the percentage of cells in the G0 phase rose in MAE-treated cells (12.3 ± 2.3 versus 5.3 ± 1.7 in control cells). The percentage of cells in the G1 phase increased with this (53.2 ± 5.8 vs. 29 ± 7.9 in control cells). The findings imply that MAE promotes a cell cycle arrest during the G0/G1 phase. The percentage of cells in the S and G2-M phases decreased along with this, with 15.7 ± 2.7 and 17.6 ± 3.4 for control cells and 3.4 ± 1.2 and 8.2 ± 3.14, respectively, indicating that MAE causes a cell cycle arrest at the G1 phase and prevents cell entry into the S phase.

The p53 gene is a tumor suppressor; that is, when it is active, tumors cannot grow[31]. Phosphorylated p53 levels rose in response to MAE treatment in a concentration-dependent manner; at 200 µg/mL, there was a significant increase (1.53±0.02-fold change in the control) (Figure 9B). This data implies that cell cycle arrest may result from MAE-induced p53 activation. Moreover, the p38 MAPK (mitogen-activated protein kinase) pathway is frequently associated with preventing cell proliferation by regulating the progression of the cell cycle and inducing apoptosis[32], Western blotting was used to measure the protein levels of the active phosphorylated form of p38 (P-p38). The findings indicate that MAE increased P-p38 levels in a concentration-dependent manner; following treatment with MAE, the cells increased by 1.3 ± 0.09 at 100 μg/mL and significantly by 1.5 ± 0.15-fold at 200 μg/mL (Figure 9B). Moreover, MAE significantly increased the levels of CDK inhibitors p21 and p27, which are downstream effectors of p38, to 1.61±0.21 and 1.51±0.26 at 200 μg/mL, respectively. In addition to being a downstream effector of p38 signaling, retinoblastoma protein (Rb) plays a part in differentiation, tumor suppression, cell cycle regulation, and apoptosis control[33] . P-rb levels were markedly decreased by MAE in a concentration-dependent manner (Figure 8B). Rb phosphorylation was significantly decreased by 200μg/mL to 0.29±0.1-fold compared to the vehicle control. The impact of MAE on cell cycle dynamics is further supported by these findings.

2.11. MAE Reduces iNOS and COX-2 Levels and Inhibits Angiogenesis in Ovo

Angiogenesis is the process by which new blood vessels are created to supply cells with oxygen and nutrients as a result of tumor cells spreading from the primary tumor site and metastasizing into secondary sites. As a result, stopping angiogenesis is crucial to preventing[34]. The chick-embryo chorioallantoic membrane (CAM) assay was used to investigate how MAE influences angiogenesis. This was done by applying MAE to the surface of the widely vascularized CAM membrane over 24 hours. By reducing the overall vessel length and number of junctions, MAE treatment of 200µg/mL significantly suppressed angiogenesis, compared to the control, and resulted in a decrease in the number of junctons of 72.92 ± 3.44% and a reduction in the total vessel length of 44.56 ± 9.4% as seen in Figure 10B. Moreover, inducible nitric oxide synthase (iNOS), a crucial producer of nitric oxide (NO)[35], and cyclooxygenase 2 (COX-2), an enzyme that produces prostaglandin E2 (PGE2)[36] are both mediators of angiogenesis. Our findings demonstrated that 200 µg/mL MAE significantly decreased the levels of COX-2 and iNOS proteins by 0.58 ± 0.038 and 0.52 ± 0.11, respectively compared to the control. According to these findings, MAE suppresses angiogenesis by focusing on the synthesis of PGE2 and NO (Figure 10A).

