Copper-overload promotes ferroptosis in cervical cancer cells by upregulating HMOX1 expression

Abstract

Cuproptosis is a newly defined regulated cell death model and is considered as a potential approach for cancer treatment. We have previously found that cervical cancer cells have the capability of anti-ferroptosis. A recent study has reported that elesclomol (ES) is able to induce copper-dependent ferroptosis. However, its effect on cervical cancer cell ferroptosis is still unclear. In this study, we found that the expression levels of copper metabolism-related genes ATP7A and ATP7B were decreased in cervical cancer tissues. In cervical cancer cells, combined treatment of ES and Cu²⁺ inhibited cell proliferation and promoted cell death, but did not promote cuproptosis. However, ES-Cu²⁺ treatment led to the accumulation of cellular reactive oxygen species, increased cellular Fe²⁺ levels, and decreased expression of GPX4. Moreover, the expression levels of HMOX1 and FTH1 were increased, while the expression level of TFRC was decreased after co-treated with ES-Cu²⁺ in cervical cancer cells. The decreased expression of GPX4 induced by ES-Cu²⁺ was attenuated by ferroptosis inhibitors ferrostatin-1 and DFO. Knockdown of HMOX1 could alleviate ES-Cu²⁺-induced GPX4 downregulation. The expression level of HMOX1 was upregulated in cervical cancer tissues and was negatively associated with ATP7B. In conclusion, copper metabolism is altered in cervical cancer tissues. ES-Cu²⁺ co-treatment induce ferroptosis in cervical cancer cells by upregulating HMOX1. Our study provides new insights into the relation between cuproptosis and ferroptosis, which may be potential for clinical therapies of cervical cancer.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12672-025-03421-2.

Introduction

Cervical cancer is the fourth most prevalent cancer and the third leading cause of cancer death in women worldwide, with more than 500,000 new cases and 200,000 deaths per year globally [1]. The major risk factor of cervical cancer is the persistent infection of high-risk human papilloma virus (HPV) [2]. Although the increasing coverage of HPV vaccination and screening test for cervical cancer have improved detection rates, cervical cancer is often detected in advanced stages in underdeveloped area. Moreover, advanced-stages cervical cancer patients usually have a poor prognosis [3]. Thus, understanding the pathogenesis of cervical cancer is critical to develop new approaches for the diagnosis and clinical therapy.

Copper is an essential trace element which serves as the catalytic cofactor of various enzymes. It plays important roles in mitochondrial respiration, antioxidant defense, and bio-compound synthesis [4]. Copper is usually at a low level in normal cells to avoid producing excess free oxygen radicals [5]. Copper accumulation is related to a verity of diseases, especially cancer [4]. In different cancer types, high copper levels promoted the proliferation and metastasis of cancer cells [6–8]. Recently, a new cell death model named cuproptosis was discovered [9]. In cuproptosis, copper ionophore, such as elesclomol (ES) mediated Cu accumulation, causing the aggregation the lipoylated proteins in tricarboxylic acid (TCA) cycle and inducing the instability of iron-sulfur cluster protein, ultimately leading cell death [9]. Previous researches have reported that Cu level was elevated in cervical cancer tissues [10, 11], suggesting cuproptiosis might be invoked as a potential therapeutic approach for cervical cancer. Moreover, some researchers suggested that ES could also induce copper-depended ferroptosis [12, 13]. Ferroptosis is a distinct form of iron and reactive oxygen species (ROS)-dependent cell death pathway characterized by iron accumulation and lipid peroxidation [14]. In cervical cancer, we have previously reported that the persistent ferroptosis in squamous intraepithelial lesions causes anti-ferroptotic effects of cervical cancer cells [14]. We also reported that cervical cancer cells suppressed ferritinophagy and ferroptosis by inhibiting 25-hydroxycholesterol production [15]. Whether ES and copper-induced ferroptosis could reverse the anti-ferroptotic effects in cervical cancer cells is need to be explored. So, investigating the antitumor effect of ES-mediated copper overload in cervical cancer may contribute to cervical cancer therapy in the future.

