Disulfiram (DSF) was purchased from Selleck (Cat. No: S1680, Houston, TX, USA) and CuCl${}_2$ from Sigma-Aldrich (Cat. No: 7447-39-4, St. Louis, MO, USA). They were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 50 mM and stored at –80 °C prior to use. CCK-8 solution was purchased from US Everbright® Inc (Cat. No: C6005M, Silicon Valley, CA, USA). TRIzol reagent was purchased from Invitrogen (Cat. No: 15596026, Carlsbad, CA, USA). SYBR® Premix Ex Taq TM reagents were purchased from TaKaRa (Cat. No: DRR041A, Tokyo, Japan). The Light Cycle® 96 Real-Time PCR System was purchased from Roche (Basel, Switzerland). HMOX1 antibody was purchased from Abcam (Cat. No: ab13243, Cambridge, UK) and GAPDH antibodies from Proteintech (Cat. No: 60004-1-Ig, Wuhan, Hubei, China). The Glutathione Assay Kit and Lipid Peroxidation MDA Assay Kit were purchased from Beyotime (Cat. No: S0058, S0131M, Shanghai, China). The JC-1 MitoMP Detection Kit (Cat. No: MT09) and FerroOrange were purchased from Dojindo Molecular Technologies Company (Cat. No: F374, Kumamoto, Kyushu, Japan).

DSF and Cu were freshly mixed at a ratio of 1:1.7 prior to use. The human TNBC cell line MDA-MB-231 (Cat. No. CL-0150B) was purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, Hubei, China). MDA-MB-231 was grown in RPMI 1640 medium (Cat. No: R8758-500ML, Gibco, Big Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Cat. No: F8687) and 1% penicillin-streptomycin solution (Cat. No: SV30010, Gibco, Big Island, NY, USA). All cells were cultured at 37 °C in a 5% CO${}_2$ atmosphere. The cell lines were authenticated using Short Tandem Repeat (STR) profiling within the last three years. All experiments were performed with mycoplasma-free cells.

MDA-MB-231 cells was seeded into 6-well plates at 2 $\times$ 10${}^5$ cells per well. After 24 h, cells were treated with DSF/Cu (0.2 µM/0.2 µM) and incubated for 12 or 24 h. Cells were then harvested and fixed in a droplet of 2% glutaraldehyde overnight at 4 °C. Samples were processed and observed by TEM (HT7700, HITACHI, Tokyo, Japan).

Cells were assessed for mitochondrial function using a fluorescent JC-1 probe according to the manufacturer’s protocol [18]. JC-1 accumulates in healthy mitochondria and is red fluorescent. At the collapse of mitochondrial potential, the red/green fluorescent intensity ratio reflects membrane depolarization. TNBC cells (2.5 $\times$ 10${}^5$ cells per well) were seeded into 6-well plates and incubated with culture medium. The next day, cells were treated with 0.1% DMSO or DSF/Cu and incubated for 24 h. The medium was then removed and 10 µg/mL JC-1 was added for a 10-minute incubation. After washing thrice, the cells were examined using a fluorescent microscope (Ti2-U, Nikon, Tokyo, Japan) equipped with a digital camera. All pictures were taken using the same microscope settings and exposure times.

To determine the transcriptome changes caused by DSF/Cu, RNA was extracted by TRIzol reagent from MDA-MB-231 cells following 24 h treatment with DSF/Cu. The construction and RNA-Seq analysis were carried out by Lianchuan Company (Hangzhou, Zhejiang, China).

Total RNA was extracted from MDA-MB-231 TNBC cells using TRIzol reagent according to the manufacturer’s instructions. A total of 500 ng of RNA per sample was reverse-transcribed into cDNA with TransScript All-in-One-First-Strand cDNA Synthesis Super Mix for qPCR (Cat. No: AT321-01, TRAN, TRAN, Shanghai, China). Briefly, qPCR was performed using FastStart Essential DNA Green Master (Cat. No: 4913914001, Roche, Basel, USA). The 2${}^{-\mathrm{\Delta }\mathrm{\Delta }\text{Ct}}$ method was used to calculate the average relative fold-change in mRNA expression. Primers sequences are shown in Supplementary Table 1.

