Increased NFκ-B Activity in HCT116 Colorectal Cancer Cell Line Harboring TLR4 Asp299Gly Variant

Toll-Like Receptor 4 (TLR4), considered one of  the most important TLR, recognizes lipopolysaccharide of  gram-negative bacteria. Recognition  of  ligands by  TLRs  induces signaling pathways resulting in activation of transcriptional factors such as NF-κB which are involved in  the  expression of  inflammatory cytokines and  chemokines. To  prevent  an inappropriate immune response, a complex network of molecules negatively regulates TLRs and their associated signaling pathways. Two cosegregating single nucleotide polymorphisms of  the human TLR4 gene, namely Asp299Gly and Thr399Ile, have been associated with hyporesponsiveness to inhaled LPS. The purpose of  this study was to determine the impact of  TLR4 gene variant on NF-κB activity in colorectal cancer cell line. HCT116  cells were transfected  with wild-type and mutants  Flag-CMV1-TLR4 expression vectors.  Western  blot  analysis was performed  to evaluate selected molecules involved in TLR4 signaling. NF-κB activity was assessed by dual- luciferase reporter  assay and cytokine profiles were evaluated by ELISA  and Cytometric Bead Array method. Results showed that the activity of pNF-κB was higher in cells harboring 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 about four fold higher level of IL-8 in cells with wild-type TLR4. This study suggest that TLR4 Asp299Gly gene variant has an impact on TLR4 signaling and potentially on intestinal homeostasis due to impaired control signals at the epithelial cell level  which  may  lead  to   chronic  intestinal  inflammation  and  interrupted   intestinal homeostasis and may eventually lead to colorectal cancer.