A novel recombinant arginine deiminase from Limosilactobacillus reuteri DSM 20016 demonstrates anticancer activity against colon cancer cells.

[BACKGROUND] Colon cancer remains one of the most prevalent and aggressive malignancies, underscoring the demand for targeted and biologically safe therapeutic strategies. Arginine deiminase (ADI) has gained increasing interest as a potential anticancer agent due to its ability to modulate cellular arginine availability and inhibit tumor progression.

[METHODS AND RESULTS] In this study, we evaluated the anticancer efficacy of a recombinant ADI derived from the probiotic bacterium Limosilactobacillus reuteri (LrADI) against HCT116 human colorectal cancer. LrADI exhibited potent, dose-dependent cytotoxicity with a half-maximal inhibitory concentration (IC) of 0.1 µg/mL, while demonstrating negligible toxicity toward normal colon fibroblasts (CCD-18Co) at the same concentration. Hemolysis assays further established its biocompatibility, showing minimal erythrocyte lysis even at elevated concentrations. Beyond cytotoxicity induction, LrADI significantly reduced clonogenic survival, indicating strong inhibition of long-term tumorigenic potential. Wound-healing assays revealed suppression of cell migration, suggesting that LrADI impairs the migration of colon cancer cells, potentially limiting metastatic potential. LrADI treatment reduced the number and size of colonospheroids, showing its inhibitory effect on colon cancer stem cells. Nuclear staining with Hoechst 33342 revealed prominent apoptotic morphology, including chromatin condensation and fragmentation, while AO/EtBr dual staining confirmed a substantial increase in apoptotic cell populations following LrADI treatment. The flow cytometric analysis employing Annexin V-FITC/Propidium iodide (PI) staining provided quantitative validation of apoptosis induction, demonstrating an increase in both early and late apoptotic cell populations within the LrADI-treated groups compared with the untreated controls.

[CONCLUSIONS] Collectively, these findings demonstrate that LrADI exerts selective, multi-faceted anticancer effects through reduction in cell viability, cell migration, and apoptosis induction, with minimal impact on non-cancerous cells. Owing to its probiotic origin, safety profile, and potent activity, LrADI represents a promising candidate for further development as a therapeutic agent for colorectal cancer.