Honeybee associated Aspergillus niger AW17 as a source of selective anticancer compounds with cytotoxicity evaluation in human cancer cell lines

Cancer remains a leading cause of death worldwide, necessitating the discovery of novel therapeutic agents with improved efficacy and selectivity. This study investigated the anticancer potential of Aspergillus niger strain AW17 isolated from honeybees, a previously unexplored ecological niche for anticancer drug discovery. The fungal isolate was characterized through morphological, microscopic, and molecular methods, confirming its identity as Aspergillus niger. Chemical profiling using GC-MS and UPLC-MS/MS revealed a complex composition including high oleic acid content (28.88%) and unique compounds like pachymic acid (18.93%), rarely associated with A. niger. The extract's anticancer activity was evaluated against colorectal (Caco-2), liver (HepG2), breast (MCF-7), and lung (A549) cancer cell lines using MTT assay, confocal microscopy with AO/PI staining, cell cycle analysis, and apoptosis detection. Results demonstrated remarkable selective cytotoxicity of the Aspergillus niger extract against cancer cell lines, following the sensitivity hierarchy HepG2 (IC50 = 5.22 µg/ml) > Caco-2 (26.78 µg/ml) > A549 (34.18 µg/ml) > MCF-7 (55.91 µg/ml), while sparing normal cell lines significantly, with IC50 values of 1454.7 µg/ml for WI-38 and 668.3 µg/ml for HFB-4, indicating high cancer cell selectivity. Confocal microscopy confirmed membrane integrity loss as a primary cytotoxic mechanism. Cell cycle analysis revealed cell-type specific arrest patterns, with Caco-2 and HepG2 exhibiting G1 arrest, while MCF-7 and A549 showed S phase accumulation. Flow cytometry indicated distinct death mechanisms, with Caco-2 undergoing early apoptosis and substantial necrosis, HepG2 showing predominant late apoptosis, and A549 displaying primary necrotic death. These findings highlight the potential of honeybee-associated fungi as sources of novel anticancer compounds with remarkable selectivity, providing new avenues for targeted cancer therapy development and demonstrating the value of exploring specialized ecological niches in natural product discovery.