Non-small cell lung cancer (NSCLC) cell lines A549, NCIH358, NCIH460, and Calu-1, human colorectal cancer cell line HCT116, human fibrosarcoma cell line HT-1080 were obtained from China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China). Cells were cultured in RPMI-1640 medium (Hyclone, Logan, UT) with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY), supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin (Hyclone). Cells were maintained in a humidified incubator at 37°C, in the presence of 5% CO₂. All cell lines were cultured to limited passage before implantation and were routinely screened to confirm the absence of contamination.

Cells were under the exposure to several kinds of reagents. The amount and conditions of those reagents were as follows: cisplatin (Sigma, St. Louis, MO) was dissolved in normal saline (NS) with a final working concentration of 5 μg/mL, 5-FU (Sigma) was dissolved in NS with a final working concentration of 20 μg/mL, adriamycin (Meilunbio, Dalian, China) was prepared in dimethyl sulfoxide (DMSO; Sigma) with a final working concentration of 10 μg/mL, paclitaxel (Meilunbio) was prepared in DMSO with a final working concentration of 10 μmol/L, and sulfasalazine (Sigma) was prepared in DMSO with a final working concentration of 0.8 mmol/L. Erastin (Selleck, Houston, TX) was prepared in DMSO with a final working concentration of 10 μmol/L, ferrostatin-1 (Sigma) was prepared in DMSO with a final working concentration of 0.5 μmol/L, deferoxamine (Sigma) was prepared in DMSO with a final working concentration of 50 μmol/L, a stock solution of β-mercaptoethanol (Sigma) was diluted into a final working concentration of 50 μmol/L, a stock solution of z-vad-fmk (Beyotime, Haimen, China) was diluted into a final working concentration of 20 μmol/L, necrostatin-1 (Santa Cruz Biotechnology, Santa Cruz, CA) was prepared in DMSO with a final working concentration of 30 μmol/L, chloroquine (Meilunbio) was dissolved in NS with a final working concentration of 5 μg/mL.

Cells (5×10³) cultured in RPMI-1640 medium with 10% FBS were seeded in 96-well plates and received treatments for a given period. The cytotoxic effect of different treatments was determined by the methylthiazoltetrazlium (MTT; Biosharp, Carlsbad, CA) dye uptake method. MTT solution (20 μL, 5 mg/mL) was added to each well. After incubation for 4 hours at 37°C, the supernatants were removed and 150 μL DMSO were added to each well, then incubated at 37°C for 15 minutes. Optical density (OD) was detected with a microplate reader (Biotech, New York, NY). Inhibition rate (IR) (%)=[1–(OD of the experimental sample/OD of the control)]×100%, Q=IR_A+B/(IR_A+IR_B–IR_A×IR_B) (IR_A was the IR of drug A, IR_B was the IR of drug B, IR_A+B was the IR of drug A+drug B), and Q > 1.15 was a symbol for significant synergistic effect of drug A and drug B.

The 2×10⁵ of cells were seeded in 6-well plates in advance. The next day, cells were incubated in 2 mL media with different drugs for 48 hours. Then, the culture media was replaced by serum-free media containing 10 μmol/L 2',7'-dichlorodihydrofluorescein diacetate (Sigma) and placed in dark for 30 minutes, gently shaken every 5 minutes. Cells were harvested in 15 mL tubes by centrifuging at 1,000 rpm for 5 minutes and washed 3 times with serum-free media followed by re-suspending in serum-free media, then incubated with 5 μL 7-aminoactinomycin D (KeyGEN Bio-TECH, Nanjing, China) in dark for 5 minutes. Fluorescence was determined by flow cytometry (BD Biosciences, San Jose, CA) at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The average intensity of fluorescence in each group indicated the amount of ROS within cells.

