Introduction
Cell death is an essential physiological process that is key in tissue homeostasis, aging, development and various pathological states (1). Ferroptosis is a newly identified type of cell death that is characterized by the iron-dependent accumulation of lipid peroxides to lethal levels, which is genetically, biochemically and morphologically distinct from autophagy, necroptosis and apoptosis (2). Ferroptosis can be prevented or reversed by iron chelation (3) and has been previously shown to be involved in the pathological progression of carcinogenesis (4), renal ischemia/reperfusion injury (5) and oxidative stress-induced cell death (6).
Herceptin (trastuzumab) is a chemotherapeutic agent that targets HER2 and is commonly utilized for the treatment of patients with HER2+ breast cancer (7). However, the clinical use of Herceptin is limited due to its reported cardiotoxic effects, including congestive heart failure and irreversible degenerative cardiomyopathy (8-10). Death of terminally differentiated cardiomyocytes is one of the major pathogenic causes of heart injury (11). Therefore, preventing cardiomyocyte cell death is proposed to be an effective cardioprotective approach (11).
Iron homeostasis is sustained by several mechanisms, including hepcidin and iron regulatory proteins (IRP), including IRP1 and IRP2, at systemic and cellular levels (12). Disruption of iron homeostasis leads to excessive intracellular iron accumulation, thereby causing the oxidation and modification of lipids, proteins and DNA through the production of free radicals and oxidative stress (13). Aberrant dysregulation of iron metabolism is associated with in a number diseases, including neurodegenerative diseases (Alzheimer's disease, Parkinson's disease and Huntington's disease) (14,15), cancer (lung cancer, colon cancer and breast cancer) (16,17) and cardiovascular diseases (atherosclerosis and myocardial ischemia-reperfusion injury) (18,19). Dysregulation of iron homeostasis results in excessive iron deposition in several organs, leading to progressive tissue damage (20). Iron overload serves a pathogenic role in cardiomyopathy by generating ROS via the Fenton reaction and exerts harmful influences (18,21). In 2012, iron-dependent cell death (ferroptosis) was discovered for the first time by Dixon et al (2). In animal models of cardiomyopathy and patients with heart failure, iron overload has been revealed to mediate the pathogenesis of cardiotoxicity under numerous cardiopathic conditions, where doxorubicin toxicity typically occurs by the preferential accumulation of iron specifically in the mitochondria independent of the topoisomerase-2β (a well-known target of doxorubicin) pathway (22,23). However, little is known regarding the underlying mechanism of iron accumulation or its toxicity. The present study therefore aimed to investigate the effects of targeting ferroptosis on Herceptin-induced heart failure in an in vitro model.
Materials and methods
Cell culture and in vitro treatment
H9c2 rat cardiomyocytes were purchased from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. Cells were incubated in DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% heat-inactivated FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a humidified atmosphere containing 5% CO2.
Herceptin [0, 0.2, 0.5, 1 and 10 µM; Genentech Inc.; Roche Pharma (Schweiz) Ltd.] was administrated for 12, 24 or 48 h at 37°C (24). The ferroptosis inhibitor, ferrostatin-1 (Fer-1; Sigma-Aldrich; Merck KGaA), a lipid reactive oxygen species (ROS) scavenger, was initially dissolved in DMSO and diluted to a final concentration of 10 µM (25). Deferoxamine (DFO; cat. no. HY-B0988; MedChem Express), which is an iron chelator, was diluted to a final concentration of 100 µM (26). H9c2 rat cardiomyocytes were randomly divided into the following four groups: i) Control; ii) Herceptin; iii) Herceptin + Fer-1; and iv) Herceptin + DFO. Fer-1 or DFO was co-administrated with Herceptin. Samples were collected at the indicated time points after Herceptin treatment.
Cell viability
Cell viability was assessed using MTT assay. Briefly, H9c2 cells (3×104 cells/well) were seeded into 96-well plates and incubated at 37°C for 24 h. Following the aforementioned treatments, H9c2 cells were added with a MTT solution (5 mg/ml) and incubated for 4 h at 37°C. Next, DMSO was added into each well to lyse the cells and dissolve the formazan crystals after the removal of supernatant. The absorbance at 570 nm was recorded in each well with a Flexstation® 3 microplate reader (Molecular Devices, LLC). The optical density value was reported as the ratio to control group (set as 100%).
