Abstract

Background

   Doxorubicin (DOX), a classical chemotherapeutic agent, faces
   significant limitations because of its well-documented risk of inducing
   cardiotoxicity. Effective prevention of DOX-induced cardiotoxicity is
   urgently needed. Given that alterations in metabolic pathways have been
   observed in both cardiovascular diseases and cancer, targeting specific
   metabolic pathways may offer dual benefits by mitigating DOX-induced
   cardiotoxicity while simultaneously enhancing its antitumor efficacy.

Objectives

   This study sought to explore the characteristic metabolic alterations
   associated with early DOX-induced cardiotoxicity and identify a
   therapeutic target that simultaneously inhibits cancer and protects the
   myocardium.

Methods

   Metabolomic and transcriptomic analyses were performed on heart tissues
   from murine models of DOX-induced cardiotoxicity to identify the most
   significantly altered metabolic pathway. The most altered metabolite
   involved in the candidate pathway was chosen and verified in both mice
   and humans. The protective effects of the chosen metabolite against
   DOX-induced cardiotoxicity and its antitumor effects were evaluated.
   Potential mechanisms were explored using C57BL/6J mice, OPLAH global
   knockout mice, BALB/c nude mice and NSG mice.

Results

   The glutathione metabolic pathway was identified as the most altered
   pathway in heart tissues with DOX-induced cardiotoxicity. The
   5-oxoproline/OPLAH (5-oxoprolinase) axis was the most critical node.
   Downregulation of 5-oxoproline was observed in serum samples from both
   humans and mice. Exogenous 5-oxoproline supplementation effectively
   restored myocardial 5-oxoproline levels, subsequently mitigating
   DOX-induced cardiac dysfunction. Interestingly, we observed an
   additional inhibitory effect of 5-oxoproline on tumor proliferation in
   tumor-bearing mice treated with DOX. Mechanistically, 5-oxoproline
   exerts its cardioprotective effects by restoring glutathione metabolic
   homeostasis through the modulation of its downstream enzyme OPLAH while
   simultaneously suppressing tumor proliferation by inhibiting its
   upstream enzyme, gamma-glutamyl cyclotransferase (GGCT).

Conclusions

   This study reveals a previously unrecognized dual role of 5-oxoproline,
   which functions both as an early biomarker for DOX-induced
   cardiotoxicity detection and as a therapeutic target that
   simultaneously inhibits cancer growth and protects the myocardium.

   Keywords: Cardio-oncology, 5-oxoproline, Cardiotoxicity, Heart failure,
   Doxorubicin, Breast cancer

Highlights

     * •
       5-oxoproline is a promising prevention of DOX-induced
       cardiotoxicity.
     * •
       5-oxoproline supplement may serve as an adjuvant treatment for
       tumors.
     * •
       5-oxoproline is potential for early detection of DOX-induced
       cardiotoxicity.

1. Introduction

   Cardiovascular mortality has emerged as the leading cause of
   noncancer-related death among cancer patients [[47]1]. The emergence of
   cardio-oncology has increased interdisciplinary collaboration between
   oncologists and cardiologists, driving rapid advancements in preventing
   the side effects of antitumor chemotherapies. This field aims to
   improve patient prognosis through early detection, monitoring and
   treatment of cardiotoxicity.

   Doxorubicin (DOX), a classical anthracycline antibiotic, is widely used
   because of its potent antitumor effects [[48]2]. However, its clinical
   application is substantially limited by dose-dependent cardiotoxicity,
   known as DOX-induced cardiotoxicity, which leads to irreversible
   cardiac damage following cardiac fibrosis development. Early
   intervention in DOX-induced cardiotoxicity, even in asymptomatic
   patients, is crucial for preventing cardiac dysfunction and reducing
   adverse cardiac events [[49]3]. Dexrazoxane is currently the only
   FDA-approved agent for preventing DOX-induced cardiotoxicity. However,
   its use is limited by an elevated risk of secondary malignancies
   [[50]4]. Consequently, exploring early biomarkers of DOX-induced
   cardiotoxicity and developing preventive strategies to mitigate
   DOX-induced cardiotoxicity without compromising antitumor efficacy are
   urgently needed.

   Protein inhibitors, such as THZ1 and TP-10, as well as gene mutations
   in PI3Kγ and Mfn2, have shown promising cardioprotective and
   antineoplastic effects during DOX chemotherapy [[51][5], [52][6],
   [53][7], [54][8]]. However, these inhibitors are exogenous compounds
   rather than endogenous substances within the body. In contrast,
   biologically active metabolites are closely linked to human disease
   phenotypes, and they play crucial roles in regulating
   pathophysiological functions, such as anti-inflammatory, antioxidant,
   and metabolic regulation [[55][9], [56][10], [57][11]]. Various
   research groups, including ours, have reported that small-molecule
   metabolites have potential for the prevention and treatment of
   cardiovascular diseases [[58][12], [59][13], [60][14]]. Nonetheless,
   evidence of the therapeutic value of metabolites in DOX-induced
   cardiotoxicity remains scarce. Moreover, no studies have reported the
   dual effects of metabolite supplementation on both cardiac and tumor
   tissues during DOX therapy. This approach may represent a potential
   adjunctive therapy for cancers requiring anthracycline-based
   chemotherapy.

   In the present study, we explored metabolic alterations associated with
   early DOX-induced cardiotoxicity and identified therapeutic targets
   that simultaneously inhibit cancer progression and protect the
   myocardium. We found that glutathione synthesis was disrupted in early
   DOX-induced cardiotoxicity and identified 5-oxoproline as a biomarker
   for this condition. 5-Oxoproline exhibits antitumor effects while
   concurrently protecting the heart from DOX-induced cardiotoxicity.
   Mechanistically, 5-oxoproline exerts cardioprotective effects by
   enhancing glutathione synthesis via 5-oxoprolinase (OPLAH) and
   suppresses tumor growth through the downregulation of gamma-glutamyl
   cyclotransferase (GGCT) expression.

2. Methods

   The Supplemental Methods and Materials include details on the following
   methods: DOX-induced cardiotoxicity models and tumor models; culture
   and treatment of neonatal rat cardiomyocytes (NRCMs) and cancer cells;
   in vivo imaging analysis; histological analysis; terminal
   deoxynucleotidyl transferase dUTP nick-end labeling staining; seahorse
   respirometry; transmission electron microscopy; mitochondrial assays;
   C11 and DHE staining; detection of cell proliferation; measurement of
   MDA and GSH/GSSG; RNA sequencing; metabolomic profiling; 5-oxoproline
   analysis; liquid chromatography‒tandem mass spectrometry (LC‒MS);
   Western blot analysis; real-time quantitative polymerase chain reaction
   (RT‒qPCR); and ELISA.

