Riccardo Autelli

Oxidative stress activates a positive feedback between the γ‐ and β‐secretase cleavages of the β‐amyloid precursor protein

Sequential cleavages of the β‐amyloid precursor protein cleaving enzyme 1 (BACE1) by β‐secretase and γ‐secretase generate the amyloid β‐peptides, believed to be responsible of synaptic dysfunction and neuronal cell death in Alzheimer’s disease (AD). Levels of BACE1 are increased in vulnerable regions of the AD brain, but the underlying mechanism is unknown. Here we show that oxidative stress (OS) stimulates BACE1 expression by a mechanism requiring γ‐secretase activity involving the c‐jun N‐terminal kinase (JNK)/c‐jun pathway. BACE1 levels are increased in response to OS in normal cells, but not in cells lacking presenilins or amyloid precursor protein. Moreover, BACE1 is induced in association with OS in the brains of mice subjected to cerebral ischaemia/reperfusion. The OS‐induced BACE1 expression correlates with an activation of JNK and c‐jun, but is absent in cultured cells or mice lacking JNK. Our findings suggest a mechanism by which OS induces BACE1 transcription, thereby promoting production of pathological levels of amyloid β in AD.

The up-regulation of BACE1 mediated by hypoxia and ischemic injury: role of oxidative stress and HIF1α

While it is well established that stroke and cerebral hypoperfusion are both significant risk factors for Alzheimer’s disease, the molecular link between ischemia and amyloid precursor protein processing has only been recently established. Specifically, hypoxia significantly increases β‐site APP cleaving enzyme (BACE1) gene transcription through the over‐expression of hypoxia inducible factor 1α, resulting in increased BACE1 secretase activity and amyloid‐β production. In this study, we significantly extend these findings both in vitro, in differentiated SK‐N‐BE neuroblastoma cells, and in vivo, in rats subjected to cerebral ischemia, showing that hypoxia up‐regulates BACE1 expression through a biphasic mechanism. The early post‐hypoxic up‐regulation of BACE1 depends on the production of reactive oxygen species mediated by the sudden interruption of the mitochondrial electron transport chain, while the later expression of BACE1 is caused by hypoxia inducible factor 1α activation. The involvement of reactive oxygen species released by mitochondria in the BACE1 up‐regulation was confirmed by the complete protection exerted by complex I inhibitors such as rotenone and diphenyl‐phenylen iodonium. Moreover, the oxidative stress‐mediated up‐regulation of BACE1 is mediated by c‐jun N terminal kinase pathway as demonstrated by the protection exerted by the silencing of c‐jun N‐terminal kinase isoforms 1 and 2. Our study strengthens the hypothesis that oxidative stress is a basic common mechanism of amyloid‐β accumulation.

Overexpression of Bcl-2 by activated human hepatic stellate cells: resistance to apoptosis as a mechanism of progressive hepatic fibrogenesis in humans

Background and aims: Myofibroblast-like cells, originating from activation of hepatic stellate cells (HSC/MFs), play a key role in liver fibrosis, a potentially reversible process that may rely on induction of HSC/MFs apoptosis. While this possibility has been shown in cultured rat HSC, very limited data are currently available for human HSC/MFs. Methods: Cultured human HSC/MFs were exposed to several proapoptotic stimuli, including those known to induce apoptosis in rat HSC/MFs, and induction of cell death and related mechanisms were investigated using morphology, molecular biology, and biochemical techniques. Results: In this study we report that fully activated human HSC/MFs did not undergo spontaneous apoptosis and survived to prolonged serum deprivation, Fas activation, or exposure to nerve growth factor, tumour necrosis factor α (TNF-α), oxidative stress mediators, doxorubicin, and etoposide. Induction of caspase dependent, mitochondria driven apoptosis in HSC/MFs was observed only when protein synthesis or transcription were inhibited. Importantly, the process of HSC activation was accompanied by changes in expression of a set of genes involved in apoptosis control. In particular, activated human HSC/MFs in culture overexpressed Bcl-2. The role of Bcl-2 was crucial as Bcl-2 silenced cells became susceptible to TNF-α induced apoptosis. Finally, Bcl-2 was markedly expressed in HSC/MFs present in liver tissue obtained from patients with hepatitis C virus related cirrhosis. Conclusions: Human activated HSC/MFs are resistant to most proapoptotic stimuli due to Bcl-2 overexpression and this feature may play a key role in the progression of fibrosis in chronic liver diseases.