The 2×10⁵ of cells were seeded in 6-well plates in advance. The next day, cells received different treatment for 48 hours followed by harvesting to determine cell number. Nearly 6×104 live cells from each sample were transferred to new tubes, washed in phosphate buffered saline (PBS) and centrifuged at 1,200 rpm at 4°C for 5 minutes twice. The cell pellet was resuspended in 8 μL protein removal solution, thoroughly incorporated, and placed in –70°C and 37°C sequentially for fast freezing and thawing, then placed in 4°C for 5 minutes and centrifuged at 10,000 ×g for 10 minutes. The supernatant was used to determine the amount of GSH in the sample. We used the GSH and GSSG Assay Kit (product No. S0053, Beyotime) and followed the product instructions to determine GSH levels [10]. Briefly, GSH assay buffer, GSH reductase, 5,5′-dithio-bis 2-nitrobenzoic acid solution and supernatant sample were mixed together and incubated at 25°C for 5 minutes, then NADPH was added into this system to trigger the reaction. The increase in the absorbance of 5-thio-2-nitrobenzoic acid was measured at 412 nm, and the GSH levels were calculated following the product instructions.

Cells with or without compound treatment were harvested and washed with PBS twice, resuspended in 50 μL cell lysis solution and homogenized at 4°C for 15 minutes. The resulting crude lysates were cleared by centrifuging at 12,000 ×g for 10 minutes, and the supernatant was kept for next step. The total amount of protein in the supernatant was determined first through BCA method (BCA Protein Detection Kit, Google Bio, China). Then, Total Glutathione Peroxidase Assay Kit (product No. S0056, Beyotime) was used to measure the activity of GPX [10]. In microtubes, GPX assay buffer, NADPH, GSH, GSH reductase and supernatant sample were transferred and completely mixed according to the manufacturer’s guidelines. GPX reaction was started by adding peroxide reagent tert-butylhydroperoxide (t-Bu-OOH) to the mixture above. The decrease in the absorbance of NADPH was determined at 340 nm. The amount of enzyme which oxidizes 1 μmol of NADPH per min at 25°C is defined as one unit of enzyme.

Cells were examined at the ultrastructural level using transmission electron microscopy (Olympus, Tokyo, Japan). At least three independent fields were acquired for each experimental condition. Representative photographs from one field of view are shown.

Small interfering RNAs (siRNA) targeting IREB2 (siRNA IREB2-7499, GCUGUGAAAUUGUUUCGAAtt; siRNA IREB2-7500, GGAACAUUUUCUUCGCAGAtt) and siNeg (random sequence) were obtained from Genepharma Technologies (Shanghai, China). Six thousand cells were seeded in 96-well plates and incubated at 37°C for 24 hours. Reverse transfection was performed by preparing two solutions of siRNA-Optimem (siRNA:Optimem=1:100) and lipo2000-Optimem (lipo2000:Optimem=1:100, Lipofectamine 2000 and optimum media were both purchased from Invitrogen, Carlsbad, CA). After stayed at room temperature for 5 minutes, the two solutions were mixed up at a ratio of 1:1 and placed for 25 minutes at 37°C. Next, 50 μL siRNA-lipo2000-Optimem mixture and 50 μL serum-containing media were added to each well and incubated for 6 hours at 37°C. Following incubation, 100 μL serum-containing media were transferred to each well and incubated at 37°C. Real-time polymerase chain reaction and western blot were performed 48 hours later to verify the efficiency of transfection. Then, drug treatment experiments were performed 24 hours after the transfection.

Cells received siRNA transfection were harvested and RNA was purified using the Total RNA extraction Trizol kits (Invitrogen) according to the manufacturer’s instructions, the concentration of RNA was determined by spectrophotometer (Eppendorf, Hamburg, Germany). The 800 ng total RNA per sample was subsequently used in a reverse transcription reaction using the Reverse Transcription Kit (Takara, Tokyo, Japan). The primers for polymerase chain reaction (PCR) were designed with Primer Express (upstream sequence, 5′-CTACCTGCCGAGGATCTTGTGA-3′downstream sequence, 5′-TGATGAGCCATTCCAGTTCCAG-3′).

Quantitative PCR was performed on triplicate samples, and the relative amount of the target gene in experimental and control conditions was computed using the 2^-ΔΔCT method.

