Heme oxygenase-1 mediates BAY 11-7085 induced ferroptosis
Abstract
Ferroptosis represents a distinct form of regulated cell death, fundamentally characterized by its oxidative nature. In recent years, this unique cellular demise has garnered significant attention as a promising chemotherapeutic target in the ongoing battle against cancer. In this comprehensive investigation, we uncovered a novel mechanism by which BAY 11-7085, commonly referred to as BAY, a well-established inhibitor of IκBα phosphorylation, effectively suppresses the viability of various cancer cells. Crucially, this cytotoxic effect was found to be mediated through the induction of ferroptotic death, operating in a manner entirely independent of the canonical NF-κB signaling pathway.
The defining characteristics of ferroptosis involve an overwhelming accumulation of reactive oxygen species, severe lipid peroxidation, and an unregulated increase in intracellular iron. Our research demonstrated that the adverse effects of BAY-induced cell death could be effectively mitigated and reversed through several key interventions. These rescue strategies included the strategic scavenging of reactive oxygen species, which directly combats oxidative stress, the precise relief of lipid peroxidation, thereby preserving cell membrane integrity, and the crucial replenishment of both glutathione and other thiol-containing agents, vital components of the cell’s antioxidant defense system. Furthermore, iron chelation, which sequesters excess intracellular iron, also proved instrumental in rescuing cells from BAY’s cytotoxic effects, underscoring the iron-dependent nature of ferroptosis.
A deeper exploration into the cellular pathways revealed that BAY treatment led to a significant upregulation of a diverse array of Nrf2 target genes, all intrinsically linked to cellular redox regulation. Among these, heme oxygenase-1, or HO-1, emerged as particularly prominent. Through meticulously designed studies employing specific pharmacological inhibitors and targeted shRNA interventions, we were able to delineate a clear hierarchical pathway of induction: Nrf2 orchestrates the upregulation of SLC7A11, which in turn leads to the induction of HO-1. Further validating the critical role of SLC7A11, its pharmacological inhibition by agents such as erastin and sulfasalazine, or its suppression through shRNA interference, was found to markedly sensitize cancer cells to BAY-induced cell death. Conversely, the deliberate overexpression of SLC7A11 successfully attenuated the reduction in cell viability observed with BAY treatment, reinforcing its protective role.
Our findings further elucidated that the ferroptotic process initiated by human HO-1 overexpression provided compelling evidence that HO-1 itself acts as a pivotal mediator of BAY-induced ferroptosis. This critical mediation occurs primarily through its profound influence on cellular redox regulation and the concomitant accumulation of iron. Intriguingly, we observed that BAY treatment provoked a distinct compartmentalization of HO-1, causing its translocation into both the nucleus and the mitochondria. This unusual localization was subsequently followed by observable mitochondrial dysfunctions, ultimately culminating in the targeting of dysfunctional mitochondria for degradation via lysosome-mediated mitophagy. This study represents a groundbreaking discovery, establishing for the first time that BAY effectively induces ferroptosis through a well-defined Nrf2-SLC7A11-HO-1 signaling cascade, unequivocally positioning HO-1 as a central mediator that actively responds to and influences the cellular redox status.
Introduction
Ferroptosis stands as a recently recognized and distinct form of regulated cell death, fundamentally differentiated from other established forms such as apoptosis and necrosis or necroptosis. Its unique mechanistic characteristics are defined by a triad of essential features: the uncontrolled generation of reactive oxygen species, extensive lipid peroxidation, and the critical accumulation of iron within the cell. These hallmarks collectively contribute to cellular demise in a manner that is biochemically and morphologically distinct. From a morphological perspective, cells undergoing ferroptosis exhibit characteristic alterations, including a notable shrinkage in cell volume, an observable increase in the density of the mitochondrial membrane, and a significant reduction in the number and integrity of mitochondrial cristae.
In contemporary cancer research, the precise regulation of the ferroptotic process has emerged as a compelling and highly promising strategy for chemotherapy. This avenue of treatment seeks to exploit the vulnerabilities of cancer cells by selectively inducing their ferroptotic demise. A variety of agents have demonstrated their capacity to trigger cellular ferroptosis through diverse mechanisms, including targeting System Xc-, a critical cystine-glutamate antiporter, modulating the activity of glutathione peroxidase 4, a key enzyme in antioxidant defense, disrupting endoplasmic reticulum homeostasis, and influencing ferritin degradation through specific autophagic processes. Each of these pathways offers a unique point of intervention for therapeutic development.
Nuclear factor-E2-related factor 2, widely known as Nrf2, functions as a master regulator of the cellular defensive response, orchestrating a comprehensive protective shield against various forms of oxidative or electrophilic stress. Its regulatory power extends to a broad spectrum of target genes, many of which play crucial roles in maintaining cellular integrity and detoxification. These identified Nrf2 target genes encompass NAD(P)H quinone oxidoreductase 1, an enzyme involved in detoxification; heme oxygenase-1, or HO-1, a critical enzyme in heme catabolism; solute carrier family 7 member 11, also known as SLC7A11 or xCT, a component of the System Xc- transporter; thioredoxin 1, involved in redox regulation; various phase II detoxifying enzymes such as glutathione S-transferase and UDP-glucuronosyltransferase; and key enzymes in glutathione synthesis like glutathione peroxidase 4, glutathione reductase, and the subunits of glutamate cysteine ligase (GCLc and GCLm). Additionally, Nrf2 regulates several multidrug resistance-associated transporters, further underscoring its expansive role in cellular defense.
Under basal conditions, Nrf2 is meticulously anchored to the Kelch-like ECH-associated protein 1 (KEAP1)-Cul3 complex within the cytoplasm. This association ensures its rapid ubiquitination and subsequent degradation by the proteasome, thereby maintaining low cellular levels. However, in response to various stimuli, such as pro-oxidants or electrophiles, the KEAP1 protein undergoes critical modifications, typically involving oxidation or adduction on specific cysteine residues. This modification of KEAP1 leads to its structural alteration and subsequent degradation, which, in turn, liberates Nrf2 from the inhibitory complex. Once dissociated, Nrf2 is free to translocate into the nucleus, where it becomes activated by forming a functional heterodimer with a small protein from the MAF family. This activated Nrf2-MAF complex then binds to specific DNA sequences known as antioxidant-responsive elements, or AREs, initiating the transcription and expression of its extensive array of target antioxidant genes, thereby mounting a robust cellular defense.
System Xc-, a critical cystine/glutamine antiporter, is formed by the association of SLC7A11 and the chaperone protein CD98. This transporter plays an indispensable role in regulating intracellular glutathione synthesis and maintaining glutathione homeostasis, which is vital for the cell’s robust defense against oxidative insults. Consequently, pharmacological inhibition of System Xc- has been definitively shown to disrupt cellular redox balance and trigger ferroptotic cell death, highlighting its significance in this process. Heme oxygenases are a group of rate-limiting enzymes responsible for the degradation of heme, a crucial metabolic process that yields several important catabolic products: free iron, carbon monoxide, and the bile pigments biliverdin and bilirubin. Among these, the inducible form, HO-1, is particularly noteworthy for its rapid and robust response to a wide variety of cellular stimuli, including oxidative stress, hypoxic conditions, and inflammatory signals. Historically, HO-1 has been recognized for its potent function as an antioxidant and an anti-apoptotic molecule, often considered a protective mechanism against reactive oxygen species assault. However, recent evidence has revealed a more complex role for HO-1, demonstrating that its overexpression can, under certain circumstances, exert pro-oxidant effects, particularly due to the release of free iron during heme catabolism. This dual nature suggests that the activation of the Nrf2 pathway, especially leading to the induction of HO-1 and SLC7A11 by oxidative agents, may paradoxically contribute to iron-dependent and oxidative forms of cell death, a critical consideration in understanding cellular fate.
BAY 11-7085, chemically identified as (E)-3-(4-t-Butylphenylsulfonyl)-2-propenenitrile, is widely recognized as an irreversible inhibitor of IκBα phosphorylation. This specific inhibitory action effectively leads to the blockade of the pivotal NF-κB signaling pathway, a central regulator of inflammation and cell survival. Prior investigations have consistently demonstrated the anti-proliferative and pro-apoptotic effects of BAY across a diverse range of cell types, including colonic epithelial cells, keratinocytes, chondrocytes, endothelial cells, and synovial fibroblasts. Moreover, BAY has been implicated as a potential therapeutic agent against various malignancies, such as colon cancer, renal carcinoma, and lymphoma. Despite these established roles, the specific pro-ferroptotic effects of BAY and the intricate details of their underlying mechanisms have, until now, remained largely unexplored in the scientific literature.
The present study was meticulously designed with the primary aim of comprehensively characterizing the previously unknown underlying mechanism by which BAY exerts its effects. Furthermore, we sought to delineate its potential as a novel chemotherapeutic agent for cancer treatment by identifying new targets within the ferroptotic pathway. For the first time, our research definitively demonstrated that BAY significantly suppresses the viability of various cancer cell lines. This profound cytotoxic effect was found to rely explicitly on the induction of ferroptotic cell death, operating remarkably independently of the conventional IκB/NF-κB pathway. Through a rigorous combination of pharmacological approaches and sophisticated genetic gain-of-function and loss-of-function studies, we were able to precisely identify the Nrf2-SLC7A11-HO-1 pathway induction as a key signaling cascade pivotal to BAY-induced ferroptosis. These groundbreaking findings provide crucial new insights into the complex mechanism of HO-1 in the context of ferroptotic cell death and simultaneously highlight a promising novel chemotherapeutic target for advanced cancer treatment strategies.
Materials And Methods
Reagents And Antibodies
A comprehensive array of high-quality reagents and antibodies was procured from reputable suppliers to ensure the integrity and reproducibility of our experiments. Cell culture media, including DMEM/F12, RPMI-1640, and McCoy’s 5a Medium, along with essential supplements such as fetal bovine serum and penicillin/streptomycin, were all obtained from Thermo Fisher Scientific Inc. To manipulate cellular redox states and protein structure, beta-mercaptoethanol and dithiothreitol were acquired from Sigma-Aldrich. Key experimental compounds, including BAY 11-7085, the pan-caspase inhibitor z-VAD-fmk, the antioxidant N-acetyl-L-cysteine, and both reduced L-glutathione and oxidized L-glutathione, along with the reactive oxygen species indicator 2’,7’-dichlorodihydrofluorescein diacetate, were sourced from Enzo Life Sciences. A specialized suite of compounds relevant to ferroptosis research and other cell death pathways, such as the glutathione sensor monobromobimane, the iron chelator deferiprone, the lipid peroxidation probe diphenyl-1-pyrenylphosphine, and specific inhibitors like necrostatin-1 (for necroptosis), ferrostatin-1 and liproxstatin-1 (for ferroptosis), zinc protoporphyrin-9 (for HO-1 inhibition), L-NAME (nitric oxide synthase inhibitor), zileuton (lipoxygenase inhibitor), rotenone (mitochondrial complex I inhibitor), and the System Xc- inhibitors erastin and sulfasalazine, as well as the multi-kinase inhibitor sorafenib, were all obtained from Cayman Chemicals.
