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Acetylshikonin induces ferroptosis via the lipid peroxidation pathway in osteosarcoma cells

  • Authors:
    • Ji-Ying Chen
    • Guo-Shou Wang
    • Tsung-Ming Chang
    • Ju-Fang Liu
  • View Affiliations / Copyright

    Affiliations: Department of Medicine, MacKay Medical College, New Taipei City 252005, Taiwan, R.O.C., School of Dental Technology, College of Oral Medicine, Taipei Medical University, Taipei 110301, Taiwan, R.O.C., Translational Medicine Center, Shin‑Kong Wu Ho‑Su Memorial Hospital, Taipei 111045, Taiwan, R.O.C.
    Copyright: © Chen et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 55
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    Published online on: November 27, 2025
       https://doi.org/10.3892/mmr.2025.13765
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Abstract

Osteosarcoma, a prevalent primary malignant bone tumor, primarily affects adolescents and young adults. Current treatment strategies involve a combination of surgical intervention and chemotherapy. However, the effectiveness of chemotherapy is constrained by considerable challenges, such as drug resistance and insensitivity. Ferroptosis, a form of programmed cell death that is distinct from apoptosis, presents a promising alternative target for cancer therapy. Ferroptosis is characterized by iron‑dependent lipid peroxidation, producing reactive oxygen species (ROS) and suppressing glutathione peroxidase 4 (GPX4). Notably, ferroptosis circumvents the conventional mechanisms associated with apoptosis. Inducing ferroptosis in cancer cells may help overcome drug resistance and enhance the effectiveness of existing treatments, including chemotherapy, radiotherapy and immunotherapy. Acetylshikonin is a derivative of naphthoquinone that possesses anticancer properties. However, the effects of acetylshikonin on the treatment of osteosarcoma and the mechanisms by which it induces cancer cell death remain unclear. The present study aimed to investigate the potential of acetylshikonin to induce apoptosis in osteosarcoma cells. Using cell viability assays, ROS detection, mitochondrial membrane potential analysis and ferroptosis inhibitor rescue experiments, the results demonstrated that acetylshikonin significantly reduced the viability of osteosarcoma cell lines while exhibiting low toxicity to normal cells. Mechanistically, acetylshikonin induced the production of ROS, disrupted the mitochondrial membrane potential and promoted lipid peroxidation, ultimately leading to ferroptosis. Additionally, treatment with acetylshikonin led to decreased levels of GPX4 and increased intracellular ferrous ion (Fe²+) concentrations, further supporting its role in the induction of ferroptosis. In conclusion, the current study emphasized the potential of acetylshikonin as an effective agent in inducing ferroptosis in osteosarcoma cells. Acetylshikonin reduced osteosarcoma cell viability and selectively promoted ferroptosis by increasing ROS production, disrupting mitochondrial function and enhancing lipid peroxidation. Furthermore, its ability to downregulate GPX4 and increase intracellular Fe2+ levels indicated its role in triggering ferroptosis. These findings suggest that acetylshikonin may be a valuable therapeutic candidate for the treatment of osteosarcoma, potentially improving outcomes and addressing the limitations of current therapies.
View Figures

Figure 1

Acetylshikonin reduces osteosarcoma
cell viability and increases membrane permeability. (A) Molecular
structure of acetylshikonin. (B) CCK-8 assay results showing the
viability of hFOB 1.19 cells following exposure to acetylshikonin
(0.5–3 µM) for 24 h (n=4). (C) MG63, (D) HOS and (E) U2OS cell
viability was assessed using the CCK-8 assay following treatment
with acetylshikonin (0.05–20 µM) for 24 and 48 h (n=4). (F)
Phase-contrast microscopy images depicting morphological changes in
osteosarcoma cells treated with acetylshikonin (3 µM) for 24 h
(n=4). (G) Fluorescence microscopy images showing nuclear staining
with Hoechst 33342, membrane integrity with PI and viability with
Calcein-AM in osteosarcoma cells treated with acetylshikonin
(0.5–10 µM) for 24 h (n=4). Data are presented as the mean ± SD.
*P<0.05 vs. untreated control. CCK-8, Cell Counting Kit-8; PI,
propidium iodide.

Figure 2

Acetylshikonin induces DNA
fragmentation in osteosarcoma cells. Osteosarcoma cells
(5×105) were treated with acetylshikonin (0.1–3 µM) for
24 h, then underwent the TUNEL assay. Fluorescence was analyzed by
flow cytometry (n=4). Untreated cells were used as controls. Data
are presented as the mean ± SD. *P<0.05, **P<0.01 vs.
untreated control.

Figure 3

Acetylshikonin induces apoptosis in
osteosarcoma cells. Osteosarcoma cells (5×105) were
treated with acetylshikonin (0.1–3 µM) for 24 h, then underwent the
Annexin V/PI assay (n=4). Untreated cells were used as controls.
Data are presented as the mean ± SD. *P<0.05 vs. untreated
control. PI, propidium iodide.

Figure 4

Acetylshikonin promotes cell cycle
arrest in osteosarcoma cells. Cell cycle distribution of
osteosarcoma cells treated with acetylshikonin (0.1–3 µM) for 24 h,
was assessed by propidium iodide staining and flow cytometry.
Untreated cells were used as controls. Data are presented as the
mean ± SD. *P<0.05 vs. untreated control.

