Cell cycle arrest and anti-cancer potential of probiotic <i>Lactobacillus rhamnosus</i> against HT-29 cancer cells

<i><b>Introduction:</b></i> Nowadays, probiotic bacteria have been considered as a factor in the prevention and treatment of cancer, especially by induction of apoptosis. This study aimed to evaluate the cytotoxic, anti-proliferative, and apoptotic effects of the supernatant of probiotic <i>Lactobacillus rhamnosus</i> on HT-29 cell line. <b><i>Methods</i></b> : Molecular identification of probiotic <i>L. rhamnosus</i> was carried out using specific primers of 16S rRNA gene and sequencing. HT-29 cells were treated with different concentrations of bacterial supernatants at 24, 48, and 72 hours. MTT assay, Annexin V-FITC, real-time PCR, cell cycle analysis, and DAPI staining tests were conducted to evaluate the induction of apoptosis. The level of cyclin D1 protein was measured by immunocytochemistry method. <i><b>Results:</b></i> The supernatant of <i>L. rhamnosus</i> inhibited the growth of HT-29 cancer cells in a dose- and time-dependent manner. The results of flow cytometry confirmed apoptotic cell death. Probiotic bacterial supernatant caused up-regulation of pro-apoptotic genes including caspase-3, caspase-9, and Bax. In addition, they resulted in down-regulation of Bcl2 and a decrease in expression levels of cyclin D1, cyclin E, and ERBB2 genes. Cancer cells were arrested in the G0/G1 phase of the cell cycle. The results of immunocytochemistry showed significant down-regulation of cyclin D1 protein during the 48 hours treatment with bacterial supernatant compared to the untreated cells. <i><b>Conclusion:</b></i> The supernatant of probiotic <i>L. rhamnosus</i> has a great potential to inhibit the proliferation of HT-29 cells and the induction of apoptosis. <i>L. rhamnosus</i> might be used as a biological anti-cancer factor in the prevention and treatment of colon cancer.