Henning Schulze-Bergkamen

Cyclooxygenase-2 inhibition induces apoptosis signaling via death receptors and mitochondria in hepatocellular carcinoma.

Inhibition of cyclooxygenase (COX)-2 elicits chemopreventive and therapeutic effects in solid tumors that are coupled with the induction of apoptosis in tumor cells. We investigated the mechanisms by which COX-2 inhibition induces apoptosis in hepatocellular carcinoma (HCC) cells. COX-2 inhibition triggered expression of the CD95, tumor necrosis factor (TNF)-R, and TNF-related apoptosis-inducing ligand (TRAIL)-R1 and TRAIL-R2 death receptors. Addition of the respective specific ligands further increased apoptosis, indicating that COX-2 inhibition induced the expression of functional death receptors. Overexpression of a dominant-negative Fas-associated death domain mutant reduced COX-2 inhibitor-mediated apoptosis. Furthermore, our findings showed a link between COX-2 inhibition and the mitochondrial apoptosis pathway. COX-2 inhibition led to a rapid down-regulation of myeloid cell leukemia-1 (Mcl-1), an antiapoptotic member of the Bcl-2 family, followed by translocation of Bax to mitochondria and cytochrome c release from mitochondria. Consequently, overexpression of Mcl-1 led to inhibition of COX-2 inhibitor-mediated apoptosis. Furthermore, blocking endogenous Mcl-1 function using a small-interfering RNA approach enhanced COX-2 inhibitor-mediated apoptosis. It is of clinical importance that celecoxib acted synergistically with chemotherapeutic drugs in the induction of apoptosis in HCC cells. The clinical relevance of these results is further substantiated by the finding that COX-2 inhibitors did not sensitize primary human hepatocytes toward chemotherapy-induced apoptosis. In conclusion, COX-2 inhibition engages different apoptosis pathways in HCC cells stimulating death receptor signaling, activation of caspases, and apoptosis originating from mitochondria.

Knockout of myeloid cell leukemia‐1 induces liver damage and increases apoptosis susceptibility of murine hepatocytes

Myeloid cell leukemia‐1 (Mcl‐1) is an antiapoptotic member of the Bcl‐2 protein family. It interacts with proapoptotic Bcl‐2 family members, thereby inhibiting mitochondrial activation and induction of apoptosis. Mcl‐1 is essential for embryonal development and the maintenance of B cells, T cells, and hematopoietic stem cells. We have recently shown that induction of Mcl‐1 by growth factors rescues primary human hepatocytes from CD95‐mediated apoptosis. This prompted us to further analyze the relevance of Mcl‐1 for hepatocellular homeostasis. Therefore, we generated a hepatocyte‐specific Mcl‐1 knockout mouse (Mcl‐1flox/flox‐AlbCre). Deletion of Mcl‐1 in hepatocytes results in liver cell damage caused by spontaneous induction of apoptosis. Livers of Mcl‐1flox/flox‐AlbCre mice are smaller compared to control littermates, due to higher apoptosis rates. As a compensatory mechanism, proliferation of hepatocytes is enhanced in the absence of Mcl‐1. Importantly, hepatic pericellular fibrosis occurs in Mcl‐1 negative livers in response to chronic liver damage. Furthermore, Mcl‐1flox/flox‐AlbCre mice are more susceptible to hepatocellular damage induced by agonistic anti‐CD95 antibodies or concanavalin A. Conclusion: The present study provides in vivo evidence that Mcl‐1 is a crucial antiapoptotic factor for the liver, contributing to hepatocellular homeostasis and protecting hepatocytes from apoptosis induction. (HEPATOLOGY 2009.)

