Peri-weaning cholera toxin consumption suppresses chemically-induced carcinogenesis in mice.

Gastrointestinal bacteria are known to have an impact on local and systemic immunity, and consequently either promote or suppress cancer development. Following the notion that perinatal bacterial exposure might confer immune system competency for life, we investigated whether early-life administration of cholera-toxin (CT), a protein exotoxin of the small intestine pathogenic bacterium Vibrio cholerae, may shape local and systemic immunity to impart a protective effect against tumor development in epithelia distantly located from the gut. For that, newborn mice were orally treated with low non-pathogenic doses of CT and later challenged with the carcinogen 7,12-dimethylbenzanthracene (DMBA), known to cause mainly mammary, but also skin, lung and stomach cancer. Our results revealed that CT suppressed the overall incidence and multiplicity of tumors, with varying efficiencies among cancer types, and promoted survival. Harvesting mouse tissues at an earlier time-point (105 instead of 294 days), showed that CT does not prevent preneoplastic lesions per se but it rather hinders their evolution into tumors. CT pretreatment universally increased apoptosis in the cancer-prone mammary, lung and nonglandular stomach, and altered the expression of several cancer-related molecules. Moreover, CT had a long-term effect on immune system cells and factors, the most prominent being the systemic neutrophil decrease. Finally, CT treatment significantly affected gut bacterial flora composition, leading among others to a major shift from Clostridia to Bacilli class abundance. Overall, these results support the notion that early-life CT consumption is able to affect host's immune, microbiome and gene expression profiles toward the prevention of cancer.

Alterations in Faecal Microbiota and Elevated Levels of Intestinal IgA Following Oral Administration of Lacticaseibacillus casei in mice.

The intestinal microbiota has been identified as a crucial regulator of the overall health, with studies describing its influence in a variety of disorders and developmental processes throughout the body. A widely accepted approach of influencing the microbiota and regulating its functionality in health or disease is the consumption of probiotics. In this study, we aimed to identify the impact of probiotic Lacticaseibacillus casei ATCC393 on the intestinal microbiota of mice and circulating soluble products of microbial origin or the immune system. Investigation of the gut microflora using next-generation sequencing analysis revealed alterations in the microbial populations following consumption of the probiotic. Abundance of taxa classified as Muribaculaceae was increased in lactobacilli-fed animals, while abundance of taxa classified as Lachnospiraceae and Oscillospiraceae was decreased. In addition, the composition of the intestinal microbiota was modified by the administration of L. casei, as evident by the clustering of test subjects when inspecting beta diversity, without however any significant effect on the alpha diversity of the animals. Finally, production of IgA in the intestinal lumen of mice that had received the microorganism was significantly increased, as was the concentration of lactic acid, while levels of acetic acid were noticeably lower in the L. casei group. The findings suggest that L. casei can be considered a potential candidate strain for the modulation of intestinal homeostasis and a component of dietary interventions aiming to improve overall health.

Anticancer Activity of Biogenic Selenium Nanoparticles: Apoptotic and Immunogenic Cell Death Markers in Colon Cancer Cells.

