Rita M. Ramalho

Hepatocyte apoptosis, expression of death receptors, and activation of NF-κB in the liver of nonalcoholic and alcoholic steatohepatitis patients

OBJECTIVES:The increasing incidence of nonalcoholic (NASH) and alcoholic steatohepatitis (ASH), associated with lack of effective treatment, has prompted intensive studies on disease pathogenesis. Apoptosis is recognized as common in liver injury and may also contribute to tissue inflammation, fibrogenesis, and development of cirrhosis. In this study, we identified mechanisms of apoptosis induction in human steatohepatitis, and evaluated potential associations between apoptosis, liver pathology, and clinical presentation in NASH and ASH.METHODS:Hepatocyte apoptosis was evaluated by the TUNEL assay in 20 patients with NASH (all ambulatory), 40 with ASH (20 ambulatory, 20 hospitalized), and 20 controls. Liver biopsies were also graded for inflammation and fibrosis. Immunohistochemistry was performed for death receptors (Fas and TNF-R1), activated caspase-3, NF-κB p65, antiapoptotic Bcl-2 protein, and uncoupling protein 2 (UCP-2).RESULTS:TUNEL-positive hepatocytes were markedly increased in NASH (p < 0.05) and ASH (p < 0.01). Similar results were obtained for activated caspase-3, confirming the occurrence of apoptosis. The Fas receptor was upregulated in ASH, especially in hospitalized patients (p < 0.01), whereas TNF-R1 was expressed both in NASH and ASH (p < 0.01). In addition, patients with ASH showed a remarkable expression of active NF-κB, as compared to NASH and controls (p < 0.01). Degrees of inflammation and fibrosis correlated with NF-κB p65 expression, which in turn coincided with apoptosis albeit Bcl-2 and UCP-2 expression.CONCLUSIONS:Liver injury in NASH and ASH is associated with increased hepatocyte apoptosis mediated by death receptors. Further, apoptosis correlates with active NF-κB expression, and disease severity. This potential mechanistic link might provide multiple interesting targets for therapeutic intervention.

Bile acids: regulation of apoptosis by ursodeoxycholic acid

Bile acids are a group of molecular species of acidic steroids with peculiar physical-chemical and biological characteristics. At high concentrations they become toxic to mammalian cells, and their presence is pertinent in the pathogenesis of several liver diseases and colon cancer. Bile acid cytoxicity has been related to membrane damage, but also to nondetergent effects, such as oxidative stress and apoptosis. Strikingly, hydrophilic ursodeoxycholic acid (UDCA), and its taurine-conjugated form (TUDCA), show profound cytoprotective properties. Indeed, these molecules have been described as potent inhibitors of classic pathways of apoptosis, although their precise mode of action remains to be clarified. UDCA, originally used for cholesterol gallstone dissolution, is currently considered the first choice therapy for several forms of cholestatic syndromes. However, the beneficial effects of both UDCA and TUDCA have been tested in other experimental pathological conditions with deregulated levels of apoptosis, including neurological disorders, such as Alzheimers, Parkinsons, and Huntingtons diseases. Here, we review the role of bile acids in modulating the apoptosis process, emphasizing the anti-apoptotic effects of UDCA and TUDCA, as well as their potential use as novel and alternate therapeutic agents for the treatment of apoptosis-related diseases.

Apoptosis and Bcl-2 expression in the livers of patients with steatohepatitis.

Objectives Apoptosis may play a role in the pathogenesis of alcoholic (ASH) and non-alcoholic steatohepatitis (NASH). In this study, we investigated the modulation of apoptosis-related liver proteins in steatohepatitis. Methods Hepatocyte apoptosis was evaluated by the TUNEL assay in liver tissue of 12 patients with NASH, 12 with ASH and in histologically normal controls. In addition, caspase-3 processing was evaluated by immunoblot analysis. Expression of death receptors, Bcl-2 family members, and NF-κB inhibitor (IκB) were determined by western blot. Liver biopsies were also graded for inflammation and fibrosis. Results Apoptotic hepatocytes were markedly increased in NASH (P<0.05) and ASH (P<0.001) as compared to controls. Active caspase-3 was also elevated in steatohepatitis (P<0.01), coinciding with upregulation of pro-apoptotic Bax (P<0.001). Further, production of tumour necrosis factor-receptor 1 was increased up to 4-fold (P<0.05). Degradation of IκB increased >70% in steatohepatitis (P<0.001). Notably, Bcl-2 was also strongly expressed (>100-fold; P<0.001). These data were significantly correlated with relative degrees of portal and lobular inflammation. Conclusion The results show that liver injury in NASH and ASH is associated with apoptosis and NF-κB activation. Anti-apoptotic Bcl-2 is strongly expressed, probably reflecting an adaptive response to obesity or alcohol-related stress.