Figure 5

Acetylshikonin promotes intracellular
ROS accumulation. Osteosarcoma cells (5×105) were
treated with acetylshikonin (0.1–3 µM) for 1 h, then stained with 1
µM H2DCFDA. Fluorescence was analyzed by flow cytometry
(n=4). Untreated cells served as controls. Data are presented as
the mean ± SD. *P<0.05, **P<0.01 vs. untreated control. ROS,
reactive oxygen species.

Figure 6

Acetylshikonin disrupts mitochondrial
membrane potential. Cells were incubated with acetylshikonin (1 µM)
for (A) 2 or (B) 8 h and subsequently stained with JC-1 (n=4). (C)
Western blot analysis of Bcl-2, Bcl-xl, Bax and Bak protein
expression in osteosarcoma cells following acetylshikonin treatment
(0.1–3 µM) for 8 h (n=4). Untreated cells served as controls. Data
are presented as the mean ± SD.

Figure 7

Acetylshikonin decreases
mitochondrial volume and enhances lipid peroxidation in
osteosarcoma cells. (A) Transmission electron microscopy images of
HOS cells treated with acetylshikonin (3 µM) for 24 h, showing a
reduction in mitochondrial volume (blue triangles). The red arrow
indicates the endoplasmic reticulum. (B) Fluorescence microscopy
analysis of lipid peroxidation in osteosarcoma cells treated with
acetylshikonin (3 µM) and C11-BODIPY™ 581/591 (n=4). (C-E) Flow
cytometric analyses showing lipid peroxidation in osteosarcoma
cells incubated with acetylshikonin (0.1–3 µM) and C11-BODIPY
(581/591) for 30 min (n=4). (F-H) Intracellular Fe2+
levels in osteosarcoma cells treated with acetylshikonin (3 µM) for
24 h were quantified using an Fe2+ detection reagent and
microplate reader. (I-K) Western blot analysis of GPX4 protein
expression in osteosarcoma cells treated with acetylshikonin (0.1–3
µM) for 8 h (n=4). Untreated cells served as controls. Data are
presented as the mean ± SD. *P<0.05 vs. untreated control. GPX4,
glutathione peroxidase 4; Fe2+, ferrous ion.

Figure 8

Acetylshikonin induces
ferroptosis-mediated cell death. (A-C) CCK-8 assay results of
osteosarcoma cells pretreated with ferrostatin-1 (10 µM),
z-DEVD-FMK (10 µM), necrostatin-1 (10 µM), IM54 (10 µM) and
liproxstain-1 (1 µM) for 1 h before exposure to acetylshikonin (3
µM) for 24 h (n=4). (D) Flow cytometric analysis of Annexin V/PI
staining in osteosarcoma cells pretreated with ferrostatin-1 (10
µM) for 1 h followed by acetylshikonin (3 µM) for 24 h (n=4). (E)
CCK-8 assay results of osteosarcoma cells treated with
acetylshikonin (3 µM), erastin (3 µM) and RSL3 (3 µM) for 24 h
(n=4). Untreated cells served as controls. Data are presented as
the mean ± SD. *P<0.05 vs. untreated control;
#P<0.05 vs. acetylshikonin-treated group. CCK-8, Cell
Counting Kit-8.
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Copy and paste a formatted citation
Spandidos Publications style
Chen J, Wang G, Chang T and Liu J: Acetylshikonin induces ferroptosis via the lipid peroxidation pathway in osteosarcoma cells. Mol Med Rep 33: 55, 2026.
APA
Chen, J., Wang, G., Chang, T., & Liu, J. (2026). Acetylshikonin induces ferroptosis via the lipid peroxidation pathway in osteosarcoma cells. Molecular Medicine Reports, 33, 55. https://doi.org/10.3892/mmr.2025.13765
MLA
Chen, J., Wang, G., Chang, T., Liu, J."Acetylshikonin induces ferroptosis via the lipid peroxidation pathway in osteosarcoma cells". Molecular Medicine Reports 33.2 (2026): 55.
Chicago
Chen, J., Wang, G., Chang, T., Liu, J."Acetylshikonin induces ferroptosis via the lipid peroxidation pathway in osteosarcoma cells". Molecular Medicine Reports 33, no. 2 (2026): 55. https://doi.org/10.3892/mmr.2025.13765
Copy and paste a formatted citation
x
Spandidos Publications style
Chen J, Wang G, Chang T and Liu J: Acetylshikonin induces ferroptosis via the lipid peroxidation pathway in osteosarcoma cells. Mol Med Rep 33: 55, 2026.
APA
Chen, J., Wang, G., Chang, T., & Liu, J. (2026). Acetylshikonin induces ferroptosis via the lipid peroxidation pathway in osteosarcoma cells. Molecular Medicine Reports, 33, 55. https://doi.org/10.3892/mmr.2025.13765
MLA
Chen, J., Wang, G., Chang, T., Liu, J."Acetylshikonin induces ferroptosis via the lipid peroxidation pathway in osteosarcoma cells". Molecular Medicine Reports 33.2 (2026): 55.
Chicago
Chen, J., Wang, G., Chang, T., Liu, J."Acetylshikonin induces ferroptosis via the lipid peroxidation pathway in osteosarcoma cells". Molecular Medicine Reports 33, no. 2 (2026): 55. https://doi.org/10.3892/mmr.2025.13765
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