In this study, we accessed the expression levels of copper metabolism genes and evaluated the role of ES and Cu²⁺ in cervical cancer cells. We found that copper metabolism is altered in cervical cancer tissues. ES-mediated Cu²⁺-overload inhibited the proliferation of cervical cancer cells and promoted cell death. Furthermore, we identified HMOX1 as the target gene for ES-Cu²⁺-induced GPX4 reduction and thus trigger ferroptosis. Overall, our study provides new insight into the role of ES-mediated copper overload in ferroptosis and strongly implies its potential for developing novel clinical therapies for cervical cancer.

Materials and methods

Reagents and antibodies

Elesclomol (S1052), Ferrostatin-1 (S7243) and DFO (S5742) were purchased from Selleckchem (Shanghai, China). Cucl₂.2H₂O (805300) was purchased from Macklin (Shanghai, China). Dulbecco’s modified Eagle medium (DMEM, BC-M-005), FBS (BC-SE-FBS01), trypsin (BC-CE-005), penicillin and streptomycin (BC-CE-007) were purchased from Biochannel (Nanjing, China). Antibodies for ATP7A (ER62772), ATP7B (ER62663) and FDX1 (HA721329) were purchased from HuaAn biotechnology (Hangzhou, China). SLC31A1 (67221-1) and LIAS (11577-1-AP) were obtained from Proteintech biotechnology (Wuhan, China). HMOX1 (A19062), GPX4 (A11243), TFRC (A5865), NCOA4 (A5695), FTH1 (A19544), β-Actin (AC026) and goat anti-rabbit second antibody (AS014) were purchased from Abclonal technology (Wuhan, China). Cell counting kit-8 (CCK-8, CK04), ROS Assay Kit (R252) and FerroOrange (F374) were purchased from Dojindo (Kumamoto, Japan). Cytotoxicity Assay Kit was purchased from Byotime (C2015S, Nantong, China).

Cell culture

SiHa, Caski and HeLa cell lines were purchased and verified by short tandem repeat (STR) genotyping from Hysigen (Suzhou, China). Cells were cultured in Dulbecco’s modified Eagle medium (DMEM, Biochannel, China) supplemented with 10% FBS (Biochannel, China), 1% penicillin and streptomycin (Biochannel, China) at 37 °C in an incubator containing 5% CO₂.

Tissue samples

Cervical cancer tissues and non-cancer cervical tissues used in this research were excess biopsy samples from patients undergoing cervical conization in Department of Gynecology and Obstetrics at Nanjing Jiangning Hospital during 2021. All the tissues were confirmed by pathologist. Specimens were stored in liquid nitrogen immediately after operation until RNA extraction.

Quantitative real‑time PCR (qRT‑PCR)

Total RNA was extracted from tissues and cells using FastPure Cell/Tissue Total RNA Isolation Kit (Q341-02, Vazyme, Nanjing, China). cDNA was synthesized using HiScript III All-in-one RT SuperMix Perfect for qPCR(Q333-01, Vazyme, Nanjing, China). PCR was performed by using ChamQ SYBR qPCR Master Mix (Q341-02, Vazyme, Nanjing, China). The primers are listed in supplementary data (Table S1). The reaction conditions were as follows: 95 °C for 30s, 95 °C for 10s and 60 °C for 30s for 40 cycles. The 2^−δδCT method was used to calculate the relative expression level of target genes.

