Penicilazaphilone C triggers ferroptosis in triple-negative breast cancer cells via the MDM2/p53/SLC7 A11/GPX4 pathway

Abstract

Penicilazaphilone C (PAC) is a novel azaphilone isolated by our research team. While known for its antitumor properties, the potential of PAC to trigger ferroptosis in triple-negative breast cancer (TNBC) cells remains unexplored. This study aims to assess the ferroptotic induction capability of PAC and elucidate the underlying molecular mechanisms. Our results showed that treatment with PAC demonstrated a dose- and time-dependent inhibition of growth in MDA-MB-231 and MDA-MB-436 cells. Flow cytometry analysis and lactate dehydrogenase assay revealed various forms of cell death induced by PAC in both cell lines. Specifically, flow cytometry analyzed 7-AAD-stained dead cells, and ferroptosis markers, such as lipid peroxidation BODIPY-C11 and Fe²⁺ ions, identified ferroptosis as the major type of cell death pathway induced by PAC. Moreover, co-administration of the ferroptosis inhibitor Fer-1 notably mitigated PAC-induced cell death, further highlighting ferroptosis as the primary mechanism through which PAC inhibits TNBC cell growth and proliferation. Further exploration of the molecular mechanisms unveiled that PAC modulated the expression of Mouse double minute 2 (MDM2), p53, SLC7A11, and GPX4. In vivo experiments using nude mouse models implanted with MDA-MB-231 and MDA-MB-436 cells demonstrated that PAC treatment effectively suppressed tumor growth, increased levels of BODIPY-C11 and Fe²⁺ ions in isolated single tumor cells, downregulated MDM2 expression, and upregulated p53, SLC7A11, and GPX4 expression. These results suggest that PAC hinders TNBC cell growth and proliferation by modulating the MDM2/p53/SLC7A11/GPX4 axis to induce ferroptosis, positioning PAC as a promising azaphilone candidate for TNBC therapy.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12672-025-02687-w.

Introduction

Breast cancer is currently the most common cancer worldwide[1], accounting for 11.7% of all cancer cases. This is mainly due to changes in lifestyle and the increasing number of women working in industries, leading to delayed childbirth and lower birth rates[2, 3]. Triple-negative breast cancer (TNBC) is a highly aggressive form of breast cancer characterized by the absence of three specific receptors: the estrogen receptor, the progesterone receptor, and the human epidermal growth factor receptor 2 (HER2)[4]. This subtype of breast cancer has a high tendency to metastasize and is associated with a poor prognosis, making surgery and chemotherapy less effective[5–7]. Approximately 15–20% of breast cancer cases are TNBC, which is linked to a poorer prognosis, with about one-third of patients experiencing distant recurrence and eventual death[8–10]. Despite the heterogeneity, several emerging molecular targets such as PI3 K/AKT/mTOR, PIM1, and PD-L1 are under investigation[11–14]. However, clinical and preclinical studies have shown that targeting these pathways often results in limited efficacy, due to factors such as tumor heterogeneity, resistance, and the low prevalence of actionable mutations[11–14]. This highlights the need to explore alternative treatment approaches to improve outcomes for TNBC patients[15, 16].

Ferroptosis is an iron-dependent form of cell death characterized by lipid peroxide accumulation[17, 18]. Unlike apoptosis and necrosis, ferroptosis is marked by increased cell membrane permeability and is implicated in various medical conditions, including cancer, neurodegenerative diseases, and tissue damage[19–25]. Recent research suggests its potential as a therapeutic strategy for drug-resistant tumors, such as TNBC[26–30]. The precise molecular mechanisms of ferroptosis are still under investigation, but studies have shown decreased expression of ferroptosis-inhibitory proteins, such as solute carrier family 7 member 11 (SLC7 A11) and glutathione peroxidase 4 (GPX4), in TNBC[31–34]. The E3 ubiquitin ligase MDM2, known for negatively regulating the tumor suppressor protein p53, is implicated in this process[35–37]. Additionally, p53 plays a crucial role in modulating ferroptosis in cancer cells by directly inhibiting the expression of SLC7 A11[38, 39], a gene that encodes xCT, a cystine-glutamate antiporter crucial for importing cystine into cells[40]. Cystine is then converted to cysteine intracellularly, a precursor that is essential for glutathione (GSH) synthesis[41, 42]. GSH, which relies on cysteine as the primary substrate, plays a pivotal role in preventing ferroptosis[43–45]. Consequently, the inhibition of SLC7 A11 can induce cell death through ferroptosis[46–49].

Penicilazaphilone C (PAC) is a novel natural compound isolated from the marine fungus Penicillium M-22 by our research team[50]. PAC, a nitrogen-containing derivative, features a distinctive pyranonaphthoquinone bicyclic core structure (Fig. 1A). Previous research has highlighted the diverse biological activities of nitrogen-containing phenyl ketone compounds[51–53]. Studies have shown that PAC selectively induces cell death in various cancer types and possesses anti-inflammatory properties[50, 54, 55]. However, the precise molecular mechanism underlying PAC's inhibitory effects on breast cancer cells remains unclear. This study aims to investigate PAC's potential to trigger ferroptosis in TNBC cells. Our findings demonstrate that PAC effectively induces ferroptosis in TNBC cells both in vitro and in vivo. Specifically, PAC suppresses MDM2 expression, leading to increased p53 protein levels, subsequently reducing SLC7 A11 and GPX4 levels, thereby decreasing resistance to ferroptosis. These results suggest that PAC could be a promising therapeutic agent for TNBC through ferroptosis induction, warranting further investigation into its clinical translational potential.