Measurement of intracellular and mitochondrial ROS
The intracellular and mitochondrial ROS levels were quantified by measuring the fluorescence of DCFH-DA-A (cat. no. S0033; Beyotime Institute of Biotechnology) and MitoSOX™ Red (Mitochondrial Superoxide Indicator; cat. no. M36008; Thermo Fisher Scientific, Inc.), respectively. After the aforementioned treatments, H9c2 cells were collected and washed three times with 1X PBS, followed by incubation with 5 µM DCFH-DA-A or MitoSOX™ Red for 45 min at 37°C in the dark. H9c2 cells were then washed three times with 1X PBS and detached with trypsin/EDTA. The relative level of cellular fluorescence was quantified by flow cytometry (BD FACSCanto™; BD Biosciences). Data were analyzed using FlowJo version ×0.7 (FlowJo LLC).
Measurement of glutathione (GSH)/oxidized glutathione (GSSG) ratio
H9c2 cells (5×104 cells/well) were seeded into 96-well white-walled multiwall luminometer plates. After treatment with Herceptin (0, 0.2, 0.5, 1 and 10 µM) for 12, 24 and 48 h at 37°C, the GSH/GSSG ratio was detected using a GSH/GSSG Ratio Detection Assay kit (cat. no. ab138881; Abcam), following the manufacturer's protocols.
Western blot analysis
Total protein was extracted from H9c2 cells via homogenization in RIPA buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) containing protease inhibitors. Protein concentration was evaluated using a Pierce BCA protein assay kit (Thermo Fisher Scientific, Inc.). In total, 20 µg protein was separated by 10-12% SDS-PAGE and subsequently transferred onto nitrocellulose membranes. The membranes were blocked with 5% BSA (Thermo Fisher Scientific, Inc.) in TBS containing 0.2% Tween-20 for 1 h, followed by an overnight incubation at 4°C with primary antibodies against glutathione peroxidase (GPX)4 (cat. no. DF6701; dilution 1:1,000; Affinity Biosciences), recombinant solute carrier family 7 member 11 (SLC7A11; cat. no. A13685; dilution 1:1,000; ABclonal Biotech Co., Ltd.), acyl-CoA synthetase long chain family member 4 (ACSL4; cat. no. DF12141; dilution 1:1,000; Affinity Biosciences), voltage-dependent anion-selective channel (VDAC2/3; cat. nos. 11663-1-AP and 14451-1-AP; dilution 1:1,000; Proteintech Group, Inc.), cytochrome c (Cyto c; cat. no. 10993-1-AP; dilution 1:1,000; Proteintech Group, Inc.), mitochondrial fission 1 protein (fis1; cat. no. 10956-1-AP; dilution 1:1,000; Proteintech Group, Inc.), mitofusin (Mfn1/2; cat. no. 13798-4-AP; dilution 1:1,000; Proteintech Group, Inc.), dynamin-related protein (Drp1; cat. no. ab184247; dilution 1:1,000; Abcam), mitochondrial optic atrophy (OPA1-1/2; cat. no. 27733-1-AP; dilution 1:2,000; Proteintech Group, Inc.), mitochondrial ferritin (Mtf; cat. no. LS0-C293861; dilution 1:500; Lifespan BioSciences, Inc.) and GAPDH (cat. no. AB-P-R001; dilution 1:1,000; Hangzhou Xianzhi Biological Technology Co., Ltd.). The membranes were then incubated for 1 h at room temperature with an HRP-conjugated secondary antibody (cat. no. BA1054; dilution 1:5,000; Boster Biological Technology). Finally, the bands were detected using the Pierce ECL system (Thermo Fisher Scientific, Inc.) and band intensity quantified using the ImageJ software (version 1.8.0; National Institutes of Health).
