Apoptosis of liver-derived cells induced by parvovirus B19 nonstructural protein

Parvovirus B19 has been implicated in some cases of acute fulminant non-A, non-B, non-C, non-G liver failure. Our laboratory previously demonstrated that B19 infection of hepatocytes induces apoptosis and that the B19 viral nonstructural protein, NS1, may play a critical role. To study the involvement of NS1 in apoptosis of liver cells, we generated a fusion protein of NS1 with enhanced green fluorescent protein (eGFP) in a system allowing for inducible gene expression. Transfection of the liver-derived cell line HepG2 with the eGFP/NS1 vector allowed expression of the fusion protein, which was visualized by fluorescence microscopy and demonstrated by immunoblotting. The fusion protein localized to discrete domains in the nucleus. Transfection of HepG2 cells with the eGFP/NS1 vector led to apoptosis of 35% of transfected cells, a sevenfold increase over cells transfected with the parent eGFP expression vector. Mutation of the eGFP/NS1 vector to eliminate the nucleoside triphosphate-binding site of NS1 significantly decreased apoptosis, as did treatment of transfected cells with inhibitors of caspase 3 or 9. Neutralization of tumor necrosis factor alpha or Fas ligand had no effect on apoptosis. These results demonstrate that NS1 is sufficient to induce apoptosis in liver-derived cells and that it does so through the initiation of an intrinsic caspase pathway.     

Apoptosis participates to liver damage in HSV-induced fulminant hepatitis

Background: HSV fulminant hepatitis is a rare pathology. Rapid hepatic failure, as a consequence of extended liver damage, has generally been attributed to necrosis. As apoptosis can constitute another way for hepatocytes to die, we decided to investigate whether programmed cell death took place during HSV fulminant hepatitis. Methods: Liver sections were obtained from two cases of fulminant herpetic hepatitis as well as from hepatitis B virus and Rickettsia-infected livers. Herpes simplex virus infection was confirmed using in situ hybridization. Apoptosis was assessed by histopathological examination, p53, activated-caspase 3 and Fas immunohistochemistry and TUNEL labeling. Results: We report that the number of cells expressing activated-caspase 3 was largely increased in fulminant herpes simplex virus hepatitis, when compared to livers chronically infected by hepatitis B virus or from a Rickettsial acute hepatitis. Apoptosis of hepatocytes was confirmed by a positive double-staining for activated-caspase 3 and hepatocytes. Finally, the apoptotic process has progressed beyond the step of nuclear DNA cleavage as demonstrated by TUNEL labeling. Conclusion: These data as a whole show that apoptosis is responsible, at least partially, for liver damage during HSV fulminant hepatitis.     

Possible Interactions between the NS-1 Protein and Tumor Necrosis Factor Alpha Pathways in Erythroid Cell Apoptosis Induced by Human Parvovirus B19

Human erythroid progenitor cells are the main target cells of the human parvovirus B19 (B19), and B19 infection induces a transient erythroid aplastic crisis. Several authors have reported that the nonstructural protein 1 (NS-1) encoded by this virus has a cytotoxic effect, but the underlying mechanism of NS-1-induced primary erythroid cell death is still not clear. In human erythroid progenitor cells, we investigated the molecular mechanisms leading to apoptosis after natural infection of these cells by the B19 virus. The cytotoxicity of NS-1 was concomitantly evaluated in transfected erythroid cells. B19 infection and NS-1 expression induced DNA fragmentation characteristic of apoptosis, and the commitment of erythroid cells to undergo apoptosis was combined with their accumulation in the G(2) phase of the cell cycle. Since B19- and NS-1-induced apoptosis was inhibited by caspase 3, 6, and 8 inhibitors, and substantial caspase 3, 6, and 8 activities were induced by NS-1 expression, there may have been interactions between NS-1 and the apoptotic pathways of the death receptors tumor necrosis factor receptor 1 and Fas. Our results suggest that Fas-FasL interaction was not involved in NS-1- or B19-induced apoptosis in erythroid cells. In contrast, these cells were sensitized to tumor necrosis factor alpha (TNF-alpha)-induced apoptosis. Moreover, the ceramide level was enhanced by B19 infection and NS-1 expression. Therefore, our results suggest that there may be a connection between the respective apoptotic pathways activated by TNF-alpha and NS-1 in human erythroid cells.     