3. Discussion

Plants and plant-derived metabolites still have major roles in the development of drugs for the prevention or treatment of diseases. Currently, there is growing interest in screening plants to identify therapeutic agents. However, there are scarce investigations on the therapeutic potential of its therapeutic bioactivities. Plant-derived drugs are the source of many of the current cancer chemotherapy agents[37]. In this context, the Food and Drug Administration (FDA) has authorized three herbal mixture-based treatments, including antiallergenic, anticancer, and anti-psoriatic medications, in recent years[38]. Plants are rich in secondary metabolites with a variety of biological functions that are essential to the growth and development of the plant, in addition to serving as a reservoir of phytochemicals that shield the plant from environmental constraints and aid in adaptation. It is well known that many of these secondary metabolites have potent anti-inflammatory, anti-cancer, and antioxidant therapeutic qualities[39]. However, Mandragora autumnalis's medicinal potential has not been thoroughly studied. In this current study, we assessed the ethanolic extract of Mandragora autumnalis (MAE) for antioxidant potential. We used LC-MS to qualitatively and quantitatively analyze the phytochemical composition of the extract. Furthermore, we examined MAE's potential as an anticancer agent by evaluating its impact on key indicators of cancer, such as adhesion, aggregation, invasion, migration, and proliferation, compared to the aggressive triple-negative breast cancer cell subtype and the underlying molecular mechanisms. Numerous classes of phytochemical compounds, such as flavonoids, phenols, tannins, steroids, and essential oils, were found in MAE, according to the qualitative phytochemical analysis. Our findings concur with earlier research indicating the presence of coumarins and lipid-like compounds in extracts from Mandragora species[17]. As a result of its capacity to scavenge DPPH radicals, the ethanolic extract of Mandragora autumnalis demonstrated good antioxidant potential in vitro[40,41]. Moreover, phenols and flavonoids are recognized for their anticancer activity, anti-inflammatory, and antioxidant function[42]. The LC-MS profiling of the tested plant extract revealed a diverse set of bioactive phytochemicals, many of which have been documented in other medicinal plants for their promising anticancer properties, particularly against triple-negative breast cancer (TNBC). TNBC is an aggressive breast cancer subtype lacking estrogen, progesterone, and HER2 receptors, often exhibiting poor prognosis and limited therapeutic options[43]. Hence, phytochemicals capable of multi-targeted anticancer action offer significant therapeutic value. In this study, the LC-MS analysis revealed a diverse profile of bioactive compounds, including alkaloids (hyoscyamine, tropine, tropinone, solacaproine), phenolic acids (chlorogenic acid), flavonoids (quercetin, chrysin), fatty acid derivatives (hexadecanamide, ethyl palmitate, linoleic acid, ethyl linolenate), and esters (ethyl hydrocinnamate, ethyl 3-hydroxybutanoate). Similar phytochemical compositions have been reported in medicinal plants like Datura stramonium, Atropa belladonna, and Withania somnifera, which are known for their rich alkaloid and flavonoid contents, targeted anticancer therapies. Among the major identified compounds, chlorogenic acid, scopoletin, caffeic acid, rutin, hyperoside, and linolenic acid have garnered substantial interest for their roles in inhibiting TNBC cell viability and metastasis. For instance, chlorogenic acid—widely found in Coffea arabica, Lonicera japonica, and Solanum nigrum—has demonstrated the ability to suppress TNBC cell proliferation, migration, and invasion through the modulation of key signaling pathways such as NF-κB and PI3K/Akt[44]. In MDA-MB-231 TNBC cells, chlorogenic acid downregulated MMP-9 expression and induced apoptosis, suggesting its chemopreventive potential[45]. Scopoletin, found in Morinda citrifolia, Artemisia absinthium, and Scopolia carniolica, appeared at high intensities in the sample, indicating a major role in bioactivity. This coumarin compound has been reported to inhibit TNBC cell growth by inducing oxidative stress-mediated apoptosis and enhancing chemosensitivity[46]. Rutin and hyperoside, both flavonol glycosides, are common in Sophora japonica, Ginkgo biloba, and Hypericum perforatum and exhibit anticancer potential by inhibiting epithelial-mesenchymal transition (EMT), a critical event in TNBC metastasis. Rutin has also shown synergistic effects with doxorubicin in reducing TNBC tumor burden[47]. Similarly, hyperoside inhibits the proliferation and migration of MDA-MB-231 cells by regulating miRNA expression and downregulating MMPs. Additionally, 3-methyl-2,5-furandione, a furan derivative, though less studied, shares structural similarity with compounds known for antiproliferative effects and may represent a novel candidate for anti-TNBC activity[48]. Collectively, the presence of these compounds, many of which are found in medicinal plants traditionally used in cancer therapy, supports the potential of this extract as a multi-targeted approach against cancer, especially TNBC.

Cancer is marked by uncontrolled proliferation, resulting from improper cell division and death regulation[49]. We found that MAE extract had significant anti-proliferative activity against MDA-MB-231 cells in a concentration-dependent manner. This coincided with a substantial decrease in Ki67, a highly expressed proliferation marker that correlates with tumor severity[50]. Inducing apoptosis is an effective strategy for controlling cancer cell proliferation, and targeting apoptotic pathways is becoming increasingly important in cancer treatment development [51]. To better understand the anti-proliferative mechanism of the MAE extract, we studied apoptosis induction. MAE significantly inhibited MDA-MB-231 cell proliferation in a dose-dependent manner. Previous research has shown that Mandragora autumnalis inhibits the proliferation of various cancer cell lines [15]. This study is the first to evaluate the impact of MAE on the triple-negative subtype of breast cancer, including its underlying molecular mechanisms and cancer phenotypes.

In cancer therapy, inducing apoptosis in cancer cells is an essential tactic in addition to preventing unchecked proliferation [52]. TNBC is among the cancer cells that frequently avoid apoptosis, which promotes tumor growth and treatment failure[53]. Consequently, activating apoptotic pathways is a highly effective method of treating cancer. The downregulation of pro-caspase 3, the anti-apoptotic Bcl-2 protein, and the upregulation of the pro-apoptotic Bax protein and caspase 3 in our investigation all suggested that MAE induced intrinsic apoptosis. This comes in line with previous studies on other plant extracts, including Ziziphus nummularia, and Haludule uninervis, on triple-negative breast cancer cells[54,55].

