Ononin suppresses tumor-induced platelet activation and invasion and enhances cell-cycle arrest and apoptosis in triple-negative breast cancer cells.

Introduction
Metastasis remains the primary cause of death in cancer patients and results from intricate communication between tumor cells and their surrounding microenvironment1. Among the critical players in this process are platelets. It has been shown that platelets support tumor cell migration, invasion, survival in circulation, and eventual colonization of distant organs. When activated by tumor cells, platelets can drive epithelial–mesenchymal transition (EMT) through the release of pro-metastatic factors such as TGF-β1. They also form protective clusters with circulating tumor cells, shielding them from immune attack and facilitating vascular extravasation2,3. Moreover, tumor-derived nucleic acids recruit and activate platelets at dysregulated vasculature, fostering immune suppression. Notably, disrupting platelet–leukocyte interactions inhibited tumor progression with efficacy comparable to systemic checkpoint blockade4.
Platelet–cancer interactions are reciprocal: platelets internalize tumor-derived vesicles and become tumor-educated platelets, while cancer cells receive signals from platelet-derived extracellular vesicles, including microparticles and exosomes, which modulate gene expression and enhance metastatic traits5,6. In addition, platelet-derived cytokines and growth factors facilitate metastatic outgrowth7,8.
Taken together, these finding can be concluded that drugs that inhibit tumor-induced platelet activation, as well as agents that act directly on platelets offer therapeutic benefits in cancer treatment. Tumor-induced platelet inhibitors specifically block the interaction between cancer cells and platelets, thereby reducing platelet-mediated protection of circulating tumor cells, reduce metastatic effect, and enhance tumor sensitivity to chemotherapy and immune-mediated clearance. Importantly, this strategy is largely tumor-specific, minimizing the risk of widespread bleeding. In contrast, direct platelet inhibitors act systemically to reduce platelet activity, preventing platelet-assisted tumor growth through multiple pathways and providing a protective effect against cancer-associated thrombosis.
Triple-negative breast cancer (TNBC) is an aggressive tumor that is highly metastatic9,10. Previous studies have shown that the MDA-MB-231 TNBC cell line exhibits highly invasive, metastatic behavior facilitated by platelet activation, which promotes EMT, TGF-β1 secretion, and immune evasion11. Given the pivotal role of platelets in promoting EMT, invasion, and immune evasion in TNBC, targeting both platelet activation and tumor aggressiveness represents a promising therapeutic strategy. While agents such as diallyl trisulfide and aspirin have shown dual inhibitory effects on platelet activation and MDA-MB-231 invasion11,12., the potential of other naturally derived compounds remains underexplored.
Ononin, an isoflavone glycoside of the Fabaceae family and a constituent of Astragali Radix, has emerged as a promising anticancer agent with the ability to sensitize MDA-MB-231 cells to doxorubicin13. However, its role in modulating platelet–cancer cell interaction has not been elucidated. Building on this foundation, the present study evaluates ononin’s effects on MDA-MB-231 cells across four key axes: inhibition of invasion, suppression of tumor cell-induced platelet activation, induction of cell-cycle arrest, and promotion of apoptosis, aiming to establish its therapeutic potential in mitigating TNBC progression, particularly metastasis driven by platelet–cancer interactions.

Materials and methods

Ethical approval and blood sampling
This study received ethical approval from the Research Ethics Committee at Al-Ahliyya Amman University (Approval No. IRB: AAU/5/14/2021–2022). Written informed consents were obtained from all patients prior to the ablation procedure. This study was conducted in accordance with the principles of the Declaration of Helsinki. Samples of blood were drawn from healthy donors aged between 19 and 30 years and having normal platelet count. Exclusion criteria included pregnancy, contraceptive pill use, a history of any disease, smoking, or medication use within the past two weeks.

