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Exploring the mechanism of action of glaucocalyxin D against acute myeloid leukemia using network pharmacology, molecular docking and cellular experiments

  • Authors:
    • Lian Chen
    • Weixian Yang
    • Weiqing Zhang
    • Heng Luo
    • Chen Yan
  • View Affiliations / Copyright

    Affiliations: College of Pharmaceutical Science, Guizhou University of Traditional Chinese Medicine, Guiyang, Guizhou 550025, P.R. China, Department of Pharmacy, Anshun City People's Hospital, Anshun, Guizhou 561000, P.R. China, State Key Laboratory of Discovery and Utilization of Functional Components in Traditional Chinese Medicine, Natural Products Research Center of Guizhou Province, Guizhou Medical University, Guiyang, Guizhou 550014, P.R. China
    Copyright: © Chen et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 267
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    Published online on: April 27, 2026
       https://doi.org/10.3892/ol.2026.15622
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Abstract

Glaucocalyxin D (GLD), an ent‑kaurane diterpenoid isolated from Isodon species, exhibits extensive pharmacological potential; however, its mechanism of action against acute myeloid leukemia (AML) remains to be elucidated. The present study employed a combined network pharmacology and experimental approach to elucidate the anti‑AML mechanisms of GLD. Potential targets were identified using database mining and a protein‑protein interaction network was constructed. The Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes enrichment analyses highlighted the JAK‑STAT signaling pathway as central to action by GLD. Molecular docking predicted stable binding of GLD to core targets, including STAT3. Experimental validation in HEL and K562 AML cells demonstrated that GLD potently and dose‑dependently inhibits cell proliferation, with efficacy similar to the standard chemotherapeutic agent doxorubicin. Mechanistically, GLD suppressed the phosphorylation of JAK2 and STAT3. GLD also induced mitochondrial apoptosis by modulating the Bcl‑2/Bax ratio and triggered G2/M phase arrest by downregulating cyclin B1 and CDK1. These findings delineated a coherent mechanism whereby GLD exerts anti‑leukemic effects by inhibiting the JAK‑STAT pathway, supporting its potential as a novel lead compound for AML therapy in the future.
View Figures

Figure 1

Potential AML-associated targets of
GLD. (A) Venn diagram of AML-associated target interactions. The
numbers of targets retrieved from the OMIM, GeneCards and TTD
databases are shown: OMIM, 92; GeneCards, 5,446; TTD, 16. (B) Venn
diagram of GLD and AML target intersections. A total of 74
GLD-associated targets and 5,576 AML-associated targets were
analyzed, yielding 55 overlapping targets. (C) Construction of the
GLD and AML gene-protein interaction network. The network was
generated using the STRING database and visualized in Cytoscape.
(D) Screening of core targets for GLD treatment of AML using the
cytoHubba plugin. (E) Screening of core targets using the MCODE
plugin. (F) Combined core target network diagram. AML, acute
myeloid leukemia; GLD, glaucocalyxin D; OMIM, Online Mendelian
Inheritance in Man; TTD, Therapeutic Target Database.

Figure 2

Enrichment analysis. (A) GO
biological function analysis; (B) KEGG pathway enrichment analysis:
Top 20 significantly enriched pathways [-log10 (P-value)]. GO, Gene
Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 3

Docking results of glaucocalyxin D
with 10 core target molecules. (A) PNPT11, (B) HSP90AB1, (C) MAPK1,
(D) STAT1, (E) STAT3, (F) HIF1A, (G) GRB2, (H) TLR4, (I) mTOR, (J)
NFKB1. Binding energies (kcal/mol) and detailed interactions
(hydrogen bonds, hydrophobic interactions, salt bridges,
π-stacking) for each target are presented in Table II. In the docking analysis, ‘-’
indicates that no corresponding interaction was detected.

Figure 4

GLD inhibits the proliferation of AML
cells. (A) Chemical structure of GLD. (B) IC50 values. (C)
Inhibition rate in HEL cells. (D) Inhibition rate in K562 cells.
(E) Hoechst 33258 staining with morphological changes observed
under an inverted microscope (×100 magnification; scale bar, 100
µm). GLD, glaucocalyxin D; AML, acute myeloid leukemia.

