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Investigation into the synergistic effect of atorvastatin combined with ultrasound stimulation for anti‑glioma therapy

  • Authors:
    • Meng Ting Lin
    • Tsai-Yun Chan
    • Wei-Hao Liao
    • Chueh-Hung Wu
    • Tai-Horng Young
    • Wen-Shiang Chen
  • View Affiliations / Copyright

    Affiliations: Department of Biomedical Engineering, College of Medicine, National Taiwan University, Taipei 100, Taiwan, R.O.C., Department of Physical Medicine and Rehabilitation, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei 100, Taiwan, R.O.C.
    Copyright: © Lin et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 467
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    Published online on: August 1, 2025
       https://doi.org/10.3892/ol.2025.15213
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Abstract

Glioblastoma (GBM) is an aggressive, malignant brain tumor marked by rapid growth and invasiveness. Ultrasound (US) stimulation has emerged as a potential therapeutic approach for the management of GBM. Atorvastatin (ATO), a drug widely used to treat hyperlipidemia, has also been recognized for its anticancer properties, including inhibition of cell proliferation, induction of cell cycle arrest and promotion of apoptosis. Despite these promising attributes, the combined effectiveness of ATO and US stimulation in GBM treatment remains unclear. The present study aimed to explore the potential synergistic effects of ATO and US stimulation on C6 glioma cells. The optimal concentration of ATO and calibrated US parameters were determined in the present study. The cells were treated with ATO or US, followed by assessments of cellular viability and reactive oxygen species (ROS) to establish the most effective ATO dose and US parameters. The cells were then treated with ATO, US or their combination, and cellular viability, ROS levels, ATP production, tumor cell migration and the impact on downstream molecular pathways, particularly the AKT/mTOR signaling pathway, which is key for cell survival and proliferation, were assessed. The present findings revealed that ATO independently suppressed glioma cell viability by elevating ROS levels and reducing ATP production, and it showed a trend toward impairing tumor cell migration. These effects were notably associated with downregulation of the AKT axis, which indicated disruption of key survival mechanisms within the tumor cells. However, the anticipated synergistic effect of combining ATO with US stimulation was not observed under the tested conditions, thus suggesting that US stimulation did not further augment the therapeutic effect of ATO. While the combination therapy did not yield additive benefits, ATO alone exhibited notable potential as a therapeutic agent against glioma. In conclusion, the present study highlighted the need for further research on the role of ATO to further harness its anticancer properties in the context of GBM treatment.
View Figures

Figure 1

Effect of ATO on the viability of C6
glioma cells. (A) Flow diagram. Effects of 1, 5 and 10 µM ATO on
cell (B) viability (n=4) and (C) morphology at 24 h. (D) Extended
dose-response analysis at 24 h, which demonstrated survival rates
(n=3) and (E) cellular morphology following treatment with 1, 2,
2.5, 3 and 5 µM ATO. Scale bar, 200 µm. **P<0.01, ***P<0.001,
****P<0.0001. ATO, atorvastatin.

Figure 2

Effect of ATO on the ROS levels of C6
glioma cells. (A) Flow diagram. (B) ROS levels of C6 cells treated
with 1, 2, 2.5, 3 and 5 µM, determined by flow cytometry (n=3). (C)
Flow cytometry scatter diagrams. **P<0.01, ****P<0.0001.
DCFDA, 2′,7′-dichlorodihydrofluorescein diacetate; ATO,
atorvastatin; ROS, reactive oxygen species.

Figure 3

Effect of US stimulation on the
viability of C6 cells (n=3). US, ultrasound.

Figure 4

Effect of US stimulation on the ROS
levels of C6 glioma cells. (A) Flow diagram. (B) ROS levels of C6
cells treated with 1, 1.5, 2, 2.5 and 3 W/cm2 US,
determined by flow cytometry (n=3). (C) Flow cytometry scatter
diagrams. DCFDA, 2′,7′-dichlorodihydrofluorescein diacetate; ROS,
reactive oxygen species; US, ultrasound.

