Efeitos do tratamento com probióticos e vitamina D na carcinogênese do cólon

Abstract

The properties of vitamin D on the modulation of cell differentiation and proliferation and the role of probiotics in the intestinal microbiota and immunogenic response have generated interest in the application of both in the context of chemotherapeutics and chemoprevention of colorectal tumors. Colorectal cancer (CRC) has late diagnosis and treatment with low response rates, coupled with chemoresistance and adverse effects. In this regard, the present study aimed to investigate the effects of isolated and/or combined treatment of vitamin D3 and probiotics on colorectal carcinogenesis. In vitro, colon adenocarcinoma cells (HCT-8) were treated for 48 hours with vitamin D, probiotics (Bifidobacterium bifidum and Lactobacillus gasseri) and doxorubicin, alone and in combination. Selectivity was assessed using a normal keratinocyte cell line (HaCat), and the synergistic or antagonistic effect of the combinations was evaluated using the method of Chou and Talalay. In vivo, Wistar rats were treated with the probiotics and vitamin D alone and in combination in three different treatment approaches (pre-, post-, and simultaneous) for a period of 12 weeks, and pre-neoplastic lesions were induced with 1,2-dimethylhydrazine (DMH, 40 mg/kg body weight). Toxicological monitoring (water consumption and weight gain), evaluation of the frequency of aberrant crypt foci (ACF) and aberrant crypts (AC), fecal microbiome analysis, biochemical evaluation, immunohistochemical and RT-PCR assays were performed. The in vitro results indicate a selective and dose-dependent effect on viability reduction in both probiotic and vitamin D treatments alone in HCT-8, and reduced IC 50 values in combination, with confirmation of the synergistic effect of the combination of vitamin D with 5-fluorouracil and with doxorubicin. The in vivo results indicate protective action of vitamin D at a dose of 250 IU in the pre-treatment model, at a dose of 2000 IU (International Units) in the post-treatment, and at both doses in the simultaneous model. Probiotics at a dose of 10 7 CFU (Colony Forming Units) have significantly reduced FCA in all approaches studied. Similar effect was reached by the combined treatments. The analysis of the fecal microbiota demonstrated predominance of different phyla in the groups exposed to probiotics and vitamin D, evidencing the modulatory effect of the microbiota presented by the treatments. The evaluation of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine and urea ratified the safety of the treatments. The data provided by RT-PCR and

immunohistochemistry assays for the isolated or combined treatments point to the action of probiotics and vitamin D on important pathways for carcinogenesis in its different stages, including NFE2 like bZIP transcription factor 2 (Nrf2) signaling and Glutathione S-trasferase (GST) activity, Ciclooxigenase 2 (COX2) and Inducible Nitric Oxide Sintase (iNOS) inflammatory regulation, and also proliferation and transcriptional modulation pathways such as the Wnt/β-catenin pathway and Proliferating Cell Nuclear Antigen (PCNA). Under the experimental conditions evaluated, the results obtained suggest that both vitamin D and probiotics have an effect on different pathways involved in colorectal carcinogenesis, which provides new perspectives for the improvement of established therapies.