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Nobiletin enhances Doxorubicin sensitivity in osteosarcoma through ER stress-induced apoptosis mediated by the PI3K/AKT pathway
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  • Published: 19 March 2026

Nobiletin enhances Doxorubicin sensitivity in osteosarcoma through ER stress-induced apoptosis mediated by the PI3K/AKT pathway

  • Fei Liu1 na1,
  • Daotong Yuan2 na1,
  • Zhimeng Zhang3,
  • Rui Gong3,
  • Ximin Jin3,
  • Chaolu Wang1,
  • Wenpeng Xie4 &
  • …
  • Yongkui Zhang4 

Scientific Reports , Article number:  (2026) Cite this article

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We are providing an unedited version of this manuscript to give early access to its findings. Before final publication, the manuscript will undergo further editing. Please note there may be errors present which affect the content, and all legal disclaimers apply.

Subjects

  • Bone cancer
  • Pharmacology

Abstract

Osteosarcoma (OS) is the most prevalent primary malignant bone tumor, and chemotherapy resistance represents one of the primary challenges in its treatment. Nobiletin (Nob), a natural compound with anti-tumor properties, has an unclear mechanism of action but may increase the sensitivity of tumor cells to chemotherapy agents. This study explores how Nob enhances the effects of Doxorubicin (Dox) on OS cells. Two OS cell lines (143B and U2OS) were used in vitro experiments, where different concentrations of Nob and Dox were combined to treat the cells. The CCK-8 was used to calculate the cell proliferation inhibition rate, and SynergyFinder 3.0 was employed to assess the synergistic concentration of the interventions. Colony formation and cell scratch assays were used to assess the proliferation and migration potential of the cells. Apoptosis levels were identified by flow cytometry, while the expression of proteins related to apoptosis, endoplasmic reticulum stress (ERS), and the PI3K-AKT pathway was detected by Western blotting and immunofluorescence. Moreover, qPCR was employed to measure the expression levels of associated mRNAs. GO and KEGG analyses were used to confirm the differential biological processes and signaling pathways in OS cells. A tumor xenograft model, complemented with H&E and immunohistochemistry staining, were employed to validate the anti-tumor effects and underlying biological mechanisms of the combined treatment with Nob and Dox. The combined treatment of Nob and Dox markedly inhibited both the proliferation and migration of OS cells, exhibiting a pronounced synergistic effect. The combination treatment led to an increased apoptosis rate, characterized by reduced expression of Bcl-2 and elevated levels of Bax and Caspase3. Additionally, the combination group significantly upregulated ERS-related proteins GRP78, CRT, CHOP and ATF6, while the activity of the PI3K-AKT pathway was notably diminished. In a tumor xenograft model, the combination of Nob and Dox significantly inhibited tumor growth, inducing ERS and apoptosis. This study reveals that Nob enhances Dox-induced ERS and apoptosis by inhibiting the PI3K-AKT pathway, thereby increasing the sensitivity of OS cells to Dox. This discovery offers a promising new strategy for the treatment of OS.

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Data availability

The raw data supporting the conclusions requests should be directed to the corresponding author at 71000356@sdutcm.edu.cn.

Abbreviations

OS:

Osteosarcoma

Nob:

Nobiletin

Dox:

Doxorubicin

ERS:

endoplasmic reticulum stress

ER:

endoplasmic reticulum

UPR:

unfolded protein response

IF:

immunofluorescence

IHC:

immunohistochemistry

GO:

Gene Ontology

KEGG:

Kyoto Encyclopedia of Genes and Genomes

BCL-2:

B-cell lymphoma-2

Bax:

Bcl-2-associated X protein

GRP78:

Glucose-regulated protein

CRT:

Calreticulin

ATF6:

Activating transcription factor 6

CHOP:

C/EBP homologous protein

PI3K:

Phosphoinositide 3-Kinase

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Funding

This work was supported by the Natural Science Foundation of Shandong Province (NO. ZR2023MH236, ZR2019MH114), Medical and Health Science and Technology Development Project of Shandong Province (202404070421), Development Plan of Shandong Medical and Health Technology (2019WS580), and Cultivation Project of Qilu Health and Wellness Leading Talent.

Author information

Author notes

  1. These authors contributed equally: Fei Liu and Daotong Yuan.

Authors and Affiliations

  1. Department of orthopedics, Wangjing Hospital of China Academy of Chinese Medical Sciences, Beijing, 100102, China

    Fei Liu & Chaolu Wang

  2. School of Integrated Traditional Chinese and Western Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin, China

    Daotong Yuan

  3. First Clinical Medical College, Shandong University of Traditional Chinese Medicine, Jinan, 250355, Shandong, China

    Zhimeng Zhang, Rui Gong & Ximin Jin

  4. Department of Orthopedics, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan, 250355, Shandong, China

    Wenpeng Xie & Yongkui Zhang

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Contributions

Yongkui Zhang and Wenpeng Xie: Conceptualization, Funding acquisition, Supervision, Project administration, Writing–review & editing. Fei Liu, Daotong Yuan: Writing – original draft, Methodology, Data curation, Formal analysis. Zhimeng Zhang and Rui Gong: Resources, Software, Visualization. Ximin Jin and Chaolu Wang: Validation, Investigation.

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Correspondence to Wenpeng Xie or Yongkui Zhang.

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Liu, F., Yuan, D., Zhang, Z. et al. Nobiletin enhances Doxorubicin sensitivity in osteosarcoma through ER stress-induced apoptosis mediated by the PI3K/AKT pathway. Sci Rep (2026). https://doi.org/10.1038/s41598-026-44757-1

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  • Received: 18 February 2025

  • Accepted: 13 March 2026

  • Published: 19 March 2026

  • DOI: https://doi.org/10.1038/s41598-026-44757-1

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Keywords

  • Osteosarcoma
  • Nobiletin
  • Doxorubicin
  • Chemoresistance
  • Apoptosis
  • Endoplasmic reticulum stress
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