Measurement of intracellular labile iron pool (LIP)
H9c2 cells (1×104 cells/well) were seeded into a 96-well plate and grown overnight. The H9c2 cells were then harvested, washed and resuspended in a buffer containing 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 5.6 mM glucose, 1.5 mM CaCl2 and 20 mM HEPES (pH 7.4), followed by the addition of 0.25 mM Calcein-AM (cat. no. C2012; Beyotime Institute of Biotechnology). This reaction mixture was incubated for 30 min at 37°C and washed three times with the buffer aforementioned prior to the resuspension of H9c2 cells. The fluorescence intensity of Calcein-AM was quantified using a microplate reader (Synergy H4; 488 nm excitation and 525 nm emission; BioTek China). After the baseline became stable, salicylaldehyde isonicotinoyl hydrazone (SIH, 100 µM) which was synthetized by Schiff base condensation between 2-hydroxybenzaldehyde and isonicotinic acid hydrazide (Shanghai Nafu Biotechnology Co., Ltd.; https://nayuansu.company.lookchem.cn/) was added. The reaction mixture was subsequently incubated for 30 min at 37°C before the fluorescence intensity was detected. Calcein-AM itself has no fluorescence. After entering the H9c2 cells, it is hydrolyzed by endogenous esterase in the H9c2 cells to produce Calcein, a polar molecule with strong negative charge that cannot penetrate the cell membrane, which is retained in the cell and emit strong green fluorescence. The increase in fluorescence intensity indicates the level of Calcein-bound iron. The results were presented as fluorescence intensity/mg protein.
Measurement of mitochondrial iron
Mitochondria were isolated from H9c2 cells (1×106 cells/dish) using a Mitochondria Isolation kit for Cultured Cells (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The levels of iron in the mitochondria was then determined using an Iron Colorimetric assay kit (cat. no. BIV-K390-100; BioVision, Inc.) according to the manufacturer's protocol. The absorbance was measured at 593 nm using a TECAN infinite M200 microplate reader (Tecan Group, Ltd.).
5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbo- cyanine iodide (JC-1) staining
Following JC-1 staining (cat. no. C2006; Beyotime Institute of Biotechnology) at 37°C for 10 min, H9c2 cells (5×104/well) were washed with the JC-1 staining buffer three times and detected via flow cytometry (BD FACSCanto™; BD Biosciences). Data were analyzed using FlowJo version ×0.7 (FlowJo LLC). In the healthy mitochondria, JC-1 aggregated to form a polymer in the mitochondrial matrix, which emits intense red fluorescence (Excitation, 585 nm; Emission, 590 nm). In the unhealthy mitochondria, JC-1 monomers presented in the cytoplasm due to the decline/loss of mitochondrial membrane potential, which generate green fluorescence (Excitation, 514 nm; Emission, 529 nm). Therefore, changes in ratio of H1-UL (red)/H1-UR (green) reflected the change in mitochondrial membrane potential.
ATP content
H9c2 cells (1×104 cells/well) were lysed by ATP lysis buffer on ice, exposed to the ATP substrate solution and cellular ATP content was determined by bioluminescent assay kit (cat. no. ab113849; Abcam).
Statistical analysis
Data were repeated ≥ three times independently, analyzed using GraphPad Prism software (version 6.01, GraphPad Software, Inc.) and are presented as the mean ± SD. Groups were compared using one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.
Results
Herceptin induces cell injury and oxidative stress in H9c2 cells
Herceptin (0, 0.2, 0.5, 1 and 10 µM) was found to reduce H9c2 cell viability in a dose-dependent manner (Fig. 1A). In addition, 10 µM Herceptin reduced H9c2 cell viability in a time-dependent manner from 12 to 48 h (Fig. 1B). Herceptin (0, 0.2, 0.5, 1 and 10 µM) also dose-dependently increased intracellular (Fig. 1C) and mitochondrial (Fig. 1E) ROS levels in H9c2 cells. Furthermore, Herceptin (10 µM) increased intracellular (Fig. 1D) and mitochondrial (Fig. 1F) ROS levels in H9c2 cells from 12 to 48 h, peaking at 24 h.
Herceptin induces ferroptosis in H9c2 cells
Herceptin (0, 0.2, 0.5, 1 and 10 µM) dose-dependently reduced the protein expression of GPX4 in H9c2 cells (Fig. 2A), whilst at 10 µM Herceptin also inhibited GPX4 protein expression in H9c2 cells in a time-dependent manner from 12 to 48 h (Fig. 2B). Herceptin (0, 0.2, 0.5, 1 and 10 µM) decreased GSH content (Fig. 2C) and increased the GSSG content (Fig. 2D), thereby decreasing the GSH/GSSG ratio in H9c2 cells in a dose-dependent manner (Fig. 2E). Furthermore, Herceptin at 10 µM decreased the GSH content (Fig. 2F) whilst increasing that of GSSG (Fig. 2G), which resulted in the reduction in the GSH/GSSG ratio in H9c2 cells in a time-dependent manner from 12 to 48 h (Fig. 2H).
