Gerald Künstle

ICE-PROTEASE INHIBITORS BLOCK MURINE LIVER INJURY AND APOPTOSIS CAUSED BY CD95 OR BY TNF-ALPHA

The two apoptosis receptors of mammalian cells, i.e. the 55 kDa TNF receptor (TNF-R1) and CD95 (Fas/APO1) are activated independently of each other, however, their signaling involves a variety of ICE-related proteases [I]. We used a cell-permeable inhibitor of ICE-like protease activity to examine in vivo whether post-receptor signaling of TNF and CD95 are fully independent processes. Mice pretreated with the inhibitor, Z-VAD-fluoromethylketone (FMK) were dose-dependently protected from liver injury caused by CD95 activation as determined by plasma alanine aminotransferase and also from hepatocyte apoptosis assessed by DNA fragmentation (ID50 = 0.1 mg/kg). A dose of 10 mg/kg protected mice also from liver injury induced by TNF-alpha. Similar results were found when apoptosis was initiated via TNF-alpha or via CD95 in primary murine hepatocytes (IC50 = 1.5 nM) or in various human cell lines. In addition to prevention, an arrest of cell death by Z-VAD-FMK was demonstrated in vivo and in vitro after stimulation of apoptosis receptors. These findings show in vitro and in vivo in mammals that CD95 and the TNF-alpha receptor share a distal proteolytic apoptosis signal.

Pre-apoptotic Alterations in Hepatocytes of TNF α-treated Galactosamine-sensitized Mice:

Tumor necrosis factor (TNF) induces apoptotic death of hepatocytes in the galactosamine (GalN)-sensitized mouse liver after 5 hr. In our study, the most remarkable sign of the early stage of apoptosis was the focal rupture of the outer mitochondrial membrane. Parts of the inner membrane extended through the gap of the outer membrane, whereas the rest of the inner membrane still formed the cristae. This feature appeared in hepatocytes before chromatin condensation. With the diaminobenzidine technique for localization of cytochrome oxidase activity, the reaction product was detectable by light and electron microscopy. Ten percent of the hepatocytes were apoptotic, with condensed chromatin and high enzyme activity, 37% were pre-apoptotic, without chromatin condensation but high enzyme activity, and 53% had neither condensed chromatin nor a remarkable reaction product of cytochrome oxidase activity. Fas (APO-1, CD95) molecules on the plasma membrane of hepatocytes increased and were represented immunohistochemically in cells without chromatin condensation. DNA strand breaks were also detectable before chromatin aggregation. The results of this study indicate that mitochondria play a pivotal role in pre-apoptotic hepatocytes, together with an increase of the Fas molecule on the plasma membrane and with the occurrence of DNA strand breaks in the nucleus.

Kupffer Cell-Expressed Membrane-Bound TNF Mediates Melphalan Hepatotoxicity via Activation of Both TNF Receptors

Isolated hepatic perfusion of nonresectable liver cancer using the combination of TNF and melphalan can be associated with a treatment-related hepatotoxicity. We investigated whether, apart from TNF, also melphalan is cytotoxic in primary murine liver cells in vitro and investigated mediators, mode of cell death, and cell types involved. Melphalan induced a caspase-dependent apoptosis in hepatocytes, which was not seen in liver cell preparations depleted of Kupffer cells. Neutralization of TNF prevented melphalan-induced apoptosis and liver cells derived from mice genetically deficient in either TNFR 1 or 2, but not from lpr mice lacking a functional CD95 receptor, were completely resistant. Cell-cell contact between hepatocytes and Kupffer cells was required for apoptosis to occur. Melphalan increased membrane-bound but not secreted TNF in Kupffer cells and inhibited recombinant TNF-α converting enzyme in vitro. Melphalan induced also severe hepatotoxicity in the isolated recirculating perfused mouse liver from wild-type mice but not from TNFR 1 or 2 knockout mice. In conclusion, this study shows that melphalan elicits membrane TNF on Kupffer cells due to inhibition of TNF processing and thereby initiates apoptosis of hepatocytes via obligatory activation of both TNFRs. The identification of this novel mechanism allows a causal understanding of melphalan-induced hepatotoxicity.

