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Article Open Access

Hesperadin sensitizes gastric cancer cells to cisplatin via NOX1‑dependent oxidative stress

  • Authors:
    • Wanjing Zhu
    • Qiyin Li
    • Zhenglei Ji
    • Di Wu
    • Jingya Chen
    • Yunli Zhao
    • Huazhang Wu
  • View Affiliations / Copyright

    Affiliations: School of Life Science, Anhui Provincial Key Laboratory of Tumor Evolution and Intelligent Diagnosis and Treatment, Bengbu Medical University, Bengbu, Anhui 233030, P.R. China, School of Public Health, Bengbu Medical University, Bengbu, Anhui 233030, P.R. China
    Copyright: © Zhu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 3
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    Published online on: October 27, 2025
       https://doi.org/10.3892/br.2025.2076
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Abstract

The therapeutic efficacy of cisplatin in gastric cancer (GC) is frequently constrained by chemoresistance and systemic toxicity. Targeted small molecules hold promise for providing synergistic effects that enhance treatment outcomes. GC cell viability was assessed via a Cell Counting Kit‑8 (CCK‑8) assay. Apoptosis, intracellular reactive oxygen species (ROS) levels, and the mitochondrial membrane potential were evaluated via flow cytometry and JC‑1 staining. Transcriptomic analysis [RNA‑sequencing (RNA‑Seq)] was subsequently conducted to identify differentially regulated pathways, followed by validation via reverse transcription‑quantitative PCR and western blotting. DNA damage was measured by γ‑H2AX immunofluorescence, and clonogenic survival was examined. The NOX1‑specific inhibitor ML171 was used to verify its mechanistic involvement. Hesperadin markedly suppressed GC cell proliferation in a dose‑dependent manner and induced mitochondrial apoptosis by regulating the Bcl‑2/Bax ratio and activating caspase pathways. RNA‑Seq analysis revealed significant upregulation of NOX1 and activation of oxidative stress‑associated pathways following Hesperadin treatment. Cotreatment with Hesperadin and cisplatin markedly reduced the IC50 of cisplatin, increased ROS accumulation, aggravated DNA damage, and potentiated apoptotic cell death. Notably, ML171 attenuated ROS generation and apoptotic effects, confirming the occurrence of a NOX1‑dependent mechanism. In conclusion, Hesperadin enhances the sensitivity of GC cells to cisplatin by inducing NOX1‑mediated oxidative stress and promoting mitochondrial dysfunction‑driven apoptosis, underscoring its potential as an effective combinatorial therapeutic strategy for GC treatment.
View Figures

Figure 1

Hesperadin inhibits proliferation and
induces death in gastric cancer cells. (A) After the cells were
treated with graded concentrations of hesperadin (0, 10, 20, 30,
40, 50, 60, 70, 80 and 90 nM) for 48 h, cell viability was assessed
via the CCK-8 assay, and the IC50 was calculated. (B) A
CCK-8 assay was used to evaluate the viability of AGS and HGC-27
cells following 24, 48 and 72 h of Hesperadin treatment. (C) Flow
cytometric analysis was conducted to assess cell mortality after 48
h of Hesperadin treatment. (D) The expression levels of the
apoptosis-related proteins BCL-2, BAX and cleaved caspase-3 were
analyzed via western blotting. β-ACTIN was used as a loading
control. Compared with the control group, the high-concentration
group was compared with the low-concentration group;
*P<0.05, **P<0.01 and
***P<0.001. CCK-8, Cell Counting Kit.

Figure 2

Transcriptomic analysis of
Hesperadin-induced model cell lines. (A) Volcano plot showing the
DEGs. (B) Heatmap illustration of significant DEGs. (C) Gene
Ontology enrichment analysis of DEGs. (D) Kyoto Encyclopedia of
Genes and Genomes pathway enrichment analysis of DEGs. (E) GSEA of
DEGs. (F) GSVA enrichment analysis. DEGs, differentially expressed
genes; GSEA, gene set enrichment analysis; GSVA, gene set variation
analysis; FDR, false discovery rate.

Figure 3

Hesperadin induces cell death in GC
cells by activating oxidative stress via ROS generation. (A)
DCFH-DA fluorescence staining was utilized to measure the
production of ROS in GC cells following Hesperadin treatment (green
fluorescence indicates ROS; scale bar, 50 µm). (B) Flow cytometry
was used to quantify the ROS levels (shown as the mean DCF
fluorescence intensity) in the groups treated with Hesperadin.
Compared with the control group, the high-concentration group was
compared with the low-concentration group; **P<0.01
and ***P<0.001. GC, gastric cancer; ROS, reactive
oxygen species.

