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Article

Fatty acid‑induced lipid accumulation promotes radiosensitization in Huh7 hepatocellular carcinoma cells

  • Authors:
    • Haruto Tanaka
    • Yasushi Mariya
    • Satoru Monzen
  • View Affiliations / Copyright

    Affiliations: Department of Radiation Science, Hirosaki University Graduate School of Health Sciences, Hirosaki, Aomori 036‑8564, Japan, Center for Cancer Treatment and Examination, Aomori Rosai Hospital, Hachinohe, Aomori 031‑8551, Japan
  • Article Number: 134
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    Published online on: February 11, 2026
       https://doi.org/10.3892/ol.2026.15487
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Abstract

 Hepatocellular carcinoma (HCC), a leading cause of cancer‑related mortality, represents a substantial global health burden, and the therapeutic efficacy of radiotherapy remains highly variable among patients with the condition. Metabolic alterations, particularly in lipid metabolism, may modulate radiosensitivity, although the underlying mechanisms are not fully understood. The present study investigated the impact of fatty acid uptake on radiosensitivity in HCC using the Huh7 cell line. Oleic acid (OA), a monounsaturated fatty acid, was used to induce intracellular lipid accumulation. Flow cytometry analyses revealed that OA treatment (1 mM; 18 h) considerably increased lipid content without inducing cytotoxicity. When combined with X‑ray irradiation (10 Gy), OA pretreatment considerably enhanced cell death, as indicated by an increased proportion of propidium iodide‑positive cells. This effect was associated with elevated levels of lipid hydroperoxides and reactive oxygen species, suggesting oxidative stress‑mediated mechanisms. Furthermore, mRNA expression analyses revealed marked upregulation of ChaC glutathione specific γ‑glutamylcyclotransferase 1, a gene involved in glutathione degradation and ferroptosis, in OA‑treated cells. The expression levels of glutathione peroxidase 4 and glutamate‑cysteine ligase catalytic subunit, key antioxidant defense genes, were also upregulated by OA and irradiation. These findings indicate that OA‑induced lipid accumulation sensitized HCC cells to radiation through enhanced oxidative stress and lipid peroxidation. However, as the present study was based on an in vitro model using a single cell line, the potential clinical relevance of these findings remains speculative and requires further validation in in vivo models and clinical studies.
View Figures

Figure 1

Radiation-induced G2/M
arrest in Huh7 cells. (A) Cell cycle distribution of Huh7 cells
following 5 Gy X-ray irradiation. The proportion of cells in the
G2/M phase was determined at 3, 6, 12, 18 and 24 h
post-irradiation using flow cytometry. Data are presented as the
mean ± SD from four independent experiments. *P<0.05,
**P<0.01 and ***P<0.001 vs. non-irradiated control. (B)
Representative histograms of DNA content assessed by PI staining at
each time point. Statistical analysis was performed using one-way
ANOVA followed by the Tukey-Kramer post hoc test.

Figure 2

OA-induced time- and dose-dependent
lipid accumulation in Huh7 cells. (A) Time-course analysis of
intracellular lipid accumulation following treatment with 1 mM OA.
Lipid content was measured at 3, 6, 12, 18, 24, 36 and 48 h using
Nile red staining and flow cytometry. Data are presented as the
mean ± SD from four independent experiments. *P<0.001 vs.
untreated control (0 h). (B) Representative histograms of Nile red
fluorescence showing a time-dependent increase in lipid
accumulation. The dashed line represents the untreated control (0
h). (C) Quantification of cell viability following 18-h treatment
with 0.5, 1 or 2 mM OA, determined using the trypan blue exclusion
assay, in which viable and non-viable cells were manually counted
under a light microscope using a Bürker-Türk hemocytometer.
Continuous data are presented as the mean ± SD of three independent
experiments. *P<0.001 vs. 0 mM, #P<0.001 vs. 0.5
mM and $P<0.001 vs. 1 mM. (D) Quantification of
intracellular lipid content following 18-h treatment with 0.5, 1 or
2 mM OA. Data are presented as the mean ± SD of three independent
experiments. *P<0.001 vs. 0 mM, #P<0.001 vs. 0.5
mM and $P<0.001 vs. 1 mM. Lipid content was assessed
by Nile red staining. (E) Representative histograms of Nile red
fluorescence showing dose-dependent lipid accumulation. The dashed
line represents the untreated control (0 mM). Statistical analysis
was performed using one-way ANOVA followed by the Tukey-Kramer post
hoc test. OA, oleic acid.

Figure 3

Cytotoxic effect of OA in Huh7 cells.
(A) Quantification of PI-positive cells following 18-h treatment
with 0.5, 0.75, 1 or 2 mM OA using annexin V/PI staining and flow
cytometry. Continuous data are presented as the mean ± SD from
three independent experiments. *P<0.05 and **P<0.01 as
indicated. (B) Representative flow cytometry plots illustrating
annexin V and PI staining profiles across different OA
concentrations. Statistical analysis was performed using one-way
ANOVA followed by the Tukey-Kramer post hoc test. OA, oleic
acid.

