Triple-negative breast cancer (TNBC), accounting for approximately 15–20% of all breast cancer cases, is characterized by the absence of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 expression (1–3). Due to the lack of actionable molecular targets, patients with TNBC are largely reliant on chemotherapy and radiotherapy for locoregional disease control (4). Radiotherapy remains a cornerstone in the management of TNBC, capable of significantly reducing local recurrence and improving overall survival (4). However, irradiation of the chest wall or breast inevitably exposes the heart to ionizing radiation, placing patients at risk for radiation-induced myocardial injury (RIMI) (5).

Although adult cardiomyocytes exhibit low proliferative activity and are considered less radiosensitive than cancer cells, their extremely limited regenerative capacity renders the heart vulnerable to even minor radiation-induced insults (6). Clinically, RIMI can manifest as progressive myocardial fibrosis, contractile dysfunction, arrhythmias, or even heart failure, which may develop days to months after treatment (7). The cardiotoxicity of radiotherapy is therefore a growing concern, particularly in TNBC patients who may receive aggressive thoracic irradiation (8).

The primary antitumor mechanism of radiotherapy is the induction of DNA double-strand breaks, which trigger irreparable genomic damage and apoptosis (9). However, rapidly proliferating tumor cells, particularly those in S phase, possess robust DNA repair capacity via high-fidelity mechanisms such as homologous recombination (10). Notably, preclinical studies have demonstrated that pharmacological blockade of the G1/S transition using CDK4/6 inhibitors not only impedes tumor growth but also enhances radiosensitivity, highlighting the potential of cell cycle modulation as a radiosensitization strategy (11).

Ellagic acid is a natural polyphenolic compound abundant in berries, pomegranates, and nuts (12, 13). It possesses two key properties that make it an attractive candidate in radiotherapy contexts: Potent antioxidant activity – The multiple hydroxyl groups on its aromatic rings allow efficient electron donation, enabling effective scavenging of radiation-induced reactive oxygen species (ROS), thus mitigating oxidative damage to normal tissues (14); Cell cycle regulatory activity – ellagic acid has been shown to inhibit the histone arginine methyltransferase CARM1, specifically blocking H3R17 dimethylation, which suppresses CDK4 transcription (15–17). Remarkably, ellagic acid exhibits stronger inhibitory effects on the CARM1–CDK4 axis than selective small-molecule probes such as TP-064, resulting in robust G1 arrest and prevention of S-phase entry (15).

Based on these features, we hypothesized that ellagic acid may exert a dual radiomodulatory effect: (1) enhancing radiosensitivity in tumor cells by preventing entry into the DNA-repair–competent S phase, and (2) protecting cardiomyocytes from radiation-induced oxidative injury through ROS scavenging. Despite the therapeutic promise of this dual mechanism, no prior studies have evaluated ellagic acid as both a radiosensitizer and a cardioprotective agent.

In this study, we comprehensively investigated the dual-function potential of ellagic acid using in vitro assays and a murine 4T1 TNBC model. Specifically, we examined its antioxidant capacity, cell cycle effects, tumor response to combined ellagic acid and radiotherapy treatment, and the extent of radiotherapy-induced cardiac injury. Our findings reveal a paradoxical yet advantageous therapeutic profile—ellagic acid sensitizes tumors to radiation while protecting the heart—supporting its translational potential in TNBC radiotherapy.

This study is the first to demonstrate that ellagic acid, a naturally occurring polyphenol, exerts dual modulatory effects during radiotherapy: it enhances tumor radiosensitivity in TNBC while simultaneously protecting the myocardium from RIMI. This unique therapeutic profile, driven by tissue-specific mechanisms, addresses two critical challenges in TNBC radiotherapy—tumor radioresistance and treatment-associated cardiotoxicity—and holds significant translational potential.

Mechanistically, ellagic acid enhances the radiosensitivity of TNBC cells by inducing G1-phase arrest and impeding cell cycle progression (15–17). Our results show that ellagic acid markedly downregulates CDK4 expression and increases the proportion of cells in G1 phase, thereby limiting progression into the S phase—a cell cycle stage associated with enhanced DNA repair capacity and radioresistance. This finding aligns with current strategies that utilize CDK4/6 inhibitors to potentiate radiation efficacy by impairing DNA damage recovery (27). Importantly, ellagic acid achieves this effect via a distinct epigenetic mechanism: by inhibiting CARM1-mediated histone H3R17 dimethylation, ellagic acid disrupts the transcriptional activation of CDK4 at an upstream level (15–17). This mode of action not only deepens its regulatory influence over the cell cycle but also suggests broader chromatin-modifying capabilities that may benefit oncologic therapy.

