Poly I:C enhances cycloheximide-induced apoptosis of tumor cells through TLR3 pathway

<p>Abstract</p> <p>Background</p> <p>Toll-like receptor 3 (TLR3) is a critical component of the innate immune response to dsRNA viruses, which was considered to be mainly expressed in immune cells and some endothelial cells. In this study, we investigated the expression and proapoptotic activity of TLR3 in human and murine tumor cell lines.</p> <p>Methods</p> <p>RT-PCR and FACS analysis were used to detect expression of TLR3 in various human and murine tumor cell lines. All tumor cell lines were cultured with poly I:C, CHX, or both for 12 h, 24 h, 72 h, and then the cell viability was analyzed with CellTiter 96^®AQueous One Solution, the apoptosis was measured by FACS with Annexin V and PI staining. Production of Type I IFN in poly I:C/CHX mediated apoptosis were detected through western blotting. TLR3 antibodies and IFN-β antibodies were used in Blockade and Neutralization Assay.</p> <p>Results</p> <p>We show that TLR3 are widely expressed on human and murine tumor cell lines, and activation of TLR3 signaling in cancerous cells by poly I:C made Hela cells (human cervical cancer) and MCA38 cells (murine colon cancer) become dose-dependently sensitive to protein synthesis inhibitor cycloheximide (CHX)-induced apoptosis. Blockade of TLR3 recognition with anti-TLR3 antibody greatly attenuated the proapoptotic effects of poly I:C on tumor cells cultured with CHX. IFN-β production was induced after poly I:C/CHX treatment and neutralization of IFN-β slightly reduced poly I:C/CHX -induced apoptosis.</p> <p>Conclusion</p> <p>Our study demonstrated the proapoptotic activity of TLR3 expressed by various tumor cells, which may open a new range of clinical applications for TLR3 agonists as an adjuvant of certain cancer chemotherapy.</p

Characterization of HCV Interactions with Toll-Like Receptors and RIG-I in Liver Cells

The aim of this study was to examine the mechanisms of IFN induction and viral escape. In order to accomplish the goal we compared our new hepatoma cell line LH86, which has intact TLR3 and RIG-I expression and responds to HCV by inducing IFN, with Huh7.5 cells which lack those features.The initial interaction of LH86 cells, Huh7.5 cells or their transfected counter parts (LH86 siRIG-I, siTLR3 or siTLR7 and Huh7.5 RIG-I, TLR3 or TLR7) after infection with HCV (strain JFH-1) was studied by measuring the expression levels of IFNβ, TRAIL, DR4, DR5 and their correlation to viral replication.HCV replicating RNA induces IFN in LH86 cells. The IFN induction system is functional in LH86, and the expression of the RIG-I and TLR3 in LH86 is comparable to the primary hepatocytes. Both proteins appear to play important roles in suppression of viral replication. We found that innate immunity against HCV is associated with the induction of apoptosis by RIG-I through the TRAIL pathway and the establishment of an antiviral state by TLR3. HCV envelope proteins interfere with the expression of TLR3 and RIG-I.These findings correlate with the lower expression level of PRRs in HCV chronic patients and highlight the importance of the PRRs in the initial interaction of the virus and its host cells. This work represents a novel mechanism of viral pathogenesis for HCV and demonstrates the role of PRRs in viral infection

Comprehensive Gene and microRNA Expression Profiling Reveals a Role for microRNAs in Human Liver Development

BACKGROUND AND AIMS: microRNAs (miRNAs) are small noncoding RNAs that regulate cognate mRNAs post-transcriptionally. miRNAs have been implicated in regulating gene expression in embryonic developmental processes, including proliferation and differentiation. The liver is a multifunctional organ, which undergoes rapid changes during the developmental period and relies on tightly-regulated gene expression. Little is known regarding the complex expression patterns of both mRNAs and miRNAs during the early stages of human liver development, and the role of miRNAs in the regulation of this process has not been studied. The aim of this work was to study the impact of miRNAs on gene expression during early human liver development. METHODS: Global gene and miRNA expression were profiled in adult and in 9-12w human embryonic livers, using high-density microarrays and quantitative RT-PCR. RESULTS: Embryonic liver samples exhibited a gene expression profile that differentiated upon progression in the developmental process, and revealed multiple regulated genes. miRNA expression profiling revealed four major expression patterns that correlated with the known function of regulated miRNAs. Comparison of the expression of the most regulated miRNAs to that of their putative targets using a novel algorithm revealed a significant anti-correlation for several miRNAs, and identified the most active miRNAs in embryonic and in adult liver. Furthermore, our algorithm facilitated the identification of TGFbeta-R1 as a novel target gene of let-7. CONCLUSIONS: Our results uncover multiple regulated miRNAs and genes throughout human liver development, and our algorithm assists in identification of novel miRNA targets with potential roles in liver development

TLR3-mediated apoptosis and activation of phosphorylated Akt in the salivary gland epithelial cells of primary Sjögren’s syndrome patients

This study aimed at ascertain whether innate immunity is involved in the apoptosis of primary cultured salivary gland epithelial cells (SGECs) in primary Sjögren\u27s syndrome (pSS). Induction of apoptosis of SGECs was performed using a TLR3 ligand, poly (I:C). Activation of phosphorylated-Akt (pAkt) and cleaved-caspase 3 was determined by Western blotting or immunofluorescence. Expression of TLR2 and TLR3 with pAkt was observed in cultured SGECs after 24-h stimulation with each ligand. Compared with stimulation with the peptidoglycan or lipopolysaccharide, that with poly (I:C) induced significant nuclear fragmentation, as determined by Hoechst staining (p = 0.0098). Apoptosis was confirmed by terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) staining of SGECs from pSS patients and a normal subject. A significant increase in TUNEL-positive cells was observed by the addition of a PI3K inhibitor, LY294002. Poly (I:C) phosphorylated stress-activated protein kinase/Jun-terminal kinase and p44/42 MAP kinase as well as Akt. Furthermore, poly (I:C)-induced caspase 3 cleavage in SGECs was also inhibited by LY294002. Similar results were obtained using SGECs obtained from a normal subject. The results demonstrated for the first time that TLR3 induces the apoptotic cell death of SGECs via the PI3K-Akt signaling pathway