Lysosomal degradation ensures accurate chromosomal segregation to prevent chromosomal instability.

Lysosomes, as primary degradative organelles, are the endpoint of different converging pathways, including macroautophagy. To date, lysosome degradative function has been mainly studied in interphase cells, while their role during mitosis remains controversial. Mitosis dictates the faithful transmission of genetic material among generations, and perturbations of mitotic division lead to chromosomal instability, a hallmark of cancer. Heretofore, correct mitotic progression relies on the orchestrated degradation of mitotic factors, which was mainly attributed to ubiquitin-triggered proteasome-dependent degradation. Here, we show that mitotic transition also relies on lysosome-dependent degradation, as impairment of lysosomes increases mitotic timing and leads to mitotic errors, thus promoting chromosomal instability. Furthermore, we identified several putative lysosomal targets in mitotic cells. Among them, WAPL, a cohesin regulatory protein, emerged as a novel SQSTM1-interacting protein for targeted lysosomal degradation. Finally, we characterized an atypical nuclear phenotype, the toroidal nucleus, as a novel biomarker for genotoxic screenings. Our results establish lysosome-dependent degradation as an essential event to prevent chromosomal instability.Abbreviations: 3D: three-dimensional; APC/C: anaphase-promoting complex; ARL8B: ADP ribosylation factor like GTPase 8B; ATG: autophagy-related; BORC: BLOC-one-related complex; CDK: cyclin-dependent kinase; CENPE: centromere protein E; CIN: chromosomal instability; ConcA: concanamycin A; CQ: chloroquine; DAPI: 4,6-diamidino-2-penylinole; FTI: farnesyltransferase inhibitors; GFP: green fluorescent protein; H2B: histone 2B; KIF: kinesin family member; LAMP2: lysosomal associated membrane protein 2; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; MTOR: mechanistic target of rapamycin kinase; PDS5B: PDS5 cohesin associated factor B; SAC: spindle assembly checkpoint; PLEKHM2: pleckstrin homology and RUN domain containing M2; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; ULK1: unc-51 like autophagy activating kinase 1; UPS: ubiquitin-proteasome system; v-ATPase: vacuolar-type H+-translocating ATPase; WAPL: WAPL cohesion release factor.

Phosphofructokinases Axis Controls Glucose-Dependent mTORC1 Activation Driven by E2F1.

Cancer cells rely on mTORC1 activity to coordinate mitogenic signaling with nutrients availability for growth. Based on the metabolic function of E2F1, we hypothesize that glucose catabolism driven by E2F1 could participate on mTORC1 activation. Here, we demonstrate that glucose potentiates E2F1-induced mTORC1 activation by promoting mTORC1 translocation to lysosomes, a process that occurs independently of AMPK activation. We showed that E2F1 regulates glucose metabolism by increasing aerobic glycolysis and identified the PFKFB3 regulatory enzyme as an E2F1-regulated gene important for mTORC1 activation. Furthermore, PFKFB3 and PFK1 were found associated to lysosomes and we demonstrated that modulation of PFKFB3 activity, either by substrate accessibility or expression, regulates the translocation of mTORC1 to lysosomes by direct interaction with Rag B and subsequent mTORC1 activity. Our results support a model whereby a glycolytic metabolon containing phosphofructokinases transiently interacts with the lysosome acting as a sensor platform for glucose catabolism toward mTORC1 activity.

Mechanisms of CPT1C-Dependent AMPAR Trafficking Enhancement.

In neurons, AMPA receptor (AMPAR) function depends essentially on their constituent components:the ion channel forming subunits and ion channel associated proteins. On the other hand, AMPAR trafficking is tightly regulated by a vast number of intracellular neuronal proteins that bind to AMPAR subunits. It has been recently shown that the interaction between the GluA1 subunit of AMPARs and carnitine palmitoyltransferase 1C (CPT1C), a novel protein partner of AMPARs, is important in modulating surface expression of these ionotropic glutamate receptors. Indeed, synaptic transmission in CPT1C knockout (KO) mice is diminished supporting a positive trafficking role for that protein. However, the molecular mechanisms of such modulation remain unknown although a putative role of CPT1C in depalmitoylating GluA1 has been hypothesized. Here, we explore that possibility and show that CPT1C effect on AMPARs is likely due to changes in the palmitoylation state of GluA1. Based on in silico analysis, Ser 252, His 470 and Asp 474 are predicted to be the catalytic triad responsible for CPT1C palmitoyl thioesterase (PTE) activity. When these residues are mutated or when PTE activity is inhibited, the CPT1C effect on AMPAR trafficking is abolished, validating the CPT1C catalytic triad as being responsible for PTE activity on AMPAR. Moreover, the histidine residue (His 470) of CPT1C is crucial for the increase in GluA1 surface expression in neurons and the H470A mutation impairs the depalmitoylating catalytic activity of CPT1C. Finally, we show that CPT1C effect seems to be specific for this CPT1 isoform and it takes place solely at endoplasmic reticulum (ER). This work adds another facet to the impressive degree of molecular mechanisms regulating AMPAR physiology.