3. Human studies

   All human samples used in this study were obtained according to
   protocols approved by the institutional review board of Shanghai Jiao
   Tong University (KY2023-070-C). Written informed consent was obtained
   from all participants, with approval from the Ethics Committee of Renji
   Hospital, School of Medicine, Shanghai Jiao Tong University. The study
   adhered to the guidelines of the Declaration of Helsinki (2013).

   Patients were included in this study if they met all the following
   criteria [[61]1]: female patients aged between 18 and 70 years [[62]2];
   newly diagnosed with histologically confirmed breast cancer (stage
   I-IIIC) and scheduled to receive chemotherapy on the basis of
   evaluation by breast specialists [[63]3]; absence of cardiac symptoms
   or discomfort [[64]4]; no documented heart diseases [[65]5]; normal
   range of cardiac biomarkers, including troponin I (TnI) and brain
   natriuretic peptide (BNP), and left ventricular ejection fraction
   (LVEF) ≥ 50 % as determined on echocardiography [[66]6]; ability to
   complete face‒to-face follow-up visits; and [[67]7] willingness to
   provide written informed consent.

   The exclusion criteria were as follows [[68]1]: prior history of
   radiotherapy [[69]2]; another concurrent tumor [[70]3]; cardiovascular
   disease identified during screening at enrollment [[71]4]; uncontrolled
   blood pressure [[72]5]; severe chronic or acute renal failure
   (glomerular filtration rate <30 ml/min/1.73 m^2) [[73]6]; severe liver
   disease [[74]7]; history of immunodeficiency or organ transplantation;
   or [[75]8] any other medical condition assessed by the investigator as
   inappropriate for participation.

   One hundred eighteen plasma samples were collected from September 10,
   2023, to July 15, 2024. Clinical data, including age, sex, history of
   hypertension, diabetes mellitus, hyperlipidemia, and current smoking,
   were collected. All patients received a predefined treatment protocol
   consisting of doxorubicin (80 mg/m^2) in 4 cycles, administered every 3
   weeks. Echocardiography was performed at baseline of DOX therapy and 3
   weeks after the final cycle. Plasma samples were separated by
   centrifugation at 400×g for 10 min at 4 °C and stored at −80 °C until
   further analysis.

4. Animal studies

   For the animal experiments, age-, sex-, and genetic background-matched
   mice were randomly separated into the indicated groups. All animal
   experiments were conducted in accordance with the National Institutes
   of Health Guidelines on the Care and Use of Laboratory Animals (NIH
   Publication, 8th Edition, 2011) and were approved by the Animal Ethics
   Committee of Renji Hospital (RJ 2023-148A). All mice were housed under
   specific pathogen-free conditions in temperature- and
   humidity-controlled rooms with a 12-h light/dark cycle and provided ad
   libitum access to food and water.

   Chronic Protocol: For the DOX-induced cardiac dysfunction model,
   8-week-old male and female C57BL/6J mice received DOX (5 mg/kg) on days
   0, 7, 14, and 21 [[76]15]. For the preventive test, three days before
   DOX treatment, mice were pretreated with 0.05 mg/kg/day 5-oxoproline or
   the same volume of vehicle (saline solution) via intraperitoneal
   injection and then continuously received the same treatment for another
   28 days. For the therapeutic test, 0.05 mg/kg/day 5-oxoproline or the
   same volume of saline was intraperitoneally injected for another 4
   weeks after 4 weeks of DOX treatment. Acute Protocol: In the preventive
   protocol, mice received 5-oxoproline (0.05 mg/kg/day, i.p.) or vehicle
   for 3 days prior to DOX administration, and treatment continued for an
   additional 7 days. DOX (15 mg/kg, i.p.) was administered as a single
   bolus to induce cardiotoxicity. In the rescue protocol, DOX (15 mg/kg,
   i.p.) was administered first, followed by 5-oxoproline treatment
   starting on day 7 and continuing for 7 consecutive days. For the
   syngeneic breast tumor model, 4T1 cells (1 × 10^6) mixed with 100 μL of
   Matrigel matrix were subcutaneously injected into female BALB/c nude
   mice aged 8 weeks. For the xenograft assays, 2 × 10^6 MDA-MB-231 cells
   were transplanted subcutaneously into the flanks of NSG mice aged 6
   weeks. When the tumor volume reached 50–100 mm^3, the animals were
   randomly divided into 4 groups. DOX (5 mg/kg/week) and 5-oxoproline
   (0.05 mg/kg/day) were administered intraperitoneally as described
   above. Tumor size was measured twice a week using a caliper and was
   calculated according to formula (L × W^2)/2, with L and W being the
   longest and shortest tumor diameters, respectively, in millimeters.
   Cardiac function was evaluated via echocardiography 1 week after the
   last DOX administration. Images of the tumor were taken, and the final
   tumor weight was measured and recorded. To explore the mechanisms
   underlying DOX-induced cardiac dysfunction, male and female C57BL/6J
   OPLAH^+/+ or OPLAH^−/− mice aged 8 weeks were treated with DOX
   (5 mg/kg/week) and 5-oxoproline (0.05 mg/kg/day) as described above.
   Global OPLAH knockout (OPLAH^−/−) mice on a C57BL/6J background were
   generated using the clustered regularly interspaced short palindromic
   repeat/CRISPR-associated nuclease 9 (CRISPR/Cas9) system at Cyagen
   Biosciences, Inc. (China). The details are described in the
   Supplemental Material. OPLAH^+/+ littermates of the same age and sex
   were used as controls.

5. Statistical analysis

   The exact sample size of each group is listed in [77]Supplementary
   Table S1 or mentioned in the corresponding figure legends. Continuous
   variables are expressed as the means ± SEMs for parametric data or
   medians (interquartile ranges) for nonparametric data. Categorical
   variables are presented as counts and percentages. The normality of
   data distributions was assessed using the Shapiro‒Wilk test. For
   two-group comparisons, Levene's test was performed to assess the
   equality of variances. Normally distributed data were compared using
   the Student's t-test (equal variances) or Welch's t-test (unequal
   variances), whereas nonnormally distributed data were analyzed using
   the Mann‒Whitney U test. For comparisons involving more than two
   groups, homogeneity of variance was assessed with the Brown-Forsythe
   test. Normally distributed data were analyzed using one-way or two-way
   ANOVA, followed by Tukey's, Šídák's, or Bonferroni's post hoc
   correction, as appropriate [[78]12]. Multiway ANOVA was used when more
   than two independent variables were included, with Holm‒Šídák post hoc
   testing for pairwise comparisons. For nonparametric data, the
   Kruskal‒Wallis test was applied. For paired comparisons in human
   anthracycline-treated patients, the Wilcoxon matched-pairs signed-rank
   test was used. Correlations between continuous variables were assessed
   using Pearson's correlation analysis. Survival analyses were performed
   using the Kaplan‒Meier method, and the results were compared by
   log-rank tests. All statistical analyses were conducted using GraphPad
   Prism version 8.0 (GraphPad Software Inc., San Diego, CA, USA). A
   two-sided P value < 0.05 was considered to indicate a statistically
   significant difference.

