Enzymatic inhibition of MICA sheddase ADAM17 by lomofungin in hepatocellular carcinoma cells.

In our previous study on hepatocellular carcinoma (HCC) susceptibility genes in chronic hepatitis patients, we identified the MHC class I polypeptide-related sequence A (MICA). Natural killer cells eliminate various cancer cells, including HCC, by suppressing MICA shedding. Therefore, we investigated MICA sheddases and inhibitors for HCC immunotherapy. In this study, HepG2, PLC/PRF/5, and Hep3B were treated with the siRNA of a disintegrin and metalloproteases (ADAMs) and matrix metalloproteases to measure the concentration of soluble MICA (sMICA) by ELISA to detect the therapeutic target. Furthermore, an FDA-approved drug library was tested for the enzymatic inhibition of the targeted enzyme in an in vitro drug screening assay system. ADAM17 knockdown reduced sMICA levels and increased membrane-bound MICA (mMICA) expression in HCC cells. In an in vitro drug screen using an FDA-approved drug library, lomofungin, an antifungal drug, was found to strongly decrease ADAM17 activity. In HCC cells, mMICA expression was induced and sMICA production was inhibited in a dose-dependent manner. These effects were cancelled upon ADAM17 knockdown, suggesting that lomofungin targeted ADAM17. Analysis of lomofungin analogs revealed the responsible functional groups. In summary, we suggest lomofungin to be an attractive agent for the immunological control of HCC, via the suppression of ADAM17.

Predominance of regorafenib over sorafenib: Restoration of membrane-bound MICA in hepatocellular carcinoma cells.

BACKGROUND AND AIM: The multi-kinase inhibitor regorafenib (REG) was recently demonstrated to be effective in patients with sorafenib (SOR)-resistant hepatocellular carcinoma (HCC). Interestingly, SOR is known to enhance the accumulation of membrane-bound MHC class I polypeptide-related sequence A (mMICA) in HCC cells and to block the production of soluble MICA (sMICA), an immunological decoy. In addition, MICA is associated with HCC in patients with chronic hepatitis C. We have now compared the impact of REG and SOR on MICA in HCC cells, as well as the immunotherapeutic implications thereof. METHODS: HepG2 and PLC/PRF/5 cells were exposed to REG and SOR, and levels of sMICA and mMICA were measured by ELISA and flow cytometry, respectively. The drugs were also tested in vitro for inhibitory activity against recombinant human A disintegrin and metalloprotease 9 (ADAM9), a sheddase that releases MICA from the membrane. RESULTS: To a greater extent than SOR, but without marked difference in cytotoxicity, REG significantly suppressed mRNA and protein expression of ADAM9 and ADAM10, thereby decreasing production of sMICA and boosting accumulation of mMICA. Accumulation of mMICA in response to REG was reversed by siRNA against ADAM9. However, the drugs did not inhibit the enzymatic activity of ADAM9 in vitro. CONCLUSIONS: The clinical superiority of REG over SOR is partially attributable to reduced MICA shedding via transcriptional suppression of ADAM9 and ADAM10.

Antagonism of sphingosine 1-phosphate receptor 2 causes a selective reduction of portal vein pressure in bile duct-ligated rodents.

UNLABELLED: Sinusoidal vasoconstriction, in which hepatic stellate cells operate as contractile machinery, has been suggested to play a pivotal role in the pathophysiology of portal hypertension. We investigated whether sphingosine 1-phosphate (S1P) stimulates contractility of those cells and enhances portal vein pressure in isolated perfused rat livers with Rho activation by way of S1P receptor 2 (S1P(2) ). Rho and its effector, Rho kinase, reportedly contribute to the pathophysiology of portal hypertension. Thus, a potential effect of S1P(2) antagonism on portal hypertension was examined. Intravenous infusion of the S1P(2) antagonist, JTE-013, at 1 mg/kg body weight reduced portal vein pressure by 24% without affecting mean arterial pressure in cirrhotic rats induced by bile duct ligation at 4 weeks after the operation, whereas the same amount of S1P(2) antagonist did not alter portal vein pressure and mean arterial pressure in control sham-operated rats. Rho kinase activity in the livers was enhanced in bile duct-ligated rats compared to sham-operated rats, and this enhanced Rho kinase activity in bile duct-ligated livers was reduced after infusion of the S1P(2) antagonist. S1P(2) messenger RNA (mRNA) expression, but not S1P(1) or S1P(3) , was increased in bile duct-ligated livers of rats and mice and also in culture-activated rat hepatic stellate cells. S1P(2) expression, determined in S1P 2LacZ/+ mice, was highly increased in hepatic stellate cells of bile duct-ligated livers. Furthermore, the increase of Rho kinase activity in bile duct-ligated livers was observed as early as 7 days after the operation in wildtype mice, but was less in S1P 2-/- mice. CONCLUSION: S1P may play an important role in the pathophysiology of portal hypertension with Rho kinase activation by way of S1P(2) . The S1P(2) antagonist merits consideration as a novel therapeutic agent for portal hypertension.

