Identification and characterization of toll-like receptor (TLR) 2 and 4 mutations in colorectal

• Main Author: Davoodi, Homa
• Format: Thesis
• Language: English
• Published: 2009
• Online Access: http://psasir.upm.edu.my/id/eprint/49958/1/FPSK%28p%29%202009%209RR.pdf; http://psasir.upm.edu.my/id/eprint/49958/
• Institution: Universiti Putra Malaysia

Toll-like receptor (TLRs) are the most important receptors in innate immunity that have been identified as a major class of pattern-recognition receptors. The TLR family comprises at least eleven members, these TLRs recognize a limited but highly conserved set of molecular structures, so called pathogen-associated molecular patterns (PAMPs). For example, TLR4 recognizes LPS, which is unique to gram-negative bacteria. Increasing evidence suggest that the ability of certain individuals to respond properly to TLR ligands may be impaired by single nucleotide polymorphisms (SNPs) within TLR genes. The role of TLRs signaling and effect of SNPs mutations on cancer outcome and survival is not exactly determined yet. The objectives of this study were firstly to detect the two most common TLR2 (Arg677Trp, Arg753Gln) and TLR4 SNPs (Asp299Gly, Thr3991lle) in colorectal cancer (CRC) secondly to evaluate the TLR2 and 4 expressions in colorectal cancer cell lines and the effect of polymorphisms on the TLR4 expression. The cytokine profiles secreted by colorectal cancer cells with mutany and non-mutant TLR4 and the expression of some signal transduction molecules involved in TLR4 signalling were also determined. Lastly, the impact of these SNPs on the cytotoxicity and apoptosis induction of the 5-Fluorouracil (5-FU) was also evaluated. PCR-RFLP was carried out on fifty normal blood samples and sixty human colorectal cancer paraffin-embedded blocks to determine the incidence of TLR2 and TLR4 mutations. The results showed two individuals were heterozygous for the Asp299Gly (D299G) and Thr399IIe (T3991) polymorphisms in the TLR4 gene. However, all samples in control group were the wild-type form. Since we could not find any TLR2 mutations in our samples, our study focused on the TLR4 gene. In Vitro studies were performed on HCT116 cell line transfected with mutant and wild-type TLR4 genotype. A series of experiments were conducted to examine the effect of TLR4 variations on the expression of TLR4 , LPS responsiveness and the response of the cells to the 5-FU as a chemotherapeutic agent. FACS analysis of TLR4 expression on transfected HCT116 cells showed that the expression of wild-type was higher than mutant TLR4. LPS induced TLR4 expression on transfected cells and the response of wild-type genotype to the LPS was more significant compared to the mutants. Western blot analysis and Dual Luciferase assay showed that the activity of pNF-kB was higher in cells transfected with plasmid for TLR4 D299G compared to the other cells. However, the activity of pAKT, pERK1 and pIRAK was higher in wild-type. The results of cytokine measurements showed that IL-8 levels were increased in wild-type and basal VEGF was high in un-transfected cells. Secreted VEGF levels was decreased by LPS in wild-type cells but increased in un-transfected cells. IL-17 was secreted by transfected cells at a low level and was not significantly affected by LPS. The results of MTT assay showed that the cytotoxicity effect of 5-Fu an tranfected cells expressing D299G TLR4 mutant was lower compared to the other cells. 5-Fu increased TLR4 expression on transfected cells and LPS has a synergistic effect with 5-FU. LPS increased the apoptosis induced by 5-FU and suggesting that it may be useful as an adjuvant in chemotherapy. HMGBI, an endogenous ligand for TLR4, was secreted by 5-FU-treated cells and also detected in cell lysate. TLR4 is functionally active on transfected HT116 cell line. The increased activity of pNF-kB in cells transfected with plasmid expressing TLR4 D299G may lead to decreased-cytotoxicity effect of 5-FU in this variant. Therefore, it is possible that the CRC patients who harbor this polymorphism tend to be more resistant to drug compared to the wild-type. High level of pNF-kB can also explain the association of this polymorphism with Inflammatory Bowel Disease (IBD) susceptibility to infectious disease and even cancer.