Cell-free supernatant of Limosilactobacillus reuteri enhances 5-fluorouracil-induced apoptosis in SW-480 colorectal cancer cells: an in vitro study

5-fluorouracil (5-Fu) is a main treatment for colorectal cancer (CRC) but shows limited efficacy and serious side effects, creating a need for novel adjunct therapies. Recent evidence suggests Limosilactobacillus reuteri derivatives may have anticancer properties. Accordingly, this study examined the proapoptotic effects of live (LLr), heat-killed (HLr), and cell-free supernatant (CFS) of L. reuteri (IBRC-M 10755), alone or combined with 5-Fu, in an in vitro CRC model. SW-480 cells were treated with different concentrations of LLr, HLr (MOI 1, 10, 100), CFS of L. reuteri (1, 10, 25, 50 µg/mL total protein), alone or concurrently with 5-Fu (1-100 µM), for 24-48 h. Cell viability was assessed using MTT assay. Expression of apoptosis-related genes (Bax, Bcl-2, caspase-3, caspase-8, and caspase-9) and apoptosis rate were measured by RT-qPCR and flow cytometry, respectively. The mRNA levels of inflammatory markers (IL-1β, TNF-α) were assessed by RT-qPCR, and secreted protein levels were measured by ELISA. LLr had no effect on cell viability at any concentration, whereas HLr (MOI 100) and CFS at 25 and 50 μg/mL significantly reduced cell viability by up to 85%, 78%, and 65%, respectively ( P <0.05 vs. untreated cells). Treatment of cells with LLr, HLr (MOI 100), and CFS (25-50 μg/mL total protein) significantly increased Bax, caspase-3, caspase-8, and caspase-9 expression; CFS also increased the apoptosis rate dose-dependently, confirmed by flow cytometry ( P <0.05 vs. untreated cells). Co-treatment with CFS and 5-Fu (10 µM) produced greater apoptosis-related gene expression and apoptosis rate than either agent alone. This combination also significantly downregulated 5-Fu-induced TNF-α and IL-1β mRNA, though this was not consistently reflected in cytokine levels as determined by ELISA. L. reuteri CFS exhibits proapoptotic effects, enhances 5-Fu-induced apoptosis, and modulates 5-Fu-induced inflammation in vitro . Further studies, including formal combination-index analysis and in vivo validation, are needed to establish the therapeutic potential of this combination for treating CRC.