Peter L. M. Jansen

Increased cancer risk after liver transplantation: a population-based study

BACKGROUND/AIMS Development of de novo malignancies emerges as a serious long term complication after liver transplantation. METHODS We reviewed the medical records of 174 adult one-year survivors for de novo malignancies. The observed cancer rates were compared with the expected cancer rates in the Dutch population. RESULTS Twenty-one of the 174 patients developed 23 malignancies (12%). Skin and lip cancer accounted for 12 of the 23 malignancies (52%). Only one patient had a B-cell lymphoma. The cumulative risk for de novo malignancy was 6, 20, and 55% at 5, 10, and 15 years after transplantation, respectively. The overall relative risk (RR) as compared with the general population was 4.3 (95% confidence interval 2.4-7.1). Significantly increased RRs were observed for non-melanoma skin cancer (RR 70.0), non-skin solid cancer (RR 2.7), renal cell cancer (RR 30.0), and colon cancer (RR 12.5). Multivariate analysis showed that an age > 40 years and pretransplant use of immunosuppression were significant risk factors. CONCLUSIONS An increased risk of cancer exists after liver transplantation, for both for skin/lip cancer, and other solid tumors. Older age and the use of immunosuppression are risk factors.

Antiviral activity of telaprevir (VX‐950) and peginterferon alfa‐2a in patients with hepatitis C

Telaprevir (VX‐950), an inhibitor of the hepatitis C virus (HCV) NS3/4A protease, substantially decreased plasma HCV RNA levels in a prior clinical study. The present study evaluated viral kinetics and safety during dosing with telaprevir alone and in combination with peginterferon alfa‐2a for 14 days. Previously untreated patients with genotype 1 hepatitis C were randomized to receive placebo and peginterferon alfa‐2a (n = 4); telaprevir (n = 8); or telaprevir and peginterferon alfa‐2a (n = 8). Telaprevir was given as 750 mg oral doses every 8 hours; peginterferon alfa‐2a was given as weekly 180 μg subcutaneous injections. The median change in HCV RNA from baseline to day 15 was −1.09 log10 (range, −2.08 to −0.46) in the placebo and peginterferon alfa‐2a group; −3.99 log10 (range, −5.28 to −1.26) in the telaprevir group, and −5.49 log10 (range, −6.54 to −4.30) in the telaprevir and peginterferon alfa‐2a group. Day 15 HCV RNA levels were undetectable in 4 patients who received telaprevir and peginterferon alfa‐2a and in 1 patient who received telaprevir alone. No viral breakthrough occurred in patients who received telaprevir and peginterferon alfa‐2a. The majority of adverse events were mild. There were no serious adverse events or premature discontinuations. Twelve weeks after starting off‐study standard therapy, HCV RNA was undetectable in all 8 patients in the telaprevir and peginterferon alfa‐2a group, 5 patients in the telaprevir group, and 1 patient in the placebo and peginterferon alfa‐2a group. Conclusion: This study confirmed the substantial antiviral effects of telaprevir and showed an increased antiviral effect of telaprevir combined with peginterferon alfa‐2a. (HEPATOLOGY 2007;46:640–648.)

ATP‐ and glutathione‐dependent transport of chemotherapeutic drugs by the multidrug resistance protein MRP1

