Roland Sonntag

Pro-apoptotic Sorafenib signaling in murine hepatocytes depends on malignancy and is associated with PUMA expression in vitro and in vivo

The multi-kinase inhibitor Sorafenib increases the survival of patients with advanced hepatocellular carcinoma (HCC). Current data suggest that Sorafenib inhibits cellular proliferation and angiogenesis and promotes apoptosis. However, the underlying pro-apoptotic molecular mechanisms are incompletely understood. Here we compared the pro-apoptotic and anti-proliferative properties of Sorafenib in murine hepatoma cells and syngeneic healthy hepatocytes in vitro and in animal models of HCC and liver regeneration in vivo. In vitro, we demonstrate that cell cycle activity and expression of anti-apoptotic Bcl-2 like proteins are similarly downregulated by Sorafenib in Hepa1-6 hepatoma cells and in syngeneic primary hepatocytes. However, Sorafenib-mediated activation of caspase-3 and induction of apoptosis were exclusively found in hepatoma cells, but not in matching primary hepatocytes. We validated these findings in vivo by applying an isograft HCC transplantation model and partial hepatectomy (PH) in C57BL/6 mice. Sorafenib treatment activated caspase-3 and thus apoptosis selectively in small tumor foci that originated from implanted Hepa1-6 cells but not in surrounding healthy hepatocytes. Similarly, Sorafenib did not induce apoptosis after PH. However, Sorafenib treatment transiently inhibited cell cycle progression and resulted in mitotic catastrophe and enhanced non-apoptotic liver injury during regeneration. Importantly, Sorafenib-mediated apoptosis in hepatoma cells was associated with the expression of p53-upregulated-modulator-of-apoptosis (PUMA). In contrast, regenerating livers after PH revealed downregulation of PUMA and were completely protected from Sorafenib-mediated apoptosis. We conclude that Sorafenib induces apoptosis selectively in hepatoma cells but not in healthy hepatocytes and can additionally increase non-apoptotic hepatocyte injury in the regenerating liver.

Loss of caspase-8 in hepatocytes accelerates the onset of liver regeneration in mice through premature NF-?B activation

The cytokine tumor necrosis factor alpha (TNF‐α; TNF) plays a critical role early in liver regeneration following partial hepatectomy (PH). TNF stimulates at least three different pathways leading to nuclear factor kappa B (NF‐κB) activation, apoptosis signaling by way of caspase‐8 (Casp8), and activation of cJun N‐terminal kinases (JNK). The present study aimed to better define the role of Casp8 during liver regeneration. We performed PH in mice lacking Casp8 specifically in hepatocytes (Casp8Δhepa) and determined their liver regeneration capacity by measuring liver mass restoration and kinetics of cell cycle progression. Casp8Δhepa mice showed an accelerated onset of DNA synthesis after PH, delayed hepatocyte mitosis, but overall normal liver mass restoration. Analysis of immediate TNF‐dependent signaling pathways revealed that loss of Casp8 prevents proteolytic cleavage of the receptor‐interacting protein 1 (RIP1) in hepatocytes and subsequently triggers premature activation of NF‐κB and JNK/cJun related signals. In order to define the role of NF‐κB in this setting we blocked NF‐κB activation in Casp8Δhepa mice by concomitant inactivation of the NF‐κB essential modulator (NEMO) in hepatocytes. Lack of NEMO largely reverted aberrant DNA synthesis in Casp8Δhepa mice but resulted in incomplete termination of the regeneration process and hepatomegaly. Conclusion: Casp8 comprises a nonapoptotic function during liver regeneration by balancing RIP1, NF‐κB, and JNK activation. While loss of Casp8 triggers NF‐κB activation and thus improves liver regeneration, combined loss of Casp8 and NEMO impairs a controlled regenerative response and drives hepatomegaly. (Hepatology 2013;58:1779–1789)

Loss of caspase‐8 in hepatocytes accelerates the onset of liver regeneration in mice through premature nuclear factor kappa B activation

The cytokine tumor necrosis factor alpha (TNF‐α; TNF) plays a critical role early in liver regeneration following partial hepatectomy (PH). TNF stimulates at least three different pathways leading to nuclear factor kappa B (NF‐κB) activation, apoptosis signaling by way of caspase‐8 (Casp8), and activation of cJun N‐terminal kinases (JNK). The present study aimed to better define the role of Casp8 during liver regeneration. We performed PH in mice lacking Casp8 specifically in hepatocytes (Casp8Δhepa) and determined their liver regeneration capacity by measuring liver mass restoration and kinetics of cell cycle progression. Casp8Δhepa mice showed an accelerated onset of DNA synthesis after PH, delayed hepatocyte mitosis, but overall normal liver mass restoration. Analysis of immediate TNF‐dependent signaling pathways revealed that loss of Casp8 prevents proteolytic cleavage of the receptor‐interacting protein 1 (RIP1) in hepatocytes and subsequently triggers premature activation of NF‐κB and JNK/cJun related signals. In order to define the role of NF‐κB in this setting we blocked NF‐κB activation in Casp8Δhepa mice by concomitant inactivation of the NF‐κB essential modulator (NEMO) in hepatocytes. Lack of NEMO largely reverted aberrant DNA synthesis in Casp8Δhepa mice but resulted in incomplete termination of the regeneration process and hepatomegaly. Conclusion: Casp8 comprises a nonapoptotic function during liver regeneration by balancing RIP1, NF‐κB, and JNK activation. While loss of Casp8 triggers NF‐κB activation and thus improves liver regeneration, combined loss of Casp8 and NEMO impairs a controlled regenerative response and drives hepatomegaly. (Hepatology 2013;58:1779–1789)

