thromboplastin and Carcinoma--Hepatocellular

Selectively inducing tumor thrombosis and subsequent necrosis is a novel and promising antitumor strategy. We have previously designed a targeting procoagulant protein, called tTF-EG3287, which is a fusion of a truncated tissue factor (tTF) with EG3287, a short peptide against the neuropilin-1 (NRP1) binding site of vascular endothelial growth factor-A 165 (VEGF-A 165). However, off-target effects and high-dose requirements limit the further use of tTF-EG3287 in antitumor therapy. Therefore, we encapsulated tTF-EG3287 into poly(2-ethyl-2-oxazoline)-distearoyl phosphatidyl ethanolamine (PEOz-DSPE)-modified liposomes to construct pH-responsive liposomes as a novel vascular embolization agent, called tTF-EG3287@Liposomes. The liposomes had an average particle size of about 100 nm and showed considerable drug-loading capacity, encapsulation efficiency, and biocompatibility. Under the stimulation of acidic microenvironments (pH 6.5), the lipid membrane of tTF-EG3287@Liposomes collapsed, and the cumulative drug release rate within 72 h was 83 ± 1.26%. When administered to a mouse model of hepatocellular carcinoma (HCC), tTF-EG3287@Liposomes showed prolonged retention and enhanced accumulation in the tumor as well as a superior antitumor effec, compared with tTF-EG3287. This study demonstrates the potential of tTF-EG3287@Liposomes as a novel embolic agent for solid tumors and provides a new strategy for tumor-targeted infarction therapy.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Hydrogen-Ion Concentration; Liposomes; Liver Neoplasms; Mice; Thromboplastin; Tumor Microenvironment; Vascular Endothelial Growth Factor A

In recent decades, selectively inducing tumor vascular thrombosis, followed by necrosis of tumor tissues has been a promising and potential anticancer strategy. In this report, we prepared a kind of vascular targeting drug that consists of anti-neuropilin-1 monoclonal antibody (anti-NRP-1 mAb) and truncated tissue factor (tTF). Anti-NRP-1 mAb could guide tTF to the surface of tumor vascular endothelial cells and lead to subsequent vascular embolization. This vascular targeting drug, which is also one of the antibody drug conjugates, was generated using a coupling method with water-soluble 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccimide. Afterwards, in-vitro and in-vivo assays were performed to characterize its potential coagulation ability and antitumor activity. In-vitro experiments indicated that tTF-anti-NRP-1 monoclonal antibody (tTF-mAb) retained both the targeting activity of anti-NRP-1 mAb and the procoagulant activity of tTF. Live imaging system was used to assess its biodistribution and tumor-binding capability, which also yielded promising results. Furthermore, in-vivo studies showed that tTF-mAb was capable of significantly inducing tumor vascular thrombosis and inhibiting tumor growth in nude mice bearing subcutaneous xenografts, and histopathologic changes were rarely observed in normal organs.

Topics: Animals; Antibodies, Monoclonal; Apoptosis; Carcinoma, Hepatocellular; Cell Proliferation; Humans; Liver Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Neovascularization, Pathologic; Neuropilin-1; Thromboplastin; Thrombosis; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

In patients with chronic liver diseases, hypercoagulability can contribute to the progression of fibrosis and complications of cirrhosis. Tissue factor (TF) is a transmembrane glycoprotein that initiates the extrinsic pathway of blood coagulation. Recent investigations have established that TF is elevated in patients with pancreatic cancer, blood disorders, diabetes, and cardiovascular disease. Alternatively spliced tissue factor (asTF), a secreted form of TF, induces angiogenesis and exhibits low-level procoagulant activity. The aim of this study was to investigate whether the circulating levels of asTF are elevated in the plasma of patients with liver disease.. In a single-center study, we retrospectively analyzed asTF plasma levels in healthy participants and patients having stage F0-F3 liver fibrosis, liver cirrhosis, as well as hepatocellular carcinoma (HCC). AsTF plasma levels were measured using a sandwich enzyme-linked immunosorbent assay. Values were expressed as median with interquartile range (IQR).. The lowest median plasma asTF concentration (94 pg/ml, IQR: 33-275) was found in the healthy control group. The patients with low-grade liver fibrosis (F0-F1 group) displayed the highest median asTF concentration (404 pg/ml, IQR: 277-789). Significant differences between the asTF levels in the plasma of healthy participants and those in patients with grade F0-F1 fibrosis (P<0.001), patients with grade F2-F3 fibrosis (P=0.019), patients with cirrhosis (P=0.004), and patients with HCC (P<0.001) were found using a Wilcoxon rank-sum test. Treatment-naive patients with HCC had significantly higher asTF levels (P=0.018) than those receiving treatment. AsTF levels were found to increase with worsening Child-Pugh scores and heightened liver disease activity.. AsTF levels are elevated in patients with chronic liver diseases, which increase with worsening Child-Pugh scores and decrease following HCC therapy.

Topics: Adult; Alternative Splicing; Biomarkers; Carcinoma, Hepatocellular; Chronic Disease; Female; Humans; Liver Cirrhosis; Liver Diseases; Liver Neoplasms; Male; Middle Aged; Retrospective Studies; Severity of Illness Index; Thromboplastin

The general prognosis of patients with hepatocellular carcinoma (HCC) remains extremely dismal, due to the high frequency of metastasis. Since 2003, our research group has explored the gene expression profiles of metastasized HCC tissue samples and identified a significant upregulation of CCN3. However, the role and precise pathological function of CCN3 remains elusive. We showed that CCN3 is associated with the poor prognosis of patients with HCC, the malignant phenotype of HCC, and vascular thrombosis. We further evaluated the negative roles of CCN3 in vitro and in vivo, and identified osteopontin (OPN), and coagulation factors tissue factor (TF) and thrombin as the leading genes downstream of CCN3, that are positively associated with HCC cell stemness. We demonstrated that overexpressed CCN3 in HCC cells leads to enhanced survival and increased number of pulmonary metastases in vivo. The elevated levels of OPN and TF were associated with signal activation of nuclear factor κB (NFκB) and extracellular signal-regulated kinases (ERK). Our findings suggest CCN3 is a potential therapeutic target that would affect the upregulation of OPN and coagulation factors, which would lead to an enhanced stemness and blood coagulation microenvironment in HCC tissue.