Fig. 1.

PAC inhibits the cell viability and proliferation of TNBC cells. A The chemical structure of PAC. B Cell viability of MDA-MB-231 and MDA-MB-436 cells treated with varying concentrations of PAC was determined using a CCK-8 assay. C The impact of a specific concentration of PAC (0.4 μM) on the cell viability of these cells over different time intervals was assessed. D FCM was used to analyze the percentage of EdU-positive MDA-MB-231 and MDA-MB-436 cells treated with PAC, with representative images provided (left). The data, representing the mean ± standard deviation of five or ten independent replicates, were subjected to statistical analysis using a two-way ANOVA and post hoc multiple comparisons. Nonsignificant differences are denoted by identical letters above columns, while distinct letters indicate significant differences with a p-value below 0.05

Materials and methods

Cell culture and reagents The TNBC cell lines, MDA-MB-231 and MDA-MB-436, utilized in this research were procured from the American Type Culture Collection (ATCC) and maintained in the laboratory. MDA-MB-231 cells were cultured in RPMI 1640 (11875119, GIBCO, Beijing, China), while MDA-MB-436 cells were maintained in Dulbecco's Modified Eagle Medium (DMEM, 11965092, GIBCO, Beijing, China). Both media were supplemented with 10% fetal bovine serum (FBS), 1% penicillin (15140122, GIBCO, Beijing, China), and 1% streptomycin (P0781, Sigma, Shanghai, China). To ensure optimal cell growth and viability, the cells were incubated at 37 °C in a 5% carbon dioxide environment.The antibodies and reagents utilized in this study adhered to necessary scientific standards. Z-VAD-FMK (HY-16658B), 3-MA (HY-19312), and ferrostatin-1 (HY-100579) were purchased from MedChemExpress (Shanghai, China) and dissolved in dimethyl sulfoxide (DMSO), with a final DMSO concentration not exceeding 0.1% in assays. These compounds were applied at a working concentration of 0.6 μM along with PAC to assess their inhibitory effects on various cell death pathways. Control groups maintained equivalent DMSO concentrations to control for any solvent-related effects. 7-AAD (HY-D1020) was acquired from MedChemExpress (Shanghai, China) and dissolved in phosphate-buffered saline (PBS) as per the manufacturer's instructions. Hoechst nuclear dye (H10325) was sourced from Thermo Fisher Scientific (Waltham, Massachusetts, USA).The mouse monoclonal anti-p53 antibody (DO-7, GA61661-2, Dako, Denmark) and the mouse monoclonal anti-MDM2 antibody (SMP14, Santa Cruz Biotechnology) were unconjugated and detected using species-specific secondary antibodies conjugated to horseradish peroxidase (HRP) or fluorophores, depending on the assay. The rabbit monoclonal anti-SLC7 A11 antibody (ab37185) and the rabbit monoclonal anti-glutathione peroxidase 4 (GPX4) antibody (ab125066) from Abcam were also unconjugated and required secondary antibodies for detection.

Detection of cell viability and proliferation In this study, cell viability and proliferation were assessed using CCK-8 and ethynyl-2′-deoxyuridine (EdU) assays, respectively. Cell viability was evaluated by following the manufacturer's instructions for the CCK-8 assay kit (C0037, Beyotime, Shanghai, China). Specifically, 5000 cells were seeded in a 96-well plate and exposed to varying concentrations of PAC (0, 0.1, 0.2, 0.3, 0.4, 0.6, and 0.7 μM) and other relevant reagents for 24 h. After the incubation period, 10% CCK-8 solution was added, and the absorbance at 450 nm was measured using a microplate reader (ELX808IU, BioTek, VT, USA). The IC50 values were determined through non-linear curve fitting, yielding 0.379 μM for MDA-MB-231 cells and 0.414 μM for MDA-MB-436 cells. Based on these results, a concentration of 0.4 μM PAC—which is close to the determined IC50 values—was selected for subsequent experiments. Subsequently, both cell lines were cultured with 0.4 μM PAC for 12, 24, 36, and 48 h, and cell viability was determined using the same CCK-8 assay protocol. To identify proliferating cells with EdU-incorporated nuclei, an EdU cell proliferation assay kit with Alexa Fluor 488 (C0071S, Beyotime) was used. Cells were stained with the EdU solution according to the manufacturer's protocol and analyzed using a flow cytometer (CyFlow Cube 6, Sysmex, Japan).