Drugs and chemicals
Mouse monoclonal anti-human antibodies CD62P (FITC-conjugated, catalog # BBA34) was purchased from Bio-techne/R&D systems (USA) and CD42b/GPIb alpha antibody (MM2/174) (PE-conjugated, catalog # NBP1-28503) was purchased from Bio-techne/Novus biologicals (USA). A stock solution of ononin (Sigma-Aldrich, USP; cat. no. 486-62-4) was prepared by dissolving it in dimethyl sulfoxide (DMSO) (ChemCruz, USA) and diluted to obtain the desired concentrations so that the final DMSO concentration did not surpass 0.1%.

Platelet and cancer cell co-culture
The MDA-MB-231 cell line was purchased from the American Type Culture Collection (ATCC, USA) and cultured in high-glucose RPMI medium (Euroclone, Italy) enriched with 10% fetal bovine serum, 10 U/mL penicillin-streptomycin, and 10 g/L L-glutamine. Cells were maintained at 37 °C in a humidified incubator with 5% CO₂. MDA-MB-231 breast cancer cells (passage 22) were seeded at a density of 1 × 10⁵ cells per well in 1 mL of culture medium in 24-well plates and incubated at 37 °C with 5% CO₂ for 24 h to promote adhesion and monolayer formation. The IC50 of ononin was found to be 50 ± 5.00 µM as previously reported in our laboratory14. After 24 h, fresh media containing ononin at 37 µM (0.75 IC50), and 25 µM (0.5 IC50) were prepared from a 1 mM stock solution using M1V1 = M2V2, and MDA-MB-231 cells were treated for 72 h and incubated at 37 °C.
To minimize platelet activation during phlebotomy, a 21-gauge needle was used. Whole blood samples were collected into tubes containing 3.8% sodium citrate as anticoagulant (9:1 volume/volume ratio) (Vacupro, Jordan). PRP was prepared by centrifuging whole blood at 200 × g for 15 min under stringent conditions to prevent platelet activation as reported earlier15. Blood was taken from three independent donors and each donor sample was run in triplicate technical wells.
Following drug treatment, media were removed, and cells were washed twice with PBS. Then, cells were incubated with 200 µL of diluted platelet-rich plasma (PRP) (1:1 in PBS) for 20 min. PRP was diluted with PBS to prevent platelet aggregation. After the incubation period, PRP was collected. The MDA-MB-231 cells were washed twice with PBS and the wash fractions were pooled with the collected PRP to recover platelets adhering to the cell surface, ensuring accurate platelet counts and activation status. Then, the collected PRP was centrifuged, and the resulting pellet was resuspended in 150 µL of PBS for dual immunolabeling. Mouse monoclonal anti-human CD42b antibody and CD62P antibody were added at a concentration of (1:100 dilution). Staining was performed at room temperature in the dark for 10 min. The experiment included PRP alone (blank for autofluorescence), PRP + arachidonic acid (positive control for platelet activation), PRP + MDA-MB-231 (negative control for ononin effect) and single-stained samples for compensation. Following labeling, cells were fixed in 1% paraformaldehyde for 10 min, centrifuged at 200 × g for 5 min, and then resuspended in 500 µL of PBS. Samples were transferred to flow cytometry tubes and kept at 4 °C prior to analysis.
Flow cytometry was conducted using the FACSCanto™ II system (BD Biosciences) at the Cell Therapy Center (CTC), Jordan. Platelets were analyzed by flow cytometry with PMT voltages set to FSC 200, SSC 350, FITC 500, and PE 500, with platelets identified by their FSC/SSC characteristics and, when applicable, FITC/PE fluorescence using fixed gates applied to all samples. Gating of platelet population is provided in Figure S1. For each sample, the cytometer was configured to acquire 10,000 events within the platelet gate, and data were exported for subsequent analysis of platelet positivity and/or median fluorescence intensity. Flow cytometry gating was applied to CD42b-positive cells to identify platelets. Activation of platelets was assessed as the fraction of events double-positive for CD42b and CD62P (CD42b⁺/CD62P⁺). Mean fluorescence intensity was determined with PE-labelled (CD42b) on the Y-axis and FITC-labelled (CD62P, P-selectin) on the X-axis. Activation percentage was calculated as: Activation % = Q2 / (Q4 + Q2) where: Q4 = CD42b⁺/CD62P⁻ (resting platelets) and Q2 = CD42b⁺/CD62P⁺ (activated platelets).

