The ovarian cancer cell lines Hey and SKOV3 were purchased from Procell Life Science & Technology Co., Ltd. A human normal ovarian epithelial cell line (IOSE80) was purchased from BioVector NTCC, Inc. Cell line authentication was performed using short tandem repeats. Hey and SKOV3 cells were maintained in DMEM (HyClone; Cytiva) supplemented with 10% fetal bovine serum (FBS; HyClone; Cytiva) and 1% antibiotics (penicillin and streptomycin; HyClone; Cytiva). IOSE80 cells were cultured in 90% RPMI-1640 medium (HyClone; Cytiva) and 10% FBS (HyClone; Cytiva). All cells were maintained in an incubator and 5% CO₂ at 37°C.

In brief, SKOV3 and Hey cells were seeded in 96-well plates at a density of 3,000 cells/well overnight at 37°C. Thereafter, SKOV3 and Hey cells were incubated with 100, 200, 300, 400 and 500 µg/ml CMP for 24 h at 37°C. Then, 10 µl CCK-8 (Beijing Solarbio Science & Technology Co., Ltd.) was added to each well and incubated at 37°C for 4 h. The absorbance was determined at 450 nm using a microplate reader (Thermo Fisher Scientific, Inc.).

SKOV3 and Hey cells were seeded in 6-well plates at a density of 10,000 cells/well overnight at 37°C. Then, SKOV3 and Hey cells were treated with 100 and 200 µg/ml CMP for 24 h at 37°C. Cell death was analyzed using an Annexin V-PE/7-AAD Apoptosis Detection kit (cat. no. WE0328; Beijing Biolab Technology Co., Ltd.) according to the manufacturer's instructions. In brief, the cells were centrifuged at 1,000 × g for 5 min at 37°C and collected. Then, 250 µl binding buffer was added, and the cell density was adjusted to 10⁶ cells/ml. Next, the cells were incubated with 5 µl Annexin V-PE and 10 µl 7-AAD for 15 min at room temperature. Subsequently, 400 µl PBS was added, and the data were analyzed using a CytoFLEX V2-B4-R2 flow cytometer (cat. no. C02945; Beckman Coulter, Inc.). The data [early (Q3) + late (Q2) apoptotic cells] were analyzed using FlowJo v10 (FlowJo LLC).

SKOV3 and Hey cells were seeded in 6-well plates at a density of 10,000 cells/well overnight at 37°C. Then, SKOV3 and Hey cells were treated with 100 and 200 µg/ml CMP for 24 h at 37°C in the presence or absence of ferrostatin-1 (Fer-1, MCE). Total RNA was isolated from SKOV3 and Hey cells using RNAVzol LS (Vigorous Biotechnology Beijing Co., Ltd.) according to the manufacturer's protocol. The concentration and purity of RNA samples were determined by measuring the optical density (OD) 260/OD280.

RT-PCR was performed using a Takara PrimeScript One Step RT-PCR kit (Takara Bio, Inc.) according to the manufacture's protocols with three experiments replicated. The PCR amplifications were performed in a 50-µl reaction system containing 20 µl RNase Free ddH₂O, 25 µl 2X Step Buffer, 2 µl PrimeScript 1 Step Enzyme Mix, 1 µl upstream primer (1 µM), and 1 µl downstream primer (1 µM). The PCR was as follows: 50°C for 30 min; 94°C for 2 min; and 30 cycles of 94°C for 30 sec, 55°C for 30 sec, 72°C for 1 min. GAPDH was used as an internal control using the 2^−∆∆Cq method (17). The primers were designed using Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome) and the sequences were listed in Table I.

