GSTP1-mediated inhibition of ACSL4-dependent ferroptosis via JNK pathway in DOX-induced cardiomyopathy

Abstract

Background:

Doxorubicin hydrochloride (DOX) is extensively used in the treatment of various tumors. However, its clinical application is limited due to dose-dependent cardiotoxicity. Currently, few effective strategies exist to mitigate or eliminate DOX-induced cardiomyopathy (DIC). Although ferroptosis is implicated in DIC and its inhibition partially alleviates the condition, the direct targets of DOX in the progression of cardiotoxicity remain unclear. This study aimed to discover the direct targets of DOX in ferroptosis-mediated DIC.

Methods:

A DOX pulldown assay was performed to identify proteins specifically binding to DOX in murine hearts, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify candidate proteins. A cardiac injury mouse model was established by DOX treatment. Based on this, multiple ferroptosis biomarkers were detected by flow cytometry, quantitative real-time polymerase chain reaction, western blotting, immunochemistry, etc. Besides, specific activator and inhibitor of signaling pathways were applied to illuminate molecular mechanisms.

Results:

Glutathione S-transferase P1 (GSTP1) was identified as a DOX target. GSTP1 activity was inhibited in DOX-treated cardiomyocytes, while its overexpression significantly alleviated DIC. Moreover, GSTP1 overexpression inhibited acyl-CoA synthetase long-chain family member 4 (ACSL4)-dependent ferroptosis. Mechanistically, GSTP1 overexpression suppressed c-Jun N-terminal kinase (JNK) phosphorylation, thereby reducing reactive oxygen species (ROS) production and inhibiting ferroptosis in DIC.

Conclusions:

This study identifies the DOX/GSTP1/JNK axis as a critical pathway mediating ACSL4-dependent ferroptosis in DIC. GSTP1 is highlighted as a potential key mediator of ferroptosis and a promising therapeutic target for DIC.

Introduction

Doxorubicin hydrochloride (DOX), a potent antitumor agent, is widely used in the treatment of various cancers.^[1] However, its clinical application is constrained by systemic adverse effects, including heart failure, hypertension, and structural cardiac damage. Notably, many patients experience cardiac side effects even at lower dosages of DOX. Previous studies have suggested that DOX-induced cardiomyopathy (DIC) is caused by a combination of factors, including reactive oxygen species (ROS), mitochondrial dysfunction, iron regulatory protein dysregulation, and cell death pathways such as apoptosis, autophagy, and necrosis.^[2,3,4,5,6] Among these, apoptosis-induced cardiomyocyte dysfunction and oxidative stress have been identified as primary contributors to DIC.^[1,7]

Recent advances in understanding DIC pathogenesis have highlighted the pivotal role of ROS production and iron dysregulation, particularly in the novel cell death pathway termed ferroptosis.^[8] Ferroptosis has also been implicated in other cardiac conditions, such as diabetic cardiomyopathy, underscoring its relevance in cardiac pathophysiology.^[9] These findings suggest that targeting ferroptosis may represent a promising strategy for mitigating DOX-induced cardiac dysfunction. Ferroptosis primarily arises from imbalances in ROS homeostasis and lipid metabolism.^{[10,11,12,13,14]} Over the past few years, various molecular pathways involved in ferroptosis have been identified.^{[15,16,17,18,19,20]} Key regulators include glutathione peroxidase 4 (GPX4), nuclear receptor coactivator 4 (NCOA4), FUN14 domain-containing 2 (FUNDC2), and acyl-CoA synthetase long-chain family (ACSL4).^{[21,22,23,24]} Among this, ACSL4 has been shown to promote ferroptosis in various diseases, including DIC.^{[23,25,26,27,28]} Recent findings further identified the ACSL4/ferritin heavy chain 1 (FTH1) axis-dependent ferroptosis as a therapeutic target in DIC.^[28]

Glutathione S-transferase P1 (GSTP1), a member of the glutathione S-transferase (GST) enzyme system, is a dimeric protein comprising two identical subunits.^[29,30,31] GSTP1 catalyzes intracellular detoxification reactions, thereby mitigating ROS levels.^[32,33,34] By facilitating the conjugation of gutathione (GSH) with ROS for clearance, GSTP1 plays a critical role in maintaining ROS homeostasis and regulating cell proliferation.^{[31,35,36,37]} Recent studies have suggested that GSTP1 may be involved in ferroptosis. However, the downstream pathways through which GSTP1 influences ferroptosis remain unclear. Although GSTP1 has been identified as a key mediator of the c-Jun N-terminal kinase (JNK) signaling pathway in various diseases,^[38,39,40] its involvement in ferroptosis through the GSTP1/JNK axis has not yet been directly established. Therefore, this study aimed to discover whether GSTP1 could influence ferroptosis via JNK in DIC.

Methods

Animals

Male C57BL/6 mice aged 6–8 weeks were used in this study (Huafukang Co., Beijing, China) and housed under specific pathogen-free conditions in the animal facility at Chengdu Medical College in accordance with animal care and ethical regulations (No. 20220225138). The DIC model was induced 3 weeks later through a single intraperitoneal injection of DOX (No. S1208, Selleck Chemicals, Houston, Texas, USA) at a dose of 20 mg/kg. To achieve cardiomyocyte-specific overexpression of GSTP1 in vivo, adeno-associated virus serotype 9 (AAV9) vectors encoding GSTP1 (AAV-GSTP1) or negative control (AAV-NC) were administered via tail vein injection at a dose of 2 × 10¹¹ vector genomes per mouse (Genechem Biosciences, Shanghai, China). For rescue experiments, ferrostatin-1 (Fer-1; No. S7243, Selleck Chemicals) was administered intraperitoneally 2 h before DOX injection, followed by daily administration at 2 mg/kg. Mice were euthanized by cervical dislocation for subsequent analysis.