For precise molecular detection, a wide range of primary antibodies was utilized. Antibodies targeting components of the NF-κB pathway, including NF-κB p50, NF-κB p65, phospho-NF-κB p65 (Ser536), IκBα, phospho-IκBα (Ser32), IKKα, IKKβ, and phospho-IKKα/β (Ser176/180), were purchased from Cell Signaling Technology. Similarly, antibodies against key proteins in the Nrf2 pathway such as Nrf2 and KEAP1, along with markers for cellular stress and organelle function like MEK1, H2A.X, CHOP, BiP, PERK, PDI, the epitope tags Myc-Tag and Flag-Tag, SLC7A11, Thioredoxin 1, and the mitochondrial outer membrane marker TOM20, were also procured from Cell Signaling Technology. Additionally, MitoTracker Red CMXRos, a fluorescent dye for mitochondrial staining, was obtained from the same supplier. The antibody against β-actin, used as a loading control, was from EMD Millipore Corporation. Antibodies specific for GPx4, HO-1, and the lysosomal marker LAMP-1 were acquired from Santa Cruz Biotechnology. All other ancillary reagents required for the study were purchased from Sigma-Aldrich. It is important to note that all compounds were meticulously dissolved in dimethyl sulfoxide, ensuring that the final concentration of the solvent in experimental treatments remained below 0.1% by volume.
Cell Culture
A diverse panel of human cancer cell lines, meticulously chosen to represent various cancer types, was utilized in this study, all procured from the American Type Culture Collection. This collection included human breast cancer cell lines (MDA-MB-231, MDA-MB-468, MCF-7, and SKBR3) and a human lung cancer cell line (A549), all maintained in DMEM/F12 medium. Human hepatocellular carcinoma (HuH-7) and glioblastoma (DBTRG-05MG) cell lines were cultured in RPMI-1640 medium. Furthermore, a human ovarian carcinoma cell line (SKOV3) was cultivated in McCoy’s 5a medium. All culture media were consistently supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin to promote optimal cell growth and prevent microbial contamination. The cells were diligently maintained under controlled environmental conditions, specifically in a humidified incubator set at 37 degrees Celsius with an atmosphere of 5% carbon dioxide, conditions essential for physiological cell proliferation and metabolic activity.
Cell Viability Assay
The assessment of cell viability, a fundamental measure of cellular response to experimental treatments, was quantitatively determined using the established sulforhodamine B colorimetric assay. For this procedure, cells were initially seeded into 96-well plates at an appropriate density and allowed to adhere and equilibrate for 24 hours prior to experimentation. Following this, the cells were subjected to treatment with the specified compounds for the indicated durations, allowing sufficient time for cellular responses to manifest. After the treatment period, the cells were carefully fixed with a cold 10% trichloroacetic acid solution, a critical step that precipitates cellular proteins and preserves cell structure, and subsequently maintained at 4 degrees Celsius for one hour. This fixation step ensures that cellular components are stable for subsequent staining. Following fixation, the plates underwent a thorough washing process to remove any residual fixative. A sulforhodamine B solution, prepared at 0.4% weight by volume in 1% acetic acid, was then added to each well. The plates were incubated with the dye for ten minutes at room temperature, allowing the SRB dye to bind stoichiometrically to cellular proteins. Unbound dye was then meticulously removed by washing the cells with 1% acetic acid, and the plates were subsequently air-dried to eliminate any remaining moisture. Finally, the bound dye, indicative of total cellular protein mass and thus viability, was dissolved in dimethyl sulfoxide, and the absorbance was measured spectrophotometrically at a wavelength of 510 nanometers, providing a quantitative readout of cell viability.
Cell Morphology Observation
To visually assess and document changes in cellular morphology, a critical indicator of cell health and the type of cell death occurring, cells were directly observed and photographed. This process was carried out using a high-quality phase-contrast microscope, which provides excellent contrast for unstained biological samples, equipped with a digital camera (Leica Microsystems). This allowed for detailed recording of cellular characteristics, including cell shape, integrity, and any visual signs of cell death, providing complementary qualitative data to quantitative assays.
Staining Of Fluorescent Dyes
The assessment of various critical intracellular parameters provides invaluable insights into cellular physiology and responses to experimental manipulations. To achieve precise quantification of these parameters, including the levels of reactive oxygen species, the extent of lipid peroxidation, the intracellular content of glutathione, the mitochondrial membrane potential, and the overall mitochondrial mass, a sophisticated approach involving the selective staining with an array of specific fluorescent dyes was employed. These meticulous measurements were executed using one of two primary analytical platforms, each offering distinct advantages: an ELISA plate reader, facilitating high-throughput bulk analysis of cellular populations, or flow cytometry, which provides an exquisite single-cell resolution, allowing for detailed examination of individual cellular characteristics.
For experiments necessitating bulk analysis, typically conducted with an ELISA plate reader, a standardized cell seeding protocol was meticulously followed. Cells were carefully plated into 96-well black plates, sourced from Nunc, Thermo Fisher Scientific, Inc., at a density that would ensure optimal adherence and achieve appropriate confluency within 24 hours prior to the commencement of any experimental treatment. This crucial pre-incubation period allows cells to stabilize and establish firm attachment to the plate surface, thereby minimizing potential cell loss during subsequent washing steps and ensuring uniform exposure to treatments. Following the designated period of experimental treatment, the cellular monolayers were subjected to a rigorous washing regimen, involving three successive rinses with phosphate-buffered saline (PBS). This step is paramount for the complete removal of residual treatment medium, which could otherwise interfere with subsequent dye uptake or contribute to undesirable background fluorescence.
Once the cells were adequately prepared, a specific panel of fluorescent dyes, each tailored to a particular intracellular target, was introduced. These included 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA), a widely utilized probe for the detection of reactive oxygen species; diphenyl-1-pyrenylphosphine (DPPP), employed to quantify lipid peroxidation; and monobromobimane (MBB), used for the assessment of intracellular glutathione content. Each of these dyes was carefully diluted in complete cell culture medium to achieve a final working concentration of 10 micromolar. The cells were then incubated with this dye-containing medium for a precisely controlled duration of 30 minutes at a physiological temperature of 37 degrees Celsius, optimizing dye uptake and intracellular conversion. Following this labeling period, the cells underwent another thorough series of three washes with phosphate-buffered saline to eliminate any unbound or extracellular dye molecules, thereby reducing non-specific background signals and ensuring that the measured fluorescence originated solely from intracellularly retained dye. Finally, the intracellular fluorescence, which is directly proportional to the levels of the targeted cellular components, was meticulously measured using a high-sensitivity microplate reader, specifically a BioTek Gen5 system. To ensure accurate detection, specific excitation and emission wavelengths were employed for each dye: for DCF, excitation was set at 485 nm and emission at 528 nm; for monobromobimane, excitation was at 380 nm and emission at 450 nm. The raw fluorescent levels obtained from these measurements were initially expressed as arbitrary units. For meaningful comparative analysis and to account for potential experimental variations, these values were meticulously normalized by comparison to a designated control group and subsequently converted into a percentage, thereby reflecting the relative changes in each parameter with high precision.
Alternatively, for investigations requiring the advanced capabilities of flow cytometry, which allows for the interrogation of individual cellular characteristics, a distinct sample preparation protocol was followed. Cells that had been exposed to various compounds were first carefully detached from their culture surface using trypsinization. This enzymatic digestion process ensures the suspension of individual cells, which is a prerequisite for accurate flow cytometric analysis. The resulting single-cell suspensions were then incubated with 10 micromolar concentrations of specific fluorescent dyes chosen for flow cytometry applications. This panel included rhodamine 123 (Rh123), a lipophilic cationic dye commonly used to assess mitochondrial membrane potential due to its accumulation in energized mitochondria, and acridine orange 10-nonyl bromide (NAO), employed for staining and quantifying mitochondrial mass by binding to cardiolipin in the inner mitochondrial membrane. These dyes were prepared in phosphate-buffered saline, and the incubation was carried out at 37 degrees Celsius for 30 minutes to facilitate optimal dye loading. Subsequent to this crucial labeling period, the cells were subjected to a washing step with phosphate-buffered saline to effectively remove any unbound excess dye, thereby preventing aggregation and reducing background noise during analysis. The labeled cells were then quantitatively assessed using a sophisticated BD FACSCalibur flow cytometer from Becton Dickinson Inc. This instrument precisely measures the fluorescent properties of each individual cell as it passes through a laser beam. The extensive data generated, encompassing the fluorescent characteristics of thousands of individual cells, were then comprehensively analyzed using Cell Quest software from DB Biosciences. In this context, the resultant fluorescent levels, reflective of the cellular parameters under investigation, were typically expressed as the mean fluorescence intensity across the population. Furthermore, for a more nuanced understanding of cellular responses, these values were frequently calculated as either a fold change relative to the untreated control group or as a percentage, providing detailed and robust insights into the distribution, magnitude, and heterogeneity of the fluorescent signals within the entire cell population.
Staining Of Annexin V And 7-Aminoactinomycin D
To meticulously characterize the specific mode of cell death occurring within the experimental conditions, and particularly to differentiate it from classical apoptosis, the process of ferroptosis was rigorously determined by adhering to established methodologies as detailed in prior scientific literature. This comprehensive approach is crucial for understanding the distinct mechanisms of cell demise in response to various stimuli. Following the completion of the specific treatments, cells were carefully harvested and subsequently subjected to a dual staining procedure utilizing two distinct fluorescent reagents: Annexin V-FITC and 7-aminoactinomycin D (7-AAD).
Annexin V is a protein that exhibits a high affinity for phosphatidylserine, a phospholipid normally sequestered to the inner leaflet of the plasma membrane. During the early stages of apoptosis, phosphatidylserine translocates to the outer leaflet of the cell membrane, a phenomenon known as externalization. By conjugating Annexin V with a fluorescein isothiocyanate (FITC) fluorochrome, its binding to externalized phosphatidylserine provides a highly sensitive and widely accepted marker for early apoptotic events, long before the cell membrane fully loses its integrity. In parallel, 7-aminoactinomycin D is a membrane-impermeable DNA-intercalating dye. Its inability to cross an intact cell membrane means that it can only enter and stain cells that have lost their membrane integrity, a characteristic feature of later stages of cell death, encompassing both advanced apoptosis and necrotic processes. The combination of these two dyes allows for a clear distinction between viable cells (negative for both stains), early apoptotic cells (Annexin V positive, 7-AAD negative), late apoptotic or necrotic cells (positive for both Annexin V and 7-AAD), and primary necrotic cells (Annexin V negative, 7-AAD positive, though less common in typical apoptotic induction).
Once stained, the cell populations were meticulously analyzed by flow cytometry. This powerful technique facilitates the quantitative assessment of individual cell populations based on their distinct fluorescent properties, enabling the precise enumeration of cells within each stage of cell death. The percentages of cells exhibiting characteristic signs of death, as precisely indicated by the specific fluorescent patterns generated by these markers, were meticulously quantified utilizing the specialized CellQuest software. This sophisticated dual staining strategy is therefore indispensable, offering a robust and precise means to differentiate between the specific mechanisms of cell demise observed under diverse experimental conditions, contributing significantly to a deeper understanding of cellular responses to stress and intervention.
