Doxorubicin Induces Ferroptosis via Targeting GSTP1 and Activating JNK Signaling Pathway in Cardiomyopathy

Doxorubicin(Dox), a kind of anthracyclines, is widely used for various tumors, but its clinical usage is limited by dosage-dependent cardiotoxicity. However, there is still few effective ways to relieve or even eliminate DOX-induced cardiomyopathy (DIC). Although ferroptosis play an important role in DIC, and the inhibitors of ferroptosis could partly relieve DIC, but more direct targets of DOX in the progression of cardiotoxicity remains to be revealed. In this study, we disclosed glutathione S-transferase P1(GSTP1) as a target of DOX, which not reported before in the murine heart under DIC. GSTP1 is an isoenzyme in the glutathione-S transferases (GSTs) enzyme system, which is closely related to the regulation of cell oxidative stress. We found that the activity of GSTP1 was markedly inhibited in DOX-treated cardiomyocytes. Besides, GSTP1 overexpression significantly relieved DIC in vivo and in vitro. We further revealed that ferroptosis, rather than other cell death, participated in the process of improved DIC by GSTP1 overexpression. Mechanistically, our results showed that GSTP1 overexpression inhibit the phosphorylation of c-Jun N-terminal kinase (JNK), which restricted the generation of reactive oxygen species (ROS) and subsequently suppressed the ferroptosis during DIC. In general, our study revealed that DOX targets to GSTP1 and activates JNK signal pathway to accelerate ferroptosis in DIC, indicating that GSTP1 may be a key mediator of ferroptosis and potential target in the therapeutic strategy against the development of DIC.\n\nDoxorubicin (Dox), an effective antineoplastic agent, is widely used for multiple cancer types(1). However, its wide use was retained by various systemic adverse effects, which is characterized by heart failure (HF), structural damage, and hypertension. Patients receiving long-term DOX therapies develop heart dysfunction even at a lower dosage. It was estimated that approximately 26% of patients progressed to heart failure when receiving DOX at a cumulative dose over 550 mg/m2. Previous studies suggested that the pathogenesis of DOX-induced cardiomyopathy (DIC) was caused by multiple factors, including reactive oxygen species (ROS), mitochondrial damage, dysfunction of iron regulatory protein and cell death(2, 3). However, the detailed mechanism of DIC has not been fully elucidated, and therapeutic strategies to prevent DIC have also not been established. Thus, understanding factors and pathways that enhance the myocardial recovery induced by DOX could provide clinical implications on cardiac protection against heart failure.\n\nPrevious studies reported that apoptosis-mediated loss of cardiomyocytes and oxidative stress is the main cause of DIC(1, 4). However, a recent study revealed that ferroptosis, a type of iron-dependent programmed cell death, is an essential process mediated the progression of DIC(5). Ferroptosis is mainly characterized by iron accumulation and lipid peroxidation. And lipid peroxidation is mainly caused by imbalance of ROS (reactive oxygen species) homeostasis(6). There are various key molecular pathways involved in ferroptosis have been reported over past few years(7-11). For example, GPX4 (glutathione peroxidase 4) and NCOA4 (nuclear receptor coactivator 4) has been identified as a key regulator of ferroptosis(12, 13). GPX4 can apply its catalytic activity to reduce the toxicity of lipid peroxides and maintain membrane lipid bilayer homeostasis. RSL3 can inactivate GPX4 to trigger ferroptosis(14, 15). Wang Y et al showed that DOX down-regulates GPX4 and cause ferroptosis, leading to DIC(16). The knockdown of NCOA4 can block lipid peroxidation and ferroptosis by reducing the amount of bioavailable intracellular labile iron pools(17, 18). In addition, Na Ta et al reported that FUNDC2 (FUN14 domain–containing 2, also known as HCBP6) interacts with SLC25A11 to mediate mitochondrial GSH homeostasis imbalance, regulating ferroptosis in the process of DIC(19). Moreover, several recent researches showed that loss of TRIM21(tripartite motif-containing protein 21)(20), ATF4 (activating transcription factor 4)(21), ATF3 (activating transcription factor 3)(22), and exosomal thioredoxin-1(23) alleviates DIC by suppressing ferroptosis. Herein, it is needed to investigate the possibility of defending against ferroptosis under DIC, which benefits the clinical therapy strategies of DIC.\n\nThe glutathione S-transferase P1(GSTP1) is an isoenzyme in the glutathione-S transferases (GSTs) enzyme system. It can catalyze intracellular detoxification reactions so that play role in buffering ROS(24, 25). It is a dimeric protein composed of two same subunits with a molecular weight of 22.5 kDa(26). Recent researches showed that GSTP1 plays a vital role in maintaining the balance of cell oxidation and regulating cell proliferation(27, 28). For its related molecular mechanism, it is reported that GSTP1 binds to JNK to inhibit the activation of JNK phosphorylation, regulating apoptosis and autophagy in multiple diseases(29, 30). Interestingly, there is a recent review predicting the possibility of GSTP1 involving in the ferroptosis which may function as an important role in the lung cancer radiotherapy(31). Besides, owing to its notable role in regulating the correlation between GSH and ROS, and lipid peroxidation mediated by dysregulation of GSH and ROS is an essential characteristic in the process of ferroptosis, GSTP1 is highly possible to participate in the ferroptosis. However, whether GSTP1 participates in the process of ferroptosis and even ferroptosis-mediated DIC still remains to be verified and illuminated.\n\nIn this study, we firstly revealed that DOX could target to GSTP1 specifically and inhibit its activity whether in vitro or in vivo. Regarding this new finding, we next investigated the detailed role of GSTP1 in DIC. We found that overexpression of GSTP1 significantly relieved the heart disorders in DIC, and this process was mediated by ferroptosis. Mechanically, overexpression of GSTP1 inhibited the JNK pathway, further inhibited the ferroptosis, improving DIC. In addition, we found that overexpression of GSTP1 inhibits ferroptosis induced by RSL3 in 293T cells, which suggested that GSTP1 may be a major regulator of ferroptosis. In general, our findings revealed a novel mediator of ferroptosis and may provide therapeutic targets for the treatment strategies of DIC.