Probiotics are being investigated for their different health beneficial effects. Anticancer or cancer-preventive effects of different probiotic species are the most beneficial properties of them and according to this knowledge, the consumption of probiotics or prebiotics is suggested as a promising approach for the prevention and treatment of CRC. In this way, different mechanisms have been proposed for anticancer properties of probiotics such as, production of antitumorigenic or anti-mutagenic compounds, antitoxin effects against different toxins, antimicrobial effects, production of short chain fatty acids (SCFA), trophic effects on enterocytes, and improvement of intestinal barrier function and microflora (11, 21, 29).
It is recently reported that probiotic microorganisms, especially Lactobacillus and Bifidobacterium species exert anticancer effects through the production of antioxidative enzymes, binding to reactive oxygen species, chelating heavy metals, and neutralizing different carcinogens. Furthermore, they can regulate the cell cycle in cancer cells and inhibit their proliferation and make them susceptible to apoptosis (13, 27, 30).
Apoptosis is a controlled process in which some defective cells are selectively eliminated and plays an important role in controlling cell numbers. Many cancer cell types including CRC are resistant to apoptosis and have uncontrolled proliferation. As well, different studies showed that bifidobacteria can interrupt this resistance via upregulation and downregulation of effective genes with pro-apoptotic and anti-apoptotic activities (30, 31). Therefore, the regulation of cell proliferation and apoptosis can be one of the main cancer treatment strategies, and probiotics are reported to be involved in regulation of cell apoptosis and proliferation (11, 31).
To the best of our knowledge, this is the first study that investigated the effects of secretion metabolites from different bifidobacteria species on colorectal cancer cell lines. In the present study, we investigated the potential anticancer activities of five species of bifidobacteria on HT-29 and Caco-2 cell lines. Our findings showed that the secretion metabolites of bifidobacteria species can induce intrinsic and extrinsic apoptosis pathways in human colorectal cancer cells. Also, in HT-29 cancer cell lines, the highest and lowest levels of apoptosis were induced by B. bifidum and B. angulatum, respectively. The highest percentages of induced apoptosis in Caco-2 cells belonged to B. bifidum, followed by B. animalis subsp. Lactis and B. animalis subsp. animalis. Similarly, You et al. and Ku et al. suggested that B.bifidum strains inhibited the growth of several cancer cell lines including HT-29 (32, 33). B. bifidum has been previously reported to be anti-proliferative and protective effects against preneoplastic lesions in animal models of colorectal carcinogenesis (8). The anti-proliferative properties of B. bifidum on cancer cell lines are considered as an example of the interaction between Bifidobacterium spp. and host cells (34). The interactions between B. bifidum and colon cancerous cells leads to the suppression of cancer cell growth that indicates anticancer or antitumor effects of it. It is reported that whole peptidoglycan, a metabolite produced by B.bifidum, was capable to activate macrophages to produce large amounts of cytotoxic molecules including TNF-α, IL-6, and IL-12 (35). By considering the antitumor activity of these mediators, the cytotoxic molecules secreted by activated macrophages mediate the antitumor effects of the whole peptidoglycan. However, in this study, the main effective metabolites of B .bifidum was not characterized, and warrants further study. Further studies are necessary to elucidate the role of B. bifidum as a preventive or therapeutic agent.
The results of the present study indicated that B. adolescentis significantly inhibited the proliferation of human colon cancer cell lines including HT-29, and Caco- 2, which are similar to the results of kim et al (36). Similarly, Lee et al. demonstrated that the butanol extract of B. adolescentis SPM0212 can decrease the proliferation of three human colon cancer cell lines, Caco2, HT-29, and SW480 in a dose-dependent manner. In addition, their findings showed that the treatment of cancer cells with butanol extract of B. adolescentis SPM0212 triggers macrophage activation and significantly enhances the production of TNF-α and NO as two mediators of the immune system with cytotoxic effects on tumor cells (21). Also, Asadollahi et al. demonstrated that the treatment of LS174T cancer cells and CRC mice model by a cocktail of 5 strains of Bifidobacteria has significant protective and anti-cancer properties via downregulation of effective genes such as EGFR, HER-2, and PTGS-2 (COX-2) and suggested as the most efficient treatment in CRC (20). Moreover, administration of live Lactobacillus casei ATCC 393 on murine (CT26) and HT-29 colon cancer cell lines significantly decreased the cell viability and showed potent anti-proliferative effects. Besides, the tumor-suppressive effects of Lactobacillus casei were associated to 60 fold higher mRNA expression of TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand), as effective gene in activation of extrinsic apoptosis signaling pathways, and 10 fold lower mRNA expression of cyclin D1 (a protein required for progression through the G1 phase of the cell cycle), and BIRC5 (Baculoviral IAP repeat-containing 5), that encode the anti-apoptotic protein Survivin (27). likewise, Bibalan et al. examined the antiproliferative and anti-pathogenic effects of Lactic acid bacteria isolated from fecal samples of healthy humans on HT-29 cell line. They showed that amongst the 13 Lactobacillus isolates, L. plantarum 03 has the significant and maximum antiproliferative activities. In addition, they suggested that administration of combination of Lactobacillus species is more effective and required for activation of biological defense system (37).