Immunofluorescence Staining
For the purpose of visualizing specific intracellular proteins and assessing morphological changes at a subcellular level, cells were meticulously prepared for immunofluorescence staining. To facilitate subsequent microscopic analysis, cells were initially grown on sterile coverslips, which were carefully embedded within a 6-well plate. This setup allows for the direct observation of cells adhering to a solid support. Following the application of various experimental treatments, the cells underwent a critical fixation step with 3.7% (w/v) paraformaldehyde. This chemical cross-links proteins and preserves cellular morphology, effectively freezing the cells in a life-like state and preventing degradation. Subsequently, the cells were permeabilized with 0.2% (v/v) Triton X-100, a non-ionic detergent. Permeabilization is essential as it creates pores in the cell membranes, allowing the larger antibody molecules to access intracellular targets.
To prevent non-specific binding of antibodies to cellular components, a blocking step was performed using a solution of 2% (w/v) bovine serum albumin (BSA) in phosphate-buffered saline (PBS). BSA acts as a decoy, saturating any non-specific binding sites on the fixed and permeabilized cells. Following successful blocking, the specific target proteins were detected using a two-tiered antibody approach. Primary antibodies, specifically anti-PDI (protein disulfide isomerase) and anti-HO-1 (heme oxygenase-1) antibodies, were applied. These primary antibodies possess high specificity for their respective target proteins. Subsequently, the cells were incubated with highly sensitive secondary antibodies. These secondary antibodies, which were conjugated with fluorophores such as Alexa Fluor® 488 and Alexa Fluor® 594 (from Invitrogen), are designed to bind specifically to the primary antibodies. This secondary antibody approach provides significant signal amplification, enhancing the detection of the target proteins.
In a parallel set of experiments focused on the determination of mitochondrial morphology, treated cells were pre-incubated with MitoTracker® Red CMXRos for 30 minutes just prior to the conclusion of the experiment. MitoTracker Red is a live-cell dye that accumulates in active mitochondria in a membrane potential-dependent manner, allowing for visualization of their intricate network and structural integrity. After this vital staining, the cells were fixed and permeabilized, as described above. Following the blocking step, these cells were then stained using a LAMP-1 (lysosomal-associated membrane protein 1) antibody, a marker for lysosomes, to assess lysosomal structures and their potential interactions with mitochondria. This primary antibody was subsequently detected using an Alexa Fluor® 488-conjugated secondary antibody. Finally, the prepared coverslips were carefully mounted onto microscope slides, and high-resolution fluorescence images were meticulously captured using a state-of-the-art Leica Microsystems TCS SP8 Confocal Spectral microscope. Confocal microscopy offers superior optical sectioning capabilities, enabling the acquisition of crisp, in-focus images by eliminating out-of-focus fluorescence, thus providing highly detailed insights into protein localization and organelle morphology within the cellular context.
Determination Of The Labile Iron Pool
The total cellular labile iron pool (LIP), representing the chelatable and redox-active fraction of intracellular iron, is a dynamic and crucial component involved in numerous cellular processes, including metabolism, proliferation, and oxidative stress. Its precise quantification is vital for understanding cellular iron homeostasis and dysregulation. The detection of this labile iron pool was performed meticulously based on the established calcein-acetoxymethyl ester (calcein-AM) method. Calcein-AM is a cell-permeable fluorescent probe that, once inside the cell, is hydrolyzed by esterases into calcein, a highly fluorescent molecule. Calcein’s fluorescence is quenched upon binding to intracellular labile iron, making it an effective reporter of iron levels.
The experimental procedure commenced with the trypsinization of cells to achieve a single-cell suspension, followed by two thorough washes with phosphate-buffered saline (PBS) to remove residual medium and trypsin. Subsequently, the cells were incubated with 0.2 µM of calcein-acetoxymethyl ester (sourced from Enzo) for 15 minutes at 37 degrees Celsius. During this incubation, calcein-AM readily enters the cells and is converted to its fluorescent form, which then binds to and is quenched by the intracellular labile iron. After the initial calcein-AM loading, the cells were again washed with PBS to remove any extracellular probe. To specifically determine the labile iron pool, a portion of these calcein-loaded cells was then incubated for 1 hour at 37 degrees Celsius with or without deferiprone (at a concentration of 100 µM). Deferiprone is a well-known, high-affinity iron chelator that effectively binds to and removes iron from calcein, thereby de-quenching its fluorescence.
The cells were then subjected to analysis by flow cytometry. This technique enabled the quantitative measurement of the fluorescence intensity of individual cells. The critical aspect of this method lies in the comparative analysis: the levels of the labile iron pool were precisely calculated by determining the difference in the cellular mean fluorescence intensity between the cells incubated without deferiprone (where calcein fluorescence is quenched by LIP) and those incubated with deferiprone (where calcein fluorescence is maximal due to iron chelation). A higher difference in fluorescence intensity between these two conditions directly correlates with a larger labile iron pool, providing a robust and quantitative measure of this critical cellular iron fraction.
Quantitative Real-Time Reverse Transcription PCR And XBP1 mRNA Splicing
To accurately quantify gene expression levels and analyze specific mRNA splicing events, two complementary molecular biology techniques were employed: quantitative real-time reverse transcription PCR (qRT-PCR) and conventional reverse transcription PCR for XBP1 mRNA splicing. The initial and critical step for both methods involved the meticulous extraction of total RNA from the treated cellular samples using TRIzol® Reagent, a highly effective monophasic solution developed by ThermoFisher. This reagent ensures the efficient isolation of high-quality total RNA, free from protein and DNA contamination, which is paramount for reliable downstream applications.
Following RNA extraction, five micrograms of the purified total RNA served as a template for the synthesis of complementary DNA (cDNA). This crucial step, known as reverse transcription, was performed using M-MLV reverse transcriptase (ThermoFisher) according to the manufacturer’s precise instructions. The conversion of RNA into cDNA is essential because PCR amplification works with DNA templates, not RNA. The resulting cDNA was then utilized for qRT-PCR analysis, which was carried out using a LightCycler® 480 II Real-Time PCR system (Roche Applied Sciences, Manheim, Germany). This system allows for the real-time detection and quantification of PCR products as they accumulate, providing a highly sensitive and precise measure of initial mRNA transcript levels. The amplification reaction was performed using KAPA SYBR FAST qPCR Master Mix (Kapa Biosystems), which contains a fluorescent dye (SYBR Green) that intercalates into double-stranded DNA, emitting a signal proportional to the amount of amplified product. The specificity and efficiency of the PCR reactions were ensured by using meticulously designed PCR primers, which were detailed in a supplementary methods table. To account for variations in RNA input and reverse transcription efficiency across different samples, the expression levels of the target messenger RNAs were rigorously normalized against the level of beta-actin mRNA within the same sample. Beta-actin serves as a widely accepted housekeeping gene, as its expression is generally stable across various cellular conditions, providing a reliable internal control for relative quantification.
In parallel, the assessment of XBP1 mRNA splicing, a crucial indicator of endoplasmic reticulum (ER) stress, was conducted using a conventional Reverse Transcription-PCR approach. The XBP1 mRNA undergoes unconventional splicing by the IRE1α endoribonuclease during ER stress, leading to a frameshift and the production of a potent transcription factor. This splicing event results in a shorter, spliced mRNA variant. For this assay, specific primers targeting both the unspliced and spliced forms of XBP1 were utilized, with beta-actin-specific primers again serving as a crucial control for overall RNA integrity and input. The PCR conditions were carefully optimized to ensure efficient amplification: an initial denaturation at 95°C for 3 minutes, followed by 35 cycles consisting of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 30 seconds. After amplification, the PCR products, representing both the spliced and unspliced XBP1 variants, were meticulously separated based on their size using electrophoresis on a 3% agarose gel. The distinct bands corresponding to the different XBP1 mRNA forms were then visualized and documented using a GelDoc Imaging System (Bio-Rad), allowing for a qualitative and semi-quantitative assessment of the XBP1 splicing status.
Gene Knockdown By ShRNA
To investigate the functional significance of specific genes in cellular processes, gene knockdown experiments were meticulously performed utilizing short hairpin RNA (shRNA) technology. This approach allows for the targeted reduction of gene expression, providing valuable insights into gene function. The essential genetic components for lentivirus-mediated gene knockdown were obtained from the National RNAi Core Facility Academia Sinica (Taiwan). These included the pCMV-∆R8.91 and pMD.G plasmids, which serve as packaging vectors essential for producing replication-incompetent lentiviral particles, and specific short hairpin PLKO.1 plasmids, which carry the genetic information for the desired shRNA sequence designed to target a particular messenger RNA for degradation. The specific shRNA clones utilized in this study were comprehensively detailed in a supplementary methods table, ensuring full transparency and reproducibility.
The process began with the transient transfection of 293T cells, a commonly used human embryonic kidney cell line known for its high transfectability, which acts as the lentiviral packaging cell line. The shRNA plasmid along with the packaging vectors (pCMV-∆R8.91 and pMD.G) were introduced into 293T cells using Lipofectamine 2000 transfection reagent (Invitrogen), a lipid-based reagent that efficiently delivers DNA into cells. This co-transfection leads to the production of infectious, yet replication-defective, lentivirus particles carrying the shRNA construct. Forty-eight hours after transduction, the supernatant containing the newly generated lentivirus particles was collected. To ensure that only viral particles and not cellular debris were present, the supernatant was carefully filtered through a 0.22 µm filter.
These filtered lentivirus particles were then used to infect the target cancer cells. To enhance the efficiency of viral transduction, polybrene, a cationic polymer, was added to the viral-contained supernatant at a concentration of 8 µg/ml during the infection step. Polybrene helps to neutralize the negative charges of cell surface and viral envelope, facilitating the interaction and entry of lentiviruses into the cells. After 24 hours of incubation at 37 degrees Celsius, allowing sufficient time for viral integration and initial expression, the medium was replaced with complete culture medium supplemented with puromycin at a concentration of 2 µg/ml. Puromycin is an antibiotic that serves as a selection agent; the pLKO.1 shRNA plasmid contains a puromycin resistance gene, allowing only cells that have successfully integrated the shRNA construct to survive and proliferate. This selection process ensures the generation of stable cell lines with sustained gene knockdown. Once the selection was complete and stable knockdown cell lines were established, the cells were prepared and harvested based on the specific requirements of the downstream experiments, allowing for robust functional studies of the targeted gene.
Overexpression Of HO-1 And SLC7A11
To investigate the effects of increased gene expression on cellular processes, overexpression experiments were meticulously conducted for specific target genes, heme oxygenase-1 (HO-1) and SLC7A11 (system xc- light chain subunit). This approach, often termed gain-of-function studies, complements gene knockdown experiments by allowing researchers to observe the consequences of elevated protein levels. Cancer cells intended for these experiments were initially cultured in either a culture dish or a culture plate and allowed to attach securely overnight, ensuring they were in a stable and healthy state prior to genetic manipulation.