Mechanism of staurosporine-induced apoptosis in murine hepatocytes

Staurosporine (STS) induces apoptosis in various cell lines. We report in this study that primary cultured mouse hepatocytes are less sensitive to STS compared with Jurkat cells and Huh-7 cells. In contrast to the cell lines, no apparent release of cytochrome c or loss of mitochondrial transmembrane potential was detected in primary hepatocytes undergoing STS-induced apoptosis. Caspase-3 was activated in primary hepatocytes by STS treatment, but caspase-9 and -12 were not activated, and caspase-3 activation is not dependent on caspase-8. These findings point to a novel pathway for caspase-3 activation by STS in primary hepatocytes. Pretreatment with caspase inhibitor converted STS-induced apoptosis of hepatocytes to necrotic cell death without significantly changing total cell death. Thus STS causes hepatocytes to commit to death upstream of the activation of caspases. We also demonstrated that STS dramatically sensitized primary hepatocytes to tumor necrosis factor-alpha-induced apoptosis. STS activated I kappa B kinase and nuclear factor-kappa B (NF-kappa B) nuclear translocation and DNA binding but inhibited transactivation of I kappa B-alpha, inducible nitric oxide synthase, and inhibitor of apoptosis protein-1 in hepatocytes and NF-kappa B reporter in transfected Huh-7 cells.     

Suppression of E-cadherin Mediates Gallotannin Induced Apoptosis in Hep G2 Hepatocelluar Carcinoma Cells

Though gallotannin was known to have anti-oxidant and antitumor activity, the underlying antitumor mechanism of gallotannin still remains unclear. Thus, in the present study, antitumor mechanism of gallotannin was elucidated in hepatocellular carcinoma cells. Gallotannin significantly exerted cytotoxicity against Hep G2 and Chang hepatocellular carcinoma cells with the accumulation of the sub-G1 population and increase of terminal deoxynucleotidyltransferasedUTP nick end labeling (TUNEL) positive cells as an apoptotic feature. Also, gallotannin attenuated the expression of pro-caspase9, pro-caspase3, Bcl2 and integrin β1 and cleaved poly(ADP)-ribose polymerase (PARP) in Hep G2 and Chang cancer cells. Furthermore, gallotannin suppressed cell repair motility by wound healing assay and also inhibited cell adhesion in Hep G2 cells. Of note, gallotannin attenuated the expression of epithelial cadherin (E-cadherin) to form cell-cell adhesion from the early stage, and also beta-catenin at late phase in Hep G2 cells. Consistently, Immunofluorescence assay showed that E-cadherin or β-catenin expression was suppressed in a time dependent manner by gallotannin. Furthermore, silencing of E-cadherin by siRNA transfection method enhanced PAPR cleavage, caspase 3 activation and sub G1 population and attenuated the cell adhesion induced by gallotannin in Hep G2 cells. Overall, our findings demonstrate that the disruption of cell adhesion junction by suppression of E-cadherin mediates gallotannin enhanced apoptosis in Hep G2 liver cancer cells.     