Western blot assay

Cells were digested with trypsin (Biochannel, China) and lysated in RIPA lysis buffer (P0013B, Beyotime, China), and the protein was collected by centrifuged at 13,000 g in 4 °C. Protein concentration was detected using the BCA assay (P0012S, Beyotime, China) at a microplate reader (Infinite 200 Pro, Tecan Austria GmbH, Austria). Equal amounts of protein samples were separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE, Genscript, China). Following electrophoresis, the entire gel was cut into different sizes according to the molecular weight of the target genes. The corresponding gels were transferred onto appropriately sized PVDF membranes (IPVH00010, Milipore, Germany). After blocking in QuickBlock™ Blocking Buffer (P0252, Beyotime, China) for 20 min, the membranes were incubated with specific primary antibodies at 4℃ overnight. On the following day, the membranes were washed and incubated with appropriate secondary antibodies. Protein bands were visualized by SuperSignal West Pico PLUS (34577, Thermo, American) at FluorChem E (ProteinSimple, American) after washing with wash buffer. Each experiment was repeated three times.

Immunohistochemistry (IHC)

Paraffin blocks were obtained from the Department of Pathology at Nanjing Jiangning Hospital. IHC was performed as previously described [15].

Cell proliferation assay

Cell proliferation was detected using a cell counting kit in 96 well plates. After reaching 80% confluence, the cells were treated with different drugs. 24 h later, the medium was replaced by 100ul DMEM containing 10ul CCK-8, the absorbance of the supernatant was measured after 2 h incubation at 450 nm using a microplate reader (Infinite 200 Pro, Tecan Austria GmbH, Austria).

Cytotoxicity assay

Cells were cultured in 12 well plates. After treatments, cells were stained by propidium iodide (PI, C2015S, Beyotime, China) and cultured at 37 °C in an incubator for 30 min. Dead cells were imaged by using a fluorescence microscope (Olympus, Japan).

Reactive oxygen species (ROS) assay

Cells were seeded in 6-well plate and treated with ES and Cu²⁺ for 18 h. Highly sensitive DCFH-DA dye was used to detect the cellular ROS level according to the manufacture’s guidelines in a flow cytometer (MACSQuant Analyzer 10, Miltenyi, Germany).

Iron assay

The cellular Fe²⁺ level was determined by FerroOrange. Cells were seeded in 24-well plate and incubated with ES and Cu²⁺ for 24 h. Then, cells were cultured in 1 μm FerroOrange for 30 min. The images were captured by fluorescence microscopy (Olympus, Japan).

RNA interference

The HMOX1 siRNA sequences were designed and synthesized in Tsingke Biotech (Beingjing, China). Before transfection, cells were treated with ES and Cu²⁺ for 6 h. The transfection was performed by using GP-transfect-Mate (G04008, Genepharma, China) following the manufacture’s instruction, and the final concentration of the siRNA was 50nM.

Statistical analysis

The data are expressed as the mean ± standard deviation. Student’s t test or one-way ANOVA analysis was used for significance testing. P < 0.05 was regarded as the threshold of difference significance.

Results

Copper metabolism is altered in cervical cancer tissues

To explore the expression pattern of copper metabolism genes in cervical cancer, we reanalyzed our previous RNA-seq data [16] of three cervical cancer tissues and three adjacent normal tissues. Principal component analysis (PCA) showed distinct differences in gene between cervical cancer tissues and adjacent normal tissues (Fig. 1A). Significant transcriptional changes were detected with 657 differentially expressed genes (DEGs) out of 16,048 detectable genes (|log2FC | >1 and p < 0.05), of which 293 were upregulated and 364 were downregulated in the cervical cancer tissues (Fig. 1B). GO enrichment analysis revealed that the enriched clusters were related to protein maturation by copper ion transfer in biological processes and metal ion binding and ferric iron transmembrane transporter activity in molecular functions (Fig. 1C). KEGG analysis showed enrichment in ferroptosis, glutathione metabolism and oxidative phosphorylation (Fig. 1D). Further analysis revealed that copper importer SLC31A1 and copper-transporting ATPases ATP7A and ATP7B showed aberrant expression (Fig. 1E). Compared with control tissues, ATP7A was downregulated in cervical cancer tissues, while ATP7B and SLC31A1 were upregulated (Fig. 1E). These results suggested that, in addition to iron metabolism, the copper metabolism is also altered in cervical cancer tissues.

Fig. 1.