Topics: Animals; Apoptosis; Biomarkers, Tumor; Blood Coagulation; Carcinoma, Hepatocellular; Cell Adhesion; Cell Movement; Cell Proliferation; Female; Follow-Up Studies; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Neoplasm Invasiveness; Neoplastic Stem Cells; Nephroblastoma Overexpressed Protein; NF-kappa B; Osteopontin; Prognosis; Survival Rate; Thromboplastin; Tumor Cells, Cultured; Tumor Microenvironment; Xenograft Model Antitumor Assays

Topics: Carcinoma, Hepatocellular; Factor VII; Humans; Liver Neoplasms; Receptor, PAR-2; RNA Interference; RNA, Small Interfering; Signal Transduction; Thromboplastin

Autophagy has an important role in tumor biology of hepatocellular carcinoma (HCC). Recent studies demonstrated that tissue factor (TF) combined with coagulation factor VII (FVII) has a pathological role by activating a G-protein-coupled receptor called protease-activated receptor 2 (PAR2) for tumor growth. The present study aimed to investigate the interactions of autophagy and the coagulation cascade in HCC. Seventy HCC patients who underwent curative liver resection were recruited. Immunohistochemical staining and western blotting were performed to determine TF, FVII, PAR2 and light chain 3 (LC3A/B) expressions in tumors and their contiguous normal regions. We found that the levels of autophagic marker LC3A/B-II and coagulation proteins (TF, FVII and PAR2) were inversely correlated in human HCC tissues. Treatments with TF, FVII or PAR2 agonist downregulated LC3A/B-II with an increased level of mTOR in Hep3B cells; in contrast, knockdown of TF, FVII or PAR2 increased LC3A/B. Furthermore, mTOR silencing restored the impaired expression of LC3A/B-II in TF-, FVII- or PAR2-treated Hep3B cells and activated autophagy. Last, as an in vivo correlate, we administered TF, FVII or PAR2 agonist in a NOD/severe combined immunodeficiency xenograft model and showed decreased LC3A/B protein levels in HepG2 tumors with treatments. Overall, our present study demonstrated that TF, FVII and PAR2 regulated autophagy mainly via mTOR signaling. The interaction of coagulation and autophagic pathways may provide potential targets for further therapeutic application in HCC.

Topics: Animals; Antineoplastic Agents; Autophagy; Blood Coagulation; Carcinoma, Hepatocellular; Cell Proliferation; Factor VII; Hep G2 Cells; Humans; Liver Neoplasms; Mice; Mice, Inbred NOD; Mice, SCID; Microtubule-Associated Proteins; Oligopeptides; Receptor, PAR-2; RNA Interference; Signal Transduction; Thromboplastin; Time Factors; TOR Serine-Threonine Kinases; Transfection; Tumor Burden; Xenograft Model Antitumor Assays

To investigate the inhibitory effects of humanized monoclonal antibody-3 (huTNT-3) mediated truncated tissue factor (tTF) on the H(22) hepatoma-bearing mice, and to explore its mechanisms.. The coagulation activity of the huTNT-3/tTF fusion protein was detected by clotting assay and clotting factor X (FX) activation test in vitro. Mouse hepatoma cell line H(22) cells were inoculated subcutaneously into mice to establish the mouse models of hepatoma. The mice were randomly divided into two groups to be injected once with huTNT-3/tTF fusion protein or tTF protein labeled with rhodamine B isothiocyanate (RBITC), respectively. The localization of huTNT-3/tTF fusion protein in the mouse hepatoma tissue was analyzed by confocal laser scanning microscopy 24 hour after the injection. Fifteen mice were randomly divided into three groups to be injected with the huTNT-3/tTF fusion protein, tTF protein or phosphate buffered saline (PBS) once, respectively. The tumor size was measured every two days to calculate the tumor volume. Ten days after the injection the mice were sacrificed. Samples of the tumor, heart, livers, spleen, lung, kidney and brains of the mice were taken for histopathological examination.. Both the huTNT-3/tTF fusion protein and tTF protein effectively promoted blood coagulation. Under the conditions of Ca(2+), the coagulation time in the 1.5, 3, 6 µmol/L huTNT-3/tTF groups was (12.90 ± 0.60) min, (10.39 ± 0.40) min and(8.15 ± 0.24) min, respectively, and the coagulation time of the 1.5, 3, 6 µmol/L tTF groups was (14.23 ± 0.46) min, (12.10 ± 0.49) min and (9.83 ± 0.52) min, respectively, the difference between the two groups was not significant (F = 0.145, P = 0.705). The huTNT-3/tTF fusion protein was similar to the tTF protein in the ability of activating FX (t = 0.101, P > 0.05). The confocal laser scanning microscopic analysis showed that RBITC-fluorescence labeled huTNT-3/tTF fusion protein was enriched in the hepatoma tissue. The tumor volume of the huTNT-3/tTF fusion protein group was significantly lower than that of the tTF and PBS groups (both P < 0.001), however, there was not significant difference between the tTF and PBS groups (t = -0.616, P > 0.05). The survival time of the huTNT-3/tTF group was (25.5 ± 2.5) d, significantly longer than that of the PBS group (17.3 ± 1.9) d and the tTF group (18.6 ± 1.9) d, (both P < 0.05).. The huTNT-3/tTF fusion protein retains the coagulation ability and has the capability of targeting to tumor vasculature, and induces thrombosis in the tumor vessels, thus to suppress the growth of hepatoma in the mice.