4-Hydroxynonenal as a selective pro-fibrogenic stimulus for activated human hepatic stellate cells

BACKGROUND/AIMSn4-Hydroxynonenal (HNE) is a putative pro-fibrogenic product of oxidative stress able to elicit apoptosis and cytotoxicity in several cell types. This study has been performed to evaluate its in vivo levels in injured liver and whether HNE may induce apoptosis and/or affect selected phenotypic responses in activated human hepatic stellate cells (HSC/MF).nnnMETHODS/RESULTSnDuring the development of acute liver injury induced by CCl(4), liver tissue HNE levels were in the range 0.5-10 microM, as shown by high performance liquid chromatography analysis. Cultured human HSC/MF, developed cytotoxicity only if exposed to very high HNE concentrations (25-50 microM) without any sign of induction of classic, caspase-dependent apoptosis, as assessed by evaluating morphology and biochemical parameters of cell death. HNE, at non-cytotoxic doses, up-regulated procollagen type I and tissue inhibitor of metalloproteinases-1 gene expression and/or protein synthesis without significantly affecting chemotaxis (wound healing and haptotaxis assay), matrix metalloproteinases 1 and 2 mRNA expression and activity as well as basal DNA synthesis.nnnCONCLUSIONSnHNE, at concentrations compatible with those detected in vivo, does not elicit HSC/MF classic apoptosis but, rather, may act as a potent pro-fibrogenic stimulus for the expression of genes involved in excess extracellular matrix deposition and proposed as survival signals for HSC/MF.

JNK and ERK1/2 pathways have a dual opposite effect on the expression of BACE1

The activity of beta-secretase (BACE1), the endo-protease essential for the production of amyloid beta (Abeta) peptides, is increased in brain of late-onset sporadic Alzheimers disease (AD), and oxidative stress is the potential cause of this event. Oxidative stress up-regulates the expression and the activity of BACE1 in cellular and animal models, through a mechanism that involves the increase of gamma-secretase cleavage on APP and the activation of c-jun N-terminal kinase/activator protein 1 (JNK/AP1) pathway. We further characterized the cellular pathways that control BACE1 expression under oxidative stress. We investigated the involvement of extracellular signal regulated MAP kinase (ERK1/2) pathway in the regulation of BACE1 expression, since it has been recently shown that ERK1/2 is an endogenous regulator of the gamma-secretase activity. We found that ERK1/2 pathway negatively modulates BACE1 expression and activity. Moreover, we observed that conditions that abrogate the gamma-secretase activity favor the activation of signalling pathways that promote cell survival, such as ERK1/2 and the serine/threonine kinase Akt/protein kinase B (Akt). These data suggest that the positive or negative cellular responses to oxidative stress parallel the activities of the beta- and the gamma-secretase. ERK1/2 and JNK pathways are involved in this bipartite response, which can lead to neurodegeneration or neuroprotection depending on the cellular and environmental conditions or cooperation with other signalling pathways such as Akt cascade.

Expression of Cox-2 in human breast cancer cells as a critical determinant of epithelial-to-mesenchymal transition and invasiveness

Introduction: Cyclooxygenase-2 (COX-2) is overexpressed in several malignancies and is implicated in breast cancer progression. Objectives: We investigated whether changes in COX-2 expression may affect epithelial-to-mesenchymal transition (EMT) and then invasive potential of human breast cancer cells, in relationship with hypoxia. COX-2-null MCF-7 human breast cancer cells, MCF-7 cells transiently expressing COX-2 and COX-2-expressing MDA-MB-231 cells were employed. Results: COX-2 overexpression resulted in downregulation of E-cadherin and β-catenin, upregulation of vimentin, N-cadherin and SNAI1, suggesting EMT occurrence. COX-2-overexpressing MCF-7 cells were also characterized by increased invasiveness and release of matrix-metalloproteinase-9. The above-mentioned characteristics, homologous to those detected in highly invasive MDA-MB-231 cells, were reverted by treatment of COX-2-overexpressing MCF-7 cells with celecoxib, a COX-2-specific inhibitor, partly through the inhibition of COX-2-related intracellular generation of reactive oxygen species. Hypoxia further exacerbated COX-2 expression, EMT changes and invasive ability in both COX-2-overexpressing MCF-7 cells and MDA-MB-231 cells. Finally, immunohistochemistry performed on samples from normal and neoplastic human breast tissues revealed that COX-2-positive malignant cells were also positive for EMT-related antigens, hypoxia-inducible factor (HIF)-2α and the oxidative stress marker heme oxygenase. Conclusions: These findings support the existence of a direct link between COX-2 overexpression, EMT and invasiveness in human breast cancer cells, emphasizing the role of hypoxic microenvironment.