The expression levels of Cu metabolism-related genes in cervical cancer tissues and adjacent normal tissues. A Principal component analysis (PCA) showed distinct differences in gene between cervical cancer tissues and adjacent normal tissues. B Heatmap showed differential expressed genes in three pairs of cervical cancer and adjacent normal tissues. GO enrichment analysis (C) and KEGG analysis (D) displayed the enriched cluster and pathways of the differential expressed genes in cervical cancer tissues. E Heatmap of differential expressed cuproptosis regulators in cervical cancer and adjacent normal tissues. Ca: cervical cancer tissues, adja: adjacent normal tissues

Copper-transporting ATPases ATP7A and ATP7B are down-regulated in cervical cancer tissues

To verify the mRNA expression levels of SLC31A1, ATP7A and ATP7B, we collected 16 cases of cervical cancer tissue and 10 cases of non-cancer cervical tissue from patients. The mRNA levels of ATP7A and ATP7B were significantly decreased in cervical cancer group than that in control group, while SLC31A1 showed no significant changes (Fig. 2A). The protein levels of SLC31A1, ATP7A and ATP7B in 10 cases of cervical cancer tissue and 8 cases of non-cancer cervical tissue were detected using IHC experiment. The results showed that the expression levels of ATP7A and ATP7B were decreased, while the expression level of SLC31A1 was increased in cervical cancer group than that in control group (Fig. 2B), suggesting that copper export is inhibited in cervical cancer and further indicating that cervical cancer might sensitive to copper-overload.

Fig. 2.

The expression levels of Cu metabolism-related genes in cervical cancer tissues. The relative mRNA (A) and protein (B) expression levels of SLC31A1, ATP7A and ATP7B in cervical cancer tissues and control tissues were examined by qRT-PCR and IHC assay. * p < 0.05, ns: not significant

Copper-overload inhibits cell proliferation and promotes cell death in cervical cancer cell lines

To evaluate the sensitivity of cervical cancer cells to copper, three types of cervical cancer cell lines were used for treatment. After the cervical cancer cells were treated with different concentration of Cu²⁺ for 24 h, we found that the cell viability of the three types of cervical cancer cells were decreased as the dose of Cu²⁺ increased (Fig. 3A), indicating that copper treatment led to dose-dependent inhibition in the proliferation of the SiHa, Caski and HeLa cell lines. Then we evaluated the effect of ES (a cuproptosis inducer), we found that the cell viability of the three types of cervical cancer cells were decreased as the dose of ES increased, indicating that ES could increase the uptake of bovine serum derived-copper and exhibited a dose-dependent effect in the SiHa, Caski and HeLa cell lines (Fig. 3B). Moreover, we found that adding exogenous Cu²⁺ (0.5 µg/ml) in culture medium with presence of ES could significantly inhibit cell proliferation as compared with ES treatment (Fig. 3B). Based on the cell viability assays, we chose 40 nm ES and 0.5 µg/ml Cu²⁺ for SiHa, 10 nm ES and 0.5 µg/ml Cu²⁺ for Caski and 20 nm ES and 0.5 µg/ml Cu²⁺ for HeLa in the following experiments. Propidium iodide (PI) staining showed that combined treatment with ES and Cu²⁺ significantly promoted the cell death of SiHa, Caski and HeLa cell lines (Fig. 3C).To better observe the effect of copper overload, we treated the cells with the combination of ES-Cu²⁺ and detected the expression levels of genes related to copper metabolism and curoptosis in SiHa, Caski and HeLa cells. We found that ATP7A was significantly decreased after ES-Cu²⁺ treated Caski and HeLa cells, while ATP7B was significantly decreased after ES-Cu²⁺ treatment in SiHa and Caski cell lines. Compared with control groups, FDX1, LIPT1, DLAT and PDHA1 were significantly downregulated while MTF1 was significantly upregulated in ES-Cu²⁺ co-treated SiHa cells. In Caski cells, combinational treatment of ES and Cu²⁺ significantly downregulated the expression levels of FDX1, DLAT and PDHA1. In HeLa cells, DLAT were significantly downregulated while MTF1 was significantly upregulated after ES-Cu²⁺ treatment (Fig. 3D). The results suggested that ES-Cu²⁺ co-treatment inhibited cooper export and cuproptosis might not the direct reason of copper-overload induced cell death in cervical cancer cells.

