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P052. Inflammation, Toll-like receptors and tumorigenic processes in the colon: Studies in murine models

E. Eyre1, J.F. Burgueno1, E. Fernandez1

1Universitat Autonoma de Barcelona, Biologia Celular, Fisiologia i Immunologia, Bellaterra (Cerdanyola del Valles), Spain

Background: Inflammation seems to play important roles in the development and progression of cancer. Colorectal cancer (CRC) is one of the most serious complications of IBD, particularly in Ulcerative Colitis (UC). TLRs signalling through different pathways (NF-kB, Wnt, beta-catenin) may bias regulation of cell proliferation and apoptosis and eventually modify the development and progression of Colitis associated CRC.

Methods: C57BL/6 female mice were given a single dose of azoxymethane (AOM; 10 mg/kg i.p.). Some of these mice were additionally given Dextran Sulphate Sodium (3 cycles of DSS; 2% for 7 days in drinking water, alternating with 2 weeks of plain water). A group of healthy control mice of matched age was also used. Two end-points were pre-established, i.e. 11 or 12 weeks. Spleen and colon were macro and microscopically inspected, and TLR2, PCNA, beta-catenin and Caspase‑3 analized by immunohistochemistry (IHC). Quantitative RT-PCR was used to measure the expression of TLR and inflammation, apoptosis and proliferation related factors (tlr2, tlr4, myd88, cyclin D1, bcl2 and tgf‑beta 1).

Results: Mice treated with AOM (WA mice) showed dysplasic signs of low severity and incidence. In contrast, mice treated with AOM and DSS (WAD mice) showed an important inflammation, leukocyte infiltration, crypt loss. Tumour incidence and size was highly increased in the WAD group, with findings such as squamous metaplasia, and eventual carcinoma but no metastasis. IHC analysis evidenced decreased Caspase‑3 and beta-catenin, increased PCNA, and scattered TLR2 immunoreactivity. Striking differences were found in the gene expression at the two chosen endpoints. Very subtle changes were found in animals euthanized at week 11. At week 12 tlr2, tlr4, cyclin D1 and myd88 expression was much higher in WA than in WAD tumour tissue. Bcl2 expression also tended to decrease in WAD compared to WA tumour tissue. In contrast, the expression of tgf-beta 1 expression was not significantly changed. Gene expression in healthy tissue of both WA and WAD mice was increased compared to healthy tissue of control mice.

Conclusions: These results suggest a stepwise rather than a linear progression of the tumorigenic process. High levels of TLR2 and TLR4 expression correlate with a lower tumour incidence and score supporting the concept that tumour progression involves different pathways on an inflammatory background.