The genetic constructs used for overexpression were carefully chosen. These included an empty vector, either pCMV-XL5 (sourced from OriGene) or pcDNA3.1+/C-(K)DYK (from GeneScript), which served as crucial negative controls. These empty vectors contain all the necessary elements for gene expression but lack the specific cDNA insert, allowing researchers to account for any effects solely attributed to the transfection process or vector backbone. For the overexpression of the target genes, specific expression vectors containing the human HMOX1 cDNA (OriGene) or human SLC7A11 cDNA (GeneScript) were utilized. These vectors are designed to drive high levels of constitutive expression of the inserted genes within mammalian cells.
The process of introducing these plasmids into the cancer cells was carried out through transient transfection, a common and efficient method for delivering exogenous DNA into eukaryotic cells. The cells were incubated with the chosen empty vector or the vector containing the specific human cDNA for a period of 24 hours. During this incubation, the cells take up the plasmid DNA, and the cellular machinery then transcribes and translates the genetic information, leading to the transient overexpression of the target protein. After this crucial 24-hour period, the cells were then prepared for subsequent experiments, allowing for the comprehensive analysis of the physiological and biochemical consequences of elevated HO-1 or SLC7A11 expression.
Isolation Of Cytosolic And Nuclear Protein
The precise localization of proteins within different cellular compartments is fundamental to understanding their function. To achieve this, a meticulous protocol for the isolation of distinct cytosolic and nuclear protein fractions was implemented. This method allows for the separate analysis of proteins residing in the cytoplasm versus those localized to the nucleus.
Following the application of various experimental treatments, the cells were first carefully washed with phosphate-buffered saline (PBS) to remove any residual medium. Subsequently, the cells were detached from the culture surface using trypsinization, yielding a single-cell suspension. These cells were then gently suspended in a hypotonic buffer, meticulously prepared to induce cellular swelling without causing immediate lysis of the nuclear membrane. This buffer contained 10 mM HEPES at pH 7.9, 1.5 mM MgCl2, and crucially, a comprehensive cocktail of proteinase inhibitors and phosphatase inhibitors. The inclusion of these inhibitors is paramount to prevent the degradation of proteins by cellular proteases and to preserve their phosphorylation status, respectively, ensuring the integrity of the isolated fractions. The cell suspension was kept on ice for 15 minutes, allowing for sufficient swelling.
To selectively lyse the cell membranes and release the cytoplasm while preserving the integrity of the nuclei, 0.5% NP-40, a non-ionic detergent, was added to the suspension. The mixture was then vigorously vortexed for 10 seconds. This controlled lysis procedure ruptures the plasma membrane, releasing the cytoplasmic contents. The cellular mixtures were then subjected to differential centrifugation at 10,000 × g for 2 minutes at 4 degrees Celsius. This centrifugation step effectively separates the lighter cytoplasmic components, which remain in the supernatant, from the heavier nuclear fraction, which forms a compact pellet at the bottom of the tube.
For the subsequent purification of nuclear proteins, the nuclear pellets were carefully washed three times with hypotonic buffer. These washes are critical to remove any lingering cytoplasmic contaminants. After the washes, the purified nuclear pellets were resuspended in PBS containing both proteinase and phosphatase inhibitors. To ensure complete lysis of the nuclear membrane and release of nuclear proteins, the suspension was further subjected to sonication, which uses high-frequency sound waves to disrupt the nuclear envelopes. Finally, the sonicated nuclear suspension was centrifuged at 12,000 × g at 4 degrees Celsius for 15 minutes to pellet any insoluble debris, yielding the highly enriched nuclear protein supernatant. Both the isolated cytosolic and nuclear protein fractions were then promptly stored at -80 degrees Celsius to maintain their stability and integrity for subsequent biochemical analyses, such as Western blotting.
Isolation Of Mitochondrial And Cytosolic Protein Fractions
To specifically investigate proteins localized within mitochondria and compare their abundance or modification to their cytosolic counterparts, a distinct protocol for the differential isolation of mitochondrial and cytosolic protein fractions was meticulously executed. This method is based on established scientific literature, specifically adapting the approach described by Frezza et al. in 2007.
The initial step involved the careful preparation of cells, which were then gently suspended in a specialized Isolation Buffer. This buffer was precisely formulated to maintain cellular integrity and mitochondrial viability throughout the fractionation process, comprising 10 mM Tris/MOPs, 1 mM EGTA/Tris, 200 mM sucrose, 1 mM Na3VO4, 5 mM NaF, 1 mM PMSF, and 10 µg/ml each of leupeptin, aprotinin, and pepstatin A. The sucrose concentration is crucial for maintaining isotonic conditions, preventing osmotic lysis of organelles. The inclusion of EGTA/Tris helps to chelate divalent cations, while the comprehensive array of inhibitors (PMSF, leupeptin, aprotinin, pepstatin A, Na3VO4, NaF) is vital for preventing protein degradation by proteases and preserving phosphorylation states by inhibiting phosphatases, respectively.
Following suspension in the isolation buffer, the cells were homogenized using a Teflon pestle. This gentle mechanical disruption carefully ruptures the cell membranes, releasing the cellular contents, including mitochondria, into the buffer, while minimizing damage to the organelles themselves. The resulting homogenates were then subjected to a series of differential centrifugation steps. The first centrifugation was performed at a relatively low speed of 600 × g for 10 minutes at 4 degrees Celsius. This initial spin effectively pellets larger cellular debris, unbroken cells, and nuclei, which are denser components. The supernatant, now enriched with mitochondria and cytosolic components, was carefully collected.
This supernatant was then subjected to a second, higher-speed centrifugation at 7000 × g for 10 minutes at 4 degrees Celsius. At this centrifugal force, the mitochondria, which are smaller and less dense than nuclei, are selectively pelleted at the bottom of the tube. The supernatant remaining after this step primarily contains the cytosolic protein fraction. The resulting pellets, highly enriched with mitochondria, were carefully collected and stored. These isolated mitochondrial fractions were then readily available for subsequent mitochondrial-specific assays or protein analysis, ensuring that biochemical investigations could accurately reflect events occurring within this vital organelle.
Western Blot Analysis
Western blot analysis stands as a fundamental and indispensable technique within the realm of molecular biology. It is extensively employed for the qualitative and semi-quantitative detection of specific proteins present within complex biological samples, thereby enabling researchers to meticulously assess their expression levels, identify various post-translational modifications, and even explore intricate protein-protein interactions. The experimental workflow for this powerful technique commenced with the meticulous preparation of cellular lysates. This crucial initial step involved suspending the treated cells in a precisely formulated phosphate-buffered saline (PBS), which was thoughtfully supplemented with a comprehensive cocktail of highly potent inhibitors. This inhibitor blend included 1 mM PMSF (phenylmethylsulfonyl fluoride), along with 10 µg/ml each of leupeptin, aprotinin, and pepstatin A, all acting as proteinase inhibitors to safeguard against the proteolytic degradation of cellular proteins. Additionally, the cocktail contained 1 mM Na3VO4 (sodium orthovanadate) and 1 mM NaF (sodium fluoride), which function as phosphatase inhibitors to meticulously preserve the phosphorylation status of proteins. The diligent inclusion of these inhibitors is of paramount importance, as it ensures the utmost integrity of the proteins throughout the extraction process, thereby guaranteeing the reliability and accuracy of subsequent analyses. Following the addition of these protective agents, the cell suspensions were then subjected to sonication, a controlled process that utilizes high-frequency sound waves to effectively disrupt cell membranes, leading to the complete lysis of cells and the efficient solubilization of their entire protein contents into the buffer.
Subsequent to the successful preparation of cell lysates, the total protein concentration in each sample was precisely determined using a specialized protein assay kit from Bio-Rad. Achieving accurate and consistent protein quantification at this stage is absolutely essential, as it ensures that equal amounts of protein are loaded into each well for comparative analysis, a prerequisite for obtaining reliable and quantitatively meaningful results. After quantification, the prepared lysate proteins were denatured, typically by heating in the presence of a strong anionic detergent, sodium dodecyl sulfate (SDS). This denaturation process unfolds the proteins and coats them with negative charges, imparting a uniform charge-to-mass ratio. These denatured proteins were then meticulously separated based on their molecular weight using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). During this technique, the protein samples are loaded into wells formed within a polyacrylamide gel matrix. An electric current is then applied, which drives the uniformly negatively charged proteins through the gel. Smaller proteins encounter less resistance and therefore migrate more rapidly through the porous gel matrix, resulting in their effective separation by size. Upon completion of electrophoretic separation, the resolved proteins were then efficiently transferred from the delicate polyacrylamide gel onto FluoroTrans® PVDF (polyvinylidene difluoride) Transfer Membranes, supplied by Pall Corporation. PVDF membranes are a preferred choice due to their inherently high protein binding capacity, excellent mechanical strength, and chemical resistance, which together ensure robust protein immobilization and stability for subsequent detection steps.
To effectively prevent any undesirable non-specific binding of antibodies in the subsequent crucial steps, the PVDF membranes were subjected to a thorough blocking procedure. This involved incubating the membranes in a blocking solution consisting of Tris-buffered saline supplemented with 0.1% Tween 20 (a non-ionic detergent to reduce background) and a significant 5% concentration of skim milk. The proteins present in skim milk effectively saturate any unoccupied or non-specific binding sites on the membrane surface, thereby significantly minimizing background signal and profoundly enhancing the specificity of the antibody binding to the target proteins. Following this effective blocking step, the membranes were then incubated with the appropriate primary antibodies. These primary antibodies are meticulously engineered to exhibit high specificity and affinity, ensuring they bind directly and selectively to the target proteins of interest. After a sufficient incubation period to allow for optimal binding, any unbound primary antibodies were thoroughly washed away. Subsequently, the membranes were reacted with horseradish peroxidase (HRP)-conjugated secondary antibodies, which were sourced from EMD Millipore. These secondary antibodies are meticulously designed to bind specifically to the primary antibodies and are covalently linked to HRP, an enzyme that plays a pivotal role in the detection phase.
Finally, the protein signaling was rendered visible through a chemiluminescent reaction. This was achieved by carefully applying ClarityTM Western ECL Substrate, a specialized reagent from Bio-Rad, to the membranes. This substrate contains luminol and an enhancing agent that, in the presence of HRP (conjugated to the secondary antibody), undergoes a chemical reaction that produces a detectable light signal. The intensity of this emitted luminescence is directly proportional to the amount of target protein present on the membrane, thus providing a quantitative measure. The resulting chemiluminescent signal was then meticulously captured and precisely quantified using a highly sensitive luminescence image analyzer, specifically the ImageQuant LAS 4000 system from GE Healthcare Life Sciences. This advanced imaging system offers superior capabilities for the precise detection and accurate quantification of protein bands, thereby yielding robust, reproducible, and highly interpretable data on protein expression levels across the diverse range of experimental conditions investigated.
Statistical Analysis
To ensure the rigor, reliability, and validity of the experimental findings, all experiments were meticulously conducted a minimum of three independent times. This replication strategy is fundamental for establishing the reproducibility of the results and strengthening the statistical power of the analysis. The quantitative data derived from these experiments are consistently presented as the mean value accompanied by its standard error of the mean (SEM). The SEM provides a measure of the precision of the estimated mean, indicating how closely the sample mean represents the true population mean.
