Screening For Anticancer Effect Of Probiotics And The Mechanism Of Induce Apoptosis In HT-29Cells

Colon cancer is one of the most common types of cancer with high morbidity and mortality in the world. Many causes can induce colon cancer, especially dietary habits. Many studies showed food intake imbalance, rich in calories and animal fat and low in fibre, is a factor that fosters the occurrence of colon cancer. Meanwhile, a great deal of data confirmed by lactobacilli exerted protective effects against cancer. However, their mechanism was obscure.In our study,138lactobacillus strains were isolated from traditional fermented foods of minority nationalities or infant and adult faeces, and were examined for anti-proliferative activity. We obtained11strains via MTT assay, L. rhamnosus GG (LGG, ATCC43121, USA) was used as positive. Further study showed that4strains (M5, X11, X12and K14) exerted significantly higher adhering capability on HT-29cells, and better resistance to biological barriers (acid and bile salts), as compared to LGG (P<0.05). The nucleotide sequence of16S rDNA showed that M5and X12strains were members of the genus L. paracasei subp. paracasei, X11and K14strains were L. casei.The cell wall and cytoplasm extracts from4lactobacillus strains (X11, X12, M5and K14) were tested for anti-cancer effects. Among these strains, the stronger anti-proliferative activity was observed with cell wall extracts from X11, X12, M5and K14strains and cytoplasm extracts from M5and K14strains, as compared to LGG (P<0.05). Further analysis (SCGE assay, DNA ladder, Annexin-V and PI staining, cell cycle and mitochondrial membrane potential analysis) revealed that cell walls extracted from X12, M5and K14strains displayed the high anti-cancer and DNA damage activity. In addition, they were determined to be less harmful to noncancerous (Vero) cells than to human colon cancer HT-29cells (P<0.05).We investigated the anti-cancer effects of WPG (whole peptidoglycan) by detecting WPG exerted anti-proliferative activity against HT-29cells. WPG that were extracted from X12and M5strains exerted significantly anti-proliferative activity against HT-29cells through MTT assay and TBE assay (P<0.05). SDS-PAGE analysis confirmed the presence of WPG with dominant bands of approximately14.4kDa. Further analysis revealed that amino acids present in the WPG consisted of alanine, glycine, glutamic acid and lysine. The physical structure of WPG was unvaried during the isolation and chemical purification procedures and retained the shape of the whole cells completely, which were observed by Scanning electron microscopy (SEM) and transmission electron microscope (TEM). HT-29 cell apoptosis was confirmed via flow cytometry analysis (Annexin V-FITC/PIstaining and cell cycle distribution).Futhermore, the study was focused on revealing the detailed mechanism of WPG inducing apoptotic, definitively. We found that WPG from X12and M5strains caused the breakdown of△m and induced ROS formation through flow cytometry analysis. Rhodamine123staining showed80μg/mL and160μg/mL WPG from M5strain caused the breakdown of△m (13.63%and16.23%) comparison with the untreated control cells (P<0.05),80μg/mL and160μg/mL of WPG from X12strain caused the breakdown of△m (27.79%and30.60%), comparison with the untreated control cells (P<0.05). DCFH-DA staining showed the highest level of ROS was observed at160μg/mL of WPG from M5strain and320μg/mL of WPG from X12strain, intracellular ROS levels were significantly higher than control cells,42.10%and66.71%, respectively. ELISA analysis showed that the increased of Cyto-C amount in cytosol and decreased in mitochondria (P<0.05), accompanied by the dose-dependent after treatment with80-1000μg/mL of WPG. Semi-quantitative RT-PCR analysis showed WPG up-regulated pro-apoptotic genes (Bax and Bad) and down-regulated anti-apoptotic gene (Bcl-xl), activated Caspase-3, and was accompanied by the dose-dependent. Moreover, western blot analysis finds WPG from X12strain increased expression of Bax protein, and decreased expression of Bcl-2protein, accelerated the proteolytic processing of the inactive form to the active form of Caspase3protein, as observed by a decline of Procaspase3(32kDa) band. Simultaneously, WPG exerted only minor toxic activity on noncancerous cell line (Vero cell).In conclusion, the results demonstrated that WPG from M5and X12strains obtained could exert anti-cancer effects, and they could induce apoptosis in HT-29in a dose-dependent manner. The apoptotic pathway triggered by WPG was dependent on mitochondria pathway.