The gene expression profiles (RNA-seq) and related clinical information of 1226 breast cancer patients were downloaded from UCSC Xena (http://xena.ucsc.edu/) [19]. 110 TNBC patients (breast carcinoma estrogen receptor status = ‘Negative’, breast carcinoma progesterone receptor status = ‘Negative’, lab proc her2 neu immunohistochemistry receptor status = ‘Negative’) were selected for further analysis. The transcription expression value from RNA seq was shown as log${}_2$ (x + 1) transformed RSEM normalized count. The expression level of each selected genes in each sample was defined as high or low by using “survminer” package (Release 0.4.9) [20] in R software (version 4.2.1) [21]. And then, survival analysis was conducted using the Kaplan–Meier plot and Cox’s proportional hazard regression (HR) model, R package “survival” (Release 3.4-0) [22] was used to accomplish this part. HR (95% CI) and p value of each gene was modified based on profile penalized maximum likelihood bias reduction method by using R package “coxphf” (Release 1.13.1, https://CRAN.R-project.org/package=coxphf). The results were considered statistically significant if Log rank p value was less than 0.05.

Cells were collected by 0.25% trypsinization, washed in ice-cold phosphate buffer saline (PBS) and then lysed in radio immunoprecipitation assay lysis (RIPA) buffer. The protein concentration was quantified using a BCA Protein Assay Kit purchased from Thermo Fisher Scientific (Cat. No. 23227, Waltham, MA, USA). Western blot analysis was conducted using the antibodies described above and the immunoblotting method described in our previous study [23]. Briefly, equal amounts of protein were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes purchased from Merck Millipore (Cat. No: IPVH15150, Darmstadt, Germany). The membranes were blocked with 5% BSA or fat-free milk and incubated at 4 °C overnight with primary antibodies (1:1000 dilution) before washing three times in TBS-T. The membranes were then incubated with secondary antibody (1:2000) at room temperature for 1 h and washed three times in TBS-T. Immunoreactive protein bands were visualized using High-sig ECL Western Blotting Substrate (Cat. No: 180-5001, Tanon, Shanghai, China).

The method described in our previous study [23]. Cells were grown on cover slides in a 24-well plate and then treated with DSF/Cu (0.2 µM/0.34 µM) for 48 h. After treatment, cells were fixed with 4% paraformaldehyde for 30 min and then permeabilized with 0.1% Triton X-100 for 15 min. The cells were blocked with 2% bovine serum albumin (BSA) for 1 h and then incubated overnight at 4 °C with primary antibodies. After washing thrice, the cells were incubated with a Goat Anti-Rabbit/Mouse IgG (Proteintech, Wuhan, Hubei, China) secondary antibody in the dark for 1.5 h and then stained with 0.5 µg/mL 4,6-diamino-2-phenyl indole (DAPI) for 15 min. The samples were examined using a fluorescent microscope (Ti2-U, Nikon, Tokyo, Japan) equipped with a digital camera. All pictures were taken using the same microscope settings and exposure times.

The MDA-MB-231 cells were treated with DSF/Cu for 6, 12 or 24 h. The culture medium was then removed and the cells washed three times with PBS. FerroOrange working solution diluted in serum-free medium to a concentration of 1 µM was added and incubated at 37 °C in a 5% CO${}_2$ incubator for 30 min. The samples were examined using a fluorescent microscope (Ti2-U, Nikon, Tokyo, Japan) equipped with a digital camera. All images were taken using the same microscope settings and exposure times.