Cells received siRNA transfection were incubated with 50 μL cell lysis solution for 30 minutes, then transferred into new tubes and centrifuged at 16,000 rpm at 4°C for 15 minutes. The amount of protein in the supernatant was determined by BCA method (BCA Protein Detection Kit, Google Bio). After quantification, samples were mixed with sodium dodecyl sulfate (SDS) loading buffer and separated by SDS-polyacrylamide gel electrophoresis (Western Blotting Kit, Google Bio). Western transfer was performed using the iBlot system (Invitrogen). PVDF membranes (Millipore, Billerica, MA) were blocked for 1 hour in Tris-buffered saline (pH 7.4) with 1% Tween-20 (TBS-T) with 5% milk and incubated in primary antibody overnight at 4°C. Following washed about 10 minutes in TBS-T for three times, the membrane was incubated with secondary antibodies for 1 hour at 37°C. The membrane was washed again in TBS-T thrice for 10 minutes prior to visualization using enhanced chemiluminescence (ECL, Thermo Fisher Scientific, Waltham, MA). Antibody for IREB2 (ab10-6926, Abcam, Cambridge, MA) were used at 1:1,000 dilution, and detected using a goat mouse horseradish peroxidase conjugate secondary antibody (Santa Cruz Biotechnology) at 1:5,000 dilution.

All assays were conducted 3 times and found to be reproducible. Data were expressed as mean±standard deviation and analyzed by GraphPad Prism 5 software (GraphPad Software Inc., San Diego, CA). Differences were considered statistically significant at p < 0.05.

To test our hypothesis that ferroptosis might exist in the process of cell death induced by classical therapeutic drugs, we designed our exploration using six kinds of cancer cells and five typical chemotherapeutic drugs. Ferrostatin-1 has been proved to be a potent specific inhibitor of ferroptosis, and its mechanism of action is to prevent ferroptosis-induced accumulation of cytosolic and lipid ROS [5]. NSCLC cell lines A549, NCIH358, NCIH460, Calu-1, human colorectal cancer cell line HCT116, and human fibrosarcoma cell line HT-1080 received treatments of cisplatin, 5-FU, adriamycin, paclitaxel, and sulfasalazine for 48 hours, respectively. We found that only cisplatin induced cell death could be partially reversed by ferrostatin-1 in both A549 and HCT116 cells, while other drugs showed no response to ferrostatin-1 (Fig. 1A). Later, we applied several different kinds of cell death inhibitors together with cisplatin to A549 and HCT116 cells, including caspase inhibitor z-vad-fmk, necrosis inhibitor necrostatin-1, autophagy inhibitor chloroquine, ferroptosis specific inhibitor ferrostatin-1 and iron chelator deferoxamine. The results demonstrated that the cell death induced by cisplatin could be partially reversed by deferoxamine, ferrostatin-1, z-vad-fmk, and the phenomena became more obvious when ferrostatin-1 and z-vad-fmk were combined together (Fig. 1B). Thus, we thought that cisplatin induced cell death was a result from the joint action of both ferroptosis and apoptosis. Besides, we investigated the appropriate concentration and action time for cisplatin and found out that ferroptosis were more obvious when the concentration of cisplatin was higher than 5 μg/mL and the treatment was longer than 48 hours (Fig. 1C).

