Glycochenodeoxycholic acid (GCDC) induced hepatocyte apoptosis is associated with early modulation of intracellular PKC activity

The effect of GCDC-induced apoptosis on PKC activity and PKC's role in GCDC-induced hepatocyte apoptosis is unclear. The specific aims of this study were to determine if GCDC-induced apoptosis changed intracellular PKC activity and if modulation of PKC activity affected GCDC-induced hepatocyte apoptosis.Apoptosis was induced in isolated hepatocytes using GCDC. PKC activity was measured and specific PKC and calpain inhibitors were used to study the effects of PKC and calpain modulation on GCDC-induced apoptosis.After 4 h exposure, 50 M GCDC induced apoptosis in 42% of hepatocytes. Intracellular PKC activity decreased to 44% of controls 2 h after exposure of hepatocytes to GCDC (p < 0.001). Pre-incubation of hepatocytes with the calpain protease inhibitor restored PKC activity in GCDC exposed hepatocytes to 91± 5% of control cells. Pre-incubation of hepatocytes with a calpain inhibitor decreased GCDC-induced apoptosis as did pre-incubation with the PKC activating phorbol ester, PMA. The combination of calpain inhibition and PMA further reduced GCDC-induced apoptosis but caused low level hepatic apoptosis. Inhibition of PKC with chelerythrine also substantially reduced GCDC-induced hepatocyte apoptosis.GCDC-induced apoptosis is associated with decreases in total cellular PKC activity, which appear to be dependent on intracellular calpain-like protease activity. The combination of protease inhibition and phorbol ester pretreatment preserved total cellular PKC activity and decreased GCDC-induced apoptosis but induced low level apoptosis in the absence of GCDC exposure. PKC inhibition also decreased GCDC-induced hepatocyte apoptosis highlighting the complex interactions of PKC and proteases during GCDC-induced apoptosis.     

iNOS/NO signaling regulates apoptosis induced by glycochenodeoxycholate in hepatocytes

Inducible nitric oxide synthase (iNOS) and nitric oxide (NO) can ameliorate apoptosis induced by toxic glycochenodeoxycholate (GCDC) in hepatocytes. However, the underlying molecular mechanisms are not yet understood in detail. This study is to clarify the function of iNOS/NO and its mechanisms during the apoptotic process. The apoptosis was brought about by GCDC in rat primary hepatocytes. iNOS/NO signaling was then investigated. iNOS inhibitor 1400 W enhanced the GCDC-induced apoptosis as reflected by caspase-3 activity and TUNEL assay. Exogenous NO regulated the apoptosis subsequent to NO donor S-nitroso-N-acetyl-penicillamine (SNAP) or sodium nitroprusside (SNP). The GCDC-induced apoptosis was decreased with 0.1 mM SNAP or 0.15 mM SNP, while it was increased with 0.8 mM SNAP or 1.2 mM SNP. The endogenous iNOS inhibited apoptosis, but the exogenous NO played a dual role during the GCDC-induced apoptosis. There was a potential iNOS/Akt/survivin axis that inhibited the hepatocyte apoptosis in low doses of NO donors. In contrast, high doses of NO donors activated CHOP through p38MAP-kinase (p38MAPK), upregulated TRAIL receptor DR5, and suppressed survivin. Consequently the high doses of NO donors promoted the apoptosis in hepatocytes. Our data suggest that the iNOS/NO signaling can modulate Akt/survivin and p38MAPK/CHOP pathways to mediate the GCDC-induced the apoptosis in hepatocytes. These signaling pathways may serve as targets for therapeutic intervention in cholestatic liver disease.     