Redox control of hepatic cell death

In necrotic liver failure like upon acetaminophen overdose, loss of the major intracellular thiol antioxidant glutathione was shown to be causal for hepatic dysfunction. In sharp contrast, fulminant apoptotic liver destruction upon overstimulation of the death receptors TNFR1 and CD95 was not associated with reduced hepatic glutathione levels. In view of the importance of the role of reactive oxygen intermediates versus antioxidants for apoptosis, we investigated the effect of phorone-induced enzymatic GSH depletion on the sensitivity of the liver towards CD95- or TNFR1-mediated hepatotoxicity. Our findings demonstrate in vivo that receptor-mediated hepatic apoptosis is disabled when glutathione is depleted, i.e. that an intact glutathione status is a critical determinant for the execution of apoptosis. In vitro, we did mechanistic studies in lymphoid cell lines and found that pro-caspase-8 at the CD95 death receptor and the mitochondrial activation of pro-caspase-9 are the enzyme targets that require sufficient intracellular reduced glutathione for their activation.

Topoisomerase inhibitor camptothecin sensitizes mouse hepatocytes in vitro and in vivo to TNF‐mediated apoptosis

Topoisomerases are nuclear enzymes that maintain and modulate DNA structure. Inhibitors of topoisomerases like camptothecin (CPT), etoposide, and others are widely used antitumor drugs that interfere with transcription, induce DNA strand breaks, and trigger apoptosis preferentially in dividing cells. Because transcription inhibitors (actinomycin D, galactosamine, α‐amanitin) sensitize primary hepatocytes to the cytotoxic action of tumor necrosis factor (TNF), we reasoned whether topoisomerase inhibitors would act similarly. CPT alone was not toxic to primary cultured murine hepatocytes. When incubated with CPT, murine hepatocytes displayed an inhibition of protein synthesis and were thereby rendered sensitive to apoptosis induction by TNF. Apoptosis was characterized by morphology (condensed/fragmented nuclei, membrane blebbing), caspase‐3‐like protease activity, fragmentation of nuclear DNA, and late cytolysis. Hepatocytes derived from TNF receptor‐1 knockout mice were resistant to CPT/TNF‐induced apoptosis. CPT treatment completely abrogated the TNF‐induced NF‐kappa B activation, and mRNA expression of the antiapoptotic factors TNF‐receptor associated factor 2, FLICE‐inhibitory protein, and X‐linked inhibitor of apoptosis protein was also inhibited by CPT. The caspase inhibitors benzyloxycarbonyl‐Val‐Ala‐Asp‐(OMe)‐fluoromethylketone (zVAD‐fmk) and benzyloxycarbonyl‐Asp(OMe)‐Glu(OMe)‐Val‐Asp(OMe)‐chloromethylketone (zDEVD‐fmk), as well as depletion of intracellular ATP by fructose prevented CPT/TNF‐induced apoptosis. In vivo, CPT treatment sensitized mice to TNF‐induced liver damage. In conclusion, the combination of topoisomerase inhibition and TNF blocks survival signaling and elicits a type of hepatocyte death similar to actinomycin D/TNF or galactosamine/TNF. During antitumor treatment with topoisomerase inhibitors, an impaired immune function often results in opportunistic infections, a situation where the systemic presence of TNF might be critical for the hepatotoxicity reported in clinical topoisomerase inhibitor studies. (HEPATOLOGY 2004;39:1311–1320.)

ATP-Depleting Carbohydrates Prevent Tumor Necrosis Factor Receptor 1-Dependent Apoptotic and Necrotic Liver Injury in Mice