Figure 5

Flow cytometric analysis. (A) HEL and
K562 cells treated with GLD at various concentrations (0.25, 0.5
and 1 µmol/l; DMSO as control) for 48 h. (B and C) Quantification
of apoptotic cell percentage. (D) Analysis by flow cytometry of the
effects of GLD on the cell cycle of HEL and K562 cells after 48 h
(G1 phase, green; S phase, yellow; G2/M
phase, blue). (E and F) Cell cycle statistics. Data are presented
as mean ± SD (n=3). *P<0.05, **P<0.01 and ***P<0.001
compared with the control group. GLD, glaucocalyxin D.

Figure 6

GLD exerts its anti-AML effects by
targeting the JAK-STAT signaling pathway. (A) HEL cells were
treated with the indicated concentrations of GLD (0, 0.25, 0.5 and
1 µmol/l) for 48 h. The protein expression levels of Bax, Bcl-2,
CDK1, cyclin B1, JAK2, p-JAK2, STAT3 and p-STAT3 were detected by
western blotting. β-actin and GAPDH were used as loading controls.
(B) K562 cells were treated with GLD (0, 0.25, 0.5 and 1 µmol/l)
for 48 h and the protein expression levels were detected as
described in (A). (C) Quantification of the protein bands presented
in (A) and (B). The relative protein expression levels were
normalized to the loading controls, and p-JAK2 and p-STAT3 were
further normalized to total JAK2 and STAT3, respectively. Data are
presented as the mean ± SD (n=3). *P<0.05, **P<0.01 and
***P<0.001 vs. the control group. p, phosphorylated; AML, acute
myeloid leukemia; GLD, glaucocalyxin D.
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Copy and paste a formatted citation
Spandidos Publications style
Chen L, Yang W, Zhang W, Luo H and Yan C: Exploring the mechanism of action of glaucocalyxin D against acute myeloid leukemia using network pharmacology, molecular docking and cellular experiments. Oncol Lett 31: 267, 2026.
APA
Chen, L., Yang, W., Zhang, W., Luo, H., & Yan, C. (2026). Exploring the mechanism of action of glaucocalyxin D against acute myeloid leukemia using network pharmacology, molecular docking and cellular experiments. Oncology Letters, 31, 267. https://doi.org/10.3892/ol.2026.15622
MLA
Chen, L., Yang, W., Zhang, W., Luo, H., Yan, C."Exploring the mechanism of action of glaucocalyxin D against acute myeloid leukemia using network pharmacology, molecular docking and cellular experiments". Oncology Letters 31.6 (2026): 267.
Chicago
Chen, L., Yang, W., Zhang, W., Luo, H., Yan, C."Exploring the mechanism of action of glaucocalyxin D against acute myeloid leukemia using network pharmacology, molecular docking and cellular experiments". Oncology Letters 31, no. 6 (2026): 267. https://doi.org/10.3892/ol.2026.15622
Copy and paste a formatted citation
x
Spandidos Publications style
Chen L, Yang W, Zhang W, Luo H and Yan C: Exploring the mechanism of action of glaucocalyxin D against acute myeloid leukemia using network pharmacology, molecular docking and cellular experiments. Oncol Lett 31: 267, 2026.
APA
Chen, L., Yang, W., Zhang, W., Luo, H., & Yan, C. (2026). Exploring the mechanism of action of glaucocalyxin D against acute myeloid leukemia using network pharmacology, molecular docking and cellular experiments. Oncology Letters, 31, 267. https://doi.org/10.3892/ol.2026.15622
MLA
Chen, L., Yang, W., Zhang, W., Luo, H., Yan, C."Exploring the mechanism of action of glaucocalyxin D against acute myeloid leukemia using network pharmacology, molecular docking and cellular experiments". Oncology Letters 31.6 (2026): 267.
Chicago
Chen, L., Yang, W., Zhang, W., Luo, H., Yan, C."Exploring the mechanism of action of glaucocalyxin D against acute myeloid leukemia using network pharmacology, molecular docking and cellular experiments". Oncology Letters 31, no. 6 (2026): 267. https://doi.org/10.3892/ol.2026.15622
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