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Anticancer effects of tangeretin associated with reactive oxygen species generation, mitochondrial dysfunction and apoptosis in CaSki cells

  • Authors:
    • Seung-Hyeon Ahn
    • Zeeshan Ahmad Bhutta
    • Hwayoung Na
    • Hong Kyu Lee
    • Kyung-Chul Choi
  • View Affiliations / Copyright

    Affiliations: Laboratory of Biochemistry and Immunology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungcheongbuk 28644, Republic of Korea, Department of Companion Animal Health, College of Biomedical Science and Health, Inje University, Gimhae, Gyeongsangnam 50834, Republic of Korea
    Copyright: © Ahn et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 166
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    Published online on: July 24, 2026
       https://doi.org/10.3892/or.2026.9171
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Abstract

Cervical cancer is the fourth most common cancer and the fourth leading cause of cancer‑related mortality among women worldwide. Tangeretin (TAN), a polymethoxylated flavonoid derived from citrus fruit peel, exhibits relatively high structural stability due to its methoxy groups and exerts anticancer effects in various malignancies, including lung, liver and breast cancer. However, to the best of our knowledge, the anticancer effects of TAN in cervical cancer remain insufficiently explored. The present study investigated the mechanisms underlying the anticancer effects of TAN on the CaSki cervical cancer cell line. Cell viability was evaluated using the EZ‑Cytox cell viability assay. The colony formation and cell cycle arrest assays demonstrated that TAN inhibited cell proliferation by inducing G1 phase arrest. The wound‑healing and Transwell migration assays demonstrated that TAN could reduce the migratory ability of CaSki cells, and the Annexin V/propidium iodide staining assay revealed that TAN increased the apoptotic cell population. Mitochondrial reactive oxygen species (ROS) were identified using MitoSOX™ staining and the mitochondrial membrane potential (MMP) was detected using JC‑1 staining. The findings of these assays suggested that TAN could increase mitochondrial ROS levels and decrease mitochondrial MMP in CaSki cells. Western blot analysis showed that TAN upregulated the protein expression levels of E‑cadherin and Bax. In addition, TAN restored the tumor suppressor protein p53. Collectively, these findings suggested that may exhibit anticancer activity in CaSki cells.
View Figures

Figure 1

TAN decreases the viability and
proliferation of CaSki cells. (A) Cell viability was assessed using
an EZ-Cytox cell viability assay following TAN treatment. (B)
Inhibitory effect of TAN on cell proliferation was assessed using a
colony formation assay. (C) Colony formation was semi-quantified as
the colony area and expressed as a percentage of the control. Data
are presented as the mean ± SD of technical triplicates.
**P<0.01 vs. control. TAN, tangeretin.

Figure 2

TAN induces G1 cell cycle
arrest in CaSki cells. (A) Cell cycle distribution was measured by
flow cytometry after Hoechst 33342 staining. (B) Percentage of
cells in each cell cycle phase was quantified using FlowJo
software. Data are presented as the mean ± SD of technical
triplicates. **P<0.01 vs. control. TAN, tangeretin.

Figure 3

TAN suppresses the migratory ability
of CaSki cells. (A) Representative images of the wound-healing
assay showing the effect of TAN on the migration of CaSki cells.
Scale bar, 500 µm. (B) Semi-quantification of wound closure at 24
and 48 h, expressed as the percentage of wound area relative to 0
h. (C) Representative images of the Transwell migration assay.
Scale bar, 500 µm (D) Semi-quantification of migratory cells in the
Transwell migration assay. (E) Protein expression levels of
E-cadherin were analyzed by western blot analysis. (F) E-cadherin
expression was semi-quantified by densitometric analysis and
normalized to GAPDH. Data are presented as the mean ± SD of
technical triplicates. *P<0.05, **P<0.01 vs. control. TAN,
tangeretin.

Figure 4

TAN induces the intrinsic apoptosis
of CaSki cells. (A) Representative flow cytometry plots of Annexin
V and PI staining showing apoptosis in CaSki cells following TAN
treatment. (B) Quantification of apoptotic cell populations. (C)
Representative western blot analysis images of Bax protein
expression. (D) Semi-quantification of Bax protein expression by
densitometric analysis normalized to GAPDH. Data are presented as
the mean ± SD of technical triplicates. *P<0.05, **P<0.01 vs.
control. PI, propidium iodide; TAN, tangeretin.

