cGMP favors the interaction between APP and BACE1 by inhibiting Rab5 GTPase activity.

We previously demonstrated that cyclic guanosine monophosphate (cGMP) stimulates amyloid precursor protein (APP) and beta-secretase (BACE1) approximation in neuronal endo-lysosomal compartments, thus boosting the production of amyloid-ß (Aß) peptides and enhancing synaptic plasticity and memory. Here, we further investigated the mechanism by which cGMP regulates the subcellular localization of APP and BACE1, finding that the cyclic nucleotide inhibits the activity of Rab5, a small GTPase associated with the plasma membrane and early endosomes. Accordingly, we also found that expression of a dominant-negative Rab5 mutant increases both APP-BACE1 approximation and Aß extracellular levels, therefore mimicking the effects induced by cGMP. These results reveal a functional correlation between the cGMP/Aß pathway and the activity of Rab5 that may contribute to the understanding of Alzheimer's disease pathophysiology.

HO-1 downregulation favors BRAFV600 melanoma cell death induced by Vemurafenib/PLX4032 and increases NK recognition.

Heme oxygenase 1 (HO-1) plays a pivotal role in preventing cell damage. Indeed, through the antioxidant, antiapoptotic and anti-inflammatory properties of its metabolic products, it favors cell adaptation against different stressors. However, HO-1 induction has also been related to the gain of resistance to therapy in different types of cancers and its involvement in cancer immune-escape has been hypothesized. We have investigated the role of HO-1 expression in Vemurafenib-treated BRAFV600 melanoma cells in modulating their susceptibility to NK cell-mediated recognition. Different cell lines, isolated in house from melanoma patients, have been exposed to 1-10 µM PLX4032, which efficiently reduced ERK phosphorylation. In three lines, Vemurafenib was able to induce only a limited decrease in cell viability, while HO-1 expression was upregulated. HO-1 silencing/inhibition was able to induce a further significant reduction of Vemurafenib-treated melanoma viability. Moreover, while NK cell degranulation and killing activity were decreased upon interaction with melanoma exposed to Vemurafenib, HO-1 silencing was able to completely restore NK cell ability to degranulate and kill. Furthermore, melanoma cell treatment with Vemurafenib downregulated the expression of ligands of NKp30 and NKG2D activating receptors, and HO-1 silencing/inhibition was able to restore their expression. Our results indicate that HO-1 downregulation can both improve the efficacy of Vemurafenib on melanoma cells and favor melanoma susceptibility to NK cell-mediated recognition and killing.

microRNA-494 Favors HO-1 Expression in Neuroblastoma Cells Exposed to Oxidative Stress in a Bach1-Independent Way.

Heme oxygenase 1 (HO-1) is crucially involved in cell adaptation to oxidative stress and has been demonstrated to play an important role in cancer progression and resistance to therapies. We recently highlighted that undifferentiated neuroblastoma (NB) cells are prone to counteract oxidative stress through the induction of HO-1. Conversely, differentiated NB cells were more sensitive to oxidative stress since HO-1 was scarcely upregulated. In this work, we investigated the role played by miR-494, which has been proved to be involved in cancer biology and in the modulation of oxidative stress, in the upregulation of HO-1. We showed that NB differentiation downregulates miR-494 level. In addition, endogenous miR-494 inhibition in undifferentiated cells impairs HO-1 induction in response to exposure to 500 µM H2O2, reducing the number of viable cells. The analysis of Bach1 expression did not reveal any significant modifications in any experimental conditions tested, proving that the impairment of HO-1 induction observed in cells treated with miR-494 inhibitor and exposed to H2O2 is independent from Bach1. Our results underline the role played by miR-494 in favoring HO-1 induction and cell adaptation to oxidative stress and contribute to the discovery of new potential pharmacological targets to improve anticancer therapies.

Investigating the amyloid-beta enhancing effect of cGMP in neuro2a cells.

Long-term potentiation (LTP) and the process of memory formation require activation of cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) pathways. Notably, recent evidence indicated that both cyclic nucleotides boost the production of amyloid-beta (Aß) peptides. In particular, cAMP was shown to favor hippocampal LTP by stimulating the synthesis of the amyloid precursor protein APP, whereas cGMP was found to enhance LTP and to improve memory by increasing Aß levels without affecting the expression of APP. The results of the present study substantiate that cGMP has a role in the endocytic pathway of APP and suggest a scenario where the cyclic nucleotide enhances the production of Aß by favoring the trafficking of APP from the cell cortex to the endolysosomal compartment.