Bile acids and apoptosis modulation: an emerging role in experimental Alzheimer's disease

The potential role of apoptosis in Alzheimers disease (AD) has been an area of intense research in recent years. Ursodeoxycholic acid (UDCA) and its taurine-conjugate, tauroursodeoxycholic acid (TUDCA) are endogenous bile acids that act as potent inhibitors of apoptosis. Their therapeutic effects have been tested in many experimental pathological conditions, including neurological disorders, such as AD. TUDCA regulates precise transcriptional and post-transcriptional events that impact mitochondrial function in neurons. TUDCA not only stabilizes the mitochondrial membrane and prevents Bax translocation, inhibiting the release of cytochrome c and the activation of caspases, but also interferes with upstream factors, including cell cycle-related proteins. In addition, TUDCA is capable of inducing survival pathways. Here, we review the role of apoptosis in AD and discuss the therapeutic potential of TUDCA in treating this disease.

Nuclear translocation of UDCA by the glucocorticoid receptor is required to reduce TGF-β1–induced apoptosis in rat hepatocytes†

Ursodeoxycholic acid (UDCA) inhibits classical mitochondrial pathways of apoptosis by either directly stabilizing mitochondrial membranes or modulating specific upstream targets. Furthermore, UDCA regulates apoptosis‐related genes from transforming growth factor β1 (TGF‐β1)–induced hepatocyte apoptosis by a nuclear steroid receptor (NSR)–dependent mechanism. In this study, we further investigated the potential role of the glucocorticoid receptor (GR) in the antiapoptotic function of UDCA. Our results with short interference RNA (siRNA) technology confirmed that UDCA significantly reduces TGF‐β1–induced apoptosis of primary rat hepatocytes through a GR‐dependent effect. Immunoprecipitation assays and confocal microscopy showed that UDCA enhanced free GR levels with subsequent GR nuclear translocation. Interestingly, when a carboxy‐terminus deleted form of GR was used, UDCA no longer increased free GR and/or GR translocation, nor did it protect against TGF‐β1–induced apoptosis. In co‐transfection experiments with GR response element reporter and overexpression constructs, UDCA did not enhance the transactivation of GR with TGF‐β1. Finally, using a flourescently labeled UDCA molecule, the bile acid appeared diffuse in the cytosol but was aggregated in the nucleus of hepatocytes. Both siRNA assays and transfection experiments with either wild‐type or mutant forms of GR showed that nuclear trafficking occurs through a GR‐dependent mechanism. In conclusion, these results further clarify the antiapoptotic mechanism(s) of UDCA and suggest that GR is crucial for the nuclear translocation of this bile acid for reducing apoptosis. (HEPATOLOGY 2005;42:925–934.)

Inhibition of the E2F-1/p53/Bax pathway by tauroursodeoxycholic acid in amyloid β-peptide-induced apoptosis of PC12 cells

Amyloid β‐peptide (Aβ)‐induced cell death may involve activation of the E2F‐1 transcription factor and other cell cycle‐related proteins. In previous studies, we have shown that tauroursodeoxycholic acid (TUDCA), an endogenous bile acid, modulates Aβ‐induced apoptosis by interfering with crucial events of the mitochondrial pathway. In this study, we examined the role of E2F and p53 activation in the induction of apoptosis by Aβ, and investigated novel molecular targets for TUDCA. The results showed that despite Bcl‐2 up‐regulation, PC12 neuronal cells underwent significant apoptosis after incubation with the active fragment Aβ (25–35), as assessed by DNA fragmentation, nuclear morphology and caspase‐3‐like activation. In addition, transcription through the E2F‐1 promoter was significantly induced and associated with loss of the retinoblastoma protein. In contrast, levels of E2F‐1, p53 and Bax proteins were markedly increased. Overexpression of E2F‐1 in PC12 cells was sufficient to induce p53 and Bax proteins, as well as nuclear fragmentation. Notably, TUDCA modulated Aβ‐induced apoptosis, E2F‐1 induction, p53 stabilization and Bax expression. Further, TUDCA protected PC12 cells against p53‐ and Bax‐dependent apoptosis induced by E2F‐1 and p53 overexpression, respectively. In conclusion, the results demonstrate that Aβ‐induced apoptosis of PC12 cells proceeds through an E2F‐1/p53/Bax pathway, which, in turn, can be specifically inhibited by TUDCA, thus underscoring its potential therapeutic use.

Tauroursodeoxycholic acid modulates p53-mediated apoptosis in Alzheimer's disease mutant neuroblastoma cells.