Cellular GSH levels were measured using the Glutathione Assay Kit (S0053, Beyotime, Shanghai, China) as recommended by the manufacturer. Briefly, MDA-MB-231 cells were seeded into 6-well plates at 2 $\times$ 10${}^5$ cells per well. The next day, cells were treated with DMSO or DSF/Cu and incubated for 24 h. Cells were then washed with PBS and collected into a 1.5 mL EP tube. Cell pellets (10 µL) were mixed with 30 µL of the protein removal reagent M solution. The samples were then frozen and thawed twice using liquid nitrogen and 37 °C water. They were centrifuged at 10,000 g for 10 min and the supernatant used for GSH measurement with a Varioskan Flash Multimode Reader (Varioskan LUX, Thermo Scientific, Waltham, MA, USA) at 412 nm. All assays were performed in triplicate.

The cells MDA concentration was assessed using Lipid Peroxidation MDA Assay Kit (Cat.No.S0131S, Beyotime, Shanghai, China). In brief, MDA-MB-231 cells were seeded in a 6-well cell plates at 2 $\times$ 10${}^5$ cells. The next day, cells were treated with DMSO or DSF/Cu and incubated for 24 h. Cells were washed with PBS, then collected into a 1.5 mL EP tube and lysed in RIPA buffer. The lysed cells were centrifuged at 10,000 g for 10 min and the supernatant was collected. Then, the supernatant was incubated with an equal volume of thiobarbituric acid buffer (TAB) at 100 °C for 15 minutes. The MDA levels were measured by a Varioskan Flash Multimode Reader (Varioskan LUX, Thermo Scientific, Waltham, MA, USA) at 532 nm. All assays were replicated in triplicate.

All experimental data are expressed as the mean value $\pm$ standard deviation (SD). qPCR data were analyzed with Prism 7 (GraphPad, LA Jolla, CA, USA) by Student’s t-test and one-way ANOVA. p 0.05 was considered statistically significant. For the gene integration analysis, a false discovery rate (FDR)-adjusted p-value of $\le$0.05 and an absolute value of log2 fold-change (FC) $\ge$2 was used as thresholds to define significant differences in gene expression.

To examine the inhibitory effect of DSF/Cu on TNBC cells, MDA-MB-231 cells were treated with 0.2 µM DSF and 0.34 µM CuCl${}_2$ for various times. The optimal concentration of DSF/Cu was determined in our previous study [23]. The effect of DSF/Cu on the morphology of TNBC cells was investigated using an inverted microscope (Fig. 1A). Compared to the control group, MDA-MB-231 cells treated with DSF/Cu for 6 h, 12 h, 24 h and 48 h showed varying degrees of shrinkage, roundness, and multidirectional. We measured cell survival by CCK-8 assay. Six thousand cells per well were plated and cultured in a 96-well dish for 24 h. Then, the cells were treated with different concentrations of 6-TG and DSF/Cu for 48 h. Compared with that of the control cell lines, the proliferation of the cell lines treated with the DSF/Cu was significantly inhibited. (Fig. 1B). Furthermore, we used wound healing assays to examine the effect of DSF/Cu on MDA-MB-231 cells. The metastatic capacity of cells treated with DSF/Cu was significantly lower than that of cells treated with DMSO (Fig. 1C,D).

Fig. 1.

DSF/Cu impaired the mitochondria in TNBC cells. (A) DSF/Cu impairs the mitochondria in TNBC cells. The cellular morphology of MDA-MB-231 cells following treatment with DMSO or DSF/Cu (0.2 µM/0.34 µM) for 12 h, 24 h or 48 h. Scale bar: 10 µm. (B) Relative cell viability in MDA-MB-231 cells was measured by CCK-8 assay (mean $\pm$ SD, n = 3) after 48 h treatment with 6-TG or/and DSF/Cu. (C,D) Wound healing assays were used to examine the cell migration in MDA-MB-231 cells following exposure to DSF/Cu (0.2 µM/0.34 µM) for 24 h, 48 h. Scale bar: 10 µm. (E) Transmission electron microscopy images of MDA-MB-231 cells after treatment with DSF/Cu for 24 h. Arrows indicate the mitochondria in cells. Scale bar: 1 µm. (F) Representative fluorescence images of JC-1 staining in MDA-MB-231 cells treated with DSF/Cu (0.2 µM/0.34 µM) for 12 h and 24 h. The data are presented as the mean $\pm$ SD of three replicates. *p value 0.1, **p value 0.01, ***p value 0.001 and ****p value 0.0001 compared to controls.