Biacore

A Biacore T200 surface plasmon resonance (SPR) sensor (GE HealthCare, Chicago, USA) was used to measure the binding constant between DOX and GSTP1, following a previously reported method.^[41] Recombinant GSTP1 was diluted to 10 mg/mL using sodium acetate buffer (pH = 4.5) and immobilized on a CM5 chip via amine coupling, achieving approximately 5000 response units (RUs) per flow cell. DOX was diluted in phosphate buffered saline (PBS) at various concentrations and applied to measure response values. Data were analyzed using Biacore Analysis Software 3.0 (GE HealthCare).

Cell culture and treatment

H9c2 and HEK-293T cells were cultured in Dulbecco’s modified Eagle medium (DMEM, HyClone, Utah, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37°C in a humidified incubator with 5% CO₂. For experiments involving DOX or RAS-selective lethal compound 3 (RSL3, Selleck Chemicals) treatment, cells were infected with the indicated adenovirus for 48 h, followed by treatment with DOX or RSL3 for an additional 24 h.

Cell viability analysis

Cell viability was assessed using the Cell Counting Kit-8 (No. B34304, Selleck Chemicals) according to the manufacturer’s instructions. Briefly, cells were seeded in 96-well plates at an appropriate density and incubated overnight. After treatment with the specified agents, the test compound was added to the cell culture for 2 h at room temperature. Absorbance at 450 nm was measured using a plate reader.

Lentivirus infection

Lentiviral vectors encoding GSTP1 (Lenti-GSTP1) or negative control (Lenti-NC) were used to infect H9c2 cells in vitro. All lentiviruses were provided by Genechem Biosciences. Cells were seeded in 10-cm dishes at an appropriate density, incubated overnight, and infected with lentiviruses at a multiplicity of infection (MOI) of 10 for 48 h. Successfully infected cells were selected using puromycin (No. HY-B1743A, MCE, New Jersey, USA) at a concentration of 4 μg/mL. After infection, cells were treated with DOX (5 μmol/L) or Fer-1 at the indicated concentrations for further analysis.

Small interfering RNA (siRNA) transfection

The siRNA sequences for silencing GSTP1 and ACSL4 were synthesized by Ribobio (Guangzhou, China). The siRNA sequences used were listed in the Supplementary Table 1, http://links.lww.com/CM9/C564.

For transfection, Lipo293 reagent (No. C0521, Beyotime, Shanghai, China) was used following the manufacturer’s protocol. Cells were seeded in 10 cm dishes at an appropriate density, incubated overnight, and transfected with siRNAs mixed with Opti-MEM (No. 31985070, Thermo Fisher Scientific, Waltham,USA) for 48 h before analysis.

Echocardiography assessment

Transthoracic echocardiography was performed 14 days after left anterior descending artery (LAD) surgery using the Vevo 3100 ultrasound system (Fujifilm VisualSonics, Ontario, Canada). Mice were anesthetized with 2% isoflurane, maintaining a heart rate of approximately 400 beats per minute (bpm). B-mode cardiac imaging was obtained from parasternal long- and short-axis views, with papillary muscles serving as landmarks for short-axis imaging. M-mode recordings of the left ventricle (LV) were acquired, and data were analyzed using VevoLAB software (Version 5.6.1, Fujifilm VisualSonics, Ontario, Canada) following standardized guidelines.

Histological analysis

Heart tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned into 4 μm slices. Additional slices were subjected to immunofluorescence and immunohistochemical (IHC) staining based on established protocols.^[42,43] Briefly, antigen retrieval was performed under high temperature and pressure for 10 minutes, followed by blocking and incubation with primary antibodies or specific dyes, including JNK (No. 9252S, CST, Danvers, USA), phosphorylated JNK (p-JNK; No. 9251S, CST), 4-Hydroxynonenal (4-HNE, No. bs-6313R, Bioss, Beijing, China), and FerroOrange (No. F374, DOJINDO, Kumamoto Prefecture on Kyushu Island, Japan) at 4°C overnight. Finally, slices were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 minutes at room temperature. Images were captured using an Olympus microscope (Olympus, Tokyo, Japan).

Sirius Red and Masson staining

Heart tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned into 4 μm slices. Selected slices were stained using Masson staining kit (No. G1340, Solarbio, Beijing, China) and Sirius Red staining kit (No.G1472, Solarbio, Beijing, China) respectively, according to the instructions. Images were captured using an Olympus microscope.

RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was isolated from all samples using TRIzol^TM reagent (No. 15596026, Invitrogen, MA, USA). A total of 2 µg of RNA was reverse-transcribed into complementary DNA (cDNA) using the PrimeScript^TM RT reagent kit (No. RR047A, Takara, Kyoto, Japan). Quantitative polymerase chain reaction (qPCR) was subsequently performed using SYBR Green Pro Taq HS (No. AG11733, Accurate Biology, Shenzhen, China), with Gapdh serving as the internal control. The primer sequences used in the study were listed in the Supplementary Table 1, http://links.lww.com/CM9/C564. Data analysis was conducted using the 2^−ΔΔCt method.

Co-immunoprecipitation (Co-IP) and Western blotting

For Co-IP, cell or tissue lysates prepared with IP lysis buffer were incubated with specific primary antibodies or immunoglobulin G (IgG) as a negative control. Protein A/G beads were added to the mixture, which was incubated overnight at 4°C. The beads were subsequently washed and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting, as described previously.^[42,43] For Western blotting, the protein incubated with primary antibodies, including GSTP1 (No. 3369, CST), JNK, p-JNK, GAPDH (No. AC001, ABclonal, Beijing, China), tubulin (No. AC021, ABclonal), ACSL4 (No. 22401-1-AP, Proteintech, Chicago, USA), and IgG (No. AS029, ABclonal). After incubation with appropriate secondary antibodies, protein bands were visualized using a Clinx imaging system (Clinx, Shanghai, China).

