The human HCC cell line Huh7 was obtained from the RIKEN BioResource Research Center. Cells were cultured in low-glucose Dulbecco's modified Eagle medium (DMEM; FUJIFILM Wako Pure Chemical Corporation) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Japan Bioserum) and 1% penicillin-streptomycin (Thermo Fisher Scientific, Inc.). Cultures were maintained at 37°C in a humidified incubator with 5% CO₂ and 95% air. To induce lipid accumulation, Huh7 cells were cultured in low-glucose DMEM supplemented with 5% heat-inactivated FBS and 1% penicillin-streptomycin and treated with OA at concentrations ranging from 0.5 to 2 mM. OA was dissolved in 70% ethanol and added to the medium at the desired final concentrations. Control cells received an equivalent volume of 70% ethanol without OA. OA was dissolved in 70% ethanol, and an equivalent concentration of ethanol was added to all control groups. Ethanol alone did not significantly affect cell viability, lipid accumulation, or oxidative stress parameters under the experimental conditions used.

X-ray irradiation was performed using an X-ray generator (MBR-1520R-3; Hitachi Medical Co., Ltd.) operated at 150 kVp and 20 mA, with 0.5-mm aluminum and 0.3-mm copper filters. The source-to-sample distance was set at 45 cm. The delivered dose was measured using a thimble-type ionization chamber dosimeter placed adjacent to the samples during irradiation. The dose rate was 1 Gy/min. The 10 Gy dose was selected as a mechanistic in vitro model to reliably induce oxidative stress and lipid peroxidation, based on prior radiobiological studies and preliminary dose-response experiments, rather than to directly mimic clinical dose regimens.

Cells were seeded in 35-mm culture dishes (2 ml of medium per dish) and subjected to X-ray irradiation at a dose of 5 Gy. Following irradiation, the cells were incubated for 3, 6, 12, 18, or 24 h. At each time point, cells were harvested and fixed with 70% ethanol precooled to −20°C. After fixation, the cells were treated with RNase I (3.6 µg/ml; Merck KGaA) at 37°C for 20 min in the dark. Propidium iodide (PI; 37 µg/ml) was then added, and DNA content was analyzed immediately by flow cytometry using a Cell Lab Quanta™ SC MPL system (Beckman Coulter, Inc.). The proportions of cells in the Sub-G1, G0/G1, S, and G2/M phases were calculated using Kaluza analysis software (version 2.1; Beckman Coulter, Inc.).

Intracellular lipid accumulation was quantified using Nile red stain (FUJIFILM Wako Pure Chemical Corporation). A 1 mM stock solution of Nile red was prepared by dissolving the dye in DMSO. To assess time-dependent lipid accumulation, cells were seeded in 60-mm culture dishes (4 ml medium per dish) and treated the following day with 1 mM OA. Cells were incubated for 3, 6, 12, 18, 24, 36, or 48 h. After incubation, cells were harvested, fixed with 4% paraformaldehyde, resuspended in PBS, and stored at −20°C. For measurement, cells were incubated with Nile red at a final concentration of 0.1 µg/ml in PBS at 37°C for 15 min in the dark. Fluorescence intensity was measured by flow cytometry using the Cell Lab Quanta™ SC MPL system (Beckman Coulter, Inc.) and analyzed using Kaluza analysis software (version 2.1; Beckman Coulter, Inc.). To assess concentration-dependent lipid accumulation, cells were similarly seeded in 60-mm dishes and treated the following day with 0.5, 1, or 2 mM OA for 18 h. Cells were then fixed with 4% paraformaldehyde, washed with PBS, and stored at −20°C. Staining and fluorescence measurement were performed as described above.

To evaluate the cytotoxicity of OA, cell viability was assessed using the trypan blue exclusion assay. Cells were collected from the same dishes used in the concentration-dependent lipid accumulation experiments.

Cell death induced by OA treatment or X-ray irradiation was analyzed using direct immunofluorescence-based flow cytometry with the Cell Lab Quanta™ SC MPL system (Beckman Coulter, Inc.). Cells collected using a single pipette were washed twice with Annexin V binding buffer (cat. no. 422201; BioLegend, Inc.), after which they were stained according to the manufacturer's protocol. Fluorescein isothiocyanate (FITC)-conjugated Annexin V (cat. no. 640906; BioLegend, Inc.) and PI (MilliporeSigma) were added to the cell suspension. Fluorescence intensity was quantified by flow cytometry and analyzed using Kaluza analysis software (version 2.1; Beckman Coulter, Inc.).