Figure 5

Combined effect of ATO and US on cell
viability. (A) Flow diagram. (B) Cell survival in response to ATO
combined with high US intensity (1.5 W/cm2, duty cycle
5%) (n=3). (C) Cell survival in response to ATO combined with low
US intensity (0.2 W/cm2, duty cycle 20%) (n=3).
**P<0.01, ***P<0.001, ****P<0.0001. US, ultrasound; ATO,
atorvastatin.

Figure 6

Combined effect of ATO and US on ROS
levels. (A) ROS levels (n=3). (B) Flow cytometry scatter diagrams.
US intensity was set as 1.5 W/cm2, duty cycle 5%.
**P<0.01, ***P<0.001, ****P<0.0001. ATO, atorvastatin;
ROS, reactive oxygen species; US, ultrasound; DCFDA,
2′,7′-dichlorodihydrofluorescein diacetate.

Figure 7

Combined effect of ATO and US on ATP
levels (n=3). **P<0.01, ***P<0.001, ****P<0.0001. ATO,
atorvastatin; US, ultrasound.

Figure 8

Tumor cell migration experiments. (A)
Gap closure assay was used to assess tumor cell migration. Scale
bar, 200 µm. (B) Quantification of tumor migration coverage after
ATO treatment with or without US (n=6). ATO, atorvastatin; US,
ultrasound.

Figure 9

Western blot analysis results. (A)
Western blot analysis of proteins associated with the AKT/mTOR
pathway. Semi-quantification of (B) p-AKT/AKT (n=3) and (C)
p-mTOR/mTOR (n=3). *P<0.05, **P<0.01. ATO, atorvastatin; US,
ultrasound; p-, phosphorylated.
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Copy and paste a formatted citation
Spandidos Publications style
Lin MT, Chan T, Liao W, Wu C, Young T and Chen W: Investigation into the synergistic effect of atorvastatin combined with ultrasound stimulation for anti‑glioma therapy. Oncol Lett 30: 467, 2025.
APA
Lin, M.T., Chan, T., Liao, W., Wu, C., Young, T., & Chen, W. (2025). Investigation into the synergistic effect of atorvastatin combined with ultrasound stimulation for anti‑glioma therapy. Oncology Letters, 30, 467. https://doi.org/10.3892/ol.2025.15213
MLA
Lin, M. T., Chan, T., Liao, W., Wu, C., Young, T., Chen, W."Investigation into the synergistic effect of atorvastatin combined with ultrasound stimulation for anti‑glioma therapy". Oncology Letters 30.4 (2025): 467.
Chicago
Lin, M. T., Chan, T., Liao, W., Wu, C., Young, T., Chen, W."Investigation into the synergistic effect of atorvastatin combined with ultrasound stimulation for anti‑glioma therapy". Oncology Letters 30, no. 4 (2025): 467. https://doi.org/10.3892/ol.2025.15213
Copy and paste a formatted citation
x
Spandidos Publications style
Lin MT, Chan T, Liao W, Wu C, Young T and Chen W: Investigation into the synergistic effect of atorvastatin combined with ultrasound stimulation for anti‑glioma therapy. Oncol Lett 30: 467, 2025.
APA
Lin, M.T., Chan, T., Liao, W., Wu, C., Young, T., & Chen, W. (2025). Investigation into the synergistic effect of atorvastatin combined with ultrasound stimulation for anti‑glioma therapy. Oncology Letters, 30, 467. https://doi.org/10.3892/ol.2025.15213
MLA
Lin, M. T., Chan, T., Liao, W., Wu, C., Young, T., Chen, W."Investigation into the synergistic effect of atorvastatin combined with ultrasound stimulation for anti‑glioma therapy". Oncology Letters 30.4 (2025): 467.
Chicago
Lin, M. T., Chan, T., Liao, W., Wu, C., Young, T., Chen, W."Investigation into the synergistic effect of atorvastatin combined with ultrasound stimulation for anti‑glioma therapy". Oncology Letters 30, no. 4 (2025): 467. https://doi.org/10.3892/ol.2025.15213
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