Proapoptotic Effects Of TLR9Agnoists On Human Hepatoma Cells And Its Cross-talk With TLR3Agnois

ObjectiveToll-like receptors (TLRs) are a family of pattern recognition receptors. Until now, at least11mammalian TLRs have been identified TLRs are expressed on many kinds of immune cells, especially APC including DCs, macrophages and B cells. Numerous researches demonstrated TLRs play a crucial role in the innate immune response and the subsequent induction of adaptive immune responses against microbial infection or tissue injury.Due to the pivotal role of TLR in immune responses to pathogens, most researches aimed at understanding the function and biological importance of TLR on immune cells in the early time. However, TLRs are also expressed on many kinds of tumor cells, such as melanoma cells, brain tumor and colon tumor. Preliminary evidences suggest that TLRs expressed on tumor cells may play an important role in tumor development. For example, triggering of TLR4expressed on human head and neck squamous cell carcinoma promotes tumor development and protects the tumor from immune attack. Activation of TLR5on breast cancer cells by flagellin suppresses cell proliferation and tumor growth. These indicated different TLRs play various roles on tumor progression. So, it is of great importance to investigate the roles of TLRs expressed on tumors.Hepatocellular carcinoma (HCC) is one of the most frequent human malignancies all over the world and has ranked the third in cancer mortality in China. It has become a serious threat to the health and lives of people. It is imminent to find a more effective strategy for treating HCCs. HCC cells expressed a broad repertoire of TLR genes. Publications reported that TLR3agonist polyI:C promotes HCC cell apoptosis and results in cell survival impaired. Triggering of TLR9on tumor cells lead to cell growth inhibition and apoptosis induction. But the anti-tumor effect of TLR9agonist and TLR3agonist in combination with TLR9agonist is rarely studied. The aim of this work was to illustrate the anti-HCC effects of TLR9agonist and TLR3agonist in combination with TLR9agonist.Methods1. RT-PCR assay was used to detecte the expression of TLRs on HCC cells;2. Transfection:ODN M362Ctrl, ODN M362, ODN2006-G5, polyI:C and TLR9-specific siRNA were transfected into HepG2and H7402cells by using Lipofectamine2000;3. MTT asssy was used to determined cell proliferation changes after transfect ion of ODN M362and ODN M362Ctrl;4. FACS was used to analyze the cell apoptosis and cell cycle;5. Real-time PCR was used to analysis the expression of cytokines, receptors and apoptosis associated genes;6. Western Blot assay was used to analyze the expression of RIG-I, NF-κB, p-NF-κB and IκB-α;7. Detection of fluorescence:Fluorescence was used to analysis the cell apoptosis and detect the the efficiency of siRNA or pEGFP-Nl entry cells;8. Mice were subcutaneously (s.c.) injected with H7402cells;9. ODN M362-liposome solution was injected intratumorally.Results1. TLR9agonist induce hepatocellular carcinoma cells apoptosis independent on TLR91.1TLRs are expressed by hepatocellular carcinoma except for TLR7and TLR8. All three tested tumor cell lines including HepG2, H7402and PLC/RPF/5showed high levels of TLR9expression.1.2ODN M362and ODN M362Ctrl inhibited HCC proliferation especially for H7402cells.1.3ODN M362and ODN M362Ctrl didn’t affect the cell cycle of HCC cells, but, apoptotic peak was found before G0/G1peak in cells treated with ODN M362Ctrl and ODN M362. 1.4ODN M362and ODN M362Ctrl induced apoptosis of HCC especially for H7402.1.5ODN M362and ODN M362Ctrl up-regulated the expression of inflammatory cytokines including TNF-a, IL-6, IL-8, IFN-a and IFN-β.1.6The expression of IκB-α, phosphorylation of NFκB was not affected after challenging with ODN M362Ctrl and ODN M362. ODN M362and ODN M362Ctrl didn’t activate NFκB.1.7The apoptosis rate of the cell induced by ODN M362Ctrl and ODN M362after silencing TLR9was not affected.ODN M362and ODN M362Ctrl mediated HCC apoptosis was not dependent on TLR9.1.8Cells treated by ODN M362-liposome did not form tumor. ODN M362inhibited the formulation of tumor.1.9ODN M362inhibited tumor growth in vivo.2. Phosphorothioate-modification of TLR9ligands inhibits TLR3ligand-induced apoptosis of hepatocellular carcinoma by entry blockade2.1TLR9agonist ODN M362inhibited HCC cells proliferation by inducing cell apoptosis.2.2TLR9agonist ODN M362suppressed polyI:C-induced apoptosis when co-transfected simultaneously.2.3ODN M362depressed the expression of receptor recginzed polyI:C including RIG-I, MDA5, LGP2and TLR3.2.4ODN M362depressed the expression of gene related to apoptosis induced by polyI:C including Noxa, puma and so on.2.5ODN M362depressed the expression of inflammatory cytokine induced by polyI:C including TNF-α, IL-6, IL-8, IFN-a and IFN-β.2.6ODN M362also antagonized polyI:C induced apoptosis in colon cancer cell line.2.7Both ODN M362Ctrl and ODN M362suppressed polyI:C-induced apoptosis, however, ODN2006-G5didn’t. CpG ODN blocked polyI:C-induced apoptosis depends on the phosphorothioate modification. 2.8Phosphorothioate-modified CpG ODN could block the entry of fluorescently labeled siRNA and pEGFP-N1plasmid. Phosphorothioate-modified CpG ODN suppressed polyI:C induced-apoptosis by blocking polyI:C entry.2.9The pro-apoptosis effect induced by polyI:C would be improved by pre-treatment HCC with CpG ODN.ConclusionTLRs were expressed on HCC cells. Activation of TLR9expressed in the cytoplasm could induce cell apoptosis and inhibit cell proliferation by both ODN M362and ODN M362Ctrl. The expression of TLR9and pro-inflammatory cytokines were also upregulated, but didn’t activate NFκB. ODN M362inhibited the formulation of tumor and tumor growth in vivo.We also found that simultaneous transfection of polyI:C and ODN M362exhibited lower apoptotic effect on HCCs when compared to polyI:C alone, accompanying with down-regulation of poly I:C-related innate receptors, pro-inflammatory cytokines and apoptotic genes induced by polyI:C. Further studies indicated that these effects were partly due to phosphorothioate-modification of TLR9ligands which blocked the entry of polyI:C, and the blockage would be avoided by using polyI:C after CpG ODN. Moreover, polyI:C mediated apoptotic effects would be enhanced by pre-treating HCC cells with CpG ODN. Our findings suggest when TLR3agonists and TLR9agonists were combinationally used for cancer therapy, polyI:C should be used after CpG ODN to avoid the blocking effect of phosphorothioate-modified TLR9ligands. These findings provided beneficial experimental evidence for clinical reasonable and effective use of TLRs agonists.