The present study was performed to investigate the ability of the multidrug resistance protein (MRP1) to transport different cationic substrates in comparison with MDR1‐P‐glycoprotein (MDR1). Transport studies were performed with isolated membrane vesicles from in vitro selected multidrug resistant cell lines overexpressing MDR1 (A2780AD) or MRP1 (GLC4/Adr) and a MRP1‐transfected cell line (S1(MRP)). As substrates we used 3H‐labelled derivatives of the hydrophilic monoquaternary cation N‐(4′,4′‐azo‐n‐pentyl)‐21‐deoxy‐ajmalinium (APDA), the basic drug vincristine and the more hydrophobic basic drug daunorubicin. All three are known MDR1‐substrates. MRP1 did not mediate transport of these substrates per se. In the presence of reduced glutathione (GSH), there was an ATP‐dependent uptake of vincristine and daunorubicin, but not of APDA, into GLC4/Adr and S1(MRP) membrane vesicles which could be inhibited by the MRP1‐inhibitor MK571. ATP‐ and GSH‐dependent transport of daunorubicin and vincristine into GLC4/Adr membrane vesicles was inhibited by the MRP1‐specific monoclonal antibody QCRL‐3. MRP1‐mediated daunorubicin transport rates were dependent on the concentration of GSH and were maximal at concentrations 10 mM. The apparent KM value for GSH was 2.7 mM. Transport of daunorubicin in the presence of 10 mM GSH was inhibited by MK571 with an IC50 of 0.4 μM. In conclusion, these results demonstrate that MRP1 transports vincristine and daunorubicin in an ATP‐ and GSH‐dependent manner. APDA is not a substrate for MRP1.

Bile acid receptors as targets for drug development

The intracellular nuclear receptor farnesoid X receptor and the transmembrane G protein-coupled receptor TGR5 respond to bile acids by activating transcriptional networks and/or signalling cascades. These cascades affect the expression of a great number of target genes relevant for bile acid, cholesterol, lipid and carbohydrate metabolism, as well as genes involved in inflammation, fibrosis and carcinogenesis. Pregnane X receptor, vitamin D receptor and constitutive androstane receptor are additional nuclear receptors that respond to bile acids, albeit to a more restricted set of species of bile acids. Recognition of dedicated bile acid receptors prompted the development of semi-synthetic bile acid analogues and nonsteroidal compounds that target these receptors. These agents hold promise to become a new class of drugs for the treatment of chronic liver disease, hepatocellular cancer and extrahepatic inflammatory and metabolic diseases. This Review discusses the relevant bile acid receptors, the new drugs that target bile acid signalling and their possible applications.

High expression of the bile salt‐homeostatic hormone fibroblast growth factor 19 in the liver of patients with extrahepatic cholestasis

Fibroblast growth factor 19 (FGF19) is an endocrine factor produced by the small intestine in response to uptake of luminal bile salts. In the liver, FGF19 binds to FGF receptor‐4, resulting in down‐regulation of cytochrome P (CYP) 7A1 and reduced bile salt synthesis. Down‐regulation of CYP7A1 under cholestatic conditions has been attributed to bile salt–mediated induction of the transcriptional repressor short heterodimer partner (SHP), because the interrupted enterohepatic cycle of bile salts is thought to abrogate intestinal FGF19 production and thus result in lowering of plasma FGF19 levels. Unexpectedly, we observed marked elevation of plasma FGF19 in patients with extrahepatic cholestasis caused by a pancreatic tumor (2.3 ± 2.3 in cholestatic versus 0.40 ± 0.25 ng/mL and 0.29 ± 0.12 ng/mL in postcholestatic patients who received preoperative drainage by biliary stenting, P = 0.004, and noncholestatic control patients, P = 0.04, respectively). Although FGF19 messenger RNA (mRNA) is virtually absent in normal liver, FGF19 mRNA was strongly increased (31‐fold to 374‐fold, P < 0.001) in the liver of cholestatic patients in comparison with drained and control patients. In the absence of changes in SHP mRNA, CYP7A1 mRNA was strongly reduced (7.2‐fold to 24‐fold, P < 0.005) in the liver of cholestatic patients in comparison with drained and control patients, indicating an alternative regulatory pathway. Alterations in transcripts encoding hepatobiliary transporters [adenosine triphosphate–binding cassette, subfamily C, member 3 (ABCC3)/multidrug resistance protein 3 (MRP3), organic solute transporter α/β (OSTα/β), organic anion‐transporting polypeptide (OATP1B1)] further suggest that bile salts are secreted via a nonbiliary route in patients with extrahepatic cholestasis. Conclusion: The liver expresses FGF19 under conditions of extrahepatic cholestasis. This is accompanied by a number of adaptations aimed at protecting the liver against bile salt toxicity. FGF19 signaling may be involved in some of these adaptations. (HEPATOLOGY 2009.)

