The Role Of Fgl2 In Tumor Growth And Angiogenesis In Hepatocellular Carcinoma

[BACKGROUND&OBJECTIVE] Fibrinogen-like protein 2(fg12)/fibroleukin, also called fgl2 prothrombinase, belongs to fibrinogen-related protein superfamily. Fg12 prothrombinase has serine protease activity, which can directly catalyze prothrombinase into activated thrombinase, initiating cascade coagulating reaction. In our previous study, increased fg12 expression was detected in the tumor tissues, located mainly in the tumor cells, interstitial infiltrating cells and vascular endothelial cells. The fibrin deposition can be seen in the close area to the fg2 positive cells. In this study, we aim to investigate the role of fg12 expression in tumor growth and tumor angiogenesis through in vitro study of human highly invasive hepatocellular carcinoma cell line HCCLM6 and human monocyte macrophage cell line THP-1, which might shed light on novel target for the treatment of liver cancer.[METHODS] The stable fg12 knock down HCCLM6 and THP-1 cell clones were established by RNA interference technique. Semi-quantitive RT-PCR were used to detect the expression of fg12 mRNA in HCCLM6 cells in rest status or induced by cytokines including interleukin 1β(IL-1β), tumor necrosis factor-α(TNF-α) and interferon-γ(IFN-γ) to select the cytokine that induces strongest fg12 expression in HCCLM6 cells. The effect of fg12 knock down on cell proliferation and cell apoptosis of HCCLM6 and cell surface antigen expression pattern of THP-1 cells were analyzed by flow cytometry. ELISA and real-time quantitative PCR were used to detect the protein and mRNA expression of IL-8,VEGF,TNF-αand TGF-β. Transwell tissue culture technology was used to detect the effect of fg12 knock down on THP-1 cell migration and HCCLM6 cell invasion. Flow cytometry beads array was used to detect the phosphorylation of cell signaling moleculars in HCCLM6 cells and THP-1 cells. Immunohistochemical staining was used to detect the correlation of fg12 expression with phosphorylation of MAPKs in tumor tissues of subcutaneously transplanted HCC tumor.[RESULTS] Fg12 expression was found markedly up-regulated in response to proinflammatory cytokines TNF-a. Inhibition of fg12 expression markedly reduced HCCLM6 cell proliferation and led HCCLM6 cells highly susceptible to TNF-αinduced apoptosis. Moreover, fg12 knock down on HCCLM6 cells significantly decreased the VEGF and IL-8 expression both in protein level and mRNA level. Fg12 knockdown in HCCLM6 cells resulted in a dramatic decrease in TNF-αinduced activation of ERK,JNK and p38-MAPK but no apparent direct effect on activation of PLC-γor stat3. Exogenous recombinant fg12 protein caused transient P38-MAPK activation and subsequently sustained ERK1/2-MAPK activation through thrombin dependent pathway. The fgl2 expression was correlated to ERK phosphprylation in tumor issue of HCC.FGL2 knockdown on THP-1 cells significantly decreased the expressing of CD11b and CD38, while elicited an up-regulation of HLA-DR expression and unchanged CD 14 expression. At the same time, the antigen expression pattern changes correlated with inhibited migration and less IL-8 and VEGF expression in fgl2(-)THP-1. HCCLM6 cells co-cultured with fg12(-)THP-1 cells showed less invasive ability than those co-cultured with THP-1 cells. Soluble fg12 protein induced phosphorylation of p38-MAPK but down-regulated phosphorylation of JNK in THP-1 cells, membrane combined fg12 induced both significantly p38-MAPK and JNK phosphorylation but only moderate ERK phosphorylation in THP-1 cells through thrombin generation.[CONCLUSION] Fg12 expression was up-regulated by cytokines in tumor micro-environment both in tumor cells and tumor infiltrating immune cells. Fgl2 plays an important role in tumor growth as well as tumor angiogenesis of HCC through direct or indirect induction of MAPK pathway. [Objective] To investigate the regulation of fibrinogen-like protein 2/fibroleukin(fg12) expression in human highly invasive hepatocellular carcinoma cell line (HCCLM6) and the role of fg12 in tumor growth and tumor angiogenesis of HCC.[Method] Semi-quantitive RT-PCR was used to detect the expression of fg12-mRNA in HCCLM6 cells in rest status or induced by cytokines including interleukin 1β(IL-1β), tumor necrosis factor-α(TNF-α) and interferon-γ(IFN-γ). The stable fg12 knock down HCCLM6 cell clone was established by RNA interference technique. The effect of fgl2 knock down on cell proliferation index, cell cycle and cell apoptosis of HCCLM6 were analyzed by flow cytometry. The expression of IL-8 and VEGF was detected by real time RT-PCR or ELISA on mRNA level or protein level respectively. Matrigel coated transwell was used to detetct the invasive ability of HCCLM6 cells.