JNK inhibition sensitises hepatocellular carcinoma cells but not normal hepatocytes to the TNF-related apoptosis-inducing ligand.

BACKGROUND: cJun terminal kinase (JNK) is constitutively activated in most hepatocellular carcinomas (HCCs), yet its exact role in carcinogenesis remains controversial. While tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is known as a major mediator of acquired immune tumour surveillance, and is currently being tested in clinical trials as a novel cancer therapy, the resistance of many tumours to TRAIL and concerns about its toxicity in vivo represent obstacles to its clinical application. In this study we investigated whether JNK activity in HCC could contribute to the resistance to apoptosis in these tumours. METHODS: The effect of JNK/Jun inhibition on receptor-mediated apoptosis was analysed by pharmacological inhibition or RNA interference in cancer cells and non-tumour cells isolated from human liver or transgenic mice lacking a phosphorylation site for Jun. RESULTS: JNK inhibition caused cell cycle arrest, enhanced caspase recruitment, and greatly sensitised HCC cells but not normal hepatocytes to TRAIL. TRAIL-induced activation of JNK could be effectively interrupted by administration of the JNK inhibitor SP600125. CONCLUSIONS: Expression and TRAIL-dependent feedback activation of JNK likely represent a mechanism by which cancer cells escape TRAIL-mediated tumour surveillance. JNK inhibition might represent a novel strategy for specifically sensitising HCC cells to TRAIL thus opening promising therapeutic perspectives for safe and effective use of TRAIL in cancer treatment.

Ethanol-induced apoptosis in hepatoma cells proceeds via intracellular Ca(2+) elevation, activation of TLCK-sensitive proteases, and cytochrome c release.

Ethanol is known to induce apoptosis in hepatocytes. However, intracellular signaling events of ethanol-induced death are still only partially understood. We studied such processes in ethanol-induced apoptosis in HepG2 cells as a model system for human liver cells. We determined the incidence of apoptosis by DNA fragmentation and tested the effects of various known inhibitors. Ethanol induces apoptosis in HepG2 cells in a dose- and time-dependent manner as well as in rat primary hepatocytes. This effect was not mediated through the death receptor CD95 and the tumor necrosis factor receptors. It was efficiently inhibited by the caspase inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (zVAD-fmk), the Ca(2+) chelator EGTA, and the serine protease inhibitor N-p-tosyl-l-lysine chloromethyl ketone (TLCK). Upon ethanol treatment, the intracellular calcium ion concentration was increased and cytochrome c was released from the mitochondria, and caspases were activated. EGTA and TLCK could inhibit cytochrome c release from the mitochondria. Furthermore, overexpression of Bcl-x(L) saved cells from ethanol-induced apoptosis. These data suggest that ethanol-induced apoptosis in liver cells is initiated by the intracellular Ca(2+) elevation in the cytoplasm and activation of TLCK-sensitive serine proteases. Our data provide new insight into ethanol-induced apoptosis in liver cells and may lead to therapeutic strategies to prevent liver damage.

The chemotherapeutic drug 5-fluorouracil induces apoptosis in mouse thymocytes in vivo via activation of the CD95(APO-1/Fas) system.

The CD95/CD95 ligand (CD95L) system has been shown to mediate chemotherapeutic drug-induced apoptosis in vitro. However, the contribution of the CD95 pathway to drug-induced apoptosis is controversial. We have shown previously that 5-fluorouracil (5-FU) induces apoptosis in vitro via the activation of the CD95/CD95L system. To study the effects of the chemotherapeutic drug 5-FU and the contribution of the CD95 system to 5-FU-induced apoptosis in vivo, we gave mice an i.p. injection of 5-FU. Apoptotic cell death peaked in thymocytes at 18 h after administration of 5-FU. Total organ weight and cell number in the thymus were reduced by approximately 40%. This cell loss was due to apoptosis, as measured in cell suspensions by measuring hypodiploid DNA content and by terminal deoxynucleotidyl transferase-mediated nick end labeling staining of tissue sections. The number of apoptotic cells correlated with the extent of weight loss and cell attrition of the organs. Furthermore, in the thymi of 5-FU-treated animals, CD95L was strongly up-regulated. Apoptosis of thymocytes was blocked in vivo with neutralizing anti-CD95L antibodies. In addition, cell loss in the thymus was negligible in lpr mice in comparison with wild-type mice. Thus, a significant portion of apoptosis of thymocytes in vivo on treatment with 5-FU is mediated via the CD95/CD95L pathway. Our findings therefore contribute to the understanding how chemotherapeutic drugs exert their effects in vivo.

