Human cervical cancer HeLa cells and human cervical squamous cell carcinoma SiHa cells were purchased from Procell Life Science & Technology Co., Ltd. All cell lines were authenticated using short tandem repeat profiling and were verified to be free of mycoplasma contamination before use in the present study. Cells were routinely cultured in DMEM (cat. no. 11995065; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% (v/v) fetal bovine serum (FBS; cat. no. 10099141; Gibco; Thermo Fisher Scientific, Inc.) and a penicillin-streptomycin mixture (100 U/ml and 100 µg/ml, respectively) in a humidified incubator maintained at 37°C with 5% CO₂.

X-ray irradiation was performed using an X-RAD 320 irradiator (Precision X-Ray, Inc.), with a source-to-surface distance of 50 cm and a field size of 20×20 cm², at a dose rate of 2.0 Gy/min. The irradiation duration was 1 min for 2 Gy, 2 min for 4 Gy, 3 min for 6 Gy, 4 min for 8 Gy and 5 min for 10 Gy. All irradiations were performed at room temperature and the culture plates were positioned to ensure uniform dose delivery across the entire well surface. For all experiments, cells were divided into four groups: i) DMSO control group, in which cells were treated with an equal volume of DMSO as that used to dissolve LOP in the LOP and R + LOP groups (final concentration <0.1%, v/v) for 48 h at 37°C; ii) R group, in which cells were exposed to 6 Gy X-ray irradiation alone (dose rate, 2.0 Gy/min; exposure duration, 3 min) at room temperature; iii) LOP group, in which cells were treated with 15 µM LOP (cat. no. HY-B0418A; MedChemExpress) alone for 48 h at 37°C; and iv) R + LOP group, in which cells were treated with 15 µM LOP for 4 h at 37°C prior to exposure to 6 Gy X-ray irradiation (dose rate, 2.0 Gy/min; exposure duration, 3 min) and maintained in the presence of the drug throughout the remainder of each experiment. Unless otherwise indicated, cells were cultured for a total of 48 h before analysis.

Cells were counted and seeded into 96-well microplates at a density of 3,000 cells/well. On the following day, two sets of experiments were performed. For the LOP cytotoxicity assay, cells were treated with LOP alone for 48 h at 37°C without irradiation; the concentrations used were 0, 5, 10, 20, 40 and 80 µM for SiHa cells, and 0, 10, 20, 40, 80 and 160 µM for HeLa cells. For the combination experiments, cells were treated with LOP at 0, 5, 10, 20, 40 and 80 µM for 4 h at 37°C, followed by X-ray irradiation at 0, 2, 4, 6 8 and 10 Gy (dose rate, 2.0 Gy/min; exposure duration, 1, 2, 3, 4 and 5 min, respectively). LOP was dissolved in DMSO with a final concentration of <0.1% (v/v), which had no notable effect on cell viability. After 48 h of culture, the culture medium was removed, and 100 µl fresh DMEM containing 10 µl CCK-8 solution was added to each well, followed by an additional 2 h incubation at 37°C. The optical density value at 450 nm was determined using a multifunctional microplate reader (SpectraMax 190; Molecular Devices, LLC). The half-maximal inhibitory concentration (IC₅₀) was calculated accordingly. The combination index (CI) was calculated using CompuSyn software (version 1.0; ComboSyn, Inc.) based on the Chou-Talalay method (27). The fraction affected (Fa) represents the proportion of cells inhibited by the treatment, and the Fa-CI plot was generated to evaluate the synergistic effect across different Fa levels. CI<1 indicates synergy, CI=1 indicates additivity and CI>1 indicates antagonism.

Exponentially growing cells were counted and seeded into 24-well plates at a density of 10,000 cells/well, with three replicate wells for both the control and experimental groups. After 24 h, when cells reached 50–70% confluence, they were subjected to the aforementioned treatments (DMSO, LOP, R or R + LOP). Following 48 h of treatment, the culture medium was discarded, and 200 µl of a 100:1 mixture of medium and EdU solution was added to each well, followed by incubation for 6 h at 37°C. The mixture was then removed, and the cells were washed three times with PBS, fixed with 4% paraformaldehyde (Biosharp Life Sciences) for 30 min at room temperature and washed once with PBS. Subsequently, 2 mg/ml glycine was added, incubated for 5 min at room temperature and the cells were washed with PBS. The cells were then treated with 0.5% Triton X-100 for 20 min at room temperature and washed three times with PBS. Subsequently, the EdU reaction solution was prepared according to the manufacturer's instructions (kFluor488-EdU Cell Proliferation Detection Kit; cat. no. KGA9608-100; Nanjing KeyGEN Biotech Co., Ltd.), added at 100 µl/well and incubated in the dark for 30 min at room temperature. After three washes with PBS, the cells were stained with DAPI staining solution for 10 min at room temperature, washed three times with PBS and images were captured under an inverted fluorescence microscope.