Topics: Animals; Antibodies, Monoclonal, Humanized; Blood Coagulation; Carcinoma, Hepatocellular; Cell Line, Tumor; Factor X; Liver Neoplasms; Male; Mice; Neoplasm Transplantation; Random Allocation; Recombinant Fusion Proteins; Thromboplastin; Tumor Burden

Fibrinogen-like protein 2 (FGL2), which directly generates thrombin from prothrombin without activation of the conventional coagulation cascade, was shown to be overexpressed in various human malignant tumours.. Herein, we aimed to investigate its expression pattern, biological function and mechanism of action in hepatocellular carcinoma (HCC).. FGL2 expression and colocalization with fibrin was examined in 15 HCC tissues. FGL2 downregulation was performed by targeting microRNA in a HCCLM6 cell line in which FGL2 was highly expressed in xenografts of nude mice. The effects of FGL2 knockdown on tumour growth and angiogenesis were evaluated in vitro and in vivo. Cytometric bead arrays were employed to identify FGL2-regulated signalling pathways.. FGL2 was overexpressed in HCC tissues and colocalized with fibrin deposition. Knockdown of FGL2 expression in HCCLM6 cells (hFGL2(low) HCCLM6) resulted in delayed xenografts tumour growth within an observation period of 42 days and decreased vascularization, which was accompanied by decreased phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). In vitro hFGL2(low) HCCLM6 cells exhibited decreased proliferation without significant induction of apoptosis. Overexpression of FGL2 in HCCLM6 cells or addition of recombinant hFGL2 protein induced phosphorylation of p38-MAPK and ERK1/2 involving protease-activated receptors (PARs).activation.. FGL2 contributes to HCC tumour growth and angiogenesis in a thrombin-dependent manner, and downregulation of its expression might be of therapeutic significance in HCC.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Extracellular Signal-Regulated MAP Kinases; Fibrin; Fibrinogen; Flow Cytometry; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Immunohistochemistry; JNK Mitogen-Activated Protein Kinases; Liver Neoplasms; Mice; Mice, Nude; MicroRNAs; Neovascularization, Pathologic; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Thromboplastin

To study the effect of tissue factor (TF) on human hepatocellular carcinoma cells when their inner TF gene was knocked down by RNA interference.. The eukaryotic expression vector bearing siRNA sequence that targeted at TF gene was transfected into human hepatocellular carcinoma cell line HepG2. RT-PCR and Western blot were used to detect the changes of TF gene expression. Transwell invasion assay invitro were used to show the invasive ability of HepG2 cells with transfected pGPU6/GFP/Neo-TFsiRNA.. RT-PCR and Western blot analysis showed that HepG2 cells with pGPU6/GFP/Neo-TFsiRNA transfected decreased the endogenous TF gene expression. Correspondingly the mean invading cells was 14 ± 10 for pGPU6/GFP/Neo-TFsiRNA transfection and 128 ± 18 for the NCsiRNA transfection, respectively by the Transwell invasion test in vitro (P < 0.05). The results indicated that the invasive ability of the HepG2 cells with pGPU6/GFP/Neo-TFsiRNA transfected decreased obviously.. TF is involved in the progression of hepatocellular carcinoma and inhibiting the expression of TF can decrease the invasion and metastasis ability of tumor cell in vitro.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Humans; Liver Neoplasms; RNA Interference; RNA, Small Interfering; Thromboplastin; Transfection

Numerous studies indicate that tissue factor (TF), namely tissue thromboplastin, has a close relationship with malignant tumor genesis and progress. It contributes to blood coagulation as well as the regulation of cellular differentiation, the formation of blood vessels, and also tumor recurrence and metastasis. The present study aimed to detect TF expression in hepatocellular carcinoma (HCC) patients and to elucidate its association with prognosis and clinical features of the disease.. The plasma TF levels of 50 HCC patients and 30 controls were assayed by ELISA. The expressions of TF mRNA and protein in HCC tissues, adjacent tissues and normal tissues were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. The acquired data were analyzed with related clinic-pathological documents. The patients were followed up for five years, and the relationship between TF and prognosis was analyzed.. The plasma TF levels were significantly increased in HCC compared to the controls (P < 0.05), presenting a close relationship with differentiation level, tumor size and hepatocirrhosis occurrence (P < 0.05). There were remarkably higher values in cases of lymphatic metastasis, extrahepatic metastasis and portal tumor thrombus (PTT) (P < 0.05) compared to non-metastasis or non-tumor thrombus, but no significant difference with different focus number or envelope (P > 0.05). The positive rates and the relative expression of TF mRNA in HCC tissue were 63.0% (17/27) and 0.567 ± 0.268, respectively, significantly higher than that in adjacent tissues or normal tissues (P < 0.05). In the patients with positive results, the relative expression intensity varied significantly with different tumor size and index of local invasion and metastasis (P < 0.05). The positive rates and the relative expression intensities of TF protein in HCC tissue were 74.1% (20/27) and 4.093 ± 1.256, respectively, significantly higher than those in adjacent tissue or normal tissue (P < 0.05). In the patients with positive results, the relative expression intensity showed significant difference in different tumor size, differentiation level, and index of local invasion and metastasis (P < 0.05).. The TF levels were significantly higher in plasma and tissues of HCC patients, presenting a close relationship with the index of invasion and metastasis. It indicated that TF might be related to differentiation and metastasis of HCC.