Additionally, the p53 pathway was investigated to better understand the mechanism of MAE's anti-proliferative effects. p53 is a tumor suppressor protein encoded by the TP53 gene; its primary biological function appears to be to protect the DNA integrity of the cell. TP53 has additional functions in development, aging, and cell differentiation [56,57]. Activated p53 promotes cell cycle arrest to allow DNA repair and apoptosis to prevent the spread of cells with severe DNA damage by transactivating target genes involved in cell cycle arrest and apoptosis[58]. Mutations in the p53 gene (mtp53) are associated with a variety of cancers, including 70-80% of TNBC [59]. Interestingly, the phosphorylation of Ser15 (mediated primarily by the ATM and ATR protein kinases in response to genotoxic stress) acts as a nucleation event that promotes or permits subsequent sequential modification of many residues[60]. Furthermore, ATM-induced phosphorylation promotes the recruitment of histone/lysine acetyltransferases (HAT), such as p300 and CBP, resulting in the acetylation of multiple lysine residues in p53's DNA binding (DBD) and carboxy-terminal domains. These modifications are thought to contribute to the stabilization of p53 by blocking ubiquitylation and suggest that p53 restores its tumor-suppressive activity[61]. MAE induced p53 phosphorylation in MDA-MB-231 cells, potentially restoring its wild-type conformation and increasing transcriptional activity. This suggests a promising therapeutic strategy for preventing cancer cell proliferation. Moreover, to gain insight into the mechanism of MAE-induced anti-proliferative effects, the involvement of the p38 mitogen-activated protein kinase (MAPK) pathway was investigated. P38 is known to play a crucial role in maintaining cellular homeostasis by regulating various cellular processes, including cell cycle progression, apoptosis, and cellular response to stress[62]. Our results showed that MAE increased the levels of p38, which is by other studies reporting the relationship between p38 activation and the induction of cell cycle arrest[63]. In addition to that, the levels of the cell cycle inhibitor protein p21, a downstream effector of p38, and p27 were significantly increased by MAE. Moreover, MAE significantly reduced the phosphorylation of Rb, further implicating the p38 MAPK pathway in the proliferation of MDA-MB-231 cells. This can be explained by the activation of p38 MAPK, which stabilizes CDK inhibitors (e.g., p21 and p27) , delaying Rb phosphorylation, which keeps Rb in its active, growth-suppressing form and causes cell cycle arrest in the G1 phase[64,65]. As a result of TNBC, increasing the levels of activity of p27, p21, p38, Rb, and p53 leads to a significant extension of the G0 and G1 phases of the cell cycle, preventing cancer cells from entering the DNA synthesis S phase. This cell cycle arrest is a desirable therapeutic effect, as it limits tumor cell proliferation and promotes apoptosis or senescence. Targeting this network of tumor suppressors and stress response proteins holds promise for developing more effective treatments for TNBC, which currently lacks targeted therapies.

Reactive oxygen species (ROS) are elevated in nearly all cancers, where they support various aspects of the growth and spread of the tumor. The delicate regulation of intracellular ROS signaling to efficiently rob cells of ROS-induced tumor-promoting events and tip the scales in favor of ROS-induced apoptotic signaling will present a challenge for innovative therapeutic approaches. On the other hand, therapeutic antioxidants may stop critical early stages of tumor development before ROS are produced[66]. In this study, MAE reduction of MDA-MB-231 viability was attenuated by suppressing ROS levels with a potent antioxidant like NAC. All of these points to a scenario in which MAE causes ROS levels to rise, which in turn triggers anti-proliferative signaling pathways in MDA-MB-231 cells. It's possible that this signaling involves the ROS-p53 axis. Similar findings stated that when hysapubescin B, a withanolide derived from Physalis pubescens, was applied to HCT116 colorectal cancer cells, it produced reactive oxygen species (ROS) that inhibited mTORC1, which in turn activated autophagy and caused cell death[67] Notably, MAE stimulates the production of reactive oxygen species (ROS), suggesting that MAE contributes to oxidative stress in MDA cells. One of the main causes of DNA damage and a threat to genomic integrity is oxidative stress, which activates members of the p53 family, most notably p53, which is essential for tumor suppression[68]. Upcoming experiments using p53 inhibitors and ROS scavengers should provide insight into the mode of interaction between ROS and p53, including whether p53 acts upstream or downstream of ROS or whether there is a bidirectional crosstalk.

Furthermore, we demonstrated that MAE impacted MDA's ability to migrate and adhere to one another. These functions are essential to the epithelial-mesenchymal transition (EMT) and serve as a marker of the cancer's spread to metastases. Cancer cells can migrate and invade more areas with less cell-to-cell adhesion[69].