A novel AP-1 element in the CD95 ligand promoter is required for induction of apoptosis in hepatocellular carcinoma cells upon treatment with anticancer drugs.

The CD95 (also called APO-1 or Fas) system plays a major role in the induction of apoptosis in lymphoid and nonlymphoid tissues in response to a variety of extracellular signals, including chemotherapeutic drugs. Here we report that the CD95 ligand (CD95L) is upregulated in hepatoma cells upon treatment with antineoplastic drugs. Upregulation by different chemotherapeutic drugs is functionally relevant for drug-induced apoptosis and is mediated by transcriptional mechanisms. The MEKK1/JNKK pathway and a novel AP-1 element in the CD95L promoter downstream of the TATA box are required for CD95L upregulation. Thus, understanding the mechanisms of CD95-mediated apoptosis through CD95L upregulation upon treatment of hepatocellular carcinomas with chemotherapeutic drugs may contribute to the improvement of anticancer chemotherapy.

Expression and regulation of cell adhesion molecules by hepatic stellate cells (HSC) of rat liver: involvement of HSC in recruitment of inflammatory cells during hepatic tissue repair.

Hepatic stellate cells (HSC), a pericyte-like nonparenchymal liver cell population, are regarded as the principal matrix-synthesizing cells of fibrotic liver. They might also play a role during liver inflammation. The present study analyzed (i) expression of cell adhesion molecules (CAMs) mediating cell infiltration, like intercellular adhesion molecule-1 (I-CAM-1) and vascular cell adhesion molecule-1 (V-CAM-1), by HSC, (ii) CAM regulation in HSC by growth factors and inflammatory cytokines, and (iii) CAM expression in situ during liver inflammation, using immunochemistry and Northern blot analysis. I-CAM-1 and V-CAM-1 expression was present in HSC in vitro and in cells located in the sinusoidal/perisinusoidal area of normal liver. Growth factors, eg, transforming growth factor-beta1, down-regulated I-CAM-1- and V-CAM-1-coding mRNAs and stimulated N-CAM expression of HSC. In contrast, inflammatory cytokines like tumor necrosis factor-alpha reduced N-CAM-coding mRNAs, whereas induction of I-CAM-1- and V-CAM-1-specific transcripts increased several fold. In situ, messengers specific for I-CAM-1 and V-CAM-1 were induced 3 hours after CCl4 treatment (thereby preceding mononuclear cell infiltration starting at 12 hours), were expressed at maximal levels 9-12 hours after CCl4 application, and decreased afterwards. I-CAM-1 and V-CAM-1 immunoreactivity increased in a linear fashion starting 3 hours after CCl4-induced liver injury, was detected in highest amounts at 24-48 hours characterized by maximal cell infiltration, and returned to baseline values at 96 hours. Interestingly, the induction/repression of CAM-specific messengers paralleled the time kinetics of tumor necrosis factor-alpha transforming growth factor-beta1 expression in injured liver. HSC might be important during the onset of hepatic tissue injury as proinflammatory elements and might interact with I-CAM-1 and V-CAM-1 ligand-bearing cells, namely lymphocyte function-associated antigen-1- or Mac-1/very late activation antigen-4-positive inflammatory cells, thereby modulating the recruitment and migration of mononuclear cells within the perisinusoidal space of diseased livers.

Sinusoidal intercellular adhesion molecule-1 up-regulation precedes the accumulation of leukocyte function antigen-1-positive cells and tissue necrosis in a model of carbontetrachloride-induced acute rat liver injury.

