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AZD1080, a specific inhibitor of GSK‑3β, inhibits stemness and malignancies in osteosarcoma cancer stem‑like cells

  • Authors:
    • Peiyu Guo
    • Zhenkai Lou
    • Hongda Gong
    • Xiaodong Hou
    • Chunqiang Zhang
    • Bing Wang
    • Kaili Du
  • View Affiliations / Copyright

    Affiliations: Department of Orthopedics, The First Affiliated Hospital of Kunming Medical University, Kunming, Yunnan 650032, P.R. China
    Copyright: © Guo et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 248
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    Published online on: July 8, 2025
       https://doi.org/10.3892/mmr.2025.13613
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Abstract

Osteosarcoma is the most common primary bone cancer, primarily affecting children and young adults. Cancer stem cells (CSCs), a subpopulation presenting stemness, critically influence prognosis and promote recurrence and metastasis. Due to the crucial role of glycogen synthase kinase‑3 beta (GSK‑3β) in maintaining stemness, it is considered as an important target for drug development. The aim of the present study was to evaluate the inhibitory effect of AZD1080, a GSK‑3β inhibitor, on osteosarcoma CSCs. AZD1080 treatment clearly inhibited sphere formation in U2OS and 143B cells and dissociated spheres in CSCs derived from U2OS and 143B; in both processes, stemness markers OCT4 and SOX2 were markedly decreased, without affecting cell proliferation or apoptosis. AZD1080 treatment inhibited phosphorylation of GSK‑3β and its downstream regulated genes, including HEY1, HES1, CyclinD1 and β‑catenin. It was also observed that GSK‑3β activity was critical for the inhibitory effects of AZD1080 treatment on sphere formation and stemness. Moreover, GSK‑3β knockdown inhibited sphere formation and invasion capacity, indicating that AZD1080 exerts inhibitory roles in a GSK‑3β‑dependent manner. Taken together, the results showed AZD1080 as a specific inhibitor of CSC stemness, without cytotoxicity, and indicated it was a promising therapeutic agent that targeted GSK‑3β signaling in osteosarcoma.
View Figures

Figure 1

AZD1080 presented no obvious effect
on proliferation or apoptosis in CSCs derived from U2OS and 143B.
(A) Following culture in SFM for 5 and 10 days, cell morphology was
observed. (B) After 5 days of culture in SFM, cells were collected
and total protein was employed for western blot analysis for OCT4
and SOX2. *P<0.05, vs. PCs group. After 1, 2 or 3 days of
co-culture with 10 µmol/l AZD1080, cell viability was detected by
performing (C) CCK-8 assay, (D) cell cycle distribution was
detected by flow cytometry assay following PI staining and (E) cell
apoptosis was detected by performing flow cytometry assay following
FITC/PI double staining. CSCs, cancer stem cells; PCs, parental
cells; SFM, serum-free culture medium.

Figure 2

Addition of AZD1080 promoted
dissociation of spheres and cell attachment. (A) 100 Spheres >50
µm in diameter were co-cultured with or without 10 µmol/l AZD1080
for 1–3 days in SFM and sphere morphology and attachment capacity
were observed. (B) Total protein of CSCs co-cultured with or
without 10 µmol/l AZD1080 for 1, 2 or 3 days was collected and
analyzed by western blotting to detect OCT4 and SOX2. *P<0.05,
vs. mock group. CSCs, cancer stem cells.

Figure 3

Addition of AZD1080 inhibited sphere
formation without affecting cell attachment. (A) AZD1080 (10
µmol/l) was added in SFM for U2OS and 143B culture for 1–4 days and
cell morphology and sphere formation were imaged. (B) Total protein
of cells co-cultured with or without 10 µmol/l AZD1080 for 1 and 4
days was collected and analyzed by western blotting to detect OCT4
and SOX2. *P<0.05, vs. mock group. SFM, serum-free culture
medium.

Figure 4

Addition of AZD1080 inhibited
invasion and tumor formation in soft agar of CSCs derived from U2OS
and 143B. Spheres were collected and dissociated to obtain single
cells of CSCs, which were then co-cultured with 10 µmol/l AZD1080
for 24 h. (A) The invasion capacity of single cells of CSCs were
detected by performing Transwell assay. (B) Tumor formation assay
was measured in soft agar. **P<0.01, ***P<0.001,
****P<0.0001 vs. mock group. CSCs, cancer stem cells.

Figure 5

AZD1080 inhibited GSK-3β
phosphorylation and its downstream regulated signaling. Spheres
were collected and dissociated to obtain single cells of CSCs,
which were then co-cultured with 10 µmol/l AZD1080 for 24 h. Total
protein was collected to detect GSK-3β total protein and
phosphorylated protein, including its downstream regulated
proteins, CYCLIND1, β-catenin, HEY1, HES1 in (A) U2OS CSCs and (B)
143B cells. *P<0.05 vs. mock group. GSK-3β, glycogen synthase
kinase-3β; CSCs, cancer stem cells.