Invasion assay
Cells were cultured as spheroids and incubated for 48 h using the hanging drop method, then seeded in triplicate into a collagen matrix (pH 7.4) within an 8-well glass chamber (Nunc, Lab-Tek, Thermo Scientific). The spheroids were layered with an equal volume of collagen to the base matrix and set for 1 h at 37 °C to polymerize. After that, treatment was prepared in culture media with concentrations of 25 µM and 37 µM of ononin added over the polymerized collagen and incubated at 37 °C, 5% CO2, and 90% relative humidity for five days. Spheroids were observed and microscopic images were captured daily at a magnification of 40x using an LCD light microscopic screen. Spheroid diameter was measured using a free web version of ImageJ software, version 1.54p 1.x (https://imagej.net/ij/download.html), and invasion area was calculated using the following equation: Invasion area = total area at day 5 – core area at day 1.

Cell cycle
Cells were seeded in 6-well plates at a density of 5 × 10⁵ cells per well and incubated at 37 °C with 5% CO₂ and 90% humidity for 24 h. Subsequently, the cells were treated with 25 µM and 37 µM concentrations of ononin and incubated for an additional 48 h under the same conditions. After treatment, the medium was removed, and cells were collected by centrifugation at 300 × g for 5 min. The resulting pellets were resuspended in PBS and slowly added dropwise to cold ethanol for fixation, followed by a second centrifugation step. The fixed cell pellets were then resuspended in 300 µL of propidium iodide (PI) staining buffer (Sigma-Aldrich/USA, CAS number 25535-16-4) and incubated for one hour. PI fluorescence was measured at an emission wavelength of 535 nm using a BD FACSCanto™ II flow cytometer (USA). All experiments were conducted in triplicate with data analysis carried out using FCS Express 7.

Apoptosis
Cells were seeded in 12-well plates at a density of 2 × 10⁵ cells per well and incubated at 37 °C with 5% CO₂ and 90% humidity for 24 h. Later, cells were treated with ononin (25 µM and 37 µM) and incubated for 48 h. Afterward, the cells were collected and incubated with PI and FITC staining buffers using the TACS™ Annexin V-FITC Apoptosis Detection Kit (R&D Systems, USA; Cat # K4830-01-K) according to the manufacturer’s instructions. Apoptotic populations were measured in triplicate using a FACS Canto II flow cytometer, detecting emission peaks at 525 nm and 578 nm for FITC and PI, respectively. Data analysis was performed with BD FACSDiva™ software version 8.0.

Statistical analysis
All statistical analyses were conducted using GraphPad Prism version 8. One-way ANOVA followed by Tukey’s post hoc test was used to test for significant differences between the three groups (control, high and low dose ononin) in flow cytometric analysis of platelet activation and Annexin V/PI assay. On the other hand, two-way ANOVA was used for cell cycle analysis and invasion assay followed by Tukey’s post hoc test. Results are presented as mean ± standard deviation (SD), and significance was assigned to p-values below 0.05.