Exponentially growing cells were plated into 6-well plates at a density of 1,000 cells/well. For the irradiation dose-response experiments, cells were exposed to 0, 2, 4, 6, 8 or 10 Gy X-ray irradiation at room temperature (dose rate, 2.0 Gy/min; exposure duration, 1, 2, 3, 4 and 5 min, respectively) to determine the optimal irradiation dose. For the combination experiments, cells were subjected to the aforementioned treatments (DMSO, LOP, R or R + LOP) after small colonies (~30 cells) had formed. The culture medium and drug were replaced every 3 days. After 6–8 days, when colonies (>50 cells) in the control group were clearly visible under microscopy, the medium was discarded. The cells were then washed three times with PBS, fixed with 4% paraformaldehyde for 30 min at room temperature and washed another three times with PBS. Subsequently, the cells were stained with crystal violet for 30 min at room temperature, washed several times with PBS, air-dried and images were captured.

Exponentially growing cells were digested with trypsin to form a single-cell suspension and seeded into 6-well culture plates. The cells were cultured at 37°C for 24 h until they reached 100% confluence, after which, a vertical scratch was created using a 200-µl pipette tip. After washing three times with PBS to remove detached cells, the cells were cultured in DMEM supplemented with 2% (v/v) FBS. Images were captured under a ×4 objective lens to confirm a straight and centered scratch. Subsequently, the cells were subjected to the aforementioned treatments (DMSO, LOP, R or R + LOP). Images were acquired at 24 and 48 h post-treatment, and the wound-healing rate was quantified using ImageJ software (version 1.53t; National Institutes of Health).

Cells were serum-starved for 12 h, harvested and seeded into the upper chambers of 24-well Transwell inserts (pore size, 8 µm; Corning, Inc.) at a density of 2×10⁴ cells/well. The lower chambers were filled with complete medium containing 20% FBS as a chemoattractant. The cells were subjected to the aforementioned treatments (DMSO, LOP, R or R + LOP), and then incubated for 48 h. Non-migrated cells on the upper surface were removed with cotton swabs, whereas cells that had migrated to the lower membrane surface were fixed with 4% paraformaldehyde for 30 min at room temperature, stained with 0.1% crystal violet for 30 min at room temperature and counted under a light microscope in five randomly selected fields per well.

Cells were seeded into 6-well plates, subjected to the aforementioned treatments (DMSO, LOP, R or R + LOP), and cultured for 48 h. Cell culture supernatants were then collected. The adherent cells were then digested with EDTA-free trypsin, and the resulting cell suspension was combined with the previously collected supernatants to ensure that both adherent and detached (apoptotic) cells were harvested. The combined suspension was centrifuged at 179 × g for 4 min at room temperature to harvest cell pellets. Following two washes with pre-chilled PBS and centrifugation at 179 × g for 4 min at room temperature, the cells were gently resuspended in pre-chilled 1X Binding Buffer (Annexin V-EGFP Apoptosis Detection Kit; cat. no. C1067S; Beyotime Biotechnology) to adjust the cell density to 1×10⁶ cells/ml, as recommended by the manufacturer. Subsequently, 100 µl cell suspension was incubated with 5 µl Annexin V-EGFP and 5 µl PI for 8–10 min at room temperature in the dark. After the addition of 400 µl pre-chilled 1X Binding Buffer and gentle mixing, flow cytometric analysis was performed within 1 h using a CytoFLEX flow cytometer (Beckman Coulter, Inc.) equipped with CytExpert software (version 2.4.0.28; Beckman Coulter, Inc.). The gating strategy included forward/side scatter gating to exclude debris and doublets, and auto-compensation was performed using single-stained controls. A total of 10,000 events were acquired per sample.

