The human intestinal flora is home to a diverse range of bacteria, many of which are vital for optimizing digestion. Among these, probiotics stand out for their ability not only to enhance the breakdown of complex molecules but also to synthesize key compounds, such as vitamins and beneficial antibiotics, thereby promoting overall health. Probiotics play an essential role in stimulating, regulating, and modulating various functions, including digestion, metabolism, immune defense, pathogen exclusion, and the complex communication between the gut and brain, all through their adherence to the intestinal lining. Lactic acid bacteria, a diverse group of Gram-positive, non-toxic, facultative anaerobes, are particularly important in this process, as they efficiently convert carbohydrates into lactic acid, This characteristic makes them invaluable as starter cultures in food fermentation (Su et al., 2020).

Most probiotics recommended for human use are naturally occurring residents of the complex gastrointestinal ecosystem, primarily from two groups of lactic acid-producing microorganisms: Bifidobacteria and lactic acid bacteria (LAB). Probiotics are associated with various health benefits, including the modulation of gut flora, suppression of excessive allergic responses, and tumor-suppressive effects. Abnormal changes in the gut flora of colon cancer patients have been reported, indicating that disturbances in the gut microbiota are closely related to the initiation and progression of colon cancer. Considering that, several studies have shown that the consumption of some probiotic microorganisms can reduce the risk of cancer development. Therefore, changes in the regulation of intestinal microbiota may play a role in the prevention of intestinal cancer Morsli et al., (2025)). Numerous in vitro and in vivo studies have demonstrated that Lactobacillus species can suppress or prevent colorectal cancer development through various mechanisms. These include antimicrobial effects against carcinogen-producing microbes, anti-genotoxic activities that combat both internal and external carcinogens, activation of the gut mucosal immune system, and the regulation of cell apoptosis and proliferation (Hwang et al., 2022).

The key characteristics of cancer include uncontrolled cell growth, unchecked proliferation, and resistance to apoptosis. Apoptosis is a tightly regulated cellular process that controls cell numbers through the activation of caspases. Caspases are a family of enzymes that play a central role in both the initiation and execution phases of apoptosis. Once activated, these enzymes target specific substrates, leading to various biochemical and morphological changes in apoptotic cells. As such, the assessment of caspase activity serves as a key biochemical marker for apoptosis. Research has shown that the consumption of probiotics may positively influence the apoptosis mechanism, potentially supporting cancer prevention and treatment (Mousavi Jam et al., 2020). Apoptosis is an active cellular process in which damaged or mutated cells undergo self-destruction, helping the organism to regulate its normal growth. Each step of apoptosis requires different proteins, including caspases. One of the beneficial roles of caspases is to prevent tumor progression.

Given that the anticancer activity of probiotics may affect metabolic, immune, and protective functions in the colon and may even induce apoptosis of tumor cells, the use of probiotics, including bifidobacteria, could be beneficial. Although the exact molecular mechanisms and pathways through which bifidobacteria affect tumor cells are still unclear (Saffar et al., 2024).

Therefore, in the present study, the cytotoxic effects of Bifidobacterium cell extracts on colon cancer cells (SW948) were investigated. Also, the apoptotic signaling pathways (caspase 1, caspase 3, caspase 9 and BAX) were investigated in the strains that showed the highest cytotoxic potential against colon cancer cells.