Results

Platelet activation by cancer cells is inhibited by pretreatment of cells with Ononin
The flow cytometry scatter plots (Fig. S1A–E) depict platelet populations defined by CD42 expression (y-axis) and CD62 expression (x-axis). Figure S1A shows platelet-rich plasma (PRP) that was not incubated with cancer cells. Most events are clustered in Q4 (CD42⁺/CD62P⁻), indicating that the platelets are predominantly in a resting state with minimal activation, with a total population of 0.0%, 3.2%, 0.5%, and 96.3% in Q1, Q2, Q3, and Q4, respectively. In Fig. S1B, which represents platelets exposed to vehicle-treated MDA-MB-231, a distinct rightward shift toward Q2 (CD42⁺/CD62P⁺) is observed, reflecting the emergence of activated platelets, with total populations of 0.3%, 71.1%, 0.1%, and 28.5% in Q1, Q2, Q3, and Q4, respectively. Figure S1C and D, which represent platelets co-cultured with ononin-exposed cancer cells, show a scatter distribution of the double-positive population (CD42⁺/CD62P⁺), indicating both platelet activation and population heterogeneity. In response to treatment with 37 µM ononin, a total population of 0.2%, 95.4%, 0.0%, and 4.3% was obtained for Q1, Q2, Q3, and Q4, respectively (Fig. S1C). Whereas in response to 25 µM ononin (Fig. S1D), a total population of 0.1%, 93.0%, 0.0%, and 6.9% was obtained in Q1, Q2, Q3, and Q4, respectively. In Fig. S1E, which represents platelets exposed to arachidonic acid (positive control), a substantial proportion of events accumulate in Q2, confirming robust platelet activation, with 0.0%, 65.6%, 0.6%, 33.8% of populations in Q1, Q2, Q3, and Q4, respectively.
Flow cytometry analysis (Fig. 1) showed a significant reduction in platelet activation with ononin treatment—62% at the high dose and 63.9% at the low dose—compared with 80% in vehicle-treated cells (p < 0.01 for each). However, no significant difference was found between the two doses of ononin.

Invasion assay
Spheroid gel invasion assay demonstrated that the invasion area of MDA-MB-231 monitored for five days in response to two concentrations of ononin (25 µM and 37 µM) was reduced significantly (Table S1). Two-way ANOVA revealed that the treatment factor accounted for 63.1% of the total variation, while the time factor contributed 15.1%. The interaction between treatment and time explained an additional 21.6% of the variation, (p-value < 0.0001 for all) (Fig. 2).

Spheroid images of MDA-MB-231 shown in Fig. 3 indicate invasion containment in response to ononin treatment. The untreated, control spheroids show increased spreading of cells with increased spheroid diameter (Fig. 3A). In contrast, spheroids treated with both concentrations of ononin (25 µM and 37 µM) exhibit diameter retention and reduced cell spreading at the interface of the spheroids, reflecting the inhibitory effect of ononin on TNBC cellular invasion, with further reduction in spheroid size at increasing ononin concentration (37 µM) (Fig. 3B and C).

Effect of ononin on cell cycle
Cell cycle analysis showed that ononin induced concentration-dependent G1 arrest. As shown in Fig. 4A, cells treated with ononin exhibited a significant increase in the proportion of cells in the G1 phase (p < 0.0001 for both), accompanied by a marked reduction in the S phase population (p < 0.001 and p < 0.0001, respectively) and a significant decrease in the G2 phase (p < 0.0001 for both). The pie chart (Fig. 4B) further confirms this shift, demonstrating an accumulation of cells in G1 at the expense of both S and G2 phases. Specifically, the control group showed 76.41% in G1, 19.56% in S, and 4.03% in G2, whereas 25 µM ononin increased G1 to 79.35% with almost complete loss of the G2 population. At 37 µM ononin, the effect was more pronounced, with 85.49% of cells arrested in G1 and only 14.51% remaining in S. Further details on DNA histograms and gating of cellular populations of MDA-MB-231 are provided in Figure S2.

Effect of ononin on apoptosis
The percentage of viable cells significantly decreased in both ononin-treated groups (25 µM and 37 µM) compared to the control group (p = 0.0025 for both). Apoptotic cells increased significantly at both ononin concentrations compared to the untreated group (p = 0.0019). Necrotic cell percentage also increased significantly in the ononin treated groups compared to control (p = 0.0170). Comparisons in apoptotic and necrotic populations between the two concentrations of ononin (25 µM and 37 µM) show no significant difference (p ≥ 0.05), indicating that ononin exerts similar apoptotic and necrotic effects at both concentrations (Fig. 5).