6. Results

6.1. Glutathione metabolism is dysregulated in early DOX-induced
cardiotoxicity

   To identify candidate therapeutic targets, we established a standard
   DOX-induced cardiac dysfunction mouse model and monitored dynamic
   cardiac functions ([79]Fig. 1A and [80]Figure S1, A-B). Since early
   detection of cardiac dysfunction is imperative, we used the global
   longitudinal strain (GLS) as an indicator of early cardiotoxicity in
   accordance with the European Society of Cardiology recommendation
   [[81]16]. One week after DOX administration was set as the early phase
   of myocardial impairment [[82]17], based on significant GLS reduction
   ([83]Fig. 1B) in the absence of alterations in traditional systolic
   function parameters, including the LVEF and left ventricular
   end-systolic diameter ([84]Figure S1, C-D).

Fig. 1.

   [85]Fig. 1
   [86]Open in a new tab

   Glutathione metabolism is dysregulated, and 5-oxoproline is decreased
   in the early phase of DOX-induced cardiotoxicity. A. Schematic
   protocol: Wild-type mice were treated with 4 rounds (once per week) of
   intraperitoneal DOX (5 mg/kg). Hearts were collected 24 h after the
   second administration of DOX for analysis of the early stage of
   DOX-induced cardiotoxicity. B. Global longitudinal strain and the
   global longitudinal strain rate were measured at 1 week and 4 weeks;
   n = 7. C. Volcano plot revealing the genes with altered expression in
   the control group and the group treated with DOX for 1 week (DOX-1W);
   n = 3. D. Volcano plot for Gene Ontology (GO) analysis of
   differentially expressed genes (DEGs). E. The top 10 Kyoto Encyclopedia
   Genes and Genomes (KEGG) pathways of the DEGs. F. Heatmap of altered
   cardiac tissue metabolites in the control group and DOX-1W group;
   n = 3. G. The top 15 KEGG pathways of the differentially expressed
   metabolites. H. Multiomic joint pathway analysis of transcriptomes and
   metabolomes. The highest-ranked pathway was glutathione metabolism. I.
   Schematic illustration of metabolites and enzymes involved in
   glutathione metabolism. J. 5-Oxoproline levels in the serum and heart
   tissues of the control and DOX-1W groups determined by liquid
   chromatography–mass spectrometry/MS analysis; n = 6. K. Association
   between 5-oxoproline (plasma and myocardium) and global longitudinal
   strain. Each symbol represents a single analysis; n = 12. The data are
   presented as the means ± SEMs. B. One-way ANOVA followed by
   Bonferroni's multiple comparison test. J. Student's t-test. K. Pearson
   correlation analysis.

   To investigate the early myocardial impairment induced by DOX, we
   performed bulk RNA sequencing and metabolomic analyses on heart samples
   from wild-type mice treated with either DOX or vehicle for 1 week
   [[87]17]. The differentially expressed genes (DEGs) identified between
   the two groups ([88]Fig. 1C) demonstrated that the response to
   oxidative stress represented the most significantly altered biological
   process according to Gene Ontology ([89]Fig. 1D). Glutathione
   metabolism was among the top 10 enriched pathways according to Kyoto
   Encyclopedia of Genes and Genomes (KEGG) analysis ([90]Fig. 1E).
   Furthermore, differentially expressed metabolites were confirmed in
   cardiac tissues between the two groups by metabolomic analyses. KEGG
   pathway enrichment analysis revealed that several biological pathways
   were significantly affected by DOX treatment, with glutathione
   metabolism emerging as the predominant altered pathway ([91]Fig. 1F–G).
   To further explore the regulatory pathway, we performed a combined
   analysis of transcriptomes (differentially expressed genes,
   Benjamini‒Hochberg adjusted P < 0.05 and |log2[foldchange]|≥1) and
   metabolomics (metabolites, variable importance in projection >2 using
   orthogonal partial least squares‒discriminant analysis) with the
   MetaboAnalyst online tool ([92]https://www.metaboanalyst.ca) [[93]18].
   As shown in [94]Fig. 1H, the glutathione metabolism pathway ranked as
   the top pathway according to the joint pathway analysis. These findings
   suggest that the modulation of glutathione metabolism might play a
   critical role in the pathogenesis of DOX-induced cardiotoxicity.

6.2. 5-Oxoproline declines in the early phase of DOX therapy

   The main regulators contributing to this DOX-induced glutathione
   metabolism pathway alteration were further explored. 5-Oxoproline and
   its associated catabolic enzyme, OPLAH ([95]Fig. 1I), were identified
   as the candidate metabolite and gene, respectively, owing to their most
   pronounced alterations. The levels of 5-oxoproline were evaluated by
   targeted metabolic measurements. A significant decrease in 5-oxoproline
   levels was observed in both the plasma and heart tissues of mice with
   DOX-induced cardiotoxicity as early as 1 week after modeling ([96]Fig.
   1J). In addition, plasma and cardiac 5-oxoproline levels were inversely
   correlated with the GLS ([97]Fig. 1K), indicating a systemic metabolic
   response to early DOX-induced cardiotoxicity.

6.3. 5-Oxoproline supplementation alleviates DOX-induced cardiac
cardiotoxicity and dysfunction in mice

   To determine whether 5-oxoproline supplementation attenuates
   DOX-induced cardiotoxicity, a pilot study was performed to confirm the
   optimal concentration of 5-oxoproline. As a result, supplementation at
   0.05 mg/kg/day was shown to restore 5-oxoproline levels in both
   myocardial tissues and serum to physiological levels in mice ([98]Fig.
   S2A). 5-Oxoproline supplementation significantly attenuated the
   DOX-induced decreases in body weight ([99]Fig. 2A), heart weight
   ([100]Fig. 2B), and cardiac systolic function ([101]Fig. 2C–D).
   Additionally, it improved cardiac morphology ([102]Fig. S2B) and
   decreased the levels of myocardial damage biomarkers ([103]Fig. 2E–H).

Fig. 2.