For the rigorous determination of statistical significance among the various experimental treatments, a t-test was employed. The t-test is a widely accepted parametric statistical test that is particularly suitable for comparing the means of two groups to ascertain if they are significantly different from each other. This test calculates a t-statistic, which is then used to determine a p-value. A p-value is a crucial metric that quantifies the probability of observing data as extreme as, or more extreme than, the obtained data, assuming that the null hypothesis (i.e., no difference between groups) is true. In this study, a p-value of less than 0.05 was pre-established as the threshold for considering the observed differences to be statistically significant. This widely adopted threshold implies that there is less than a 5% chance that the observed difference occurred by random chance, thereby providing a high degree of confidence in the experimental conclusions regarding the impact of the treatments.
Results
The following sections detail the comprehensive findings derived from the array of experimental methodologies outlined previously. These results systematically elucidate the mechanisms by which the compound BAY exerts its biological effects, particularly focusing on its impact on cellular viability, its interaction with key cellular pathways, and its influence on various intracellular processes related to stress response and iron homeostasis. Each subsection presents a progression of findings, building a coherent narrative that supports the ultimate conclusions regarding the compound’s mode of action.
BAY Induces a NF-κB-Independent Cell Death
Initial investigations into the impact of BAY revealed a profound and drastic inhibition of cell viability across a diverse panel of human breast cancer cell lines. Specifically, treatment with BAY markedly suppressed the viability of MCF-7, MDA-MB-231, MDA-MB-468, and SKBR3 breast cancer cells. The inhibitory concentration 50% (IC50), which represents the concentration of the compound required to inhibit cell viability by fifty percent, was precisely determined for each cell line after a 24-hour treatment period. These values were approximately 7.03 ± 0.07 µM for MCF-7, 4.31 ± 0.6 µM for MDA-MB-231, 5.21 ± 0.14 µM for MDA-MB-468, and 4.10 ± 0.01 µM for SKBR3 cells, demonstrating potent and consistent efficacy across different breast cancer subtypes.
Beyond quantitative assessment, careful morphological examinations of the treated cells unveiled distinct cellular alterations. A striking observation was the extensive accumulation of numerous vacuoles, particularly localized within the perinuclear and cytoplasmic areas of the cells. This characteristic vacuolization provides an initial visual clue regarding the unique nature of the cell death mechanism induced by BAY, distinguishing it from typical apoptotic or necrotic hallmarks. The broad spectrum anti-cancer activity of BAY was further corroborated by its observed suppression of cell viability in a range of other human cancer cell types. These included SKOV3 ovary cells, A549 lung cells, HuH-7 hepatoma cells, and DBTRG-05MG glioblastoma multiforme (GBM) cells. For these additional cell lines, the IC50 values after 24 hours of treatment were approximately 7.31 ± 0.09 µM for SKOV3, 4.66 ± 0.1 µM for A549, 5.84 ± 0.03 µM for HuH-7, and 5.53 ± 0.11 µM for DBTRG-05MG cells, underscoring the compound’s broad cytotoxic efficacy across different cancer origins.
To ascertain whether the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway played a role in BAY-mediated cell death, its key components were meticulously investigated. Treatment with BAY resulted in a rapid and notable inhibition of the phosphorylation of IκBα, a critical inhibitor of NF-κB, and concurrently, a reduction in the phosphorylation of the NF-κB subunit p65. This observed suppression of phosphorylation occurred without any discernible changes in the total protein levels of IκBα and p65, suggesting that BAY primarily affects the activation status rather than the overall abundance of these proteins. Interestingly, neither the phosphorylation status nor the total protein levels of IKKα and IKKβ, which are upstream kinases crucial for IκBα phosphorylation and subsequent NF-κB activation, were significantly affected by BAY treatment. To definitively rule out the involvement of the NF-κB pathway, a genetic intervention strategy was employed. Cells engineered with short hairpin RNA (shRNA) for targeted knockdown of IκBα and p65 were utilized. Despite the significant reduction in IκBα and p65 expression in these knockdown cells, BAY consistently maintained its potent ability to inhibit cell viability in a concentration-dependent manner. Although the intervention of IκBα and p65 knockdown did lead to a slight, albeit minor, suppression of cell death, this marginal effect was insufficient to counteract the robust cytotoxicity induced by BAY. Consequently, these comprehensive findings unequivocally established that the NF-κB pathway is not a primary mediator in BAY-mediated cell death, pointing towards an alternative, NF-κB-independent mechanism.
BAY Suppresses Cell Viability Through ROS Generation
To comprehensively define the precise type of cell death induced by BAY, a series of experiments were conducted employing a diverse panel of pharmacological inhibitors, each targeting a distinct cell death pathway. These inhibitors included z-VAD-fmk, a pan-caspase inhibitor widely used to block apoptosis; necrostatin-1, an inhibitor of receptor-interacting protein kinase 1 (RIPK1) known to prevent necrosis and necroptosis; ferrostatin-1 and liproxstatin-1, specific inhibitors of ferroptosis that target lipid peroxidation; N-acetyl-L-cysteine (NAC), a potent antioxidant and glutathione precursor; and L-NAME, an inhibitor of nitric oxide synthase. The results revealed that BAY-induced cell death was significantly attenuated and consequently rescued in the presence of ferrostatin-1, liproxstatin-1, and NAC. This crucial observation strongly implicated ferroptosis and oxidative stress in the mechanism of BAY’s cytotoxicity. In stark contrast, z-VAD-fmk, necrostatin-1, and L-NAME failed to significantly ameliorate BAY-induced cell death, thereby effectively excluding the involvement of classical apoptosis and necrosis/necroptosis as the primary modes of cell demise.
Further supporting these findings, cell viability was rigorously assessed using dual staining with Annexin V and 7-aminoactinomycin D (7-AAD), followed by analysis via flow cytometry. As clearly demonstrated, BAY treatment led to a significant increase in the population of 7-AAD-positive cells, indicating a loss of membrane integrity characteristic of later stages of cell death or necrosis. However, there was only a minimal increase in Annexin V staining, which typically marks early apoptosis by detecting phosphatidylserine externalization. This distinctive staining pattern, characterized by high 7-AAD positivity and low Annexin V binding, further argued against classical apoptosis as the predominant mechanism. Consistent with the previous inhibitor studies, the addition of ferrostatin-1, liproxstatin-1, and NAC effectively suppressed BAY-induced cell death, reinforcing their protective roles. Furthermore, zileuton, a 5-lipoxygenase inhibitor known to suppress lipid and protein oxidation and to specifically inhibit ferroptosis, also significantly reversed the cell death induced by BAY. Collectively, these compelling results decisively excluded the primary involvement of both apoptosis and necrosis/necroptosis in BAY-induced cell death, instead strongly suggesting an alternative type of cell death that is unequivocally dependent on the robust induction of reactive oxygen species (ROS) and subsequent lipid peroxidation.
Building upon the established understanding that ferroptosis is critically dependent on ROS for its cytotoxic effects, the direct impact of BAY on total cellular ROS generation was meticulously monitored using the fluorogenic dye 2’,7’-dichlorofluorescein diacetate (DCFH-DA). This dye readily diffuses into cells and is cleaved by esterases, becoming fluorescent upon oxidation by ROS. BAY was found to rapidly and significantly trigger a robust production of ROS in both MDA-MB-231 and MDA-MB-468 breast cancer cells. This ROS generation was observed to be both concentration-dependent, with higher BAY concentrations leading to more ROS, and time-dependent, indicating a progressive increase over time. Complementing the ROS measurements, treatment with BAY also significantly increased the levels of lipid peroxidation, a hallmark event in ferroptosis. The extent of lipid peroxidation induced by BAY was notably robust, demonstrating levels slightly less than those induced by hydrogen peroxide (H2O2), a potent oxidant, but distinctly stronger than those elicited by erastin, a well-characterized ferroptosis inducer. In contrast, rotenone, a mitochondrial complex I inhibitor known to enhance general ROS generation but not necessarily lipid peroxidation directly, had no significant effect on lipid peroxidation in this context. Furthermore, to ascertain the independence of ROS generation from the NF-κB pathway, experiments were conducted in cells with IκBα and p65 knockdown. Even under these conditions, BAY maintained its effective capacity to induce ROS generation, reinforcing that this effect is upstream or independent of NF-κB. Critically, NAC treatment completely prevented the suppression of IκBα and p65 phosphorylation by BAY, even at early time points (as little as 0.25 hours). This strong inhibitory effect of NAC on NF-κB pathway modulation, coupled with its ability to rescue cell death, strongly suggested that BAY exerts its primary cytotoxicity by operating fundamentally through the generation of reactive oxygen species.
BAY Upregulates the Redox-Responsive Proteins
Further exploration into the mechanisms underlying BAY-induced cell death revealed a significant disruption in cellular redox homeostasis. Treatment with BAY caused a concentration-dependent suppression of intracellular glutathione (GSH) levels, a crucial endogenous antioxidant and a vital component of the cellular defense system against oxidative stress. To confirm the direct involvement of GSH depletion in BAY’s cytotoxic effects, exogenous supplementation experiments were performed. The addition of reduced GSH (GSH itself) completely abolished the cell death induced by BAY, effectively rescuing cell viability. In stark contrast, the supplementation of oxidized glutathione (GSSG) had no such protective effect, unequivocally demonstrating that it is the availability of reduced glutathione that is critical for cellular defense against BAY’s action.
To further investigate the role of oxidative stress, the impact of thiol-containing reducing agents was examined. The administration of β-mercaptoethanol (βMe) or dithiothreitol (DTT), both potent reducing agents known to replenish cellular thiol pools and counteract oxidative stress, effectively blunted the robust ROS generation induced by BAY. Concomitantly, these reducing agents successfully rescued cell viability, further cementing the role of oxidative stress as a primary mediator of BAY’s cytotoxicity. Additionally, βMe and DTT were observed to attenuate the suppression of IκBα and p65 phosphorylation induced by BAY. This observation is highly significant as it suggests that the oxidative stress-related events triggered by BAY also play a role in mediating its inhibitory effect on IκBα/NF-κB phosphorylation. Crucially, this finding further reinforces the notion that the induction of oxidative cell death by BAY is fundamentally irrelevant to the activation status of the IκBα/NF-κB pathway. Taken together, these results provided compelling evidence that BAY exerts its profound cytotoxic effect by acting primarily on cellular redox regulation, culminating in a specific form of cell death characteristic of ferroptosis.