Biochemical detection

Following euthanasia via cervical dislocation, serum was collected through eyeball blood sampling. The levels of creatine kinase isoenzymes (CK-MB; No. CSB-E14404m, CUSABIO), lactate dehydrogenase (LDH; No. CSB-E11723m, CUSABIO), lipid peroxidation (malondialdehyde [MDA], No. BC0025, Solarbio), and aspartate aminotransferase (AST; No. CSB-E12649m, CUSABIO) were quantified using the respective kits, according to the manufacturers’ protocols and previous studies.^[28,44]

Iron assay

Intracellular ferrous iron (Fe²⁺) levels were measured using FerroOrange, following the manufacturer’s instructions and previous studies.^[45,46] Briefly, cells were seeded into 24-well plates and incubated overnight. After treatment with the indicated agents, cells were stained with 1 μmol/L FerroOrange for 30 minutes at 37°C under 5% CO₂. Following washing 3 times with PBS, fluorescent images were captured using a microscope (Nikon Microsystems, Tokyo, Japan).

DOX pulldown

DOX was conjugated with epoxy-activated μSphere resin (No. P057A, Sinopae, Wuxi, China) via incubation in radioimmunoprecipitation assay (RIPA) lysis buffer for 12 h at 4°C. The resin-DOX complex was washed three times and subsequently incubated with heart tissue lysates prepared from 7-day-old C57BL/6 mice. The lysates were centrifuged at 4°C (12,000 r/min, r = 10 cm, 10 min), and the supernatant was incubated with resin-DOX at 4°C for 12 h. Differential proteins were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.

Measurement of GSTP1 activity

The enzymatic activity of GSTP1 was determined using GST activity assay kits (No. ab65325, Abcam, Cambridge, England), following the manufacturer’s guidelines and previous reports.^[47,48] GST activity was calculated based on the linear relationship between absorbance and reaction time.

Molecular docking

Molecular docking studies were conducted using the 3D structure of GSTP1 (PDB code: 14GS) and DOX, employing the CB-Dock2 platform for cavity detection-based blind docking (a cavity detection-guided protein–ligand blind docking web server, https://cadd.labshare.cn/cb-dock2/php/blinddock.php#job_list_load).^[49,50] The PDB file of GSTP1 was uploaded to the server, along with the SDF file of DOX, to generate the GSTP1-DOX complex structure in PDB format.

Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay

Apoptosis levels in cardiomyocytes and heart tissues were evaluated using a TUNEL assay kit (No. T2130, Solarbio), following the manufacturer’s instructions and prior studies.^[51] Briefly, cells were treated with DOX or not for 24 h, then fixed in 4% paraformaldehyde and washed twice with PBS. Permeabilization was performed using 0.2% Triton X-100 for 20 minutes. Following this, 50 μL of TUNEL reaction buffer was added and incubated at 37°C for 1 h. After incubation, the reaction buffer was removed, and the samples were washed twice with PBS. Images were captured using a microscope.

Measurement of ROS level

ROS levels were measured using diacetyldichlorofluorescein (DCFH-DA; No. S0033M, Beyotime) staining in cells and DHE (No. ID3560, Solarbio) staining in heart tissues. For DCFH-DA staining, cells were washed with PBS and incubated with DCFH-DA for 30 minutes at 37°C in the dark. After incubation, the cells were washed three times with PBS, and ROS levels were analyzed via flow cytometry (BD Biosciences, East Rutherford, USA). For DHE staining, tissue sections were stained with DHE for 20 minutes at 37°C in the dark, followed by two PBS washes. Fluorescence images were captured using a fluorescence microscope (Olympus).

Lipid ROS assay

Lipid ROS levels were assessed by collecting treated cells, washing them with PBS, and incubating them with BODIPY581/591-C11 (No. D3816, Thermo Fisher Scientific, Waltham, USA) for 20 minutes at 37°C in the dark. The cells were subsequently washed with PBS, resuspended, and analyzed using flow cytometry (BD Biosciences).

Transmission electron microscopy (TEM)

Tissues and cells were immediately immersed in aldehyde fixing solution after treatment. Samples were dehydrated and embedded at room temperature and sectioned into ultrathin slices. These sections were stained with uranyl acetate and lead citrate. Imaging was performed using a transmission electron microscope (Thermo Fisher Scientific, Waltham, Massachusetts, USA).

Statistical analysis

All statistical analyses were performed using GraphPad Prism 10.0 (GraphPad Software, California, USA). Data are presented as mean ± standard error of the mean (SEM). A Student’s t-test was used for comparisons between two groups, whereas one-way analysis of variance (ANOVA) test followed by Dunnett’s multiple comparisons test was used for comparisons among more than two groups. All experiments were conducted at least three times. A P <0.05 was considered statistically significant.

Results

DOX targets GSTP1 and inhibits its activity

To investigate the potential targets of DOX in the pathogenesis of DIC, DOX pulldown assays were performed to identify proteins specifically binding to DOX in murine hearts. GSTP1 was selected for further study owing to its close association with myocardial damage and the established relationship between ROS imbalance and DIC.^[52,53] SDS-PAGE and LC-MS/MS analyses identified that recombinant GSTP1 protein demonstrated direct binding with DOX [Supplementary Figure 1A and B, http://links.lww.com/CM9/C564]. Furthermore, a Biacore assay confirmed the specific binding of GSTP1 with DOX at varying concentrations in vitro [Figure 1A]. A DIC mouse model was established through intraperitoneal administration of DOX. Cardiac systolic and diastolic function exhibited a downward trend in the DOX-treated group [Figure 1B]. Sirius red staining revealed significant cardiac fibrosis in DOX-treated mice compared with the saline control [Figure 1C]. These results confirmed the successful establishment of the DIC model.