Total proteins were isolated from SKOV3 and Hey cells using a total protein extraction kit (Beijing Solarbio Science & Technology Co., Ltd.) and collected following centrifugation at 12,000 × g for 30 min at 4°C. A BCA protein assay kit (Pierce; Thermo Fisher Scientific, Inc.) was used to determine the protein concentration. A total of 20 µg protein was separated using 12% SDS-PAGE (F15012Gel; ACE Biotechnology Co., Ltd.), transferred onto polyvinylidene difluoride membranes (Pierce; Thermo Fisher Scientific, Inc.) and blocked with 5% fat-free milk at room temperature for 2 h. The membrane was incubated with the following primary antibodies: Nrf2 (cat. no. 12721; 1:1,000; Cell Signaling Technology, Inc.), HO-1 (cat. no. Ab52947; 1:1,000; Abcam), xCT (cat. no. 12691 for human, cat. no. 98051 for mouse; 1:1,000; Cell Signaling Technology, Inc.), GPX4 (cat. no. ab125066; Abcam) and GAPDH (cat. no. 5174; Cell Signaling Technology, Inc.), at 4°C overnight. Then, the membranes were washed with PBST thrice. Next, the membrane was incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (both 1:5,000; cat. no. ZB-2301; OriGene Technologies, Inc.) for 2 h at room temperature. Enhanced chemiluminescence (MilliporeSigma) was used to determine the protein concentrations according to the manufacturer's protocol. Signals were detected using a Super ECL Plus kit (Nanjing KeyGen Biotech Co., Ltd.) Quantitative analysis was performed using UVP 7.0 software (UVP LLC). Relative protein expression was normalized to GAPDH. All experiments were repeated thrice. ImageJ v1.43b software (National Institutes of Health) was used for densitometry analysis.

The intracellular levels of Fe²⁺, MDA, SOD and GSH were determined using colorimetric assay kits, including an iron assay kit (cat. no. EC-BC-K304-S; Elabscience Biotechnology, Inc.), Lipid Peroxidation MDA Assay kit (cat. no. A003-1; Nanjing Jiancheng Biotechnology Co. Ltd.), superoxide dismutase (SOD) Assay kit (cat. no. A001-3-1; Nanjing Jiancheng Biotechnology Co. Ltd.) and Reduced Glutathione Assay kit (cat. no. A005-1; Nanjing Jiancheng Biotechnology Co. Ltd.) according to the manufacturer's instructions.

SKOV3 and Hey cells were seeded in 6-well plates at a density of 10,000 cells/well overnight. Then, SKOV3 and Hey cells were treated with 100 and 200 µg/ml CMP for 24 h at 37°C. Then, the cells were incubated with 1 ml DCFH-DA (1:1,000) at room temperature for 20 min. Cells were washed with DMEM culture without FBS thrice. Representative images were obtained under a fluorescence microscope (magnification, ×20; CKX53; Olympus Corporation).

A total of eight nude female BALB/cA-nu mice (6 weeks old, weighing 20.1±1.8 g) were purchased from SPF (Beijing) Biotechnology Co., Ltd. (n=4 per group). The mice were kept in a 12 h light/dark cycle with controlled humidity (50–70%) and temperature (20–24°C) with free access to food and water. They were randomly assigned to two experimental groups (n=4 per group). All experiments were approved according to the Ethics Committee of Tengzhou Central People's Hospital (Tengzhou, China; approval number TZH2020AH6) and were performed according to the National Institute of Health guidelines. SKOV3 cells (2×10⁶ cells in 100 µl PBS) were subcutaneously injected into the flanks of 6-week-old female nude mice to induce tumor formation. After 24 h, mice in the control group were orally administered distilled water (20 ml/kg, 1 time/day for 28 days). Mice in the therapy group were orally administered CMP (50 mg/kg, 1 time/day for 28 days). All mice were sacrificed 28 days after injection by resection of the decapitation under deep isoflurane anesthesia (5%) (1). The successful induction of anesthesia was confirmed by observation of the following parameters: respiration decreased in frequency and increased in depth, eyelid and cornea reflexes disappeared, muscle tension and the reflex response reduced, and no response to pain or other stimulation was exhibited. Tumor grafts were excised, weighed, and harvested for further analysis. Tumor diameters were measured at regular intervals, and the tumor volume was calculated using the following formula: volume = length × width²/2.