Topics: Adult; Aged; Blotting, Western; Carcinoma, Hepatocellular; Enzyme-Linked Immunosorbent Assay; Female; Humans; Liver Neoplasms; Male; Middle Aged; Neoplasm Metastasis; Reverse Transcriptase Polymerase Chain Reaction; Thromboplastin

To examine the role of Fibrinogen-like protein 2 (fgl2)/fibroleukin in tumor development. Fgl2 has been reported to play a vital role in the pathogenesis in MHV-3 (mouse hepatitis virus) induced fulminant and severe hepatitis, spontaneous abortion, allo- and xeno- graft rejection by mediating "immune coagulation".. Tumor tissues from 133 patients with six types of distinct cancers and the animal tumor tissues from human hepatocellular carcinoma (HCC) model on nude mice (established from high metastasis HCC cell line MHCC97LM6) were obtained.. Hfgl2 was detected in tumor tissues from 127 out of 133 patients as well as tumor tissues collected from human HCC nude mice. Hfgl2 was highly expressed both in cancer cells and interstitial inflammatory cells including macrophages, NK cells, and CD8(+) T lymphocytes and vascular endothelial cells. Hfgl2 mRNA was localized in cells that expressed hfgl2 protein. Fibrin (nogen) co-localization with hfgl2 expression was determined by dual immunohistochemical staining. In vitro, IL-2 and IFN-gamma increased hfgl2 mRNA by 10-100 folds and protein expression in both THP-1 and HUVEC cell lines. One-stage clotting assays demonstrated that THP-1 and HUVEC cells expressing hfgl2 had increased procoagulant activity following cytokines stimulation.. The hfg12 contributes to the hypercoagulability in cancer and may induce tumor angiogenesis and metastasis via cytokine induction.

Topics: Adult; Aged; Animals; Carcinoma, Hepatocellular; Cell Line; Cell Line, Tumor; Disease Models, Animal; Endothelial Cells; Female; Fibrinogen; Humans; Interferon-gamma; Interleukin-2; Leukemia, Monocytic, Acute; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; RNA, Messenger; Thrombophilia; Thromboplastin

Thrombin, acting via protease-activated receptors (PARs), and tissue factor (TF) are involved in inflammation, tissue repair and tumorigenesis. Hepatocellular carcinomas (HCCs) usually complicate chronic liver diseases characterised by inflammation and fibrosis. The aim of this study was to describe the expression of PARs and TF in normal liver, cirrhosis and HCCs. We performed an immunohistochemical detection of PAR-1, PAR-3, PAR-4 and human TF in human tissue samples from 19 subnormal livers, 33 cirrhosis and 30 HCCs. PAR-1 was found on endothelial cells of sinusoids and larger vessels. In cirrhosis, spindle-shaped cells within septa and T lymphocytes were PAR-1 positive. A few PAR-1-positive tumour cells were found in 10% of HCCs. PAR-4 expression was restricted to macrophages, B lymphocytes and nerves. PAR-3 expression was rare. Unexpectedly, TF was expressed in 95% of normal livers and in 94% of cirrhosis but only in 50% of HCCs (p<0.001). Staining was mostly hepatocellular. No association existed between TF labelling and clinicopathological characteristics of HCCs. In conclusion, the pattern of expression of PARs is compatible with its role in chronic liver disease by promoting inflammation via immune cells and neurogenic stimulation. However, our data do not support a role for PARs or TF in HCC progression.

Topics: Carcinoma, Hepatocellular; Female; Humans; Immunohistochemistry; Liver; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Receptors, Proteinase-Activated; Thromboplastin

Tissue factor (TF) is a transmembrane glycoprotein that serves as the prime initiator of blood coagulation and plays a critical role in thrombosis and hemostasis. In addition, a variety of tumor cells overexpress cell-surface TF, which appears to be important for tumor angiogenesis and metastasis. To elucidate the mechanism involved in the upregulation of TF in human tumor cells, a comprehensive analysis of TF mRNA from various normal and tumor cells was performed. The results of these studies indicate that, in addition to possessing a normal full-length TF transcript and minor levels of an alternatively spliced transcript known as alternatively-spliced tissue factor (asTF), human tumor cells express additional full-length TF transcripts that are also generated by alternative splicing. Reverse transcriptase-polymerase chain reaction (RT-PCR) and 5'-rapid amplification of cDNA ends- (5'-RACE) based analyses of cytoplasmic RNA from normal and tumor cells revealed that there is alternative splicing of the first intron between exon I and exon II resulting in 2 additional TF transcripts. One of the transcripts has an extended exon I with inclusion of most of the first TF intron (955 bp), while the second transcript is formed by the insertion of a 495 bp sequence, referred to as exon IA, derived from an internal sequence of the first intron. The full length TF transcript with alternatively spliced novel exon IA, referred to as alternative exon 1A-tissue factor (TF-A), represented approximately 1% of the total TF transcripts in normal cells, but constituted 7-10% of the total TF transcript in tumor cells. Quantitative real-time RT-PCR analysis indicated that cultured human tumor cells contain 10-25-fold more copy numbers of TF-A in comparison to normal, untransformed cells. We propose that high-level expression of the novel TF-A transcript, preferentially in tumor cells, may have utility in the diagnosis and staging of a variety of solid tumors.

