Circulating mortalin autoantibody—a new serological marker of liver cirrhosis

Mortalin is a stress chaperone belonging to the Hsp70 family of proteins. Frequently enriched in cancers, it is a multifunctional protein and regulates cell proliferation, apoptosis, mitochondrial functions, and the activity of tumor suppressor protein p53. In the present study, we investigated circulating mortalin and its autoantibody in normal, cirrhosis, and cancerous liver. We found that although mortalin is enriched in liver cancer cells and tumors, it is not detected in the serum of either the liver cirrhosis or cancer patients. In contrast, mortalin autoantibody was detected in patients’ sera and showed significant correlation with the occurrence of cirrhosis. It is suggested as a potential noninvasive marker for liver cirrhosis.     

Zinc supplementation inhibits hepatic apoptosis in mice subjected to a long-term ethanol exposure

Hepatocyte apoptosis has been documented in both clinical and experimental alcoholic liver disease. This study was undertaken to examine the effect of dietary zinc supplementation on hepatic apoptosis in mice subjected to a long-term ethanol exposure. Male adult 129S6 mice fed an ethanol-containing liquid diet for 6 months developed hepatitis, as indicated by neutrophil infiltration and elevation of hepatic keratinocyte chemoattractant (KC) and monocyte chemoattractant protein-1 (MCP-1) levels. Apoptotic cell death was detected in ethanol-exposed mice by a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and was confirmed by the increased activities of caspase-3 and -8. Zinc supplementation attenuated alcoholic hepatitis and reduced the number of TUNEL-positive cells in association with inhibition of caspase activities. Ethanol exposure caused oxidative stress, as indicated by reactive oxygen species accumulation, mitochondrial glutathione depletion, and decreased metallothionein levels in the liver, which were suppressed by zinc supplementation. The mRNA levels of tumor necrosis factor (TNF)-alpha, TNF-R1, FasL, Fas, Fas-associated factor-1, and caspase-3 in the liver were upregulated by ethanol exposure, which were attenuated by zinc supplementation. Zinc supplementation also prevented ethanol-elevated serum and hepatic TNF-alpha levels and TNF-R1 and Fas proteins in the liver. In conclusion, zinc supplementation prevented hepatocyte apoptosis in mice subjected to long-term ethanol exposure, and the action of zinc is likely through suppression of oxidative stress and death receptor-mediated pathways.     

p53与细胞死亡

p53是一种重要的肿瘤抑制因子,是迄今发现与人类肿瘤相关性最高的分子之一。超过50%的人类肿瘤含有p53基因突变。因此,p53是肿瘤治疗中的重要分子靶点。p53依赖的细胞凋亡是其抑制肿瘤的重要机制之一。然而,最近研究发现,p53不仅参与细胞凋亡,还与程序性细胞坏死、细胞自噬以及铁诱导的细胞死亡等细胞死亡途径相关。促使肿瘤细胞死亡是肿瘤治疗的重要目标。因此,进一步了解p53与细胞死亡之间的关系,将有助于探索以p53为靶点的肿瘤治疗和p53相关肿瘤细胞耐药机制。     

Apoptosis and apoptosis related proteins in chronic viral liver disease

Background: Apoptosis may be an important mechanism of hepatocyte death in chronic viral liver disease. Methods: We studied apoptosis in liver biopsies from 30 patients with chronic viral hepatitis and 8 patients with viral cirrhosis by the TUNEL method. 12 cases of non-alcoholic steatohepatitis and 12 cases of primary biliary cirrhosis were used as non-viral disease controls. Immunohistochemical expression of p53, p21/waf1, bcl-2 and mdm-2 proteins was also studied in the same patients. Results: A statistically significant increase of apoptotic liver cells was found in severe chronic viral hepatitis (5.3 ± 0.3%), cirrhosis (3.4 ± 0.5%) and PBC (4.4 ± 0.4%) cases compared to patients with non-alcoholic steatohepatitis (0.8 ± 0.3%). The expression of p53 protein was increased in the cases of viral cirrhosis and in chronic severe viral hepatitis whereas in the cases of chronic mild hepatitis, PBC and non-alcoholic steatohepatitis we found no expression of p53. P21/waf1 expression was increased in severe chronic hepatitis, cirrhosis and PBC cases compared to mild hepatitis and non-alcoholic steatohepatitis cases. However no induction of mdm-2 was observed in the subgroups of chronic liver disease. Bcl-2 was expressed only in epithelium of bile ducts and mononuclear cells of the portal tracts and liver lobules. A weaker Bcl-2 expression was noted in the epithelium of bile ducts of 7/12 PBC cases. Conclusion: Our results provide evidence of increased apoptosis in severe chronic viral liver disease, suggesting that apoptotic cell death might be involved in the pathogenesis of hepatocellular damage of viral hepatitis and cirrhosis. Furthermore we analysed part of the apoptotic pathways implicated in the above process and found an increased expression of p21/waf1, probably p53 mediated, without overexpression of the apoptosis inhibiting bcl-2 and mdm-2 proteins. By contrast p21/waf1 overexpression in PBC seems to be propagated by a p53 independent mechanism.     

