Dehghani Najme, Tafvizi Farzaneh, Jafari Parvaneh
Department of Biology, Parand Branch, Islamic Azad University, Parand, Iran.
Microbiology Department, Faculty of Science, Arak Branch, Islamic Azad University, Arak, Iran.
Bioimpacts. 2021;11(4):245-252. doi: 10.34172/bi.2021.32. Epub 2020 Aug 10.
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 on HT-29 cell line. : Molecular identification of probiotic 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. The supernatant of 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. The supernatant of probiotic has a great potential to inhibit the proliferation of HT-29 cells and the induction of apoptosis. might be used as a biological anti-cancer factor in the prevention and treatment of colon cancer.
如今,益生菌已被视为癌症预防和治疗的一个因素,尤其是通过诱导细胞凋亡。本研究旨在评估益生菌上清液对HT-29细胞系的细胞毒性、抗增殖和凋亡作用。:使用16S rRNA基因的特异性引物并进行测序,对益生菌进行分子鉴定。HT-29细胞在24、48和72小时时用不同浓度的细菌上清液处理。进行MTT试验、膜联蛋白V-异硫氰酸荧光素、实时聚合酶链反应、细胞周期分析和4',6-二脒基-2-苯基吲哚染色试验以评估细胞凋亡的诱导情况。通过免疫细胞化学方法测量细胞周期蛋白D1的蛋白水平。益生菌上清液以剂量和时间依赖性方式抑制HT-29癌细胞的生长。流式细胞术结果证实了凋亡性细胞死亡。益生菌细菌上清液导致促凋亡基因包括半胱天冬酶-3、半胱天冬酶-9和Bax的上调。此外,它们导致Bcl2的下调以及细胞周期蛋白D1、细胞周期蛋白E和ERBB2基因表达水平的降低。癌细胞停滞在细胞周期的G0/G1期。免疫细胞化学结果显示,与未处理细胞相比,在用细菌上清液处理48小时期间细胞周期蛋白D1蛋白显著下调。益生菌上清液具有抑制HT-29细胞增殖和诱导凋亡的巨大潜力。益生菌可能用作结肠癌预防和治疗中的一种生物抗癌因子。