Salirasib sensitizes hepatocarcinoma cells to TRAIL-induced apoptosis through DR5 and survivin-dependent mechanisms

Ras activation is a frequent event in human hepatocarcinoma that may contribute to resistance towards apoptosis. Salirasib is a ras and mTOR inhibitor that induces a pro-apoptotic phenotype in human hepatocarcinoma cell lines. In this work, we evaluate whether salirasib sensitizes those cells to TRAIL-induced apoptosis. Cell viability, cell death and apoptosis were evaluated in vitro in HepG2, Hep3B and Huh7 cells treated with DMSO, salirasib and YM155 (a survivin inhibitor), alone or in combination with recombinant TRAIL. Our results show that pretreatment with salirasib sensitized human hepatocarcinoma cell lines, but not normal human hepatocytes, to TRAIL-induced apoptosis. Indeed, FACS analysis showed that 25 (Huh7) to 50 (HepG2 and Hep3B) percent of the cells treated with both drugs were apoptotic. This occurred through activation of the extrinsic and the intrinsic pathways, as evidenced by a marked increase in caspase 3/7 (five to ninefold), caspase 8 (four to sevenfold) and caspase 9 (eight to 12-fold) activities in cells treated with salirasib and TRAIL compared with control. Survivin inhibition had an important role in this process and was sufficient to sensitize hepatocarcinoma cells to apoptosis. Furthermore, TRAIL-induced apoptosis in HCC cells pretreated with salirasib was dependent on activation of death receptor (DR) 5. In conclusion, salirasib sensitizes hepatocarcinoma cells to TRAIL-induced apoptosis by a mechanism involving the DR5 receptor and survivin inhibition. These results in human hepatocarcinoma cell lines and primary hepatocytes provide a rationale for testing the combination of salirasib and TRAIL agonists in human hepatocarcinoma.     

Mechanism of Arctigenin-Induced Specific Cytotoxicity against Human Hepatocellular Carcinoma Cell Lines: Hep G2 and SMMC7721

Arctigenin (ARG) has been previously reported to exert high biological activities including anti-inflammatory, antiviral and anticancer. In this study, the anti-tumor mechanism of ARG towards human hepatocellular carcinoma (HCC) was firstly investigated. We demonstrated that ARG could induce apoptosis in Hep G2 and SMMC7721 cells but not in normal hepatic cells, and its apoptotic effect on Hep G2 was stronger than that on SMMC7721. Furthermore, the following study showed that ARG treatment led to a loss in the mitochondrial out membrane potential, up-regulation of Bax, down-regulation of Bcl-2, a release of cytochrome c, caspase-9 and caspase-3 activation and a cleavage of poly (ADP-ribose) polymerase in both Hep G2 and SMMC7721 cells, suggesting ARG-induced apoptosis was associated with the mitochondria mediated pathway. Moreover, the activation of caspase-8 and the increased expression levels of Fas/FasL and TNF-α revealed that the Fas/FasL-related pathway was also involved in this process. Additionally, ARG induced apoptosis was accompanied by a deactivation of PI3K/p-Akt pathway, an accumulation of p53 protein and an inhibition of NF-κB nuclear translocation especially in Hep G2 cells, which might be the reason that Hep G2 was more sensitive than SMMC7721 cells to ARG treatment.     

In vitro and in vivo anticarcinogenic effects of RNase MC2, a ribonuclease isolated from dietary bitter gourd, toward human liver cancer cells

Hepatocellular carcinoma (HCC) constitutes a predominant part of primary liver cancer which ranks as the fifth most common cancer as well as the third most common cause of cancer mortality. In view of the poor prognosis of unresectable liver cancers, it is of pivotal importance to develop novel chemotherapeutical regimens. RNase MC2 is a 14-kDa ribonuclease isolated from dietary bitter gourd (Momordica charantia) that manifested antitumor potential against breast cancers. In this study, we investigated the potential application of RNase MC2 on Hep G2 cells. We showed that RNase MC2 inhibited cell proliferation and induced cell apoptosis in both in vitro and in vivo studies. RNase MC2 treatment caused cell cycle arrest predominantly at the S-phase and apoptosis, which is associated with the activation of both caspase-8 and caspase-9 regulated caspase pathways. Our further investigation disclosed that RNase MC2 down-regulated the anti-apoptotic protein Bcl-2 and increased the expression of pro-apoptotic protein Bak. Moreover, the phosphorylation of ERK and JNK was involved in the apoptosis process. Importantly, RNase MC2 significantly suppressed the growth of Hep G2 xenograft-bearing nude mice by inducing apoptosis. This notion is supported by data indicating an increased number of caspase-3- and PARP-positive cells, and TUNEL-positive cells in RNase MC2-treated tumor tissues. In summary, we have revealed the antitumor potential of RNase MC2 toward Hep G2 cells. Considering that bitter gourd is a common dietary component in many countries, this study may help to prompt the clinical application of RNase MC2.     