   [104]Fig. 2
   [105]Open in a new tab

   5-Oxoproline relieved DOX-induced cardiotoxicity in mice. A-B. Body
   weight and heart weight/body weight evaluated in groups treated with or
   without DOX and 5-oxoproline; n = 5. C-D. Representative images and
   quantification of echocardiography data; n = 5. E-H. Plasma levels of
   LDH, AST, TnT, and CK-MB; n = 5. I. Representative images of
   hematoxylin and eosin-stained hearts. Scale bars, 1 mm. J-K.
   Representative images and quantification of WGA staining. Scale bars,
   40 μm; n = 5. L-M. Representative images and quantification of
   interstitial myocardial fibrosis. Scale bars, 50 μm; n = 5. N.
   Representative images and quantification of TUNEL staining. The arrows
   indicate apoptotic cardiomyocytes. Scale bars, 20 μm; n = 5. The data
   are presented as the means ± SEMs. A. Two-way ANOVA followed by the
   Šídák post hoc multicomparison test. B through N. Two-way ANOVA
   followed by the Bonferroni multiple comparison test. WGA = wheat germ
   agglutinin; 5-OXO = 5-oxoproline; LDH = lactate dehydrogenase;
   AST = aspartate aminotransferase; TnT = troponin T; CK-MB = creatine
   kinase-myocardial band.

   Moreover, DOX has been shown to elicit severe cardiac atrophy,
   fibrosis, and apoptosis [[106]17,[107]19]. Accordingly, we evaluated
   the effect of 5-oxoproline and found that it significantly attenuated
   DOX-induced myocardial atrophy, interstitial fibrosis, and
   cardiomyocyte apoptosis ([108]Fig. 2I–N). Furthermore, 5-oxoproline
   ameliorated cardiomyocyte apoptosis, as evidenced by a decrease in the
   proapoptotic factor Bax and cleaved caspase-3 and an increase in the
   antiapoptotic factor Bcl2 ([109]Fig. S2C).

   DOX treatment led to notable generation of reactive oxygen species
   (ROS), which are key inducers of oxidative stress and ferroptosis
   [[110]20]. The glutathione system plays a crucial role in regulating
   ferroptosis, as its activation can mitigate oxidative damage and
   protect against ferroptotic cell death in myocardial injury [[111]21].
   Histological and biochemical analyses revealed that DOX treatment
   increased the levels of MDA and 4-HNE and decreased the levels of
   GSH/GSSG, indicating excessive lipid peroxidation. Interestingly, these
   changes were mitigated by 5-oxoproline ([112]Fig. 3A–C), which
   indicated that 5-oxoproline was involved in the modulation of
   ferroptosis in DOX-treated cardiomyocytes. In addition, we assessed
   markers related to ferroptosis, including GPX4 (the end terminator of
   ferroptosis) [[113]22], ACSL4 (an instigator of ferroptotic signaling)
   [[114]23], COX2 (an oxidative enzyme) [[115]24], and FTH1 (an iron
   scavenger) [[116]25]. Decreases in GPX4 and FTH1 and increases in ACSL4
   and COX2 were simultaneously observed in cardiomyocytes following DOX
   treatment, and these changes were attenuated by 5-oxoproline ([117]Fig.
   3D). Furthermore, we examined Nrf2, a crucial transcription factor for
   maintaining redox homeostasis [[118]26]. 5-Oxoproline restored Nrf2
   protein levels in hearts subjected to DOX treatment, along with the
   levels of the downstream proteins HO-1 and SOD2, while reducing the
   production of protein oxidative byproducts, specifically
   3-nitrotyrosine (3-NT) ([119]Fig. 3E). Moreover, DOX treatment led to
   pronounced mitochondrial damage in cardiomyocytes, characterized by
   evident mitochondrial swelling, vacuolization, and crista
   disintegration and lysis, and these features were markedly alleviated
   by exogenous 5-oxoproline supplementation ([120]Fig. 3F). In addition,
   necroptosis, a recently discovered form of programmed cell death, was
   also upregulated by doxorubicin, leading to cardiomyocyte death and
   cardiac dysfunction [[121]27]. Thus, we also evaluated necroptosis in
   our study. As shown in [122]Fig. 3G and H, the ratio of phosphorylated
   RIP1 to total RIP1 was significantly elevated following DOX
   administration, whereas cotreatment with 5-oxoproline effectively
   reduced this increase. Similarly, DOX treatment resulted in increased
   phosphorylation of RIP3 and MLKL, as reflected by increased
   p-RIP3/total RIP3 and p-MLKL/total MLKL ratios, both of which were
   attenuated upon 5-oxoproline treatment.

Fig. 3.

   [123]Fig. 3
   [124]Open in a new tab

   5-Oxoproline inhibits DOX-induced ferroptosis, oxidative stress and
   necroptosis in vivo. A. Representative images and statistical data of
   4-HNE staining. Scale bars, 50 μm; n = 5. B. Quantification of
   myocardial MDA; n = 5. C. Quantification of the myocardial GSH/GSSG
   ratio; n = 5. D. Representative images and statistical results of
   Western blot analysis of ferroptotic indicators (ACSL4, FTH1, GPX4, and
   COX2); n = 4. E. Representative images and statistical results of
   Western blot analysis of oxidative stress indicators (3-NT, SOD2, Nrf2,
   and HO-1); n = 5. F. Representative transmission electron microscopy
   images of the mitochondrial morphology of cardiac muscle. Scale bars,
   1 μm. G-H. Representative images and statistical results of Western
   blot analysis of necroptosis indicators (p-RIP1/RIP1, p-RIP3/RIP3, and
   p-MLKL/MLKL); n = 5. The data are presented as the means ± SEMs. The
   data were analyzed by two-way ANOVA followed by the Bonferroni multiple
   comparison test. GSH = glutathione; MDA = malondialdehyde;
   4-HNE = 4-hydroxynonenal; GSSG = oxidized glutathione; other
   abbreviations as in [125]Fig. 2.

   Importantly, in addition to its prophylactic effects, 5-oxoproline also
   demonstrated therapeutic efficacy. When administered after the onset of
   DOX-induced cardiac dysfunction, 5-oxoproline significantly improved
   cardiac function, reduced pathological remodeling and oxidative stress,
   and mitigated both programmed cell death and mitochondrial structural
   abnormalities compared with DOX alone ([126]Figs. S3 and S4). In
   addition, both the preventive and therapeutic effects of 5-oxoproline
   were independently validated in an acute DOX-induced cardiotoxicity
   model ([127]Figs. S5 and S6). Collectively, these findings indicate
   that 5-oxoproline confers both preventive and therapeutic protection
   against DOX-induced cardiotoxicity in both chronic and acute models,
   likely through the restoration of myocardial 5-oxoproline levels and
   the associated glutathione metabolic balance.

6.4. 5-Oxoproline attenuates DOX-induced cardiomyocyte death and
mitochondrial dysfunction In vitro

   To further validate the role of 5-oxoproline, we investigated its
   effects on DOX-induced cardiotoxicity in vitro in neonatal rat
   cardiomyocytes (NRCMs). First, the optimal concentration of
   5-oxoproline was determined to be 5 μM, as this was the lowest
   concentration that restored maximal cell viability ([128]Fig. S7A).
   Consistent with the results of the animal models, TUNEL staining
   ([129]Fig. S7B) and Western blot analysis ([130]Fig. S7C) demonstrated
   that 5-oxoproline significantly inhibited DOX-induced cell apoptosis
   and decreased the levels of apoptotic markers.

