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

Introduction: 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 Lactobacillus rhamnosus on HT-29 cell line. Methods: Molecular identification of probiotic L. rhamnosus 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. Results: The supernatant of L. rhamnosus 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. Conclusion: The supernatant of probiotic L. rhamnosus has a great potential to inhibit the proliferation of HT-29 cells and the induction of apoptosis. L. rhamnosus might be used as a biological anti-cancer factor in the prevention and treatment of colon cancer.