Intracellular LOOH levels were measured using Liperfluo staining solution (Dojindo Laboratories, Inc.). Untreated cells or cells pretreated with OA and/or exposed to X-ray irradiation were incubated with 1 µM Liperfluo at 37°C for 30 min in the dark. After staining, cells were collected and analyzed by flow cytometry using the Cell Lab Quanta™ SC MPL system (Beckman Coulter, Inc.). Fluorescence intensity was quantified using Kaluza analysis software (version 2.1; Beckman Coulter, Inc.).

Intracellular ROS levels were measured using the fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA; Dojindo Laboratories, Inc.). Cells were washed twice with Hanks' balanced salt solution (HBSS) and then incubated with a working solution of DCFH-DA at 37°C in a humidified atmosphere of 95% air and 5% CO₂ for 30 min. After incubation, cells were washed twice with HBSS and collected. ROS levels were analyzed by flow cytometry using the Cell Lab Quanta™ SC MPL system (Beckman Coulter, Inc.). The excitation and emission wavelengths were set at 488 and 530 nm, respectively.

Total RNA was extracted from cultured cells using the RNeasy^® Plus Mini Kit (Qiagen), and RNA concentrations and purity were determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Inc.). First-strand complementary DNA (cDNA) was synthesized from total RNA using the ReverTra Ace^® qPCR RT Master Mix (Toyobo Co., Ltd.) according to the manufacturer's instructions.

Quantitative PCR was performed using the Power SYBR™ Green PCR Master Mix (Applied Biosystems, Thermo Fisher Scientific) and the SmartCycler^® II system (Takara Bio Inc.). Thermal cycling was performed as follows: initial denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. The relative expression levels of glutamate-cysteine ligase catalytic subunit (GCLC), glutathione peroxidase 4 (GPX4), and ChaC glutathione specific γ-glutamylcyclotransferase 1 (CHAC1) were normalized to those of β-actin (ACTB) and calculated using the 2^−ΔΔCq method (17).

Gene-specific primers were designed using Primer3 software and synthesized by Eurofins Genomics. Gene sequences were obtained from the NCBI Gene database (https://www.ncbi.nlm.nih.gov/gene/), and the following accession numbers were used: GCLC (NM_001498.4), GPX4 (NM_002085.5), CHAC1 (NM_024111.6), and ACTB (NM_001101.5) (Table I).

All statistical analyses were performed using R software (version 4.4.1; R Foundation for Statistical Computing). For comparisons among multiple groups, one-way analysis of variance (ANOVA) was used, followed by the Tukey-Kramer post hoc test. Normality and homogeneity of variance were confirmed prior to ANOVA. Analyses were applied to datasets including cell cycle distribution, lipid accumulation, cytotoxicity, LOOH levels, ROS levels, and mRNA expression. A P-value of <0.05 was considered statistically significant. All experiments were independently repeated using separate cell cultures (biological replicates). For each biological replicate, measurements were performed in duplicate or triplicate as technical replicates where applicable.

To investigate the effect of IR on cell cycle progression in HCC cells, Huh7 cells were exposed to 5 Gy X-ray irradiation, and the cell cycle distribution was assessed at 3, 6, 12, 18, and 24 h post-irradiation using PI staining and flow cytometry. A marked accumulation of cells in the G2/M phase was observed at 12 h (37.7±3.5%, P<0.001) and 18 h (35.6±2.0%, P<0.001) compared with the non-irradiated controls (19.5±2.1%) (Fig. 1A). In contrast, no remarkable changes were detected at 3 h or 6 h, whereas a nonsignificant increase was noted at 24 h. Representative histograms of PI-stained cells are shown in Fig. 1B. These findings indicate that Huh7 cells undergo a robust G2/M phase arrest following DNA damage, supporting their use as a reliable in vitro model for studying radiotherapeutic responses. Based on these results, a post-irradiation time point of 12 h was selected for downstream analyses to capture the peak DNA damage response.

Huh7 cells were treated with 1 mM OA, and intracellular lipid accumulation was assessed at 3, 6, 12, 18, 24, 36, and 48 h using Nile red staining and flow cytometry. Compared to the untreated control (2.9±0.1), OA-treated cells exhibited remarkably increased lipid levels at all time points (3 h: 6.3±0.4; 6 h: 8.0±0.7; 12 h: 16.5±0.3; 18 h: 23.5±0.7; 24 h: 14.7±0.2; 36 h: 17.6±0.3; 48 h: 13.3±0.6; P<0.001 for all) (Fig. 2A and B). The highest lipid accumulation was observed at 18 h. Next, cells were exposed to OA at concentrations of 0.5, 1 or 2 mM for 18 h, and intracellular lipid levels were analyzed. Cell viability was evaluated using the trypan blue exclusion assay under the same conditions. A marked reduction in viability was detected at 2 mM OA (61.2±3.8%) compared with 0 mM (91.9±2.1%), 0.5 mM (90.0±3.6%) and 1 mM (86.1±6.2%) (P<0.001) (Fig. 2C). In addition, a dose-dependent increase in intracellular lipid-associated Nile red fluorescence intensity was observed (0 mM: 3.04; 0.5 mM: 12.7; 1 mM: 20.5; 2 mM: 29.6) (Fig. 2D and E).

