Modulation of the wnt/β-catenin signal pathway in vitro by apoE COG1410 in IEC-18 after 5-FU injury

Introduction: The understanding of signaling pathways and their alterations involved in the pathophysiology of cancer and during its treatment is fundamental to identify pharmacological targets capable of reducing uncontrolled growth of neoplastic cells. The Intestinal mucositis is one of the main side effects of 5-fluorouracil (5-FU), a chemotherapy treatment for colorectal cancer. Recent studies have shown that apolipoprotein E (ApoE) exerts anti-inflammatory effects on the intestinal mucositis in animal models. This study evaluated the involvement of Wnt/β-catenin signaling in the recovery of intestinal epithelial cells IEC-18 after 5-FU-induced injury, with or without treatment of ApoE-mimetic peptide COG1410. Methods: IEC-18 cells were exposed to 1 mM of 5-FU in glutamine-free medium, with or without ApoE peptide COG1410 (0.3, 1.0 and 3.0μM). The standard medium with glutamine was used as control. The IEC-18 viability was assessed at 12, 24 and 48 hours following the 5-FU challenge, from the tetrazolium salt assay. Cell migration assay was performed in a monolayer healing model of IEC-18 24 h after 5-FU challenge. Moreover, IEC-18 were obtained 24 hours after 5-FU challenge for gene expression analysis of mRNA by RT-qPCR for β-catenin, axin, APC, TCF4 and LEF, c-Myc and cyclin D1. Results: ApoE COG1410 at doses of 1.0 and 3.0 μm increased the viability rate in standard medium with glutamine at 12 and 24 h (p < 0.05). After 5-FU there was a reduction in the number of viable IEC-18 in the challenged groups, 12 and 24 h after exposure to 5-FU as compared to the group supplemented with glutamine, which was not exposed to 5-FU (p < 0.001). ApoE COG1410 at the three dosages was able to significantly increase the viability rate after 5-FU (p < 0.001) in glutamine-free media, at all measured times, compared to the untreated challenged control. In the model of wound-healing monolayer, the groups challenged with 5-FU presented a reduction in IEC-18 migration in medium with and without glutamine in comparison with the control group, 24 h after the 5-FU challenge. COG1410 increased cell migration at the dose of 1μM in the medium with and without glutamine. After being challenged with 5-FU, all the peptide doses increased cell migration rates after 24 h. Furthermore, the challenge with 5-FU significantly increased the levels of mRNA for axin, APC and TCF4 as compared to control; however, the peptide was able to increase LEF mRNA expression. 5-FU significantly reduced the expression of β-catenin transcribed in the control group. COG1410 significantly increased the β-catenin transcript compared to 5-FU. 5-FU reduced the transcripts for c-Myc and cyclin D1, an effect that was reversed by the peptide. Conclusion: The results found in IEC-18 cells suggest a protective role of COG1410 after challenge by 5-FU, through the indirect activation of the Wnt/β-catenin pathway.