Long-term exposure of Lactobacillus rhamnosus to quercetin enhances its anti-angiogenic and anti-colorectal cancer efficacy

Abstract Colorectal cancer (CRC) is the second leading cause of cancer death worldwide, with rising incidence projected over the coming decades. Here we show that prolonged exposure of Lactobacillus rhamnosus PTCC 1637 to quercetin (up to 200 µg/mL) improves its probiotic traits and markedly boosts the anticancer activity of its cell-free supernatant (que-CFS). Acid tolerance increased, and antibacterial zones against Staphylococcus aureus , Salmonella typhi, and Escherichia coli grew by 2–13 % using que-CFS compared with untreated CFS. In SW-480 colorectal adenocarcinoma cells, que-CFS lowered the 48-h IC₅₀ to 28.36 % (v/v) versus 65.7 % for CFS. Flow cytometry revealed 87.4 % apoptosis with que-CFS; DAPI staining confirmed greater nuclear fragmentation and a 57.9 % reduction in nuclear area. Que-CFS suppressed wound closure by 26.5 % at 48 h, reduced 3-D spheroid volume by 27 % at 96 h, and inhibited angiogenesis in the chick chorioallantoic membrane by 36.3 % versus 23.1 % for CFS. qRT-PCR showed stronger upregulation of Caspase-8, BAX and TP53. Proteinase K abolished antibacterial activity, confirming proteinaceous mediators. These data reveal that quercetin reprograms L. rhamnosus metabolism to yield metabolites with superior cytotoxic, anti-migratory, anti-spheroid, and anti-angiogenic effects, opening a dietary route to potentiate probiotic anticancer therapy.