Exopolysaccharides from Bifidobacterium pseudocatenulatum ATCC 27919 induced autophagy and apoptosis in caco-2 cells / Husna Zulkipli

The application of probiotics and its components as an adjuvant or alternative treatments for cancer is an ongoing research. Exopolysaccharide (EPS) is one of the probiotic components which have been reported to play vital parts in the modulation of cell cycle and apoptosis in cancer cells. In this regards, EPS from Bifidobacterium pseudocatenulatum was explored for its potential to induce autophagy and apoptosis in human colon cancer (Caco-2) cells. Studies on EPS specifically from Bifidobacterium pseudocatenulatum ATCC 27919 has yet to be comprehensively examined, especially on the underlying mechanism of anti-cancer potential. Therefore, this study aimed to determine the effects of EPS from Bifidobacterium pseudocatenulatum ATCC 27919 exposure in Caco-2 cells on cell death mechanisms that include apoptosis and associate it with autophagy. The anticancer potentiality of the EPS were evaluated using cytotoxicity assay and microscopy observation in the Caco-2 cells after exposure to 1, 5, and 10 mg/ml EPS for 24 and 48 hours. Additionally, the effects of EPS on both apoptosis (cell cycle assay, annexin V and dead cell assay, DAPI staining and immunoassay (ELISA)) and autophagy (LC-3 assay and immunoassay) were determined respectively. At molecular level, the alteration in gene expression of apoptotic and autophagic mechanisms were quantified by qPCR. Furthermore, the association between the apoptosis and autophagy mechanisms was also quantified by qPCR. The obtained data confirmed the cytotoxicity effects of EPS in the Caco-2 cells by significantly (p<0.001) reducing the viability from 67 to 9%. From the viability assay and IC50 calculated, 5 mg/ml of EPS was chosen for further experiments. Thirty (30) μM of Rapamycin was used as the positive control and showed a more significant (p<0.001) reduction in cell viability which is 38%. Microscopy observation and morphological assessments exhibited typical morphological features of apoptosis in EPS-exposed Caco-2 cells, such as shrinkage of cells, condensed nuclei, and loss of shape. An early phase of apoptosis and cell cycle arrest at the G2/M phase were detected in EPS-exposed cells. qPCR assay showed a significant increase in mRNA of apoptosis markers such as cleaved Caspase-3, Bcl-2-associated X (BAX), and Poly (ADP-ribose) polymerase -1 (PARP-1). They were enhanced relative to Rapamycin-exposed cells compared to unexposed cells. Besides, autophagy was also detected, whereby autophagic LC3-II protein was observed after 24 hours of exposure to EPS on Caco-2 cells. In contrast, mRNA expression of Beclin-1 (upregulated) and Bcl-2 (downregulated) by qPCR assay also indicated the occurrence of autophagy. Detection of protein showed that Sequestosome 1 (SQSTM1) /p62 protein was also enhanced in EPS-exposed cells at 24 hours. The upstream activation of the autophagy event was evident by the increased phosphorylated AMP-activated protein kinase (AMPK) protein with the reduced phosphorylated mechanistic Target Of Rapamycin (mTOR) protein. The potential association node between the mechanisms was demonstrated by examining GRP78 mRNA expression. GRP78 has been recognized to be involved in Endoplasmic reticulum (ER) stress-induced autophagy. GRP78 was significantly upregulated (p<0.001) in EPS-exposed Caco-2 cells. In conclusion, autophagy was suggested as a cytoprotective response in Caco-2 cells against stress, as it preceded the apoptosis upon EPS exposure in Caco-2 cells. In this study, it is also suggested that both autophagy and apoptosis are associated with antagonistic effects in which suppressing autophagy has enhanced the apoptosis mechanism in the Caco-2 cells.