Suppression of Mcl-1 via RNA interference sensitizes human hepatocellular carcinoma cells towards apoptosis induction

BackgroundHepatocelluar carcinoma (HCC) is one of the most common cancers worldwide and a major cause of cancer-related mortality. HCC is highly resistant to currently available chemotherapeutic drugs. Defects in apoptosis signaling contribute to this resistance. Myeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic member of the Bcl-2 protein family which interferes with mitochondrial activation. In a previous study we have shown that Mcl-1 is highly expressed in tissues of human HCC. In this study, we manipulated expression of the Mcl-1 protein in HCC cells by RNA interference and analyzed its impact on apoptosis sensitivity of HCC cells in vitro.MethodsRNA interference was performed by transfecting siRNA to specifically knock down Mcl-1 expression in HCC cells. Mcl-1 expression was measured by quantitative real-time PCR and Western blot. Induction of apoptosis and caspase activity after treatment with chemotherapeutic drugs and different targeted therapies were measured by flow cytometry and fluorometric analysis, respectively.ResultsHere we demonstrate that Mcl-1 expressing HCC cell lines show low sensitivity towards treatment with a panel of chemotherapeutic drugs. However, treatment with the anthracycline derivative epirubicin resulted in comparatively high apoptosis rates in HCC cells. Inhibition of the kinase PI3K significantly increased apoptosis induction by chemotherapy. RNA interference efficiently downregulated Mcl-1 expression in HCC cells. Mcl-1 downregulation sensitized HCC cells to different chemotherapeutic agents. Sensitization was accompanied by profound activation of caspase-3 and -9. In addition, Mcl-1 downregulation also increased apoptosis rates after treatment with PI3K inhibitors and, to a lower extent, after treatment with mTOR, Raf I and VEGF/PDGF kinase inhibitors. TRAIL-induced apoptosis did not markedly respond to Mcl-1 knockdown. Additionally, knockdown of Mcl-1 efficiently enhanced apoptosis sensitivity towards combined treatment modalities: Mcl-1 knockdown significantly augmented apoptosis sensitivity of HCC cells towards chemotherapy combined with PI3K inhibition.ConclusionOur data suggest that specific downregulation of Mcl-1 by RNA interference is a promising approach to sensitize HCC cells towards chemotherapy and molecularly targeted therapies.

Primary human hepatocytes – a valuable tool for investigation of apoptosis and hepatitis B virus infection

BACKGROUND/AIMS Apoptosis is a key event in the pathophysiology of many liver diseases. Primary human hepatocytes (PHH) provide a useful model to study physiological and pathophysiological processes in the liver. Our aim was to optimize PHH cultures to allow studies on induction of apoptosis and of hepatitis B virus (HBV) infection. METHODS PHH were isolated from human liver tissue by two-step collagenase perfusion. PHH and hepatoma cells were treated with different apoptosis-inducing agents in parallel. PHH cultures were infected with wild type HBV and transduced with HBV genomes using adenoviral vectors. RESULTS PHH were successfully isolated from 40 different tissue samples with high viability and purity. Perfusion time and seeding density turned out to be critical parameters for optimal cell yield and culture conditions, respectively. Serum addition to the medium reduced viability of PHH. PHH allowed reproducible studies of CD95-dependent and -independent apoptosis. Sensitivity towards CD95-mediated apoptosis was markedly higher than in hepatoma cells. PHH could efficiently be infected with HBV, but infection did neither induce apoptosis nor prevent CD95-induced cell death. CONCLUSIONS Our data show that PHH provide an excellent tool for the investigation of apoptosis induced by agents like death receptor-ligands and hepatotropic viruses.

Hepatocyte‐specific deletion of the antiapoptotic protein myeloid cell leukemia‐1 triggers proliferation and hepatocarcinogenesis in mice

