TLR4 Signaling Promotes The Immunosuppressive Cytokine Production Of Human Hepatocellular Carcinoma Cells HepG-2

Introduction and ObjectiveHepatocellular carcinoma remains a major health problem. Viral hepatitis,aflatoxins and water contamination are the most recognized risk factors for this malignancy. Chronic inflammation is a risk factor for the development of cancer. Progression of cancer is often associated with a generalized immunosuppression of the host. Tumors evade immune surveillance by multiple mechanisms, including the production of factors such as transforming growth factor-β(TGF-β1),vascular endothelial growth factor(VEGF) and interleukin-6(IL-6), which inhibit dendritic cell activation and impair tumor-specific T cell immunity. However, the mechanisms regulating expression by tumor cells of factors/molecules that are important for tumor cell immune evasion are not well understood. Toll-like receptors(TLRs) play a crucial role in the inflammation and innate host defense against invading microorganisms by recognizing conserved motifs of microbial origin, also known as pathogen-associated molecular patterns(PAMPs). Some study suggest that lipopolysaccharide(LPS) and heat shock protein 70(HSP70) is the exogenous and endogenous ligand of Toll-like receptor 4 respectively. The TLR4 signaling pathway activates several different signaling elements, including nuclear factorκB(NF-κB), which regulate the release of pro-inflammatory cytokines. Coincidentally, many of these same signaling elements are also involved in tumorigenesis and tumor growth, suggesting that TLR4 may affect tumor growth. Recently, Toll-like receptors 4(TLR4) has been found to be expressed by some kinds of tumor cells. However, what is the biological function of TLR4 on tumor cells and whether human hepatocellular carcinoma cells can express TLR4 remain to be fully understood. In this study, we would detect the expression of TLR4 on human hepatocellular carcinoma cells HepG-2 and find the biological function of TLR4 signal system which was activated by LPS and HSP70-peptide complex. Methods1. To detect the expression of TLR4 mRNA and protein on human hepatocellular carcinoma cells HepG-2 by RT-PCR and Western-blot.2. To detect the expression of TLR4 protein on human hepatocellular carcinoma tissue by SP immunohistochemical method.3. To detect the level of TGF-β1, VEGF, IL-6 mRNA and protein before and after the treatment of LPS by RT-PCR and Western-blot, and inhibit the TLR4, detect the level of TGF-β1, VEGF, IL-6 mRNA and protein after the treatment of LPS by RT-PCR and Western-blot.4. To detect the level of NF-κB mRNA and protein before and after the treatment of LPS by RT-PCR and Western-blot, and inhibit the TLR4, detect the level of NF-κB mRNA and protein after the treatment of LPS by RT-PCR and Western-blot.5. To purify HSP70-peptide complex: the mixed protein drived from hepatocellular carcinoma tissues by means of spliting, centrifuge and was separated by affinity chromatography on concanavalin A-Sepharose and DEAE-Sephacel exchange chromatography. The qualitative and quantitative analysis of HSP70-peptide complex: the molecular weight and identity of the HSP70 was confirmed by SDS-PAGE and Western-blot, the concentration was measured by the means of Bradford.6. To detect the level of TGF-β1, VEGF, IL-6 mRNA and protein before and after the treatment of HSP70-peptide complex by RT-PCR and Western-blot, and inhibit the TLR4, detect the level of TGF-β1, VEGF, IL-6 mRNA and protein after the treatment of HSP70-peptide complex by RT-PCR and Western-blot.7. To detect the level of NF-κB mRNA and protein before and after the treatment of HSP70-peptide complex by RT-PCR and Western-blot, and inhibit the TLR4, detect the level of NF-κB mRNA and protein after the treatment of HSP70-peptide complex by RT-PCR and Western-blot.Results1. The TLR4 mRNA can be detected by RT-PCR on human hepatocellular carcinoma cells HepG-2. The TLR4 protein can be detected by Western-blot on human heaptocellular carcinoma cells HepG-2. 2. Cells staining for TLR4 show yellow or brow granules located on cell membrane in human hepatocellular carcinoma tissue. The positive rate is 76.7%(23/30).3. The levels of TGF-β1, VEGF, IL-6 mRNA and protein were all upregulated after the treatment of LPS on human hepatocellular carcinoma cells HepG-2 and then downregulated after the inhibition of TLR4.4. The levels of NF-κB mRNA and protein were all upregulated after the treatment of LPS on human hepatocellular carcinoma cells HepG-2 and then downregulated after the inhibition of TLR4.5. A sharp stained protein band with a molecular weight of about 70kD was obtained and shown to be HSP70 as confirmed by Western blot, the quantity of HSP70 obtained from 10g hepatocellular carcinoma tissues was 8mg by the means of Bradford.6. The levels of TGF-β1, VEGF, IL-6 mRNA and protein were all upregulated after the treatment of HSP70-peptide complex on human hepatocellular carcinoma cells HepG-2 and then downregulated after the inhibition of TLR4.7. The levels of NF-κB mRNA and protein were all upregulated after the treatment of HSP70-peptide complex on human hepatocellular carcinoma cells HepG-2 and then downregulated after the inhibition of TLR4.Conclusion1. Human hepatocellular carcinoma cells HepG-2 and Cells of human hepatocellular carcinoma tissues have the expression of TLR4.2. As the exogenous ligand of Toll-like receptor 4, LPS can stimulate the activation of NF-κB signaling pathway and promote the production of pro-inflammatory cytokines TGF-β1, VEGF, IL-6 in human heaptocellular carcinoma cells.3. The pure-peptide complex can be obtained through the methods of affinity chromatography on concanavalin A-Sepharose and DEAE-Sephacel exchange chromatography.4. As the endogenous ligand of Toll-like receptor 4, HSP70 can stimulate the activation of NF-κB signaling pathway and promote the production of pro-inflammatory cytokines TGF-β1, VEGF, IL-6 in human heaptocellular carcinoma cells.