   Subsequently, intracellular ROS and lipid peroxidation were assessed in
   vitro in NRCMs. The results demonstrated that in NRCMs, DOX treatment
   markedly increased intracellular ROS ([131]Fig. 4A), lipid peroxidation
   ([132]Fig. 4B), and MDA levels ([133]Fig. S8A), while significantly
   decreasing the GSH/GSSG ratio ([134]Fig. S8B). However, all these
   detrimental effects were reversed by 5-oxoproline. Moreover,
   mitochondrial ROS levels were markedly increased following DOX
   treatment, leading to a significant decrease in the mitochondrial
   membrane potential (MMP), as indicated by elevated MitoSOX fluorescence
   and an increased JC-1 monomer/aggregate ratio ([135]Fig. 4C–F). These
   alterations were substantially attenuated by 5-oxoproline
   supplementation. To further evaluate mitochondrial function, real-time
   mitochondrial respiration was assessed using extracellular flux
   analysis. DOX exposure significantly reduced the oxygen consumption
   rate (OCR), including basal and maximal respiration, spare respiratory
   capacity, and ATP production ([136]Fig. 4G). Cotreatment with
   5-oxoproline effectively ameliorated these impairments, indicating a
   protective effect on mitochondrial bioenergetics. Additionally, the
   downregulation of Nrf2, HO-1, SOD2, GPX4, and FTH1, along with the
   upregulation of 3-NT, p67phox, ACSL4, COX2 and necroptosis-related
   proteins (p-RIP1/RIP1, p-RIP3/RIP3, p-MLKL/MLKL) induced by DOX, was
   effectively prevented by 5-oxoproline ([137]Fig. S8C–E). Collectively,
   these findings demonstrate that 5-oxoproline effectively ameliorated
   DOX-induced cardiac dysfunction via the suppression of oxidative stress
   and ferroptosis.

Fig. 4.

   [138]Fig. 4
   [139]Open in a new tab

   5-Oxoproline suppresses oxidative stress, ferroptosis and mitochondrial
   damage in vitro. A. Representative images and quantifications of DHE
   staining in NRCMs; n = 5. Scale bars, 150 μm. B. Analysis of lipid-ROS
   accumulation using C11 BODIPY 581/591 fluorescence staining; n = 5.
   Scale bars, 20 μm. C-D. Representative images and quantification of
   MitoSOX staining in NRCMs; n = 6. Scale bars, 20 μm. E-F. JC-1 staining
   for assessment of MMP expression in NRCMs. Representative images and
   quantification of JC-1 monomers/aggregates; n = 6. Scale bars, 20 μm.
   G. Mitochondrial OCR of NRCMs. Representative images and quantification
   of basal respiration, maximal respiration, spare respiratory capacity,
   and ATP production; n = 6. The data are presented as the means ± SEMs.
   The data were analyzed by two-way ANOVA followed by the Bonferroni
   multiple comparison test. NRCMs = neonatal rat cardiac myocytes;
   MMP = mitochondrial membrane potential; OCR = oxygen consumption rate;
   ROS = reactive oxygen species; DHE = dihydroethidium; other
   abbreviations as in [140]Fig. 2.

6.5. 5-Oxoproline inhibits tumor growth in addition to exerting
cardioprotective effects

   The potential of 5-oxoproline as a cardioprotective agent in the
   context of anthracycline therapy was further evaluated in 4T1
   tumor-bearing nude mice ([141]Fig. 5A). Consistently, 5-oxoproline
   supplementation significantly improved the survival rate and mitigated
   DOX-induced cardiotoxicity, including cardiac function, cardiac
   atrophy, fibrosis, and apoptosis ([142]Fig. 5B–E; [143]Fig. S9), in 4T1
   tumor-bearing nude mice treated with DOX. Unexpectedly, 5-oxoproline
   delayed tumor growth in vivo ([144]Fig. 5F–H) and suppressed 4T1 cell
   proliferation both in vivo and in vitro ([145]Fig. 5I–K). Its antitumor
   effects were further enhanced by cotreatment with DOX. In addition to
   syngeneic tumor models, we also investigated the dual cardioprotective
   and antitumor efficacy of 5-oxoproline in xenograft assays. Consistent
   with the findings in the syngeneic tumor models, 5-oxoproline
   concurrently significantly inhibited tumor growth in human-derived
   xenografts while protecting against DOX-induced cardiotoxicity
   ([146]Figs. S10 and S11). Collectively, these findings reveal that
   5-oxoproline is a novel adjuvant that not only protects against
   DOX-induced cardiotoxicity but also attenuates tumor growth.

Fig. 5.

   [147]Fig. 5
   [148]Open in a new tab

   5-Oxoproline inhibits 4T1 tumor growth and simultaneously alleviates
   DOX-induced cardiotoxicity. A. Experimental scheme of 4T1 cell
   transplantation into the flank of BALB/c nude mice and treatment with
   either vehicle or 5-oxoproline (0.05 mg/kg/day) intraperitoneally 3
   days before saline or DOX (5 mg/kg/week) administration continued for 4
   weeks. The tumor volume was measured twice a week for 30 days in total.
   B. Survival curves in the indicated groups; n = 20. C. Heart
   weight/body weight; n = 6. D. Representative M-mode echocardiographic
   images. E. Ejection fraction and fractional shortening after 4 weeks of
   5-oxoproline administration; n = 6. F‒G. The volume and weight of
   tumors in the indicated groups; n = 6. H. Photographs of the excised
   tumors in the indicated groups. I. Representative images and
   quantification of Ki67 staining; n = 6. Scale bars, 100 μm. J. CCK-8
   activity of 4T1 cells treated with DOX or 5-oxoproline; n = 20. K. EdU
   assay of 4T1 cells; n = 6. Scale bars, 50 μm. The data are presented as
   the means ± SEMs. B. Survival data were analyzed by the Kaplan‒Meier
   method and compared by log-rank tests. F. Two-way ANOVA followed by the
   Šídák post hoc multicomparison test. C, E, G, I, K. Two-way ANOVA
   followed by the Bonferroni multiple comparison test. J. One-way ANOVA
   followed by the Bonferroni multiple comparison test.
   GGCT = gamma-glutamyl cyclotransferase; other abbreviations as in
   [149]Fig. 2, [150]Fig. 3.