Regulation of hepatocellular apoptosis is crucial for liver homeostasis. Increased sensitivity of hepatocytes toward apoptosis results in chronic liver injury, whereas apoptosis resistance is linked to hepatocarcinogenesis and nonresponsiveness to therapy‐induced cell death. Recently, we have demonstrated an essential role of the antiapoptotic Bcl‐2 family member Myeloid cell leukemia‐1 (Mcl‐1) in hepatocyte survival. In mice lacking Mcl‐1 specifically in hepatocytes (Mcl‐1Δhep), spontaneous apoptosis caused severe liver damage. Here, we demonstrate that chronically increased apoptosis of hepatocytes coincides with strong hepatocyte proliferation resulting in hepatocellular carcinoma (HCC). Liver cell tumor formation was observed in >50% of Mcl‐1Δhep mice already by the age of 8 months, whereas 12‐month‐old wild‐type (wt) and heterozygous Mcl‐1flox/wt mice lacked tumors. Tumors revealed a heterogenous spectrum ranging from small dysplastic nodules to HCC. The neoplastic nature of the tumors was confirmed by histology, expression of the HCC marker glutamine synthetase and chromosomal aberrations. Liver carcinogenesis in Mcl‐1Δhep mice was paralleled by markedly increased levels of Survivin, an important regulator of mitosis which is selectively overexpressed in common human cancers. Conclusion: This study provides in vivo evidence that increased apoptosis of hepatocytes not only impairs liver homeostasis but is also accompanied by hepatocyte proliferation and hepatocarcinogenesis. Our findings might have implications for understanding apoptosis‐related human liver diseases. (HEPATOLOGY 2010.)

IFN-α–Induced Apoptosis in Hepatocellular Carcinoma Involves Promyelocytic Leukemia Protein and TRAIL Independently of p53

IFNs are pleiotropic cytokines that have been shown to be important regulators of cell growth. IFN-alpha has recently been recognized to harbor therapeutic potential in prevention and treatment of hepatocellular carcinoma (HCC). However, HCC cells respond differentially to IFN treatment, the mechanism of which is largely unknown. To address this issue, we analyzed the effect of IFN-alpha on different liver tumor cell lines. We found that growth inhibiting effects of IFN-alpha in hepatoma cells require PML-NB induction and, moreover, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression on the mRNA and protein level. RNAi silencing of PML down-regulates TRAIL expression in hepatoma cells and correspondingly blocks IFN-alpha-induced apoptosis. In addition, PML-deficient primary hepatocytes fail to up-regulate TRAIL upon IFN-alpha-treatment in contrast to their wild-type counterparts. These data identify TRAIL as a novel downstream transcriptional target of PML-mediated apoptosis in hepatomas and suggest that PML and TRAIL play important roles in IFN-regulated apoptosis in HCC. Furthermore, the mechanism is independent of the p53 status of the tumor cells. In summary, our results identify central molecules mediating IFN-alpha induced apoptosis in liver tumors, shed light on the differential response of hepatoma cells to IFN exposure and, thus, may contribute to an efficient application of this substance in the treatment of liver cancer.

Trends in epidemiology, treatment, and survival of hepatocellular carcinoma patients between 1998 and 2009: an analysis of 1066 cases of a German HCC Registry.

Goals: The aim of this study was to analyze clinical presentation, course of disease, and management of patients with hepatocellular carcinoma (HCC) in a German referral center between 1998 and 2009. Background: HCC is a rare tumor in Germany, but its incidence has increased over the last 30 years. New therapies such as chemoembolization with drug-eluting beads, selective internal radiotherapy, and sorafenib were introduced recently; however, the impact on clinical management and overall survival (OS) is unclear. Study: In this retrospective analysis, 1066 patients with HCC, separated into two 6-year periods (n=385; 1998 to 2003 and n=681; 2004 to 2009) were evaluated. Results: The number of patients presenting each year (64 vs. 114 per year), with an age over 80 years or with nonalcoholic steatohepatitis increased significantly between periods. The main risk factors were alcoholic liver disease in 51.7%, chronic hepatitis C virus in 28.2%, and chronic hepatitis B virus in 13.4% of patients with liver cirrhosis and HCC. Patients presented with more advanced tumor stages and with worse liver function in period 2. The majority (61.6%) of patients received local treatment over a spectrum of Barcelona Clinic Liver-Cancer (BCLC) stages, whereas systemic therapy was offered to a minority (8.8%) and limited to BCLC stage C patients only. OS decreased in BCLC stage A and D and improved in BCLC stage B and C and decreased for all patients from 16.5 to 15.3 months between periods. Conclusions: No improvement of OS was observed when comparing time periods, partly because of the more advanced stage of HCC and because of the increasing age in the second time period. Improved and new therapeutic options and the intensification of surveillance programs are likely to increase survival of HCC patients in the future.