Figure 1.

DOX targets GSTP1 and inhibits its activity. (A) Biacore determination of DOX and GSTP1. Affinity/kinetic characterization was performed by concentration gradient injection of DOX. (B and C) Cardiac function was tested by echocardiography (B) and Sirius-red staining (C) in the mouse model of DIC. (D) IHC staining of GSTP1 of heart tissues under DOX treatment or not. (E) Western blotting analysis of GSTP1 in heart tissues in the mouse model of DIC. (F) Western blotting analysis of GSTP1 in H9c2 cells treated with DOX for 24h. (G) Detection of GSTP1 activity in H9c2 cells with DOX. (H) Detection of recombinant GSTP1 activity in vitro with DOX. (I) Molecular docking results of DOX binding sites. The cartoon representation of GSTP1 homodimer, subunit A and B are colored as green and cyan, respectively. The DOX is shown as a brown stick, and the GSH is shown as a purple stick. The detailed interactions between ligands and GSTP1 residues are labeled with blue dashed lines. All data are expressed as mean ± SEM, ^*P <0.001. DIC: DOX-induced cardiomyopathy; DOX: Doxorubicin hydrochloride; GSH: Gutathione; GSTP1: Glutathione S-transferase P1; IHC: Immunohistochemistry; ns: No significance; SEM: Standard error of the mean.

Considering the specific interaction between DOX and GSTP1 in the murine heart, GSTP1 expression was assessed using Western blotting and IHC. No significant changes in GSTP1 levels were observed following DOX treatment in vivo [Figure 1D and E]. Similarly, GSTP1 expression remained unaffected under gradient DOX treatments in H9c2 cells by Western blotting [Figure 1F].

Subsequent analyses showed that DOX significantly inhibited GSTP1 activity in H9c2 cells [Figure 1G]. Recombinant GSTP1 protein exhibited similar results in vitro [Figure 1H]. Treatment with the GSTP1-specific inhibitor, TLK199, confirmed this inhibitory effect, reducing GSTP1 activity both intracellularly (GSTP1 activity of H9c2 cells) and extracellularly (activity of recombinant GSTP1 protein) [Supplementary Figure 1C and D, http://links.lww.com/CM9/C564]. Moreover, TLK199 treatment decreased H9c2 cell viability in a dose-dependent manner [Supplementary Figure 1E, http://links.lww.com/CM9/C564], implying that GSTP1 inhibition contributes to cardiac damage. Although DOX did not alter GSTP1 expression, its binding to GSTP1 and potential impact on DIC warranted further investigation. To assess whether DOX binding impacts GSTP1 activity, DOX was docked into GSTP1 using the CB-Dock2 online server, employing cavity-detection blind docking.^[50,54] The docking model revealed that DOX localized within GSTP1’s cavity and established strong interactions with residues W38, Q51, L52, R100, N204, and G205. In addition, the anthracene moiety of DOX formed a π-π stacking interaction with F8. Comparatively, the structure of GSTP1 bound to GSH (PDB ID: 3GUS) indicated that GSH interacts with residues E9, R13, W38, K44, Q51, Q64, S65, and N66. The overlapping binding regions, including shared residues W38 and Q51, suggest that DOX binding may interfere with GSTP1’s recognition of GSH [Figure 1I].

Overall, these findings reveal that DOX specifically targets GSTP1, disrupts its interaction with GSH, and inhibits its enzymatic activity.

GSTP1 protects against DIC

Since GSTP1 inhibition (via TLK199 treatment) exacerbates cardiomyocyte damage, GSTP1 was hypothesized to play a critical role in mitigating DIC. To elucidate the role of GSTP1 in vivo, AAV9 was used to modulate GSTP1 expression. Overexpression of GSTP1 using AAV9-GSTP1 was confirmed by Western blotting and was unaffected by DOX treatment [Figure 2A].

Figure 2.

GSTP1 protects against DIC. (A) Western blotting analysis of relative expression levels of GSTP1 in different groups of mice. (B) Cardiac function was tested by echocardiography after DOX treatment. (C–F) EF (C), FS (D), heart rate (E), and stroke volume (F) were measured in different groups of mice (all n = 6). (G) Collagen fibrosis of slices from mice heart tissue was visualized by Sirius-red staining (scale bars, 100 μm). (H) Cell viability of heart tissues was detected by TUNEL staining (scale bars, 100 μm). (I) Representative fluorescence microscopy images of zebrafish embryos with green fluorescent protein (EGFP) specifically expressed in the myocardial cells. (Scale bars, 50 μm). All data are expressed as mean ± SEM, ^*P <0.01, ^†P <0.05. AAV: Adeno-associated virus; CTL: Control; DAPI: 4’,6-diamidino-2-phenylindole; DIC: DOX-induced cardiomyopathy; DOX: Doxorubicin hydrochloride; EF: Ejection fraction; FS: Fractional shortening; GSTP1: Glutathione S-transferase P1; SEM: Standard error of the mean; TUNEL:Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.