Figure 4

OA enhances radiation-induced cell
death in Huh7 cells. (A) Quantification of PI-positive cells
following 1–10 Gy X-ray irradiation with or without OA pretreatment
(1 mM for 18 h). Cell death was assessed by annexin V/PI staining
12 h post-irradiation. Continuous data are presented as the mean ±
SD from eight independent experiments. *P<0.05 vs. 10 Gy without
OA. (B) Representative flow cytometry plots of annexin V and PI
staining in irradiated cells with or without OA pretreatment.
Statistical analysis was performed using one-way ANOVA followed by
the Tukey-Kramer post hoc test. OA, oleic acid.

Figure 5

OA enhances radiation-induced lipid
peroxidation in Huh7 cells. (A) Quantification of intracellular
LOOH using Liperfluo staining following treatment with OA (1 mM; 18
h), 10 Gy X-ray irradiation or both. LOOH levels were measured 12 h
post-irradiation by flow cytometry. Continuous data are presented
as the mean ± SD from six independent experiments. *P<0.05,
**P<0.01 and ***P<0.001 as indicated. (B) Representative
histograms of Liperfluo fluorescence intensity depicting the LOOH
distribution across treatment groups. Statistical analysis was
performed using one-way ANOVA followed by the Tukey-Kramer post hoc
test. LOOH, lipid hydroperoxides; OA, oleic acid.

Figure 6

OA increases intracellular ROS levels
in Huh7 cells. (A) Quantification of ROS levels following treatment
with OA (1 mM; 18 h), 10 Gy X-ray irradiation or both. ROS levels
were measured using the DCFH-DA fluorescent probe and analyzed by
flow cytometry 12 h post-irradiation. Continuous data are presented
as the mean ± SD of six independent experiments. *P<0.05 vs.
untreated control. (B) Representative histograms of DCFH-DA
fluorescence intensity showing the ROS distribution across
treatment groups. Statistical analysis was performed using one-way
ANOVA followed by the Tukey-Kramer post hoc test. OA, oleic acid;
ROS, reactive oxygen species; DCFH-DA,
2′,7′-dichlorodihydrofluorescein diacetate.

Figure 7

Modulation of oxidative
stress-related gene expression by OA and irradiation in Huh7 cells.
Relative mRNA expression levels of (A) GCLC, (B) GPX4
and (C) CHAC1 in Huh7 cells treated with OA (1 mM; 18 h),
X-ray irradiation (10 Gy) or their combination. Gene expression was
quantified by reverse transcription-quantitative PCR and normalized
to ACTB using the 2−ΔΔCq method. Continuous data
are presented as the mean ± SD of three independent experiments.
Statistical analysis was performed using one-way ANOVA followed by
the Tukey-Kramer post hoc test. *P<0.05, **P<0.01 and
***P<0.001 as indicated. CHAC1, ChaC glutathione specific
γ-glutamylcyclotransferase 1; GCLC, glutamate-cysteine
ligase catalytic subunit; GPX4, glutathione peroxidase 4;
OA, oleic acid.
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Spandidos Publications style
Tanaka H, Mariya Y and Monzen S: Fatty acid‑induced lipid accumulation promotes radiosensitization in Huh7 hepatocellular carcinoma cells. Oncol Lett 31: 134, 2026.
APA
Tanaka, H., Mariya, Y., & Monzen, S. (2026). Fatty acid‑induced lipid accumulation promotes radiosensitization in Huh7 hepatocellular carcinoma cells. Oncology Letters, 31, 134. https://doi.org/10.3892/ol.2026.15487
MLA
Tanaka, H., Mariya, Y., Monzen, S."Fatty acid‑induced lipid accumulation promotes radiosensitization in Huh7 hepatocellular carcinoma cells". Oncology Letters 31.4 (2026): 134.
Chicago
Tanaka, H., Mariya, Y., Monzen, S."Fatty acid‑induced lipid accumulation promotes radiosensitization in Huh7 hepatocellular carcinoma cells". Oncology Letters 31, no. 4 (2026): 134. https://doi.org/10.3892/ol.2026.15487
Copy and paste a formatted citation
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Spandidos Publications style
Tanaka H, Mariya Y and Monzen S: Fatty acid‑induced lipid accumulation promotes radiosensitization in Huh7 hepatocellular carcinoma cells. Oncol Lett 31: 134, 2026.
APA
Tanaka, H., Mariya, Y., & Monzen, S. (2026). Fatty acid‑induced lipid accumulation promotes radiosensitization in Huh7 hepatocellular carcinoma cells. Oncology Letters, 31, 134. https://doi.org/10.3892/ol.2026.15487
MLA
Tanaka, H., Mariya, Y., Monzen, S."Fatty acid‑induced lipid accumulation promotes radiosensitization in Huh7 hepatocellular carcinoma cells". Oncology Letters 31.4 (2026): 134.
Chicago
Tanaka, H., Mariya, Y., Monzen, S."Fatty acid‑induced lipid accumulation promotes radiosensitization in Huh7 hepatocellular carcinoma cells". Oncology Letters 31, no. 4 (2026): 134. https://doi.org/10.3892/ol.2026.15487
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