Flow cytometry analysis FCM was employed to evaluate multiple cellular parameters in the study, encompassing the detection of EdU-positive cells, mitochondrial potential, 7-AAD-positive deceased cells, levels of lipid peroxidation (BODIPY-C11), and Fe²⁺. MDA-MB-231 and MDA-MB-436 cells were treated with PAC and relevant reagents for 24 h before undergoing flow cytometry analysis. Subsequently, the cells were exposed to specific markers such as EdU, 7-AAD, JC-1, BODIPY-C11, or FerroOrange (F374, Dojindo, Japan) live cell dye for 30 min at 37 °C. FerroOrange, a fluorescent probe specifically designed to detect intracellular Fe²⁺, was used to measure Fe²⁺ levels. Data acquisition was performed on a CyFlow Cube 6 system (Sysmex, Japan) under standardized conditions, with voltage settings of FSC at 350 V, SSC at 300 V, FL1 (EdU) at 450 V, FL2 (JC-1 green) at 500 V, FL3 (7-AAD) at 550 V, FL4 (BODIPY-C11) at 500 V, and FL5 (FerroOrange) at 520 V. These settings were chosen to optimize the resolution of positive cell populations while minimizing background noise, ensuring high-quality and reproducible data. FCM analysis was conducted to quantify the fluorescence intensity of stained cells. The resulting FCS files were analyzed using FlowJo software to extract precise data and images. The percentage of positive cells was determined for EdU-positive (reflecting DNA synthesis and cell proliferation), JC-1 green (indicating mitochondrial membrane potential), and 7-AAD-positive cells (representing dead or late apoptotic cells), while the fold change in mean fluorescence intensity (MFI) in relation to the control group was calculated for lipid peroxidation and Fe²⁺ levels. Additional information regarding cell treatments can be found in subsequent sections.

5-Ethynyl-2′-deoxyuridine assay The 5-ethynyl-2′-deoxyuridine (EdU) assay was performed using the Cell Light EdU Apollo 567 in vitro imaging kit (RiboBio, Guangzhou, China) according to the manufacturer's instructions. Cells were seeded at a density of 2 × 10^6 cells per well in a 24-well plate and treated with PAC or other specified agents for 24 h. Subsequently, 50 µM EdU was added to the cell culture and incubated at 37 °C for 30 min. After that, the cells were washed with PBS, fixed in 4% anhydrous methanol at room temperature for 30 min, treated with 2 mg/ml glycine for 5 min, and then washed with PBS. The cells were stained with Hoechst 33342 for 30 min at room temperature, washed three times with PBS, and examined under a fluorescence microscope (LX83, Olympus, Tokyo, Japan). FCM analysis was performed using a CyFlow Cube 6 instrument (Sysmex, Japan).

Lactate dehydrogenase release assay The LDH release assay kit (ab102526, Abcam) was used to assess the cytotoxic impact of PAC on TNBC cells according to the manufacturer's instructions. Specifically, 100 μL of cells at a concentration of 10^5 cells/mL were plated in a 96-well plate and treated with 20 μL of 0.4 mol/L lactic acid solution, 20 μL of 4 mmol/L 2-p-iodophenyl-3-p-nitrophenyltetrazolium chloride, and 20 μL of reaction solution. The samples were then incubated at room temperature for 30 min, and the optical density (OD) was measured at a detection wavelength of 492 nm and a reference wavelength of 650 nm using an enzyme-linked immunosorbent assay reader (ELX808IU, Bio-Tek). The fold change in OD for each sample, relative to the control group, was calculated by dividing the OD value of each sample by the average OD value of the control group, as previously reported.

Western blot analysis The Western blot analysis was conducted following previously reported methods. In brief, TNBC cells were lysed using RIPA buffer containing 1 mM EDTA, 50 mM Tris, 0.1% Triton X-100, 150 mM NaCl, 1% sodium deoxycholate, and 1 mM PMSF. Protein concentrations in the cell lysates were determined using a BCA protein assay kit (ab102536, Abcam). Equal amounts of protein were separated on a 10–12% SDS-PAGE gel and transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was then blocked with 10% skim milk at 4 °C for 1 h to prevent non-specific binding.After blocking, the PVDF membrane was incubated overnight at 4 °C with primary antibodies diluted at 1:300. Following this, the membrane was incubated with HRP-conjugated secondary antibodies diluted at 1:5000. Finally, the membrane was treated with an enhanced chemiluminescence (ECL) substrate for 30 min, and the chemiluminescent signals were captured using an ECL system (Amersham Biosciences, UK) for visualization and analysis of the protein bands.

Analyzing MDM2-related genes for ferroptosis using the STRING database Access the STRING database at https://cn.string-db.org/. Enter"MDM2"in the"Protein by name"field and select"Homo sapiens"as the Organism.Click"CONTINUE"to proceed. On the interaction network page, open"Settings". Under"2nd Shell", choose"Custom Value", enter 50 as the maximum number of interactors, and click"UPDATE". In the"Exports"module, select"Export your current network"and download it as"Short Tabular Text Output"to obtain the interaction list for MDM2. Download the network image if required.

