Enzymatically derived chitooligosaccharides exhibit enhanced therapeutic efficacy against estrogen receptor-positive and triple-negative breast cancer cells.

This study investigates the enzymatic production of chitooligosaccharides (E-NACOS) from Alcaligenes faecalis chitinase and evaluates their potential therapeutic relevance in breast cancer models. Electrophoretic protein profiling combined with activity-based zymography of the crude supernatant identified four chitinase isoforms (∼45-150 kDa) exhibiting complementary endo- and exo-hydrolytic activities. HPLC analysis of colloidal chitin hydrolysates confirmed the generation of N-acetylglucosamine (GlcNAc) and short-chain oligomers (dimers to tetramers), reflecting the broad substrate specificity of the enzyme system. When compared with commercially available chitooligosaccharides (C-NACOS), E-NACOS demonstrated lower IC₅₀ values (9.4 mg/mL for MCF-7 and 2.4 mg/mL for MDA-MB-468) and stronger cytotoxic responses in both ER-positive (MCF-7) and ER-negative (MDA-MB-468) breast cancer cell lines. Functional assays further revealed that E-NACOS effectively reduced cell migration and invasion, enhanced intracellular ROS accumulation, disrupted normal cell-cycle distribution and increased apoptotic sub-G1 fractions, and increased sub-G1 populations. Flow cytometric analysis indicated significant apoptosis (91.3% in MCF-7; 47.8% in MDA-MB-468), while fluorescence microscopy exhibited cytoskeletal disruption and nuclear condensation, supporting the loss of cellular integrity. In summary, these findings suggest that E-NACOS exert relevant anti-migratory and pro-apoptotic effects in breast cancer cell models, potentially through oxidative stress associated mechanisms. Although further mechanistic insights and in vivo validation are required, A. faecalis-derived E-NACOS represent a promising bioactive preparation worthy of continued exploration for prospective applications in breast cancer management.