To further examine the effect of DSF/Cu on the ultrastructure of TNBC cells, MDA-MB-231 cells were treated for 12 h and 24 h and then observed by TEM. Treated cells did not show the characteristic morphological hallmarks of apoptosis. The mitochondria of TNBC cells became smaller and showed increased membrane density, while the mitochondrial cristae decreased in size or disappeared, suggesting that DSF/Cu may cause ferroptosis in these cells (Fig. 1E). To further examine the effect of DSF/Cu on mitochondrial function, JC-1 staining was used to visualize the mitochondrial membrane potential (MMP). JC-1 accumulates in healthy mitochondria and exhibits red fluorescence. When the mitochondrial potential collapses, the fluorescence changes from red to green. The red fluorescence JC-1 polymer and green fluorescence JC-1 monomer were photographed by fluorescence microscope. ImageJ software (version: V1.8.0.112, LOCI, University of Wisconsin, Madison, WI, USA) for mixed channels. MMP was evaluated by the fluorescence intensity ratio of red to green. Immunofluorescence analysis showed that DSF/Cu decreased the MMP in MDA-MB-231 cells compared to control cells (Fig. 1F).

Lipid peroxidation driven by oxidative stress is an important cytotoxic factor in ferroptosis. We therefore used the fluorochrome Liperfluo to examine the effect of DSF/Cu on lipid peroxidation levels in MDA-MB-231 cells. Treatment with DSF/Cu for 24 h was observed to increase lipid peroxidation in MDA-MB-231 cells, while co-treatment with the ROS scavenger N-acetyl-L-cysteine (NAC) reversed this increase (Fig. 2A). Furthermore, we also found that ROS scavenger NAC could partially reverse the death of MDA-MB-231 cells induced by DSF/Cu (Fig. 2B). Furthermore, NAC partially prevented the death of MDA-MB-231 cells induced by DSF/Cu (Fig. 2B). These results indicate that DSF/Cu can induce lipid peroxidation and morphological changes in mitochondria.

Fig. 2.

DSF/Cu-induced cytotoxicity in TNBC cells is mediated by ROS. (A) Cellular lipid ROS detected by the Liperfluo fluorescent probe. DSF/Cu treatment increased the lipid peroxidation level. (B) MDA-MB-231 cells pretreated with 1- or 2-mM NAC were co-incubated with DSF/Cu (0.1/0.17 or 0.2/0.34 µM) for 24 h and their viability then tested using the CCK8 kit. Results are presented as the mean $\pm$ SD of three replicates. ****p value 0.0001 compared to controls.

Key biomarkers of ferroptosis are iron accumulation, GSH depletion, and lipid peroxidation [24]. To further investigate whether ferroptosis is implicated in DSF/Cu-induced tumor inhibition, we evaluated the levels of intracellular labile Fe (Ⅱ), GSH, and the oxidative stress marker malondialdehyde (MDA) in MDA-MB-231 treated with DSF/Cu. The FerroOrange probe was used to examine the intracellular labile Fe (Ⅱ) content in MDA-MB-231 cells after DSF/Cu treatment for 6, 12 and 24 h. In MDA-MB-231 cells, a time-dependent increase in labile Fe (Ⅱ) was observed in response to DSF/Cu (Fig. 3A).

Fig. 3.