Cell transfection was performed to overexpress MDM2 in MDA-MB-231 and MDA-MB-436 cells by transfecting the pCMV-MDM2 plasmid. The pCMV-MDM2 plasmid was obtained from Addgene (plasmid number: 10838). Cells were seeded at a density of 5 × 10⁵ cells/well in a 6-well plate and cultured overnight at 37 °C with 5% CO₂ until reaching 70–80% confluency. Prior to transfection, the medium was replaced with antibiotic-free medium. Transfection of the plasmid was carried out using Lipofectamine 3000 transfection reagent (Invitrogen, USA) following the manufacturer's protocol. In each well, 2 μg of pCMV-MDM2 plasmid and 5 μL of Lipofectamine 3000 were complexed in 250 μL of Opti-MEM medium (Gibco, USA), then added to the cell culture wells after 15 min of incubation at room temperature, followed by further incubation in serum-free medium for 6 h. Subsequently, the medium was replaced with complete medium containing 10% fetal bovine serum (FBS), and cells were further cultured for 24 h. After 24 h of transfection, cell lysates were collected for Western blot analysis to detect the expression levels of MDM2 and p53 proteins to confirm transfection efficiency. Successfully transfected cells were used for subsequent CCK-8 cell proliferation assays and flow cytometry (FCM) to assess the impact of PAC treatment on cell death and ferroptosis. A control group was established using the empty vector pCMV plasmid (Addgene, plasmid number: 82000) to exclude nonspecific effects of the plasmid vector on cell proliferation and death. All experiments were repeated three times to ensure the reliability and reproducibility of the results.

Establishment of the mouse TNBC tumor models and treatment with PAC TNBC tumor models in mice were established using MDA-MB-231 and MDA-MB-436 cell lines, as previously described[56]. Briefly, 6-week-old female BALB/c nude mice (weighing approximately 18–20 g) were obtained from the Hunan Animal Center in Changsha, China (certification No. SCXK2021-0241) and housed in a controlled environment free of pathogens. TNBC cells (1 × 10^6) were injected subcutaneously into the right flank of the mice to establish the TNBC mouse model. Once the tumor volume reached around 10 mm3, the mice were divided into two groups for further observation of tumor growth and immunohistochemical analysis: the control (Ctrl) group and the PAC group. The Ctrl group received saline treatment, while the PAC group received PAC at a dosage of 30 mg/kg alone via tail vein injection once every three days, for a total of seven treatments, as previously described[54]. Tumor images and volumes were monitored every three days using a portable imaging device named TM900 produced by Peira in Belgium. After 28 days, the mice were humanely euthanized via an intraperitoneal injection of pentobarbital sodium (150 mg/kg) followed by cervical dislocation, and the tumors were collected for further analysis, including weighing and immunohistochemical experiments. All animal experiments were carried out in accordance with the guidelines established by the Ethics Committee for Animal Care and Use at Hainan Medical University (approval No. HYLL-2022-598).

Isolate single cells from tumor tissue for flow cytometry analysis Prior to isolating single cells from the tumor tissues, a tumor digestion solution was prepared. The solution, comprising 10 mL, included 1000 μL of collagenase/hyaluronidase at a concentration of 1 mg/mL, 1500 μL of DNase I solution, and 3.75 mL of RPMI 1640 medium. This solution was thoroughly mixed and equilibrated to room temperature (15–25 °C). Subsequently, the minced tumor tissue was placed in a 14 mm round-bottom tube with 5 mL of the tumor digestion solution and incubated at 37 °C on a shaker for 25 min. Following this, a 70 μm cell strainer was positioned on a 50 mL conical tube. The isolated cells were then drawn into a syringe plunger and passed through the filter into the 14 mm round-bottom tube. The cells were washed with PBS in the 50 mL conical tube, resuspended in 50 mL of PBS, and centrifuged at 300×g at room temperature for 10 min with a low brake setting. The supernatant was carefully removed, and the cell pellet was mixed with a 10 mL ammonium chloride solution, followed by an incubation at room temperature for 1–2 min. Finally, the isolated single cells were resuspended in Dulbecco’s Eagle’s Medium (DMEM) medium at a concentration of 5 × 10^7 cells/mL. These single cells were ultimately used for the analysis of lipid peroxidation and ferrous ions using the aforementioned FCM technique.

Statistical analysis The experimental data were analyzed using GraphPad Prism v6.0 software (GraphPad Software, San Diego, CA).

One-way or two-way ANOVA with Tukey's post hoc test was employed for comparisons among multiple groups. To determine the difference between two groups, an unpaired t-test was used. Significance was denoted as ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05. Non-significant differences were not labeled or were indicated as “ns” (not significant). All data are presented as the mean ± standard deviation (SD).

Results

PAC treatment suppresses cell proliferation in TNBC cells In this study, we investigated the impact of PAC treatment on the proliferation of TNBC cell lines MDA-MB-231 and MDA-MB-436 through CCK-8 and EdU assays. The CCK-8 results illustrated a dose- and time-dependent decrease in cell viability after 24 h of PAC treatment (Fig. 1B, C), with IC₅₀ values calculated at 0.379 μM for MDA-MB-231 cells and 0.414 μM for MDA-MB-436 cells through non-linear curve fitting. Subsequently, flow cytometry (FCM) analysis was employed for quantitative evaluation of EdU-positive cells to assess TNBC cell proliferation. The results revealed a significant reduction in the percentage of proliferating cells following PAC treatment, exhibiting a dose-dependent trend. Quantitative analysis of the FCM data indicated a marked decline in the proportion of EdU-positive proliferating cells in both TNBC cell lines, which correlated with increasing PAC concentrations compared to the control group (Fig. 1D).