Cells were seeded into 6-well plates at a density of 2×10⁵ cells/well and subjected to the aforementioned treatments (DMSO, LOP, R or R + LOP) and cultured for 48 h. The cells were then trypsinized, centrifuged at 179 × g for 4 min at room temperature and washed three times with pre-chilled PBS. Subsequently, the cells were incubated with DCFH-DA (Reactive Oxygen Species Assay Kit; cat. no. S0033S; Beyotime Biotechnology) and BODIPY-C11 (Lipid Peroxidation Assay Kit; cat. no. S0043S; Beyotime Biotechnology) probes (both diluted 1:1,000 in serum-free DMEM) at 37°C for 30 min. After three washes with pre-chilled PBS, 500 µl serum-free DMEM was added and flow cytometric analysis was performed within 1 h using a CytoFLEX flow cytometer equipped with CytExpert software (version 2.4.0.28).

Cells were seeded into 6-well plates at a density of 2×10⁵ cells/well and cultured overnight at 37°C. The cells were then subjected to the aforementioned treatments and cultured for a total of 48 h (for the R + LOP group, LOP was added 4 h before irradiation and maintained until 48 h after the start of treatment) before analysis. Subsequently, the cells were trypsinized and centrifuged at 179 × g for 4 min at room temperature. IP lysis buffer (cat. no. P0013; Beyotime Biotechnology) was then added and the cells were lysed on ice for 30 min. After lysis, the supernatant was collected by centrifugation at 12,000 × g for 10 min at 4°C. The MDA content was determined using a Lipid Peroxidation MDA Assay Kit (cat. no. S0131S; Beyotime Biotechnology) by measuring the absorbance at 532 nm, and the results were expressed as µmol/mg protein. The GSH and GSH disulfide (GSSG) contents were determined using a GSH and GSSG Assay Kit (cat. no. S0053; Beyotime Biotechnology) according to the manufacturers' instructions. Briefly, total GSH was measured by the DTNB-based colorimetric reaction at 412 nm after incubation at 25°C, and GSSG was measured after GSH was eliminated using the GSH scavenging reagent. The GSH content was calculated as GSH=total GSH-GSSG × 2, and the GSH/GSSG ratio was calculated accordingly. All results were normalized to protein concentration.

Cells were pretreated with 10 µM Ferrostatin-1 (Fer-1; cat. no. HY-100579; MedChemExpress) or 0.1 µM liproxstatin-1 (Lip-1; cat. no. HY-12726; MedChemExpress) for 2 h at 37°C prior to the aforementioned treatments, and maintained throughout the subsequent 48-h culture period. Erastin (5 µM; cat. no. HY-15763; MedChemExpress) was used alone as a positive control for ferroptosis induction, and cells were treated for 48 h at 37°C. These pretreatments were applied in the MDA/GSH, Transwell migration and colony formation assays.

Cells were plated into 6-well plates and subjected to the aforementioned treatments (DMSO, LOP, R, or R + LOP) and cultured for a total of 48 h before analysis. For the R + LOP group, LOP was added 4 h before irradiation and maintained until the end of the 48-h period. Total RNA was then extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Subsequently, 1 µg total RNA was reverse-transcribed into cDNA using a RT kit (HiScript III RT SuperMix for qPCR; cat. no. R323-01; Vazyme Biotech Co., Ltd.) according to the manufacturer's instructions. qPCR was performed using ChamQ Universal SYBR qPCR Master Mix (cat. no. Q711-02; Vazyme Biotech Co., Ltd.) to assess the mRNA expression levels of GPX4 and SLC7A11, and GAPDH was used as an internal reference gene. The thermocycling conditions were as follows: Initial denaturation at 95°C for 30 sec, followed by 40 cycles at 95°C for 10 sec, 58°C for 30 sec and 72°C for 30 sec, with a final melting curve analysis. The relative mRNA expression levels were determined using the 2^−ΔΔCq method (28). The primer sequences were as follows: SLC7A11, forward 5′-TCATTGGAGCAGGAATCTTCA-3′, reverse 5′-TTCAGCATAAGACAAAGCTCCA-3′; GPX4, forward 5′-CCCGATACGCTGAGTGTGGTTTG-3′, reverse 5′-TGTTCGTTACTCCCTGGCTCCTG-3′; GAPDH, forward 5′-TGTGGGCATCAATGGATTTGG-3′ and reverse 5′-ACACCATGTATTCCGGGTCAAT-3′.