Fer-1 protects H9c2 cells against Herceptin-induced cell injury and ferroptosis
Fer-1 treatment was found to reverse the Herceptin-induced reduction in cell viability (Fig. 3A), restored GPX4 and SLC7A11 expression that was previously suppressed by Herceptin whilst reversing the Herceptin-induced increase in ACSL14 expression (Fig. 3B). Fer-1 was also revealed to partially restore GSH production that was previously suppressed by Herceptin without affecting the GSSG content, which in turn lead to the reversal of the Herceptin-induced reduction in GSH/GSSG ratio in H9c2 cells (Fig. 3C-E). Additionally, Fer-1 reversed the Herceptin-induced increase in intracellular (Fig. 3F) and mitochondrial (Fig. 3G) iron levels in H9c2 cells. However, compared with those mediated by DFO, the effects of Fer-1 were less potent.
Fer-1 protects H9c2 cells from Herceptin-induced mitochondrial dysfunction
Fer-1 was demonstrated to reverse the Herceptin-induced reduction in ATP production (Fig. 4A). In addition, Fer-1 could negate the Herceptin-mediated increases in mitochondrial ROS production (Fig. 4B) whilst reversing the Herceptin-induced decrease in mitochondrial membrane potential (Fig. 4C). In terms of protein expression, Herceptin increased Cyto c, VDAC2/3, fis1, Drp1 and Mtf protein expression but decreased OPA1-1/2 and Mfn1/2 expression (Fig. 4D), all of which were reversed by Fer-1 co-treatment. However, compared with DFO, the effects mediated by Fer-1 were not as potent.
Discussion
The present study discovered that Herceptin induced injury, oxidative stress, mitochondrial dysfunction and ferroptosis in H9c2 cells, which could be attenuated by Fer-1. Oxidative stress has been frequently reported to mediate cellular and molecular damage resulting from excessive ROS production caused by the inhibition of antioxidant enzyme systems and/or insufficient antioxidants production (27). This can cause numerous diseases, including cardiovascular diseases (27). The overproduction of ROS leads to the dysregulation of cell cycle progression, breaking of DNA strands and/or cell death (28). In the present study, it was demonstrated that Herceptin reduced cell viability and increased both intracellular and mitochondrial ROS generation in H9c2 cells. This suggest that Herceptin induced cell injury and oxidative stress in H9c2 cells, which is consistent with a previous report that also showed the involvement of oxidative stress in Herceptin-mediated cardiotoxicity (29).
Ferroptosis is characterized by the iron-dependent accumulation of lipid peroxides to lethal levels (2). Excessive ROS accumulation can induce damage in numerous cell types, which is associated with decreased GSH content (30). GSH is a water-soluble tripeptide that consists of the amino acid triad of glutamine, cysteine and glycine, which exists in either the oxidized GSSG state or the reduced GSH state inside the cell (31). Therefore, it is crucial to sustain optimal GSH/GSSG ratios in the cells for survival (31). Mechanistically, GSH is a key antioxidant enzyme that participates in the clearance of ROS (32) and detoxification of various electrophilic compounds and peroxides by glutathione S-transferases and GPX through catalysis (31). In the present study, Herceptin was observed to decrease GPX4 expression and the GSH/GSSG ratio in H9c2 cells, suggesting that Herceptin induced ferroptosis in H9c2 cells, consistent with a previously published report that also demonstrated that ferroptosis may mediate Herceptin-induced cardiotoxicity (33).