HO-1 Induction in Cancer Progression: A Matter of Cell Adaptation.

The upregulation of heme oxygenase-1 (HO-1) is one of the most important mechanisms of cell adaptation to stress. Indeed, the redox sensitive transcription factor Nrf2 is the pivotal regulator of HO-1 induction. Through the antioxidant, antiapoptotic, and antinflammatory properties of its metabolic products, HO-1 plays a key role in healthy cells in maintaining redox homeostasis and in preventing carcinogenesis. Nevertheless, several lines of evidence have highlighted the role of HO-1 in cancer progression and its expression correlates with tumor growth, aggressiveness, metastatic and angiogenetic potential, resistance to therapy, tumor escape, and poor prognosis, even though a tumor- and tissue-specific activity has been observed. In this review, we summarize the current literature regarding the pro-tumorigenic role of HO-1 dependent tumor progression as a promising target in anticancer strategy.

Amyloid ß: Walking on the dark side of the moon.

For some decades, amyloid ß (Aß) has only been considered as a cytotoxic peptide, putative cause and marker of Alzheimer's disease (AD). Today, however, a considerable amount of evidence goes against the classical amyloid hypothesis and illustrates a new picture in which the Aß loss of function, rather than its accumulation, has a pathogenic role in AD. In this concise review, we summarize some highlights of a collection of research pointing to the physiological function of Aß and its role in the mechanisms of memory formation.

Monomeric Aß1-42 and RAGE: key players in neuronal differentiation.

The aggregation of amyloid-ß (Aß) peptides plays a crucial role in the onset and progression of Alzheimer's disease. Monomeric form of Aß, indeed, could exert a physiological role. Considering the anti-oligomerization property of all-trans retinoic acid (ATRA), the involvement of monomeric Aß1-42 in ATRA-induced neuronal differentiation has been investigated. Four-day ATRA treatment increases ß-secretase 1 (BACE1) level, Aß1-42 production, and receptor for advanced glycation end-products (RAGE) expression. RAGE is a well-recognized receptor for Aß, and the block of both RAGE and Aß1-42 with specific antibodies strongly impairs neurite formation in ATRA-treated cells. The involvement of Aß1-42 and RAGE in ATRA-induced morphologic changes has been confirmed treating undifferentiated cells with different molecular assemblies of peptide: 1 µM monomeric, but not oligomeric, Aß1-42 increases RAGE expression and favors neurite elongation. The block of RAGE completely prevents this effect. Furthermore, our data underline the involvement of the RAGE-dependent adhesion molecule amphoterin-induced gene and open reading frame-1 as downstream effector of both ATRA and Aß1-42. In conclusion, our findings identify a novel physiological role for monomeric Aß1-42 and RAGE in neuronal differentiation.

Structural alterations of human serum albumin caused by glycative and oxidative stressors revealed by circular dichroism analysis.

The aim of this work was to evaluate the ability of oxidative and glycative stressors to modify properties of human serum albumin (HSA) by analyzing markers of glycation (pentosidine) and oxidation (advanced oxidative protein products (AOPPs)) and assessing fluorescence and circular dichroism. HSA was incubated for up to 21 days with ribose, ascorbic acid (AA) and diethylenetriamine pentacetate (DTPA) in various combinations in order to evaluate influences of these substances on the structure of HSA. Ribose was included as a strong glycative molecule, AA as a modulator of oxidative stress, and DTPA as an inhibitor of metal-catalyzed oxidation. Ribose induced a significant increase in pentosidine levels. AA and DTPA prevented the accumulation of pentosidine, especially at later time points. Ribose induced a mild increase in AOPP formation, while AA was a strong inducer of AOPP formation. Ribose, in combination with AA, further increased the formation of AOPP. DTPA prevented the AA-induced generation of AOPP. Ribose was also a potent inducer of fluorescence at 335nm ex/385nm em, which is typical of pentosidine. AA and DTPA prevented this fluorescence. Circular dichroism showed complex results, in which AA and DTPA were strong modifiers of the percentages of the alpha-helical structure of HSA, while ribose affected the structure of HSA only at later time points.