In the DIC mouse model, DOX administration led to significant declines in ejection fraction (EF), fractional shortening (FS), heart rate (bpm), and stroke volume compared with controls (AAV-NC). These detrimental changes were attenuated by cardiac-specific GSTP1 overexpression [Figure 2B–F]. In addition, Sirius red staining revealed that DOX-induced cardiac fibrosis was alleviated by GSTP1 overexpression [Figure 2G]. TUNEL staining further demonstrated that cardiomyocyte injury caused by DOX was mitigated by GSTP1 overexpression in murine heart tissues [Figure 2H].

Using a previously established model,^[55] transgenic zebrafish embryos overexpressing GSTP1 (cmlc2: GSTP1-HA-p2a-EGFP) were evaluated for DIC. GSTP1 overexpression reduced DOX-induced morphological abnormalities, such as elongation of the heart shape and thinning of atrial walls [Figure 2I].

In vitro experiments further corroborated these findings. H9c2 cells overexpressing GSTP1, generated via lentiviral transduction, exhibited reduced cytotoxicity under DOX treatment compared with control cells (Lenti-NC), as observed through microscopic examination [Supplementary Figure 2A and B, http://links.lww.com/CM9/C564]. DOX-induced cytotoxicity and inhibition of GSTP1 activity were dose-dependent [Supplementary Figure 2C and D, http://links.lww.com/CM9/C564]. Overexpression of GSTP1 alleviated these effects, whereas GSTP1 silencing exacerbated them [Supplementary Figure 2C, E and F, http://links.lww.com/CM9/C564].

In summary, these results indicate that GSTP1 overexpression enhances its activity, mitigating DIC-associated cytotoxicity and cardiac damage.

GSTP1 ameliorates DOX-induced ferroptosis in vitro

Previous studies have indicated that ferroptosis plays a significant role in DIC, and selective inhibitors of ferroptosis markedly improve outcomes following DOX treatment.^[8,56] In order to determine whether GSTP1 is involved in DOX-induced ferroptosis, markers of ferroptosis were evaluated in H9c2 cells following DOX treatment.

Initial findings from cell viability assays demonstrated that GSTP1 overexpression alleviated DOX-induced cytotoxicity [Figure 3A]. Compared with Lenti-NC cells, overexpression of GSTP1 (Lenti-GSTP1) suppressed DOX-induced production of ROS and lipid ROS [Figure 3B–D]. Furthermore, DOX-induced mitochondrial injury was significantly mitigated by GSTP1 overexpression [Figure 3E and F]. FerroOrange staining showed that GSTP1 overexpression reversed the increased Fe²⁺ levels caused by DOX [Figure 3G].

Figure 3.

GSTP1 ameliorates DOX-induced ferroptosis in H9c2 cells. (A) Cell viability was detected by cell counting kit-8. (B and C) ROS (B) and Lipid-ROS (C) were detected with flow cytometry. (D) Microscope detection of BODIPY581/591-C11 staining (scale bars, 20 μm). (E) Representative TEM images of cardiac tissues (scale bars, 1 μm). (F) Representative images of cellular JC-1 fluorescence, green indicates the monomer JC-1, red indicates the aggravated JC-1, and orange indicates merged cells (scale bars, 50 μm). (G) Cellular level of Fe²⁺ was determined by FerroOrange staining (scale bars, 20 μm). (H) Relative PTGS2 mRNA expression level was determined (n = 3). (I) GSH/GSSG ratio was detected. All data are expressed as mean ± SEM, ^*P <0.001, ^†P <0.01. CTL: Control; DOX: Doxorubicin hydrochloride; GSTP1: Glutathione S-transferase P1; GSH/GSSG: Reduced glutathione/oxidized glutathione disulfide; Lenti-GSTP1: Lentiviral vectors encoding GSTP1; Lenti-NC: Lentiviral vectors encoding negative control; ROS: Reactive oxygen species; SEM: Standard error of the mean; TEM: Transmission electron microscopy.

In addition, the elevated expression of PTGS2, a biochemical marker of ferroptosis, induced by DOX was reduced by GSTP1 overexpression [Figure 3H]. The abnormal increase in the total GSH/glutathione disulfide (GSSG) ratio observed in DOX-treated cardiomyocytes was also mitigated by GSTP1 overexpression [Figure 3I].

To further elucidate the role of GSTP1 in ferroptosis, GSTP1 expression was silenced in H9c2 cells using siRNA, and ferroptosis markers were subsequently assessed. Silencing GSTP1 exacerbated DOX-induced cytotoxicity and ferroptosis [Supplementary Figure 3, http://links.lww.com/CM9/C564]. Similar results were observed when H9c2 cells were treated with the GSTP1 inhibitor TLK199, which intensified ferroptosis markers in a manner consistent with GSTP1 silencing [Supplementary Figure 4, http://links.lww.com/CM9/C564].

These findings collectively indicate that GSTP1 is a critical mediator of ferroptosis, attenuating DOX-induced cardiomyocyte injury.

GSTP1 relieved RSL3-induced ferroptosis and is a potential mediator of ferroptosis

To further investigate whether GSTP1 regulates ferroptosis, various parameters of ferroptosis were evaluated in H9c2 cells. Treatment with RSL3, a ferroptosis activator, resulted in a marked reduction in cell survival. Interestingly, GSTP1 overexpression mitigated this reduction, while GSTP1 silencing exacerbated it, suggesting GSTP1’s involvement in ferroptosis [Figure 4A].

Figure 4.

