Caffeic acid adaptation alters the antibacterial and anticancer activities of Lactobacillus reuteri cell-free supernatant

Colorectal cancer (CRC) as a malignancy that develops in the colon and the rectum, is considered one of the most serious health concerns worldwide, with high prevalence and mortality rates. Probiotics are beneficial bacteria commonly found in the gut and have recently attracted scientific attention, due to their potential to prevent or slow the progression of CRC. Probiotics can break down dietary bioactive compounds, such as polyphenols. These compounds may can promote probiotic characteristics, such as antibacterial and anticancer activities, while preventing disorders like cancer. This study aimed to assess the effects of caffeic acid treatment on Lactobacillus reuteri and to determine the impact of the cell-free supernatants obtained from caffeic acid-treated bacteria (CAF-CFS) on the inhibition of growth and proliferation of colorectal cancer cells (HCT-116) compared with the supernatant of untreated bacteria (CFS). This study aimed to determine the potential effect of caffeic acid treatment on the acid resistance and antibacterial properties of L. reuteri . To achieve this, various assays were conducted, including low pH tolerance, minimum inhibitory concentration (MIC), and antibiofilm formation assays. Additionally, to evaluate the effect of CAF-CFS on colorectal cancer cell viability, apoptosis induction, and migration suppression, MTT assay, Annexin V-FITC/PI staining, cell cycle, colony formation, and scratch (wound healing) assay were performed. Eventually, the expression levels of genes associated with these pathways were determined using the qRT-PCR technique. The acid tolerance assay results indicated that caffeic acid adapted bacteria exhibited elevated acid tolerance compared to the untreated bacteria. Furthermore, MIC and antibiofilm assays revealed that CFS from caffeic acid-adapted L. reuteri showed a significant antibacterial activity relative to the control. Plus, based on MTT assay results, the half-maximal inhibitory concentration (IC50) of the supernatant from caffeic acid-treated bacteria was determined to be 95.25% (v/v). At 100% (v/v), CAF-CFS reduced cell viability to approximately 38%, compared with approximately 57% following treatment with CFS. Flow cytometry analysis revealed a significant increase in the apoptotic population in cells exposed to CAF-CFS compared to those treated with standard CFS. CFS treatment increased the hypodiploid sub-G1 population, a finding consistent with DNA fragmentation and apoptotic cell death. additionally, the migratory and colony-forming capacities of CAF-CFS-treated cells were significantly dropped compared to both CFS-treated and untreated groups. Finally, qRT-PCR findings demonstrated that HCT-116 cells treated with CAF-CFS exhibited upregulation of Bax and Caspase-3/9 genes, alongside downregulation of MMP3, MMP9, and Bcl-2 genes compared to cells treated with CFS from non-adapted bacteria. Collectively, these findings confirm that caffeic acid-adapted bacteria exert a more potent anticancer effect than the control group. This study showed that caffeic acid adaptation of L. reuteri enhanced the antibacterial properties of its cell-free supernatant and increased the anticancer activity of CAF-CFS against HCT-116 colorectal cancer cells compared with CFS derived from untreated bacteria.