Thomas Pleli

Serum microRNA-1 and microRNA-122 are prognostic markers in patients with hepatocellular carcinoma

BACKGROUND The identification of new biomarkers to predict the aggressiveness of hepatocellular carcinoma (HCC) and supplement the current set of prognosis and treatment algorithms is an important clinical need. Extracellular microRNAs (miRNAs) circulating in the blood are a new class of highly promising disease markers. AIM Here we investigated the prognostic potential of miR-1 and miR-122 in sera from patients with HCC. METHODS RNA was extracted from 195 sera of HCC patients and 54 patients with liver cirrhosis, obtained at the time of study enrolment. miR-1 and miR-122 levels were correlated with overall survival (OS), Cancer of the Liver Italian Program (CLIP) score, Barcelona Clinic Liver Cancer stage, clinical chemistry parameters and tumor specific treatment. RESULTS Patients with higher miR-1 and miR-122 serum levels showed longer OS than individuals with lower miR-1 and miR-122 serum concentrations (hazard ratio [HR] 0.440, 95% confidence interval [CI] 0.233-0.831, P=0.011 for miR-1 and HR 0.493, 95% CI 0.254-0.956, P=0.036 for miR-122, respectively). Serum miR-1 and miR-122 concentrations did not differ significantly between patients with HCC and liver cirrhosis. An age-, sex-, tumor stage and treatment-adjusted multivariate Cox regression analysis revealed that miR-1 serum levels (HR 0.451, 95% CI 0.228-0.856, P=0.015) were independently associated with OS, whereas serum miR-122 was not. miR-1 serum levels showed no relevant correlation with clinical chemistry liver parameters, whereas serum miR-122 correlated with clinical chemistry parameters of hepatic necroinflammation, liver function and synthetic capacity. CONCLUSION Our data indicate that serum miR-1 is a new independent parameter of OS in HCC patients and may therefore improve the predictive value of classical HCC staging scores.

Serum microRNA-122 levels in different groups of patients with chronic hepatitis B virus infection

Summary.  miR‐122 is a liver‐specific microRNA, which also circulates in the blood. The levels of miR‐122 in serum and plasma correlate with hepatic necroinflammation in patients with hepatitis B virus (HBV) infection. Here, we investigated whether miR‐122 levels correlate with surrogate markers for viral replication and translation. Furthermore, we examined whether miR‐122 levels differ in the different groups of HBV‐infected patients and whether miR‐122 levels may be useful to identify patients with higher or lower risk for liver disease progression. Therefore, RNA was extracted from sera of therapy‐naïve patients with HBV infection (n = 89) and from healthy volunteers (n = 19). The concentration of miR‐122 was assessed by quantitative real‐time reverse‐transcription PCR. HBs antigen and HBV DNA levels were quantified as surrogate parameters for HBV replication and translation. Liver biopsies were examined according to the histological activity index and the degree of fibrosis was assessed. We found that the miR‐122 serum concentration correlated with the level of ALT, HBV DNA and HBs antigen (r = 0.259, P < 0.05; r = 0.225, P < 0.05; r = 0.508, P < 0.001, respectively). The miR‐122 serum levels discriminated the different patient groups infected with HBV from healthy subjects (P < 0.001), and inactive carrier patients with high (>3500 IU/mL) or low (<3500 IU/mL) levels of HBs antigen could be differentiated by the miR‐122 serum concentration (P < 0.05). As serum miR‐122 levels strongly correlated with HBs antigen, it might be an indicator for viral translation. Furthermore, serum miR‐122 levels discriminated HBV carrier patients with high or low risk for disease progression and may, thus, be an additional marker for risk stratification.