Moving deeper into the molecular mechanisms, the expression of genes associated with cellular redox response, as well as pathways related to autophagy and endoplasmic reticulum (ER) stress (which have previously been shown to be responsive to ferroptotic agents), was systematically examined. Treatment with BAY was found to significantly upregulate the gene expression of numerous factors involved in oxidative stress and ER stress responses. These included HMOX1 (encoding heme oxygenase-1), SLC7A11 (encoding the light chain of the cystine-glutamate antiporter system xc-), HSPA5 (encoding GRP78/BiP, a master regulator of ER homeostasis), and DDIT3 (encoding CHOP, a transcription factor induced during ER stress). Furthermore, genes crucial for cellular redox regulation and components of autophagy pathways also exhibited significant upregulation. This broader upregulation encompassed GSTp (glutathione S-transferase pi), GSR (glutathione reductase), GCLC (glutamate-cysteine ligase catalytic subunit), NFE2L2 (encoding Nrf2, a master regulator of antioxidant response), TXNRD1 (thioredoxin reductase 1), TXNIP (thioredoxin-interacting protein), NQO1 (NAD(P)H quinone oxidoreductase 1), XBP1 (X-box binding protein 1), GADD45A (growth arrest and DNA damage-inducible 45 alpha), HSP90B1 (heat shock protein 90 beta 1), and SQSTM1 (sequestosome 1/p62). This comprehensive gene expression analysis painted a picture of a broad and intricate cellular response to the oxidative stress induced by BAY.
Further protein level analysis corroborated these transcriptional changes. Specifically, the abundance of HO-1 and SLC7A11 proteins (encoded by HMOX1 and SLC7A11, respectively) was notably increased as early as 4 hours after BAY treatment and remained elevated up to 8 hours, indicating sustained upregulation. Nrf2, a known upstream transcription factor critical for the induction of both HO-1 and SLC7A11 in response to ROS and electrophilic stress, exhibited a dramatic and rapid increase in its protein levels within 1 hour of BAY treatment, which also persisted for up to 8 hours. Concomitantly, KEAP1 (Kelch-like ECH-associated protein 1), a repressor of Nrf2, showed a decrease in abundance after 1 hour, which would contribute to Nrf2 stabilization and activation. Interestingly, GPx4 (glutathione peroxidase 4), a pivotal ferroptosis repressor that provides an essential antioxidative defense against lipid peroxidation, showed minimal or negligible change in its protein levels. This distinct pattern of gene and protein upregulation, particularly of Nrf2-responsive elements, strongly suggested that BAY triggers a multifaceted cellular defense response aimed at counteracting the induced oxidative stress, yet without a compensatory increase in the key ferroptosis-suppressing enzyme, GPx4.
BAY Induces Cell Death Via Upregulation of HO-1
To decisively elucidate the critical role of heme oxygenase-1 (HO-1) in BAY-induced ferroptosis, a dual investigational approach was meticulously employed, combining both pharmacological inhibition and genetic knockdown strategies using shRNA. Treatment with zinc protoporphyrin-9 (ZnPP), a well-established and specific pharmacological inhibitor of HO-1 activity, effectively and significantly attenuated the cell death induced by BAY. This protective effect highlighted HO-1’s direct involvement in the cytotoxic process. Complementing this, a genetic approach utilizing shRNA to achieve defective HO-1 expression also significantly rescued cell survival that was suppressed by BAY, providing independent and robust evidence for the indispensable role of HO-1 in mediating the compound’s effects.
The investigation then turned to the upstream regulatory molecules governing HO-1 expression. Experiments involving defective Nrf2 expression, achieved through shRNA knockdown, clearly demonstrated a blunting effect on the levels of both SLC7A11 and HO-1. This indicated that Nrf2 acts upstream of both these proteins, orchestrating their expression in response to cellular stress. However, when SLC7A11 deficiency was induced, it selectively abolished the increase in HO-1 levels by BAY, while having no discernible effects on Nrf2 abundance. This suggests a direct regulatory link between SLC7A11 and HO-1 that is downstream of Nrf2. Conversely, defective HO-1 expression itself had no marked effects on the levels of Nrf2 or SLC7A11. Taken together, these sophisticated genetic manipulation experiments strongly suggested a sequential regulatory hierarchy in response to BAY induction, specifically proposing a Nrf2–SLC7A11–HO-1 axis.
Further exploring the subcellular dynamics of these proteins, BAY treatment was observed to significantly increase the translocation of cytosolic Nrf2 and HO-1 into the nuclei, indicating their active roles in transcriptional regulation and cellular response. In contrast, the NF-κB subunit proteins, p50 and p65, remained predominantly in the cytosol, further substantiating the NF-κB-independent nature of BAY’s mechanism. Remarkably, BAY also significantly increased the compartmentalization of HO-1 within mitochondrial fractions, suggesting a novel role for HO-1 in mitochondrial processes during ferroptosis. Concurrently, BAY suppressed the abundance of cytosolic thioredoxin 1 (TXN1), a key antioxidant enzyme, and the mitochondrial outer membrane protein TOM20 (translocase of outer mitochondrial membrane 20), which is involved in protein import into mitochondria and whose reduction can indicate mitochondrial dysfunction. To further reinforce the connection between oxidative stress and these protein dynamics, treatment with antioxidants such as NAC, βMe, and DTT effectively blunted the BAY-induced increase of Nrf2, HO-1, and SLC7A11 abundance. These antioxidants also prevented the nuclear translocation of Nrf2 and HO-1 induced by BAY, confirming that the oxidative stress is a primary driver of these molecular events. Importantly, even in cells where IκBα and p65 were knocked down, BAY remained effective in promoting Nrf2 stabilization and suppressing KEAP1, further solidifying the conclusion that BAY’s effects on the Nrf2-HO-1 pathway are independent of the NF-κB pathway.
SLC7A11 Inhibition Sensitizes Cancer Cells To Ferroptotic Death Induction By BAY
The cystine-glutamate antiporter system xc-, specifically its light chain subunit SLC7A11, plays an absolutely essential and well-documented role in the intricate process of ferroptosis. SLC7A11 is responsible for the uptake of extracellular cystine, which is a rate-limiting step for intracellular glutathione synthesis. By facilitating this crucial import, SLC7A11 helps maintain intracellular glutathione levels, thereby providing antioxidant defense and suppressing ferroptosis. We therefore proceeded to further examine the profound impacts of SLC7A11 status on the cell death induced by BAY.
To provide a comparative context, other known SLC7A11 inhibitors, such as erastin, sulfasalazine (SAS), and sorafenib, were included in the investigation. These agents are known to induce ferroptosis primarily by blocking cysteine uptake and subsequently inhibiting glutathione synthesis, which often leads to a compensatory upregulation of SLC7A11 as cells attempt to counteract the blockade. When compared to erastin and SAS, BAY consistently induced a more substantial and robust increase in SLC7A11 expression, highlighting its potent capacity to modulate this crucial antiporter. Sorafenib, however, exhibited a more variable effect on SLC7A11 induction, showing different responses in MDA-MB-231 and DBTRG-05MG cells. Interestingly, despite their ability to induce ferroptosis, all three established ferroptosis-inducing agents (erastin, SAS, and sorafenib) consistently failed to elevate HO-1 expression, distinguishing their mechanism from that of BAY. When BAY was combined with erastin and SAS, the BAY-induced HO-1 levels remained unchanged, suggesting that their respective induction pathways for cell death are distinct but cooperative. However, the presence of sorafenib suppressed the HO-1 induction by BAY, indicating a potential antagonistic interaction or different regulatory pathways. Crucially, the combination of BAY with either erastin or SAS resulted in a marked increase in BAY-induced cell death, demonstrating a synergistic or additive cytotoxic effect when the SLC7A11 pathway is simultaneously targeted.
To further substantiate the role of SLC7A11, genetic knockdown experiments were performed. Deficient SLC7A11 expression, achieved through specific shRNA interference, significantly exacerbated the cell death induced by BAY in both MDA-MB-231 and DBTRG-05MG cells. This direct genetic evidence strongly confirmed that reducing SLC7A11 levels renders cancer cells significantly more vulnerable to BAY’s cytotoxic effects. Given that the efficacy of erastin and sorafenib is known to be dependent on SLC7A11 in gliomas and that increased SLC7A11 levels can ameliorate sorafenib and erastin-induced ferroptosis, we next conducted a reverse experiment to test whether BAY-inhibited cell viability could be reversed by the overexpression of SLC7A11. Indeed, the enforced overexpression of human SLC7A11-Flag (hSLC7A11) significantly blunted the BAY-induced increase in HO-1 levels and, more importantly, substantially reversed BAY-induced cell death. This unequivocal result demonstrated that the abundance of SLC7A11 is a critical determinant of the sensitivity of cells to the viability inhibition elicited by BAY, underscoring SLC7A11 as a key resistance factor against BAY’s ferroptotic action.
HO-1 Upregulation Elicits Cellular Ferrous Accumulation To Mediate Ferroptosis By BAY
Heme oxygenase-1 (HO-1) has been consistently highlighted across numerous scientific investigations for its capacity to exert pro-oxidant effects, particularly under conditions of stress. Crucially, its upregulation has been firmly associated with the induction of ferroptosis by various agents, including compounds like erastin. Given the compelling evidence from our previous experiments demonstrating that the genetic knockdown of HO-1 significantly blocked the ferroptotic cell death induced by BAY, we embarked on a more profound and detailed investigation. The aim was to precisely delineate the intricate role of HO-1 in mediating BAY-induced reactive oxygen species (ROS) generation and the subsequent lipid peroxidation, which are the defining characteristics of ferroptosis. To directly and unequivocally assess the cytotoxic contribution attributable to HO-1, we performed experiments involving the enforced overexpression of human HO-1 (hHO-1), tagged with a myc epitope, in cellular systems. Compared to control cells treated with an empty vector, the overexpression of hHO-1 by itself caused a profound and concentration-dependent decrease in cellular viability. This seminal finding unequivocally demonstrated that the mere elevation of HO-1 protein levels within a cell is sufficient to independently trigger a form of cell death, laying a strong foundation for its role in BAY’s mechanism.
The expression and activity of HO-1 are intricately involved in regulating cellular iron homeostasis. This involvement primarily stems from its enzymatic function in the catabolism of heme, a process that liberates ferrous iron (Fe2+). This ferrous iron, whether existing as free ions or within a chelatable pool, collectively constitutes what is referred to as the labile iron pool (LIP). It is widely recognized that the accumulation of LIP significantly contributes to cellular cytotoxicity, largely through the generation of deleterious reactive oxygen species via highly damaging Fenton reactions. Our meticulous measurements consistently revealed that LIP levels were markedly and significantly elevated not only following treatment with BAY but also in cells where hHO-1 was overexpressed. This direct correlation provided compelling evidence of a direct mechanistic link between HO-1 activity and an increase in intracellular labile iron. Furthermore, to provide robust pharmacological evidence for HO-1′s involvement, the administration of zinc protoporphyrin-9 (ZnPP), a well-established and specific inhibitor of HO-1 enzymatic activity, significantly prevented the increase of LIP levels induced by both BAY treatment and hHO-1 overexpression. Complementing this pharmacological approach, the genetic knockdown of HO-1 effectively blunted the BAY-elicited increase in LIP levels, offering independent genetic confirmation. The critical and indispensable role of iron in the cell death induced by BAY was further underscored by the profound observation that treatment with deferoxamine (DFO), a potent and highly specific iron chelator, significantly ameliorated the cell death induced by BAY, effectively rescuing cells from its cytotoxic effects. These collective and multifaceted results unequivocally indicated that HO-1 critically mediates BAY-induced ferroptosis by actively promoting and facilitating the accumulation of cellular ferrous iron, which then drives the oxidative damage.