Effect of low doses of actinomycin D on neuroblastoma cell lines.

BACKGROUND: Neuroblastoma is a malignant embryonal tumor occurring in young children, consisting of undifferentiated neuroectodermal cells derived from the neural crest. Current therapies for high-risk neuroblastoma are insufficient, resulting in high mortality rates and high incidence of relapse. With the intent to find new therapies for neuroblastomas, we investigated the efficacy of low-doses of actinomycin D, which at low concentrations preferentially inhibit RNA polymerase I-dependent rRNA trasncription and therefore, ribosome biogenesis. METHODS: Neuroblastoma cell lines with different p53 genetic background were employed to determine the response on cell viability and apoptosis of low-dose of actinomycin D. Subcutaneously-implanted SK-N-JD derived neuroblastoma tumors were used to assess the effect of low-doses of actinomycin D on tumor formation. RESULTS: Low-dose actinomycin D treatment causes a reduction of cell viability in neuroblastoma cell lines and that this effect is stronger in cells that are wild-type for p53. MYCN overexpression contributes to enhance this effect, confirming the importance of this oncogene in ribosome biogenesis. In the wild-type SK-N-JD cell line, apoptosis was the major mechanism responsible for the reduction in viability and we demonstrate that treatment with the MDM2 inhibitor Nutlin-3, had a similar effect to that of actinomycin D. Apoptosis was also detected in p53(-/-)deficient LA1-55n cells treated with actinomycin D, however, only a small recovery of cell viability was found when apoptosis was inhibited by a pan-caspase inhibitor, suggesting that the treatment could activate an apoptosis-independent cell death pathway in these cells. We also determined whether actinomycin D could increase the efficacy of the histone deacetylase inhibitor, SAHA, which is in being used in neuroblastoma clinical trials. We show that actinomycin D synergizes with SAHA in neuroblastoma cell lines. Moreover, on subcutaneously-implanted neuroblastoma tumors derived from SK-N-JD cells, actinomycin D led to tumor regression, an effect enhanced in combination with SAHA. CONCLUSIONS: The results presented in this work demonstrate that actinomycin D, at low concentrations, inhibits proliferation and induces cell death in vitro, as well as tumor regression in vivo. From this study, we propose that use of ribosome biogenesis inhibitors should be clinically considered as a potential therapy to treat neuroblastomas.

Dopamine induces mitochondrial depolarization without activating PINK1-mediated mitophagy.

Parkinson's disease (PD) is one of the most prevalent neurodegenerative disorders, characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta. PD mostly occurs sporadically and its cause remains unknown, nevertheless the discovery of familiar forms of PD, characterized by mutations of genes encoding proteins associated with mitochondria homeostasis, suggests a strong implication of the mitochondrial quality control system in PD. We investigated the effect of dopamine cytosolic accumulation in undifferentiated SH-SY5Y cells, an in vitro model widely used to reproduce impairment of dopamine homeostasis, an early step in PD pathogenesis. A strong depolarization of the mitochondrial membrane was observed after dopamine exposure. Nevertheless, mitochondrial network resulted to assume a peculiar morphology with a distinct pattern of OPA1 and MFN1, key regulators of mitochondrial dynamics. Moreover, selective elimination of dysfunctional mitochondria did not take place, suggesting an impairment of the mitophagic machinery induced by dopamine. Indeed, PINK1 did not accumulate on the outer mitochondrial membrane, nor was parkin recruited to depolarized mitochondria. Altogether, our results indicate that an improper handling of dysfunctional mitochondria may be a leading event in PD pathogenesis. Impaired dopamine (DA) homeostasis and oxidative stress play a key role in the pathogenesis of Parkinson's disease. Free cytosolic dopamine undergoes spontaneous oxidation and generates semiquinonic and quinonic species (DAQ) with the concurrent production of reactive oxygen species (ROS). Dopamine dissipates mitochondrial potential (Δψm ) with a peculiar alteration of the mitochondrial network. However, PINK1-dependent mitophagy is not activated by dopamine toxicity and dysfunctional mitochondria accumulate inside the cell.