Differential Stability of Cell-Free Circulating microRNAs: Implications for Their Utilization as Biomarkers

Background MicroRNAs circulating in the blood, stabilized by complexation with proteins and/or additionally by encapsulation in lipid vesicles, are currently being evaluated as biomarkers. The consequences of their differential association with lipids/vesicles for their stability and use as biomarkers are largely unexplored and are subject of the present study. Methods The levels of a set of selected microRNAs were determined by quantitative reverse-transcription PCR after extraction from sera or vesicle- and non-vesicle fractions prepared from sera. The stability of these microRNAs after incubation with RNase A or RNase inhibitor, an inhibitor of RNase A family enzymes was studied. Results The levels of microRNA-1 and microRNA-122, but not those of microRNA-16, microRNA-21 and microRNA-142-3p, declined significantly during a 5-h incubation of the sera. RNase inhibitor prevented the loss of microRNAs in serum as well as the degradation of microRNA-122, a microRNA not expressed in blood cells, in whole blood. Stabilization of microRNA-122 was also achieved by hemolysis. Prolonged incubation of the sera led to enrichment of vesicle-associated relative to non-vesicle-associated microRNAs. Vesicle-associated microRNAs were more resistant to RNase A treatment than the respective microRNAs not associated with vesicles. Conclusions Serum microRNAs showed differential stability upon prolonged incubation. RNase inhibitor might be useful to robustly preserve the pattern of cell-free circulating microRNAs. In the case of microRNAs not expressed in blood cells this can also be achieved by hemolysis. Vesicle-associated microRNAs appeared to be more stable than those not associated with vesicles, which might be useful to disclose additional biomarker properties of miRNAs.

Improving Drug Penetrability with iRGD Leverages the Therapeutic Response to Sorafenib and Doxorubicin in Hepatocellular Carcinoma.

iRGD is a derivative of the integrin-binding peptide RGD, which selectively increases the penetrability of tumor tissue to various coadministered substances in several preclinical models. In this study, we investigated the ability of iRGD to improve the delivery of sorafenib and doxorubicin therapy in hepatocellular carcinoma (HCC) using established mouse models of the disease. A contrast-enhanced MRI method was developed in parallel to assess the in vivo effects of iRGD in this setting. We found that iRGD improved the delivery of marker substances to the tumors of HCC-bearing mice about three-fold without a parallel increase in normal tissues. Control peptides lacking the critical CendR motif had no effect. Similarly, iRGD also selectively increased the signal intensity from tumors in Gd-DTPA-enhanced MRI. In terms of antitumor efficacy, iRGD coadministration significantly augmented the individual inhibitory effects of sorafenib and doxorubicin without increasing systemic toxicity. Overall, our results offered a preclinical proof of concept for the use of iRGD coadministration as a strategy to widen the therapeutic window for HCC chemotherapy, as monitored by Gd-DTPA-enhanced MRI as a noninvasive, clinically applicable method to identify iRGD-reactive tumors.

Serum Autotaxin Is a Parameter for the Severity of Liver Cirrhosis and Overall Survival in Patients with Liver Cirrhosis – A Prospective Cohort Study

Background Autotaxin (ATX) and its product lysophosphatidic acid (LPA) are considered to be involved in the development of liver fibrosis and elevated levels of serum ATX have been found in patients with hepatitis C virus associated liver fibrosis. However, the clinical role of systemic ATX in the stages of liver cirrhosis was unknown. Here we investigated the relation of ATX serum levels and severity of cirrhosis as well as prognosis of cirrhotic patients. Methods Patients with liver cirrhosis were prospectively enrolled and followed until death, liver transplantation or last contact. Blood samples drawn at the day of inclusion in the study were assessed for ATX content by an enzyme-linked immunosorbent assay. ATX levels were correlated with the stage as well as complications of cirrhosis. The prognostic value of ATX was investigated by uni- and multivariate Cox regression analyses. LPA concentration was determined by liquid chromatography-tandem mass spectrometry. Results 270 patients were enrolled. Subjects with liver cirrhosis showed elevated serum levels of ATX as compared to healthy subjects (0.814±0.42 mg/l vs. 0.258±0.40 mg/l, P<0.001). Serum ATX levels correlated with the Child-Pugh stage and the MELD (model of end stage liver disease) score and LPA levels (r = 0.493, P = 0.027). Patients with hepatic encephalopathy (P = 0.006), esophageal varices (P = 0.002) and portal hypertensive gastropathy (P = 0.008) had higher ATX levels than patients without these complications. Low ATX levels were a parameter independently associated with longer overall survival (hazard ratio 0.575, 95% confidence interval 0.365–0.905, P = 0.017). Conclusion Serum ATX is an indicator for the severity of liver disease and the prognosis of cirrhotic patients.