Cyclic adenosine monophosphate as an endogenous modulator of the amyloid-ß precursor protein metabolism.

Besides playing a pathogenic role in Alzheimer disease, amyloid-beta peptides are normally produced in low amounts in the brain, and several lines of evidence suggest that they can modulate synaptic plasticity and memory. As cyclic adenosine monophosphate (cAMP) is known to be involved in the same processes and the blockade of its degradation by phosphodiesterase 4 inhibitors has consistently shown beneficial effects on cognition, we investigated the possible correlation between this second messenger and Aß peptides in neuronal N2a cells overexpressing the amyloid-ß precursor protein (APP). We herein report that the elevation of endogenous cAMP by rolipram increased APP protein expression and both its amyloidogenic and nonamyloidogenic processing. The effects of rolipram were reproduced by both the cAMP membrane-permeant analog 8Br-cAMP and the forskolin-induced activation of adenylyl cyclase but were not affected by the PKA inhibitor H-89. Our results demonstrate that, in neuronal cells, APP metabolism is physiologically modulated by cAMP and suggest that this might represent an additional mechanism through which the second messenger could influence memory functions.

Evaluation of the cytotoxic effects of humid lightweight coal ash derived from the disposal of waste on normal human keratinocyte and endothelial cell lines in 2-D and 3-D culture.

The presence of waste in the environment has frequently been indicated as a significant risk to human health. Therefore, landfill sites and the disposal of urban solid and non-hazardous waste by incineration are subject to much environmental monitoring, in addition to the regulations already in place. However, little action has been taken, and consequently no specific legislation exists, in relation to the assessment of the real biological risk of various substances, including chemical mixtures and ashes, derived from the incineration processes. This study assessed the cytotoxic potential of humid lightweight coal ash (LA) derived from incineration processes and waste management, on two cell lines: NCTC 2544 normal human keratinocytes and HECV endothelial cells. To reach this goal and to assess more-realistic methods for animal replacement, we employed different in vitro experimental approaches: acute and longer exposure to LA, by direct and indirect contact (0-2mg/ml and 16mg, respectively), both in 2-D and 3-D cultures. In 2-D HECV cultures, we observed a decrease in the viability index, but only during direct contact with LA doses higher than 0.1mg/ml. Moreover, some striking differences in cytotoxicity were observed between the 2-D and 3-D models. Taken together, these observations indicate that, for studying pollutant toxicity during longer exposure times, 3-D cultures in direct contact with the pollutant seem to offer a more suitable approach - they mimic the in vivo behaviour of cells more realistically and under strictly controlled conditions. Thus, in readiness for possible forthcoming European regulations, we believe that the proposed study, even in its preliminary phase, can provide new advice on the assessment of the toxic and biological potential of particular chemical compounds derived from waste management processes.

Cholesterol and Alzheimer's disease: a still poorly understood correlation.

A large amount of evidence suggests a pathogenic link between cholesterol homeostasis dysregulation and Alzheimer's disease (AD). In cell culture systems, the production of amyloid-ß (Aß) is modulated by cholesterol, and studies on animal models have consistently demonstrated that hypercholesterolemia is associated with an increased deposition of cerebral Aß peptides. Consequently, a number of epidemiological studies have examined the effects of cholesterol-lowering drugs (i.e., statins) in the prevention and the treatment of AD. However, while retrospective studies suggested a potential benefit of statin therapy, clinical trials produced inconsistent results. Here, we summarize the main findings from in vitro and in vivo research where the correlation between cholesterol and the neurodegenerative disorder was investigated. Recognition of this correlation could be an important step forward for our understanding of AD pathogenesis and, possibly, for the development of new therapeutic strategies.

Cholesterol and amyloid-ß: evidence for a cross-talk between astrocytes and neuronal cells.

Accumulating data supports the concept that alterations of cholesterol metabolism might influence the development of Alzheimer's disease (AD), a neurodegenerative disorder characterized by progressive accumulation of amyloid-ß (Aß) peptides in the brain. Changes in the neuronal production of Aß have been described as a function of cholesterol levels, thus suggesting a causal link between cholesterol homeostasis dysregulation and AD pathogenesis. Under physiological conditions, cholesterol uptake in the brain is efficiently prevented by the blood-brain barrier, and mature neurons are thought to rely on glial cells for their cholesterol supply. In the present study, we tested the hypothesis that Aß may serve as a signaling molecule capable of informing the astroglial network about the neuronal need for cholesterol. Collectively, our data bolster this hypothesis and demonstrate, for the first time, that Aß(42) exerts an inhibitory effect on the expression of the cholesterol transporter ABCA1 in cultured astrocytes. Accordingly, we also show that ABCA1 expression is reduced in the brain of AßPP/PS1 transgenic mice. These results provide a biological function for Aß peptides and may help to define the pathogenic relationship between cholesterol metabolism in brain and AD.