Overexpression of GSTP1 inhibits RSL3-induced ferroptosis in H9c2 cells. (A) Cell viability was detected. (B and C) The ROS (B) and lipid ROS (C) level was determined with flow cytometry. (D) Ptgs2 mRNA expression level was detected. (E and F) MDA (E) and GSH/GSSG ratio (F) were detected. (G) Representative images of cellular JC-1 fluorescence in H9c2 cells, in which green indicates the monomer JC-1, red indicates the aggravated JC-1, and orange indicates merged cells (scale bars, 50 μm). (H) Cellular level of Fe²⁺ was determined by FerroOrange staining (scale bars, 20 μm). (I) Representative TEM images of H9c2 cells were obtained (scale bars, 1 μm). All data are expressed as mean ± SEM, ^*P <0.001. GSH/GSSG: Reduced glutathione/oxidized glutathione disulfide; GSTP1: Glutathione S-transferase P1; Lenti-GSTP1: Lentiviral vectors encoding GSTP1; Lenti-NC: Lentiviral vectors encoding negative control; Lipid-ROS: Lipid reactive oxygen species; MDA: Malondialdehyde; Ptgs2: Prostaglandin-endoperoxide synthase 2; ROS: reactive oxygen species; SEM: Standard error of the mean; TEM: Transmission electron microscopy.

RSL3-induced production of ROS and lipid ROS, elevated PTGS2 expression, increased malondialdehyde (MDA) levels, and a higher GSH/GSSG ratio were all alleviated by GSTP1 overexpression [Figure 4B–F]. Immunofluorescence analysis and TEM were performed to assess mitochondrial damage caused by RSL3. Severe mitochondrial injury and elevated Fe²⁺ levels induced by RSL3 were attenuated by GSTP1 overexpression [Figure 4G–I].

To validate these findings in a different model, the same parameters were examined in 293T cells. Results consistent with those in H9c2 cells confirmed GSTP1’s role in ferroptosis regulation [Supplementary Figure 5, http://links.lww.com/CM9/C564]. These findings suggest that GSTP1 may function similarly to GPX4, a key regulator of ferroptosis.

Overall, GSTP1 overexpression alleviated RSL3-induced ferroptosis, highlighting its potential role as a key mediator of ferroptosis.

Overexpression of GSTPP1 and Fer-1 administration improved DIC

To determine whether GSTP1-mediated protection against DIC is ferroptosis-dependent, Fer-1 was administered prior to DOX treatment in vivo. Both GSTP1 overexpression and Fer-1 administration significantly improved survival rates [Figure 5A] and alleviated myocardial damage [Figure 5B, Supplementary Figure 7D and E, http://links.lww.com/CM9/C564], coupled with enhanced cardiac performance. This included lower serum levels of LDH, CK-MB, and AST, as well as improved heart weight and tibia length (TL)/body weight ratios [Supplementary Figure 6A–C, Supplementary Figure 7A–F, http://links.lww.com/CM9/C564].

Figure 5.

Overexpression of GSTP1 and Fer-1 administration improved DIC in vivo. (A) Kaplan–Meier survival curves were profiled in the process of DIC model over 14 days. (B) Representative HE staining images of heart tissues. Scale bar, 1000 μm (up) and 100 μm (below). (C) Representative DHE staining images for detecting ROS level of heart tissues (scale bar, 50 μm, n = 6). (D) IHC staining of 4-HNE for detecting lipid-ROS in heart tissues (scale bar, 50 μm, n = 6). (E) Representative TEM images of cardiac tissues (scale bars, 1 μm). (F) Relative Ptgs2 mRNA expression level was determined (n = 3). (G and H) Quantitative analysis of serum MDA (G), and cardiac MDA (H) levels were measured (n = 6). (I) Immunoblot analysis of 4-HNE in heart tissues. All data are expressed as mean ± SEM, ^*P <0.05, ^†P <0.01. AAV: Adeno-associated virus; DIC: DOX-induced cardiomyopathy; DHE: Dihydroethidium; DOX: Doxorubicin hydrochloride; GSTP1: Glutathione S-transferase P1; IHC: Immunohistochemistry; MDA: Malondialdehyde; NC: Negative control; Ptgs2: Prostaglandin-Endoperoxide Synthase 2; qPCR: Quantitative polymerase chain reaction; ROS: Reactive oxygen species; SEM: Standard error of the mean; 4-HNE: 4-Hydroxynonenal.

Increased ROS levels, aberrant 4-HNE expression, mitochondrial damage (murine hearts), reduced Ptgs2 mRNA expression, and increased MDA production (serum and cardiac tissue) caused by DOX were attenuated by GSTP1 overexpression and Fer-1 administration [Figure 5C–I], mirroring the protective effects of GSTP1 overexpression.

Collectively, these findings demonstrate that GSTP1 overexpression and Fer-1 administration confer significant protective effects against DIC, underscoring the essential role of GSTP1 in ferroptosis-mediated cardio-protection.

DOX inhibits GSTP1-JNK binding and further induces ferroptosis

It has been reported that GSTP1 interacts with JNK to suppress its phosphorylation, thereby inhibiting the JNK signaling pathway. Under oxidative stress, the GSTP1-JNK complex dissociates, leading to downstream activation and induction of apoptosis.^{[56,57,58,59]} Furthermore, inhibition of GSTP1 is associated with increased JNK activity in pancreatic ductal adenocarcinoma (PDAC) cells.^[60] Based on these findings, Co-IP assays were performed to examine the interaction between GSTP1 and JNK.

Results confirmed that GSTP1 binds specifically to JNK irrespective of DOX treatment, although DOX treatment led to the downregulation of JNK expression [Figure 6A]. Under varying concentrations of DOX, GSTP1 expression remained unchanged, whereas p-JNK levels increased significantly, suggesting that DOX activates JNK phosphorylation [Figure 6B]. Immunohistochemical staining of JNK and p-JNK in murine heart tissue yielded consistent results, further demonstrating that DOX inhibits GSTP1 activity and disrupts its binding to JNK, thereby activating JNK phosphorylation [Figure 6C].

Figure 6.

