Eicosapentaenoic acid-free fatty acid prevents inflammation and cancer in colitis-associated colorectal cancer

Long-standing ulcerative colitis (UC) is associated with an increased risk of colorectal cancer (CRC). The consumption of n-3 polyunsaturated fatty acids (n-3 PUFAs) reduces the risk of CRC but clinical studies in Inflammatory Bowel Disease (IBD) have yielded conflicting results. Potential effectiveness for n-3 PUFAs in IBD derives from animal studies of intestinal inflammation. In Fat-1 transgenic mice (that converts n-6 in n-3 PUFAs) treated with DSS, colitis is suppressed, while in animals treated with AOM-DSS colonic tumors are reduced. Recently, Interleukin-6 (IL-6), produced by lamina propria macrophages, has been identified as a tumor promoter during colitis-associated cancer (CAC), enhancing proliferation and protecting intestinal pre-malignant epithelial cells (IEC) from apoptosis, through activation of Stat3. In breast cancer, IL-6 induces an up-regulation of the Notch ligand Jagged1 while in CRC Jagged1 is up-regulated by beta-catenin. The interplay between IL-6, Notch and Wnt pathways has not been addressed so far in CAC. We have recently tested a highly pure (99%) eicosapentaenoic acid as free fatty acid (EPA-FFA) on the development of polyps in the ApcMin/+ model. We obtained a surprisingly high suppression of polyps number and size, similar to those obtained with Cox-2 targeted inhibitors and, compared to other studies employing n-3 PUFAs, a 10 times higher incorporation of EPA into cellular membrane. The aim of this study is to evaluate the effectiveness of EPA-FFA in preventing inflammation and cancer in models of colitis and CAC, both in vitro and in vivo. The in vitro study will test if EPA-FFA could prevent the activation of the Notch1 pathway in IEC by preventing the survival and anti-apoptotic effect of proinflammatory cytokines released by macrophages and the up-regulation of Jagged1 either Stat3 or beta-catenin driven. The in vivo study will evaluate if EPA-FFA could repress tumor formation and inflammation in the AOM-DSS and DSS models and if this suppression is NFkB-IL-6-Stat3- Notch1 or Wnt-Notch1 mediated. For the in vitro study NCM460 will be cultured with a conditioned inflammatory medium obtained from supernatants of activated THP-1 or CD11b+ macrophages isolated from UC-patients with or without EPA-FFA. We expect that these treatments would result in Jagged1/Notch1 pathway activation driven by IL-6-Stat3 and/or beta- catenin, increasing proliferation and reducing apoptosis. Pre-treatment of macrophages with EPA-FFA would reduce the pro-inflammatory cytokine pool, in particular IL-6, resulting in abrogation of Stat3 phosphorylation and suppression of the Notch1 pathway. The high incorporation of EPA-FFA in the cellular membrane would result in reduction of PGE2 production which will prevent the nuclear translocation of beta-catenin. For the in vivo study C57BL/6 mice will be started on Ctrl or EPA-FFA 5% diets four weeks prior to DSS or AOMDSS protocols. We predict that the pre-treatment of animals with EPA-FFA not only will affect the pro-inflammatory effect of DSS, but also counteract the induction of carcinogenesis AOMmediated. Based on our experience with EPA-FFA treatment we are expecting a reduction of tumor size and incidence by at least 70% with a concomitant increase in apoptosis and decrease in proliferation of IEC and a decrease in Notch1 activation.