Effect of diets, drugs, and natural compounds on the prevention of colon carcinogenesis: the role of intestinal microbiome

Colorectal cancer (CRC) represents the second cause of cancer death in Europe. The development of disease is a multistep process (adenoma-carcinoma sequence) in which the accumulation of mutations in cancer-related genes drives the transformation of normal epithelium into cancer. The initial event in this process is represented by mutations in the oncosuppressor APC gene (Adenomatous polyposis coli), mutated in familial adenomatous polyposis (FAP) and in sporadic CRC. Besides the heritable component of CRC, its risk is strongly affected by dietary habits, with red and processed meat consumption associated with an increased risk, dietary fibres associated with a low risk, while for other foods like fish, although studies suggest a protective effect, the evidence is still inconclusive. Scientific evidence also points to the intestinal microbiome as a determinant in the development of CRC; although we know that its composition is affected by dietary habits, the role of the microbiome in determining CRC risk is not well defined. In this regard, a significant portion of my Ph.D. project focused on defining the role of the intestinal microbiome in the risk of colorectal cancer associated with diet (the project is related to Meatic, an European funded project). To this aim, a control diet (CTR), a high CRC risk red Meat-based diet (MBD), a medium CRC risk diet (MBD) supplemented with α-tocopherol (MBDT), and a low CRC risk Pesco-vegetarian diet (PVD) were tested both in humans and in two validated models of CRC: PIRC rats, mutated in Apc gene and spontaneously developing colon tumours and azoxymethane (AOM)-induced rats. We demonstrated that after 3 months of treatment, PVD caused a significant reduction in the number of intestinal tumours in PIRC rats, while in AOM-treated rats it was also observed (INRAe, France) that preneoplastic lesions were smaller in the PVD group than in the MBD. Results obtained by measuring additional CRC-related biomarkers were consistent with the carcinogenesis data. To demonstrate a causal role of the gut microbiome in determining CRC risk associated with the different diets (CTR, MBD, MBDT, and PVD), we performed a faecal microbiota transplantation (FMT) in collaboration with INRAe Micalis Institute (France). PIRC faeces collected at the end of the dietary treatments, were transplanted into germ-free (GF) rats treated with AOM, to induce CRC and were fed a standard CTR diet for 3 months. Strikingly, MBD-transplanted rats showed a higher number of preneoplastic lesions compared to PVD ones, demonstrating that the dietary-shaped microbiome was indeed able to transfer CRC risk. In collaboration with CNR-IBBA, we performed the taxonomic analysis of the intestinal microbiota in rats of the three models (PIRC, AOM, and FMT). The different diets were able to shape the microbiome composition, as demonstrated by the taxonomic distribution at the end of the treatments. In the same way, the metabolomic analysis defined specific profiles of dietary-associated metabolites. The results obtained within the Meatic project formed the basis for an experiment of chemoprevention, that is, the use of natural compounds or synthetic drugs, to block or revert the process of carcinogenesis. This experiment aimed to test the impact on CRC of a low-risk PVD diet administered in combination with the nonsteroidal antiinflammatory drugs (NSAIDs) Aspirin (ASA) and Sulindac (SU), endowed with chemopreventive activities against CRC but often associated with adverse effects. The aim was to determine if the preventive effect of PVD would be further enhanced by the use of those two NSAIDs. PIRC rats were fed with PVD or CTR diets containing ASA(800 ppm and 1600 ppm in the diet) and SU (80 ppm in the diet). After 3 months of treatment, data on colon carcinogenesis confirmed the strong protective effect of PVD compared to CTR diet, ASA in the CTR diet caused a slight but not significant reduction of colon tumours, while in the PVD-fed animals the highest dose of ASA (1600 ppm) caused a significant reduction of tumours compared to CTR group with no treatment. SU at 80 ppm was not effective. Recent data suggest that part of the chemopreventive effect of NSAIDs may be dependent on gut microbiota composition, therefore faeces were also collected from these animals and experiments are in progress to see whether ASA and SU shape intestinal microbiota also in these rats. Besides NSAIDs, natural compounds such as phytoestrogens may exert chemopreventive activity. In our previous work, PIRC rats treated with Adipol (2.5%), a patented preparation rich in phytoestrogens and fibres, showed a lower number of colonic tumours compared to Controls. Therefore, we set up an experiment treating PIRC rats animals with a combination of Adipol (2.5%) and SU (320 ppm) to evaluate a possible synergic chemopreventive effect that may allow us to use a reduced SU dose, counteracting its possible side effects. The results obtained so far indicate that animals treated with the combination of the two compounds showed a reduction in colonic tumours compared to Control rats with no treatment. We also aimed to understand the possible beneficial effects on colon tumours of an olive leaf extract enriched in Oleuropein (ORLE) in collaboration with the Pathology Dept. of the University of Florence and Piacenza. To this aim, PIRC rats aged 1 year and thus already bearing tumours were treated with ORLE (2.7% in CTR diet) for one week. At the end of the treatment, ORLE was able to increase apoptosis in tumours and in the normal mucosa, to affect the intestinal microbiome composition and to modulate the plasma metabolites. In conclusion, we clearly document the protective properties of a PVD diet in CRC and confirm the detrimental effect of MBD-diets; we further demonstrate that these impacts of the diets on carcinogenicity are, at least in part, mediated by the intestinal microbiome. The results of the experiments with PVD, NSAIDs or phytoestrogens, although preliminary, suggest that the combination of different drugs together or, the combination of a protective diet with a drug, could be an effective strategy to prevent CRC in subjects at high risk.