Objective  To explore the potential targets and mechanisms of action of Lanatoside C in the treatment of glioblastoma (GBM) based on network pharmacology and molecular docking technology.

Methods  The potential targets of Lanatoside C were identified using PharmMapper, SwissTargetPrediction, and TargetNet databases. GeneCards, DisGeNET, and TTD databases were used to obtain disease-related targets associated with GBM. The Venny 2.1.0 platform was used to cross-analyze drug targets and disease targets to identify their intersection targets. Cytoscape 3.9.1 software was employed to construct a protein-protein interaction (PPI) network diagram of the intersection targets, and core targets were identified using plugins. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted on the intersection targets based on the Metascape platform to predict the potential pathways through which Lanatoside C may act in the treatment of GBM. Molecular docking technology was applied to verify the binding ability of Lanatoside C to the core targets. To comprehensively analyze the potential mechanism of Lanatoside C in GBM, a gene association network was constructed using the GeneMANIA platform. The thiazolium blue assay [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, MTT] was performed on U-87 MG and U251 MG cells to explore the effect of Lanatoside C on GBM cell proliferation.

Results  Finally, 377 potential targets of Lanatoside C and 4 012 disease-related targets for GBM were screened. After removing duplicates, a total of 189 intersection targets were identified. PPI network topology analysis revealed 10 core targets, ranked by degree. KEGG pathway enrichment analysis showed that Lanatoside C was primarily involved in cancer signaling pathways. Molecular docking results indicated that ESR1 had the highest binding affinity for Lanatoside C. After the gene association network was constructed, each core gene was expanded to 10 co-expressed genes, and after merging and removing duplications, an expanded potential target database containing 107 targets was formed for the treatment of GBM by Lanatoside C. The MTT assay further confirmed that Lanatoside C had a concentration-dependent inhibitory effect on the proliferation of U-87 MG and U251 MG cells.

Conclusion Through network pharmacology analysis, it was found that Lanotoside C acts on GBM through multiple targets and pathways. In vitro experiments have verified that it inhibits GBM cell proliferation in a concentration-dependent manner, provideing new ideas and a theoretical basis for further research and clinical application of Lanatoside C.

1.Park YW, Vollmuth P, Foltyn-Dumitru M, et al. The 2021 WHO classification for gliomas and implications on imaging diagnosis: part 1-key points of the fifth edition and summary of imaging findings on adult-type diffuse gliomas[J]. J Magn Reson Imaging, 2023, 58(3): 677-689. DOI: 10.1002/jmri.28743.

2.Ostrom QT, Gittleman H, Truitt G, et al. CBTRUS statistical report: primary brain and other central nervous system tumors diagnosed in the United States in 2011-2015[J]. Neuro Oncol, 2018, 20(suppl_4): iv1-iv86. DOI: 10.1093/neuonc/noy131.

3.Cudalbu C, Bady P, Lai M, et al. OTME-13. Integration of metabolic and transcriptional signatures of glioblastoma invasion reveals extracellular matrix reorganization and vasculature remodeling[J]. Neuro-Oncology Advances, 2021, 3(Suppl 2): ii16. DOI: 10.1093/noajnl/vdab070.064.

4.Wirsching HG, Galanis E, Weller M. Glioblastoma[J]. Handb Clin Neurol, 2016, 134: 381-397. DOI: 10.1016/B978-0-12-802997-8.00023-2.

5.Chao MW, Chen TH, Huang HL, et al. Lanatoside C, a cardiac glycoside, acts through protein kinase Cδ to cause apoptosis of human hepatocellular carcinoma cells[J]. Sci Rep, 2017, 7: 46134. DOI: 10.1038/srep46134.

6.Ha DP, Tsai YL, Lee AS. Suppression of ER-stress induction of GRP78 as an anti-neoplastic mechanism of the cardiac glycoside Lanatoside C in pancreatic cancer: Lanatoside C suppresses GRP78 stress induction[J]. Neoplasia, 2021, 23(12): 1213-1226. DOI: 10.1016/j.neo.2021.10.004.