Our study's results showed that treating MDA extract with MAE increased the creation of cell-cell aggregates and reduced their migration and invasion, reversing the EMT phenotype and preventing metastasis as a result. The complicated process of cancer metastasis involves the cancer cells moving from the main tumor site to other organs. It contributes to the spread of cancer and accounts for a sizable share of cancer-related mortality. Because of its aggressive nature, dismal prognosis, and resistance to treatment, TNBC metastasis poses a serious clinical challenge[70]. Tumor cell invasion is commonly believed to require the epithelial-to-mesenchymal transition (EMT); however, mounting data suggests that there are other pathways through which tumor cells can spread. Using mesenchymal or amoeboid dissemination mechanisms, tumor cells can spread individually or in clusters. Mesenchymal cells move forward through the production of traction force through integrin-mediated extracellular matrix adhesion and cytoskeletal contractility[71]. With their elongated morphology, mesenchymal cells can move forward by producing traction force through integrin-mediated extracellular matrix adhesion and cytoskeletal contractility[72]. The production of migration pathways by mesenchymal tumor cells is also dependent on proteolysis-dependent ECM degradation[73]. It has been determined that epithelial tumor cells can acquire mesenchymal phenotypes through the use of EMT and hybrid EMT. However, in the absence of proteolysis-dependent ECM remodeling, amoeboid cells with a rounded and deformable morphology can fit through small pores and narrow spaces in the ECM[74]. High-speed movement is produced during this kind of movement when the cells display bleb-like protrusions powered by actomyosin contractility and maintain weak but dynamic cell adhesion to ECM[75]. Collective cell migration is a movement pattern of multiple cells that maintain cell-cell connections and migrate in unison, as opposed to single-cell motility[76].Actin dynamics, integrin-based ECM adhesion, and proteolytic cleavage of ECM are the three factors that control this kind of tumor cell migration. The diverse tumor cells that migrate in cohesive groups preserve front-rear polarity and collaborate hierarchically [77]. The increase in cancer cell migration and invasion is partly due to the dysregulation of cell-cell adhesion and the extracellular matrix (ECM) degradation by metalloproteinases (MMPs)[78]. The MDA-MB-231 cells' ability to adhere to collagen was reduced by MAE treatment, suggesting that the cell-ECM interaction had been disrupted. Furthermore, MAE reduced the amounts of integrin β1, an adhesion molecule linked to TNBC's heightened invasiveness and aggression [79]. These findings support MAE's capacity to suppress TNBC cells' capacity to spread by focusing on cell-cell and cell-ECM interactions. Boyden chamber and wound-healing assays were used in our study to examine the impact of MAE on the migration and invasion characteristics of MDA-MB-231. In this study, TNBC cell migration and invasion were markedly inhibited by MAE. Multiple investigations involving various plant extracts have demonstrated that the inhibition of MMP-2 and MMP-9 expression diminishes the migratory and invasive potential of MDA-MB-231 cells[80]. In our investigation, MAE significantly and concentration-dependently decreased the MDA-MB-231 cells' cell-cell adhesion.

This was accompanied by a decrease in integrin β1 protein levels, suggesting that MAE could block tumor migration by inhibiting cell-to-cell adhesion. All things considered, MAE may be able to reduce TNBC metastasis by reducing cell-–ECM contact, preventing cell migration, adhesion, and invasion, and boosting cell-cell aggregation. More research is needed to determine whether the downregulation of MMPs mediates the MAE-induced inhibition of migration and invasion. The existence of multiple signaling pathways involved in cancer pathogenesis emphasizes the need for additional research[81,82]. Despite progress in cancer research, providing more involved pathways and molecular targets is very important[83]. STAT3, in particular, plays important roles in a variety of cellular processes, including the cell cycle, cell proliferation, cellular apoptosis, and tumorigenesis[84]. Persistent activation of STAT3 has been reported in a variety of cancer types, and a poor prognosis of cancer may be associated with the phosphorylation level of STAT3[85]. STAT3 promotes tumor progression and metastasis in TNBC by regulating gene expression-related apoptosis, EMT, cell migration, aggregation, and invasion. STAT3 can be tightly controlled by upstream signaling molecules like Janus kinase (JAK) and epidermal growth factor receptor (EGFR)[86]. It then localizes to the nucleus of cells and binds to target DNA, regulating the expression of subsequent proteins. However, in addition to its normal cell functions, STAT3 activation in the tumor microenvironment (TME) is considered an oncogenic event. High phospho-STAT3 expression is linked to a poor prognosis in cancer patients[87]. STAT3 constitutive activation is critical for tumor formation, development, metastasis, and recurrence. These are strongly associated with cancer hallmarks and lead to poor patient outcomes[88]. Therefore, the STAT3 pathway is a promising target for cancer therapy. Based on previous findings it has been shown that plant-derived compounds can reduce cancer by inhibiting the STAT3 signaling pathway and its associated genes. As an example, Baicalein and Cantharidin inhibit the STAT3 pathway via downregulating of IL6 and EGFR levels respectively. Moreover, Hydroxy-jolkinolide B, Deoxy-2β,16-dihydroxynagilactone E, Acetoxychavicol acetate inhibit triple negative breast cancer cells by downregulating of JAK/STAT pathway[88].It has been demonstrated that phosphorylated STAT3 (p-STAT3), which was significantly decreased by MAE, downregulates important pro-metastatic factors like matrix metalloproteinase-9 (MMP-9) which was significantly decreased, effectively inhibits tumor growth. When activated, the transcription factor p-STAT3 moves into the nucleus and stimulates the expression of genes related to invasion, metastasis, and cell survival. MMP-9, an enzyme that breaks down extracellular matrix components to promote cancer cell invasion and metastasis, is one of its well-established targets[89]. Thus, blocking STAT3 phosphorylation results in a decrease in MMP-9 expression[90], which in turn reduces TNBC cells' capacity for invasion which was achievedieved by MAE in this investigation. In order to reduce tumor aggressiveness and enhance outcomes for patients with TNBC, this regulatory axis emphasizes the therapeutic potential of targeting the STAT3/MMP-9 pathway.