6.6. 5-Oxoproline alleviates cardiotoxicity via OPLAH-mediated glutathione
synthesis

   OPLAH, the enzyme responsible for converting 5-oxoproline into
   glutamate acid, is highly expressed in cardiomyocytes. Despite limited
   research, its role is critically important. Considering that glutamate
   acid is further converted into glutathione, which possesses robust
   antioxidant capacity in the heart, we hypothesized that 5-oxoproline
   might exert its cardioprotective effects through an OPLAH-mediated
   enzymatic reaction. To validate this hypothesis, OPLAH^+/+ and
   OPLAH^−/− mice ([151]Fig. 6A; [152]Fig. S12) were treated with DOX and
   administered either saline or 5-oxoproline daily for 4 weeks. As
   expected, 5-oxoproline supplementation alleviated DOX-induced
   cardiotoxicity in OPLAH^+/+ mice, as evidenced by improvements in body
   weight loss ([153]Fig. 6B), heart weight loss ([154]Fig. 6C), cardiac
   dysfunction ([155]Fig. 6D–E), cardiac injury ([156]Fig. 6F–I), cardiac
   atrophy and fibrosis ([157]Fig. 6J–N), cardiac ferroptosis ([158]Fig.
   7A–D), cardiomyocyte apoptosis ([159]Fig. 7E; [160]Fig. S13A),
   mitochondrial dysmorphology ([161]Fig. 7F), cardiomyocyte necroptosis
   ([162]Fig. 7G–H) and oxidative stress ([163]Fig. S13B). However, these
   beneficial effects were completely abrogated when OPLAH was knocked
   out.

Fig. 6.

   [164]Fig. 6
   [165]Open in a new tab

   OPLAH deficiency eliminates the protective effect of 5-oxoproline on
   DOX-induced cardiotoxicity. A. Western blot images of hearts from
   OPLAH-WT or OPLAH-KO mice; n = 3. B–C. Body weight and heart
   weight/body weight; n = 5. D-E. Representative images and statistical
   data evaluated by echocardiography; n = 5. F–I. Plasma levels of LDH,
   AST, TnT, and CK-MB; n = 5. J. Representative images of hematoxylin and
   eosin staining. Scale bars, 1 mm. K. Representative images of WGA
   staining. Scale bars, 40 μm. L. Myocardial interstitial fibrosis. Scale
   bars, 50 μm. M. Quantification of total fibrosis; n = 5. N.
   Quantification of cardiomyocyte atrophy; n = 5. The data are presented
   as the means ± SEMs. B. Two-way ANOVA followed by the Šídák post hoc
   multicomparison test. C through N. Two-way ANOVA followed by the
   Bonferroni multiple comparison test. Abbreviations as in [166]Fig. 2.

Fig. 7.

   [167]Fig. 7
   [168]Open in a new tab

   OPLAH knockout abolishes the benefits of 5-oxoproline in vivo. A.
   Representative images and statistical data of 4-HNE staining. Scale
   bars, 50 μm; n = 5. B. Quantification of myocardial MDA; n = 5. C.
   Representative images and statistical results of Western blot analysis
   of ferroptotic indicators (ACSL4, FTH1, GPX4, and COX2); n = 4. D.
   Quantification of the myocardial GSH/GSSG ratio; n = 5. E. TUNEL
   staining of OPLAH-WT or OPLAH-KO mice treated with 5-oxoproline. The
   arrows indicate apoptotic cardiomyocytes. Scale bars, 20 μm; n = 5. F.
   Representative transmission electron microscopy images of the
   mitochondrial morphology of cardiac muscle. Scale bars, 1 μm. G-H.
   Representative images and statistical results of Western blot analysis
   of necroptosis indicators (p-RIP1/RIP1, p-RIP3/RIP3, and p-MLKL/MLKL);
   n = 5. The data are presented as the means ± SEMs. The data were
   analyzed by two-way ANOVA followed by the Bonferroni multiple
   comparison test. Abbreviations as in [169]Fig. 3.

   Consistent with the in vivo findings, the protective effects of
   5-oxoproline against cellular apoptosis were significantly diminished
   by the downregulation of OPLAH in NRCMs ([170]Fig. S14A–C). Moreover,
   OPLAH knockdown impaired the inhibitory effects of 5-oxoproline on
   excessive ROS accumulation ([171]Fig. S14D–E), necroptosis ([172]Fig.
   S14F), lipid peroxidation ([173]Fig. S15A–D) and mitochondrial
   dysfunction ([174]Fig. S15E–I) in DOX-treated cardiomyocytes. These
   results indicate that 5-oxoproline exerts its cardioprotective effects
   in an OPLAH-dependent manner.

6.7. 5-Oxoproline suppresses tumor growth by inhibiting GGCT expression in
tumor cells

   Since 5-oxoproline alleviated cardiotoxicity via glutathione synthesis,
   we first measured glutathione levels in 4T1 cells following
   5-oxoproline administration. However, no significant changes in
   glutathione levels were observed after 5-oxoproline supplementation
   ([175]Fig. 8A). Unlike that in cardiomyocytes, the expression of OPLAH
   in tumor tissues was nearly 100-fold lower than that in heart tissues
   after DOX therapy ([176]Fig. 8B). In contrast to OPLAH, GGCT, the
   upstream enzyme of 5-oxoproline in the glutathione pathway, is
   upregulated in most tumors, and its depletion has been shown to inhibit
   tumor proliferation [[177]28]. In our study, higher levels of GGCT were
   associated with significantly shorter relapse-free survival in breast
   cancer patients ([178]Fig. 8C). Accordingly, we hypothesized that
   5-oxoproline exerts an inhibitory effect on tumors through negative
   feedback inhibition of GGCT expression. Consistent with this
   hypothesis, we found that GGCT expression decreased in DOX-treated mice
   and was further reduced with 5-oxoproline supplementation ([179]Fig.
   8D). Similarly, GGCT mRNA and protein levels were downregulated in 4T1
   cells following DOX treatment, and 5-oxoproline further suppressed GGCT
   expression ([180]Fig. 8E–F). Moreover, GGCT knockdown significantly
   reduced 4T1 cell growth and proliferation, as shown by cell viability
   and EdU absorption ([181]Fig. 8G–I). These findings suggest that
   5-oxoproline suppresses tumor growth by inhibiting GGCT expression in
   tumor cells.

Fig. 8.

   [182]Fig. 8
   [183]Open in a new tab

   5-Oxoproline suppresses tumor growth by downregulating GGCT in tumor
   cells. A. GSH/GSSG ratios in 4T1 cells with or without the
   administration of DOX and 5-oxoproline; n = 6. B. Relative mRNA levels
   of OPLAH in heart tissues and tumor tissues; n = 6. C. Data were
   obtained from an online database of potential cancer biomarkers
   ([184]http://www.kmplot.com) to assess the prognostic implications of
   GGCT (Affymetrix ID: 215380_s_at) expression in patients with breast
   cancer. D. Representative images of GGCT staining; n = 6. Scale bars,
   100 μm. E. Representative Western blot images and statistical results
   of GGCT in 4T1 cells with or without the administration of DOX and
   5-oxoproline; n = 6. F. Relative mRNA levels of GGCT in 4T1 cells;
   n = 6. G. Representative Western blot images and relative mRNA levels
   of GGCT in 4T1 cells after transfection with NC-siRNA or GGCT-siRNA;
   n = 6. H. CCK-8 activity of 4T1 cells after transfection with NC-siRNA
   or GGCT-siRNA; n = 6. I. EdU assay of 4T1 cells after transfection with
   NC-siRNA or GGCT-siRNA; n = 6. Scale bars, 50 μm. The data are
   presented as the means ± SEMs. A, D, E, F, H, I. Two-way ANOVA followed
   by Bonferroni multiple comparison tests. B. Mann‒Whitney U test. G.
   Student's t-test. Abbreviations as in [185]Fig. 3, [186]Fig. 5.