Activation of CD40 with platelet derived CD154 promotes reactive oxygen species dependent death of human hepatocytes during hypoxia and reoxygenation

Background

Hypoxia and hypoxia-reoxygenation (H-R) are pathogenic factors in many liver diseases that lead to hepatocyte death as a result of reactive oxygen species (ROS) accumulation. The tumor necrosis factor super-family member CD154 can also induce hepatocyte apoptosis via activation of its receptor CD40 and induction of autocrine/paracrine Fas Ligand/CD178 but the relationship between CD40 activation, ROS generation and apoptosis is poorly understood. We hypothesised that CD40 activation and ROS accumulation act synergistically to drive human hepatocyte apoptosis.

Methods

Human hepatocytes were isolated from liver tissue and exposed to an in vitro model of hypoxia and H-R in the presence or absence of CD154 and/or various inhibitors. Hepatocyte ROS production, apoptosis and necrosis were determined by labelling cells with 2′,7′-dichlorofluorescin, Annexin-V and 7-AAD respectively in a three-colour reporter flow cytometry assay.

Results

Exposure of human hepatocytes to recombinant CD154 or platelet-derived soluble CD154 augments ROS accumulation during H-R resulting in NADPH oxidase-dependent apoptosis and necrosis. The inhibition of c-Jun N-terminal Kinase and p38 attenuated CD154-mediated apoptosis but not necrosis.

Conclusions

CD154-mediated apoptosis of hepatocytes involves ROS generation that is amplified during hypoxia-reoxygenation. This finding provides a molecular mechanism to explain the role of platelets in hepatocyte death during ischemia-reperfusion injury.     

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.     

Revisiting p53 for cancer-specific chemo- and radiotherapy

Despite intense studies, highly effective therapeutic strategies against cancer have not yet been fully exploited, because few true cancer-specific targets have been identified. Most modalities, perhaps with the exception of radiation therapy, target proliferating cells, which are also abundant in normal tissues. Thus, most current cancer treatments have significant side effects. More than 10 years ago, the tumor suppressor p53 was first explored as a cancer-specific target. At the time, the approach was to introduce a normal p53 gene into mutant p53 (mp53) tumor cells to induce cell cycle arrest and apoptosis. However, this strategy did not hold up and mostly failed in subsequent clinical studies. Recent research developments have now returned p53 to the limelight. Several studies have reported that mutant or null p53 tumor cells undergo apoptosis more easily than genetically matched, normal p53 counterparts when inhibiting a specific stress kinase in combination with standard chemotherapy or when exposed to an ataxia-telangiectasia mutated (ATM) kinase inhibitor and radiation, thus achieving true cancer specificity in animal tumor models. This short review highlights several of these recent studies, discusses possible mechanism(s) for mp53-mediated “synthetic lethality,” and the implications for cancer therapy.     

Assessing DNA damage and health risk using biomarkers

Increased genomic instability has been found associated with cancer and aging. The p53 tumor suppressor protein is a major determinant of genomic instability as a regulator of cell cycle control and apoptosis in response to DNA damage. To investigate the rate of age-related mutation accumulation in the absence of p53, we crossed Trp53 null mice with transgenic mice harboring a lacZ mutational target gene. In the hybrid animals, lacZ mutation frequencies at early age (i.e. at about 2 months) were found to be the same as in the control lacZ animals. However, up until about 6 months, when the Trp53-knockout mice usually die from cancer, mutations were found to accumulate with age in the spleen, and to a lesser extent in the liver, at a more rapid rate than in the control Trp53(+/+) or Trp53(+/-), lacZ hybrid mice. Treatment of 2-3-month-old Trp53(-/-), lacZ hybrid mice with the powerful mutagen ethyl nitrosourea (ENU) resulted in a higher number of mutations induced in the liver but not in the spleen, as compared to the Trp53(+/+), lacZ mice. These results suggest that p53 is not an important determinant of gene mutation induction, either spontaneously during development or after treatment with a mutagen. The accelerated age-related accumulation of mutations in normal spleen and liver could be explained by the defect in apoptosis, which would prevent severely damaged cells from being eliminated.     

PIDDosome‐induced p53‐dependent ploidy restriction facilitates hepatocarcinogenesis

Polyploidization frequently precedes tumorigenesis but also occurs during normal development in several tissues. Hepatocyte ploidy is controlled by the PIDDosome during development and regeneration. This multi‐protein complex is activated by supernumerary centrosomes to induce p53 and restrict proliferation of polyploid cells, otherwise prone for chromosomal instability. PIDDosome deficiency in the liver results in drastically increased polyploidy. To investigate PIDDosome‐induced p53‐activation in the pathogenesis of liver cancer, we chemically induced hepatocellular carcinoma (HCC) in mice. Strikingly, PIDDosome deficiency reduced tumor number and burden, despite the inability to activate p53 in polyploid cells. Liver tumors arise primarily from cells with low ploidy, indicating an intrinsic pro‐tumorigenic effect of PIDDosome‐mediated ploidy restriction. These data suggest that hyperpolyploidization caused by PIDDosome deficiency protects from HCC. Moreover, high tumor cell density, as a surrogate marker of low ploidy, predicts poor survival of HCC patients receiving liver transplantation. Together, we show that the PIDDosome is a potential therapeutic target to manipulate hepatocyte polyploidization for HCC prevention and that tumor cell density may serve as a novel prognostic marker for recurrence‐free survival in HCC patients.