V-ATPase: a master effector of E2F1-mediated lysosomal trafficking, mTORC1 activation and autophagy.

In addition to being a master regulator of cell cycle progression, E2F1 regulates other associated biological processes, including growth and malignancy. Here, we uncover a regulatory network linking E2F1 to lysosomal trafficking and mTORC1 signaling that involves v-ATPase regulation. By immunofluorescence and time-lapse microscopy we found that E2F1 induces the movement of lysosomes to the cell periphery, and that this process is essential for E2F1-induced mTORC1 activation and repression of autophagy. Gain- and loss-of-function experiments reveal that E2F1 regulates v-ATPase activity and inhibition of v-ATPase activity repressed E2F1-induced lysosomal trafficking and mTORC1 activation. Immunoprecipitation experiments demonstrate that E2F1 induces the recruitment of v-ATPase to lysosomal RagB GTPase, suggesting that E2F1 regulates v-ATPase activity by enhancing the association of V0 and V1 v-ATPase complex. Analysis of v-ATPase subunit expression identified B subunit of V0 complex, ATP6V0B, as a transcriptional target of E2F1. Importantly, ATP6V0B ectopic-expression increased v-ATPase and mTORC1 activity, consistent with ATP6V0B being responsible for mediating the effects of E2F1 on both responses. Our findings on lysosomal trafficking, mTORC1 activation and autophagy suppression suggest that pharmacological intervention at the level of v-ATPase may be an efficacious avenue for the treatment of metastatic processes in tumors overexpressing E2F1.

MYCN concurrence with SAHA-induced cell death in human neuroblastoma cells.

BACKGROUND: In the past, the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) has been shown to induce apoptosis in several human tumor types, including neuroblastomas. Amplification and over-expression of the MYCN oncogene is a diagnostic hallmark and a poor prognostic indicator in high-risk neuroblastomas. Here, we studied the relationship between MYCN amplification and over-expression and the anti-tumor effect of SAHA to assess whether this drug may serve as a treatment option for high-risk neuroblastomas. METHODS: Different human neuroblastoma cell lines, over-expressing or not over-expressing MYCN, were used in this study. Targeted knockdown and exogenous over-expression of MYCN were employed to examine correlations between MYCN expression levels and SAHA responses. After various time periods and concentration exposures to the drug, cell viability was measured by MTS assay, and variations in MYCN mRNA and protein levels were assessed by qPCR and Western blotting, respectively. RESULTS: We found that SAHA decreased cell viability in all cell lines tested through apoptosis induction, and that SAHA had a stronger effect on cell lines carrying an amplified MYCN gene. A decrease in MYCN mRNA and protein levels was observed in the SAHA treated cell lines. Subsequent silencing and exogenous over-expression of MYCN changed the proliferation rate of the cells, but did not have any significant impact on the effect of SAHA on the viability of the cells. We also found that SAHA blocked the expression of MYCN and, by doing so, reduced the effects mediated by this protein. CONCLUSIONS: Our results suggest that SAHA may be used as a single-drug treatment option for neuroblastomas with an amplified MYCN gene, and as an adjuvant treatment option for all neuroblastomas.

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ROS production is essential for the apoptotic function of E2F1 in pheochromocytoma and neuroblastoma cell lines.