Nitric oxide ameliorates hydrophobic bile acid-induced apoptosis in isolated rat hepatocytes by non-mitochondrial pathways

Hydrophobic bile acids are toxic to isolated rat hepatocytes by mechanisms involving mitochondrial dysfunction and oxidative stress. In the current study we examined the role of nitric oxide (NO), a potential mediator of apoptosis, during bile acid-induced apoptosis. Freshly isolated rat hepatocytes and hepatic mitochondria generated NO and peroxynitrite (ONOO(-)) in a concentration- and time-dependent manner when exposed to the toxic bile salt glycochenodeoxycholate (GCDC) (25-500 microm), which was prevented by the nitric-oxide synthase (NOS) inhibitors N(G)-monomethyl-N-arginine monoacetate (l-NMMA) and 1400W. Relationships between hepatocyte NO production and apoptosis were examined by comparing the effects of NOS inhibitors with other inhibitors of GCDC-induced apoptosis. Inhibitors of caspases 8 and 9, the mitochondrial permeability transition blocker cyclosporin A, and the antioxidant idebenone reduced NO generation and apoptosis in GCDC-treated hepatocytes. In contrast, NOS inhibitors had no effect on GCDC-induced apoptosis despite marked reduction of NO and ONOO(-). However, treatment with the NO donors S-nitroso-N-acetylpenicillamine and spermine NONOate [N-(-aminoethyl)N-(2-hydroxy-2-nitrohydrazino)-1,2-ethylenediamine) inhibited apoptosis and caspase 3 activity while significantly elevating NO levels above GCDC-stimulated levels. Neither NO donors nor NOS inhibitors affected GCDC-induced mitochondrial permeability transition or cytochrome c release from liver mitochondria or GCDC-induced mitochondrial depolarization from isolated hepatocytes, suggesting that NO inhibits bile acid-induced hepatocyte apoptosis by a non-mitochondrial-dependent pathway. In conclusion, whereas NO produced from GCDC-treated hepatocytes neither mediates nor protects against bile acid-induced apoptosis, higher levels of NO inhibit GCDC-induced hepatocyte apoptosis by caspase-dependent pathways.     

The effect of capillarisin on glycochenodeoxycholic acid-induced apoptosis and heme oxygenase-1 in rat primary hepatocytes

The accumulation of hydrophobic bile acids plays a role in the induction of apoptosis and necrosis of hepatocytes during cholestasis. Glycochenodeoxycholate acid (GCDC) triggers a rapid oxidative stress response as an event of glutathione (GSH) depletion and nuclear factor kappa B (NF-κB) activation. We therefore investigated whether the bioactivity of the antioxidant capillarisin (Cap) prevents GCDC-induced hepatocyte damage. Isolated rat hepatocytes were co-incubated with 100 μM GCDC and 0.5 mg/ml Cap for 4 h. GSH depletion and thiobarbituric acid-reactive substances (TBARS, measure of lipid peroxidation) increased after GCDC exposure, but were markedly suppressed by Cap treatment. Cap protected hepatocytes from a GCDC-induced increase in reactive oxygen species (ROS) generation and mitochondrial membrane potential induction, as measured by flow cytometry analysis. In addition, Cap was shown to inhibit GCDC-mediated NF-κB activation by using electrophoretic mobility shift assays (EMSA). In contrast to GCDC, Cap not only significantly decreased cytochrome c release and caspase-3 enzyme activity, but also suppressed heme oxygenase-1 protein and mRNA expression in hepatocytes. These results demonstrate that Cap function as an antioxidant reduced hepatocyte injury caused by hydrophobic bile acids, perhaps by preventing generation of ROS and release of cytochrome c, thereby minimizing hepatocytes apoptosis.     

cAMP inhibits bile acid-induced apoptosis by blocking caspase activation and cytochrome c release

We have previously shown that cAMP protects against bile acid-induced apoptosis in cultured rat hepatocytes in a phosphoinositide 3-kinase (PI3K)-dependent manner. In the present studies, we investigated the mechanisms involved in this anti-apoptotic effect. Hepatocyte apoptosis induced by glycodeoxycholate (GCDC) was associated with mitochondrial depolarization, activation of caspases, the release of cytochrome c from the mitochondria, and translocation of BAX from the cytosol to the mitochondria. cAMP inhibited GCDC-induced apoptosis, caspase 3 and caspase 9 activation, and cytochrome c release in a PI3K-dependent manner. cAMP activated PI3K in p85 immunoprecipitates and resulted in PI3K-dependent activation of the survival kinase Akt. Chemical inhibition of Akt phosphorylation with SB-203580 partially blocked the protective effect of cAMP. cAMP resulted in wortmannin-independent phosphorylation of BAD and was associated with translocation of BAD from the mitochondria to the cytosol. These results suggest that GCDC-induced apoptosis in cultured rat hepatocytes proceeds through a caspase-dependent intracellular stress pathway and that the survival effect of cAMP is mediated in part by PI3K-dependent Akt activation at the level of the mitochondria.     