Ammonia removal using hepatoma cells in mammalian cell cultures

It was examined whether hepatocyte cell lines can be used for ammonia removal in mammalian cell cultures. It was found that there exists a critical ammonium concentration level for each hepatocyte cell to remove ammonia. Among the cells tested in this work, primary hepatocytes showed the strongest ammonia removal capability if ammonium concentration is higher than the critical level. However, primary hepatocytes lost the liver function gradually and finally died after 2-3 weeks. Because of this limitation, primary hepatocytes were not appropriate to be used for ammonia removal in long-term cultures. Hep G2 cells, which are immortal, also showed a strong ammonia removal activity. The ammonia removal activity of Hep G2 cells depended on the concentration of ammonium in the medium, as in the case of primary hepatocytes. However, urea could not be detected in the course of ammonia removal by Hep G2 cells. Instead of urea, Hep G2 cells secreted glutamine into the culture medium. The capacity for ammonia removal was higher in the absence than in the presence of glutamine. Thus we checked the activity of glutamine synthetase in the Hep G2 cells. The level of glutamine synthetase activity increased with the addition of ammonium chloride. This result accounts for the ammonium concentration dependency of Hep G2 cells in ammonia removal and glutamine synthesis. Furthermore Hep G2 cells could grow well in the absence of glutamine, which was necessarily required in mammalian cell cultures. These results prove that glutamine formation serves as the primary mechanism of detoxifying ammonia in hepatocyte cell lines as expected. In addition, it was demonstrated that ammonium level could be reduced 38% and that erythropoietin production increased 2-fold in the mixed culture of Hep G2 and recombinant CHO cells.     

Sorafenib sensitizes hepatocellular carcinoma cells to physiological apoptotic stimuli

Sorafenib increases survival rate of patients with advanced hepatocellular carcinoma (HCC). The mechanism underlying this effect is not completely understood. In this work we have analyzed the effects of sorafenib on autocrine proliferation and survival of different human HCC cell lines. Our results indicate that sorafenib in vitro counteracts autocrine growth of different tumor cells (Hep3B, HepG2, PLC-PRF-5, SK-Hep1). Arrest in S/G2/M cell cycle phases were observed coincident with cyclin D1 down-regulation. However, sorafenib's main anti-tumor activity seems to occur through cell death induction which correlated with caspase activation, increase in the percentage of hypodiploid cells, activation of BAX and BAK and cytochrome c release from mitochondria to cytosol. In addition, we observed a rise in mRNA and protein levels of the pro-apoptotic "BH3-domain only" PUMA and BIM, as well as decreased protein levels of the anti-apoptotic MCL1 and survivin. PUMA targeting knock-down, by using specific siRNAs, inhibited sorafenib-induced apoptotic features. Moreover, we obtained evidence suggesting that sorafenib also sensitizes HCC cells to the apoptotic activity of transforming growth factor-β (TGF-β) through the intrinsic pathway and to tumor necrosis factor-α (TNF) through the extrinsic pathway. Interestingly, sensitization to sorafenib-induced apoptosis is characteristic of liver tumor cells, since untransformed hepatocytes did not respond to sorafenib inducing apoptosis, either alone or in combination with TGF-β or TNF. Indeed, sorafenib effectiveness in delaying HCC late progression might be partly related to a selectively sensitization of HCC cells to apoptosis by disrupting autocrine signals that protect them from adverse conditions and pro-apoptotic physiological cytokines.     

