Demethylzeylasteral inhibits proliferation and metastasis of osteosarcoma cells by modulating the PI3K/AKT/Autophagy pathways

Abstract

Background: Osteosarcoma (OS) remains a highly aggressive malignancy with limited treatment options, necessitating the discovery of novel therapeutic agents. Demethylzeylasteral (DEM), a compound previously shown to exert anti-tumor properties in several malignancies, has not been sufficiently explored for its potential in OS treatment.

Purpose: This study focused on the anti-tumor properties of DEM on OS cells as well as the potential mechanisms.

Methods: OS cell lines (MG63 and 143B) were exposed to varying concentrations of DEM, followed by assessment of diverse cell functions. RNA sequencing was implemented to identify the molecular pathways affected by DEM exposure. The mechanistic underpinnings of DEM's action were also studied via a series of assays. Additionally, the therapeutic potential was validated utilizing xenograft models.

Results: DEM evidently repressed OS cell proliferation in a dose- and time-dependent fashion, arrested cells in G2/M phase, and facilitated apoptosis through the modulation of the BCL2/BAX ratio. Furthermore, DEM suppressed cell migration and invasion by reversing EMT-related protein expression. RNA sequencing revealed that DEM primarily affected autophagy-related pathways, particularly through the PI3K/AKT signaling. DEM treatment led to an elevation in ROS generation and enhanced autophagic activity, as demonstrated by elevated LC3B puncta formation and autophagy-related protein expression. In vivo, DEM effectively suppressed tumor growth while showing a favorable safety profile.

Conclusion: This study provides comprehensive evidence that DEM exerts potent anti-tumor properties in OS via the PI3K/AKT pathway, highlighting the significance of DEM as a therapeutic candidate for OS.

Keywords: Autophagy; Demethylzeylasteral; Osteosarcoma; PI3K/AKT pathway.

Graphical abstract

Fig. 1

Demethylzeylasteral (DEM) inhibits osteosarcoma cell proliferation and colony formation in a dose- and time-dependent manner. (A) Chemical structure of Demethylzeylasteral. (B, C) Viability of MG63 and 143B osteosarcoma cells treated with varying concentrations of DEM (0, 10, 20, and 40 uM) for 24, 48, and 72 h, assessed using a CCK8 assay. (D) Summary of IC₅₀ values calculated for MG63 and 143B cells at 24, 48, and 72 h. (E-H) Colony formation assays revealing a significant suppression of colony-forming ability in MG63 and 143B cells following DEM treatment at different concentrations (0, 10, 20, and 40 uM) in comparison to DMSO controls. *p < 0.05, **p < 0.01, ***p < 0.001. DEM, Demethylzeylasteral.

Fig. 2

DEM induces G2/M phase cell cycle arrest and promotes apoptosis in osteosarcoma cells. (A, B) Flow cytometric analysis demonstrating cell cycle alterations in MG63 and 143B cells after treatment with DMSO or DEM (0,10, 20, and 40 uM) for 48 h. (C, D) Western blot analysis showing the expression levels of cell cycle regulators (CDK2 and Cyclin B1) in MG63 and 143B cells following DEM treatment for 48 h. (E, F) Flow cytometric assessment of apoptosis in cells treated with DMSO or DEM for 48 h using Annexin V/PI staining. (G, H) Western blot analysis for apoptosis-related proteins (BCL2 and BAX), confirming the induction of apoptosis by DEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 3

DEM suppresses migration, invasion, and epithelial-mesenchymal transition (EMT) in osteosarcoma cells. (A, B) Transwell migration assays for assessing migration of 143B and MG63 cells treated with DMSO or DEM (0, 10, 20, and 40 uM) for 48 h. (C, D) Transwell invasion assays demonstrating decreased invasion potential of DEM-treated cells compared to controls. (E-H) Western blot analysis of EMT-related markers in 143B and MG63 cells, showing downregulation of mesenchymal markers (Vimentin and N-cadherin) and upregulation of the epithelial marker (E-cadherin) upon DEM treatment. *p < 0.05, **p < 0.01, ***p < 0.001. EMT, epithelial-mesenchymal transition.

Fig. 4

DEM modulates gene expression in osteosarcoma cells, affecting autophagy and the PI3K/AKT signaling. (A) Overview of differentially expressed genes in MG63 cells after 48-hour exposure to 25 uM DEM, with 723 genes upregulated and 1,356 downregulated (|log2(fold change)| ≥ 1 and Q value < 0.01). (B) Volcano plot illustrating the differentially expressed genes from RNA sequencing analysis. (C) Heatmap showing clustering of the differentially expressed genes. (D, E) KEGG pathway analysis revealing significant enrichment in autophagy-related and PI3K/AKT signaling. (F) Gene set enrichment analysis (GSEA) confirming the activation of autophagy-associated pathways. (G) RT-qPCR validation of five upregulated autophagy-related genes (ATG5, BECN1, MAP1LC3B, ATG7, and ULK1). **p < 0.01, ***p < 0.001.

Fig. 5

DEM promotes autophagy in osteosarcoma cells through the PI3K/AKT pathway. (A, B) Flow cytometric analysis of ROS levels in osteosarcoma cells treated with increasing concentrations of DEM. (C) Immunofluorescence imaging showing enhanced LC3B puncta formation in DEM-treated cells. (D-G) Western blot analysis demonstrating increases in autophagy markers (LC3B-II conversion, decreased p62 expression) and suppression of PI3K/AKT signaling (p-PI3K and p-AKT) in DEM-treated cells. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 6

In vivo efficacy of DEM in osteosarcoma xenograft models. (A) Representative tumor images showing the effect of DEM treatment on MG63 xenograft mouse models. (B) Analysis of tumor growth rates and final tumor weights, demonstrating the anti-tumor effects of DEM. (C) Tumor volume measurements showing significant reductions in the DEM-treated group compared to the vehicle control.(D) Monitoring of body weight throughout the treatment period, indicating that DEM treatment causes minimal systemic toxicity. (E) Representative images of Hematoxylin and Eosin (HE) staining (top) and Ki67 immunohistochemistry (IHC) (bottom) of tumor tissues. (F) Representative images of LC3-B immunofluorescence (IF) staining (Green: LC3-B; Blue: DAPI) in tumor tissues, showing autophagy levels. (G) Western blot analysis of p-PI3K, PI3K, p-AKT, and AKT expression in tumor tissues from each group. (H) Schematic diagram illustrating the proposed molecular mechanism of DEM in osteosarcoma cells. Data are presented as mean ± SD (n = 5 per group). * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the DMSO control group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)