Our in vivo data further support this mechanism. ellagic acid -treated tumors exhibited a significant reduction in S-phase cell populations and an increase in radiation-induced apoptosis, resulting in robust tumor growth inhibition. Concurrently, ellagic acid exhibited notable cardioprotective effects. Histopathological analyses revealed that ellagic acid alleviated radiation-induced myocardial vacuolization and structural disorganization, reduced the number of TUNEL-positive apoptotic cardiomyocytes, and preserved key functional parameters such as LVIDs and ejection fraction. Mechanistically, these protective effects are underpinned by ellagic acid’s potent antioxidant activity, as demonstrated by its free radical scavenging performance in chemical (e.g., DPPH) and cellular oxidative stress assays. Given that radiation-induced cardiac injury is primarily mediated by oxidative stress in non-proliferating cardiomyocytes, ellagic acid’s redox-modulating properties provide a biologically sound and clinically relevant basis for myocardial protection.

Remarkably, ellagic acid circumvents the conventional trade-off between radiosensitization and toxicity. Most radiosensitizers increase the risk of collateral damage to normal tissues, while radioprotectors may compromise tumor control (28, 29). Ellagic acid uniquely resolves this dilemma via cell-type–specific mechanisms: in tumor cells, it promotes radiosensitization by disrupting cell cycle progression; in cardiomyocytes, it mitigates oxidative injury without interfering with cell division (as cardiomyocytes are non-proliferative). This differential modulation represents a conceptual advance in the development of radiotherapy adjuvants and positions ellagic acid as a paradigm-shifting dual-function compound. Traditional agents like amifostine are designed solely to shield normal tissues from radiation damage. While effective, their use is often limited by systemic side effects (e.g., hypotension, nausea) and a theoretical concern of potentially protecting tumor cells, which could compromise radiotherapy efficacy (30, 31). In stark contrast, EA operates with tissue selectivity. It does not act as a blanket protector; instead, it exploits the fundamental biological difference between proliferating cancer cells and cardiomyocytes. This mechanistic dichotomy effectively decouples protection from tumor compromise, a major advancement over non-selective protectors.

The promising dual effects of EA demonstrated in our preclinical model necessitate a critical evaluation of its potential administration routes for future clinical application in TNBC radiotherapy. While intraperitoneal injection was effectively employed in our murine studies to establish proof-of-concept, translating this finding to the clinic requires strategic adaptation to meet practical, safety, and efficacy standards. The intraperitoneal route, though ensuring high bioavailability in rodents, is invasive and impractical for the repeated, long-term administration required during a typical radiotherapy course for human patients. Therefore, identifying a feasible and optimized clinical delivery method is paramount.

Oral administration stands as the most desirable and patient-compliant route. EA’s natural occurrence in certain foods suggests a favorable safety profile for oral intake (31–34). However, its notoriously poor aqueous solubility and moderate oral bioavailability present significant translational hurdles. To overcome this, future efforts must prioritize the development of advanced pharmaceutical formulations. Strategies such as nanocrystal technology, phospholipid complexes, cyclodextrin inclusion complexes, or nano-delivery systems (e.g., liposomes, polymeric micelles) could dramatically enhance EA’s solubility, stability, and intestinal absorption, thereby improving its systemic availability.

In summary, ellagic acid achieves a rare therapeutic duality: it sensitizes TNBC tumors to radiation by epigenetically suppressing CDK4-driven cell cycle progression, while protecting the heart via antioxidant-mediated mitigation of oxidative stress. This paradigm reconciles the competing goals of enhanced oncologic efficacy and reduced cardiotoxicity, offering a promising strategy to improve the therapeutic index of TNBC radiotherapy. Given its natural origin, safety, and multimodal activity, ellagic acid warrants rapid translation into clinical trials.

Cell Culture and Reagents: The 4T1 breast cancer cells were obtained from Procell (Wuhan) and cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37 °C with 5% CO₂. H9C2 cardiomyocytes were cultured under similar conditions. Ellagic acid was purchased from Yuanye (Shanghai) and dissolved in DMSO for in vitro experiments and in 0.5% CMC-Na for in vivo administration.