In this study we demonstrate that accumulation of reactive oxygen species (ROS) is essential for E2F1 mediated apoptosis in ER-E2F1 PC12 pheochromocytoma, and SH-SY5Y and SK-N-JD neuroblastoma stable cell lines. In these cells, the ER-E2F1 fusion protein is expressed in the cytosol; the addition of 4-hydroxytamoxifen (OHT) induces its translocation to the nucleus and activation of E2F1target genes. Previously we demonstrated that, in ER-E2F1 PC12 cells, OHT treatment induced apoptosis through activation of caspase-3. Here we show that caspase-8 activity did not change upon treatment with OHT. Moreover, over-expression of Bcl-xL arrested OHT-induced apoptosis; by contrast, over-expression of c-FLIP, did not have any effect on OHT-induced apoptosis. OHT addition induces BimL expression, its translocation to mitochondria and activation of Bax, which is paralleled by diminished mitochondrial enrichment of Bcl-xL. Treatment with a Bax-inhibitory peptide reduced OHT-induced apoptosis. These results point out the essential role of mitochondria on the apoptotic process driven by E2F1. ROS accumulation followed E2F1 induction and treatment with the antioxidant N-acetylcysteine, inhibited E2F1-induced Bax translocation to mitochondria and subsequent apoptosis. The role of ROS in mediating OHT-induced apoptosis was also studied in two neuroblastoma cell lines, SH-SY5Y and SK-N-JD. In SH-SY5Y cells, activation of E2F1 by the addition of OHT induced ROS production and apoptosis, whereas over-expression of E2F1 in SK-N-JD cells failed to induce either response. Transcriptional profiling revealed that many of the genes responsible for scavenging ROS were down-regulated following E2F1-induction in SH-SY5Y, but not in SK-N-JD cells. Finally, inhibition of GSK3ß blocked ROS production, Bax activation and the down regulation of ROS scavenging genes. These findings provide an explanation for the apparent contradictory role of E2F1 as an apoptotic agent versus a cell cycle activator.

Histone deacetylase inhibition induces apoptosis and autophagy in human neuroblastoma cells.

Neuroblastoma (NB) is the most common solid extracranial tumor in children. Here we showed that trichostatin A, a histone deacetylase inhibitor (HDACi), decreases cell viability in three NB cell lines of different phenotypes. The treatment leads to G2/M-phase arrest, apoptosis and autophagy. Autophagy induction accompanies apoptosis in the most proliferative, N-Myc overexpressing cells. In contrast, autophagy precedes apoptosis and acts as a protective mechanism in the less proliferative, non-N-Myc overexpressing cells. Therefore, the autophagy induction is a relevant event in the NB response to HDACis, and it should be considered in the design of new treatments for this malignancy.

A new unifying hypothesis for lathyrism, konzo and tropical ataxic neuropathy: nitriles are the causative agents.

Konzo and lathyrism are associated with consumption of cassava and grass pea, respectively. Cassava consumption has also been associated with a third disease, tropical ataxic neuropathy (TAN). This review presents a new unifying hypothesis on the causative agents for these diseases: namely, that they are nitriles, compounds containing cyano groups. The diseases may be caused by different but similar nitriles through direct neurotoxic actions not mediated by systemic cyanide release. Both cassava and Lathyrus contain nitriles, and other unidentified nitriles can be generated during food processing or in the human body. Available data indicate that several small nitriles cause a variety of neurotoxic effects. In experimental animals, 3,3'-iminodipropionitrile (IDPN), allylnitrile and cis-crotononitrile cause sensory toxicity, whereas hexadienenitrile and trans-crotononitrile induce selective neuronal degeneration in discrete brain regions. IDPN also induces a neurofilamentous axonopathy, and dimethylaminopropionitrile is known to cause autonomic (genito-urinary) neurotoxicity in both humans and rodents. Some of these actions depend on metabolic bioactivation of the parental nitriles, and sex- and species-dependent differences in susceptibility have been recorded. Recently, neuronal degeneration has been found in rats exposed to acetone cyanohydrin. Taken together, the neurotoxic properties of nitriles make them excellent candidates as causative agents for konzo, lathyrism and TAN.

Understanding autophagy in cell death control.

Autophagy is an evolutionarily conserved degradation pathway which primary functions as a cell survival adaptive mechanism during stress conditions. Autophagy is a tumor suppressor process and induction of the autophagic machinery can cause cell demise in apoptosis-resistant cancer. Thus, this metabolic pathway can act either to prevent or to promote carcinogenesis, as well as to modulate the response to anticancer therapies, included drug-induced apoptosis. Conventional therapies exert their cytotoxic activity mainly by inducing apoptosis. Massive activation of the apoptotic program in a tissue can result in cell loss providing a selective advantage for growth to displastic cells and tumor cell subpopulations with high levels of malignancy. This suggests that the activation of autophagy can counteract malignancy. On the contrary, therapeutic intervention-induced apoptosis can eliminate cells with pro-mutational biochemical alterations at risk for initiation, initiated cells and cells of focal and advanced preneoplastic and neoplastic lesions. Thus, pharmacological inhibition of autophagy may enhance apoptosis. Autophagy and apoptosis share common stimuli and signaling pathways, so that the final fate, life or death, depends on the cell response. Recently, accumulating data fuel novel potential therapeutic interventions to modulate autophagy to be beneficial in cancer therapy. This review highlights current knowledges aimed at unraveling the molecular interplay between autophagy and cell death as well as the possible therapeutic exploitation in cancer.