DOX inhibits GSTP1–JNK binding and further induces ferroptosis. (A) Co-IP and Western blotting analysis of GSTP1 binding with JNK in H9c2 cells treated with DOX or not. (B) Expression levels of JNK, p-JNK, and GAPDH were determined by western blotting in H9c2 cells. (C) IHC staining of JNK and p-JNK in heart tissues after DOX treatment (scale bar, 50 μm, n = 6). (D–G) Cell viability (D), ROS (E), Lipid-ROS (F), and MDA level (G) were detected. (H) Representative images of cellular JC-1 fluorescence in H9c2 cells (scale bar, 50 μm). Green indicates the monomer JC-1, red indicates the aggravated JC-1, and orange indicates merged cells (scale bars, 50 μm). (I) Cellular level of Fe²⁺ was determined by FerroOrange staining, and positive cells exhibited orange (scale bars, 20 μm). (J) Representative TEM images of H9c2 cells were obtained (scale bars, 1 μm). (K) Western blotting analysis of GSTP1, JNK, and p-JNK after Anisomycin and SP600126 treatment in H9c2 cells overexpressing GSTP1 or not. All data are expressed as mean ± SEM, ^*P <0.001. CTL: Control; DIC: DOX-induced cardiomyopathy; DOX: Doxorubicin hydrochloride; GSTP1: Glutathione S-transferase P1; JNK: c-Jun N-terminal kinase; Lipid-ROS: Lipid reactive oxygen species; MDA: Malondialdehyde; NC: Negative control; p-JNK: Phosphorylated JNK; ROS: reactive oxygen species; SEM: Standard error of the mean; TEM: Transmission electron microscopy.

Subsequent experiments evaluated whether the GSTP1/JNK signaling pathway mediates ferroptosis in DIC. The JNK inhibitor alleviated DOX-induced ferroptosis, as evidenced by cell viability assays showing that anisomycin (a JNK agonist) induced cytotoxicity comparable to DOX, whereas JNK inhibition and GSTP1 overexpression restored cell viability [Figure 6D]. Furthermore, DOX and anisomycin increased ROS and lipid ROS levels, which were reduced by JNK inhibition and GSTP1 overexpression [Figure 6E, F, and G].

Additional ferroptosis markers, including mitochondrial damage and Fe²⁺ overload, were assessed. GSTP1 overexpression, combined with JNK inhibition, ameliorated severe mitochondrial damage and Fe²⁺ accumulation induced by DOX and anisomycin [Figure 6H–J].

To explore the mechanism further, DOX-treated cells were exposed to anisomycin and SP600125 (a JNK inhibitor). As anticipated, anisomycin enhanced JNK phosphorylation, whereas SP600125 inhibited it, even in the presence of DOX pretreatment [Figure 6K]. In addition, GSTP1 overexpression attenuated JNK phosphorylation induced by DOX and anisomycin [Supplementary Figure 8A and B, http://links.lww.com/CM9/C564]. These findings suggest that DOX disrupts GSTP1-JNK binding, thereby promoting p-JNK activation.

These results collectively indicate that DOX targets GSTP1, activating the JNK signaling pathway to promote ferroptosis in DIC. However, these effects can be reversed by GSTP1 overexpression.

DOX activates ACSL4-dependent ferroptosis by GSTP1/JNK axis

Given the potential regulatory role of GSTP1 in ferroptosis, the specific involvement of GSTP1 in DOX-induced ferroptosis was further investigated. ACSL4, a lipid metabolism enzyme essential for lipid peroxidation,^[61] has been identified as a critical mediator of ferroptosis in DIC.^[28] In this study, ACSL4 expression was significantly elevated following DOX treatment, while GSTP1 overexpression suppressed this induction [Figure 7A]. siRNA-mediated silencing of ACSL4 (using siRNA #3) effectively reduced DOX-induced ferroptosis [Figure 7B–K], indicating that GSTP1 may mediate ACSL4-dependent ferroptosis.

Figure 7.

DOX binds to GSTP1 to activate JNK, and induces ACSL4-dependent ferroptosis in H9c2 cells. (A) Western blotting analysis of ACSL4. (B) Western blotting analysis of ACSL4 with 3 different siRNAs. (C–K) based on the silence of ACSL4, a series of markers of ferroptosis were detected including cell viability (C), ROS (D), Lipid-ROS (E), MDA level (F), JC-1 staining (G), Lipofluo (H), MitoTracker (I), FerroOrange (J), and GSH/GSSG ratio (K). (L) Western blotting analysis of ACSL4, JNK, and p-JNK. All data are expressed as mean ± SEM, ^*P <0.01, ^†P <0.001. ACSL4: Acyl-CoA synthetase long chain family member 4; CTL: Control; DOX: Doxorubicin hydrochloride; GSTP1: Glutathione S-transferase P1; GSH/GSSG: Gutathione/glutathione disulfide; JNK: c-Jun N-terminal kinase; Lipid-ROS: Lipid reactive oxygen species; MDA: Malondialdehyde; NC: Negative control; p-JNK: Phosphorylated JNK; ROS: reactive oxygen species; SEM: Standard error of the mean; TEM: Transmission electron microscopy.

To further elucidate this mechanism, H9c2 cells overexpressing ACSL4 were generated on a GSTP1-overexpression background [Supplementary Figure 9A, http://links.lww.com/CM9/C564]. Overexpression of ACSL4 reversed the protective effects of GSTP1 on DOX-induced ferroptosis, restoring ferroptosis markers such as elevated ROS, lipid ROS, and altered GSH/GSSG ratios [Supplementary Figure 9B–I, http://links.lww.com/CM9/C564].