[Result] Fg12 expression was found up-regulated in response to proinflammatory cytokines including IL-1β,IFN-γand TNF-α, the highest expression was seen under stimulation of TNF-a, and the expression level correlated to the concentration of TNF-a below the dose of 100ng/ml. Inhibition of fg12 expression by RNA interference markedly reduced the proliferation index, the percentage of cells in S-phase of cell cycle and invasive ability of HCCLM6 cells, and led HCCLM6 cells highly susceptible to TNF-a induced apoptosis. Moreover, fg12 knock down in HCCLM6 cells led to a significant reduction of TNF-a induced IL-8 and VEGF production at both mRNA level and protein level while without any effect on the expression of these pro-angiogenesis factors of the rest status.[Conclusion] Up-regulated fg12 expression can be induced by cytokines in HCCLM6 cells, and the fg12 protein in tumor cells plays an important role in tumor growth, angiogenesis and invasion as well as tolerance to TNF-a induced apoptosis. [Objective] To investigate the effect of fibrinogen-like protein 2/fibroleukin(fg12) expression on tumor infiltrating macrophages in tumor invasion and tumor angiogenesis.[Method] The stable fg12 knock down THP-1 cell clone was established by RNA interference technique; Semi-quantitive RT-PCR and flowcytometry were used to assess fg12 interference efficiency. Transwells tissue culture technology was used to examine the migration of THP-1 cells. The fg12 knock down THP-1 cells, mi-RNA control THP-1 cells and parental THP-1 cells were co-cultured with HCCLM6 cells respectively, then the antigen expression on THP-1 cells or fg12 knock THP-1 cells were analysed by flowcytometry, the cytokines such as IL-8,VEGF,TNF-α,TGF-βwere quantified by ELISA in cell-free supernatant of co-culture system. Matrigel coated transwell was used to detetct the invasive ability of HCCLM6 cells cocultured with THP-1 cells or fg12 (-)THP-1 cells respectively.[Results] A stable fgl2 knockdown THP-1 cell line was established. Compared to parental THP-1 cells and mi-RNA control THP-1 cells, the migration ability of fg12(-) THP-1 cells were significantly inhibited; When co-cultured with HCCLM6 cells, significantly decreased expression of CD11b and CD38 with upregulated HLA-DR and unchanged CD14 was observed; the secretion of the IL-8 and VEGF was decreased with unchanged TNF-αand TGF-βproduction in the supernatant of co-culture system of fg12(-)THP-1 with HCCLM6 cells compared with that of THP-1 with HCCLM6 cells. HCCLM6 cells co-cultured with THP-1 cells with the two cell populations contacted show higher invasive ability than those co-cultured with fg12(-)THP-1 cells.[Conclusions] Fg12 expression on macrophages can promote their adhesion, activation and migration abilities, the fg12 expression on tumor associated macrophages may promote tumor angiogenesis and tumor invasion. [Objective] To investigate the down-stream cell signaling transduction of fg12 in HCCLM6 cells and THP-1 cells and explore the mechanism of fg12 promoting tumor growth and tumor angiogenesis.[Methods] The phosphorylated cell signaling moleculars(such as p38-MAPK, ERK, JNK, PLC-y, stat3) of control HCCLM6 cells or fg12(-)HCCLM6 cells under stimulation of TNF-a and phosphorylated MAPKs(p38-MAPK, ERK, JNK) of HCCLM6 cells and THP-1 cells under stimulation of membrane combined fg12 or soluble fg12 were detected by CBA technique. Immunohistochemical staining was used to detect the expression of phosphorylated p38-MAPK and ERK in the subcutaneous HCC tissues of fg12 (-)HCCLM6 cells and control HCCLM6 cells.[Results] Fg12 knock down inhibited p38-MAPK, ERK and JNK phosphorylation in HCCLM6 cells at each time point of TNF-a stimulation and ERK phosphorylation at basic level. Exogenous soluble fgl2 protein and membrane combined fg12 protein stimulation alone has no effect on the phosphorylation of MAPKs in HCCLM6 cells, whereas the soluble fg12 stimulation down-regulated JNK phosphorylation in THP-1 cells, and the presence of prothrombin and Ca2+ together with membrane combined fg12 result in sequential phosphorylation of p38-MAPK and ERK in both THP-1 and HCCLM6 cells which can be completely abolished by hirudin. Immunohistochemical staining shows there is much abundant phosphorylated ERK(pERK) expression in the HCC tumor issue of HCCLM6 cells compared with that of fg12(-)HCCLM6 cells and the score of immune-reactivity of pERK correlates to fg12 protein expression level.[Conclusions] Membrane combined fg12 protein promote MAPKs phosphorylation through both thrombin dependent and in-dependent pathway, and this interaction may account for its role in tumor angiogenesis and tumor growth. Soluble fgl2 may inhibite macrophage's antigen processing function through down-regulating JNK phosphorylation.