Discussion
In this study, ononin exerted significant anticancer effects in MDA-MB-231 triple-negative breast cancer cells by inducing G1-phase cell cycle arrest and promoting both early and late apoptosis. These findings are consistent with previous reports in other cancer types, for example, in lung cancer cells, ononin inhibited the phosphorylation of PI3K, Akt, and mTOR, thereby blocking downstream survival signaling, inducing G1/S cell cycle arrest, and downregulating cyclins and cyclin-dependent kinases (CDKs)16. These results suggest that ononin exerts its anti-cancer activity by targeting critical pathways, leading to cell cycle inhibition and apoptosis. The induction of G1 arrest observed in MDA-MB-231 cells in the current study may similarly involve suppression of cyclin/CDK complexes, potentially mediated through PI3K/Akt/mTOR signaling. Future studies should investigate the precise molecular pathways involved in ononin-induced cell cycle arrest and apoptosis with full evaluation of key apoptotic markers, such as cleaved caspase-3 and PARP-1 (Poly (ADP-ribose) polymerase-1). In addition, testing ononin in vivo cancer models is required to evaluate its therapeutic potential and pharmacological relevance.
Our findings demonstrate that ononin at 25 and 37 µM reduces the proportion of viable MDA-MB-231 cells (p < 0.0001) and promotes apoptosis in a concentration-dependent manner. Late apoptotic populations increased significantly at both concentrations (p < 0.05 and p < 0.01, respectively), while early apoptosis was notably elevated at 37 µM (p < 0.05). In contrast, necrosis was more pronounced at 25 µM (p < 0.05) relative to the control group. These results are consistent with previous reports showing that ononin exerts pro-apoptotic activity in osteosarcoma cells17. Similarly, in laryngeal cancer, ononin enhanced intracellular reactive oxygen species (ROS) accumulation, leading to oxidative stress, DNA damage, and subsequent cell cycle arrest that limited the propagation of damaged cells18. Comparable mechanisms were reported in non-small cell lung cancer, where ononin triggered apoptosis through increased levels of cleaved PARP, cleaved caspase-3, and cleaved caspase-9, accompanied by elevated ROS production. Moreover, ononin has been shown to induce ferroptosis-related lipid peroxidation in TNBC by increasing malondialdehyde and ROS levels while reducing superoxide dismutase activity and downregulating key ferroptosis markers, including SLC7A11 and Nrf219. These findings support a multifaceted anticancer role for ononin.
The spheroid gel invasion assay demonstrated that ononin reduced significantly the invasion area of MDA-MB-231. In our previous study, ononin inhibited migration of MDA-MB-231 cells14. Also, a previous study reported that ononin significantly reduced the metastatic potential of breast cancer cells by inhibiting adhesion, invasiveness, motility, and reversing EMT markers. In vivo, ononin (10 and 20 mg/kg) decreased both the incidence and size of osteolytic lesions in a breast cancer bone metastasis model. Mechanistically, it suppressed proliferation, invasion, and EMT by downregulating N-cadherin, MMP-2/9, and MAPK signaling20. Similar anti-invasive effects were also observed in osteosarcoma where ononin significantly suppressed the invasion and migration of human osteosarcoma cells17.
In addition to the pro-apoptotic and anti-invasive effects, ononin was able to decrease the ability of MDA-MB-231 cancer cells to activate platelets. CD42 is a marker of platelets, whereas CD62 (P selectin) is a marker for their activation21. Flow cytometry analysis demonstrated that resting platelets (CD42⁺/CD62P⁻) predominated in PRP, whereas exposure to vehicle-treated cancer cells induced activation (CD42⁺/CD62P⁺) of platelets. Ononin significantly reduced platelet activation at both concentrations versus vehicle, with no difference between the two doses. These findings suggest that ononin modulates cancer cell–platelet interactions, shifting platelet populations toward a less activated state. This is an extremely important mechanism that helps cancer cells to metastasize7,8.
Sub-IC₅₀ concentrations of ononin (25 and 37 µM) were selected for the cancer cell–induced platelet activation assays to minimize cytotoxic effects on MDA-MB-231 cells that could otherwise confound platelet–cancer cell interactions. This approach aligns with earlier studies showing that concentrations below the IC₅₀ can reveal mechanistic effects without reducing cell viability22. Both sub-IC₅₀ concentrations of ononin significantly reduced cancer cell–induced platelet activation, with no statistically significant difference between them. In contrast, other endpoints, particularly G1-phase arrest exhibited a clearer dose-dependent pattern, with 37 µM producing more pronounced effects. Accordingly, 37 µM is preferred for mechanistic studies, while 25 µM serves as an alternative when minimal cellular disturbance is needed.
