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Probiotic Pediococcus pentosaceus GS4 produces conjugated linoleic acid (CLA) through biohydrogenation, which may be vital for colon cancer mitigation. Here, we have appraised the anti-cancer potential of probiotic GS4 against colon cancer. CLA produced by probiotic GS4 reduced the proliferation of HCT-116 in vitro rendering towards apoptosis and subsequently abrogated NF-κB and p-Akt. Probiotic GS4 efficiently mitigated colon cancer in azoxymethane (AOM)-induced colon cancer mice model, which was evident from reduced disease severity, alleviated oxidative stress and neoplastic characters. Further delineation of underlying consequences at the level of host–microbe interface, the probiotic GS4 arbitrated the gut microflora-associated biohydrogenation, which subsequently triggered apoptosis in colonocytes, apparent from PARP cleavage, caspase 3 activity, DNA fragmentation and inhibition of …