Figure 5

TAN increases mitochondrial oxidative
stress and decreases in CaSki cells. (A) Representative
fluorescence images of CaSki cells stained with MitoSOX™
and Hoechst 33342 to assess TAN-induced mitochondrial ROS
accumulation. Scale bar, 200 µm. (B) Semi-quantification of
mitochondrial ROS levels expressed as the fluorescence intensity
ratio of MitoSOX to Hoechst 33342. (C) Representative fluorescence
images of JC-1 staining showing TAN-induced changes in MMP. Scale
bar, 200 µm. (D) Semi-quantification of MMP is expressed as the
fluorescence intensity ratio of JC-1 dimers to JC-1 monomers. Data
are presented as the mean ± SD of technical triplicates.
**P<0.01 vs. control. MMP, mitochondrial membrane potential;
ROS, reactive oxygen species; TAN, tangeretin.

Figure 6

TAN upregulates the expression levels
of a tumor suppressor protein in CaSki cells. (A) Western blotting
was performed to detect the expression of p53, a tumor suppressor
protein, after TAN treatment. (B) p53 protein expression was
semi-quantified as the ratio of p53/GAPDH protein expression. Data
are presented as the mean ± SD of technical triplicates.
**P<0.01 vs. control. TAN, tangeretin.
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Copy and paste a formatted citation
Spandidos Publications style
Ahn S, Bhutta ZA, Na H, Lee HK and Choi K: Anticancer effects of tangeretin associated with reactive oxygen species generation, mitochondrial dysfunction and apoptosis in CaSki cells. Oncol Rep 56: 166, 2026.
APA
Ahn, S., Bhutta, Z.A., Na, H., Lee, H.K., & Choi, K. (2026). Anticancer effects of tangeretin associated with reactive oxygen species generation, mitochondrial dysfunction and apoptosis in CaSki cells. Oncology Reports, 56, 166. https://doi.org/10.3892/or.2026.9171
MLA
Ahn, S., Bhutta, Z. A., Na, H., Lee, H. K., Choi, K."Anticancer effects of tangeretin associated with reactive oxygen species generation, mitochondrial dysfunction and apoptosis in CaSki cells". Oncology Reports 56.4 (2026): 166.
Chicago
Ahn, S., Bhutta, Z. A., Na, H., Lee, H. K., Choi, K."Anticancer effects of tangeretin associated with reactive oxygen species generation, mitochondrial dysfunction and apoptosis in CaSki cells". Oncology Reports 56, no. 4 (2026): 166. https://doi.org/10.3892/or.2026.9171
Copy and paste a formatted citation
x
Spandidos Publications style
Ahn S, Bhutta ZA, Na H, Lee HK and Choi K: Anticancer effects of tangeretin associated with reactive oxygen species generation, mitochondrial dysfunction and apoptosis in CaSki cells. Oncol Rep 56: 166, 2026.
APA
Ahn, S., Bhutta, Z.A., Na, H., Lee, H.K., & Choi, K. (2026). Anticancer effects of tangeretin associated with reactive oxygen species generation, mitochondrial dysfunction and apoptosis in CaSki cells. Oncology Reports, 56, 166. https://doi.org/10.3892/or.2026.9171
MLA
Ahn, S., Bhutta, Z. A., Na, H., Lee, H. K., Choi, K."Anticancer effects of tangeretin associated with reactive oxygen species generation, mitochondrial dysfunction and apoptosis in CaSki cells". Oncology Reports 56.4 (2026): 166.
Chicago
Ahn, S., Bhutta, Z. A., Na, H., Lee, H. K., Choi, K."Anticancer effects of tangeretin associated with reactive oxygen species generation, mitochondrial dysfunction and apoptosis in CaSki cells". Oncology Reports 56, no. 4 (2026): 166. https://doi.org/10.3892/or.2026.9171
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