Topics: Adenocarcinoma; Alternative Splicing; Base Sequence; Carcinoma, Hepatocellular; Carcinoma, Transitional Cell; Cytoplasm; Exons; Humans; Introns; Leukemia, Promyelocytic, Acute; Liver Neoplasms; Molecular Sequence Data; Neoplasm Staging; Neoplasms; Pancreatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Messenger; Thromboplastin; Tumor Cells, Cultured; Up-Regulation

Considerable evidences showed that changes of coagulation and proteolysis factors are closely related to the genesis and growth of malignancy. This study was to detect the expression of tissue factor (TF), urokinase-type plasminogen activator (uPA), and urokinase-type plasminogen activator receptor (uPAR) in cancer tissues and plasma of patients with hepatocellular carcinoma (HCC), and analyze their prognostic significance.. Blood samples were obtained from 50 HCC patients and 30 patients with non-tumor disease. Plasma levels of TF, uPA, and uPAR were detected by ELISA. Cancer tissue and adjacent tissue samples were obtained randomly from 27 patients in HCC group; normal liver tissue samples were also collected from 27 patients in benign disease group. The mRNA levels of TF, uPA, and uPAR in all tissue samples were detected by reverse transcription-polymerase chain reaction (RT-PCR). Their correlations to clinicopathologic features of HCC were analyzed.. Plasma levels of TF, uPA, and uPAR were significantly higher in HCC group than in control group [(409.4+/-13.0) pg/ml vs. (318.8+/-69.1) pg/ml, (1.63+/-0.52) ng/ml vs. (1.20+/-0.40) ng/ml, (1.36+/-1.00) ng/ml vs. (0.68+/-0.28) ng/ml, P<0.05]. Poor differentiation, larger size, and cirrhosis of HCC increased plasma TF level (P<0.05); cirrhosis also increased plasma uPA level (P<0.05); lymphatic metastasis, extrahepatic metastasis, and portal venous tumor thrombus (PVTT) increased plasma levels of TF, uPA, and uPAR (P<0.05). The positive rates of TF, uPA, and uPAR in HCC tissues were 62.96%, 70.37%, 77.78%, respectively; the mRNA levels of TF, uPA, and uPAR were 0.57+/-0.27, 0.96+/-0.46, 0.78+/-0.32, respectively. The positive rates and mRNA levels of TF, uPA, and uPAR were all significantly higher in HCC tissues than in adjacent tissues and normal liver tissues (P<0.05). Intrahepatic metastasis and PVTT increased the positive rate and mRNA levels of TF, uPA, and uPAR in HCC (P<0.05). Pearson test showed that TF expression was positively correlated to uPA, and uPAR expression (r=0.373, P<0.01; r=0.534, P<0.01); uPA expression was positively correlated to uPAR expression (r= 0.365, P<0.01). COX regression analyses showed that TF, uPA, and uPAR were independent prognostic factors of HCC (Chi(2)=6.05, P=0.014; Chi(2)=4.29, P=0.038; Chi(2)=4.40, P=0.036).. TF, uPA, uPAR might have synergetic effect in invasion and metastasis of HCC, and they might relate to prognosis.

Topics: Adult; Aged; Carcinoma, Hepatocellular; Cell Differentiation; Female; Humans; Liver Neoplasms; Lymphatic Metastasis; Male; Middle Aged; Neoplastic Cells, Circulating; Portal Vein; Prognosis; Proportional Hazards Models; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; RNA, Messenger; Thromboplastin; Urokinase-Type Plasminogen Activator

The hypervascularity described in hepatocellular carcinoma varies according to the progression and the differentiation of the tumor, suggesting an angiogenic switch during tumor development.. We used a transgenic mouse model of hepatocellular carcinoma induced by the expression of SV40-T antigen, in which male mice developed hepatic tumors at various temporal and histological stages, whereas female mice remained tumor-free. We analyzed, by immunostaining and reverse transcription-polymerase chain reaction, factors involved in tumoral angiogenesis.. We demonstrated that tumoral angiogenesis occurred before the development of diffuse hepatocarcinoma. We showed that some SV40-T-positive cells with an endothelial phenotype are involved in angiogenic processes, suggesting a partial vasculogenic mimicry. This tumoral angiogenesis is associated with platelet activation due to tissue factor expression in endothelial cells and invading macrophages. Normal and transgenic livers exhibited different pattern of expression of hypoxia-inducible factor 1 alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) mRNA.. This model of hepatocellular carcinoma displays marked tumoral angiogenesis, with proliferation, remodeling and arterialization of hepatic sinusoids, probably associated with a partial vasculogenic mimicry. Abnormal angiogenesis observed in hepatocarcinoma was associated with platelet activation by tissue factor (TF) produced by endothelial cells and invading macrophages. In this transgenic model, HIF-1alpha, VEGF, and TF play a crucial role in tumoral angiogenesis.

Topics: Animals; Carcinoma, Hepatocellular; Disease Progression; Female; Hypoxia-Inducible Factor 1, alpha Subunit; Liver Neoplasms; Male; Mice; Mice, Transgenic; Neovascularization, Pathologic; Platelet Activation; RNA, Messenger; Thromboplastin; Tissue Distribution; Transcription Factors; Vascular Endothelial Growth Factor A

Recent studies have shown that tissue factor (TF) may be involved in tumor angiogenesis and metastasis. The role of TF in hepatocellular carcinoma (HCC) was unknown. This study evaluated whether TF expression correlates with microvessel density (MVD), vascular endothelial growth factor (VEGF) expression, tumor invasiveness, and prognosis in human HCC.. Tissue samples were obtained from 58 specimens of resected HCC. Immunohistochemical expression of TF was examined, and tumor MVD was evaluated using CD34 as the endothelial marker. TF and VEGF protein levels in the tumor cytosol were quantified by ELISA. Clinicopathologic and follow-up data of patients were prospectively collected.. The immunohistochemical expression of TF in the tumors correlated significantly with tumor MVD (P = 0.002). The median cytosolic TF protein level in the tumors was 720 pg/mg total protein (range, 67-2406 pg/mg total protein). A significant positive correlation was found between TF and VEGF levels in the tumor cytosol (r = 0.475, P < 0.001). High tumor cytosolic TF level was associated with venous invasion (P = 0.004), microsatellite nodules (P = 0.024), unencapsulated tumor (P = 0.007), and advanced tumor stage (P = 0.010). A higher than median tumor cytosolic TF level was an independent predictor of poor survival (risk ratio, 1.836; 95% confidence interval 1.130-5.312, P = 0.023).. This study shows that TF is related to tumor angiogenesis and invasiveness in HCC. Evaluation of tumor TF expression may be useful as a prognostic indicator in patients with HCC.