Stimulation by glutamine and proline of HGF production in hepatic stellate cells.

Amino acids regulate cellular functions in a variety of cell types. Most notably, leucine stimulates protein production through the mammalian target of rapamycin (mTOR)-dependent signaling pathway. We investigated the effect of amino acids on hepatocyte growth factor (HGF) production. Treatment with glutamine and proline, as well as leucine, increased HGF levels in the culture medium of a rat hepatic stellate cell clone in a dose-dependent manner. Up-regulation of phosphorylation of 70 kDa ribosomal protein S6 kinase and eukaryotic initiation factor 4E-binding protein 1 was not apparent in the cells after treatment with glutamine or proline. When rats received injections of glutamine or proline, hepatic and circulating HGF levels increased and peaked around 12h after treatment. Glutamine and proline may have the potential to stimulate HGF production but the mechanism underlying this stimulation seems not to be through the mTOR-dependent signaling pathway.

Novel chemoimmunotherapeutic strategy for hepatocellular carcinoma based on a genome-wide association study.

Pharmacotherapeutic options are limited for hepatocellular carcinoma (HCC). Recently, we identified the anti-tumor ligand MHC class I polypeptide-related sequence A (MICA) gene as a susceptibility gene for hepatitis C virus-induced HCC in a genome-wide association study (GWAS). To prove the concept of HCC immunotherapy based on the results of a GWAS, in the present study, we searched for drugs that could restore MICA expression. A screen of the FDA-approved drug library identified the anti-cancer agent vorinostat as the strongest hit, suggesting histone deacetylase inhibitors (HDACis) as potent candidates. Indeed, the HDACi-induced expression of MICA specific to HCC cells enhanced natural killer (NK) cell-mediated cytotoxicity in co-culture, which was further reinforced by treatment with an inhibitor of MICA sheddase. Similarly augmented anti-tumor activity of NK cells via NK group 2D was observed in vivo. Metabolomics analysis revealed HDACi-mediated alterations in energy supply and stresses for MICA induction and HCC inhibition, providing a mechanism for the chemoimmunotherapeutic actions. These data are indicative of promising strategies for selective HCC innate immunotherapy.

Induction of p53-dependent p21 limits proliferative activity of rat hepatocytes in the presence of hepatocyte growth factor.

BACKGROUND: Hepatocyte growth factor (HGF), a potent mitogen for hepatocytes, enhances hepatocyte function without stimulating proliferation, depending on the physiological conditions. p53, a transcription factor, suppresses the cell proliferation by expressing p21(WAF1/CIP1) in various tissues. AIM: To investigate the mechanism through which the hepatocytes maintain mitotically quiescent even in the presence of HGF. METHODS: We studied the relationship between p53 and p21 expression and the effect of p53-p21 axis on hepatocyte proliferation in primary cultured rat hepatocytes stimulated by HGF. Hepatic p21 levels are determined serially after partial hepatectomy or sham operation in rats. RESULTS: DNA synthesis was markedly increased by HGF addition in rat hepatocytes cultured at low density but not at high density. Cellular p53 levels increased in the hepatocytes cultured at both the densities. p21 levels were increased and correlated with cellular p53 levels in hepatocytes cultured at high density but not at low density. When the activity of p53 was suppressed by a chemical inhibitor for p53, cellular p21 levels were reduced, and DNA synthesis was increased. Similarly, p21 antisense oligonucleotide increased the DNA synthesis. In rats after partial hepatectomy, transient elevation of hepatic p21 levels was observed. In contrast, in sham-operated rats, hepatic p21 levels were increased on sustained time scales. CONCLUSION: p53-related induction of p21 may suppress hepatocyte proliferation in the presence of HGF in the setting that mitogenic activity of HGF is not elicitable.