Caspase-10 triggers Bid cleavage and caspase cascade activation in FasL-induced apoptosis

In contrast to caspase-8, controversy exists as to the ability of caspase-10 to mediate apoptosis in response to FasL. Herein, we have shown activation of caspase-10, -3, and -7 as well as B cell lymphoma-2-interacting domain (Bid) cleavage and cytochrome c release in caspase-8-deficient Jurkat (I9-2) cells treated with FasL. Apoptosis was clearly induced as illustrated by nuclear and DNA fragmentation. These events were inhibited by benzyloxycarbonyl-VAD-fluoromethyl ketone, a broad spectrum caspase inhibitor, indicating that caspases were functionally and actively involved. Benzyloxycarbonyl-AEVD-fluoromethyl ketone, a caspase-10 inhibitor, had a comparable effect. FasL-induced cell death was not completely abolished by caspase inhibitors in agreement with the existence of a cytotoxic caspase-independent pathway. In subpopulations of I9-2 cells displaying distinct caspase-10 expression levels, cell sensitivity to FasL correlated with caspase-10 expression. A robust caspase activation, Bid cleavage, and DNA fragmentation were observed in cells with high caspase-10 levels but not in those with low levels. In vitro, caspase-10, as well as caspase-8, could cleave Bid to generate active truncated Bid (p15). Altogether, our data strongly suggest that caspase-10 can serve as an initiator caspase in Fas signaling leading to Bid processing, caspase cascade activation, and apoptosis.     

IAP antagonists target cIAP1 to induce TNFalpha-dependent apoptosis

XIAP prevents apoptosis by binding to and inhibiting caspases, and this inhibition can be relieved by IAP antagonists, such as Smac/DIABLO. IAP antagonist compounds (IACs) have therefore been designed to inhibit XIAP to kill tumor cells. Because XIAP inhibits postmitochondrial caspases, caspase 8 inhibitors should not block killing by IACs. Instead, we show that apoptosis caused by an IAC is blocked by the caspase 8 inhibitor crmA and that IAP antagonists activate NF-kappaB signaling via inhibtion of cIAP1. In sensitive tumor lines, IAP antagonist induced NF-kappaB-stimulated production of TNFalpha that killed cells in an autocrine fashion. Inhibition of NF-kappaB reduced TNFalpha production, and blocking NF-kappaB activation or TNFalpha allowed tumor cells to survive IAC-induced apoptosis. Cells treated with an IAC, or those in which cIAP1 was deleted, became sensitive to apoptosis induced by exogenous TNFalpha, suggesting novel uses of these compounds in treating cancer.     

Role of caspases and NF-kappaB signaling in hydrogen peroxide- and superoxide-induced hepatocyte apoptosis

Reactive oxygen intermediates (ROI) have been implicated as mediators of hepatocyte death resulting from a variety of forms of liver injury. To delineate the mechanisms that underlie ROI-induced apoptosis, the roles of caspase activation and nuclear factor-kappaB (NF-kappaB) signaling were determined in the rat hepatocyte cell line RALA255-10G after treatment with H(2)O(2) or the superoxide generator menadione. By 8 h, H(2)O(2) and menadione caused 26% and 33% cell death, respectively. Death from both ROI occurred by apoptosis as indicated by morphology under fluorescence microscopy, the induction of caspase activation and DNA fragmentation, and the cleavage of poly(ADP-ribose) polymerase. Despite the presence of caspase activation in both forms of apoptosis, caspase inhibition blocked H(2)O(2)- but not menadione-induced apoptosis. In contrast, inhibition of NF-kappaB activation decreased cell death from both ROI. Different ROI, therefore, induce distinct apoptotic pathways in RALA hepatocytes that are both caspase dependent and independent. In contrast to the known protective effect of NF-kappaB activation in tumor necrosis factor-alpha-induced hepatocyte apoptosis, NF-kappaB promotes hepatocellular death from ROI in these cells.     