Fig. 3.

Combinational treatment of ES and Cu²⁺ inhibits cell proliferation and promotes cell death. SiHa, Caski and HeLa cell lines were treated with different concentration of Cu²⁺ (A), ES with or without the combination of 0.5 ug/ml Cu²⁺ (B) for 24 h followed by CCK-8 assay to detect cell viability. C PI staining assay showed the dead cell after treatment of ES, Cu²⁺ and ES-Cu²⁺ for 24 h. D The expression levels of cuproptosis-related genes were detected by qRT-PCR after treatment of ES and Cu²⁺ for 24 h. * p < 0.05, ** p < 0.01, *** p < 0.001, *****p < 0.0001, ns: not significant

Copper-overload induces iron accumulation in cervical cancer cells

Since significant cell death was observed after ES and Cu²⁺ treatment, and previous studies reported copper could promote ferroptosis [12], so we investigated whether ES-Cu²⁺ induces ferroptosis in cervical cancer cells. ROS levels and intercellular Fe²⁺ levels in SiHa and Caski cells were detected. Combined ES and Cu²⁺ treatment increased cellular ROS levels and induced cellular Fe²⁺ accumulation in SiHa and Caski cells (Fig. 4A and B). Then we examined the expression levels of some key genes related to ferroptosis. In ES-Cu²⁺ treated cells, the mRNA expression levels of HMOX1 and PTSG2 were both upregulated, while the expression level of GPX4 was downregulated. Ferritinophagy-related molecular TFRC, NCOA4 and FTH1 showed inconsistent expression levels in the two cell lines (Fig. 4C). We further verified the protein level by western blot. The results confirmed that HMOX1 and FTH1 expression were markedly increased, while GPX4 and TFRC were decreased in the dual-drug group compared with the control and single-drug groups (Fig. 4D). In addition, ferroptosis inhibitors ferrostatin-1 (Fer-1) and DFO could reverse ES-Cu²⁺-induced GPX4 downregulation (Fig. 4E) and cell proliferation inhibition in SiHa cells (Fig. 4F). Taken together, these results suggested that copper-overload could affect iron homeostasis and trigger ferroptosis.

Fig. 4.

Copper-overload induces ferroptosis in cervical cancer cells. A ROS level was determined by flow cytometer after ES and Cu²⁺ treatment. B SiHa and Caski cells were treated with ES and Cu²⁺ and then stained by ferroOrange to examine intracellular Fe²⁺. C The expression levels of ferroptosis-related genes were detected by qRT-PCR after treatment of ES and Cu²⁺. D The protein levels of TFRC, NCOA4, FTH1, HMOX1 and GPX4 were detected by western blot assay after treatment of ES and Cu²⁺. SiHa and Caski cells were treated with ES-Cu²⁺ for 6 h, and then treated with Fer-1 (10µM) or DFO (100 µM), GPX4 expression (E) was examined by western blot and cell proliferation (F) was detected by CCK-8 assay. * p < 0.05, ** p < 0.01, *** p < 0.001, *****p < 0.0001, ns: not significant