Colorectal cancer is a health problem with high mortality rates and prevalence. Thus, innovative treatment approaches need to be developed. Biogenic nanoparticles are nanomaterials that can be synthesised in biological systems and, compared to chemically synthesised nanoparticles, have better bioavailability while being more cost-effective, eco-friendlier, and less toxic. In our previous studies, the probiotic strain Lactobacillus casei ATCC 393 was used to synthesise selenium nanoparticles (SeNps), which were shown to inhibit colon cancer cell growth in vitro and in vivo. Herein, we have further investigated SeNps' pro-apoptotic activity and their ability to induce immunogenic cell death (ICD) in colon cancer cells. The SeNps' effect on Caco-2 cells growth was examined along with their potential to induce caspase activation. Moreover, the expression of typical pro-apoptotic and ICD markers were examined in SeNps-treated HT29 and CT26 cells by flow cytometry, Western blot, ELISA and fluorescence microscopy. Elevated caspase-3 activation and surface phosphatyldoserine, that subsided upon co-incubation with a pan-caspase inhibitor, were detected in SeNps-treated cells. Furthermore, nanoparticles induced modulation of the expression of various apoptosis-related proteins. We also report the detection of biomarkers involved in ICD, namely the translocation of calreticulin and ERp57, the release of HMGB1 and ATP, and the secretion of pro-inflammatory cytokines from SeNps-treated cells. Moreover, RAW246.7 macrophages exhibited a higher rate of phagocytosis against treated CT26 when compared to control cells. Taken together, our findings indicate that treatment with SeNps might be an efficient strategy to destroy tumour cells by inducing apoptotic cell death and triggering immune responses.

Biogenic selenium nanoparticles produced by Lactobacillus casei ATCC 393 inhibit colon cancer cell growth in vitro and in vivo.

Selenium compounds exhibit excellent anticancer properties but have a narrow therapeutic window. Selenium nanoparticles, however, are less toxic compared to other selenium forms, and their biogenic production leads to improved bioavailability. Herein, we used the probiotic strain Lactobacillus casei ATCC 393, previously shown to inhibit colon cancer cell growth, to synthesize biogenic selenium nanoparticles. We examined the anticancer activity of orally administered L. casei, L. casei-derived selenium nanoparticles and selenium nanoparticle-enriched L. casei, and investigated their antitumor potential in the CT26 syngeneic colorectal cancer model in BALB/c mice. Our results indicate that L. casei-derived selenium nanoparticles and selenium nanoparticle-enriched L. casei exert cancer-specific antiproliferative activity in vitro. Moreover, the nanoparticles were found to induce apoptosis and elevate reactive oxygen species levels in cancer cells. It is noteworthy that, when administered orally, selenium nanoparticle-enriched L. casei attenuated the growth of colon carcinoma in mice more effectively than the isolated nanoparticles or L. casei, suggesting a potential additive effect of the nanoparticles and the probiotic. To the best of our knowledge this is the first comparative study examining the anticancer effects of selenium nanoparticles synthesized by a microorganism, the selenium nanoparticle-enriched microorganism and the sole microorganism.

Modulation of Anti-Tumour Immune Responses by Probiotic Bacteria.

There is a growing amount of evidence to support the beneficial role of a balanced intestinal microbiota, or distinct members thereof, in the manifestation and progression of malignant tumours, not only in the gastrointestinal tract but also in distant tissues as well. Intriguingly, bacterial species have been demonstrated to be indispensable modulatory agents of widely-used immunotherapeutic or chemotherapeutic regiments. However, the exact contribution of commensal bacteria to immunity, as well as to neoplasia formation and response to treatment, has not been fully elucidated, and most of the current knowledge acquired from animal models has yet to be translated to human subjects. Here, recent advances in understanding the interaction of gut microbes with the immune system and the modulation of protective immune responses to cancer, either naturally or in the context of widely-used treatments, are reviewed, along with the implications of these observations for future therapeutic approaches. In this regard, bacterial species capable of facilitating optimal immune responses against cancer have been surveyed. According to the findings summarized here, we suggest that strategies incorporating probiotic bacteria and/or modulation of the intestinal microbiota can be used as immune adjuvants, aiming to optimize the efficacy of cancer immunotherapies and conventional anti-tumour treatments.

Immune Responses Raised in an Experimental Colon Carcinoma Model Following Oral Administration of Lactobacillus casei.