Biodegradable human serum albumin nanoparticles as contrast agents for the detection of hepatocellular carcinoma by magnetic resonance imaging.

Tumor visualization by magnetic resonance imaging (MRI) and nanoparticle-based contrast agents may improve the imaging of solid tumors such as hepatocellular carcinoma (HCC). In particular, human serum albumin (HSA) nanoparticles appear to be a suitable carrier due to their safety and feasibility of functionalization. In the present study HSA nanoparticles were conjugated with gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) using carbodiimide chemistry. The nanoparticles had a uniform spherical shape and a diameter of 235±19nm. For better optical visualization in vitro and in vivo, the HSA-Gd nanoparticles were additionally labeled with rhodamine 123. As shown by confocal microscopy and flow cytometry analysis, the fluorescent nanoparticles were readily taken up by Huh-7 hepatocellular carcinoma cells. After 24h incubation in blood serum, less than 5% of the Gd(III) was released from the particles, which suggests that this nanoparticulate system may be stable in vivo and, therefore, may serve as potentially safe T1 MRI contrast agent for MRI of hepatocellular carcinoma.

Detection of hepatocellular carcinoma in transgenic mice by Gd-DTPA- and rhodamine 123-conjugated human serum albumin nanoparticles in T1 magnetic resonance imaging

Nanoparticle (NP)-based contrast agents that enable high resolution anatomic T1-weighted magnetic resonance imaging (MRI) offer the prospect of improving differential diagnosis of liver tumors such as hepatocellular carcinoma (HCC). In the present study, we investigated the possibility of employing novel non-toxic human serum albumin nanoparticles conjugated with Gd-DTPA and rhodamine 123 (Gd-Rho-HSA-NPs) for the detection of HCC by T1-weighted MRI. In addition, the influence of surface coating of the NPs with poloxamine 908, which alters the absorptive behavior of NPs and changes their distribution between the liver and tumor was examined. MRI of transgenic mice with endogenously formed HCCs following intravenous injection of Gd-Rho-HSA-NPs revealed a strong negative contrast of the tumors. Contrasting of the HCCs by NP-enhanced MRI required less Gd as compared to gadolinium-ethoxybenzyl-diethylenetriaminepentaacetic acid-enhanced MRI, which currently provides the most sensitive detection of HCC in patients. Immunohistochemical analyses revealed that the Gd-Rho-HSA-NPs were localized to macrophages, which were - similar to HCC in patients - fewer in number in HCC as compared to the liver tissue, which is in agreement with the negative contrasting of HCC in Gd-Rho-HSA-NP-enhanced MRI. Poloxamine-coated NPs showed lower accumulation in the tumor macrophages and caused a longer lasting enhancement of the MRI signal. These data indicate that Gd-Rho-HSA-NPs enable sensitive detection of HCC by T1-weighted MRI in mice with endogenous HCC through their uptake by macrophages. Poloxamine coating of the NPs delayed the tumor localization of the NPs.

Soluble CD163 is an indicator of liver inflammation and fibrosis in patients chronically infected with the hepatitis B virus

Soluble CD163 (sCD163), a marker for macrophage activation, was found to be associated with the severity of liver cirrhosis. The aim of the current study was to investigate whether serum sCD163 levels correlate with liver inflammation and fibrosis in patients with chronic hepatitis B virus (HBV) infection. In a retrospective cohort study, serum sCD163 levels were assessed by ELISA together with clinical and laboratory data in 186 patients with chronic HBV infection and 15 healthy controls. The relation between parameters for liver fibrosis and necroinflammation and sCD163 levels was analysed. Additionally, sCD163 was quantified in a subset of follow‐up serum samples after initiation of antiviral treatment. sCD163 levels differed among phases of chronic HBV infection (P < 0.0001), and sCD163 concentrations were associated with inflammatory activity and fibrosis in the liver. sCD163 levels ≥1961 ng/l had a high specificity in the identification of subjects with substantial fibrosis (F ≥ 2). sCD163 concentrations decreased significantly after initiation of antiviral treatment. The correlation of sCD163 levels with necroinflammation and fibrosis and the sCD163 decline under treatment indicates that macrophage activation plays a role in HBV‐related liver pathogenesis.