PAC treatment induces cell death in triple-negative breast cancer cells To investigate the impact of PAC on triggering cell death in triple-negative breast cancer (TNBC) cells, we utilized JC-1 staining, 7-AAD staining, and LDH release assays. Our findings demonstrated that in the control group, both MDA-MB-231 and MDA-MB-436 cells displayed JC-1 in its polymeric state within the mitochondria, indicating a stable mitochondrial membrane potential (Fig. 2A–C). Conversely, after PAC treatment, a significant depolarization of the mitochondrial membrane potential in TNBC cells was observed, accompanied by a marked reduction in JC-1 aggregates, illustrating a dose-dependent response (Fig. 2A–C). Additionally, 7-AAD staining and FCM analysis revealed a notable increase in the percentage of 7-AAD-positive cells in both MDA-MB-231 and MDA-MB-436 cells after PAC treatment compared to the control group, indicating a substantial dose-dependent induction of cell death (Fig. 2D, E). This pattern was consistent across both MDA-MB-231 and MDA-MB-436 cells. Lastly, LDH release assay results supported the FCM analysis results, demonstrating a significant rise in LDH release after PAC treatment, further confirming cellular membrane damage (Fig. 2F). The effects of PAC treatment followed a dose-dependent trend, particularly evident at a PAC concentration of 0.6 μM, where cell death was most prominent. In summary, PAC significantly triggers cell death in TNBC cells in a dose-dependent manner. Based on these experimental results, we identified 0.6 μM PAC as the most effective concentration for inhibiting cell proliferation and inducing cell death, thus selecting this concentration for subsequent investigations.

Fig. 2.

PAC triggers various forms of cell death in TNBC cells. A–C JC-1 staining and FCM analysis were conducted on MDA-MB-231 and MDA-MB-436 cells to determine the percentage of JC-1 monomers (B) and aggregates (C). Flow cytometric analysis of 7-AAD-positive MDA-MB-231 and MDA-MB-436 cells was performed, with representative images shown (D) and quantified analysis of five samples (E). F A lactate dehydrogenase (LDH) release assay was utilized to quantify LDH levels in the supernatant of MDA-MB-231 and MDA-MB-436 cells. The data, presented as the mean ± standard deviation of five and ten (F) independent replicates, were statistically analyzed using one-way (C) or two-way ANOVA, followed by post hoc multiple comparisons. Nonsignificant differences are denoted by identical letters above the columns, while significant differences (p < 0.05) are indicated by distinct letters

PAC treatments mainly lead to ferroptosis in TNBC cells In order to determine the type of cell death induced by PAC in TNBC cells, we divided both MDA-MB-231 and MDA-MB-436 cells into five groups. The control group (Ctrl) was treated with vehicle only, the PAC group was exposed to PAC alone, and the remaining three groups were treated with PAC in combination with ferrostatin-1 (Fer-1, a ferroptosis inhibitor), Z-VAD-FMK (Z-VAD, an apoptosis inhibitor), and 3-Methyladenine (3-MA, an autophagy inhibitor), respectively. FCM analysis of 7-AAD-positive dead cells revealed a significant increase in cell death in the PAC group compared to the control (Ctrl) group, with over 50% of cells being dead (Fig. 3A). However, when PAC was co-administered with three different death inhibitors, there was a corresponding decrease in 7-AAD-positive dead cells, with the most notable reduction observed in the group treated with the ferroptosis inhibitor Fer-1 (Fig. 3A). These findings suggest that ferroptosis is the primary form of cell death induced by PAC. Ferroptosis is a novel form of cell death characterized by iron-dependent membrane lipid peroxidation, leading to membrane damage and loss of cell function. To investigate the relationship between the modes of cell death induced by PAC and ferroptosis, we assessed the levels of lipid peroxidation detected using BODIPY-C11 and ferrous ions (Fe²⁺) detected using FerroOrange in cells treated with PAC alone or in combination with multiple cell death inhibitors using FCM. Our findings revealed a significant increase of over 25-fold in BODIPY-C11 fluorescence intensity, indicating lipid peroxidation in MDA-MB-231 and MDA-MB-436 cells treated with 0.6 μM PAC alone compared to the control group (Fig. 3B). However, co-treatment with different cell death inhibitors resulted in varying degrees of reduction in BODIPY-C11 levels (Fig. 3B). Notably, the combination of PAC with the ferroptosis inhibitor Fer-1 showed the most pronounced decrease, reducing BODIPY-C11 levels by nearly two-thirds (Fig. 3B). These results further support the notion that PAC predominantly induces ferroptosis to promote cell death in triple-negative breast cancer cells. Similarly, flow cytometry analysis of cellular Fe²⁺ levels yielded similar outcomes (Fig. 3C). Collectively, these findings underscore the role of PAC in inducing ferroptosis as a mechanism to facilitate cell death in TNBC cells.

Fig. 3.