Targeting CCl4-induced liver fibrosis by RNA interference - mediated inhibition of Cyclin E1 in mice

Initiation and progression of liver fibrosis requires proliferation and activation of resting hepatic stellate cells (HSCs). Cyclin E1 (CcnE1) is the regulatory subunit of the cyclin‐dependent kinase 2 (Cdk2) and controls cell cycle re‐entry. We have recently shown that genetic inactivation of CcnE1 prevents activation, proliferation, and survival of HSCs and protects from liver fibrogenesis. The aim of the present study was to translate these findings into preclinical applications using an RNA interference (RNAi)‐based approach. CcnE1‐siRNA (small interfering RNA) efficiently inhibited CcnE1 gene expression in murine and human HSC cell lines and in primary HSCs, resulting in diminished proliferation and increased cell death. In C57BL/6 wild‐type (WT) mice, delivery of stabilized siRNA using a liposome‐based carrier targeted approximately 95% of HSCs, 70% of hepatocytes, and 40% of CD45+ cells after single injection. Acute CCl4‐mediated liver injury in WT mice induced endogenous CcnE1 expression and proliferation of surviving hepatocytes and nonparenchymal cells, including CD45+ leukocytes. Pretreatment with CcnE1‐siRNA reverted CcnE1 induction to baseline levels of healthy mice, which was associated with reduced liver injury, diminished proliferation of hepatocytes and leukocytes, and attenuated overall inflammatory response. For induction of liver fibrosis, WT mice were challenged with CCl4 for 4‐6 weeks. Co‐treatment with CcnE1‐siRNA once a week was sufficient to continuously block CcnE1 expression and cell‐cycle activity of hepatocytes and nonparenchymal cells, resulting in significantly ameliorated liver fibrosis and inflammation. Importantly, CcnE1‐siRNA also prevented progression of liver fibrosis if applied after onset of chronic liver injury. Conclusion: Therapeutic targeting of CcnE1 in vivo using RNAi is feasible and has high antifibrotic activity. (Hepatology 2017;66:1242‐1257).

Inhibition of Caspase-8 does not protect from alcohol-induced liver apoptosis but alleviates alcoholic hepatic steatosis in mice

Hepatic apoptosis is involved in the progression of alcoholic liver disease (ALD). Caspase-8, the apical initiator in death receptor-mediated apoptosis, has been implicated in acute liver injury and in non-alcoholic steatohepatitis. However, the relevance of Caspase-8 in the pathogenesis of ALD remains unclear. In the present study, we investigated the impact of Caspase-8 in human and murine alcohol-induced apoptosis and in ALD. We investigated human samples from ALD patients, primary mouse hepatocytes, and hepatocyte-specific Caspase-8 knockout (Casp8Δhepa) mice in acute and chronic models of ethanol (EtOH) administration. Caspase-8 activation was detected in liver biopsies from ALD patients, as well as in livers of wild-type (WT) mice after chronic ethanol feeding for 8 weeks using the Lieber-DeCarli model. Lack of Caspase-8 expression in Casp8Δhepa animals failed to prevent alcohol-induced liver damage and apoptosis. Instead, inhibition of Caspase-8 shifted the ethanol-induced death signals towards pronounced activation of the intrinsic, mitochondria-dependent apoptosis pathway in Casp8Δhepa livers involving enhanced release of cytochrome c, stronger Caspase-9 activation and specific morphological changes of mitochondria. In vitro and in vivo intervention using a pan-caspase inhibitor markedly attenuated alcohol-induced hepatocyte damage in a Caspase-8-independent manner. Surprisingly, EtOH-fed Casp8Δhepa mice displayed significantly attenuated steatosis and reduced hepatic triglyceride and free fatty acids content. Caspase-8 is dispensable for alcohol-induced apoptosis, but plays an unexpected role for alcohol-dependent fat metabolism. We provide evidence that simultaneous inhibition of extrinsic and intrinsic apoptosis signaling using pan-caspase inhibitors in vivo might be an optimal approach to treat alcohol-induced liver injury.