After treatment, as aforementioned (DMSO, LOP, R or R + LOP), the cells were washed three times with PBS. A lysis buffer mixture was prepared by combining RIPA buffer (cat. no. P0013B; Beyotime Biotechnology,), PMSF and a protease inhibitor cocktail (cat. no. P1005; Beyotime Biotechnology) at a ratio of 100:1:1. Subsequently, 100 µl lysis buffer was added to each well and the plates were incubated on ice for 30 min. Cells were then scraped into 1.5-ml microcentrifuge tubes using a cell scraper. After centrifugation at 11,304 × g for 15 min at 4°C, the supernatants were collected and the protein concentration was quantified using a BCA protein assay kit. Protein samples were separated by SDS-PAGE on 12% and transferred onto PVDF membranes. The membranes were then incubated overnight at 4°C with monoclonal antibodies against GPX4 (cat. no. 30388-1-AP; 1:1,000) and SLC7A11 (cat. no. 26864-1-AP; 1:1,000) (both from Proteintech Group, Inc.). After three washes with TBS containing 0.1% (v/v) Tween-20 (10 min each), the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody (cat. no. SA00001-2; 1:5,000; Proteintech Group, Inc.) for 2 h at room temperature. Protein bands were visualized using an ECL detection system (Bio-Rad Laboratories, Inc.). The band intensities were semi-quantified using ImageJ software (version 1.53t) and normalized to the loading control.

All experimental data are presented as the mean ± SD from at least three independent experiments. All experiments were performed with at least three independent biological replicates (n=3), each with three technical replicates per condition. Statistical analyses were performed using GraphPad Prism 9 software (Dotmatics). Normality was assessed using the Shapiro-Wilk test and homogeneity of variance was confirmed by Levene's test. Comparisons among multiple groups were performed using one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To determine the in vitro cytotoxicity of LOP in cervical cancer cells and to assess whether it exerts a synergistic effect with radiotherapy, the inhibitory effect of LOP on the viability of cervical cancer cells was first examined and the optimal X-ray irradiation dose identified. CCK-8 assays revealed that LOP significantly suppressed the viability of SiHa cells, with an IC₅₀ of 19.09 µM (Fig. 1A). Increasing concentrations of LOP exerted a marked inhibitory effect on SiHa cell viability in a concentration-dependent manner. Similarly, LOP significantly inhibited the viability of HeLa cells in a concentration-dependent manner, yielding an IC₅₀ value of 36.36 µM (Fig. 1B). Colony formation assays were performed to evaluate the inhibitory effect of different doses of X-ray irradiation on SiHa and HeLa cell proliferation (Fig. 1C and D). Colony formation assays revealed a dose-dependent reduction in clonogenic survival following irradiation in both cell lines. At 2–4 Gy, residual colonies remained abundant, indicating insufficient radiation damage to adequately reveal drug-sensitizing effects; at 8–10 Gy, clonogenic survival was nearly abolished, creating a plateau effect that precluded effective discrimination of further inhibition by combination treatment. At 6 Gy, however, both cell lines exhibited notable yet submaximal inhibition (50–60% survival), providing a suitable window for detecting combination effects.

To further evaluate the combined effect, SiHa and HeLa cells were treated with LOP at 0, 5, 10, 20, 40 and 80 µM, each paired with a fixed irradiation dose of 0, 2, 4, 6, 8 and 10 Gy, respectively (0 µM + 0 Gy, 5 µM + 2 Gy, 10 µM + 4 Gy, 20 µM + 6 Gy, 40 µM + 8 Gy and 80 µM + 10 Gy). Cell viability was assessed using a CCK-8 assay at 48 h post-treatment (Fig. 1E and F). CI analysis was performed using the Chou-Talalay method, and the Fa values were calculated for each single agent and their combination. Fa-CI analysis demonstrated that the combination of LOP and irradiation was synergistic (CI <1) across the evaluated Fa range (Fig. 1G and H). Among the tested combinations, 20 µM LOP combined with 6 Gy irradiation showed the strongest synergy; however, this concentration exceeded the IC₅₀ of SiHa cells (19.09 µM). Therefore, 15 µM LOP, a sub-IC₅₀ concentration that retained synergy, was selected. At this concentration, the combination with 6 Gy irradiation yielded CI values of 0.58 for SiHa cells and 0.55 for HeLa cells, both <1, confirming a synergistic interaction (Fig. 1G and H). Based on these results, 15 µM LOP and 6 Gy irradiation were selected for subsequent combination experiments in both cell lines.

