Network-based analysis and experimental validation of identified natural compounds from Yinchen Wuling San for acute myeloid leukemia

Abstract

Objective: Traditional Chinese medicine (TCM) has garnered attention for its potential in cancer therapy. Yinchen Wuling San (YWLS), a classical herbal formula, has been traditionally used for liver-related conditions, but its bioactive components and molecular mechanisms relevant to hematologic malignancies such as acute myeloid leukemia (AML) remain unclear. This study aims to identify the active compounds and potential molecular targets of Yinchen Wuling San in the context of AML through network pharmacology analysis, and to experimentally validate the effects of selected candidate compounds in AML models.

Methods: Active ingredients from six YWLS herbs were screened via the TCMSP database using oral bioavailability ≥30% and DL ≥0.18 thresholds. Targets were predicted using SwissTargetPrediction, and AML-related genes were obtained from DisGeNET and GeneCards. Key overlapping targets were analyzed via STRING PPI networks and GO/KEGG enrichment. Molecular docking was performed between three core compounds (genkwanin, isorhamnetin, quercetin) and hub proteins (e.g., SRC) using Sybyl-X. ADME profiles were predicted using SwissADME, and molecular dynamics simulations (GROMACS) assessed complex stability. These compounds were further evaluated in vitro (viability, apoptosis, cell cycle, RT-qPCR, flow cytometry) and in vivo using an AML xenograft mouse model.

Results: Of 621 YWLS targets, 113 overlapped with 1,247 AML-related genes. PPI analysis identified hub genes, including AKT1, SRC, and EGFR. Enrichment analysis highlighted PI3K-AKT, MAPK, and JAK-STAT pathways. Genkwanin, isorhamnetin, and quercetin were predicted to target SRC, with strong molecular docking affinities. ADME analysis suggested favorable pharmacokinetics, and molecular dynamics simulations confirmed structural stability. In vitro, these compounds exhibited dose-dependent cytotoxicity, induced apoptosis, modulated the cell cycle, and downregulated SRC expression. Notably, Genkwanin promoted CD8⁺ T cell proliferation and inhibited leukemia growth, improving survival in a leukemia xenograft model.

Conclusion: YWLS compounds, particularly Genkwanin, exhibit significant anti-leukemic activity via apoptosis induction, cell cycle modulation, and promote T cells proliferation. Genkwanin emerges as a promising therapeutic candidate for AML, warranting further clinical investigation.

Keywords: Yinchen Wuling San; acute myeloid leukemia; molecular docking; molecular dynamics simulation; network pharmacology; pharmacological target.

FIGURE 1

Construction of a compound-target-disease network based on the targets of active ingredients from YWLS and AML-associated genes. (A) Screening of acute myeloid leukemia related genes from DisGeNET and GeneCards databases. (B) The intersection of targets of active ingredients of YWLS and AML-related genes. (C) The active component-target-disease network. The red diamond represented the disease; The light green rectangle represented intersecting targets; The light purple diamond represented YWLS; The blue triangle represented active compounds. The edges represented the connection among active components, targets, and disease. (D) Two modules (AKT1 and PIK3CA) identified from the whole PPI network of 113 targets of active ingredients in YWLS and AML-associated genes. (E) 20 core targets determined by the degrees. Color represented the target degree.

FIGURE 2

Associations of intersecting targets with patients’ outcome. (A) Forest plot represented the associations of intersecting targets with patients’ outcome in TCGA-AML cohort. (B) Forest plot represented the associations of intersecting targets with patients’ outcome in Beat-AML cohort. (C) The Kaplan-Meier curve of SRC, CCND2, and CCND3 in TCGA-AML cohort.

FIGURE 3

Molecular docking of selected active compounds with the core AML-related target protein SRC. (A) Isorhamnetin-SRC. (B) Genkwanin-SRC. (C) Quercetin-SRC.

FIGURE 4

Molecular dynamics simulation of Genkwanin, isorhamnetin, and quercetin and SRC. (A) RMSD of Genkwanin, isorhamnetin, and quercetin in complex with SRC, respectively. (B) RMSF of amino acid residues of Genkwanin, isorhamnetin, and quercetin in complex with SRC, respectively. (C) Rg of Genkwanin, isorhamnetin, and quercetin in complex with SRC, respectively. (D) SASA of Genkwanin, isorhamnetin, and quercetin in complex with SRC, respectively. (E–G) H-bond number of isorhamnetin, Genkwanin, and quercetin in complex with SRC, respectively. (H) Gibbs binding free energy of isorhamnetin, Genkwanin, and quercetin in complex with SRC, respectively. (I) Interaction energy between the isorhamnetin, Genkwanin, and quercetin and key amino acid residues of SRC, respectively.

FIGURE 5

Effects of Genkwanin, Isorhamnetin, and Quercetin on leukemia cell apoptosis, cell cycle, and mitochondrial function. (A) Cytotoxic effects of Genkwanin, Isorhamnetin, and Quercetin on leukemic cells, presented relative to the vehicle control. (B) SRC expression in leukemic cells at 72 h following treatment with different concentrations of Genkwanin, Isorhamnetin, and Quercetin. (C) Early apoptosis of leukemic cells at 72 h after treatment with different concentrations of Genkwanin, Isorhamnetin, and Quercetin. (D) Late apoptosis of leukemic cells at 72 h after treatment with different concentrations of Genkwanin, Isorhamnetin, and Quercetin. (E) Cell cycle distribution of leukemic cells at 72 h after treatment with different concentrations of Genkwanin, Isorhamnetin, and Quercetin. (F) Reactive oxygen species levels and mitochondrial membrane potential in leukemic cells at 72 h following treatment with different concentrations of Genkwanin, Isorhamnetin, and Quercetin.

FIGURE 6

Genkwanin as a pharmacological agent in a leukemia model. (A) Schematic representation of Genkwanin treatment alone or in combination with adoptive PBMC transfer. (B) Kaplan-Meier survival curve of mice treated with Genkwanin alone or in combination with adoptive PBMC transfer. (C) Frequency of leukemia cells in the bone marrow on Day 21 post-challenge. (D) Frequency of CD3⁺CD8⁺T cells in the bone marrow in mice transferred with PBMCs alone or Genkwanin + PBMCs on Day 21 post-challenge. (E) Frequency of GZMB⁺CD8⁺T cells in the bone marrow in mice transferred with PBMCs alone or Genkwanin + PBMCs on Day 21 post-challenge. (F) Frequency of PD-1⁺CD8⁺T cells in the bone marrow in mice transferred with PBMCs alone or Genkwanin + PBMCs on Day 21 post-challenge.