PAC inhibits TNBC cell death mainly by inducing ferroptosis. A Flow cytometry analysis of the percentage of 7-AAD-positive MDA-MB-231 and MDA-MB-436 cells after treatment with PAC alone and in combination with three inhibitors, as indicated. B Flow cytometry analysis of lipid peroxidation using BODIPY-C11 was performed on MDA-MB-231 and MDA-MB-436 cells treated as described in A. C Flow cytometry assessment of intracellular Fe²⁺ levels in MDA-MB-231 and MDA-MB-436 cells treated as described in A. The data are presented as the mean ± standard deviation of five independent replicates and are analyzed using a two-way ANOVA, followed by post hoc multiple comparisons. Identical letters above columns denote nonsignificant differences, whereas distinct letters indicate significant differences with a p-value of less than 0.05

PAC induces ferroptosis by activating the MDM2/p53/SLC7 A11/GPX4 signaling axis in TNBC cells Our team previously identified that the MDM2/p53/SLC7 A11/GPX4 signaling axis suppresses ferroptosis, thereby promoting the growth and proliferation of TNBC [28]. Consequently, we further investigated whether PAC exerts its promotion of TNBC ferroptosis by inhibiting this signaling axis. Therefore, we first analyzed the relationship between MDM2 and these molecules in the ferroptosis pathway using the online STRING database. The results revealed interactions between MDM2, p53, and iron metabolism-related molecules such as GPX4 and SLC7 A11 (Fig. 4A). MDM2, as a classical E3 ubiquitin ligase of p53, negatively regulates the expression level of p53 [35–37]. Numerous studies have shown that p53 induces ferroptosis in cells by inhibiting the expression of SLC7 A11 and GPX4 [38, 39, 57, 58]. After PAC treatment of MDA-MB-231 and MDA-MB-436 cells, the protein levels of MDM2, SLC7 A11, and GPX4 significantly decreased, while p53 expression increased compared to the control group (Fig. 4B). This indicates that PAC treatment can indeed regulate the expression of MDM2, p53, SLC7 A11, and GPX4 molecules. To investigate the interplay between these molecules, TNBC cells were transfected with the MDM2 expression plasmid pMDM2 to induce MDM2 overexpression. Following a 24-h transfection period, the levels of MDM2 and p53 proteins were assessed through Western blot analysis. The results demonstrated a significant decrease in p53 expression upon pMDM2 transfection compared to PAC-treated cells (Fig. 4C), indicating MDM2's negative regulation of p53 and its involvement in ferroptosis progression. In addition, CCK-8 assay results revealed a notable reduction in growth and proliferation of MDA-MB-231 and MDA-MB-436 cells upon PAC treatment in comparison to control cells (Fig. 4D). Conversely, MDM2 overexpression via pMDM2 transfection substantially enhanced the growth and proliferation of PAC-treated cells, with cell viability increasing by almost two-thirds relative to the PAC group, approaching levels observed in control cells (Fig. 4D). However, PAC treatment after pMDM2 transfection only partially restored cellular growth and proliferation (Fig. 4D). Moreover, FCM analysis showed that MDM2 overexpression through pMDM2 transfection correlated with increased levels of lipid peroxide BODIPY-C11 (Fig. 4E) and Fe²⁺ (Fig. 4F) in the cells. Notably, cells transfected with pMDM2 exhibited a significant decrease in BODIPY-C11 (Fig. 4E) and Fe²⁺ (Fig. 4F) levels compared to cells treated with PAC alone, approaching levels similar to those in the control group. Nevertheless, treating pMDM2-transfected cells with PAC only partially restored BODIPY-C11 fluorescence intensity (Fig. 4E) and Fe²⁺ (Fig. 4F) levels. Statistical analysis indicated a significant difference in BODIPY-C11 (Fig. 4E) and Fe²⁺ (Fig. 4F) levels between the two groups (p < 0.01). These results suggest that PAC induces ferroptosis in TNBC cells through the MDM2/p53/SLC7 A11/GPX4 signaling pathway.

Fig. 4.

PAC induces ferroptosis by activating the MDM2/p53/SLC7 A11/GPX4 signaling axis. A Analysis from the STRING database reveals interactions between MDM2 and p53, as well as between SLC7 A11 and GPX4. B Western blot analysis revealed that treatment with PAC decreased the protein levels of MDM2, SLC7 A11, and GPX4, while concurrently increasing the levels of p53. C Overexpression of MDM2 led to a decrease in p53 expression. D CCK-8 assays demonstrated a reduction in cell viability upon PAC treatment, which was partially restored by overexpression of MDM2, large SD reflects biological variability and differences in transfection efficiency. Flow cytometry analysis showed an increase in lipid peroxides (E) and Fe²⁺ (F) following PAC treatment, while a significant decrease in expression occurred with MDM2 overexpression. This trend was reversed by PAC treatment. The data are presented as the mean ± standard deviation of five independent replicates and were analyzed using a two-way ANOVA, followed by post hoc multiple comparisons. Identical letters above columns denote nonsignificant differences, whereas distinct letters indicate significant differences with a p-value of less than 0.05