All data were expressed as the mean ± standard deviation. Unpaired Student's t-test was used to compare the differences between the two groups. One-way analysis of variance followed by Turkey analysis was used to analyze differences among three or more groups. Statistical analysis was performed using GraphPad Prism 8.0 Software (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

First, the cytotoxicity of CPM on a human normal ovarian epithelial cell line, IOSE80 was tested. As shown in Fig. 1A, 100 and 200 µg/ml CPM did not significantly decreased cell survival of IOSE80, but 300, 400, 500 µg/ml CPM slightly reduced IOSE80 cell survival rate. The CCK-8 assay showed that CMP significantly reduced the cell survival rate in SKOV3 and Hey cells in a dose-dependent manner (Fig. 1B and C). Compared with SKOV3 and Hey cells, CMP did not result in too much cytotoxicity in IOSE80 cells, indicating the drug was not toxic to normal cells. The IC₅₀ values of CMP in SKOV3 and Hey cells were 195.6 and 208.3 µg/ml, respectively. Optical microscopy images demonstrated that 100 and 200 µM CMP clearly reduced cell viability and increased cell death in SKOV3 and Hey cells (Fig. 1D). Furthermore, flow cytometry assays suggested that the cell death of both SKOV3 and Hey cells after 100 and 200 µg/ml CMP treatment, respectively, was significantly increased compared with that of the control (Fig. 1E and F).

SKOV3 and Hey cells were preincubated with different inhibitors, including ferroptosis inhibitors (ferrostatin-1, Fer-1), apoptosis inhibitors (Z-VAD-fluoromethylketone, Z-VAD-FMK), autophagy inhibitors (3-methyladenine, 3-MA), and necrosis inhibitors (necrostatin-1, Nec-1). As shown in Fig. 2A and B, CMP-induced cell death was largely reversed by preincubation with Fer-1 and Z-VAD-FMK but was not abolished by Nec-1 and 3-MA. Z-VAD-FMK decreased CMP-induced cell death by ~6.4%, whereas Fer-1 reduced CMP-induced cell death by ~26.1% (Fig. 2A and B). The present study further evaluated the mRNA levels of PTGS2 and CHAC1, two important ferroptosis markers, in SKOV3 and Hey cells treated with CMP and Fer-1. RT-qPCR analysis indicated that CMP significantly increased PTGS2 and CHAC1 mRNA levels, but preincubation with Fer-1 obviously reduced PTGS2 and CHAC1 mRNA levels in SKOV3 and Hey cells (Fig. 2C and D). These data indicated that CMP could induce ferroptosis in ovarian cancer cells.

DCFH-DA staining showed that both 100 and 200 µg/ml CMP elevated the intracellular SOD contents compared with those of the control in both SKOV3 and Hey cells (Fig. 3A and B). The intracellular levels of SOD, GSH, MDA and Fe²⁺ were then quantified. The data showed that 100 and 200 µg/ml CMP enhanced the production of SOD, MDA and Fe²⁺ (Fig. 3C-E) but decreased GSH levels in SKOV3 and Hey cells (Fig. 3F).

Nrf2-associated antioxidant stress plays a key role in ferroptosis inhibition (18). RT-qPCR analysis showed that CMP reduced Nrf2 and HO-1 mRNA levels but increased PTGS2 mRNA levels (Fig. 4A-C). In addition, the effects of CMP on the expression of Nrf2 and corresponding downstream target genes was tested. The data showed that Nrf2, HO-1, xCT and GPX4 protein levels were decreased in SKOV3 and Hey cells treated with CMP (Fig. 4D).

In vivo assays showed that CMP significantly suppressed tumor volume and weight compared with those of the control (Fig. 5A-C). The intracellular contents of Fe²⁺, MDA, SOD and GSH was further quantified. The data showed that CMP significantly increased the accumulation of Fe²⁺, MDA, and SOD but reduced the levels of GSH (Fig. 5D-G). In addition, Nrf2, HO-1, xCT and GPX4 protein levels were suppressed in nude mice treated with CMP compared with those of the control (Fig. 5H).