Prediction of hepatocellular carcinoma development by plasma ADAMTS13 in chronic hepatitis B and C.

BACKGROUND: Chronic liver injury evokes a wound healing response, promoting fibrosis and finally hepatocellular carcinoma (HCC), in which hepatic stellate cells play an important role. Although a blood marker of hepatic stellate cells is not known, those cells importantly contribute to the regulation of plasma a disintegrin-like and metalloproteinase with thrombospondin type-1 motifs 13 (ADAMTS13) activity, a defect of which causes thrombotic thrombocytopenic purpura. METHODS: Plasma ADAMTS13 was evaluated in chronic hepatitis B or C patients with or without HCC. RESULTS: Plasma ADAMTS13 activity significantly correlated with serum aspartate aminotransferase and alanine aminotransferase, liver stiffness value, and aspartate aminotransferase-to-platelet ratio index, irrespective of the presence of HCC, suggesting that it may reflect hepatocellular damage and subsequent wound healing and fibrosis as a result of hepatic stellate cell action. During the three-year follow-up period for patients without HCC, it developed in 10 among 81 patients. Plasma ADAMTS13 activity was significantly higher in patients with HCC development than in those without and was a significant risk for HCC development by univariate and multivariate analyses. Furthermore, during the one-year follow-up period for patients with HCC treated with radiofrequency ablation, HCC recurred in 55 among 107 patients. Plasma ADAMTS13 activity or antigen level was significantly higher in patients with HCC recurrence than in those without and was retained as a significant risk for HCC recurrence by multivariate analysis. CONCLUSIONS: Higher plasma ADAMTS13 activity and antigen level was a risk of HCC development in chronic liver disease. IMPACT: Plasma ADAMTS13 as a potential marker of hepatic stellate cells may be useful in the prediction of hepatocarcinogenesis.

Autotaxin as a novel serum marker of liver fibrosis.

BACKGROUND: The clinical significance of autotaxin (ATX), a key enzyme for the production of the bioactive lysophospholipid lysophosphatidic acid remains unknown. Serum ATX enzymatic activity reportedly increases in parallel with liver fibrosis and exhibits a gender difference. METHODS: Serum ATX antigen level, measured easier than the activity, was evaluated as a marker of liver fibrosis in 2 cohorts of chronic liver disease caused by hepatitis C virus. RESULTS: In the first cohort, serum ATX level correlated significantly with liver fibrosis stage and was the best parameter for prediction of cirrhosis with an area under the receiver operating characteristic curve (AUROC) of 0.756 in male and 0.760 in female, when compared with serum hyaluronic acid and aminotransferase-to-platelet ratio index, an established marker of liver fibrosis. In another cohort, serum ATX level correlated significantly with liver stiffness, a novel reliable marker of liver fibrosis, being the second-best parameter in male (AUROC, 0.799) and in female (AUROC, 0.876) for prediction of significant fibrosis, and the best parameter in male (AUROC, 0.863) and the third-best parameter in female (AUROC, 0.872) for prediction of cirrhosis, both of which were judged by liver stiffness. CONCLUSIONS: Serum ATX level may be a novel marker of liver fibrosis.

Expression of α-taxilin in hepatocellular carcinoma correlates with growth activity and malignant potential of the tumor.

The membrane traffic system has been recognized to be involved in carcinogenesis and tumor progression in several types of tumors. α-taxilin is a newly identified membrane traffic-related molecule, and its up-regulation has been reported in embryonic and malignant tissues of neural origin. In the present study, we analyzed the expression of α-taxilin in relation to clinicopathological features of hepatocellular carcinomas (HCC) and proliferative activity of the tumor determined by proliferating cell nuclear antigen labeling index (PCNA-LI). Twenty-nine surgically resected nodules of HCC (8 well-, 11 moderately-, and 10 poorly-differentiated) were studied. Fifteen cases showed 'strong staining', while 14 cases showed 'weak staining' for α-taxilin. A significantly higher expression of α-taxilin was observed in less-differentiated (p=0.005), and more invasive (p=0.016) HCCs. The 'strong staining' group showed significantly higher PCNA-LI than the 'weak staining' group (the medians of PCNA-LI were 59.4% vs. 14.4%, p<0.0001). We also evaluated the expression of α-taxilin in hepatoma cell lines (PLC/PRF/5, Hep G2 and HuH-6) in association with cell proliferation. The expression levels of α-taxilin protein were correlated with their growth rates. In conclusion, the expression of the α-taxilin protein was related with an increased proliferative activity and a less-differentiated histological grade of HCC. α-taxilin may be involved in cell proliferation of HCC, and its expression can be a marker of malignant potential of HCC.