6.8. Efficacy of 5-oxoproline for the early detection of
anthracycline-induced cardiotoxicity

   To further verify the decreased level of 5-oxoproline in mice, we
   extended our investigation to human participants with breast cancer
   ([187]Fig. 9A; [188]Table S1). Using liquid chromatography‒mass
   spectrometry/MS analysis, we tested 118 blood samples from 59 patients.
   Plasma levels of circulating 5-oxoproline significantly decreased after
   the first cycle of anthracycline administration, whereas cTnI levels
   did not significantly change ([189]Fig. 9B). Among these patients, 12
   were diagnosed with early cardiac dysfunction on the basis of GLS
   assessment via echocardiography. Receiver operating characteristic
   analysis revealed good performance of the 5-oxoproline prediction model
   in predicting early anthracycline-induced cardiotoxicity (AUC 0.848,
   95 % CI: 0.745–0.950) ([190]Fig. 9C). In addition, a significant
   correlation was observed between decreased 5-oxoproline levels and an
   increase in the LVEDD, as well as a decrease in the IVST at
   anthracycline cycle 4 ([191]Fig. 9D–E). These findings suggest that
   5-oxoproline could serve as a biomarker for the early detection of
   anthracycline-induced cardiotoxicity.

Fig. 9.

   [192]Fig. 9
   [193]Open in a new tab

   Role of 5-oxoproline in human anthracycline therapy. A. Schematic
   illustration of the human study design. B. Serum levels of
   5-oxoproline, BNP, and cTnI at baseline and after 1 cycle of
   anthracycline administration; n = 59. C. ROC curve of the ability of
   5-oxoproline to predict early anthracycline therapy-induced
   cardiotoxicity. D. LVEDD and IVST changes after 4 cycles of
   anthracycline administration; n = 15. E. Association between the
   5-oxoproline change and left ventricular morphological change; n = 15.
   The values are presented as the means ± SEMs. B. Mann‒Whitney U test.
   D. Wilcoxon matched-pairs signed-rank test. E. Spearman's rank
   correlation. BNP = brain natriuretic peptide; LVEDD = left ventricular
   end-diastolic diameter; IVST = interventricular septal thickness.

7. Discussion

   In the present work, we identified 5-oxoproline as a key metabolite
   associated with DOX-induced cardiotoxicity, demonstrating its dual
   cardioprotective and antitumor functions. This study presents several
   novel findings (Central Illustration). First, the glutathione pathway
   was identified as the most significantly altered metabolic pathway in
   the cardiac tissues of patients with DOX-induced cardiotoxicity, and
   the 5-oxoproline/OPLAH axis was the critical node in the pathway.
   Second, treatment with 5-oxoproline alleviated DOX-induced
   cardiotoxicity, as evidenced by improved cardiac function, reduced
   oxidative stress, and attenuated cell death, while simultaneously
   enhancing the antitumor efficacy of DOX. Third, mechanistic
   investigations revealed that 5-oxoproline exerted cardioprotective
   effects by restoring glutathione metabolic homeostasis via its
   downstream enzyme OPLAH and inhibited tumor proliferation by
   suppressing its upstream enzyme GGCT. Collectively, these findings
   provide the first evidence for a novel regulatory role of the
   GGCT/5-oxoproline/OPLAH metabolic axis in mitigating cardiovascular
   complications and suppressing tumor growth during cancer therapy.
   Finally, the level of circulating 5-oxoproline may serve as an early
   biomarker for the detection of anthracycline-induced cardiotoxicity.

   In recent years, the incidence of anthracycline-induced cardiotoxicity
   has become increasingly prominent, largely due to improved cancer
   survival rates. The development of effective cardioprotective
   strategies remains an unmet clinical need. Several therapeutic
   approaches have been explored, including conventional agents such as
   carvedilol [[194]29] and vericiguat [[195]30], as well as small
   molecule inhibitors [[196]31] and microRNAs [[197]32]. Several studies
   have also focused on mechanisms of anthracycline-induced cardiotoxicity
   involving cellular senescence [[198]33] and immune modulation
   [[199]34]. However, little attention has been given to the role of
   endogenous small-molecule metabolites in mitigating
   anthracycline-induced cardiotoxicity. Compared with supplementation
   with exogenous chemical agents, supplementation with endogenous
   metabolites may better align with physiological processes and
   potentially reduce off-target effects.

   5-Oxoproline is an important endogenous intermediate in the γ-glutamyl
   cycle, which is critical for glutathione synthesis. It is converted to
   glutamate via 5-oxoprolinase (OPLAH), thereby contributing to the
   replenishment of intracellular GSH pools [[200]35,[201]36]. Emerging
   studies have highlighted the regulatory role of the 5-oxoproline/OPLAH
   axis in maintaining both metabolic and redox homeostasis
   [[202]37,[203]38]. For example, OPLAH deficiency has been shown to
   cause oxidative stress, fibrosis, and elevated cardiac filling
   pressures [[204]39], underscoring the importance of this metabolic
   pathway in cardiovascular health. In the present study, through both
   exogenous supplementation with 5-oxoproline and in vivo experiments in
   OPLAH knockout mice, we demonstrated that 5-oxoproline confers both
   preventive and therapeutic benefits against DOX-induced cardiotoxicity
   in an OPLAH-dependent manner. Mechanistically, this protective effect
   is associated with the restoration of cardiac GSH metabolism. These
   findings reveal a previously unrecognized function of the
   5-oxoproline/OPLAH axis and suggest its potential as a therapeutic
   strategy for mitigating anthracycline-induced cardiac injury.

