Anticancer Effects of Ziziphus jujuba in Human Breast Carcinoma: An Integrated Study of Network Pharmacology, Molecular Docking and Dynamics Simulations and in-vitro Experimental Validation.

Abstract

Background: Ziziphus jujuba is a promising medicinal plant with ethnomedicinal claims and constitutes one of the important herbs in Traditional Chinese Medicine, yet it has not been explored for its anticancer effects against breast cancer (BC).

Objectives: The current study aimed to use an integrated approach involving network pharmacology, in-silico molecular docking and dynamics simulations, as well as in-vitro experimental validation, to study the anticancer effects of Z. jujuba in BC.

Methods: A total of 133 phytochemicals from Z. jujuba were screened using the TCMSP database, followed by toxicity screening (Protox 3.0) and ADMET analysis (ADMET AI). Soxhlet extraction was performed with a 70:30 ethanol:dichloromethane solvent mixture. Compound-target networks were constructed using SuperPred, SwissTargetPrediction, and Cytoscape (CytoHubba plugin, Degree method). Breast cancer targets (GeneCards, Gifts score ≥ 60%) were intersected with phytochemical targets (Venny 2.0), and a PPI network was generated (STRING, confidence ≥ 0.700). GO and KEGG analyses used ShinyGO 0.80. Expression analyses of hub genes were conducted using GEPIA2 (BRCA dataset). Molecular docking was performed with CB-Dock2, and MD simulations used iMODS (100 modes, 300 K) and Desmond. Bioassays included MTT, clonogenic, Annexin V/PI, and Western blot analyses on MDA-MB-231 and MCF-10A cells.

Results: Five phytochemicals (Spiradine A, Jujubogenin, Malkangunin, Ceanothic Acid, Moupinamide) showed no toxicity risks. The compound-target network identified 305 targets, with 160 intersecting BC targets. The PPI network (160 nodes, 568 edges, P < 1.0e-16) revealed EGFR, HSP90AA1, and STAT3 as hubs. GO/KEGG analyses linked targets to cancer pathways. EGFR, HSP90AA1, and STAT3 showed higher expression in tumors (P < 0.05), with EGFR/STAT3 linked to poorer survival (P < 0.05). Jujubogenin docking yielded binding affinities of -8.7 (EGFR), -7.5 (HSP90AA1), and -8.2 kcal/mol (STAT3). MD simulations confirmed stable EGFR/STAT3 complexes. The extract exhibited selective cytotoxicity, reduced colony formation, induced apoptosis (P < 0.05), and downregulated EGFR (70%), HSP90AA1 (60%), and STAT3 (90%) at 100 µg/mL (P < 0.01).

Conclusions: Network pharmacology revealed that Z. jujuba phytochemicals are predicted to show significant effects against BC. Out of these, Jujubogenin exhibited strong and stable interactions with network-predicted hubs. The extract showed selective cytotoxicity, induced apoptosis, inhibited colony formation, and significantly downregulated these hub proteins, validating its promising anticancer potential.