PKCδ sensitizes neuroblastoma cells to L-buthionine-sulfoximine and etoposide inducing reactive oxygen species overproduction and DNA damage.

Neuroblastoma is a type of pediatric cancer. The sensitivity of neuroblastoma (NB) cancer cells to chemotherapy and radiation is inhibited by the presence of antioxidants, such as glutathione (GSH), which is crucial in counteracting the endogenous production of reactive oxygen species (ROS). We have previously demonstrated that cells depleted of GSH undergo apoptosis via oxidative stress and Protein kinase C (PKC) δ activation. In the present study, we transfected PKCδ in NB cells resistant to oxidative death induced by L-buthionine-S,R-sulfoximine (BSO), a GSH-depleting agent. Cell responses, in terms of ROS production, apoptosis and DNA damage were evaluated. Moreover, PKCδ activation was monitored by analyzing the phosphorylation status of threonine 505 residue, carrying out PKC activity assay and investigating the subcellular localization of the kinase. The cell responses obtained in BSO-resistant cells were also compared with those obtained in BSO-sensitive cells subjected to the same experimental protocol. Our results demonstrate, for the first time, that PKCδ induces DNA oxidation and ROS overproduction leading to apoptosis of BSO-resistant NB cells and potentiates the cytotoxic effects induced by BSO in sensitive cells. Moreover, PKCδ overexpression enhances the sensitivity of NB cells to etoposide, a well-characterised drug, commonly used in neuroblastoma therapy. Altogether our data provide evidence of a pro-oxidant role of PKCδ that might be exploited to design new therapeutic strategies aimed at selective killing of cancer cells and overcoming drug resistance. However, it becomes evident that a more detailed understanding of ROS-mediated signaling in cancer cells is necessary for the development of redox-modulated therapeutic approaches.

Protein kinase C: an attractive target for cancer therapy.

Apoptosis plays an important role during all stages of carcinogenesis and the development of chemoresistance in tumor cells may be due to their selective defects in the intracellular signaling proteins, central to apoptotic pathways. Consequently, many studies have focused on rendering the chemotherapy more effective in order to prevent chemoresistance and pre-clinical and clinical data has suggested that protein kinase C (PKC) may represent an attractive target for cancer therapy. Therefore, a complete understanding of how PKC regulates apoptosis and chemoresistance may lead to obtaining a PKC-based therapy that is able to reduce drug dosages and to prevent the development of chemoresistance.

DNA oxidative damage of neoplastic rat liver lesions.

Hepatic lesions, experimentally-induced in Fisher 344 (F344) and Brown Norway (BN) rats, respectively, susceptible and resistant to liver carcinogenesis, progress differently to hepatocellular carcinoma (HCC). The mechanisms responsible for the acquisition of the resistant phenotype are not completely clear. Herein, we show that in F344 rats subjected to carcinogenic treatment, angiogenesis and DNA oxidation markers increase in preneoplastic and neoplastic liver lesions. On the contrary, in the HCCs of treated BN rats, angiogenesis and a minor DNA oxidation are accompanied by an attempt of tissue remodelling. This study suggests that DNA oxidation might be an important factor in the initiation and promotion of the events of hepatocarcinogenesis. On the other hand, the enhancement of GSH levels and the down-regulation of superoxide dismutase (SOD) expression in both rat strains suggest that antioxidant response is not involved in the acquisition of resistant phenotype.

Protein kinase C-dependent alpha-secretory processing of the amyloid precursor protein is mediated by phosphorylation of myosin II-B.

A substantial body of evidence indicates that protein kinase C (PKC) is involved in the alpha-secretory processing of the amyloid precursor protein (APP), an event that reduces the formation of the pathogenic amyloid-beta peptide. Recently, we have shown that trafficking and processing of APP are both impaired by knockdown of myosin II-B, one of the major neuronal motor proteins. Here, we provide evidence that the alpha-secretory processing of APP is mediated by PKC-dependent phosphorylation of myosin II-B. This signaling pathway provides an important link between APP and the neuronal cytoskeleton and might be crucial for the understanding of the biological and pathological roles of APP.