The development of new blood vessels, or angiogenesis, is a crucial step in the growth, invasion, and metastasis of tumors[91]. Particularly, triple-negative breast cancer (TNBC) has high angiogenic activity, which promotes fast tumor growth and makes metastasis easier. Therefore, blocking pro-angiogenic factors to target angiogenesis has become a promising therapeutic strategy for controlling the progression of TNBC[92]. Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) are two important pro-angiogenic enzymes that are upregulated in TNBC. In TNBC, their overexpression increases the risk of metastasis and vascularization[93]. Notably, treatment with MAE decreased the expression of COX-2 and iNOS in MDA-MB-231 cells. Additionally, in the chick embryo chorioallantoic membrane (CAM) assay, MAE significantly reduced vessel length and the number of junctions, demonstrating a strong anti-angiogenic effect in vivo-like environment. These results are in line with earlier studies that demonstrated that Halodule uninervis , a seagrass, also inhibits angiogenesis in TNBC[55].

As a sum up, Effective therapeutic responses in TNBC are frequently characterized by downregulation of survival and invasion markers (e.g., Bcl-2, p-STAT3, MMP-9) and upregulation of pro-apoptotic and tumor suppressor proteins (e.g., Bax, caspase-3, p21, p27, p-p53). Increases in E-cadherin and decreases in integrin/MMP-9 indicate that EMT and metastasis are suppressed. All markers studied in this investigation were listed with their change and proposed role after treatment with MAE (Table 4).

Per these findings, MAE inhibited apoptosis, adhesion, migration, invasion, cell cycle, and angiogenesis in MDA-MB-231 cells. Our research concludes that there are numerous significant plant phytochemicals in the ethanolic extract of Mandragora autumnalis leaves, many of which have been shown to have pharmacological properties. Additionally, we showed that the extract has potent anti-metastatic and anti-cancer characteristics, which may help to lessen the malignant phenotype of TNBC. The carcinogenesis process's hallmarks, such as cell proliferation, cell aggregation, cellular adhesion, migration, and invasion, were all impacted by MAE. This calls for additional investigation into MAE and the isolation of the bioactive metabolites responsible for the reported anticancer effects. These investigations could identify MAE as a new source of innovative drug candidates.

4. Materials and Methods

4.1. Collection of Mandragora autumnalis Leaves and Preparation of their Ethanolic Extract MAE

Mandragora autumnalis leaves were collected during the spring season from south Lebanon. The leaves were cleaned and dried at room temperature. The plants were identified by Mohammad Al-Zein, a resident plant taxonomist at the American University of Beirut (AUB) herbarium. A voucher specimen has been stored at the Post Herbarium, AUB, with identification number GA 2025-1. Leaves were washed and dried at room temperature and ground mechanically. The powder was suspended in 80% ethanol and incubated while shaking at 40 rpm in the dark for 72 hours. Afterward, the suspension was filtered using filter paper and lyophilized using a freeze-dryer. The obtained powder was dissolved in DMSO at a concentration of 100 mg/mL. The prepared plant extract MAE was then stored in the dark at 4 °C for further usage and analysis.

4.2. Phytochemical Analysis

The chemical composition of the Mandragora autumnalis ethanolic and aqueous extracts was investigated by performing qualitative tests to detect the presence of primary and secondary metabolites. Test for anthocyanins: A total of 0.5 g of the extract was dissolved in 5 mL of ethanol, followed by ultrasonication for 15 min at 30 ◦C. Subsequently, 1 mL of each extract was combined with 1 mL of NaOH (Sigma-Aldrich Co., St. Louis, MO, USA) and heated for 5 min at 100 ◦C. The presence of anthocyanins was indicated by the appearance of a bluish-green color. Anthraquinone test: 0.5 g of each extract (Sigma-Aldrich Co., St. Louis, MO, USA) was dissolved in 4 mL of benzene. 10% ammonia solution was added to the filtrate following filtration. The presence of anthraquinones was verified by the formation of a red or violet color. Test for cardiac glycosides: 5 mL of ethanol was used to dissolve 0.5 g of each extract. This was then ultrasonicated at 30 °C, filtered, and evaporated. Subsequently, the dehydrated extract was combined with 1 milliliter of glacial acetic acid (from Sigma-Aldrich Co., St. Louis, MO, USA) and a few drops of 2% FeCl3. Subsequently, the test tube's side was filled with 1 milliliter of concentrated sulfuric acid (H2SO4) from Sigma-Aldrich Co., St. Louis, MO, USA. The appearance of a brown ring indicated the presence of cardiac glycosides. To test for essential oils, 0.5 g of each extract was dissolved in 5 mL of ethanol, then the mixture was ultrasonically sonicated at 30 °C and filtered. 100 µL of 1MNaOH was mixed with the filtrate. A tiny amount of 1MHCl (MERCK, Darmstadt, Germany) was then added. A white precipitate's formation indicated the presence of essential oils. Test for flavonoids. One milliliter of 2% NaOH and 0.2 g of each extract were combined. When a concentrated, yellow-colored solution was achieved, a few drops of diluted acid were added to the mixture. The color in the solution vanished, indicating that flavonoids were present. Phenols test: 5 mL of ethanol was used to dissolve 0.5 g of each extract, which was then ultrasonically sonicated at 30 °C and filtered. A mixture of 2 mL distilled water and the filtrate was prepared. Subsequently, a small amount of 5% FeCl3 was added, and this caused a dark green color to form, indicating the presence of phenols. 5 mL of ethanol was used to dissolve 0.5 g of the extract, which was then ultrasonicated at 30 °C and filtered. The filtrate was then mixed with 1 mL of concentrated sulfuric acid (H₂SO4). The development of a red hue was indicative of the presence of quinones. To test for tannins, dissolve 0.5 g of each extract in 5 mL of distilled water, then filter and ultrasonicate at 80 °C. Once the filtrate had cooled to room temperature, five drops of 0.1% FeCl3 were added. A brownish-green or blue-green coloration suggested the presence of tannins. To test for terpenoids, dissolve 0.5 g of the extract in 5 mL of chloroform, then filter and ultrasonicate at 30 °C. The filtrate was then mixed with 2 mL of concentrated sulfuric acid (H₂SO4). The creation of a reddish-brown hue indicated the presence of quinones.