7.Hu Y, Yu K, Wang G, et al. Lanatoside C inhibits cell proliferation and induces apoptosis through attenuating Wnt/β-catenin/c-Myc signaling pathway in human gastric cancer cell[J]. Biochem Pharmacol, 2018, 150: 280-292. DOI: 10.1016/j.bcp.2018.02.023.

8.Yuan Z, Pan Y, Leng T, et al. Progress and prospects of research ideas and methods in the network pharmacology of traditional Chinese medicine[J]. J Pharm Pharm Sci, 2022, 25: 218-226. DOI: 10.18433/jpps32911.

9.Reddy D, Kumavath R, Ghosh P, et al. Lanatoside C induces G2/M cell cycle arrest and suppresses cancer cell growth by attenuating MAPK, Wnt, JAK-STAT, and PI3K/AKT/mTOR signaling pathways[J]. Biomolecules, 2019, 9(12): 792. DOI: 10.3390/biom9120792.

10.Liu F, Hon GC, Villa GR, et al. EGFR mutation promotes glioblastoma through epigenome and transcription factor network remodeling[J]. Mol Cell, 2015, 60(2): 307-318. DOI: 10.1016/j.molcel.2015.09.002.

11.Cataldi S, Arcuri C, Lazzarini A, et al. Effect of 1α, 25(OH)2 vitamin D3 in mutant p53 glioblastoma cells: involvement of neutral sphingomyelinase1[J]. Cancers (Basel), 2020, 12(11): 3265. DOI: 10.3390/cancers12113163.

12.Du L, Zhang Q, Li Y, et al. Research progress on the role of PTEN deletion or mutation in the immune microenvironment of glioblastoma[J]. Front Oncol, 2024, 14: 1409519. DOI: 10.3389/fonc.2024.1409519.

13.Fan HW, Ni Q, Fan YN, et al. C-type lectin domain family 5, member A (CLEC5A, MDL-1) promotes brain glioblastoma tumorigenesis by regulating PI3K/Akt signalling[J]. Cell Prolif, 2019, 52(3): e12584. DOI: 10.1111/cpr.12584.

14.Gong Y, Ma Y, Sinyuk M, et al. Insulin-mediated signaling promotes proliferation and survival of glioblastoma through Akt activation[J]. Neuro Oncol, 2016, 18(1): 48-57. DOI: 10.1093/neuonc/nov096.

15.Pan J, Sheng S, Ye L, et al. Extracellular vesicles derived from glioblastoma promote proliferation and migration of neural progenitor cells via PI3K-Akt pathway[J]. Cell Commun Signal, 2022, 20(1): 7. DOI: 10.1186/s12964-021-00760-9.

16.Nelson N, Relógio A. Molecular mechanisms of tumour development in glioblastoma: an emerging role for the circadian clock[J]. NPJ Precis Oncol, 2024, 8(1): 40. DOI: 10.1038/s41698-024-00530-z.

17.Jansson D, Dieriks VB, Rustenhoven J, et al. Cardiac glycosides target barrier inflammation of the vasculature, meninges and choroid plexus[J]. Commun Biol, 2021, 4(1): 260. DOI: 10.1038/s42003-021-01787-x.

18.Botelho AFM, Pierezan F, Soto-Blanco B, et al. A review of cardiac glycosides: structure, toxicokinetics, clinical signs, diagnosis and antineoplastic potential[J]. Toxicon, 2019, 158: 63-68. DOI: 10.1016/j.toxicon.2018.11.429.

19.Gurel E, Karvar S, Yucesan B, et al. An overview of cardenolides in Digitalis-more than a cardiotonic compound[J]. Curr Pharm Des, 2017, 23(34): 5104-5114. DOI: 10.2174/1381612823666170825125426.