Macrophages require constitutive NF-kappaB activation to maintain A1 expression and mitochondrial homeostasis

NF-kappaB is a critical mediator of macrophage inflammatory responses, but its role in regulating macrophage survival has yet to be elucidated. Here, we demonstrate that constitutive NF-kappaB activation is essential for macrophage survival. Blocking the constitutive activation of NF-kappaB with pyrrolidine dithiocarbamate or expression of IkappaBalpha induced apoptosis in macrophagelike RAW 264.7 cells and primary human macrophages. This apoptosis was independent of additional death-inducing stimuli, including Fas ligation. Suppression of NF-kappaB activation induced a time-dependent loss of mitochondrial transmembrane potential (DeltaPsi(m)) and DNA fragmentation. Examination of initiator caspases revealed the cleavage of caspase 9 but not caspase 8 or the effector caspase 3. Addition of a general caspase inhibitor, z-VAD. fmk, or a specific caspase 9 inhibitor reduced DNA fragmentation but had no effect on DeltaPsi(m) collapse, indicating this event was caspase independent. To determine the pathway leading to mitochondrial dysfunction, analysis of Bcl-2 family members established that only A1 mRNA levels were reduced prior to DeltaPsi(m) loss and that ectopic expression of A1 protected against cell death following inactivation of NF-kappaB. These data suggest that inhibition of NF-kappaB in macrophages initiates caspase 3-independent apoptosis through reduced A1 expression and mitochondrial dysfunction. Thus, constitutive NF-kappaB activation preserves macrophage viability by maintaining A1 expression and mitochondrial homeostasis.     

Licorice compounds glycyrrhizin and 18beta-glycyrrhetinic acid are potent modulators of bile acid-induced cytotoxicity in rat hepatocytes

The accumulation of hydrophobic bile acids results in cholestatic liver injury by increasing oxidative stress, mitochondrial dysfunction, and activation of cell signaling pathways. Licorice root and its constituents have been utilized as antihepatotoxic agents. The purpose of this study was to evaluate the potential modulation by a primary component of licorice root, glycyrrhizin (GL), and its metabolite, 18beta-glycyrrhetinic acid (GA), in a hepatocyte model of cholestatic liver injury. Preincubation of fresh rat hepatocyte suspensions with GL or GA reduced glycochenodeoxycholic acid (GCDC)-dependent reactive oxygen species generation, with GA more potent than GL. Interestingly, GL and GA had opposing effects toward GCDC-induced cytotoxicity; GA prevented both necrosis and apoptosis, whereas GL enhanced apoptosis. GCDC promoted activation of caspase 10, caspase 3, and PARP; all were inhibited by GA but not GL. Induction of apoptosis by GCDC was also associated with activation of JNK, which was prevented by GA. Activation of caspase 9 and dissipation of mitochondrial membrane potential were prevented by GA but not GL. In liver mitochondrial studies, GL and GA were both potent inhibitors of the mitochondrial permeability transition, reactive oxygen species generation, and cytochrome c release at submicromolar concentrations. Results from this study suggest that GL exhibits pro-apoptotic properties, whereas GA is a potent inhibitor of bile acid-induced apoptosis and necrosis in a manner consistent with its antioxidative effect.     