Inhibition of matrix metalloproteinases blocks lethal hepatitis and apoptosis induced by tumor necrosis factor and allows safe antitumor therapy.

Acute and fulminant liver failure induced by viral hepatitis, alcohol or other hepatotoxic drugs, are associated with tumor necrosis factor (TNF) production. In a mouse model of lethal hepatitis induced by TNF, apoptosis and necrosis of hepatocytes, but also lethality, hypothermia and influx of leukocytes into the liver, are prevented by a broad-spectrum matrix metalloproteinase (MMP) inhibitor, BB-94. Mice deficient in MMP-2, MMP-3 or MMP-9 had lower levels of apoptosis and necrosis of hepatocytes, and better survival. We found induction of MMP-9 activity and fibronectin degradation. Our findings suggest that several MMPs play a critical role in acute, fulminant hepatitis by degrading the extracellular matrix and allowing massive leukocyte influx in the liver. BB-94 also prevented lethality in TNF/interferon-gamma therapy in tumor-bearing mice. A broad-spectrum MMP inhibitor may be potentially useful for the treatment of patients with acute and perhaps chronic liver failure, and in cancer therapies using inflammatory cytokines.     

The role of death effector domain-containing proteins in acute oxidative cell injury in hepatocytes

Apoptosis is a mechanism that regulates hepatic tissue homeostasis and contributes to both acute and chronic injury in liver disease. The apoptotic signaling cascade involves activation of the death-inducing signaling complex (DISC) and subsequent recruitment of proteins containing death effector domains (DED), which regulate downstream effector molecules. Prominent among these are the Fas-associated death domain (FADD) and the cellular caspase 8-like inhibitory protein (cFLIP), and alterations in these proteins can lead to severe disruption of physiological processes, including acute liver failure or hepatocellular carcinoma. Their role in cell signaling events independent of the DISC remains undetermined. Oxidative stress can cause cell injury from direct effects on molecules or by activating intracellular signaling pathways including the mitogen-activated protein kinases (MAPKs). In this context, prolonged activation of the cJun N-terminal kinase (JNK)/AP-1/cJun signaling pathway promotes hepatocellular apoptosis, whereas activation of the extracellular signal-regulated kinase (Erk) exerts protection. We investigated the roles of FADD and cFLIP in acute oxidant stress induced by the superoxide generator menadione in hepatocytes. Menadione resulted in dose-dependent predominantly necrotic cell death. Hepatocytes expressing a truncated, dominant-negative FADD protein were partially protected, whereas cFLIP-deficient hepatocytes displayed increased cell death from menadione. In parallel, Erk phosphorylation was enhanced in hepatocytes expressing dnFADD and decreased in cFLIP-deficient hepatocytes. Hepatocyte injury was accompanied by increased release of proapoptotic factors and increased JNK/cJun activation. Thus, FADD and cFLIP contribute to the regulation of cell death from acute oxidant stress in hepatocytes involving MAPK signaling. This implies that DED-containing proteins are involved in the regulation of cellular survival beyond their role in cell death receptor-ligand-mediated apoptosis.     

Sub-lethal concentration of arsenic interferes with the proliferation of hepatocytes and induces in vivo apoptosis in Clarias batrachus L

We studied the hepatocellular alterations induced by sub-lethal concentrations (0.50 muM) of arsenic in Indian catfish Clarias batrachus L. Sub-lethal arsenic exposure altered serum aspartate aminotransferase and alkaline phosphatase levels and brought about significant changes in different serum biochemical parameters. Arsenic exposure reduced total hepatocyte protein content and suppressed the proliferation of hepatocytes in a time-dependent manner. Routine histological studies on liver documented arsenic-induced changes characterized by dilated sinusoids, formation of intracellular edema, megalocytosis, vacuolation and appearance of hepatic cells with distorted nuclei. Transmission electron microscopy of hepatocytes further revealed hyperplasia and hypertrophy of mitochondria, development of dilated rough endoplasmic reticulum and changes in peroxisome size with duration of arsenic exposure. Degeneration of mitochondrial cristae and condensation of chromatin was also evident in arsenic-exposed hepatocytes. A significant number of hepatocytes isolated from arsenic-exposed fish stained with annexin V and demonstrated DNA ladder characteristic of apoptosis. Single-cell gel electrophoresis of exposed hepatocytes also revealed the development of comets usually seen in apoptotic cells. Using specific inhibitors it was determined that the arsenic-induced apoptosis of hepatocytes was caspase-mediated, involving the caspase 3 pathway.     