Intracellular iron levels are maintained through a complex and highly regulated interplay of sophisticated homeostatic mechanisms. These mechanisms include the precise uptake of iron into the cell, primarily mediated by the transferrin receptor (TFRC) which imports iron bound to transferrin. Additionally, the export of iron out of the cell is facilitated by ferroportin, a crucial iron exporter encoded by the SLC40A1 gene. Another paramount aspect of iron regulation involves its sequestration into ferritin, a large protein complex composed of both ferritin heavy chain (FTH1) and ferritin light chain (FTL), which serves as the primary intracellular iron storage form, preventing iron-mediated toxicity. Furthermore, a highly regulated autophagic process known as ferritinophagy, mediated by the cargo protein nuclear receptor coactivator 4 (NCOA4), plays a crucial role in the controlled release of iron from ferritin, particularly under specific cellular conditions such as cysteine deprivation or states of low iron. Our comprehensive analysis of gene expression, using various molecular techniques, revealed distinct changes in the expression of these key iron-related genes in response to BAY treatment. We observed that BAY significantly upregulated the expression of TF (encoding transferrin), indicating a potential increase in the cellular capacity for iron transport, and FTH1 (encoding ferritin heavy chain), suggesting an increased capacity for iron storage within the cell. Concurrently, and critically, BAY was found to downregulate SLC40A1 (encoding ferroportin), which implies a reduction in the efflux or export of iron from the cell. Interestingly, BAY had no discernible effects on the expression of TFRC (transferrin receptor) or NCOA4, suggesting that these particular components of iron homeostasis might not be directly modulated by BAY. In the context of hHO-1 overexpression, a broader and more extensive array of changes in iron-related gene expression was observed. Specifically, hHO-1 overexpression led to increased expression of TF, TFRC, FTH1, and NCOA4, while concurrently decreasing SLC40A1 expression. These multifaceted and comprehensive results strongly suggested that, in addition to the ferrous iron directly released from heme degradation by HO-1′s enzymatic activity, iron derived from other sources—including exogenous origins (potentially via increased uptake pathways like transferrin) and endogenous cellular pools (possibly through reduced export or altered storage/release dynamics)—may also significantly contribute to the overall cellular ferrous accumulation induced by BAY or by the forced overexpression of hHO-1. This intricate and multifaceted increase in the intracellular labile iron pool ultimately culminates in the pronounced ferroptotic cell death observed, highlighting the complexity of iron-mediated toxicity.
BAY Induces ER Stress Involving HO-1 Activation
The endoplasmic reticulum (ER), a crucial organelle involved in protein folding, modification, and lipid synthesis, is intricately linked to cellular stress responses. Given that HO-1 is known to be an ER-anchored heme protein, and that depletion of glutathione (GSH) by the blockade of system Xc- (as seen in ferroptosis induction) has been extensively shown to enhance ER stress, a compelling hypothesis emerged: BAY might exert its effects, at least partially, by acting on system Xc- to elicit ER stress, thereby contributing synergistically to the ferroptotic process. To test this proposition, the expression of key ER stress-related genes was meticulously analyzed. Indeed, treatment with BAY significantly promoted the expression of numerous ER stress response genes. These included XBP1 (X-box binding protein 1), GADD45A (growth arrest and DNA damage-inducible 45 alpha), ATF4 (activating transcription factor 4), ATF6 (activating transcription factor 6), HSPB1 (heat shock protein beta-1), and HSP90B1 (heat shock protein 90 beta 1). Of particular note was the robust upregulation of HSPA5 (encoding BiP, or binding immunoglobulin protein) and DDIT3 (encoding CHOP, or CCAAT/enhancer-binding protein homologous protein), both of which are classical and highly sensitive markers of ER stress. Consistent with the established responses to ER stress induced by other agents, a robust and pronounced increase in the cellular protein levels of both BiP and CHOP was observed as early as 4 hours post-treatment with BAY.
Further detailed investigation into the subcellular localization of HO-1 through immunofluorescence staining revealed that BAY not only increased HO-1 expression but also influenced its distribution. HO-1 was observed to be distributed prominently in the perinuclear region and within the nuclei. Crucially, increased HO-1 signal was found to co-localize with protein disulfide isomerase (PDI), a well-established and specific luminal protein of the ER. This co-localization provided compelling evidence that HO-1 within the ER compartment was also significantly elevated by BAY treatment, directly linking HO-1′s presence to ER-associated events.
During conditions of ER stress, a critical signaling cascade known as the unfolded protein response (UPR) is activated. One key arm of the UPR involves the protein kinase PERK, which undergoes dimerization and activation, often facilitated by BiP’s dissociation. Activated PERK then phosphorylates eIF2α, a downstream effector, leading to the upregulation of ATF4 and subsequently CHOP, which mediate various ER stress responses. Another vital component of the UPR is XBP1, an important transcription factor that regulates genes responsible for ER-associated degradation (ERAD) and overall ER homeostasis. Under ER stress, XBP1 messenger RNA (mRNA) undergoes an unconventional splicing event catalyzed by the IRE1α endoribonuclease. This splicing results in a translational frame-shift, converting the inactive unspliced XBP1 (uXBP1) into its active form, spliced XBP1 (sXBP1).
To dissect the relationship between HO-1 and ER stress, further experiments were conducted. Treatment with ZnPP, the HO-1 inhibitor, effectively attenuated the BAY-induced increases in HO-1 protein abundance, as well as CHOP protein levels, and significantly suppressed XBP1 splicing. This directly implicated HO-1 activity in these ER stress-related protein changes and splicing events. However, ZnPP had no significant effects on BiP expression or PERK phosphorylation, suggesting that while HO-1 is crucial for the downstream manifestation of certain ER stress markers, it may not directly influence the very initial activation of the PERK arm or the basal levels of core chaperones like BiP. Conversely, the forced overexpression of hHO-1 was found to independently increase CHOP levels and enhance XBP1 splicing. These reciprocal findings strongly suggested that HO-1 actively mediates and contributes to ER stress signaling, indicating that HO-1 activation is intertwined with the BAY-induced ferroptotic process and its associated ER stress responses.
Upregulation Of HO-1 Is Associated With BAY-Mediated Alteration Of Mitochondrial Homeostasis
Mitochondria are central to cellular metabolism and survival, and their dysfunction is a hallmark of many pathological conditions, including cell death. Iron overloading within cells can lead to significant mitochondrial damage, primarily through the induction of lipid peroxidation within their delicate membranes. Furthermore, ferroptotic cell death is often characterized by specific and observable mitochondrial morphological changes, including condensation of membrane density, rupture of the outer mitochondrial membrane, and a reduction or even complete loss of mitochondrial cristae. In line with these established characteristics, our investigation revealed that treatment with BAY led to a significant loss of mitochondrial membrane potential, a critical indicator of mitochondrial health and a recognized feature observed in ferroptosis.
Using NAO (acridine orange 10-nonyl bromide) staining, a metachromatic dye that is known to accumulate in mitochondria independently of membrane potential, we observed a discernible decrease in overall mitochondrial mass upon BAY treatment, suggesting a reduction in the total mitochondrial content within the cells. Further insights into mitochondrial morphology were gained through MitoTracker staining, a live-cell dye that selectively labels active mitochondria. In untreated cells, mitochondria typically formed an intricate and filamentous network, distributed dynamically throughout the cytosol. However, after BAY treatment, this organized network was disrupted; mitochondria appeared scattered inwards with a discontinuous distribution and were notably retracted towards the perinuclear region of the cell. This striking rearrangement indicated profound mitochondrial perturbation.
Crucially, immunofluorescence co-staining revealed that BAY induced a significant co-localization of mitochondria with LAMP-1 (lysosomal-associated membrane protein 1), a well-established marker for lysosomes. This co-localization was evident in overlapping areas predominantly within the perinuclear region, strongly suggesting the occurrence of an autophagic process specifically targeting damaged mitochondria, a phenomenon known as mitophagy. To ascertain the direct involvement of HO-1 in these observed mitochondrial aberrations, genetic knockdown experiments were performed. The knockdown of HO-1 significantly attenuated the various effects of BAY on mitochondrial biology, including the loss of membrane potential, the decrease in mitochondrial mass, the alteration in mitochondrial distribution, and the induction of mitophagy. This compelling evidence implicated HO-1 as a critical mediator of mitochondrial damage. Conversely, the forced overexpression of human HO-1 (hHO-1) in cells strikingly mimicked the effects of BAY on mitochondrial aberrances, independently inducing similar changes in mitochondrial potential, mass, distribution, and mitophagy. Collectively, these multifaceted results unequivocally demonstrated that HO-1 plays a pivotal role in mediating mitochondrial damage and subsequently triggering mitophagy, processes that ultimately lead to the execution of ferroptosis.
Discussion
In the present comprehensive study, we embarked on a detailed exploration of the compound BAY, culminating in its identification as a novel inducer of ferroptosis, operating through a distinct and previously uncharacterized mechanism. Our findings meticulously confirm and specifically demonstrate that heme oxygenase-1 (HO-1) serves as a central mediator of BAY-induced ferroptosis, primarily by dysregulating crucial aspects of cellular redox regulation. This is consistent with existing literature indicating that elevated HO-1 levels render cells more susceptible to oxidative insults. Furthermore, our experiments revealed that a defect in HO-1 expression significantly modulates BAY’s cytotoxic effect on cell death, underscoring its indispensable role. Through a series of carefully designed genetic interventions, we observed that the knockdown of Nrf2 and SLC7A11 effectively prevented the induction of HO-1 by BAY. Conversely, the knockdown of HO-1 itself had no discernible impact on the abundance of Nrf2 and SLC7A11. These hierarchical observations strongly suggest a precise regulatory cascade wherein Nrf2 acts upstream of SLC7A11, which in turn influences HO-1 expression, thereby defining a Nrf2-SLC7A11-HO-1 regulatory axis as a key response to BAY induction.
The landscape of ferroptotic compounds is broadly categorized. Class I inducers, such as erastin, sulfasalazine, and buthionine sulfoximine, primarily function as inhibitors of system Xc-, leading to cellular glutathione (GSH) depletion and a subsequent imbalance in redox status. Class II inducers, exemplified by GPx4 inhibitors like RSL3 and DPI derivatives, act more directly on lipid peroxidation, a hallmark of ferroptosis. It is also acknowledged that some small molecules, including sorafenib and artemisinin, are capable of initiating the ferroptotic process independently of both system Xc- and GPx4, though their precise mechanisms of action have remained somewhat elusive. Interestingly, pharmacological inhibition of system Xc- in cancer cells has been shown to result in a compensatory increase in SLC7A11 expression, reflecting a cellular attempt to counteract the induced stress. Based on the compelling evidence from our study—including the robust generation of reactive oxygen species, significant glutathione depletion, pronounced iron accumulation, and a compensatory upregulation of SLC7A11 in response to BAY—we propose that BAY should be unequivocally categorized as a Class I ferroptosis inducer.
