Background: Colorectal cancer (CRC) is a serious health problem, and finding new treatments is important. There is growing evidence for the antitumor activity of probiotics. This study investigated the anti-cancer potential of a probiotic mix containing Lactobacillus plantarum, L. brevis, L. helveticus, and L. delbrueckii, alone or in combination with the chemotherapy drug 5-fluorouracil, against CRC. Methods: The research was carried out in Mashhad in 2021. The cytotoxic effect of Lactobacillus isolates on CRC cells was investigated in two-dimensional and three-dimensional cell culture models. Histological staining and molecular approaches were used to investigate the regulatory mechanism of Lactobacillus isolates on cell migration, inflammation, fibrosis, cell cycle progression, apoptosis, and tumor necrosis in the CRC mouse model. Statistical analysis was performed using SPSS software version 20 with a significance level of P<0.05. The tests employed included the Kolmogorov–Smirnov, ANOVA, Dunnett’s post hoc, and Kruskal–Wallis. Results: Lactobacillus strains effectively suppressed tumor growth in CRC by promoting cell death and inhibiting fibrosis and inflammation. These bacteria regulated apoptosis-related genes such as Bcl-2-associated protein x (P=0.0033), and BCL-2 (P=0.0029), leading to increased tumor necrosis. Treatment with bacterial supernatants reduced tumor size and fibrosis by downregulating collagen type I, alpha 1 (Col1a1) (P=0.024), Col1a2 (P=0.0231), and actin alpha 2 (P=0.0466), and transforming growth factor-beta expression. Additionally, they suppressed inflammation by decreasing tumor necrosis factoralpha (P=0.0001), interleukin 6, and IL-1β (P=0.0198) levels in tumor tissues. Furthermore, the treatment inhibited CRC cell migration by modulating epithelial cadherin (P=0.0198) and matrix metallopeptidase 2 (P=0.033) expression. Conclusion: Findings indicated that co-administration of Lactobacillus isolates with 5-FU could improve the antitumor properties of the standard drug, 5-FU, supporting the therapeutic potential of these safe isolated lactic acid bacteria for CRC patients in vivo.

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