Figure 4

Hesperadin enhances the sensitivity
of gastric cancer cells to cisplatin. (A) Changes in the
IC50 of cisplatin in AGS and HGC-27 cells after 48 h of
cotreatment with Hesperadin (10 nM) and various concentrations of
cisplatin (0, 10, 15, 20, 25, 30, 40, 60, 80, or 120 nM). (B) Cell
Counting Kit-8 assay for cell viability after 24, 48 and 72 h of
cotreatment with Hesperadin and cisplatin. (C) Colony formation
assay to assess the ability of cells to form colonies after 10 days
of cotreatment. (D) Flow cytometric analysis showing cell mortality
after cotreatment with Hesperadin and cisplatin. (E) Western blot
analysis was used to evaluate the expression of apoptosis-related
proteins (BCL-2, BAX and cleaved caspase-3). β-ACTIN was used as a
loading control. Hesperadin concentrations were 20 nM for AGS cells
and 16 nM for HGC-27 cells, whereas cisplatin concentrations were
10 µM for AGS cells and 8 µM for HGC-27 cells. Compared with all
the other groups (Control, Hes. alone, Cis. alone);
*P<0.05, **P<0.01 and
***P<0.001. Con., Control; Hes., Hesperadin; Cis.,
Cisplatin; Comb., Combination.

Figure 5

Hesperadin increases the sensitivity
of gastric cancer cells to cisplatin via oxidative stress. (A)
DCFH-DA fluorescence staining (green fluorescence represents ROS)
showing changes in ROS levels following treatment with Hesperadin
and cisplatin. (B) Quantification of ROS levels by flow cytometry.
(C) JC-1 fluorescence staining was used to detect changes in the
MMP. (D) γ-H2AX fluorescence staining was used to measure the level
of DNA damage (blue: nuclei; green: γ-H2AX); (scale bar, 100 µm).
Compared with all the other groups (Control, Hes. alone, Cis.
alone); **P<0.01 and ***P<0.001. ROS,
reactive oxygen species.

Figure 6

Hesperadin enhances the sensitivity
of GC cells to cisplatin by activating NOX1-induced oxidative
stress. (A) The mRNA expression level of NOX1 in GC cells after
Hesperadin treatment, as determined by reverse
transcription-quantitative PCR. (B-E) GC cells were pre-treated
with or without the NOX1 inhibitor ML171 (0.2 µM) for 2 h, followed
by combination treatment (Hesperadin + Cisplatin). The effects on
(B) reactive oxygen species generation, (C) cell death, (D) MMP and
(E) DNA damage were assessed using flow cytometry and
immunofluorescence analysis (scale bar, 50 µm). (F) Western blot
analysis of Cleaved Caspase-3 levels in AGS cells from the
experiment shown in C. The blot confirms the induction of apoptosis
by the combination treatment at the molecular level, corroborating
the flow cytometry data. β-ACTIN was used as a loading control.
Comb. + ML171 vs. Comb; *P<0.05,
**P<0.01 and ***P<0.001. GC, gastric
cancer; ns, not significant (P≥0.05).

Figure 7

Schematic diagram demonstrating how
hesperadin potentiates the chemosensitivity to cisplatin in GC
cells via NOX1-mediated oxidative stress. The image was created
with BioGDP.com (33). GC, gastric cancer; ROS, reactive
oxygen species.
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Spandidos Publications style
Zhu W, Li Q, Ji Z, Wu D, Chen J, Zhao Y and Wu H: Hesperadin sensitizes gastric cancer cells to cisplatin via NOX1‑dependent oxidative stress. Biomed Rep 24: 3, 2026.
APA
Zhu, W., Li, Q., Ji, Z., Wu, D., Chen, J., Zhao, Y., & Wu, H. (2026). Hesperadin sensitizes gastric cancer cells to cisplatin via NOX1‑dependent oxidative stress. Biomedical Reports, 24, 3. https://doi.org/10.3892/br.2025.2076
MLA
Zhu, W., Li, Q., Ji, Z., Wu, D., Chen, J., Zhao, Y., Wu, H."Hesperadin sensitizes gastric cancer cells to cisplatin via NOX1‑dependent oxidative stress". Biomedical Reports 24.1 (2026): 3.
Chicago
Zhu, W., Li, Q., Ji, Z., Wu, D., Chen, J., Zhao, Y., Wu, H."Hesperadin sensitizes gastric cancer cells to cisplatin via NOX1‑dependent oxidative stress". Biomedical Reports 24, no. 1 (2026): 3. https://doi.org/10.3892/br.2025.2076
Copy and paste a formatted citation
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Spandidos Publications style
Zhu W, Li Q, Ji Z, Wu D, Chen J, Zhao Y and Wu H: Hesperadin sensitizes gastric cancer cells to cisplatin via NOX1‑dependent oxidative stress. Biomed Rep 24: 3, 2026.
APA
Zhu, W., Li, Q., Ji, Z., Wu, D., Chen, J., Zhao, Y., & Wu, H. (2026). Hesperadin sensitizes gastric cancer cells to cisplatin via NOX1‑dependent oxidative stress. Biomedical Reports, 24, 3. https://doi.org/10.3892/br.2025.2076
MLA
Zhu, W., Li, Q., Ji, Z., Wu, D., Chen, J., Zhao, Y., Wu, H."Hesperadin sensitizes gastric cancer cells to cisplatin via NOX1‑dependent oxidative stress". Biomedical Reports 24.1 (2026): 3.
Chicago
Zhu, W., Li, Q., Ji, Z., Wu, D., Chen, J., Zhao, Y., Wu, H."Hesperadin sensitizes gastric cancer cells to cisplatin via NOX1‑dependent oxidative stress". Biomedical Reports 24, no. 1 (2026): 3. https://doi.org/10.3892/br.2025.2076
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