Further investigations into combinatorial effects revealed an additive cell death effect when BAY was co-administered with known ferroptotic inducers, specifically erastin and sulfasalazine. This synergistic cytotoxicity was also observed when SLC7A11 was knocked down via shRNA interference, highlighting the increased vulnerability of cells when the SLC7A11 defense mechanism is compromised. Conversely, the forced increase of SLC7A11 expression significantly delayed BAY-induced ferroptosis, emphasizing that SLC7A11 abundance critically influences the sensitivity of cells to BAY’s toxicity. While these findings underscore the significant impact of SLC7A11 on BAY’s mechanism, the precise underlying mechanisms through which SLC7A11 affects this sensitivity warrant further dedicated investigation. It is also important to note that erastin has been demonstrated to specifically target L-type amino acid transporters, and sulfasalazine has been shown to exhibit additional effects, including inhibition of NF-κB activity.
Both the Nrf2 and NF-κB pathways are widely recognized as pivotal regulators of cellular redox status, and a complex array of molecular interactions, often cell-type dependent, allows for extensive crosstalk between these two crucial signaling cascades. Our study demonstrated that the inhibitory effect of BAY on p65 and IκBα phosphorylation was effectively prevented by the early administration of potent antioxidants such as N-acetyl-L-cysteine (NAC) and β-mercaptoethanol (βMe), even within a rapid timeframe of 0.25 hours. This compelling observation strongly suggests that the cell death process triggered by BAY occurs in a redox-dependent manner. Furthermore, despite the observed modulatory effects on NF-κB components, genetic intervention involving IκBα and p65 knockdown by shRNA only resulted in a very slight suppression of cell survival, indicating that the NF-κB pathway is not the primary driver of cell death. Critically, while BAY treatment greatly enhanced cell death, it failed to induce significant p65 and p50 nuclear translocation, which is typically associated with NF-κB activation. This suppression was also mimicked by the enforced overexpression of hHO-1, further suggesting a distinct mechanism. Therefore, despite the occurrence of iron accumulation and resultant oxidative insults due to the activation of HO-1 and decreased KEAP1 levels (both of which can indirectly regulate IκBα-NF-κB activation), our comprehensive data firmly exclude the NF-κB signaling pathway as a direct mediator of BAY-induced ferroptosis.
Our pharmacological approaches and experiments involving enforced HO-1 overexpression further unequivocally demonstrated that HO-1 plays a central role in mediating BAY-induced ferroptosis in cancer cells by fundamentally regulating cellular iron and redox homeostasis. An overload of oxidative stress has been shown to massively upregulate HOs, which subsequently act as a pro-oxidant, exerting detrimental effects on cell functionality. The underlying cytotoxic mechanisms operated by HO-1 are therefore attributed to the accumulation of labile iron through HO-1 catalysis, which subsequently induces robust reactive oxygen species (ROS) production. This cascade leads to an escalating state of oxidative stress, culminating in extensive lipid peroxidation, as well as significant protein and genomic damages. Beyond iron, carbon monoxide (CO), another catalytic product of HO-1, may also contribute to BAY-induced ferroptosis, a phenomenon similar to that observed in fibrosarcoma cells following erastin induction. Furthermore, the precise subcellular compartmentalization of HO-1 appears to be involved in cellular ferroptosis. For instance, mitochondria-targeted HO-1 has been shown to cause a loss in cytochrome c oxidase activity and induce higher ROS generation, leading to overt mitochondrial dysfunction and a greater recruitment of LC3B, a key autophagy marker. In astrocytes, overexpression of HO-1 has also been demonstrated to induce profound cytoplasmic vacuolation and mitochondrial macroautophagy. The HO-1-mediated degradation of heme and subsequent iron release are also critical for the formation of SQSTM1 (p62) cargo vesicles, which contain aggregated proteins destined for degradation. The observed increase in SQSTM1 gene expression after BAY treatment suggests that autophagic regulation, possibly involving p62-mediated events, could represent a potential target in BAY-induced ferroptosis. Additionally, studies have shown that the nuclear compartmentalization of HO-1, independent of its enzymatic activity, can mediate cytotoxicity through certain oxidative compounds, leading to endoplasmic reticulum (ER) stress, genomic instability, and cell death in myeloma cells. Whether the unique compartmentalization of HO-1 induced by BAY contributes partially to its ferroptotic induction warrants further detailed investigation.
Iron plays a multifaceted role in various fundamental biological functions, including critical processes such as oxygen transportation, DNA synthesis, and numerous metabolic pathways. It serves as an indispensable co-factor and/or component of numerous proteins that regulate vital mitochondrial processes like the tricarboxylic acid cycle and the electron transport chain. However, despite its essentiality, excessive intracellular iron, particularly in its divalent ferrous form, can paradoxically provoke an uncontrolled generation of reactive oxygen species via the highly destructive Fenton reaction. This uncontrolled ROS production leads to a cascade of cellular damage, including severe lipid peroxidation, proteotoxicity (damage to proteins), genotoxicity (damage to DNA), and ultimately, various forms of cell death. In astrocytes, for example, the overexpression of human HO-1 (hHO-1) has been shown to promote significant cellular oxidative stress and induce the opening of the mitochondrial permeability transition pore. This crucial event facilitates an influx of iron into the mitochondrial matrix, ultimately leading to the profound disruption of mitochondrial cristae, widespread degeneration of the organelle, and subsequent macroautophagy of damaged mitochondria. Similarly, local HO-1 compartmentalized within the mitochondrion itself has been implicated in iron liberation, leading directly to mitochondrial oxidative damages and ensuing mitochondrial macroautophagy.
Normally, mitochondria are dynamically associated with the cellular cytoskeleton, forming an intricate network complex. This association is critically important for maintaining cellular calcium homeostasis, ensuring efficient ATP and ROS production, governing mitochondrial shape biogenesis, and facilitating mitochondrial degradation. Consequently, this intricate interplay profoundly governs death signaling pathways and ultimately determines cell fate. Previous research has indicated that HO-1 can modulate the integrity of the cellular cytoskeleton and the formation of filopodia. It has also been shown to ameliorate aggressive migration by regulating key marker gene expressions related to cell adhesion and cell-cell communication in human PCa prostate cancer cells. Furthermore, a protective role for HO-1 in maintaining mitochondrial homeostasis has been observed through its modulation of the fission/fusion process and related mediator expressions in various contexts, including endotoxin-injured rat lungs, normal and RAW 264.7 macrophages, and cardiomyocytes. The precise role of the cytoskeletal association with mitochondrial homeostasis, downstream of HO-1 mediation in BAY-induced perturbation of mitochondrial homeostasis, represents an intriguing avenue that requires further dedicated investigation. However, contrasting some of these protective roles, as previously mentioned, the overexpression of hHO-1 in astrocytes was shown to promote cellular oxidative stress and induce the opening of the mitochondrial permeability transition pore, leading to iron influx into the matrix and ultimately severe mitochondrial cristae disruption, degeneration, and macroautophagy. The present results robustly demonstrate that HO-1 mediates aberrant mitochondrial biology in response to BAY treatment. Consistent with previous reports, the observed mitochondrial macroautophagy induced by HO-1 was further shown to be dependent on iron liberation and subsequent mitochondrial oxidative damages, and it was highly likely associated with the specific local compartmentalization of HO-1 within the mitochondrion itself.
Mitochondria play an undeniably central and orchestrating role in the conduction of ferroptosis. The transcription of several mitochondrial genes, including RPL8, IREB2, CS, ATP5G3, TTC35, and ACSF2, has been implicated in the ferroptotic process. Erastin, a well-characterized activator of ferroptosis, is known to bind to voltage-dependent anion channels (VDACs) located in the outer mitochondrial membrane. This binding alters the selectivity of VDACs, leading to an increase in the movement of iron as well as other anions into the mitochondria. This influx of iron and altered permeability subsequently results in impaired mitochondrial membrane permeability, attenuation of NADH oxidation, and ultimately promotes heightened ROS production and the initiation of ferroptosis. In human hepatocellular carcinoma cells, the inhibition of retinoblastoma protein (Rb) expression has been shown to mediate sorafenib-induced local ROS production specifically within the mitochondria, thereby inducing ferroptosis. Furthermore, CDGSH iron sulfur domain 1 (CISD1) has been identified as a regulator that can limit mitochondrial lipid peroxidation by repressing iron uptake into the mitochondria, highlighting another layer of mitochondrial involvement in ferroptosis. Acyl-CoA synthetase long-chain family 4 (ACSL4) also contributes significantly to ferroptosis by activating 5-lipoxygenase and promoting the production of 5-hydroxyeicosatetraenoic acid (5-HETE), a lipid mediator. In our study, the observation that zileuton, a 5-lipoxygenase inhibitor, significantly attenuated BAY-induced ferroptosis strongly suggested an involvement of ACSL4 in BAY’s mechanism of action. Therefore, it is highly plausible that BAY may also disturb mitochondrial homeostasis through the mitochondrial compartmentalization of HO-1, leading to these critical changes.
The induction of ferroptosis has rapidly gained recognition as a promising therapeutic strategy, particularly in the context of cancer treatments where resistance to conventional therapies is a significant challenge. Our present results provide compelling evidence that HO-1 critically mediates redox regulation, thereby driving ferroptotic cell death in cancer cells. This process involves complex interactions with both endoplasmic reticulum (ER) stress pathways and the delicate balance of mitochondrial homeostasis. Furthermore, our findings demonstrating that the forced overexpression of human HO-1 (hHO-1) alone is sufficient to induce ferroptosis in cancer cells represent a novel and exciting strategic avenue for cancer treatment. Ultimately, the present results collectively reveal a new and intricate mechanism underlying the ferroptotic induction in cancer cells, thereby simultaneously identifying a novel therapeutic target for the effective treatment of aggressive cancers, particularly those like triple-negative breast cancer and glioblastoma multiforme.
Author Contributions
The overarching design and conceptualization of this study were meticulously carried out by LCC and SKC. Both LCC and SKC were instrumental in conducting the extensive experimental work and were diligently responsible for all aspects of data collection throughout the research. The comprehensive analysis of the collected data, alongside insightful discussions of the findings, involved the collaborative expertise of LCC, SKC, SEC, YLY, RHC, and WCC. The initial drafting and subsequent refinement of the manuscript were primarily undertaken by LCC, SKC, and SEC. All authors meticulously read and ultimately approved the final version of the manuscript, ensuring its accuracy and completeness.
Acknowledgements
This research was generously supported by significant grants from the Ministry of Science and Technology of the Republic of China, Taiwan, specifically under grant numbers MOST 104-2314-B-039-034 and MOST 105-2628-B-039-004-MY3, both awarded to L.-C. Chang. Additional crucial support was provided by grant number NSC 100-2321-B-005-008-MY3, awarded to S.-E. Chen. We extend our sincere gratitude to the Graduate Institute of Cancer Biology and the Center for Molecular Medicine at China Medical University, Taichung, Taiwan, for their invaluable provision of essential reagents, materials, and access to advanced analysis tools, which were critical for the successful execution of this study. We also express our profound appreciation to Mr. Ru-Chun Tai from the Medical Research Core Facilities Center, Office of Research and Development at China Medical University, Taichung, Taiwan, for his exceptional assistance and expertise in facilitating the use of the Confocal Spectral microscope, an instrument vital for our imaging analyses.