The role of the JNK signaling pathway in regulating ACSL4-dependent ferroptosis was also examined using a JNK activator and inhibitor. Activation of the JNK signaling pathway was associated with increased ACSL4 expression following DOX treatment. Conversely, GSTP1 overexpression inhibited both the JNK signaling pathway and ACSL4 expression [Figure 7L]. These findings suggest that DOX activates ACSL4 via the JNK signaling pathway, whereas GSTP1 overexpression suppresses JNK activity and, consequently, ACSL4 activation under DOX treatment.

In summary, these results demonstrate that DOX disrupts GSTP1 binding, thereby activating JNK and promoting ACSL4-dependent ferroptosis.

Discussion

In this study, we identified a novel role of GSTP1 in ferroptosis-mediated DIC. DOX specifically targets GSTP1, significantly inhibiting its activity both extracellularly and intracellularly, without altering its expression levels. Cardiac overexpression of GSTP1 alleviated DIC by regulating ferroptosis, whereas GSTP1 silencing exacerbated DIC. Furthermore, a selective ferroptosis inhibitor enhanced the protective effect of GSTP1 against DIC. Mechanistically, DOX inhibits GSTP1 activity, reducing its interaction with JNK. This reduction leads to increased phosphorylation of JNK, ultimately activating ACSL4-dependent ferroptosis. These findings underscore the critical role of the DOX/GSTP1/JNK axis in ACSL4-dependent ferroptosis during DIC.

DOX, a widely used antitumor drug, exhibits dose-dependent cardiotoxicity, often resulting in severe cardiac dysfunction. The most widely accepted mechanism of DIC involves DOX accumulation in mitochondria, leading to ROS production and subsequent cell death.^[44,62] However, the identification of specific therapeutic targets and effective strategies to counteract DIC remains a challenge. In this study, we demonstrated through Biacore assays that DOX directly targets GSTP1. Our findings demonstrated that GSTP1 overexpression correlated with reduced ROS levels and enhanced cell survival following DOX treatment. In a murine DIC model, AAV-mediated cardiac GSTP1 overexpression decreased ROS levels, mitigated cardiac injury, and improved cardiac function. Collectively, these results reveal that DOX binds to GSTP1, and GSTP1 overexpression attenuates DOX-induced cardiac dysfunction.

Recent studies have highlighted the involvement of GSTP1 in redox regulation,^[63,64,65] including findings of GSTP1 downregulation via ubiquitination during the early stages of ferroptosis and its inhibitory effect on ferroptosis in the context of immune checkpoint inhibitors.^[66] However, no prior research has elucidated the connection between DOX and GSTP1 or identified downstream pathways involved in GSTP1-mediated ferroptosis.

Given the observation that DOX targets GSTP1 in DIC, and ferroptosis has been implicated in the development of DIC,^[8] we propose that GSTP1 regulates ferroptosis, a recently identified form of cell death characterized by lipid peroxidation.^[15] Ferroptosis has been linked to various diseases, including acute kidney injury and cancer cell death. In this study, we showed that DOX caused significant disruptions in ROS, lipid ROS, Fe²⁺, GSH, mitochondrial integrity, and MDA levels. Notably, these disruptions were alleviated by GSTP1 overexpression but exacerbated by GSTP1 silencing. These findings support the conclusion that GSTP1 overexpression inhibits DIC by modulating ferroptosis, similar to the function of GPX4 in ferroptosis.

To further elucidate the role of GSTP1 in ferroptosis, we examined the interaction between GSTP1 and JNK. Previous studies have reported a direct interaction between GSTP1 and JNK in the regulation of kinase pathways.^[57,67] For example, sunitinib has been shown to induce pericyte death by inhibiting the GSTP1/JNK/autophagy pathway in myocardial tissue.^[68] In this study, we demonstrated that this signaling pathway operates in DOX-induced ferroptosis. Under DOX treatment, GSTP1 inhibition promoted JNK phosphorylation, leading to the activation of ferroptosis markers, including increased ROS, lipid ROS, and MDA levels, as well as reduced GSH levels. In addition, DOX activated the JNK signaling pathway, inducing ACSL4-dependent ferroptosis, while GSTP1 overexpression suppressed this process. Overall, our findings establish the existence of a cardiac DOX/GSTP1/JNK axis regulating ACSL4-dependent ferroptosis in DIC.

Several limitations of this study should be acknowledged. First, although AAV9-mediated GSTP1 overexpression demonstrated a protective effect against DIC in a murine DIC model, GSTP1-knockout mice were not used to confirm whether GSTP1 deficiency exacerbates DIC. Nevertheless, cellular experiments indicated that GSTP1 silencing increased DOX-induced myocardial toxicity in H9c2 cells. Second, this study focused exclusively on ferroptosis-mediated DIC, without an in-depth investigation of other forms of programmed cell death. Third, while a ferroptosis inhibitor (Fer-1) was used to alleviate DIC, other inhibitors were not evaluated to determine their potential therapeutic effects on DIC. These limitations warrant further investigation in future studies.

In conclusion, this study uncovered a novel role of the DOX/GSTP1/JNK axis in the regulation of ACSL4-dependent ferroptosis in DIC. Specifically, GSTP1 mitigates ACSL4-dependent ferroptosis through inhibition of the JNK pathway, thereby alleviating DIC. These findings advance the understanding of the molecular mechanisms underlying DOX-induced ferroptosis and suggest GSTP1 as a potential biomarker or therapeutic target for the clinical management of DIC.

Funding

This study was supported by grants from the National Natural Science Foundation of China (No. 81902786), the Fundamental Research Funds for Central Universities (No. 2022SCU12032), and the Scientific Research Foundation of Science & Technology Department of Sichuan Province (No. 2018JY0208).

Conflicts of interest

None.