Topics: Adolescent; Adult; Aged; Antigens, CD34; Carcinoma, Hepatocellular; Enzyme-Linked Immunosorbent Assay; Female; Follow-Up Studies; Humans; Immunoenzyme Techniques; Liver Neoplasms; Male; Middle Aged; Neoplasm Invasiveness; Neoplasm Staging; Neovascularization, Pathologic; Prognosis; Prospective Studies; Thromboplastin; Vascular Endothelial Growth Factor A

We have previously shown that human liver myofibroblasts promote in vitro invasion of human hepatocellular carcinoma (HCC) cells through a hepatocyte growth factor (HGF)/urokinase/plasmin-dependent mechanism. In this study, we demonstrate that myofibroblasts synthesize the serine proteinase inhibitor tissue factor pathway inhibitor-2 (TFPI-2). Despite the fact that recombinant TFPI-2 readily inhibits plasmin, we show that it potentiates HGF-induced invasion of HCC cells and is capable of inducing invasion on its own. Furthermore, HCC cells stably transfected with a TFPI-2 expression vector became spontaneously invasive. HCC cells express tissue factor and specifically factor VII. Addition of an antibody to factor VII abolished the pro-invasive effect of TFPI-2. We suggest that TFPI-2 induces invasion following binding to a tissue factor-factor VIIa complex preformed on HCC cells. Our data thus demonstrate an original mechanism of cell invasion that may be specific for liver tumor cells.

Topics: Animals; Blotting, Northern; Carcinoma, Hepatocellular; Cell Division; Cell Line; Cricetinae; DNA, Complementary; Dose-Response Relationship, Drug; Factor VII; Fibrinolysin; Gene Library; Glycoproteins; Humans; Liver; Liver Neoplasms; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Neoplasm Invasiveness; Phosphorylation; Pregnancy Proteins; Protein Isoforms; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Serine Proteinase Inhibitors; Thromboplastin; Transfection; Tumor Cells, Cultured

Topics: Carcinoma, Hepatocellular; Enzyme Activation; Enzyme Activators; Humans; Liver Neoplasms; Thromboplastin; Tumor Cells, Cultured

HGF is a powerful mitogen for both rat and human hepatocytes, epithelial cells and endothelial cells in vitro, and is angiogenic in vivo. It has considerable homology with plasminogen and has been shown to upregulate urokinase-type plasminogen activator (u-PA) in endothelial cells as well as u-PA and its receptor in kidney epithelial cells. In this study, we report that human recombinant HGF stimulates expression of plasminogen activator inhibitor type 1 (PAI-1) and tissue factor (TF) in the human hepatoma cell line HepG2. PAI-1 antigen as determined by a specific enzyme-linked immunosorbent assay increased up to threefold in conditioned media of HepG2. This increase was dose dependent with maximum stimulation achieved with a concentration of 50 ng/mL of hepatocyte growth factor (HGF). PAI-1 antigen also increased up to fourfold in the extracellular matrix in HGF treated HepG2. The production of the PAI-1 binding protein vitronectin (Vn) was not affected by HGF. In contrast, TF activity in HepG2 treated with HGF increased up to twofold. As determined by Northern blotting, PAI-1 and TF-specific mRNA were increased significantly in the presence of HGF, whereas Vn mRNA was not affected. The increase in PAI-1 and TF mRNA was also seen when HepG2 were incubated with HGF in the presence of cycloheximide, thereby indicating that de novo protein synthesis is not required to mediate the effect. u-PA could be detected neither in unstimulated or HGF-stimulated HepG2 cells on the antigen level nor on the mRNA level. In conclusion, our data give evidence that HGF, in addition to its proliferative effect for different cell types, is also involved in the local regulation of fibrinolysis and coagulation. One could speculate that HGF might modulate processes requiring matrix degradation by increasing the expression of the protease u-PA in one cell type and by upregulating the expression of the serine protease inhibitor PAI-1 in a different cell type. Because u-PA has been shown to activate latent HGF to the active form, it could furthermore be speculated that by upregulating PAI-1, which in turn could inhibit u-PA, HGF might regulate its own activation.

Topics: Carcinoma, Hepatocellular; Glycoproteins; Hepatocyte Growth Factor; Humans; Liver Neoplasms; Plasminogen Activator Inhibitor 1; Thromboplastin; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator; Vitronectin

The ability of anionic phospholipids (especially phosphatidylserine, PS) on the outer membrane leaflet of four tumour cell lines to support different stages of the extrinsic pathway of coagulation was probed using annexin V as an inhibitor. The procoagulant activity of two tumorigenic (MKN-28, human gastric carcinoma, Hep3B, human hepatoblastoma) and two non-tumorigenic (HepG2, human hepatocellular, HOC-1, human ovarian carcinoma) cell lines were observed to be inhibited by annexin V, although significant differences (observed as IC50 with respect to annexin V) were noted for each stage of coagulation and between different cell types. This was considered to suggest a restricted accessibility of PS in the vicinity of coagulation factors on the surface of the cell. PS levels, as estimated by binding of 125I-annexin V, were high on two of the cell lines tested, equivalent to 24 x 10(6) sites per cell for HepG2 (Kd 128 nM) and 6.5 x 10(6) sites per cell for MKN-28 (Kd 50 nM). During 9 days' culturing of HepG2 and MKN-28, the number of sites per cell remained constant. However, perhaps supporting a proposal of reduced availability, there was an observed fall in PS-dependent procoagulant activity of HepG2 and MKN-28 cells, subsequent to a peak on reaching confluency at 3 days. Both prothrombinase activity and total procoagulant activity fell, even though the number of 125I-annexin V binding sites remained constant.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Anions; Annexin A5; Blood Coagulation; Carcinoma, Hepatocellular; Female; Hepatoblastoma; Humans; Iodine Radioisotopes; Liver Neoplasms; Ovarian Neoplasms; Phosphatidylserines; Phospholipids; Stomach Neoplasms; Thromboplastin; Tumor Cells, Cultured

