Abstract
Objectives: This study used a combination of in silico and in-vitro methodologies to evaluate the breast cancer (BC) fighting efficacy of Chamazulene. Methods: In silico analyses utilized Protox-3.0 for toxicity prediction, SuperPred and GeneCards for target identification, and Jvenn for intersection. Protein-protein interactions were mapped with STRING and visualised in Cytoscape, followed by Cytohubba to pinpoint hub genes. Gene-ontology and KEGG-pathway enrichment were performed via DAVID and visualized with SRplot. Immune infiltration was assessed using TIMER, while UALCAN evaluated expression, promoter methylation, survival, and correlation. The MTT, clonogenic, EdU, Annexin-V/PI, cell cycle, wound healing, and Western-blotting were used to measure cytotoxicity and the mechanism of chamazulene in MDA-MB-231 cells. Results: In silico analyses indicated a safe toxicity profile and identified 53-overlapping target genes, resulting in a highly enriched PPI network. The network identified the three main hub genes: NFKB1, MAPK14, and GRB2. Enrichment analysis indicated participation in different pathways, including MAPK and HIF-1 signalling pathways. The TIMER and UALCAN investigations on BC revealed significant immune infiltration, altered gene expression, hypomethylation, and survival trends. MTT studies demonstrated a dose-dependent reduction in cancer cell viability, exceeding 50% at elevated doses. Clonogenic and EdU assays indicated reduced proliferation and DNA-synthesis, whereas apoptosis and cell cycle analyses revealed elevated cell mortality and G2/M-phase arrest. Western blotting revealed a downregulation of NFKB1, GRB2 and MAPK14, while wound healing assays suggested reduced migration. Conclusion: Chamazulene exhibits multifaceted and potent anticancer effects by specifically modifying crucial signalling-pathways and processes in aggressive BC, warranting preclinical studies to validate its therapeutic potential.