Plasma concentration of bioactive lipid mediator sphingosine 1-phosphate is reduced in patients with chronic hepatitis C.

BACKGROUND: Bioactive lipid mediator S1P has been suggested to play pathophysiological roles in various fields of clinical science as a circulating paracrine mediator. We previously established a reliable method of measuring plasma S1P concentration, and reported that the one in healthy subjects has a gender difference and a correlation with red blood cell (RBC)-parameters, however, the reports of S1P measurements in the blood in patients with a specific disease have been scarce. Because our previous evidence suggests that S1P is involved in liver pathophysiology, we examined plasma S1P concentration in chronic hepatitis C patients. METHODS: S1P assay was performed using a high-performance liquid chromatography system. RESULTS: Plasma S1P concentrations were reduced in chronic hepatitis C patients compared with in healthy subjects with the same hemoglobin concentration, irrespective of gender. Among the blood parameters, serum hyaluronic acid concentration, a surrogate marker for liver fibrosis, was most closely and inversely correlated with plasma S1P concentration. Furthermore, plasma S1P concentration decreased throughout the progression of carbon tetrachloride-induced liver fibrosis in rats. CONCLUSIONS: Plasma S1P concentration was reduced in chronic hepatitis C patients, and liver fibrosis might be involved, at least in part, in the mechanism responsible for this reduction.

Sphingosine 1-phosphate regulates regeneration and fibrosis after liver injury via sphingosine 1-phosphate receptor 2.

Sphingosine 1-phosphate (S1P), a bioactive lipid mediator, stimulates proliferation and contractility in hepatic stellate cells, the principal matrix-producing cells in the liver, and inhibits proliferation via S1P receptor 2 (S1P(2)) in hepatocytes in rats in vitro. A potential role of S1P and S1P(2) in liver regeneration and fibrosis was examined in S1P(2)-deficient mice. Nuclear 5-bromo-2'-deoxy-uridine labeling, proliferating cell nuclear antigen (PCNA) staining in hepatocytes, and the ratio of liver weight to body weight were enhanced at 48 h in S1P(2)-deficient mice after a single carbon tetrachloride (CCl(4)) injection. After dimethylnitrosamine (DMN) administration with a lethal dose, PCNA staining in hepatocytes was enhanced at 48 h and survival rate was higher in S1P(2)-deficient mice. Serum aminotransferase level was unaltered in those mice compared with wild-type mice in both CCl(4)- and DMN-induced liver injury, suggesting that S1P(2) inactivation accelerated regeneration not as a response to enhanced liver damage. After chronic CCl(4) administration, fibrosis was less apparent, with reduced expression of smooth-muscle alpha-actin-positive cells in the livers of S1P(2)-deficient mice, suggesting that S1P(2) inactivation ameliorated CCl(4)-induced fibrosis due to the decreased accumulation of hepatic stellate cells. Thus, S1P plays a significant role in regeneration and fibrosis after liver injury via S1P(2).

Leucine stimulates HGF production by hepatic stellate cells through mTOR pathway.

Branched chain amino acids modulate various cellular functions in addition to providing substrates for the production of proteins. We examined the mechanism underlying the stimulation by leucine of hepatocyte growth factor (HGF) production by hepatic stellate cells. Both p70 S6 kinase activity and phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) were up-regulated rapidly after leucine treatment of a rat hepatic stellate cell clone. No such activation was observed following treatment with valine or isoleucine. Rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), suppressed leucine-induced activation of p70 S6 kinase and 4E-BP1 and negated the stimulatory effect of leucine on HGF production. An mTOR-dependent signaling pathway mediates the stimulatory effect of leucine on the production of HGF by hepatic stellate cells.