This study investigated how prolonged adaptation of the probiotic Lactobacillus plantarum to lauric acid (LA) enhances its anticancer properties. Cell-free supernatants (CFS) were prepared from LA-treated L. plantarum (LA- CFS) and untreated cultures (CFS). Their cytotoxic and genotoxic effects were compared against HCT-116 colorectal cancer cells. Results showed that L. plantarum cultivated with LA exhibited improved tolerance to acidic conditions (pH 3.0) and enhanced antibacterial activity against pathogens such as Escherichia coli, Salmonella Typhi, Klebsiella pneumoniae, Staphylococcus aureus, and Pseudomonas aeruginosa. LA-CFS reduced the IC50 from 95.12% (v/v) to 66% (v/v) compared to untreated CFS in HCT-116 cells. Annexin V/FITC-PI staining revealed that LA-CFS significantly decreased viable cells (to 11.66%) and increased late apoptosis (57.26%) compared to CFS. DAPI staining confirmed apoptosis induction via nuclear fragmentation. LA-CFS inhibited cell migration by 18.17% more than CFS after 48 h and reduced 3D spheroid density by 18.41% and 47.77% at 48 h and 72 h, respectively. Gene expression analysis showed that LA-CFS upregulated CASP8 (1.2-fold) and BAX (1.22-fold) and downregulated AKT (2.92-fold) relative to controls in HCT-116 cells, suggesting a possible involvement of TP53-independent apoptotic signaling, potentially initiated through upstream extrinsic pathways and reinforced by mitochondrial pathway activation. The CAM assay demonstrated stronger anti-angiogenic effects with LA-CFS (34% inhibition) versus CFS (15%). These findings suggest that prolonged LA adaptation potentiates the anticancer effects of CFS derived from L. plantarum, highlighting its promising potential in colorectal cancer research.