Figure 6

AZD1080 inhibited PTEN signaling via
regulating GSK-3β activity. (A) After AZD1080 treatment for 24 h,
or GSK-3β knockdown for 48 h, total protein was collected from CSCs
derived from U2OS and 143B and western blot was performed to detect
GSK-3β, MMP2, MMP9 and PTEN. *P<0.05, vs. mock group;
#P<0.05, vs. siNC group. (B) Following AZD1080
treatment or GSK-3β knockdown, mRNA levels including GSK-3β, MMP2,
MMP9 were measured by performing reverse transcription-quantitative
PCR. *P<0.05, vs. mock group; #P<0.05, vs. siNC
group. PTEN, phosphatase with tensin homology; GSK-3β, glycogen
synthase kinase-3β; si, small interfering; NC, negative
control.

Figure 7

GSK-3β is critical for stemness
maintenance in CSCs derived from osteosarcoma. (A) Following 10
µmol/l AR-A014418 treatment for 24 h, or GSK-3β knockdown for 48 h,
U2OS or 143B cells were cultured in SFM for 10 days and sphere
formation was observed. (B) Following 24 h culture in SFM, total
protein was obtained to detect OCT4 and SOX2. *P<0.05, vs. mock
group; #P<0.05, vs. siNC group. GSK-3β, glycogen
synthase kinase-3β; CSCs, cancer stem cells; SFM, serum-free
culture medium.

Figure 8

GSK-3β is critical for invasion
capacity. (A) Following 10 µmol/l AR-A014418 treatment for 24 h, or
GSK-3β knockdown for 48 h, CSCs derived from U2OS or 143B were used
to detect invasion capacity. *P<0.05, vs. mock group;
#P<0.05, vs. siNC group. (B) Total protein was
obtained to detect E-cadherin and Vimentin. *P<0.05, vs. mock
group; #P<0.05, vs. siNC group. GSK-3β, glycogen
synthase kinase-3β; CSCs, cancer stem cells; si, small interfering;
NC, negative control.
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Copy and paste a formatted citation
Spandidos Publications style
Guo P, Lou Z, Gong H, Hou X, Zhang C, Wang B and Du K: AZD1080, a specific inhibitor of GSK‑3&beta;, inhibits stemness and malignancies in osteosarcoma cancer stem‑like cells. Mol Med Rep 32: 248, 2025.
APA
Guo, P., Lou, Z., Gong, H., Hou, X., Zhang, C., Wang, B., & Du, K. (2025). AZD1080, a specific inhibitor of GSK‑3&beta;, inhibits stemness and malignancies in osteosarcoma cancer stem‑like cells. Molecular Medicine Reports, 32, 248. https://doi.org/10.3892/mmr.2025.13613
MLA
Guo, P., Lou, Z., Gong, H., Hou, X., Zhang, C., Wang, B., Du, K."AZD1080, a specific inhibitor of GSK‑3&beta;, inhibits stemness and malignancies in osteosarcoma cancer stem‑like cells". Molecular Medicine Reports 32.3 (2025): 248.
Chicago
Guo, P., Lou, Z., Gong, H., Hou, X., Zhang, C., Wang, B., Du, K."AZD1080, a specific inhibitor of GSK‑3&beta;, inhibits stemness and malignancies in osteosarcoma cancer stem‑like cells". Molecular Medicine Reports 32, no. 3 (2025): 248. https://doi.org/10.3892/mmr.2025.13613
Copy and paste a formatted citation
x
Spandidos Publications style
Guo P, Lou Z, Gong H, Hou X, Zhang C, Wang B and Du K: AZD1080, a specific inhibitor of GSK‑3&beta;, inhibits stemness and malignancies in osteosarcoma cancer stem‑like cells. Mol Med Rep 32: 248, 2025.
APA
Guo, P., Lou, Z., Gong, H., Hou, X., Zhang, C., Wang, B., & Du, K. (2025). AZD1080, a specific inhibitor of GSK‑3&beta;, inhibits stemness and malignancies in osteosarcoma cancer stem‑like cells. Molecular Medicine Reports, 32, 248. https://doi.org/10.3892/mmr.2025.13613
MLA
Guo, P., Lou, Z., Gong, H., Hou, X., Zhang, C., Wang, B., Du, K."AZD1080, a specific inhibitor of GSK‑3&beta;, inhibits stemness and malignancies in osteosarcoma cancer stem‑like cells". Molecular Medicine Reports 32.3 (2025): 248.
Chicago
Guo, P., Lou, Z., Gong, H., Hou, X., Zhang, C., Wang, B., Du, K."AZD1080, a specific inhibitor of GSK‑3&beta;, inhibits stemness and malignancies in osteosarcoma cancer stem‑like cells". Molecular Medicine Reports 32, no. 3 (2025): 248. https://doi.org/10.3892/mmr.2025.13613
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