The mechanisms leading to the infiltration of inflammatory cells into the liver and to liver cell necrosis remain undefined. To elucidate this process, the present work analyzes the kinetics of the expression of intercellular adhesion molecule-1 (ICAM-1) and the accumulation of inflammatory leukocyte function antigen-1 (LFA-1)-positive cells in relation to the appearance of hepatocellular necrosis in the model of acute carbontetrachloride (CCl4)-induced liver injury. ICAM-1- and LFA-1-immunoreactivity was analyzed in normal livers and in livers obtained 3, 6, 9, 12, 18, 24, 48, and 72 hours after CCl4-administration, as well as in liver cells isolated 3, 6, 9, 12, 18, and 24 hours after CCl4-administration. Total RNA extracted from livers and cells was used for Northern blot analysis. ICAM-1-positivity, which was detected along the sinusoids in normal rat livers, increased 3 to 6 hours after CCl4-administration and finally accumulated in the necrotic areas (24 to 48 hours post-administration). ICAM-1 steady-state mRNA levels in liver tissue increased 3 to 6 hours after CCl4-treatment and returned to normal levels at 48 hours after treatment. Increased amounts of ICAM-1-specific transcripts could be observed in isolated sinusoidal endothelial cells and in hepatocytes as early as 3 to 6 hours after CCl4-administration. In normal rat livers, a few LFA-1-immunoreactive cells were present around the vessel walls. Starting 12 hours after CCl4-administration, the number of LFA-1-immunoreactive cells increased around the vessel walls and along the sinusoids, accumulating later in the necrotic areas. In accordance, the number of mononuclear phagocytes isolated from the liver increased 12 hours after CCl4-treatment. These data demonstrate an early up-regulation of ICAM-1 in liver cells and the accumulation of LFA-1-expressing cells prior to the development of necrotic areas. The up-regulation of ICAM-1 and accumulation of inflammatory cells seem to be critical for the induction of CCl4-induced hepatotoxicity.

Cell-type-specific expression of neural cell adhesion molecule (N-CAM) in Ito cells of rat liver. Up-regulation during in vitro activation and in hepatic tissue repair.

Ito cells (lipocytes, stellate cells) are regarded as the principle matrix-producing cell of the liver and have been shown recently to express glial fibrillary acidic protein, an intermediate filament typically found in glia cells of the nervous system. The present study examines 1) whether Ito cells of rat liver express central nervous system typical adhesion molecules, namely, neural cell adhesion molecule (N-CAM), in a cell-type-specific manner and 2) whether N-CAM expression is affected by activation of Ito cells in vitro and during rat liver injury in vivo. As assessed by reverse transcriptase polymerase chain reaction, Northern blotting, Western blotting, and immunocytochemistry of freshly isolated and cultivated hepatic cells, N-CAM expression was restricted to Ito cells and was absent in hepatocytes, Kupffer cells, and sinusoidal endothelial cells. Ito cells expressed predominantly N-CAM-coding transcripts of 6.1 and 4.8 kb in size and 140-kd isoforms of the N-CAM protein, which was localized on the cell surface membrane of Ito cells. In parallel to glial fibrillary acidic protein down-regulation and smooth muscle alpha-actin up-regulation, N-CAM expression was increased during in vitro transformation of Ito cells from resting to activated (myofibroblast-like) cells and by the fibrogenic mediator transforming growth factor-beta 1. By immunohistochemistry, N-CAM was detected in normal rat liver in the portal field as densely packed material and in a spot as well as fiber-like pattern probably representing nerve structures. However, after liver injury, N-CAM expression became detectable in mesenchymal cells within and around the necrotic area and within fibrotic septae. In serially cut tissue sections, N-CAM-positive cells were predominantly co-distributed with smooth muscle alpha-actin-positive cells rather than glial fibrillary acidic protein-positive cells, especially in fibrotic livers. The experimental results illustrate that N-CAM positivity in the liver cannot be solely ascribed to nerve endings as, among the different types of resident liver cells, Ito cells specifically express N-CAM in vitro and presumably in vivo. In addition to its role as potential cell-type-specific marker protein for activated Ito cells, the induction of N-CAM expression might illustrate a mechanism by which mesenchymal cell proliferation might be inhibited when tissue repair is concluded.