Sphingosine mediates TNFα-induced lysosomal membrane permeabilization and ensuing programmed cell death in hepatoma cells

Normally, cell proliferation and death are carefully balanced in higher eukaryotes, but one of the most important regulatory mechanisms, apoptosis, is upset in many malignancies, including hepatocellular-derived ones. Therefore, reinforcing cell death often is mandatory in anticancer therapy. We previously reported that a combination of tumor necrosis factor-α (TNF) and cycloheximide (CHX) efficiently kill HTC cells, a rat hepatoma line, in an apoptosis-like mode. Death is actively mediated by the lysosomal compartment, although lysosomal ceramide was previously shown not to be directly implicated in this process. In the present study, we show that TNF/CHX increase lysosomal ceramide that is subsequently converted into sphingosine. Although ceramide accumulation does not significantly alter the acidic compartment, the sphingosine therein generated causes lysosomal membrane permeabilization (LMP) followed by relocation of lysosomal cathepsins to the cytoplasm. TNF/CHX-induced LMP is effectively abrogated by siRNAs targeting acid sphingomyelinase or acid ceramidase, which prevent both LMP and death induced by TNF/CHX. Taken together, our results demonstrate that lysosomal accumulation of ceramide is not detrimental per se, whereas its degradation product sphingosine, which has the capacity to induce LMP, appears responsible for the observed apoptotic-like death.

Dose-Dependent Inhibition of Cell Proliferation Induced by Lipid Peroxidation Products in Rat Hepatoma Cells After Enrichment with Arachidonic Acid

Polyunsaturated fatty acids (PUFA) are important constituents of membrane phospholipids, whose levels are decreased in some tumor cells. This deficiency may cause alterations in signal transduction and an interruption of normal cellular events. The enrichment of tumor cells with PUFA may stimulate or inhibit tumor growth, probably depending on the type of PUFA and the cellular concentration of aldehydes derived from restored lipid peroxidation. We examined the effect of several doses of prooxidant on the growth of hepatoma cells with different aldehyde dehydrogenase activities, enriched with arachidonic acid. Two doses of prooxidant were sufficient to reduce growth of hepatoma cells with low aldehyde dehydrogenase activity, whereas three doses were necessary for those with high enzyme activity. In both cases, lipid peroxidation products blocked the cells in the S phase.

Inhibition of the high affinity Ca2+-ATPase activity in rat liver plasma membranes following carbon tetrachloride intoxication

In vivo administration of CCl4 (2.5 ml/kg, body wt.) to rats results in an early and then progressive inhibition of the high affinity Ca2(+)-ATPase activity in rat liver plasma membranes. The derangement to the Ca2(+)-ATPase seems to be independent on a solvent effect of the agent since the in vitro addition of increasing concentrations of either CCl4 or ethanol to control plasma membranes does not affect the enzymatic activity. By using the technique of vitamin E pretreatment of experimental animals we show that the damage to the Ca2(+)-ATPase seems to follow a two-step kinetics. The early inhibition of the enzyme is not prevented by alpha-tocopherol supplementation and seems then unrelated to lipid peroxidative processes. The same procedure is however able to affort a significant protection against the exacerbation of the damage to the Ca2(+)-ATPase becoming evident late during the course of CCl4 intoxication. The high affinity Ca2(+)-ATPase is affected in vitro by 4-hydroxy-nonenal (HNE), a major end-product of lipid peroxidation interacting with -SH groups. Similar results were obtained after the addition to the incubation medium of sulphydryl reagents. The possible mechanisms involved in Ca2(+)-ATPase inhibition are discussed in relation to the development of CCl4 toxicity and to the role of lipid peroxidative processes.

Agmatine inhibits the proliferation of rat hepatoma cells by modulation of polyamine metabolism

BACKGROUND/AIMSnPrevious experiments have shown that agmatine, the product of arginine decarboxylase, is transported in competition with putrescine into quiescent rat hepatocytes, where it promotes several effects, including marked decrease of intracellular polyamines and induction of apoptosis. The primary aim of the present study was to assess the action of agmatine on transformed and proliferating hepatic rat cells.nnnMETHODSnTo assess the effect of agmatine on hepatoma cells, analysis by flow cytometry, Western blotting, reverse transcription-polymerase chain reaction, scanning and transmission electron microscopy, immunofluorescence detection of beta-actin and alpha-tubulin were performed.nnnRESULTSnThe results showed that agmatine has antiproliferative effects on the cell lines studied (HTC, JM2, HepG2). Further experiments were performed on HTC cells. The effect was proportional to agmatine concentration (in a range between 50 and 500 microM). It was not correlated with induction of necrosis or apoptosis and was accompanied by accumulation in G(2)/M cell cycle phase and by dramatic modification of cell morphology. Spermidine reversed these effects, suggesting that the marked decrease of the polyamine pool is the main target of agmatine .nnnCONCLUSIONSnThe results obtained show a relationship between the decrease of intracellular polyamine content, the rate of cell growth and the cytoskeleton organization.