| Chronic infection and inflammation have been regarded as one of main courses of cancer pathogenesis and development. Many studies demonstrate that the presence of pro-inflammatory cytokines and persistent chronic inflammation in the tumor microenvironment may lead to cancer pathogenesis and development, for example, it has been shown that bacterial infection may drive the inflammation to cancer pathogenesis. However, the underlining mechanisms by which chronic inflammation lead to cancer pathogenesis remain to be far understood. So, the mechanistic study about the relation between chronic inflammation and cancer pathogenesis is one of hot topics in the field of tumor biology and tumor immunology. Recently, the studies about the roles of Toll-like receptors (TLRs) expressed by tumor cells in the tumor development and immune escape attract much attention.Toll was firstly found to be an essential receptor for host defense against fungal infection in Drosophila, which only has innate immunity. Innate immune cells recognize pathogen-associated molecule patterns (PAMPs) conserved in microbes by TLRs to initiate host immune response against infectious pathogens. The family of TLRs senses conserved structure found in a broad range of pathogens, causing innate immune responses that include the production of inflammatory cytokines, chemokines and interferons. Up to now, 10 human TLRs have been discovered. TLR1-9 are conserved between the human and mouse. TLR2 recognizes lipoproteins/lipopeptides from Gram-positive bacteria, associated with TLR6 discriminate diacyl lipopeptides. TLR3 is implicated in the recognition of dsRNA and viruses. TLR4 is an essential receptor for lipopolysaccharide (LPS) from Gram-negative bacteria. TLR5 recognizesflagellin, a monomeric constituent of bacterial flagella. TLR7 and TLR8 recognize a nucleic acid-like structure of the virus.TLR9 is a receptor for CpG DNA and unmethylated CpG motifs. By recognizing and discriminating various PAMPs or invading microbial pathogens, TLRs are differently activated for appropriate inflammatory cytokine production, not only determining the type and intensity of innate immunity but also controlling the development of adaptive immunity. In LPS-induced TLR4 signal transduction, both MyD88- and MyD88-independent pathways are necessary for optimal production of proinflammatory cytokines TNF-a, IL-18 and IL-6. MyD88-dependent pathway mediates early stage activation of mitogen-activated protein kinases (MAPKs) and NF-kB transcription factor, and MyD88-independent pathway mediates late stage activation of MAPKs and NF-kB transcription factor. While optimal production of the proinflammatory cytokines is necessary for eliminating invading pathogens, uncontrolled TLR activation might result in immunopathological injury to host. One of the most severe conditions is LPS-induced endotoxin shock, in which lethal amount of proinflammatory cytokines are produced and damages of tissues induced upon LPS challenge. Another issue is the pathogenesis of autoimmune diseases.In the past years, the studies of TLRs focused on the TLR expression on the immune cells and the effects of TLR signaling on the immune response and inflammation. As we know, MAPKs and NF-kB are all involved in the tumor survival and apoptosis, and such pathways are triggered by TLRs. So, in recent years, some researchers investigated the expression of TLRs on tumor cells and proposed the concept that TLR signaling in tumor cells may promote immune escape of tumor cells. Therefore, the primary aim of this study is to investigate the expression of TLRs on human lung cancer cells and what's the roles of TLR4 signaling in the tumor cell immune escape.In this study, we observed the expression of TLRs on different kinds of tumor cells, and found that TLR4 and TLR9 were expressed on all of human tumor cell line we detected, including human breast cancer cell lines MCF-7, MDA-MB231, humanlung cancer cell lines H1299, A549, human B lymphoma cell lines KG-1 > Raji. For example, we found the high mRNA expression of TLR4 in H1299, A549 cells. Consistent with the RT-PCR result, fluorescence-activated cell sorting (FACS) analysis revealed that TLR4 protein was also expressed on the surface of human lung cancer cells. Then, we selected human lung cancer cell lines H1299, A549 which express TLR4 as cellular model to investigate the roles of TLR4 signaling in the tumor cell immune escape.The high expression of TLR4 in human lung tumor cells rises up one question, that is whether this receptor is functional active in these cancer cells. We found that LPS-induced TLR4 activation of human lung cancer cells resulted in up-regulation of TGF-6, VEGF, IL-8 production. Then we investigated whether LPS can affect the proliferation of the lung cancer cells and TNF-a or TRAIL-induced apoptosis of lung cancer cells. We found that LPS has no effect on the proliferation of the lung cancer cells, but LPS pretreatment decreased the apoptosis of HI299 and A549 cells induced by TNF-a/CHX or TRAIL.Many cellular stress stimuli such as LPS can activate both NF-kB and MAPK pathways. So, we examined the activation of the TLR4 signal pathway by analyzing the phosphorylation of downstream signaling molecules such as MAPK or NF-kB in human lung cancer cells H1299, A549 after LPS stimulation. We found that there was no significant difference between phosphorylation of ERK and JNK in human lung cancer cells H1299, A549 with or without LPS stimulation. In contrast, LPS stimulation siginicantly induced MAPKp38 phosphorylation in human lung cancer cells. In addition, the pyridinyl imidazole SB203580, a specific inhibitor for p38 MAP kinase, abrogated the enhancing effect of LPS stimulation on the up-regulation of IL-8, VEGF expression in human lung cancer cells, indicating that activation of p38 pathway contributes to the LPS-induced secretion of immunosupprerssive cytokines by human lung cancer cells.The NF-kB pathway is commonly associated with cell proliferation and the prevention of apoptosis. Nuclear translocation of NF-kB in human lung cancer cell was detected 30 minutes after LPS stimulation and continued to increase for thefollowing 2 hours. And NF-kB transcription activity was confirmed by using luciferase reporter system. We found that LPS stimulation activated the NF-kB signaling pathway in human lung cancer cells. And pretreatment of PDTC, a specific inhibitor for NF-kB, could reverse apoptosis resistance of the lung cancer cells induced by LPS, and induced tumor cell apoptosis even in the presence of LPS. Also, we found that NF-kB inhibition has no effect on the LPS-induced secretion of immunosupprerssive cytokines by human lung cancer cells.Our current findings indicate that TLR4 is expressed by human lung cancer cells. TLR4 signaling induces human lung cancer cells to secret higher levels of TGF-6, VEGF, IL-8, and more resistant to apoptosis induction by TNF-a and TRAIL. LPS-induced activation of p38 MAPK, NF-kB pathways play important roles in the induction of immunosuppressive cytokines and apoptosis resistance respectively. Our results demonstrate that the expression of functionally active TLR4 in human malignant cells might affect the tumor micro-environment and lead to tumor progress by promoting tumor cell escape of immunological surveillance. |
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