Early onset familial Alzheimers disease (FAD) is linked to autosomal dominant mutations in the amyloid precursor protein (APP) and presenilin 1 and 2 (PS1 and PS2) genes. These are critical mediators of total amyloid β‐peptide (Aβ) production, inducing cell death through uncertain mechanisms. Tauroursodeoxycholic acid (TUDCA) modulates exogenous Aβ‐induced apoptosis by interfering with E2F‐1/p53/Bax. Here, we used mouse neuroblastoma cells that express either wild‐type APP, APP with the Swedish mutation (APPswe), or double‐mutated human APP and PS1 (APPswe/ΔE9), all exhibiting increased Aβ production and aggregation. Cell viability was decreased in APPswe and APPswe/ΔE9 but was partially reversed by z‐VAD.fmk. Nuclear fragmentation and caspase 2, 6 and 8 activation were also readily detected. TUDCA reduced nuclear fragmentation as well as caspase 2 and 6, but not caspase 8 activities. p53 activity, and Bcl‐2 and Bax changes, were also modulated by TUDCA. Overexpression of p53, but not mutant p53, in wild‐type and mutant neuroblastoma cells was sufficient to induce apoptosis, which, in turn, was reduced by TUDCA. In addition, inhibition of the phosphatidylinositide 3′‐OH kinase pathway reduced TUDCA protection against p53‐induced apoptosis. In conclusion, FAD mutations are associated with the activation of classical apoptotic pathways. TUDCA reduces p53‐induced apoptosis and modulates expression of Bcl‐2 family.

Tauroursodeoxycholic acid prevents E22Q Alzheimer’s Aβ toxicity in human cerebral endothelial cells

Abstract.The vasculotropic E22Q mutant of the amyloid-β (Aβ) peptide is associated with hereditary cerebral hemorrhage with amyloidosis Dutch type. The cellular mechanism(s) of toxicity and nature of the AβE22Q toxic assemblies are not completely understood. Comparative assessment of structural parameters and cell death mechanisms elicited in primary human cerebral endothelial cells by AβE22Q and wild-type Aβ revealed that only AβE22Q triggered the Bax mitochondrial pathway of apoptosis. AβE22Q neither matched the fast oligomerization kinetics of Aβ42 nor reached its predominant β-sheet structure, achieving a modest degree of oligomerization with a secondary structure that remained a mixture of β and random conformations. The endogenous molecule tauroursodeoxycholic acid (TUDCA) was a strong modulator of AβE22Q-triggered apoptosis but did not significantly change the secondary structures and fibrillogenic propensities of Aβ peptides. These data dissociate the pro-apoptotic properties of Aβ peptides from their distinct mechanisms of aggregation/fibrillization in vitro, providing new perspectives for modulation of amyloid toxicity.

Induction of apoptosis in HuH-7 cancer cells by monoterpene and β-carboline indole alkaloids isolated from the leaves of Tabernaemontana elegans

Three known (1-3) and a novel (4) monoterpene indole alkaloids have been isolated from the methanol extract of leaves of Tabernaemontana elegans and their structures were elucidated by a series of spectroscopic experiments, involving NMR, MS, UV, and IR techniques. The isolated monoterpene indole alkaloids along with previously described beta-carbolines (5-7) from the same specimen were studied for their apoptosis induction activity in human hepatoma HuH-7 cells. Methodology for apoptosis induction studies included cell viability assays, nuclear morphology assessments, and general caspase-3-like activity assays. The monoterpene indole alkaloids, tabernaemontanine (1) and vobasine (3) showed the most promising apoptosis induction profile in HuH-7 cells.

Isoflavones as Apoptosis Inducers in Human Hepatoma HuH‐7 Cells

Nine flavonoids isolated from the ethyl acetate extract of Pycnanthus angolensis were assayed for their potential apoptosis induction activities in human hepatoma HuH‐7 cells. These flavonoids include eight isoflavones, namely irilone (1), tectorigenine (2), formononetin (3), genistein (4), 2′‐hydroxybiochanin A (5), mixture of biochanin A (6) and prunetin (7), and 4′,7‐dihydroxy‐2′‐methoxyisoflavan (8), and the flavanone liguiritigentin (9). Their chemical structures were characterized by spectroscopic methods including 2D NMR experiments. Methodology for cell death detection included the LDH assay, Hoechst staining, TUNEL staining and general caspase‐3‐like activity assay. The compounds tested showed higher apoptosis induction profiles in HuH‐7 cells compared with the control. Caspase activity assays confirmed the apoptosis inducing activity of these flavonoids. Copyright