To further assess the cytotoxicity of OA, Huh7 cells were treated with 0.5, 0.75, 1, or 2 mM OA for 18 h, followed by annexin V/PI double staining and flow cytometry. A remarkable increase in the proportion of PI-positive (necrotic/late apoptotic) cells was observed only in the 2-mM group (50.0±17.1%) compared to the untreated control group (18.2±8.0%, P<0.05). No statistically significant differences were detected in the 0.5-mM (10.8±6.0%), 0.75-mM (20.6±6.8%), or 1-mM (26.8±10.1%) groups (Fig. 3). These results confirm that 1 mM OA does not induce substantial cytotoxicity, indicating its suitability for use as a sublethal dose for subsequent mechanistic studies.

To investigate whether OA-induced lipid accumulation enhances the cytotoxic effect of ionizing radiation (IR), Huh7 cells were pretreated with 1 mM OA for 18 h prior to X-ray irradiation at doses ranging from 1 to 10 Gy. Cell death was assessed 12 h post-irradiation using annexin V/PI staining and flow cytometry. At 10 Gy, the proportion of PI-positive cells was remarkably higher in the OA-pretreated group (17.9±7.8%) than in the IR-only group (10.7±0.8%, P<0.05), indicating enhanced cell membrane damage (Fig. 4A). No remarkable differences were observed at lower radiation doses. These findings suggest that OA-induced lipid accumulation sensitizes Huh7 cells to radiation-induced cell death in a dose-dependent manner, particularly at higher radiation doses. The 10-Gy dose was selected for subsequent mechanistic analyses because a significant increase in PI-positive cells was observed only at this dose when OA pretreatment was combined with irradiation, compared with irradiation alone (Fig. 4A and B). In addition, previous in vitro radiobiology studies have commonly employed single high-dose irradiation to robustly induce oxidative stress and lipid peroxidation, facilitating mechanistic evaluation of radiation-induced cell death pathways (18,19).

To elucidate the mechanism underlying OA-mediated radiosensitization, we measured the levels of intracellular oxidative stress markers, including LOOH and ROS. LOOH levels, assessed by Liperfluo staining, were not remarkably altered by OA treatment alone (473.3±14.9) compared to the untreated control. In contrast, 10 Gy X-ray irradiation remarkably increased LOOH levels (487.5±31.6, P<0.05). Notably, OA pretreatment combined with 10 Gy irradiation further elevated LOOH accumulation (541.4±2.1, P<0.05 vs. 10 Gy alone) (Fig. 5A). These findings indicate that OA treatment augments radiation-induced lipid peroxidation. Moreover, ROS levels were remarkably increased by OA alone (42.0±10.3) compared to control (24.1±8.2, P<0.05), as presented in Fig. 6.

To further elucidate the molecular mechanisms underlying OA-mediated radiosensitization, we analyzed the expression of key oxidative stress-related genes: GCLC, GPX4, and CHAC1. Irradiation alone remarkably upregulated GCLC expression (2.5±1.0-fold, P<0.05), whereas OA administration alone had no statistically significant effect. The combination of OA and irradiation did not further increase the GCLC expression beyond that induced by irradiation alone (2.2±0.4-fold, P<0.05) (Fig. 7A). In contrast, GPX4 expression was remarkably upregulated in all treatment groups-OA alone (2.4±0.1, P<0.01), IR alone (2.3±0.8, P<0.05), and the OA + IR combination (2.8±0.5, P<0.01) (Fig. 7B). Notably, the levels of CHAC1, a glutathione-degrading enzyme and marker of ferroptosis, were markedly induced by OA treatment alone (4.1±0.6-fold, P<0.01) and further elevated in the OA + IR group (6.8±1.4-fold, P<0.001); however, it was not affected by IR alone (Fig. 7C). These transcriptional changes suggest that OA enhances radiation-induced oxidative stress and may contribute to ferroptosis-related signaling pathways.