4.3. Total Phenolic Content (TPC)

With a few minor modifications, the Folin–Ciocalteu method was used to determine the total polyphenol content (TPC) of MAE[94]. The concentration of both extracts was made to be 1 mg/mL. 500 μL of the extract was separated, combined with 2.5 mL of Folin-Ciocalteu reagent, and left to oxidize for five minutes. After adding 2 mL of a 75 g/L sodium carbonate solution to neutralize the reaction, the mixture of the extract was left to incubate for 1 hour at 37° C in the dark. Following incubation, the samples' absorbance at 765 nm was compared to standards of gallic acid, which is a known polyphenol. The TPC of the extract was given as a percentage (mg GAE/g) of the total gallic acid equivalents per gram of dry leaves used in the extract's preparation. The TPC analysis was carried out three times, and the mean values ±SEM of the outcomes are shown.

2.4. Total Flavonoid Content (TFC)

Using a modified aluminum chloride colorimetric assay, the total flavonoid content (TFC) of MAE was ascertained[94]. To put it briefly, a concentration of 1 mg/mL was used to prepare the extract. Then, an aliquot (0.5 mL) of the extract was combined with 1.5 mL of 95% ethanol, 2.8 mL of ultra-pure distilled water, 0.1 mL of a 10% methanolic aluminum chloride solution, and 0.1 mL sodium acetate. Using quercetin as a standard, the absorbance was measured at 415 nm following a 30-minute dark incubation period at room temperature. The TFC was measured in (mg QE/g) of quercetin equivalents per gram of dry leaves used to prepare the extract. Three runs of this analysis were conducted, and the results are shown as mean values ± SEM.

4.5. The antioxidant Activity (DPPH) of Mandragora autumnalis Ethanolic Extract

Using the free-radical-scavenging activity of α, α-diphenyl-α-picrylhydrazyl (DPPH), the antioxidant activity of MAE was assessed. MAE at different concentrations (50, 100, 200, 400, or 600μg/mL) was combined with a 0.5 mM DPPH solution in methanol. DPPH solution (0.5 mL), methanol (3 mL), and 80% ethanol (0.5 mL) made up the blank solution, which was used for comparison. The combined samples were then exposed to darkness for 30 minutes, and a spectrophotometer was used to measure the optical density (OD) at a wavelength of 517 nm. The following formula was used to calculate the percentage of DPPH-scavenging activity for each MAE concentration. For comparison, ascorbic acid was used as the standard. Percentage of inhibition (absorbance of control − absorbance of the extract)/ (absorbance of control) × 100.

4.6. Liquid Chromatography-Mass Spectrometry

The Stock solution was prepared by dissolving 5 mg of the MAE sample in a solution of 50 µL of Dimethyl sulfoxide (DMSO) and 450 µL of methanol, then 250 µL of the solution was diluted with 1 mL of methanol and then used for identification of the metabolite in the LC-MS system. All the other reagents, Acetonitrile, methanol, water, and formic acid, used were LC/MS grade. A Bruker Daltonik (Bremen, Germany) Impact II ESI-Q-TOF System equipped with Bruker Dalotonik. The Elute UPLC system (Bremen, Germany) was used for screening compounds of interest. We used standards for identification of m/z with high-resolution Bruker TOF MS and exact retention time of each analyte after chromatographic separation. This instrument was operated using the Ion Source Apollo II ion Funnel electrospray source. The capillary voltage was 2500 V, the nebulizer gas was 2.0 bar, the dry gas (nitrogen) flow was 8 L/min, and the dry temperature was 200 ºC. The mass accuracy was ˂ 1 ppm; the mass resolution was 50000 FSR (Full Sensitivity Resolution), and the TOF repetition rate was up to 20 kHz. using Elute UHPLC coupled to a Bruker Impact II QTOFMS. Chromatographic separation was performed using Bruker Solo 2.0_C-18 UHPLC column (100 mm x 2.1 mm x 2.0 μm) at a flow rate of 0.51 mL/min and a column temperature of 40 °C. Solvents:(A) water with 0.1% methanol and (B) Methanol injection volume 3 Μl. Data analysis was performed using Bruker's MetaboScape and DataAnalysis software. Compound annotation in MetaboScape was conducted using the MetaboBase library, a list of standards, as well as a custom list of previously reported compounds in the specific plant under investigation. The raw data files were converted to the mzML format using msConvert, a C++ tool from the ProteoWizard suite designed to convert raw files into standard mass spectrometry formats. The resulting mzML files were analyzed using an in-house R script based on the xcms, CAMERA, and MSnbase packages. Chemical formulas for annotated ions were generated and checked using the Rdisop package. The isotopic pattern for annotated chemical formulas matched the theoretical pattern with more than 99%. Chromatograms and spectra were generated using both Bruker Data Analysis and the ggplot2 library in R. The analyses performed using the two methods yielded comparable results.