HMOX1 is required in copper-overload induced ferroptosis

Because our above data showed that the change of the expression level of HMOX1, which has a function of iron intake, was the most obvious among the ferroptosis genes, so we next verified whether HMOX1 participated in ES-Cu²⁺-induced GPX4 downregulation, three HMOX1 siRNA sequences were transiently transfected into cervical cancer cells. We found siHMOX1 #2 sequences was most effective in Caski and HeLa cells, but not in SiHa cells (Fig. 5A), so we used #2 siRNA sequences to knock down HMOX1 in Caski and Hela cells for subsequent experiments. Supplementary data (Figure S1) showed the effects of ES-Cu²⁺ treatment on HMOX1 and GPX4 expression in HeLa cells. After knockdown of HMOX1 in ES-Cu²⁺ treated cervical cancer cells, GPX4 expression was upregulated (Fig. 5B and C) and cell proliferation was increased (Fig. 5D), while cell death was decreased (Fig. 5E). However, HMOX1 knockdown did not significantly inhibit ES-Cu²⁺ induced Fe²⁺ accumulation (Fig. 5F). These results indicated that inhibiting HMOX1 expression could reverse the downregulated ES-Cu²⁺-induced GPX4 expression which was induced by copper-overload.

Fig. 5.

HMOX1 participates in ES-Cu²⁺-induced ferroptosis. A Three HMOX1 siRNA sequences and control sequence were transiently transfected into SiHa, Caski and HeLa cells, the expression levels of HMOX1 were detected by qRT-PCR. The mRNA (B) and protein (C) expression levels of HMOX1 and GPX4 were detected by qRT-PCR and western blot after transiently transfected with #2 HMOX1 siRNA and control sequence in ES-Cu²⁺ treated Caski and HeLa cells. Cell proliferation (D), cell death (E) and intracellular Fe²⁺ levlels (F) in Caski and HeLa cells after transfected with #2 HMOX1 siRNA in the presence of ES-Cu²⁺. ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, *****p < 0.0001

The relationship between HMOX1 and copper export in cervical cancer tissues

Our in vitro data has demonstrated the important function of HMOX1 in copper-overload related ferroptosis, we next analyzed the relationship between HMOX1 and copper export genes using a TIMER2 dataset (http://timer.cistrome.org/), we found HMOX1 was positively associated with ATP7A but negatively associated with ATP7B (Fig. 6A). We next detected the expression levels of HMOX1 in cervical cancer tissues. We found that HMOX1 was slightly up-regulated in the RNA expression levels without significant differences (Fig. 6B). The IHC images also showed deeper staining of HMOX1 in cervical cancer than that in adjacent normal tissues (Fig. 6C). These results suggested that copper overload-induced HMOX1 upregulation might be associated with the inhibition of copper export.

Fig. 6.

The correlation between HMOX1 and copper export genes ATP7A, ATP7B. A The correlation between the mRNA expression levels of HMOX1 and ATP7A, ATP7B. The mRNA (B) and protein (C) expression levels of HMOX1 in cervical cancer and control tissues were detected by qRT-PCR and IHC. ns: not significant

Discussion

In this study, we demonstrated that copper export is suppressed in cervical cancer tissues. In cervical cancer cells, ES-Cu²⁺ treatment inhibited the expression levels of genes related to copper export and promoted ferroptosis through HMOX1 upregulation.

Cu metabolism includes intestinal or tissue cell absorption, blood circulation, and tissue cell utilization, excretion or export [17]. Cu import and export directly affect intracellular copper ion levels [18]. In this study, we found that copper-transporting ATPases ATP7A and ATP7B were downregulated in cervical cancer tissues. The decreased expression of ATP7A and ATP7B might promote copper accumulation, thus influencing the metabolism of cervical cancer cells. Our study partly explains the high level of Cu in cervical cancer tissues reported in previous studies [11].

In cervical cancer cells, we found combination treatment of ES and Cu²⁺ inhibited cell proliferation and promoted cell death. ES is a mitochondrion-targeting copper ionophore developed as a chemotherapeutic agent [19], inducing cuproptosis through its high copper-transporting efficiency. Previous studies showed that ES and copper promoted cuproptosis in various diseases [20–22]. In the cuproptosis pathway, there are seven positive regulatory factors (FDX1, LIAS, LIPT1, DLD, DLAT, PDHA1, and PDHB) and three negative regulatory factors (MTF1, GLS, and CDKN2A) [9]. However, in our study, most positive regulatory factors were downregulated and negative regulatory factor MTF1 was upregulated, suggesting ES and Cu²⁺ treatment did not promote cuproptosis in cervical cancer cells. These results need further validation.