DSF/Cu regulates ferroptosis biomarkers. (A) MDA-MB-231 cells were treated with DSF/Cu (0.2 µM/0.34 µM) for 6, 12 and 24 h and then collected to visualize the intracellular Fe${}^{2+}$ ion level using the fluorescent probe FerroOrange. Scale bar: 20 µm. (B) MDA-MB-231 cells were treated with DSF/Cu (0.2 µM/0.34 µM) for 24 h and the level of glutathione was then measured. (C) MDA-MB-231 cells were treated with DSF/Cu (0.2 µM/0.34 µM) for 24 h and the level of MDA was then measured. (D) The MDA-MB-231 cells pretreated by 5, 10, 20 µM Fer-1 were co-incubated with (0.2, 0.34) µM DSF/Cu for 24 h, and the cell viability was tested by CCK8 kit. The data are presented as the mean $\pm$ SD of three replicates. **p value 0.01, ***p value 0.001 and ****p value 0.0001 compared to controls.

We next examined the levels of intracellular GSH and of the oxidative stress marker MDA. Following treatment with DSF/Cu, the GSH level showed a significant 10-fold decrease compared to controls (Fig. 3B), indicating marked GSH depletion. In contrast, the oxidative stress marker MDA showed markedly increased levels in MDA-MB-231 cells following treatment with DSF/Cu (Fig. 3C). We selected ferrostatin-1 (Fer-1), one of the ferroptosis inhibitors, and detected cell viability by CCK-8 to evaluate whether Fer-1 can save DSF/Cu-induced ferroptosis in triple negative breast cancer cells. The results showed that the addition of ferroptosis inhibitor inhibited the death of triple negative breast cancer cells caused by DSF/Cu, indicating that DSF/Cu induced the ferroptosis of triple negative breast cancer cells (Fig. 3D). Taken together, the above findings on critical ferroptosis biomarkers strongly suggest that DSF/Cu triggers ferroptosis in TNBC cells.

To investigate whether DSF/Cu triggers ferroptosis at the transcriptional level, we performed transcriptome sequencing of MDA-MB-231 cells after treatment with DSF/Cu (0.2 µM/0.34 µM) for 24 h. To reduce the number of false positives, three biological replicates were used to compare the transcriptomes of control and DSF/Cu-treated cells. The significantly differentially expressed genes in RNA Seq were counted by volcano diagram, and the more differentially expressed genes were distributed at both ends. The red dots represent genes whose expression level of control in the sample is up-regulated compared with that of the control sample, and the blue dots represent down-regulated genes, among which 874 genes are up-regulated and 764 genes are down-regulated (Fig. 4A). Subsequently, we selected the most significant up- and down-regulated top 100 gene and established heat maps to cluster the significantly different genes (Fig. 4B). We also performed KEGG analysis to identify significant pathways that had been altered after DSF/Cu treatment, and found that 20 signaling pathways were significantly enriched. The results showed that TNBC cell death induced by DSF/Cu was associated with the MAPK signaling pathway, p53 signaling pathway, mitophagy signaling pathway, and ferroptosis (Fig. 4C).

Fig. 4.

DSF/Cu treatment induced transcriptome alteration. (A) Volcano plot showing the differences in RNA expression by DSF/Cu (0.2 µM /0.34 µM) treatment, as detected in MDA-MB-231 cells using RNA Seq. The red dots represent genes whose expression level of control in the sample is up-regulated compared with that of the control sample, and the blue dots represent down-regulated genes. (B) Heat map of up-regulated and down-regulated genes with significant difference in expression after DSF/Cu treatment. (C) KEGG pathway analysis showed the significant pathways that had been altered after DSF/Cu treatment. (D) MDA-MB-231 cells were treated with DSF/Cu (0.2 µM /0.34 µM) for 12, 24 h. Ferroptosis-correlated gene including PTGS2, CHAC1, SAT1, TFRC, HMOX1 were up-regulated. Ferroptosis-correlated gene including GPX4, SLC40A1, FANCD2 were down-regulated. (E) Kaplan-Meier survival curves for TNBC patients. Data are shown as means $\pm$ SD. *p 0.05, **p 0.01, ***p 0.001 and ****p value 0.0001, n = 3.