In the present study, ononin markedly decreased the ability of MDA-MB-231 cells to induce platelet activation, as indicated by reduced CD42⁺/CD62P⁺ double-positive events; however, the direct effect of ononin on platelets was not examined and therefore cannot be inferred from the current data. In its inhibitory effect on tumor cell-induced platelet activation, ononin resembles seed oils from Kigelia africana, Ximenia caffra, and Mimusops zeyheri, which reduce the pro-aggregatory phenotype of MDA-MB-231 breast cancer cells without directly targeting platelet receptors23. This differs from the action of other natural products with direct inhibitory effects on platelets. For example, the polysaccharide fraction from Artemisia argyi selectively inhibits podoplanin-induced platelet aggregation by blocking the podoplanin– C-type lectin-like receptor 2 (CLEC-2) interaction22. Similarly, diallyl trisulfide, a garlic-derived organosulfur compound, inhibits platelet activation and aggregation, decreases TGF-β1 secretion in platelet–tumor co-cultures, and dose-dependently suppresses MDA-MB-231 cell migration and invasion11. Drugs that inhibit tumor-induced platelet activation offer a targeted way to reduce metastasis with minimal bleeding risk, while agents that act directly on platelets provide broader suppression of platelet activity and help prevent cancer-associated thrombosis and platelet-supported tumor progression. Therefore, both strategies offer therapeutic advantages. Performing experiments where platelets are incubated with ononin alone, without the presence of tumor cells, would help clarify whether ononin exerts any direct effects on platelet activation.
After interacting with platelets, epithelial cancer cells can undergo a transition to a mesenchymal phenotype, enabling them to invade distant organs more effectively. Platelets actively interact with both inflammatory and tumor cells within the bloodstream, contributing to the establishment of metastatic niches24. Additionally, platelets can directly link tumor cells to the endothelium, promoting their extravasation and facilitating the seeding and proliferation of metastatic cells7. The metastatic process depends on the presence of platelet-resident receptors and platelet secretions such as growth factors, cytokines and miRNA25. Therefore, platelet activation is linked to cancer cell migration, invasion, proliferative signaling, and cell-cycle regulation.
This study shows that ononin reduces tumor cell–induced platelet activation, which helps reduce cancer cell migration, invasion, and cell-cycle progression. Future work should measure important cytokines and growth factors in platelet–tumor co-culture and assess the mechanism in appropriate animal models. Pharmacokinetic and in vivo efficacy studies will be also needed to determine clinically safely exposure levels and dosing strategies.
A main limitation of this study is that only one TNBC cell line (MDA-MB-231) was used, which limits how broadly the findings can be applied. Future studies should test ononin in more different phenotypic breast cancer cells to gain a comprehensive understanding of its anti-cancer effects.

Conclusion
The present study demonstrates that ononin effectively suppresses MDA-MB-231 breast cancer cell-induced platelet activation, which may contribute to its anti-metastatic capacity. This isoflavone also significantly inhibits cancer cell invasion and induces G1 cell cycle arrest in a concentration-dependent manner, limiting cell proliferation. Moreover, it increases apoptosis and this supports its cytotoxic effects against TNBC cells. Collectively, these results suggest that ononin disrupts critical mechanisms involved in tumor progression in TNBC and highlights its promise as a therapeutic agent for the management of aggressive breast cancer.
Further preclinical animal studies and mechanistic investigations are needed to fully elucidate the therapeutic benefits and underlying pathways of ononin. Given its natural origin and low reported toxicity, ononin may represent a promising supplementary candidate for breast cancer patients. However, its clinical application remains cautiously feasible at present, as further preclinical validation and well-designed clinical trials are essential to determine its safety, efficacy, and therapeutic relevance.

Supplementary Information
Below is the link to the electronic supplementary material.