We demonstrated previously that depletion of hepatic ATP by endogenous metabolic shunting of phosphate after fructose treatment renders hepatocytes resistant to tumor necrosis factor (TNF)-induced apoptosis. We here address the question whether this principle extends to TNF receptor 1-mediated caspase-independent apoptotic and to necrotic liver injury. As in the apoptotic model of galactosamine/lipopolysaccharide (LPS)-induced liver damage, the necrotic hepatotoxicity initiated by sole high-dose LPS treatment was abrogated after depletion of hepatic ATP. Although systemic TNF and interferon-γ levels were suppressed, animals still were protected when ATP depletion was initiated after the peak of proinflammatory cytokines upon LPS injection, showing that fructose-induced ATP depletion affects both cytokine release and action. In T cell-dependent necrotic hepatotoxicity elicited by concanavalin A or galactosamine + staphylococcal enterotoxin B, ATP depletion prevented liver injury as well, but here without modulating cytokine release. By attenuating caspase-8 activation, ATP depletion of hepatocytes in vitro impaired TNF receptor signaling by the death-inducing signaling complex, whereas receptor internalization and nuclear factor-κB activation upon TNF stimulation were unaffected. These findings demonstrate that sufficient target cell ATP levels are required for the execution of both apoptotic and necrotic TNF-receptor 1-mediated liver cell death.

Sensitization by 5-Azacytidine toward Death Receptor-Induced Hepatic Apoptosis

5-Azacytidine (5-aza-CR) is a DNA-hypomethylating antineoplastic agent used because of its inhibitory activity on DNA methyltransferases. Today, it is approved as an epigenetically active drug therapy for treatment of myelodysplastic disorders, with a contraindication as to pre-existing liver diseases. Because the mechanism of its hepatotoxicity is still unknown, we investigated the pharmacodynamic properties of 5-aza-CR with regard to death receptor/ligand-induced apoptosis and the mode of execution of cell death. In a time- and concentration-dependent manner, primary murine, human hepatocytes and HepG2 cells exposed to 5-aza-CR became highly sensitive toward cell death induced by CD95L, tumor necrosis factor (TNF)-related apoptosis-inducing ligand, or TNF. Cell death was characterized as apoptotic by membrane blebbing, chromatin condensation, and exposure of phosphatidylserine on the outer membrane. Neither 5-aza-2′-deoxycytidine nor the common DNA methyltransferase inhibitors S-(5′-adenosyl)-l-homocysteine or RG 108 showed any significant effects under these conditions. Despite the complete protection of HepG2 by high concentrations of the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (z-VAD-fmk), effector caspase-3/7 activity was completely abolished at approximately a 20-fold lower concentration of z-VAD-fmk. Under these conditions, the serine protease inhibitors N,α-tosyl-l-phenylalanine chloromethyl ketone, N,p-tosyl-l-lysine chloromethyl ketone, and 4-(2-aminoethyl)-benzenesulfonyl fluoride, respectively, conferred protection against death receptor ligands. We conclude that this caspase-independent apoptosis is executed by a yet-unidentified serine protease.

Differential immunotoxicity of histone deacetylase inhibitors on malignant and naïve hepatocytes

Histone deacetylases (HD) represent a novel target in cancer treatment, particularly for scattered small tumours such as the hepatocellular carcinoma (HCC). However, only few studies address the toxicological impact of HD Inhibitors (HDIs) on malignantly transformed cells versus primary hepatocytes. We examined whether and how different classes of HDIs sensitise the human HCC cell line HepG2, primary healthy murine and human liver cells towards the death receptor agonists TNFα and CD95L. Apicidin, M344 (N-hydroxy-7-(-4-dimethylaminobenzol)aminoheptanamide), CBHA (m-carboxycinnamic acid bis-hydroxamide) and VPA (valproic acid) sensitised liver cell cultures towards CD95-triggered apoptosis with the following potency: apicidin > M344 ≈ CBHA ≫ VPA. Apicidin sensitised towards CD95 also in the intact organ, i.e. in the isolated perfused mouse liver. No significant sensitisation towards TNFα was found in vitro. Western blot analysis showed that all HDIs studied downregulated the anti-apoptotic protein cFLIP, but only VPA additionally affected the expression level of XIAP. Furthermore, in models of the intrinsic apoptosis pathway, i.e. in HepG2 cells treated with Melphalan and in primary hepatocytes irradiated with UV light, only VPA exhibited significant sensitisation. These findings extend the biochemical, pharmacological and toxicological basis for HDI therapy and provide a caveat for clinical use in patients with an accompanying critical inflammatory state in which the CD95 system might be pre-activated.