PAC inhibits TNBC tumorigenesis and triggers ferroptosis in vivo Our in vitro experiments have shown that PAC can induce ferroptosis and inhibit the growth of TNBC cells. To further confirm the anti-TNBC effects of PAC in vivo, tumor models were established in nude mice using MDA-MB-231 and MDA-MB-436 cell lines. The results revealed a substantial decrease in tumor volume in the PAC treatment group compared to the control group, with the PAC group exhibiting approximately half of the tumor volume of the control group 28 days after tumor cell inoculation (Fig. 5A–C), indicating a statistically significant difference between the two groups (p < 0.0001). To validate the induction of ferroptosis by PAC, tumor tissues were dissociated into single cells, and levels of the lipid peroxidation (measured by BODIPY-C11) and Fe²⁺ were quantified using FCM. FCM analysis demonstrated a marked increase in lipid peroxidation (Fig. 5D) and Fe²⁺ (Fig. 5E) levels in the tumor tissues of the PAC treatment group compared to the control group, with both lipid peroxidation and Fe²⁺ levels exhibiting an increase of over 10 times that of the control group, indicating a highly significant statistical distinction between the two groups (p < 0.0001). Additionally, Western blot analysis was performed to evaluate the expression of MDM2, p53, SLC7 A11, and GPX4 to investigate the regulatory impacts of PAC treatment on ferroptosis. The findings revealed a significant decrease in MDM2 expression in the PAC-treated tumor tissues compared to the control group, while the expressions of p53, SLC7 A11, and GPX4, which are associated with ferroptosis, were correspondingly upregulated (Fig. 5F). In conclusion, these in vivo experiments in mouse tumor models demonstrate the notable anti-tumor efficacy of PAC, which is strongly associated with the induction of ferroptosis.

Fig. 5.

PAC inhibits the development of TNBC tumors and promotes ferroptosis in vivo. After a 28-day post-inoculation period in nude mouse models injected with MDA-MB-231 and MDA-MB-436 cells, images illustrating the tumor volume (A, B) were obtained, along with precise quantitative assessments of the tumor volumes in the control and PAC treatment groups (C). Flow cytometry analysis showed an increase in lipid peroxides (D) and Fe²⁺ (E) after PAC treatment. F The expression of MDM2, p53, SLC7 A11, and GPX4 was analyzed by Western blot in tumor tissues. The data are presented as the mean ± standard deviation of five independent experiments, each with two technical replicates, and were analyzed using a t-test. Significant differences are indicated by asterisks: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. Non-significant differences are denoted by “ns” (p > 0.05)

Discussion

TNBC, a subtype of breast cancer, is recognized for its elevated risk of early distant recurrence and unfavorable prognosis[59]. The significant challenges in TNBC stem from its aggressive nature and the lack of specific molecular targets [4, 60]. Moreover, it exhibits intra-tumor and inter-tumor heterogeneity, further complicating treatment strategies[61]. TNBC shows specific patterns of metastasis, with a propensity for metastasis to the brain and lungs[62]. Given the grave prognosis of TNBC, the development of systemic therapies that can improve patient survival is crucial[4, 63]. Therefore, the identification of potential molecular biomarkers is of significant importance for enhancing TNBC diagnosis and driving the development of new therapies[64]. Currently, research is exploring ways to induce cell death in TNBC cells through autophagy, ferroptosis, and programmed necrosis to enhance treatment efficacy.

Natural compounds derived from marine fungi have garnered increasing attention as promising sources of bioactive secondary metabolites for drug discovery[65, 66]. In our exploration of novel lead compounds for anti-tumor and antibacterial agents, various fungal strains from marine sources were screened. Among these, a strain isolated from decaying tree leaves along the coast of Haikou, Hainan Province, China, was found to produce nitrogen-containing compounds, with penicilazaphilone C (PAC) identified as the primary active ingredient[50]. Previous studies have highlighted the potent anti-inflammatory and anticancer properties of penicilazaphilone C (PAC). However, the specific mechanisms by which this novel nitrogen-containing phenalenone structure regulates cancer progression remain unclear. Our study demonstrates that PAC effectively inhibits TNBC tumor growth and proliferation in vivo, indicating its therapeutic potential. In vitro experiments reveal that PAC induces ferroptosis in TNBC cells through the MDM2/p53/SLC7 A11/GPX4 pathway, underscoring its anti-tumor effects. These findings suggest that PAC, with its pyranonaphthoquinone bicyclic core structure, holds promise for TNBC treatment. Further research with a more systematic experimental design is warranted to elucidate its mechanisms comprehensively. Moreover, exploring fungi or microorganisms from tropical intertidal zones may unveil additional potent compounds for anti-tumor and anti-inflammatory applications.