Ferroptosis is a distinct form of iron and ROS-dependent RCD characterized by iron accumulation and lipid peroxidation [14]. Recently, copper has been shown to participate in ferroptosis, likely due to the regulation ROS accumulation and copper-iron interactions [12, 13, 23, 24]. Similarly, we found that Cu²⁺ accumulation produces excessive ROS in cervical cancer cells. Another interesting finding is that ES-Cu²⁺ treatment lead to increased cellular Fe²⁺ levels and decreased GPX4 expression, suggesting the emergence of ferroptosis. Thus, the anticancer roles of ES and Cu²⁺ against cervical cancer deserve further investigation.

Ferritinophagy is a biological process leading to the degradation of ferritin-iron and releases of free iron, provides Fe²⁺ to trigger ferroptosis [25]. In this study, we found the RNA expression levels of TFRC and NCOA4 were decreased, suggesting ferritinophagy might not be the primary ferroptosis pathway in ES-Cu²⁺ induced cervical cancer cell ferroptosis. Moreover, the significantly increased FTH1 protein level might result from RNA level upregulation and ferritinophagy inhibition after treatment of ES-Cu²⁺. The inconsistent RNA expression and protein expression in NCOA4 indicated possible protein modification mechanism so as its protein could not significantly change in a relatively short time, which needs further study. Moreover, the results also suggested that ferritinophagy might dynamically maintain intracellular Fe²⁺ levels within a finite range. HMGCS is a key enzyme in mevalonate pathway, which plays important roles in the regulation of ferroptosis [26]. In this study, we found the RNA level of HMGCS significantly upregulated in SiHa cells after ES-Cu²⁺ treatment. The mechanism will be further studied. We next found HMOX1 participated in ES-Cu²⁺-induced ferroptosis. HMOX1 encodes an oxidase which oxidizes cellular heme to release biliverdin, carbon monoxide (CO) and free Fe²⁺ [27]. Previous studies reported that disulfiram/Cu treatment increased HMOX1 activity in triple-negative breast cancer, and copper nanoparticles induced an increased level of HMOX-1 in HepG2 and Caco-2 cells [28–30]. In agreement with these reports, we found ES-mediated copper overload increases HMOX1 expression, and HMOX1 further promotes ferroptosis by downregulating GPX4 and increasing the labile Fe²⁺ pool in cervical cancer cells. However, we found the Fe²⁺ levels induced by ES-Cu²⁺ treatment remained unchanged when inhibited HMOX1 by siRNA. It was reported that ES treatment could indirectly elevate mitochondrial iron levels by stimulating the copper-dependent Fe importer [31]. Thus, our results suggested HMOX1 might not the only source of the labile Fe²⁺ pool in ES-Cu²⁺ treated cervical cancer cells. In cervical cancer tissues, HMOX1 expression negatively associated with ATP7B, suggesting copper overload-induced HMOX1 upregulation might be associated with the inhibition of copper export.

Although extensive researches have been carried on ferroptosis, however, there were limit publications on how copper induces ferroptosis. In this study, we found ES -Cu²⁺ treatment regulates both cuproptosis and ferroptosis. ES-Cu²⁺ promoted ferroptosis through upregulating HMOX1 in cervical cancer.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

CZ: performed experiments, formal analysis, data curation, writing—original draft, funding acquisition. TW: writing—conceptualization, review & editing, data curation. YL: data curation. YZ: data curation. JC: conceptualization, review & editing, supervision. RJ: writing—conceptualization, review & editing, project administration. All authors reviewed the manuscript.

Data availability

All data generated or analyzed during this study are included in this published article.

Declarations

Ethics approval and consent to participate

This study was approved by the ethic committee of Nanjing Jiangning Hospital (2021-03-020-K01), in accordance with the ethical guidelines of the Declaration of Helsinki. Informed consents to allow the use of their specimens in researches were signed by the patients before the study.

Competing interests

The authors declare no competing interests.