4.7. Cell Culture

Human breast cancer cells MDA-MB-231 were acquired from the American Type Culture Collection (Manassas, VA). The cells were cultured in a 37°C, 5% CO2 humidified chamber using DMEM high-glucose medium supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, United States) and 1% penicillin/streptomycin (Corning, Massachusetts, United States).

4.8. MTT Cell Availability Assay

MDA-MB-231 cells were seeded in a 96-well tissue culture plate at a density of 5 × 10³ cells/well, and they were left to grow until 30% confluence was reached. The cells were subjected to escalating concentrations of MAE (50, 100, 200, 400, 600, and 1000 μg/mL) for 72 hours. The viability of the cells was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich, St. Louis, MO, United States). The viability of the cells was determined by comparing the proportionate viability of the treated cells to the vehicle-treated cells (equivalent concentration of DMSO), whose viability was assumed to be 100%.

4.9. Migration (Scratch) Assay

In 12-well plates, MDA-MB-231 cells were cultured until a confluent cell monolayer was formed. Next, a 10 μL pipette tip was used to create a scratch across the confluent cell monolayer. After removing the cell culture medium, the cells were cleaned with PBS to get rid of any debris. Following the addition of fresh medium containing MAE at the designated concentration (100, or 200 μg/mL), cells were incubated at 37°C. With an inverted microscope (objective ×4), photomicrographs of the scratch were taken at baseline (0 hours) and 10 hours post-scratch. Using the ZEN software (Zeiss, Germany), the width of the scratch was measured and expressed as the average difference ±SEM between the measurements made at time zero and the specified time point (10 hours).

4.10. Trans-Well Migration Assay

To test MDA-MB-231 cells' migratory potential, trans-well inserts (8 µm pore size; BD Biosciences, Bedford, MA, USA) were employed. 3 × 10⁵ cells were seeded into the insert's upper chamber. After that, cells were given the indicated MAE concentrations or not. A DMEM medium with 10% FBS was added as a chemoattractant in the lower chamber. After that, the cells were cultured at 37 °C and given 24 hours to migrate. The cells on the insert's upper surface were eliminated using a sterile cotton swab. Additionally, cells that moved to the insert's lower surface were stained with DAPI (1 µg/mL) and fixed with 4% formaldehyde. and examined for quantification at a 10× magnification using a fluorescence microscope. Three assay runs were conducted, and the results are shown as mean values ± SEM.

4.11. Matrigel Invasion Assay

To evaluate MDA-MB-231 cells' invasive potential, a BD Matrigel Invasion Chamber (8µm pore size; BD Biosciences, Bedford, MA, USA) was utilized. The experiment is comparable to the trans-well migration chamber assay, except that a diluted 1:20 Matrigel matrix is added. For quantification, cells that penetrated the Matrigel layer to the insert's lower surface were fixed with 4% formaldehyde, stained with DAPI, and examined under a fluorescence microscope. Three assay runs were conducted, and the results are shown as mean values ± SEM.

4.12. Aggregation Assay

MDA-MB-231 cells were collected from confluent plates using sterile 2 mM EDTA in Ca2+/Mg2+-free PBS to measure cell aggregation. The cells were divided into individual non-adherent culture plates and subjected to MAE treatment (100 or 200 μg/mL). After three hours of gentle shaking at 90 rpm and 37°C, the cells were fixed with 1% formaldehyde. Photomicrographs were taken to be examined using an Olympus IX 71 inverted microscope.

4.13. Adhesion Assay

For a duration of 24 hours, MDA-MB-231 cells were cultured with or without different concentrations (100 or 200 μg/mL) of MAE. Following a collagen pre-coating, they were seeded onto 24-well plates and incubated for 60 minutes at 37°C. The MTT reduction assay was then used to determine the quantity of adherent cells after the cells had been washed with PBS to remove non-adherent cells.

4.14. Analysis of Apoptotic Morphological Changes

Using a phase-contrast inverted microscope, characteristics associated with apoptotic cells were observed. For this, different concentrations of MAE (100 or 200 μg/mL) were either present or absent when growing cells in 6-well plates. After 24 hours, images at 4x, 10x, and 20x magnifications were captured. 4′, 6-diamidino-2-phenylindole, dihydrochloride (DAPI) staining was used to identify changes in nuclear morphology indicative of apoptosis. For a full day, MDA-MB-231 cells were cultured in a 12-well plate with or without the indicated concentrations of MAE (100 or 200 μg/mL). Following the manufacturer's instructions, the cells were stained with DAPI (Cell Signaling #4083), fixed with 4% formaldehyde, and then fluorescence microscopy was used to visualize the apoptotic morphological alterations.

4.15. Western Blot Analysis

To create whole-cell lysates, MDA-MB-231 cells were subjected to two PBS washes before being lysed in a lysis buffer that contained 60 mM Tris and 2% SDS (pH of 6.8). After that, the lysate mixture was centrifuged for 10 minutes at 1.5 × 104 g. Bradford protein quantification kit (Biorad, Hercules, CA, United States) was used to measure the amount of protein in the resultant supernatant. Protein extracts aliquots ranging from 25 to 30 μg were resolved using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently placed onto an Immobilon PVDF membrane (Biorad).