Ablation of c-FLIP in hepatocytes enhances death-receptor mediated apoptosis and toxic liver injury in vivo.

BACKGROUND & AIMS Apoptosis is crucially involved in acute and chronic liver injury, including viral, cholestatic, toxic, and metabolic liver disease. Additionally, dysregulation of apoptosis signaling pathways has been implicated in hepatocarcinogenesis. The most prominent members of the apoptosis-mediating tumor necrosis factor receptor superfamily are the TNF-R1 (CD120a) and the CD95 (Apo-1/Fas) receptor. Although extensively studied, the intracellular signaling events in hepatocytes are only incompletely understood. METHODS To examine the role of the caspase-8 homolog cellular FLICE-inhibitory protein (c-FLIP) in liver injury, we generated mice with hepatocyte specific deletion of c-FLIP. Three models of acute liver injury were employed: the agonistic anti-CD95 antibody Jo2, d-galactosamine and LPS (GalN/LPS), and concanavalin A. RESULTS Conditional ablation of c-FLIP in hepatocytes augmented liver injury and cell death in all three models of liver injury. CD95- and GalN/LPS-induced liver injury was ameliorated by a pancaspase inhibitor, while ConA-induced injury was unaffected by caspase inhibition. Augmented activation of the MAPK JNK was observed in parallel to liver injury in c-FLIP knockout mice in all injury models; however, inhibition of JNK only affected TNF- and ConA-mediated injury. CONCLUSIONS In summary, c-FLIP is a central regulator of cell death in hepatocytes, involving increased activation of caspases and the MAPK JNK.

Glycine and Taurine Equally Prevent Fatty Livers from Kupffer Cell-Dependent Injury: An In Vivo Microscopy Study

Please cite this paper as: Bruns, Watanpour, Gebhard, Flechtenmacher, Galli, Schulze‐Bergkamen, Zorn, Büchler and Schemmer (2011). Glycine and Taurine Equally Prevent Fatty Livers from Kupffer Cell‐Dependent Injury: An In Vivo Microscopy Study. Microcirculation 18(3), 205–213.

Adjuvant therapy after resection of colorectal liver metastases: the predictive value of the MSKCC clinical risk score in the era of modern chemotherapy

BackgroundDespite introduction of effective chemotherapy protocols, it has remained uncertain, if patients with colorectal cancer (CRC) liver metastases should receive adjuvant therapy. Clinical or molecular predictors may help to select patients at high risk for disease recurrence and death who obtain a survival advantage by adjuvant chemotherapy.MethodsA total of 297 patients with potentially curative resection of CRC liver metastases were analyzed. These patients had no neoadjuvant therapy, no extrahepatic disease and negative resection margins. The primary endpoint was overall survival. Patients’ risk status was evaluated using the Memorial Sloan-Kettering Cancer Center clinical risk score (MSKCC-CRS). Multivariable analyses were performed using Cox proportional hazard models.ResultsA total of 137 (43%) patients had a MSKCC-CRS > 2. Adjuvant chemotherapy was administered to 116 (37%) patients. Patients who received adjuvant chemotherapy were of younger age (p = 0.03) with no significant difference in the presence of multiple metastases (p = 0.72) or bilobar metastases (p = 0.08). On multivariate analysis adjuvant chemotherapy was associated with improved survival in the entire cohort (Hazard ratio 0.69; 95% confidence interval 0.69–0.98). It improved survival markedly in high-risk patients with a MSKCC-CRS > 2 (HR 0.40; 95% CI 0.23–0.69), whereas it was of no benefit in patients with a MSKCC-CRS ≤ 2 (HR 0.90; 95% CI 0.57–1.43).ConclusionsThe MSKCC-CRS offers a tool to select patients for adjuvant therapy after resection of CRC liver metastases. Validation in independent patient cohorts is required.