Protective effects of intracellular reactive oxygen species generated by 6-formylpterin on tumor necrosis factor-alpha-induced apoptotic cell injury in cultured rat hepatocytes

The effects of 6-formylpterin on tumor necrosis factor (TNF)-alpha-induced apoptotic cell injury were studied in cultured rat hepatocytes. The incubation of the hepatocytes with TNF-alpha and actinomycin D (ActD) induced the apoptotic cell injury. The level of aspartate transaminase (AST) in the culture supernatant increased, and the cell viability, estimated by mitochondrial respiration (MTT assay), decreased. The DNA fragmentation and the caspase 3-like activity, which are characterized to apoptosis, increased. When the hepatocytes were incubated with 100-500 microM 6-formylpterin, the intracellular formation of reactive oxygen species (ROS) was observed, and the ratio of reduced and oxidized glutathione (GSH/GSSG) of whole cell lysate decreased. The co-incubation of the TNF-alpha/ActD-treated hepatocytes with 100-500 microM 6-formylpterin attenuated the TNF-alpha/ActD-induced apoptotic cell injury. The level of AST decreased and the cell viability increased. Both the DNA fragmentation and the caspase 3-like activity decreased. The caspases, executors of apoptosis, are known to require a reduced cystein in their active site to function, and the intact intracellular GSH/GSSG is essential for the caspase activation. Therefore, our findings suggest that intracellular ROS generated by 6-formylpterin decline the intracellular redox state to an oxidant state, which suppresses the caspase activity and prevents the apoptotic cell injury of hepatocytes.     

Hydrogen peroxide-induced apoptosis in HL-60 cells requires caspase-3 activation

Apoptosis has been associated with oxidative stress in biological systems. Caspases have been considered to play a pivotal role in the execution phase of apoptosis. However, which caspases function as executioners in reactive oxygen species (ROS)-induced apoptosis is not known. The present study was performed to identify the major caspases acting in ROS-induced apoptosis. Treatment of HL-60 cells with 50 μM hydrogen peroxide (H₂O₂) for 4 h induced the morphological changes such as condensed and/or fragmented nuclei, increase in caspase-3 subfamily protease activities, reduction of the procaspase-3 and a DNA fragmentation. To determine the role of caspases in H₂O₂-induced apoptosis, caspase inhibitors, acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone(Ac-YVAD-cmk), acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) and acetyl-Val-Glu-lle-Aspaldehyde (Ac-VEID-CHO), selective for caspase-1 subfamily, caspase-3 subfamily and caspase-6, respectively, were loaded into the cells using an osmotic lysis of pinosomes method. Of these caspase inhibitors, only Ac-DEVD-CHO completely blocked morphological changes, caspase-3 subfamily protease activation and DNA ladder formation in H₂O₂-treated HL-60 cells. This inhibitory effect was dose-dependent. These results suggest that caspase-3, but not caspase-1 is required for commitment to ROS-triggered apoptosis.     

cAMP-guanine exchange factor protection from bile acid-induced hepatocyte apoptosis involves glycogen synthase kinase regulation of c-Jun NH2-terminal kinase

Cholestatic liver disorders are accompanied by the hepatic accumulation of cytotoxic bile acids that induce cell death. Increases in cAMP protect hepatocytes from bile acid-induced apoptosis by a cAMP-guanine exchange factor (cAMP-GEF)/phosphoinositide-3-kinase (PI3K)/Akt pathway. The aim of these studies was to identify the downstream substrate in this pathway and to determine at what level in the apoptotic cascade cytoprotection occurs. Since inhibitory phosphorylation of glycogen synthase kinase-3 (GSK) occurs downstream of PI3K/Akt and this phosphorylation has been implicated in cell survival, we conducted studies to determine whether GSK was downstream in cAMP-GEF/PI3K/Akt-mediated cytoprotection. Our results show that treatment of hepatocytes with the cAMP-GEF-specific analog, 4-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cAMP, results in PI3K-dependent phosphorylation of GSK. Direct chemical inhibition of GSK in rat hepatocytes or human HUH7-NTCP cells with several structurally and functionally distinct inhibitors including bromoindirubin-3'-oxime (BIO), maleimides (SB216763, SB415286), thiadiazolidine derivatives, and LiCl attenuates apoptosis induced by glycochenodeoxycholate (GCDC). In addition, genetic silencing of the GSK β isoform with small interfering RNA attenuates GCDC apoptosis in HUH7-NTCP cells. Adenoviral inhibition of the Rap1 blocks both cAMP-GEF-mediated cytoprotection against GCDC-induced apoptosis and Akt/GSK3β phosphorylation. GCDC-induced phosphorylation of the proapoptotic kinase, c-Jun NH(2)-terminal kinase (JNK) is inhibited by GSK inhibition or cAMP-GEF activation. GCDC-induced apoptosis is accompanied by phosphorylation of the endoplasmic reticulum stress markers pIEF2α and IRE-1, and pretreatment with the cAMP-GEF analog or GSK inhibitors prevents this phosphorylation. Collectively, our results support the presence of a cAMP/cAMP-GEF/Rap1/PI3K/Akt/GSKβ survival pathway in hepatocytes that inhibits bile acid-induced JNK phosphorylation.     