Ferroptosis is a distinct form of cell death initiated by disrupted lipid metabolism and an imbalance in oxidative stress. It is characterized by the iron-dependent accumulation of lipid peroxides, which disrupt intracellular lipid metabolism and the balance of oxidative stress[17, 18]. Impairment of antioxidant defense mechanisms leads to an increase in reactive oxygen species (ROS), resulting in elevated levels of lipid peroxides and the promotion of ferroptosis[67]. Intracellular ROS levels, influenced by various factors, have the potential to induce ferroptosis, particularly in cells with compromised antioxidant capacity. Unlike apoptosis and necrosis, ferroptosis is reliant on iron and lipid peroxidation. Environmental stress, genetic mutations, or therapeutic interventions can raise ROS levels, thereby triggering ferroptosis[68, 69]. Moreover, accumulating evidence underscores the significant role of ferroptosis in impeding the proliferation of specific cancer cells[26–28]. Consequently, the discovery and development of novel compounds that stimulate ferroptosis in tumor cells bear substantial theoretical importance and translational promise for clinical use[29, 30]. In this study, our results demonstrate a dose-dependent inhibitory effect of PAC treatment on the viability and proliferation of TNBC cells, as validated by CCK-8 and EdU experiments. Furthermore, FCM and lactate dehydrogenase release assays indicate a significant increase in 7-AAD-positive dead cells and corresponding LDH levels post-PAC treatment. The detection of 7-AAD, which cannot penetrate normal cell membranes[70], and the release of LDH, predominantly from cells with membrane damage[71], collectively suggest PAC-induced membrane-permeabilizing cell death. Additionally, our findings reveal a substantial reduction in tumor cell death when PAC is co-administered with the ferroptosis inhibitor Fer-1, while co-treatment with apoptosis and autophagy inhibitors shows less pronounced effects on cell death. FCM analysis further indicates higher levels of lipid peroxidation and ferrous iron (Fe²⁺) in PAC-treated TNBC cells. These phenomena are also observed in a mouse tumor model. Taken together, these results strongly support the notion that PAC exerts its anti-tumor effects in TNBC by triggering ferroptosis, thereby inhibiting growth and proliferation. Due to the reported ability of most tumors to inhibit ferroptosis[72], it is essential to investigate whether PAC promotes ferroptosis in various other types of cancer.

In our previous research, we found that MDM2 may ubiquitinate and degrade p53, thereby regulating the expression of ferroptosis-related molecules SLC7 A11 and GPX4 to inhibit ferroptosis in TNBC cells[28]. Therefore, in this study, we primarily investigated whether PAC promotes ferroptosis in TNBC cells by modulating the MDM2/p53/SLC7 A11/GPX4 pathway. MDM2 is an E3 ubiquitin ligase that can inhibit the tumor-suppressive protein p53 by promoting its degradation[35–37]. p53, a crucial tumor suppressor, is involved in various anticancer mechanisms, including cell cycle regulation, apoptosis promotion, and induction of ferroptosis[73]. SLC7 A11 and GPX4 are important inhibitors of ferroptosis, with SLC7 A11 promoting cystine uptake to maintain glutathione (GSH) synthesis[74], and GPX4 reducing lipid peroxides by consuming GSH to inhibit ferroptosis[75, 76]. In this study, our study revealed that PAC treatment downregulates MDM2 expression, consequently stabilizing and increasing p53 protein levels, while significantly inhibiting SLC7 A11 and GPX4 levels. Hence, we hypothesize that the upregulation of p53 leads to the suppression of SLC7 A11 and GPX4 expression, ultimately increasing TNBC cell sensitivity to ferroptosis. These findings not only deepen our understanding of TNBC cell death mechanisms, but also provide a theoretical basis for PAC as a potential novel anticancer agent. However, this study primarily used Western blotting to assess the expression changes of these molecules, without further validation through corresponding molecular inhibition or gene knockout techniques. Therefore, further in-depth mechanistic studies are warranted.

In this study, we demonstrated that PAC induces ferroptosis in TNBC cells and explored its molecular mechanisms. However, further investigation is needed due to limitations. Our research relied on in vitro experiments and TNBC xenograft mouse models, providing evidence for PAC's anti-tumor effects but not fully replicating the human tumor microenvironment. Future studies should validate PAC's efficacy in additional preclinical models, such as patient-derived xenograft models, to better reflect human tumor characteristics. Additionally, while we have investigated the involvement of the MDM2/p53/SLC7 A11/GPX4 axis in PAC-induced ferroptosis, the molecular mechanisms are complex. PAC may modulate TNBC cell death through other unidentified molecules or pathways. Therefore, future investigations should explore alternative mechanisms, particularly interactions with different forms of cell death like autophagy or apoptosis. Moreover, our study did not comprehensively evaluate PAC's toxicity in normal cells, especially the impact of ferroptosis on normal tissues. Further research is essential to assess PAC's safety and toxicity in vivo, ensuring manageable potential side effects on normal cells and tissues for its clinical development as a therapeutic agent.

In summary, this study provides compelling evidence that PAC, induces ferroptosis in TNBC through modulation of the MDM2/p53/SLC7 A11/GPX4 axis, highlighting its anticancer potential. These results enhance our understanding of the molecular mechanisms involved in TNBC cell death and highlight the potential therapeutic value of PAC in TNBC treatment. Subsequent investigations should aim to further elucidate the specific molecular interactions within the ferroptosis pathway and evaluate the clinical efficacy of PAC in TNBC patients.

Author contributions

GHT, WPZ, and WTX conceived and designed the study and drafted the manuscript. GHT and WPZ offered funding support. HTL, FYH, and WTX planned and participated in data analysis and contributed primarily to manuscript writing, review, and editing. HTL, FYH, WJX, MHC, RHW, and SZD performed the experiments. FYH and GHT supervised all research, wrote, and approved the final version of the manuscript. HTL and GHT confirm the authenticity of all raw data. All authors contributed to the article and approved the final version.

Funding

This work was funded by the National Natural Science Foundation of China (32460190 and 82060639) and Hainan Provincial Natural Science Foundation (ZDKJ202003 and ZDYF2021SHFZ248).

Data availability 

All datasets generated and/or analyzed during this study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

The animal study was reviewed and approved by the Ethics Committee of Hainan Medical University (Approval No. HYLL-2019-458).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.