The biliary HCO3− umbrella: A unifying hypothesis on pathogenetic and therapeutic aspects of fibrosing cholangiopathies

This review focuses on the hypothesis that biliary HCO  3− secretion in humans serves to maintain an alkaline pH near the apical surface of hepatocytes and cholangiocytes to prevent the uncontrolled membrane permeation of protonated glycine‐conjugated bile acids. Functional impairment of this biliary HCO  3− umbrella or its regulation may lead to enhanced vulnerability of cholangiocytes and periportal hepatocytes toward the attack of apolar hydrophobic bile acids. An intact interplay of hepatocellular and cholangiocellular adenosine triphosphate (ATP) secretion, ATP/P2Y‐ and bile salt/TGR5‐mediated Cl−/ HCO  3− exchange and HCO  3− secretion, and alkaline phosphatase–mediated ATP breakdown may guarantee a stable biliary HCO  3− umbrella under physiological conditions. Genetic and acquired functional defects leading to destabilization of the biliary HCO  3− umbrella may contribute to development and progression of various forms of fibrosing/sclerosing cholangitis. (HEPATOLOGY 2010)

Hepatobiliary transport of glutathione and glutathione conjugate in rats with hereditary hyperbilirubinemia.

TR- mutant rats have an autosomal recessive mutation that is expressed as a severely impaired hepatobiliary secretion of organic anions like bilirubin-(di)glucuronide and dibromosulphthalein (DBSP). In this paper, the hepatobiliary transport of glutathione and a glutathione conjugate was studied in normal Wistar rats and TR- rats. It was shown that glutathione is virtually absent from the bile of TR- rats. In the isolated, perfused liver the secretion of glutathione and the glutathione conjugate, dinitrophenyl-glutathione (GS-DNP), from hepatocyte to bile is severely impaired, whereas the sinusoidal secretion from liver to blood is not affected. The secretion of GS-DNP was also studied in isolated hepatocytes. The secretion of GS-DNP from cells isolated from TR- rat liver was significantly slower than from normal hepatocytes. Efflux of GS-DNP was a saturable process with respect to intracellular GS-DNP concentration: Vmax and Km for efflux from TR- cells was 498 nmol/min.g dry wt and 3.3 mM, respectively, as compared with 1514 nmol/min.g dry wt and 0.92 mM in normal hepatocytes. These results suggest that the canalicular transport system for glutathione and glutathione conjugates is severely impaired in TR- rats, whereas sinusoidal efflux is unaffected. Because the defect also comes to expression in isolated hepatocytes, efflux of GS-DNP from normal hepatocytes must predominantly be mediated by the canalicular transport mechanism, which is deficient in TR- rats.

Cytokine regulation of pro- and anti-apoptotic genes in rat hepatocytes: NF-κB-regulated inhibitor of apoptosis protein 2 (cIAP2) prevents apoptosis

BACKGROUND/AIMS In acute liver failure, hepatocytes are exposed to various cytokines that activate both cell survival and apoptotic pathways. NF-kappaB is a central transcription factor in these responses. Recent studies indicate that blocking NF-kappaB causes apoptosis, indicating the existence of NF-kappaB-regulated anti-apoptotic genes. In the present study the relationship between NF-kappaB activation and apoptosis has been investigated in hepatocytes. METHODS Primary rat hepatocytes were exposed to a cytokine mixture of tumor necrosis factor alpha, interleukin-1beta, interferon-gamma and lipopolysaccharide. Modulation of signalling pathways was performed by using dominant negative adenoviral constructs. Apoptosis and NF-kappaB activation were determined by caspase-3 activity, Hoechst staining and electrophoretic mobility shift assay, respectively. Furthermore, expression and regulation of apoptosis-related genes were investigated. RESULTS (1) Inhibition of NF-kappaB activation results in apoptosis. (2) Inhibitor of apoptosis protein (IAP) family members, inhibitor of apoptosis protein1 (cIAP1), and X-chromosome-linked IAP, are expressed in rat hepatocytes. cIAP2 is induced by cytokines in an NF-kappaB-dependent manner and overexpression of cIAP2 inhibits apoptosis. (3) The anti-apoptotic Bcl-2 family member A1/Bfl-1 and the pro-apoptotic members Bak and Bid are induced by cytokines and NF-kappaB-dependent. (4) Nitric oxide inhibits caspase-3 activity in hepatocytes. CONCLUSIONS In inflammatory conditions, hepatocyte survival is dependent on NF-kappaB activation and cIAP2 contributes significantly to this protection.