Topics: Amino Acid Sequence; Autoradiography; Blotting, Western; Carcinoma, Hepatocellular; Chromatography, Affinity; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Factor VIIa; Factor Xa; Humans; Indicators and Reagents; Iodine Radioisotopes; Kinetics; Lipoproteins; Liver Neoplasms; Molecular Sequence Data; Protein Conformation; Radioisotope Dilution Technique; Thromboplastin; Tritium; Tumor Cells, Cultured

Recombinant tissue factor pathway inhibitor (rTFPI) has been expressed in four mammalian expression systems using human SK hepatoma, mouse C127, baby hamster kidney (BHK), and Chinese hamster ovary (CHO) cells as hosts. On sodium dodecyl sulfate polyacrylamide gel electrophoresis, the immunoaffinity purified rTFPIs all show broad bands and the mean molecular weight of SK hepatoma and C127 rTFPIs (M(r) approximately 38,000) appear larger than those of BHK and CHO rTFPIs (M(r) approximately 35,000). All these proteins inhibit factor Xa and appear to bind factor Xa with 1:1 stoichiometry. The ability of these proteins to inhibit tissue factor-induced coagulation in plasma was examined using a prothrombin time assay. The relative activities of SK rTFPI:C127 rTFPI:BHK rTFPI:CHO rTFPI were found to be 28:15:2.1:1. By Western blot using specific antisera against the amino- and carboxy-termini of TFPI as probes, it is found that all the immunoaffinity purified rTFPIs possess approximately equal amounts of the amino terminus, but the C127 and BHK rTFPIs are deficient in carboxy terminus and the CHO rTFPI is essentially devoid of this region of the protein. Mono S chromatography allowed separation of the full-length and the truncated molecules with high and low anticoagulant activities, respectively. The above results suggest that proteolysis of the carboxy terminus of TFPI occurs to different extent when TFPI is expressed in different cells and that the carboxy terminal region of the TFPI molecule is important for the inhibition of tissue factor-induced coagulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Blood Coagulation; Blotting, Western; Carcinoma, Hepatocellular; Cell Line; CHO Cells; Cricetinae; Electrophoresis, Polyacrylamide Gel; Female; Humans; Kidney; Lipoproteins; Liver Neoplasms; Mice; Ovary; Prothrombin Time; Recombinant Proteins; Thromboplastin; Tumor Cells, Cultured

A polyclonal antibody against a synthetic peptide corresponding to amino acids 3-25 of mature lipoprotein-associated coagulation inhibitor (LACI) was raised in rabbits. The antibody was used to study the production of LACI by Hep G2 hepatoma, Chang liver, and SK hepatoma cells, and to purify LACI from the culture media. By using an amidolytic assay for factor Xa, it was found that the culture media from these liver-derived cell lines contain inhibitors of factor Xa. In Hep G2 hepatoma culture medium, approximately 50% of Xa inhibitory activity was due to LACI. In the Chang liver and SK hepatoma culture media over 95% of the Xa inhibitory activity was due to LACI. The LACIs were purified from these media by immunoaffinity chromatography on an anti-LACI-lg-Sepharose 4B column and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified LA-CIs varied in molecular weight depending on whether the media were concentrated before chromatography. An Mr approximately 38,000 LACI was obtained by chromatography of unconcentrated media. Chromatography of concentrated media yielded a LACI of Mr approximately 35,000 with the same amino-terminal sequence, suggesting partial proteolysis in the carboxyl-terminal region. In addition, an Mr approximately 25,000 form of LACI was also present. The purified Mr approximately 38,000 and approximately 35,000 LACI species from the above cells possess similar specific activities when measured by an anti-Xa/amidolysis assay. To study the role of LACI in the control of coagulation, pooled human plasma was depleted of LACI antigen by immunoaffinity absorption and reconstituted with varying amounts of purified LACI to examine the effect on tissue factor (TF)-induced coagulation. LACI depletion shortens the time of TF-induced clotting of plasma and the clotting time is linearly related to the LACI concentration after reconstitution. These results suggest that LACI plays an important role in limiting TF-induced coagulation in human plasma. Comparison of the potencies of various purified LACIs in the prolongation of TF-induced coagulation revealed that LA-CIs from different sources are not equivalent. The plasma LACI, SK hepatoma LACI, and Chang liver LACI are approximately 7-, 6-7, and 1.3-fold higher in specific activity than Hep G2 hepatoma LACI in the TF-induced clotting assay when compared on an anti-Xa/amidolysis unit basis, suggesting possible differences in post-translational modification of these LA-CIs.