qRT-PCR was performed to confirm the results of RNA-seq. Consistent with the RNA-seq data, the mRNAs for prostaglandin-endoperoxide synthase 2 (PTGS2), cation transport regulator-like protein 1 (CHAC1), spermidine/spermine N1-acetyltransferase 1 (SAT1), transferrin receptor protein 1 (TFRC), heme oxygenase-1 (HMOX1) were found to be up-regulated after 12 h and 24 h of DSF/Cu treatment. In contrast, the mRNAs for glutathione peroxidase 4 (GPX4), ferroportin (SLC40A1) and Fanconi anemia complementation group D2 (FANCD2) were down-regulated after 12 h and 24 h of treatment (Fig. 4D). GPX4 is an antioxidant enzyme thought to be an essential suppressor of ferroptosis by using GSH as the substrate. The expression of GPX4 in DSF/Cu-treated cells decreased significantly after treatment with DSF/Cu, consistent with the observed depletion of GSH. The results of qRT-PCR suggested that among the genes involved in the ferroptosis pathway, the HMOX1 gene is the most significantly upregulated gene.

To investigate the effect of ferroptosis genes in triple negative breast cancer after DSF/Cu treatment, KM survival curve analysis was performed. Survival analysis showed that patients with higher HMOX1 (p$$ $$= 0.046, HR$$$$= 0.327), TFRC (p$$ $$= 0.03, HR$$$$= 0.28), SAT1(p$$ $$= 0.094, HR$$$$= 0.391) expression and patients with lower GPX4 (p$$ $$= 0.03, HR$$$$= 3.92) expression had an increased overall survival (Fig. 4E, Table 1).

The results of transcriptome sequencing suggested that HMOX1 was the most significantly upregulated gene amongst those involved in the ferroptosis pathway. One of the major functions of HMOX1 is the release of labile Fe (Ⅱ) [25]. To further examine this gene in MDA-MB-231 cells treated with DSF/Cu for different times (6, 12, 24, 48 h), we quantified the changes in HOMX1 mRNA level using qRT-PCR. DSF/Cu-induced HMOX1 mRNA expression was maximal at 6 h and declined thereafter (Fig. 5A). MDA-MB-231 cells were also treated with different concentrations of DSF/Cu (0.05, 0.1, and 0.2 µM) for 24 h. Interestingly, the expression of HMOX1 increased significantly only when the cells were exposed to 0.2 µM DSF/Cu (Fig. 5B). The HMOX1 protein level in MDA-MB-231 cells treated with DSF/Cu (0.2 µM/0.34 µM) for 24 h was also evaluated by Western blot. Consistent with the q-PCR data, DSF/Cu treatment increased the expression of HMOX1 protein (Fig. 5C,D). Immunofluorescence staining for HMOX1 gave a similar result (Fig. 5D). Together, these results suggest that DSF/Cu induces ferroptosis in TNBC cells by increasing the expression of HMOX1.

Fig. 5.

DSF/Cu increases cellular HMOX1 expression. (A) MDA-MB-231 cells were treated with DSF/Cu (0.2 µM/0.34 µM) for the indicated time intervals (6, 12, 24, 48 h). DSF/Cu-induced HMOX1 mRNA expression was maximal at 6 h and declined thereafter. (B) MDA-MB-231 cells were incubated with different concentrations of DSF/Cu (0, 0.1, 0.2 µM) for 24 h and the expression of HMOX1 mRNA was analyzed by q-PCR. GAPDH was used as a control. The results are presented as the mean $\pm$ SD of three replicates. (C) Western blot was used to evaluate HMOX1 protein levels in MDA-MB-231 cells treated with DSF/Cu (0.2 µM/0.34 µM) for 24 h. The results are presented as the mean $\pm$ SD of three replicates. (D) Immunofluorescence was used to detect HMOX1 expression in MDA-MB-231 cells following exposure to DSF/Cu (0.2 µM/0.34 µM). Scale bar: 10 µm. **p value 0.01, ***p value 0.001 and ****p value 0.0001 compared to the controls.