In the present study the investigation of underlying mechanisms revealed that the B. adolescentis is able to trigger apoptosis by upregulation of caspase-8, Fas-R, and BAD gene expression in HT-29 and, Caco-2 cancer cell lines. Surface receptors for extrinsic apoptosis, such as TNF-α, are produced by immune system cells and Fas, which are able to activate the cytosolic protease and caspase-8. Then, the caspase-8 activates caspase-3, caspase-6, and caspase-7 that leading to extrinsic apoptosis induction (38). In this way, Kim et al revealed that B. adolescentis potentiated the production of TNF-α, as a cytokine that induces apoptosis, and their results supported our results about the induction of apoptosis by this bacteria species (36). Furthermore, B. adolescentis has been used as a vehicle for systemic delivery of the antiangiogenic protein endostatin, and systemic administration of its spores can strongly inhibit angiogenesis and reduce tumor growth (39). Moreover, B. adolescentis effectively increased the expression level of caspase-9 in HT-29 cells and induced the intrinsic apoptosis pathway. Caspase-9 has an important role in the intrinsic apoptosis pathway and its activation is related to mitochondrial outer membrane permeabilization and release of cytochrome c (40).
Furthermore, our findings revealed that B. adolescentis significantly reduced the expression level of anti-apoptotic gene, Bcl-2, in caco-2 cell lines. Generally, apoptosis occurs via two major pathways, the intrinsic pathway (mitochondria-dependent) and the extrinsic pathways (death receptor-dependent). BCL-2 family proteins are involved in the intrinsic apoptosis pathway and composed of two groups of proteins; the first group is proteins with pro-apoptotic properties such as BAX and BAK, and the second group is proteins with anti-apoptotic properties such as BCL-2 and BCL-XL. Thus, the lower expression of BCL-2 which has anti-apoptotic activity can trigger the apoptosis pathway (40, 41).
Moreover, other species of bifidobacteria, B. animalis, has demonstrated anti-mutagenic activity during growth in the MRS broth which antagonizing the action of the carcinogen 2-amino-3-methylimidazo [4, 5-f] quinolone (42). Numerous studies have focused on the potential effects of B. animalis strain on cancer cell lines, but the precise mechanism whereby this strain exert their antitumorigenic effects remains undetermined yet (43, 44).
In the present study, B. bifidum, followed by two subspecies of B. animalis and lactis exerted the highest percentage of apoptosis in Caco-2 cells. Based on different studies, NF-κB has a pivotal role in inflammation, as well it can up-regulate several genes that suppress apoptosis, which indicates its critical role in inflammation-related carcinogenesis (45). It is reported that B. animalis subspecies lactis exert preventive effects on colitis-associated colon cancer by inhibiting NF-κB activity (46). As well, Fahmy et al. revealed that treatment of CRC mice with B. longum, isolated from women breast milk, decrease NF-κB and IL-6 concentration. On the other hand, administration of this bacteria increased IL-1β concentration and resulted in the decline of aberrant crypt foci number in CRC-mice and improve necrosis and fibrosis of the colon cells (47). Since the NF-κB is involved in cell proliferation and also plays a critical role in the inflammatory process, it provides a possible mechanistic link between inflammation and cancer (48). However, we did not evaluate the activity of NF-κB, and further studies are required to investigate the effects of Bifidobacterium strains on NF-κB signaling.
There are some limitations to the present study. Firstly, the anticancer effects of bifidobacteria species on other types of cancer cell lines were not investigated, and additionally, the specific compound(s) of the secretion metabolites of bifidobacteria species, which is involved in the antitumor activity, was not determined. Also, due to the purpose of the study and the financial constraints, the effects of Bifidobacteria species on protein levels were not investigated.
In conclusion, the present study confirmed the anticancer and apoptosis-inducing effects of secretion metabolites of bifidobacteria species on colon cancer cell lines with less adverse effects on normal epithelial cells (KDR/293). Besides, the proposed mechanisms for the CRC preventive effects of bifidobacteria species are down-regulation and up-regulation of anti-apoptotic and pro-apoptotic factors. However, performing more studies are recommended to determine the exact mechanisms of probiotics in human colon cancer.