Altered iron homeostasis results in excessive iron deposition in the heart, leading to progressive tissue damage (20). Excessive iron burden promotes oxidative stress, which serves an important role in the pathogenesis of iron overload-mediated heart disease (34), iron overload cardiomyopathy (18,35), myocardial injury and heart failure (36). However, to the best of our knowledge, the association between Herceptin-induced changes in iron homeostasis and ferroptosis in H9c2 cells has yet to be elucidated. GPX4 and SLC7A11 acts as ferroptosis inhibitors (37), GPX4 uses reduced glutathione to convert lipid hydroperoxides to lipid alcohols, thereby mitigating lipid peroxidation and inhibiting ferroptosis (38). The majority of cells obtain cysteine biologically through the import of extracellular cystine transporter SLC7A11 (39). ACSL4 is a lipid metabolism enzyme required for ferroptosis and can elevate lipid peroxidation and ferroptosis (40). In detail, inactivation of GPX4 or SLC7A11 through genetic or pharmacological means has been previously found to induce ferroptosis (2). In addition, ferroptosis can be attenuated by the inactivation of ACSL4 (41). Therefore these aforementioned indicators of ferroptosis were detected in the present study. It was demonstrated that Fer-1 treatment reversed the Herceptin-induced reduction in H9c2 cell viability, reversed Herceptin-mediated decrease in GPX4 and SLC7A11 protein expression in addition to reversing the Herceptin-induced increase in ACSL4 protein expression. Furthermore, Fer-1 reversed the inhibitory effects of Herceptin on the GSH/GSSG ratio in H9c2 cells, as well as reversing the increase in intracellular and mitochondrial iron levels in H9c2 cells induced by Herceptin. These findings suggest that targeting ferroptosis can protect H9c2 cells from Herceptin-induced cardiomyocyte injury and ferroptosis.
Oxidative stress can also result in mitochondrial dysfunction (42). Since reductions in the mitochondrial membrane potential is associated with heart failure (43), the present study also assessed the effects of Fer-1 on Herceptin-induced mitochondrial dysfunction. Under physiological conditions, adult cardiac tissues primarily rely on fatty acid oxidation for the production of ATP, with ~70% ATP being generated by fatty acid metabolism (44). However, during heart failure, the overall production of ATP is decreased (45). The present study found that Fer-1 treatment restored mitochondrial membrane potential and ATP production, both of which were previously inhibited by Herceptin. In addition, Fer1-1 reversed the Herceptin-induced increases in ROS production. Subsequently, the expression levels of the associated proteins were also detected. Drp1 (fission gene, which increases mitochondrial division), Fis1 (fission gene, which increases mitochondrial division), Mfn1/2 (fusion gene, which increases mitochondrial fusion and mitochondrial connectivity) and Opa1-1/2 (fusion gene, which increases mitochondrial fusion and mitochondrial connectivity) can regulate, maintain and remodel mammalian mitochondria dynamics and distribution (46). In addition, mitochondrial release of cytochrome c is a hallmark of cell death (47). ADP/ATP exchange is realized by VDAC2/3 on the outer mitochondrial membrane (48). Iron loss triggers mitophagy by induction of Mtf (49). It was observed that Fer-1 reversed the Herceptin-induced reduction in OPA1-1/2 and Mfn1/2 protein expression and Herceptin-induced increase in Cyto c, VDAC2/3, fis1, Drp1 and Mtf protein expression, suggesting that targeting ferroptosis protected H9c2 cells from Herceptin-induced mitochondrial dysfunction. However, compared with DFO, the effects of Fer-1 were not as potent effective.
Taken together, targeting ferroptosis can protect the cardiomyocytes from Herceptin-induced toxicity in the present H9c2 in vitro model. A previous study has shown that haploid embryonic stem cells have been used for toxicology drug screening (50), which may be useful for finding additional ferroptosis inhibitors to protect myocardial cells against chemotherapeutic drugs for cancer. The present study proposed that iron-dependent ferroptosis is one of the pathological processes underlying the development of Herceptin-induced cardiomyopathy. Mechanistically, Herceptin contributed to the release of free iron, which accumulated in the mitochondria. Furthermore, targeting ferroptosis also protected H9c2 cells from Herceptin-induced injury. Collectively, these findings provided novel insights into the pathogenic mechanisms underlying iron overload-induced cardiomyopathy and offer therapeutic targets for the development of novel strategies.
However, several limitations remain in the present study. There is a lack of in vivo evidence, rendering animal experiments a necessity for future study to verify the present findings, using techniques such as immunohistochemistry and echocardiography. Additionally, only ferroptosis was focused upon in the present study, such that other types of cell death, including apoptosis or autophagy, were not detected. Simultaneous observations of all four types of cell death should be detected in future investigations.