We further confirmed the occurrence of ferroptosis induced by cisplatin through multiple aspects. Firstly, optical microscopy images showed the protection of ferrostatin-1 from cisplatin in A549 cells (Fig. 2A). Secondly, electronic structural changes were observed. Mitochondrial changes are usually observed in ferroptosis and considered as the primary distinguishing feature [6]. Consistent with observations from previous reports, an alternation in mitochondrial ultrastructure was observed in both cisplatin and erastin (using as a positive control) treated HCT116 cells: mitochondria appeared on average smaller, less tubular, darker-staining membranes with distinct disrupted inner membrane foldings (Fig. 2B). Thirdly, the necessity of iron was determined. Ferroptosis is defined as a kind of iron-dependent cell death, so the involvement of iron is a key feature in this process. Iron-responsive element binding protein 2 (IREB2) is a master regulator of iron metabolism; silencing of IREB2 results in reciprocal changes in the iron uptake, mechanism, storage, and the inhibition of erastin induced ferroptosis [5,14,15]. We established siRNA-IREB2 (siIERB2) and transfected it into HCT116 cells. Results demonstrated that silencing IREB2 partially reversed the cytotoxicity of cisplatin, indicating the involvement of iron in cisplatin induced cell death (Fig. 3A). Fourthly, different kinds of ferroptosis specific inhibitors were applied. As displayed in our results, ferrostatin-1, deferoxamine and β-mercaptoethanol all showed suppressive effects on the anti-tumor effect of cisplatin (Fig. 3B). Fifthly, ROS levels were examined through flow cytometry analysis. Cells exposed to cisplatin displayed an obvious increase in ROS levels, and this phenomenon was partially reversed by ferrostatin-1 (Fig. 3C). Taken together, we concluded that ferroptosis was indeed involved in the process of cisplatin induced cell death, and we expected this new way of cell death could open up a new way for the utility of cisplatin in clinic.

According to previous researches on ferroptosis, biomolecules like system X_c−, GSH, GPXs and intracellular iron are referred as key targets for ferroptosis regulators [16]. Erastin is determined to inhibit the function of system X_c−, leading to the depletion of GSH and inactivation of GPX4, and finally triggers ferroptosis [5,10,16]. Platinum compounds are proved to have a high affinity to thiol-rich biomolecules, and GSH is one of the most abundant non-protein thiols in cells; in cytoplasm, a major fraction (~60%) of the intra-cellular cisplatin is conjugated with GSH to form the Pt-GS complex [17,18]. Taking consideration of this feature, we supposed that GSH and GPXs were the most possible targets for cisplatin.

There, we treated A549 and HCT116 cells with either erastin (using as a positive control) or cisplatin, and collected cells to detect changes in GSH level and GPXs activity. Both erastin and cisplatin significantly decreased GSH levels and GPXs activities in the two kinds of cells; however, the inhibition of GPXs in cells treated with cisplatin was not as strong as those treated with erastin (Fig. 4). Previous data [10,16] suggested that GPXs activity owned a more vital role in the process of ferroptosis than GSH depletion, thus the weaker inhibition of GPXs induced by cisplatin defined it as a less potent inducer of ferroptosis.

It now appears that the ability of cisplatin to trigger ferroptosis is determined mainly by direct depletion of intracellular GSH. There are eight isoforms of GPXs in humans with different tissue expression and substrate specificities, and GPX4 is proved to be a central regulator of ferroptosis induced by erastin, which one plays the vital role in cisplatin induced ferroptosis worth further discussion.

After verifying the occurrence of ferroptosis in cisplatin induced cell death, we investigated whether ferroptosis was able to become a potent assistance to cisplatin’s lethality. A549 and HCT116 cells were exposed to cisplatin alone or cisplatin together with erastin in different concentrations for 48 hours, and we found that the toxicity to cells improved a lot when the two drugs were combined together (Fig. 5A and B). The combination coefficients of cisplatin and erastin were always more than 1.15 when both drugs were in lower concentrations, which meant significant synergistic effect (Tables 1 and 2). In the subsequent experiments, we found that this improvement was significantly blocked by ferroptosis specific inhibitor ferrostatin-1, β-mercaptoethanol, and caspase inhibitor z-vadfmk in A549 and HCT116 cells, indicating the joint action of apoptosis and ferroptosis (Fig. 5C and D). Besides, elevated ROS level was observed in combination therapy and it could be significantly reversed by β-mercaptoethanol (Fig. 5E). Our data suggested that cisplatin together with erastin demonstrated better anti-tumor activity than any of them alone. Considering the completely different mechanism of ferroptosis, combining erastin together with cisplatin could be a good strategy to enhance the utility of cisplatin.