The Effects And Mechanism Of12/15-LOX,EP4and Macrophage In Colorectal Tumorigenesis

Part112/15lipoxygenase regulation of colorectal tumorigenesis is determined by the relative tumor levels of its metabolite12-HETE and13-HODE in animal modelsObjective To investigate the potential roles and mechanisms of12/15-LOX in mouse and human colorectal tumorigenesis.Methods We first Planted mouse colon adenocarcinoma MC38cells in wildtype mice and12/15-LOX knockout mice, and human colon adenocarcinoma HCA-7cells in nude mice. Then treat wildtype mice carring MC38cells with a15-LOX-1inhibitor, PD146176. Sacrificed the mice after3-4weeks of growth. Tumors were harvested and weighed at the end of experiment. Tumor tissue and culture medium eicosanoid profiling was measured by gas chromatographic/negative ion chemical ionization mass spectrometric assays using stable isotope dilution.. Intestinal macrophages from wildtype mice and12/15-LOX knockout mice were isolated, then incubated with substrate (60u M arachidonic acid or60μM linoleic acid,60min), and metabolites were measured. MC38cells and HCA-7cells were incubated with substrate (arachidonic acid or linoleic acid) with or without the presence of PD146176. siRNA technique was used to silent12/15-LOX genes in mouse MC38cells and12-LOX and15-LOX-1and15-LOX-2genes in human HCA-7cells. Medium was collected and the eicosanoid metabolites were measured.To investigate the direct effects of12-HETE,15-HETE and13-HODE on colon cancer, cells proliferation was measured by using cell proliferation assay kit. On the other hand, we evaluate15-LOX-1mRNA levels between adenomas with different sizes and their paired normal colon by qPCR. Result Pharmacologic or genetic inhibition of LOX activity had differential effects on murine and human colorectal tumorigenesis:administration of PD146176, promoted human colonic HCA-7tumor growth in association with decreased tumor levels of13-HODE, which is consistent with the observation that human15-LOX-1suppresses the development and growth of human colorectal cancer; and inhibited murine colonic MC38tumor growth in association with decreased tumor levels of both12-HETE and13-HODE; PD146176inhibited13-HODE production in HCA-7cells but inhibited12-HETE production in MC38cells;12/15-LOX deletion in the host tissue promoted MC38tumor growth in association with decreased tumor13-HODE alone;12/15-LOX deletion led to decreased production of13-HODE in isolated intestinal macrophages; and12-HETE stimulated and13-HODE inhibited both murine and human colon cancer cell proliferation.Conclusion the effect of rodent12/15LOX on colorectal tumorigenesis is complicated,and it could be affected by tumor cell type (human or mouse), relative12/15LOX activity in tumor cells and stroma as well as the relative tumor13-HODE and12-HETE levels. Part2Macrophage Prostaglandin E Receptor4(EP4) Promotes Colonic TumorigenesisObjective To investigate the effects and potential mechanisms of macrophage EP4receptor in colorectal tumorigenesis. Methods We first made ApcMin/+mouse mode with selective deletion of macrophage EP4receptor and COX-2gene and ApcMin/+mouse model treated with L-161982,which is a selective EP4receptor antagonist. Meanwhile the control group was set up. Then we observed the genetic and drug treatment in the number, size and gender differences of adenomas. We studed the potential mechanisms by using in situ hybridization,immunohistochemistry,immunofluorescence,quantitative image analysis technology, real-time quantitative PCR and Western blot.Result Inhibition of macrophage COX-2did not affect adenoma development and growth in ApcMin/+: Deletion of macrophage EP4receptors led to marked inhibition of adenoma development and growth and loss of the pro-tumorigenic M2phenotype for adenoma macrophages in ApcMin/+mice,also suppressed adenoma activities of the ERK and mTOR pathways and induced apoptosis.Antagonism of EP4receptors polarized TAMs to an anti-tumorigenic M1phenotype in ApcMin/+mice.Conclusion In the present study, we determined for the first time that selective deletion of EP4receptors in macrophages effectively inhibited intestinal adenoma development and growth.These findings suggest an important role of macrophage EP4receptor in regulation of colorectal tumorigenesis and identify EP4receptor as a possible target for prevention and/or therapy for colorectal cancer.