The role of dietary probiotic strains on host anticancer immune responses against experimental colon carcinoma was investigated. We have previously shown that Lactobacillus casei administration led to tumor growth suppression in an experimental colon cancer model. Here, we investigated the underlying immune mechanisms involved in this tumorgrowth inhibitory effect. BALB/c mice received daily live lactobacilli per os prior to the establishment of a syngeneic subcutaneous CT26 tumor. Tumor volume, cytokine production, T cell differentiation and migration, as well as tumor cell apoptosis were examined to outline potential immunomodulatory effects following L. casei oral intake. Probiotic administration in mice resulted in a significant increase in interferon gamma (IFNγ), Granzyme B and chemokine production in the tumor tissue as well as enhanced CD8+ T cell infiltration, accompanied by a suppression of tumor growth. Cytotoxic activity against cancer cells was enhanced in probioticfed compared to control mice, as evidenced by the elevation of apoptotic markers, such as cleaved caspase 3 and poly (ADPribose) polymerase 1 (PARP1), in tumor tissue. Oral administration of Lactobacillus casei induced potent Th1 immune responses and cytotoxic T cell infiltration in the tumor tissue of tumorbearing mice, resulting in tumor growth inhibition. Thus, the microorganism may hold promise as a novel dietary immunoadjuvant in raising protective anticancer immune responses.

Improving the Subcutaneous Mouse Tumor Model by Effective Manipulation of Magnetic Nanoparticles-Treated Implanted Cancer Cells.

Murine tumor models have played a fundamental role in the development of novel therapeutic interventions and are currently widely used in translational research. Specifically, strategies that aim at reducing inter-animal variability of tumor size in transplantable mouse tumor models are of particular importance. In our approach, we used magnetic nanoparticles to label and manipulate colon cancer cells for the improvement of the standard syngeneic subcutaneous mouse tumor model. Following subcutaneous injection on the scruff of the neck, magnetically-tagged implanted cancer cells were manipulated by applying an external magnetic field towards localized tumor formation. Our data provide evidence that this approach can facilitate the formation of localized tumors of similar shape, reducing thereby the tumor size's variability. For validating the proof-of-principle, a low-dose of 5-FU was administered in small animal groups as a representative anticancer therapy. Under these experimental conditions, the 5-FU-induced tumor growth inhibition was statistically significant only after the implementation of the proposed method. The presented approach is a promising strategy for studying accurately therapeutic interventions in subcutaneous experimental solid tumor models allowing for the detection of statistically significant differences between smaller experimental groups.

Lactobacillus casei Exerts Anti-Proliferative Effects Accompanied by Apoptotic Cell Death and Up-Regulation of TRAIL in Colon Carcinoma Cells.

Probiotic microorganisms such as lactic acid bacteria (LAB) exert a number of strain-specific health-promoting activities attributed to their immunomodulatory, anti-inflammatory and anti-carcinogenic properties. Despite recent attention, our understanding of the biological processes involved in the beneficial effects of LAB strains is still limited. To this end, the present study investigated the growth-inhibitory effects of Lactobacillus casei ATCC 393 against experimental colon cancer. Administration of live Lactobacillus casei (as well as bacterial components thereof) on murine (CT26) and human (HT29) colon carcinoma cell lines raised a significant concentration- and time-dependent anti-proliferative effect, determined by cell viability assays. Specifically, a dramatic decrease in viability of colon cancer cells co-incubated with 10(9) CFU/mL L. casei for 24 hours was detected (78% for HT29 and 52% for CT26 cells). In addition, live L. casei induced apoptotic cell death in both cell lines as revealed by annexin V and propidium iodide staining. The significance of the in vitro anti-proliferative effects was further confirmed in an experimental tumor model. Oral daily administration of 10(9) CFU live L. casei for 13 days significantly inhibited in vivo growth of colon carcinoma cells, resulting in approximately 80% reduction in tumor volume of treated mice. Tumor growth inhibition was accompanied by L. casei-driven up-regulation of the TNF-related apoptosis-inducing ligand TRAIL and down-regulation of Survivin. Taken together, these findings provide evidence for beneficial tumor-inhibitory, anti-proliferative and pro-apoptotic effects driven by this probiotic LAB strain.