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

Effect of cathepsin D and prostate specific antigen on latent transforming growth factor-beta in breast cancer cell lines

Transforming growth factor-beta (TGFbeta) is present, predominantly in latent forms, in normal and malignant breast tissue. The mechanisms by which latent TGFbeta is activated physiologically remain largely an enigma. The objective of this study was to assess whether the proteases, cathepsin D and prostate specific antigen (PSA) could activate latent TGFbeta1 and TGFbeta2 in conditioned media of the hormone-dependent MCF-7 and hormone-independent MDA-MB-231 human breast cancer cell lines, newly purchased from ATCC. Both of the cell lines were seeded in 6-well plates 2 days prior to treatment with varying concentrations of cathepsin D and PSA. Active TGFbeta1 and TGFbeta2 in the media were then measured by ELISA after 4, 8, 24 and 72 hours of treatment. TGFbeta1 and TGFbeta2 mRNA expression of both cell lines were measured by RT-PCR to determine whether any increase in level of active TGFbeta1 and TGFbeta2 was due to increased production. There was a significant increase in only active TGFbeta2 levels in the MDA-MB-231 cell line with both treatments. Cathepsin D and PSA did not have any effect on TGFbeta1 and TGFbeta2 mRNA expression. Cathepsin D and PSA were unable to activate latent TGFbeta1 and TGFbeta2 in these two breast cancer cell lines. A constant level of TGFbeta2 mRNA in the control and treated MDA-MB-231 cells suggests that the increase in level of active TGFbeta2 was not a result of increased production but was likely to be due to activation by a mechanism independent of cathepsin D and PSA.