To investigate whether LOP can enhance the radiosensitivity of cervical cancer cells, two cervical cancer cell lines (SiHa and HeLa) were treated with LOP alone or in combination with irradiation. The inhibitory efficiency on cell proliferation was assessed using an EdU assay. The results demonstrated that both LOP alone and LOP combined with radiotherapy significantly suppressed the proliferation of SiHa cells, and the inhibitory effect was markedly enhanced in the combination treatment group (Fig. 2A). Colony formation assays revealed that LOP significantly reduced the number of colonies formed by SiHa cells, and the inhibitory effect was further strengthened upon combined treatment with radiotherapy (Fig. 2B). Similarly, LOP combined with radiotherapy significantly inhibited the proliferation and colony formation ability of HeLa cells (Fig. 2C and D).

Tumor cell migration is a key factor in tumor invasion and metastasis (29). Accordingly, wound-healing assays were performed to determine whether LOP could enhance the inhibitory effect of radiotherapy on the migration of cervical cancer cells. The results demonstrated that, compared with in the control group, the wound closure rates of both cell lines in the LOP group, radiotherapy alone group and LOP plus radiotherapy group were significantly decreased at 24 and 48 h. Moreover, compared with in the radiotherapy alone group, cell migration was markedly arrested and almost abolished in the combination treatment group (Fig. 3A and B). Collectively, these findings indicated that LOP combined with radiotherapy may significantly suppress the migratory capacity of cervical cancer cells.

The balance between proliferation and apoptosis determines the sensitivity of tumor cells to therapy. The current study subsequently assessed whether LOP affects radiotherapy-induced apoptosis using Annexin V-PI double staining. Flow cytometric analysis in two cervical cancer cell lines (SiHa and HeLa) demonstrated that, compared with in the control group, the radiotherapy alone group, LOP alone group and LOP plus radiotherapy group all significantly increased the proportion of late apoptotic cells. Moreover, the combination of LOP and radiotherapy induced a markedly greater apoptotic effect than radiotherapy alone (Fig. 4A and B).

To explore the molecular mechanism underlying LOP-enhanced radiosensitivity in cervical cancer cells, intracellular ROS (detected using DCFH-DA) and lipid peroxidation (detected using BODIPY-C11) in HeLa cells were determined using flow cytometry. Compared with in the control group, ROS and BODIPY-C11 levels in HeLa cells were significantly elevated in the radiotherapy alone, LOP alone and combination groups, with a more marked increase observed in the combination group relative to radiotherapy alone (Fig. 5A and B). These findings indicated that LOP combined with radiotherapy may significantly promote lipid peroxidation and induce oxidative stress. In addition, MDA and GSH levels, as well as the GSH/GSSG ratio, were measured in HeLa cells. The combination treatment resulted in the highest MDA levels, and the lowest GSH levels and GSH/GSSG ratio among all groups (Fig. 5C-E).

Notably, the ferroptosis inhibitor Fer-1 partially reversed these effects induced by LOP plus radiotherapy, suggesting that ferroptosis contributes to the observed biochemical changes (Fig. 5D and E). To validate this hypothesis, RT-qPCR and western blot analysis were performed in HeLa cells. Western blot analysis demonstrated that SLC7A11 and GPX4 protein levels were decreased in all treatment groups, and the reduction was more significant in the combination group relative to radiotherapy alone (Fig. 5F). Consistently, qPCR revealed that the mRNA expression levels of SLC7A11 and GPX4 were downregulated in the treatment groups compared with in the control group, with a more pronounced reduction in the combination group than in the radiotherapy alone group (Fig. 5G). To further explore the involvement of ferroptosis in the anti-migratory effect of LOP and radiation, Transwell migration assays were performed following treatment with Fer-1 (10 µM) and Lip-1 (0.1 µM); the results demonstrated that both ferroptosis inhibitors partially reversed the inhibition of cell migration induced by the combination treatment, whereas Erastin (5 µM), a ferroptosis inducer tested alone as a positive control, inhibited cell migration to a similar extent as the combination treatment (Fig. 5H). Colony formation assays further confirmed that Fer-1 and Lip-1 partially reversed the combination-induced suppression of clonogenic survival, whereas erastin, tested alone as a positive control, reduced colony formation to a similar extent as the combination treatment (Fig. 5I). These data support the involvement of ferroptosis in the observed effects, although they do not exclude contributions from other cell death pathways. Core ferroptosis experiments (ROS, BODIPY-C11, RT-qPCR and western blotting for GPX4/SLC7A11, and Fer-1 rescue) were also performed in SiHa cells, yielding results consistent with those obtained in HeLa cells (Fig. S1).