Topics: Carcinoma, Hepatocellular; Cell Line; Electrophoresis, Polyacrylamide Gel; Factor VII; Factor Xa Inhibitors; Humans; Kinetics; Lipoproteins; Liver; Liver Neoplasms; Molecular Weight; Prothrombin Time; Thromboplastin

The lipoprotein-associated coagulation inhibitor (LACI) has been isolated from human plasma using a combination of hydrophobic, ion-exchange, and affinity chromatography. The final purification required was greater than 500,000-fold with a yield of 13%. Plasma LACI, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, contains major bands at 40 and 46 kDa and minor bands at 55, 65, 75, 90, and approximately 130 kDa. All of the molecular weight forms are recognized by antibodies to LACI's amino and carboxyl termini and are able to inhibit the factor VII(a)-tissue factor complex and factor Xa. Plasma LACI, reduced with beta-mercaptoethanol, migrates on sodium dodecyl-sulfate-polyacrylamide gel electrophoresis as a doublet at 42 kDa and has an amino-terminal sequence essentially identical to that of HepG2 LACI. The difference in size between reduced plasma LACI (42 kDa) and HepG2 LACI (47 kDa) may be related to differing degrees of N-linked glycosylation. The 46-kDa and larger forms of unreduced plasma LACI are associated with apolipoprotein A-II (apoA-II) in mixed disulfide linkages. Studies using isolated lipoproteins show that low density lipoprotein (LDL) contains primarily the 40-kDa form of LACI, whereas high density lipoprotein (HDL) contains primarily the 46-kDa form of LACI (LACI/apoA-II complexes). Gel filtration of a fresh plasma sample showed approximately 50% of plasma LACI to be associated with LDL/very low density lipoprotein, 44% with HDL, and the remaining 6% to not be associated with lipoproteins.

Topics: Amino Acid Sequence; Apolipoprotein A-II; Apolipoproteins A; Blotting, Western; Carcinoma, Hepatocellular; Chromatography; Disulfides; Electrophoresis, Polyacrylamide Gel; Factor VII; Factor VIIa; Factor Xa Inhibitors; Humans; Lipoproteins; Lipoproteins, HDL; Lipoproteins, LDL; Liver Neoplasms; Molecular Sequence Data; Molecular Weight; Thromboplastin; Tumor Cells, Cultured

Tissue factor (TF) is a lipoprotein cofactor that markedly enhances the proteolytic activation of factors IX and X by factor VIIa. The functional activity of TF is inhibited by serum in a time- and temperature-dependent fashion. The inhibitory effect is also dependent on the presence of calcium ions and can be reversed by calcium chelation (EDTA) and dilution, thus excluding direct proteolytic destruction of TF as the mechanism for inhibition. Using crude TF, serum immunodepleted of factor VII, and serum depleted of the vitamin K-dependent coagulation factors by BaSO4 absorption, it is shown that TF factor inhibition requires the presence of VII(a), X(a), and an additional moiety contained in barium-absorbed serum. When each of the other required components were at saturating concentrations, half-maximal inhibition of TF occurred in reaction mixtures containing 2% (vol/vol) of TF at a factor VII(a) concentration of 4 ng/mL (80 pmol/L), a factor X concentration of 50 ng/mL (850 pmol/L), and a concentration of barium-absorbed serum of 2.5% (vol/vol). Catalytically active factor Xa appeared to be required for the generation of optimal TF inhibition. The results are consistent with the conclusions of Hjort that barium-absorbed serum contains a moiety that inhibits the VIIa-Ca2+-TF complex. The role of factor X(a) in the generation of the inhibitory phenomenon remains to be elucidated. The inhibitor present in serum (plasma) may in part be produced by the liver in vivo since cultured human hepatoma cells (HepG2) secrete this inhibitory activity in vitro.

Topics: Barium; Blood Coagulation; Calcium; Carcinoma, Hepatocellular; Cell Line; Factor VII; Factor VIIa; Factor X; Factor Xa; Humans; Liver Neoplasms; Macromolecular Substances; Thromboplastin

Inhibition of Factor VIIa-tissue factor activity by a plasma component(s) that requires factor Xa has been described recently. In this communication, we have developed a specific radiometric assay (which utilizes 3H-Factor IX and is sensitive to less than 1% of plasma level) for this inhibitor and have measured its activity in various disease states. Strikingly, the levels of this inhibitor were found to be normal in patients with advanced chronic hepatocellular disease but low in patients with disseminated intravascular coagulation (DIC). When endotoxin was used to induce DIC in rabbits, the levels of this inhibitor fell by 25-90%. Human umbilical vein endothelial cells (HUVE), bovine pulmonary artery endothelial cells, and a human hepatoma cell line (HepG2) all synthesized and secreted this inhibitor, whereas a promyelocytic cell line (HL-60) did not and a monocytic cell line (U937) appears to synthesize only small amounts. When ammonium sulfate-fractionated human plasma and serum-free conditioned media from both HUVE and HepG2 cells were electrophoresed on sodium dodecyl sulfate acrylamide gels, two activity peaks corresponding to Mr approximately 45,000 and Mr approximately 33,000 were eluted in each case. These observations suggest that (a) the inhibitor is consumed in DIC and that (b) endothelial cells (or other cells) synthesize sufficient amounts of this inhibitor in vivo to compensate for any decreased production by liver cells.

Topics: Animals; Carcinoma, Hepatocellular; Cattle; Cells, Cultured; Chronic Disease; Disseminated Intravascular Coagulation; Factor VII; Factor VIIa; Humans; Infant, Newborn; Leukemia, Myeloid, Acute; Liver Diseases; Liver Neoplasms; Lymphoma, Large B-Cell, Diffuse; Pulmonary Artery; Rabbits; Thromboplastin; Umbilical Veins

Topics: Animals; Blood Coagulation Tests; Brain Chemistry; Carcinoma, Hepatocellular; Connective Tissue; Connective Tissue Cells; Liver; Liver Neoplasms; Rats; Sarcoma, Yoshida; Thromboplastin

Topics: Animals; Carcinoma, Hepatocellular; Fibrinolysis; Liver Neoplasms; Neoplasm Transplantation; Neoplasms, Experimental; Rats; Sarcoma, Avian; Sarcoma, Experimental; Thromboplastin