Functional role of CCL5/RANTES for HCC progression during chronic liver disease

BACKGROUND & AIMS During liver inflammation, triggering fibrogenesis and carcinogenesis immune cells play a pivotal role. In the present study we investigated the role of CCL5 in human and in murine models of chronic liver inflammation leading to hepatocellular carcinoma (HCC) development. METHODS CCL5 expression and its receptors were studied in well-defined patients with chronic liver disease (CLD) and in two murine inflammation based HCC models. The role of CCL5 in inflammation, fibrosis, tumor initiation and progression was analyzed in different cell populations of NEMOΔhepa/CCL5-/- animals and after bone marrow transplantation (BMT). For therapeutic intervention Evasin-4 was injected for 24h or 8weeks. RESULTS In CLD patients, CCL5 and its receptor CCR5 are overexpressed - an observation confirmed in the Mdr2-/- and NEMOΔhepa model. CCL5 deletion in NEMOΔhepa mice diminished hepatocyte apoptosis, compensatory proliferation and fibrogenesis due to reduced immune cell infiltration. Especially, CD45+/Ly6G+ granulocytes, CD45+/CD11b+/Gr1.1+/F4/80+ pro-inflammatory monocytes, CD4+ and CD8+ T cells were decreased. One year old NEMOΔhepa/CCL5-/- mice displayed smaller and less malignant tumors, characterized by reduced proliferative capacity and less pronounced angiogenesis. We identified hematopoietic cells as the main source of CCL5, while CCL5 deficiency did not sensitise NEMOΔhepa hepatocytes towards TNFα induced apoptosis. Finally, therapeutic intervention with Evasin-4 over a period of 8weeks ameliorated liver disease progression. CONCLUSION We identified an important role of CCL5 in human and functionally in mice with disease progression, especially HCC development. A novel approach to inhibit CCL5 in vivo thus appears encouraging for patients with CLD. LAY SUMMARY Our present study identifies the essential role of the chemoattractive cytokine CCL5 for liver disease progression and especially hepatocellular carcinoma development in men and mice. Finally, the inhibition of CCL5 appears to be encouraging for therapy of human chronic liver disease.

Sorafenib steuert differentielle Signalwege in murinen Hepatozyten und Hepatomzellen und inhibiert Leberregeneration durch Zellzyklusarrest und nicht-apoptotische Leberschädigung

Hintergrund: Der Multikinase-Inhibitor Sorafenib erhoht signifikant das Uberleben in Patienten mit fortgeschrittenem hepatozellularem Karzinom (HCC). Bisherige Daten zeigten, dass Sorafenib mitogen-aktivierte Proteinkinasen (MAPK) inhibiert und Apoptose induziert. Allerdings wurden diese Sorafenib-bedingten Effekte bislang weitestgehend in Tumorzelllinien untersucht. In dieser Studie sollte die Wirkung von Sorafenib in isogenen murinen Hepatomzellen und gesunden Hepatozyten miteinander verglichen werden, um protektive oder schadigende Einflusse in der regenerierenden Leber zu identifizieren. Methoden: Sorafenib Tosylat wurde von Bayer Pharmazeutika bereitgestellt. Fur in vitro Studien wurden murine Hepa1–6 Hepatomzellen sowie primare Hepatozyten aus C57/BL6Mausen verwendet. C57/BL6 Wildtypmause wurden vor und nach partieller Hepatektomie (PH) einmal taglich mit Sorafenib (100mg/kg, p.o.) behandelt und zu verschiedenen Zeitpunkten auf Hepatozytenproliferation, Leberschadigung und Regeneration bis zu einem Zeitpunkt von 96h nach PH analysiert. Ergebnisse: In vitro inhibierte Sorafenib gleichermasen die Zellzyklusprogression in Hepatomzellen und Hepatozyten, unter anderem durch Inhibition der Cycline D, E und A uber transkriptionelle und post-translationale Mechanismen. Zusatzlich bewirkt Sorafenib in beiden Zellspezies die Herabregulation der anti-apoptotischen Proteine Akt, Bcl-2 und Mcl-1. Interessanterweise induzierte Sorafenib Apoptose ausschlieslich in Hepatomzellen, aber nicht in primaren Hepatozyten. Diese Ergebnisse zeigen, dass die pro-apoptotische Wirkung von Sorafenib nicht allein auf einer Reduktion anti-apoptotischer Signale beruht, sondern eine maligne Zelltransformation erfordert. In vivo fuhrte die Applikation von Sorafenib uber einen Zeitraum von bis zu 4 Tagen zu einer transienten Inhibition der DNA-Synthese und Zellzyklusprogression nach PH. Allerdings wurde die Rekonstitution der Lebermasse nach PH durch Sorafenib nicht beeinflusst. In Ubereinstimmung mit den in vitro erhobenen Daten induzierte Sorafenib keine Apoptose in hepatektomierten Lebern. Allerdings erhohte sich durch die Applikation von Sorafenib die nicht-apoptotische Leberschadigung nach PH, was anhand erhohter Serumtransaminasenaktivitat sowie dem vermehrten Auftreten von Hepatozyten mit irregularen Mitosen und freier (cytoplasmatischer) kondensierter DNA nachgewiesen wurde Schlussfolgerung: Sorafenib induziert selektiv Apoptose in maligne transformierten Hepatomzellen, jedoch nicht in proliferierenden Hepatozyten in vitro oder in vivo. Weiterhin deuten unsere Daten darauf hin, dass Sorafenib in regenerierenden Lebern eine nicht-apoptotische Schadigung der Hepatozyten hervorrufen kann.