Synthesis and secretion of protein C inhibitor by the human hepatoma-derived cell line, Hep G2

The site of synthesis of protein C inhibitor, a recently identified human plasma inhibitor against activated protein C, is not known. We have studied the production and secretion of protein C inhibitor by an established human liver cell line derived from hepatocellular carcinoma (Hep G2). The concentration of protein C inhibitor, as measured by a specific radioimmunoassay, increased in the medium of Hep G2 cells with time. There was no evidence for a significant intracellular pool of this protein. Protein C inhibitor secreted from Hep G2 cells (G2 protein C inhibitor) inhibited the activity of purified activated protein C in a functional assay. De novo synthesis of protein C inhibitor was demonstrated by the presence of specific immunoprecipitable radioactivity in the medium after 5 h of labeling of the cells with [35S]methionine. Analysis of the immunoprecipitates by SDS-polyacrylamide gel electrophoresis showed a peak of radioactivity corresponding to Mr 57 000. These results indicate that the liver is a site of protein C inhibitor production.     

Downregulation of citrin, a mitochondrial AGC, is associated with apoptosis of hepatocytes

Citrin is a mitochondrial aspartate–glutamate carrier primarily expressed in liver. Adult-onset type II citrullinemia is caused by mutations in the SLC25A13 gene that encodes for citrin, and patients with this condition do not express citrin. We found apoptotic hepatocytes in one such patient. This finding prompted us to investigate the role of citrin in hepatocyte survival. Knockdown of citrin by a vector-based short-hairpin RNA technique reduced cell viability and induced apoptosis of a hepatocellular carcinoma cell line, Hep3B cells. Caspase-3/7 and caspase-9 were activated, and PARP was cleaved. Citrin knockdown also increased the expression of Bax and Bak, and reduced expression of Bcl-xL and Bcl-2. These alterations resulted in the release of cytochrome c from the mitochondria. Our results indicated that citrin downregulation induces apoptosis of hepatocytes through the mitochondrial death pathway, highlighting the importance of citrin in survival of hepatocytes and maintenance of liver function.     

Genistein enhances TRAIL-induced apoptosis through inhibition of p38 MAPK signaling in human hepatocellular carcinoma Hep3B cells

The cytotoxic effect of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is limited in some carcinoma cancer cells. However, it was found that treatment with TRAIL in combination with nontoxic concentrations of genistein sensitized TRAIL-resistant human hepatocellular carcinoma Hep3B cells to TRAIL-mediated apoptosis. Combined treatment with genistein and TRAIL-induced chromatin condensation and sub-G1 phase DNA content. These indicators of apoptosis were correlated with the induction of caspase activity that resulted in the cleavage of poly(ADP-ribose) polymerase (PARP). Both cell viability and the cleavage of PARP induced by combined treatment were significantly inhibited by caspase-3, -8 and -9 inhibitors, which demonstrates the important roles of caspases in the observed cytotoxic effects. Genistein treatment also triggered the inhibition of p38-β mitogen-activated protein kinase (MAPK) activation. Pretreatment with SB203580 resulted in significantly increased sub-G1 population and loss of mitochondrial membrane potential (MMP) in TRAIL-induced apoptosis. By contrast, overexpression of p38 MAPK protected apoptosis by co-treatment with genistein and TRAIL, suggesting that the p38 MAPK act as key regulators of apoptosis in response to treatment with a combination of genistein and TRAIL in human hepatocellular carcinoma Hep3B cells.