In Vivo Tumor Model and Treatment: Female BALB/c mice (8 weeks old, approximately 20 g) were subcutaneously inoculated with 1 × 10⁶ 4T1 cells into the left mammary fat pad, located in close proximity to the cardiac region of the thoracic wall. Once tumors reached approximately 0.1 cm³ in volume, this time point was designated as Day 0. From Day 1 onwards, mice in the ellagic acid treatment group received daily intraperitoneal injections of ellagic acid at a dose of 50 mg/kg for 14 consecutive days. For radiotherapy, mice were anesthetized and positioned supine under a linear accelerator. A single 20 Gy dose of 6 MV X-rays was delivered on Day 3, 6, 9,12 using a single anterior field, with the irradiation field encompassing both the tumor and the underlying cardiac region.

BrdU Incorporation Assay: To evaluate S-phase distribution in vivo, mice were injected intraperitoneally with BrdU (50 mg/kg) 2 hours prior to sacrifice. Tumors were harvested, fixed, embedded, and sectioned. BrdU incorporation was detected by immunofluorescence using anti-BrdU antibody (Abcam) and quantified with ImageJ software.

TUNEL Assay: Apoptotic cells in tumor and heart tissues were detected using the TUNEL apoptosis assay kit (Solarbio) according to manufacturer instructions. Nuclei were counterstained with DAPI. TUNEL-positive cells were quantified in at least five random fields per section.

Histological and Masson’s Trichrome Staining: Heart tissues were harvested on day 14, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. HE staining was performed to evaluate myocardial architecture, and Masson’s trichrome staining was used to assess fibrosis. Histological alterations were evaluated under light microscopy by blinded pathologists.

Cardiac Function Assessment: Cardiac function was evaluated using transthoracic echocardiography (Vevo2100, VisualSonics) under light anesthesia. M-mode images were obtained at the level of the papillary muscles to determine LVIDd, LVIDs, and EF%. End Volume = 7 × LVID³/(2.4 + LVID), and EF (%) = (End-Diastolic Volume - End-Systolic Volume)/End-Diastolic Volume.

Western Blotting: Cells were lysed in RIPA buffer with protease/phosphatase inhibitors. Equal protein amounts were resolved by SDS-PAGE and transferred to PVDF membranes. Membranes were incubated with primary antibodies against CDK4, followed by HRP-conjugated secondary antibodies and chemiluminescent detection.

Cell Cycle Analysis: Cell cycle distribution was assessed by propidium iodide (PI) staining followed by flow cytometry. Cells were harvested at approximately 70–80% confluence, trypsinized, and washed twice with cold phosphate-buffered saline (PBS). The cell pellets were then fixed by dropwise addition of prechilled 70% ethanol while gently vortexing and stored at –20 °C for at least 2 hours. After fixation, cells were washed once with PBS to remove residual ethanol and incubated in a staining solution containing 50 µg/mL propidium iodide (PI) and 100 µg/mL RNase A in PBS for 30 minutes at 37 °C in the dark. PI intercalates into double-stranded DNA, and the fluorescence intensity correlates directly with DNA content: G0/G1 phase cells exhibit 2N DNA content, S-phase cells show intermediate DNA content (between 2N and 4N due to active replication), and G2/M phase cells have 4N DNA content.

DPPH Free Radical Scavenging Assay: The antioxidant activity of ellagic acid was evaluated using the DPPH assay. Ellagic acid solutions of various concentrations (1–50 µM) were incubated with 0.2 mM DPPH in methanol at room temperature in the dark. Absorbance at 517 nm was measured after 30 minutes. Scavenging activity was calculated as a percentage of control.

Cell Viability and Clonogenic Survival Assay: Cells were seeded into 96-well plates at a density of 5 × 10³ cells/well and allowed to adhere overnight. Cells were then treated with ellagic acid (various concentrations) for 24 hours prior to irradiation, followed by exposure to 6 Gy X-ray. After irradiation, the culture medium was replaced with fresh medium without ellagic acid, and the cells were incubated for an additional 24 hours. Cells viability was assessed using the Cell Counting Kit-8 according to the manufacturer’s instructions. Absorbance was measured at 450 nm using a microplate reader.

Blood Biochemistry: Serum samples were collected from orbital blood on day 14. AST, ALT were measured using an automatic biochemical analyzer. cTnI levels were measured using a commercially available ELISA kit according to the manufacturer’s instructions.

Statistical Analysis: All data are presented as mean ± SEM. Statistical significance was evaluated using Student’s t-test or one-way ANOVA followed by Tukey’s post-hoc test. A p-value < 0.05 was considered statistically significant.