5-fluorouracil induce the expression of TLR4 on HCT116 colorectal cancer cell line expressing different variants of TLR4

Two common single nucleotide polymorphisms [SNPs] of the human TLR4 gene, namely Asp299Gly [D299G] and Thr399Ile [T399I], have been shown to impair the ability of certain individuals to respond properly to TLR4 ligands. 5-Fluorouracil [5-FU] is widely used for the treatment of patients with advanced colon cancers. The present study examined the impact of two common polymorphisms of the TLR4 genes on the response of the HCT116 colorectal cancer cells to 5-FU. HCT116 was transfected with Flag-CMV1-TLR4 wild-type [WT] and D299G, T399I expression plasmids. The cytotoxic effect of 5-FU on transfected cells was assessed by MTT assay. FACS analysis was performed to show the effect of 5-FU and LPS on the expression of different variants of TLR4. The lowest IC[50]-value was measured in cells expressing the WT TLR4 and non-transfected cells were more resistance to the drug compared to the other cells. 5-FU significantly induced the expression of TLR4 protein in the presence and absence of LPS. 5-FU also induced HMGB1 secretion, Cas3 and PARP activity and these effects were stronger in cells expressing WT TLR4 than the other cells. In conclusion, 5-FU-induced TLR4 expression and LPS had synergistic effect with 5-FU to induced apoptosis in colorectal cancer cells

Variant toll-like receptor4 [Asp299Gly and Thr399Ile alleles] and toll-like receptor2 [Arg753gln and Arg677Trp alleles] in colorectal cancer

The innate immune system recognizes the presence of bacterial products through the expression of a family of membrane receptors known as Toll-like receptors [TLRs]. Polymorphisms in TLRs have been shown to be associated with increased susceptibility to diseases such as inflammatory bowel disease. The aim of this study was to determine whether there was a correlation between polymorphisms of TLR4 [Asp299Gly; Thr399Ile] and TLR2 [Arg677Trp; Arg753Gln] genes and risk of colorectal cancer. DNA from 60 colorectal carcinoma patients from 3 major races in Malaysia [22 Malays, 20 Chinese and 18 Indians] and blood from 50 apparently healthy individuals were evaluated. Control group were matched to study group by race and age. The polymorphisms were determined by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism [PCR-RFLP]. Genotyping results showed two out of sixty tumor specimens [3.3%] harbored both variant TLR4 Asp299Gly and Thr399Ile alleles. In contrast, DNA isolated from blood cells of 50 apparently healthy individuals harbored wild type TLR4. In the case of TLR2 Arg753Gln genotyping, all of the fifty normal and 60 tumors were of the wild type genotype. TLR2 Arg677Trp genotyping showed a heterozygous pattern in all samples. However, this may not be a true polymorphism of the TLR2 gene as it is likely due to a variation of a duplicated [pseudogene] region. There was only a low incidence [2/60; 3.3%] of TLR4 polymorphism at the Asp299Gly and Thr399Ile alleles in colorectal cancer patients. All normal and tumor samples harbored the wild type TLR2 Arg753 allele. Our study suggests that variant TLR4 [Asp299Gly and Thr399Ile alleles] as well as TLR2 [Arg753Gln allele] are not associated with risk of colorectal cancer