The bile acid taurochenodeoxycholate activates a phosphatidylinositol 3-kinase-dependent survival signaling cascade

Liver injury during cholestasis reflects a balance between the effects of toxic and nontoxic bile acids. However, the critical distinction between a toxic and nontoxic bile acid remains subtle and unclear. For example, the glycine conjugate of chenodeoxycholate (GCDC) induces hepatocyte apoptosis, whereas the taurine conjugate (TCDC) does not. We hypothesized that the dissimilar cellular responses may reflect differential activation of a phosphatidylinositol 3-kinase (PI3K)-dependent signaling pathway. In the bile acid-transporting McNtcp.24 rat hepatoma cell line, TCDC, but not GCDC, stimulated PI3K activity. Consistent with this observation, inhibition of PI3K rendered TCDC cytotoxic, and constitutive activation of PI3K rendered GCDC nontoxic. Both Akt and the atypical protein kinase C isoform zeta (PKCzeta) have been implicated in PI3K-dependent survival signaling. However, TCDC activated PKCzeta, but not Akt. Moreover, inhibition of PKCzeta converted TCDC into a cytotoxic agent, whereas overexpression of wild-type PKCzeta blocked GCDC-induced apoptosis. We also demonstrate that TCDC activated nuclear factor kappaB (NF-kappaB) in a PI3K- and PKCzeta-dependent manner. Moreover, inhibition of NF-kappaB by an IkappaB super-repressor rendered TCDC cytotoxic, suggesting that NF-kappaB is also necessary to prevent the cytotoxic effects of TCDC. Collectively, these data suggest that some hydrophobic bile acids such as TCDC activate PI3K-dependent survival pathways, which prevent their otherwise inherent toxicity.     

Attenuating effects of heat shock against TGF-beta1-induced apoptosis in cultured rat hepatocytes

Heat shock proteins (HSPs) induction confers protection against diverse forms of cellular injury. However, the mechanism by which HSPs exert cytoprotective effects remains unclear. Treatment of rat hepatocyte with transforming growth factor-beta1 (TGF-beta1) induces growth arrest followed by extensive cell death by apoptosis. In this study, the effects of preexposure to heat on TGF-beta1-induced apoptosis of cultured hepatocytes were examined. Treatment of hepatocytes for 24 h with TGF-beta1 resulted in significant apoptotic cell death, as demonstrated by DNA fragmentation, caspase activation, and hypodiploid DNA peak. Moreover, TGF-beta1-induced cell death was accompanied by an enhanced generation of reactive oxygen species and a loss of the mitochondrial membrane potential. These effects were attenuated when the hepatocytes were subjected to 43 degrees C for 20 min prior to the cytokine stimulation. The enhancement in HSP70 expression at mRNA and protein levels induced by heat preexposure was accompanied by an increase in mRNA levels of intracellular antioxidant enzymes. Heat treatment also prevented TGF-beta1-induced activation of nuclear factor kappa B (NF-kappaB) by preventing the degradation of the inhibitory protein kappa Balpha (IkappaBalpha). In conclusion, these data indicate that in the mechanism by which a mild heat pretreatment increases the resistance of hepatocytes to TGF-beta1-induced apoptotic cell death, the upregulation of catalase expression and a decrease in ROS generation are involved.