Application of IL-36 receptor antagonist weakens CCL20 expression and impairs recovery in the late phase of murine acetaminophen-induced liver injury

Overdosing of the analgesic acetaminophen (APAP, paracetamol) is a major cause of acute liver injury. Whereas toxicity is initiated by hepatocyte necrosis, course of disease is regulated by mechanisms of innate immunity having the potential to serve in complex manner pathogenic or pro-regenerative functions. Interleukin (IL)-36γ has been identified as novel IL-1-like cytokine produced by and targeting epithelial (-like) tissues. Herein, we investigated IL-36γ in acute liver disease focusing on murine APAP-induced hepatotoxicity. Enhanced expression of hepatic IL-36γ and its prime downstream chemokine target CCL20 was detected upon liver injury. CCL20 expression coincided with the later regeneration phase of intoxication. Primary murine hepatocytes and human Huh7 hepatocellular carcinoma cells indeed displayed enhanced IL-36γ expression when exposed to inflammatory cytokines. Administration of IL-36 receptor antagonist (IL-36Ra) decreased hepatic CCL20 in APAP-treated mice. Unexpectedly, IL-36Ra likewise increased late phase hepatic injury as detected by augmented serum alanine aminotransferase activity and histological necrosis which suggests disturbed tissue recovery upon IL-36 blockage. Finally, we demonstrate induction of IL-36γ in inflamed livers of endotoxemic mice. Observations presented introduce IL-36γ as novel parameter in acute liver injury which may contribute to the decision between unleashed tissue damage and initiation of liver regeneration during late APAP toxicity.

The cytoskeletal inhibitors latrunculin A and blebbistatin exert antitumorigenic properties in human hepatocellular carcinoma cells by interfering with intracellular HuR trafficking

The impact of the RNA-binding protein HuR for the post-transcriptional deregulation of tumor-relevant genes is well established. Despite of elevations in HuR expression levels, an increase in cytoplasmic HuR abundance in many cases correlates with a high grade of malignancy. Here, we demonstrated that administration of the actin-depolymerizing macrolide latrunculin A, or blebbistatin, an inhibitor of myosin II ATPase activity, caused a dose- and time-dependent reduction in the high cytoplasmic HuR content of HepG2 and Huh7 hepatocellular carcinoma (HCC) cells. Subcellular fractionation revealed that in addition, both inhibitors strongly attenuated cytoskeletal and membrane-bound HuR abundance and conversely increased the HuR amount in nuclear cell fractions. Concomitant with changes in intracellular HuR localization, both cytoskeletal inhibitors markedly decreased the half-lives of cyclooxygenase-2 (COX-2), cyclin A and cyclin D1 encoding mRNAs resulting in a significant reduction in their expression levels in HepG2 cells. Importantly, a similar reduction in the expression of these HuR targets was achieved by a RNA interference (RNAi)-mediated knockdown of either HuR or nonmuscle myoin IIA. Using polysomal fractionation, we further demonstrate that the decrease in cytoplasmic HuR by latrunculin A or blebbistatin is accompanied by a marked change in the allocation of HuR and its mRNA cargo from polysomes to ribonucleoprotein (RNP) particles. Functionally, the basal migration and prostaglandin E2 synthesis are similarly impaired in inhibitor-treated and stable HuR-knockdown HepG2 cells. Our data demonstrate that interfering with the actomyosin-dependent HuR trafficking may comprise a valid therapeutic option for antagonizing pathologic posttranscriptional gene expression by HuR and furthermore emphasize the potential benefit of HuR inhibitory strategies for treatment of HCC.