   Glutathione, a critical nonenzymatic antioxidant in mammalian cells,
   plays a central role in redox regulation, protection against oxidative
   stress, and maintenance of cellular homeostasis. In addition to its
   well-established ability to scavenge ROS, GSH also regulates multiple
   forms of programmed cell death, including ferroptosis and apoptosis
   [[205][40], [206][41], [207][42], [208][43]]. Given the pivotal role of
   GSH in DOX-induced cardiotoxicity, GSH-related pathways—especially
   those involving ferroptosis and apoptosis—have emerged as key
   therapeutic targets for the prevention and treatment of cardiac injury
   [[209]44,[210]45]. For example, a small-molecule inhibitor targeting
   BAX has been shown to block both apoptosis and necrosis, thereby
   preventing DOX-induced cardiotoxicity [[211]31]. Similarly, the
   inhibition of ferroptosis, which is characterized by iron-dependent
   lipid peroxidation, significantly alleviates DOX-induced cardiac
   dysfunction and cardiomyocyte loss [[212]46,[213]47]. Consistent with
   these findings, our study revealed that activation of the
   5-oxoproline/OPLAH axis restores GSH metabolism and ameliorates
   DOX-induced oxidative stress and cardiomyocyte death. These results not
   only reinforce the therapeutic importance of targeting ferroptosis and
   other regulated cell death pathways but also establish a novel upstream
   regulatory mechanism of cardiac GSH homeostasis, expanding our
   understanding of redox-based cardioprotective strategies.

   Ideally, optimal cardioprotective strategies should not only mitigate
   the primary mechanisms underlying chemotherapy-induced cardiotoxicity
   but also preserve—or even potentiate—the efficacy of antitumor
   treatment. Given the substantial differences between cardiomyocytes and
   cancer cells in terms of gene expression, protein profiles, and
   metabolic signatures, it is conceivable that the same therapeutic
   interventions may exert protective effects on the heart while enhancing
   the cytotoxicity of chemotherapy toward cancer cells. Indeed, a
   previous report demonstrated that PI3Kγ inhibition synergizes with the
   antitumor effects of chemotherapy and protects against DOX-induced
   cardiac injury [[214]7]. Similarly, our study reveals a striking dual
   role for 5-oxoproline: 5-oxoproline mitigates DOX-induced cardiomyocyte
   injury by reducing oxidative stress and cell death while concurrently
   synergizing with DOX to enhance overall antitumor efficacy. These
   divergent effects may be attributed to the differential expression of
   5-oxoproline-related metabolic enzymes between cardiomyocytes and
   cancer cells. Specifically, the higher expression of OPLAH
   (5-oxoprolinase) in cardiomyocytes facilitates the conversion of
   5-oxoproline to glutamate, thereby replenishing intracellular
   glutathione and strengthening antioxidant defenses. In contrast, the
   anticancer activity of 5-oxoproline may, at least in part, be mediated
   by its feedback inhibition of γ-glutamyl cyclotransferase (GGCT), an
   upstream oncogene involved in tumor metabolism and progression
   [[215]48]. These findings highlight a promising future research
   direction, wherein high-throughput omic technologies could be employed
   to elucidate molecular distinctions between cardiomyocytes and cancer
   cells, thereby guiding the development of therapeutics that offer both
   cardioprotection and enhanced antitumor efficacy.

   Early myocardial alterations that precede overt functional decline have
   been increasingly recognized as critical windows for therapeutic
   intervention in doxorubicin (DOX)-induced cardiotoxicity [[216]49]. In
   line with this concept, our study focused on the early phase of
   DOX-induced myocardial injury, aiming to identify sensitive biomarkers
   and actionable targets for timely detection and intervention before
   irreversible cardiac dysfunction develops. Previous studies [[217]50]
   have highlighted the utility of circulating microRNAs as promising
   genomic biomarkers for the early diagnosis of DOX-related
   cardiotoxicity. Extending this line of investigation, our study is the
   first to identify plasma 5-oxoproline as a potential early biomarker
   for anthracycline-induced myocardial injury. These findings not only
   underscore the clinical importance of targeting early metabolic
   disturbances but also open new avenues for developing minimally
   invasive diagnostic tools. Nevertheless, further validation in larger
   and more diverse patient cohorts across multiple cancer types and
   treatment settings is needed to establish the clinical applicability
   and predictive value of 5-oxoproline in routine cardio-oncology
   practice.

8. Study limitations

   Several limitations of this study should be acknowledged. First, DOX
   does not fully represent the cardiotoxic effects of all
   chemotherapeutic agents, and the cardioprotective effect of
   5-oxoproline observed in this context may not be universally applicable
   to all forms of chemotherapy-induced cardiotoxicity. Second, while
   breast cancer is a major clinical context for DOX usage, we recognize
   that the findings cannot be directly generalized to other tumor types,
   which may differ in their microenvironments, metabolic dependencies,
   and responses to 5-oxoproline. Finally, our findings shed light on
   5-oxoproline supplementation as a promising therapeutic avenue for
   DOX-induced cardiotoxicity. However, more clinical research is
   warranted to confirm the benefits observed in this study.

9. Conclusions

   In conclusion, our findings suggest that 5-oxoproline may serve as a
   promising small-molecule metabolite for preventing both DOX-induced
   cardiotoxicity and tumor growth. Thus, it may serve as an incremental
   therapeutic strategy for the treatment of cancer patients requiring
   anthracycline chemotherapy. Additionally, our findings provide a
   potential biomarker for the early detection of anthracycline-induced
   cardiotoxicity.

   [218]Image 1
   [219]Open in a new tab

CRediT authorship contribution statement

   Xinning Guo: Writing – review & editing, Writing – original draft,
   Formal analysis, Data curation, Conceptualization. Meng Jiang: Writing
   – review & editing, Resources, Funding acquisition. Zhengyu Tao:
   Writing – review & editing, Formal analysis, Data curation. Huijuan
   Dai: Writing – review & editing, Project administration, Methodology.
   Chen Wu: Formal analysis, Data curation. Yinan Wang: Formal analysis,
   Data curation. Zi Wang: Data curation. Xiaoning Wang: Data curation.
   Zhixuan Zhang: Investigation. Kun Qian: Resources, Methodology.
   Shanshan Zeng: Methodology, Investigation. Yihua Bei: Writing – review
   & editing, Project administration, Investigation. Jun Pu: Writing –
   review & editing, Resources, Investigation, Conceptualization.

Central Illustration

   Schematic representation of the molecular mechanisms of 5-oxoproline in
   the heart and in tumors. In myocardial cells, 5-oxoproline regulates
   glutathione metabolism via OPLAH and alleviates cardiomyocyte
   ferroptosis caused by glutathione depletion; in tumor cells, it
   inhibits GGCT to suppress the growth of tumor cells without eliciting
   cardiac injury.

Sources of funding

   This study received funding support from the National Natural Science
   Foundation of China (82421001, U21A20341 and 82470394), the Shanghai
   Municipality Science and Technology Commission (24DZ2202700 and
   20Y11910500), the Shanghai Municipal Health Commission (202440156) and
   Basic-Clinical Collaborative Innovation Project from Shanghai Key
   Laboratory of Computational Chemistry and Nanomedicine (2025ZYB-007).

Declaration of competing interest

   The authors declare that they have no competing interests. This
   manuscript is an original contribution not previously published and not
   under consideration for publication elsewhere.

Acknowledgments