Diagnostic and therapeutic potential of miRNA signatures in patients with hepatocellular carcinoma

MicroRNAs (miRNAs) are evolutionary conserved small non-coding RNAs that regulate gene expression by mediating post-transcriptional silencing of target genes. Since miRNAs are involved in fine-tuning of physiological responses, they have become of interest for diagnosis and therapy of a number of diseases. Moreover, the role of dysregulated miRNAs in maintaining the malignant phenotype has profound implications for cancer therapy. We will review the best defined cellular miRNAs and changes in their expression profile in hepatocellular carcinoma (HCC). Cellular miRNAs can also be released into the circulation, and these miRNAs are detected in most body fluids. Circulating miRNAs are associated with HCC and are possible biomarkers. Finally, by affecting several clinically relevant targets, artificially increasing or decreasing the expression level of a given miRNA offers fascinating therapeutic perspectives. We will therefore highlight recent developments in miRNA-based gene therapy with a focus on their therapeutic potential for HCC.

Association of human liver bilirubin UDP-glucuronyltransferase activity with a polymorphism in the promoter region of the UGT1A1 gene

BACKGROUND/AIMS Gilberts syndrome is a benign form of a deficiency in bilirubin glucuronidation. It is associated with a homozygous polymorphism, A(TA)7TAA instead of A(TA)6TAA, in the TATA-box of the promoter region of the bilirubin UDP-glucuronyltransferase gene. In this study the correlation between this promoter region polymorphism and in vitro human liver bilirubin UDP-glucuronyltransferase enzyme activity was investigated. METHODS Liver samples from organ transplant donors n=39) and two known Gilberts syndrome patients were used for measuring bilirubin UDP-glucuronyltransferase enzyme activity and for isolation of DNA followed by detection of the promoter region polymorphism by polymerase chain reaction. Genotypes were assigned as follows; 6/6: homozygous for the A(TA)6TAA-allele, 7/7: homozygous for the A(TA)7TAA-allele, and 6/7: heterozygous with one of each alleles. RESULTS Seventeen out of 39 subjects (44%) had the homozygous 6/6 genotype, 18 subjects (46%) had the heterozygous 6/7 genotype, whereas four individuals (10%) and the two individuals with Gilberts syndrome had the 7/7 genotype correlated with Gilberts syndrome. This resulted in an allele frequency of 0.33 for the A(TA)7TAA-allele. The median bilirubin UDP-glucuronyltransferase enzyme activity of the 17 subjects with the 6/6 genotype (1565 nmol/g liver/h) was significantly higher than the activity of the 18 subjects with the 6/7 genotype (985 nmol/g liver/h; p<0.05) and the six individuals with the 7/7 genotype (749 nmol/g liver/h; p<0.005). No significant differences in enzyme activity were found between the 6/7 and the 7/7 genotype groups. CONCLUSIONS The results indicate a close association between the promoter region genotype and the expression of hepatic bilirubin UDP-glucuronyltransferase enzyme activity. Subjects who have a 7/7 genotype have the lowest enzyme activity, whereas subjects with the heterozygous 6/7 genotype have an intermediate enzyme activity.