PKC signaling in oxidative hepatic damage.

Protein kinase C (PKC) is a family of isoenzymes differently involved in cell response to injury and many studies describe their role as "stress sensors". Oxidative stress is strictly involved in the pathogenesis of chronic liver diseases including alcohol- or drug-induced hepatotoxicity, iron overload, hepatitis and hepatocarcinoma development, but molecular mechanisms are not really defined. A crucial role of PKC as a redox sensitive signaling molecule has been widely accepted.

Mechanisms of BSO (L-buthionine-S,R-sulfoximine)-induced cytotoxic effects in neuroblastoma.

Glutathione (GSH) depletion is widely used to sensitize cells to anticancer treatment inducing the progression of programmed cell death and overcoming chemoresistance. It has been reported that neuroblastoma cells with MYCN amplification are unable to start TRAIL-dependent death and MYCN, in concert with cytotoxic drugs, efficiently induces the mitochondrial pathway of apoptosis through oxidative mechanisms. In this study, we show that GSH loss induced by L-buthionine-S,R-sulfoximine (BSO), an inhibitor of GSH biosynthesis, leads to overproduction of reactive oxygen species (ROS) and triggers apoptosis of MYCN-amplified neuroblastoma cells. BSO susceptibility of SK-N-BE-2C, a representative example of MYCN-amplified cells, has been attributed to stimulation of total SOD activity in the absence of changes in the level and the activity of catalase. Therefore, the unbalanced intracellular redox milieu has been demonstrated to be critical for the progression of neuroblastoma cell death that was efficiently prevented by antioxidants and rottlerin. These results describe a novel pathway of apoptosis dependent on ROS formation and PKC-delta activation and independent of p53, bcl-2, and bax levels; the selective redox modulation of PKC-delta might be suggested as a potential strategy for sensitizing MYCN-amplified cells to therapeutic approaches.

In vitro effect of PPAR-gamma2 Pro12Ala polymorphism on the deposition of Alzheimer's amyloid-beta peptides.

Mounting evidence suggests that peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is involved in the modulation of pathogenic events related to Alzheimer's disease (AD). Such events would include the cerebral deposition of amyloid-beta (Abeta) and the consequent local inflammatory response. PPAR-gamma has been shown to act on both fronts, reducing either the secretion of Abeta or the expression of pro-inflammatory cytokines. Recently, the relatively common Pro12Ala polymorphism in exon 2 of PPAR-gamma has been associated with higher risk for late onset AD. Here, we compare the effect of PPAR-gamma and its genetic variant on the secretion of Abeta. Our results indicate that, in neuronal cultured cells, the Pro12Ala substitution does not affect the anti-amyloidogenic capacity of PPAR-gamma. Additional factors, PPAR-gamma related, may therefore predispose aged subjects, carrying the Ala allele, to develop the neurodegenerative disease.

Downregulation of myosin II-B by siRNA alters the subcellular localization of the amyloid precursor protein and increases amyloid-beta deposition in N2a cells.

The Alzheimer's disease (AD) brain pathology is characterized by extracellular deposits of amyloid-beta (Abeta) peptides and intraneuronal fibrillar structures. These pathological features may be functionally linked, but the mechanism by which Abeta accumulation relates to neuronal degeneration is still poorly understood. Abeta peptides are fragments cleaved from the amyloid precursor protein (APP), a transmembrane protein ubiquitously expressed in the nervous system. Although the proteolytic processing of APP has been implicated in AD, the physiological function of APP and the subcellular site of APP cleavages remain unknown. The overall structure of the protein and its fast anterograde transport along the axon support the idea that APP functions as a vesicular receptor for cytoskeletal motor proteins. In the current study, we test the hypothesis that myosin II, important contributor to the cytoskeleton of neuronal cells, may influence the trafficking and/or the processing of APP. Our results demonstrate that downregulation of myosin II-B, the major myosin isoform in neurons, is able to increase Abeta deposition, concomitantly altering the subcellular localization of APP. These new insights might be important for the understanding of the function of APP and provide a novel conceptual framework in which to analyze its pathological role.