PVDF membrane was blocked with a 5% non-fat dry milk solution in TBST (Tris-buffered saline with 0.05% Tween 20) for one hour at room temperature. To perform immunodetection, a particular primary antibody was incubated with the PVDF membrane for an entire night at 4°C. After removing the primary antibody and washing the membrane with TBST, the membrane was incubated for an hour with the secondary antibody, horseradish peroxidase-conjugated anti-IgG. Thermo Scientific, Rockford, IL, USA) supplied an enhanced chemiluminescence (ECL) substrate kit to visualize immunoreactive bands after washing the secondary antibody with TBST, by the manufacturer's instructions. Sourced from Cell Signaling (Cell Signaling Technology, Inc., Danvers, MA, United States), all primary and secondary antibodies were used.

4.16. Gelatin Zymography

In a 100mm tissue culture plate, MDA-MB-231 cells (1.0 × 10⁶) were cultivated in serum-free DMEM medium with or without varying concentrations (100, or 200 μg/mL) of MAE. The cultures' conditioned media were gathered and concentrated following a 24-hour incubation period. A 10% non-reducing polyacrylamide gel containing 0.1% gelatin was used to separate 30 µg of proteins. After electrophoresis, the gels were rinsed for one hour in 2.5% (v/v) Triton X-100 to get rid of SDS. They were then incubated for the entire night at 37°C in a solution that contained 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5 mM ZnCl₂, and 10 mM CaCl₂ to enable the media's proteases to break down the gelatin substrate enzymatically. A 0.5% solution of Coomassie brilliant blue R-250 was used to stain the resultant gel. The gel showed clear patches that showed matrix metalloproteinases (MMPs) breaking down the gelatin. Using ImageJ software, densitometry analysis was performed, and each cleared band's density was normalised to a nonspecific band on the gel that was equally loaded.

4.17. Flow Cytometry Analysis of Cell Cycle

MDA-MB-231 cells were cultivated in 100 mm tissue culture plates before incubating with or without MAE. Cells were extracted and then suspended in 500 µL of PBS after 24 hours. After that, the cells were fixed using an equivalent volume of 100% ethanol and maintained at -20°C for at least 12 hours. The cells were then centrifuged, pelleted, and washed with PBS before being resuspended in PBS containing 1 µg/mL of 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI, Cell Signaling Technology, Inc., Danvers, MA, USA) and incubated at room temperature for half an hour. After that, the cell samples were evaluated using the Becton Dickinson BD FACSCanto II Flow Cytometry System (Franklin Lakes, NJ, USA), and FACSDiva 6.1 software was used to facilitate data.

4.18. Chorioallantoic Membrane

Fertilized chicken eggs were cleaned with 70% ethanol, rotated, and incubated at 37 °C and 60% relative humidity. After a week, the air sac was revealed when the highly vascularized chorioallantoic membrane (CAM) was dropped by making an opening in the eggshell. The CAM was treated with 200 μg/mL of MAE and incubated for 24 hours to investigate its impact on blood vessel growth between treatment and control. AngioTool 0.5 software was then used to measure the lengths of the vessels and count the number of junctions in the CAM images that were taken.

4.19. Statistical Analysis

The results were assessed using the student's t-test. When comparing more than two means, one-way ANOVA followed by Dunnett's post hoc test or two-way ANOVA followed by Tukey-Kramer's post hoc test were also utilized. P-values less than 0.05 were regarded as statistically significant.

5. Conclusions

Collectively, the findings of this study unveil the Mandragora autumnalis ethanolic leaf extract as a promising phytopharmacological candidate for the management of triple-negative breast cancer, a highly aggressive and therapeutically refractory breast cancer subtype. The observed reduction in MDA-MB-231 cell viability in a dose- and time-dependent manner reflects the cytotoxic potential of MAE, aligning with prior studies on other Solanaceae species known for their bioactive alkaloids and phenolics. Notably, the significant upregulation of the tumor suppressor p53 and concomitant downregulation of proliferation and metastasis-associated markers (Ki67, MMP-9, STAT-3) reinforce the mechanistic plausibility of MAE-induced tumor suppression. These molecular events are consistent with canonical antitumor pathways modulated by natural agents. The capacity of MAE to modulate these pathways points to its potential for not only cytotoxic but also anti-metastatic and anti-angiogenic applications. Furthermore, the interference of MAE with critical metastatic processes—including cell migration, invasion, angiogenesis, aggregation, and adhesion—broadens its functional relevance. These phenotypic suppressions reflect anti-metastatic activity comparable to compounds like quercetin and apigenin. Unlike many monotherapeutic agents that narrowly target single pathways, the broad-spectrum bioactivity of MAE implies a synergistic interplay of its phytochemical constituents, which is characteristic of botanical therapeutics and presents an advantage in overcoming the molecular heterogeneity and drug resistance typical of TNBC. These results also suggest that bioactivity-guided fractionation of MAE could yield novel lead compounds with defined molecular targets, warranting further investigation through high-resolution metabolomics and in vivo tumor models.