Vulnerability of peripheral catecholaminergic neurons to MPTP is not regulated by alpha-synuclein.

Although generally considered a prototypical movement disorder, Parkinson's disease is commonly associated with a broad-spectrum of non-motor symptoms, including autonomic dysfunctions caused by significant alterations in catecholaminergic neurons of the peripheral sympathetic nervous system. Here we present evidence that alpha-synuclein is highly expressed by sympathetic ganglion neurons throughout embryonic and postnatal life and that it is found in tyrosine hydroxylase-positive sympathetic fibers innervating the heart of adult mice. However, mice deficient in alpha-synuclein do not exhibit any apparent alterations in sympathetic development. Sympathetic neurons isolated from mouse embryos and early postnatal mice are sensitive to the parkinsonian drug MPTP/MPP(+) and intoxication requires entry of the neurotoxin through the noradrenaline transporter. Furthermore, recovery of noradrenaline from cardiac sympathetic fibers is reduced in adult mice treated with MPTP systemically. However, MPP(+)-induced sympathetic neuron loss in vitro or MPTP-induced cardiac noradrenaline depletion in vivo is not modified in mice lacking alpha-synuclein. This is in clear contrast with the observation that dopaminergic neurons of the central nervous system are significantly less vulnerable to MPTP/MPP(+) in the absence of alpha-synuclein, suggesting different actions of this molecule in central and peripheral catecholaminergic neurons.

The targets of acetone cyanohydrin neurotoxicity in the rat are not the ones expected in an animal model of konzo.

Konzo is a neurotoxic motor disease caused by excess consumption of insufficiently processed cassava. Cassava contains the cyanogenic glucoside linamarin, but konzo does not present the known pathological effects of cyanide. We hypothesized that the aglycone of linamarin, acetone cyanohydrin, may be the cause of konzo. This nitrile rapidly decomposes into cyanide and acetone, but the particular exposure and nutrition conditions involved in the emergence of konzo may favor its stabilization and subsequent acute neurotoxicity. A number of preliminary observations were used to design an experiment to test this hypothesis. In the experiment, young female Long-Evans rats were given 10mM acetone cyanohydrin in drinking water for 2 weeks, and then 20mM for 6 weeks. Nutrition deficits associated with konzo were modeled by providing tapioca (cassava starch) as food for the last 3 of these weeks. After this period, rats were fasted for 24h in order to increase endogenous acetone synthesis, and then exposed to 0 (control group) or 50 micromol/kg-h of acetone cyanohydrin for 24h (treated group) through subcutaneous osmotic minipump infusion (n=6/group). Motor activity and gait were evaluated before exposure (pre-test), and 1 and 6 days after exposure. Brains (n=4) were stained for neuronal degeneration by fluoro-jade B. Rats exposed to 50 micromol/kg-h of acetone cyanohydrin showed acute signs of toxicity, but no persistent motor deficits. Two animals showed fluoro-jade staining in discrete thalamic nuclei, including the paraventricular and the ventral reuniens nuclei; one also exhibited labeling of the dorsal endopiriform nucleus. Similar effects were not elicited by equimolar KCN exposure. Therefore, acetone cyanohydrin may cause selective neuronal degeneration in the rat, but the affected areas are not those expected in an animal model of konzo.

Effects of dopamine on LC3-II activation as a marker of autophagy in a neuroblastoma cell model.

Dopamine at 100-500 microM has toxic effects on human SH-SY5Y neuroblastoma cells, manifested as apoptotic cell loss and strong autophagy. The molecular mechanisms and types of dopamine-induced cell death are not yet well known. Their identification is important in the study of neurodegenerative diseases that specifically involve dopaminergic neurons. We looked for changes in expression and content of proteins involved in apoptosis and autophagy after dopamine treatment. All the changes found were prevented by avoiding dopamine oxidation with N-acetylcysteine, indicating a key role for the products of dopamine oxidation in dopamine toxicity. As early as 1-2h after treatment we found an increase in hypoxia-inducible factor-1alpha (HIF-1alpha) and an accumulation of ubiquitinated proteins. Proteins regulated by HIF-1alpha and involved in apoptosis and/or autophagy, such as p53, Puma and Bnip3, were subsequently increased. However, apoptotic parameters (caspase-3, caspase-7, PARP) were only activated after 12h of 500muM dopamine treatment. Autophagy, monitored by the LC3-II increase after LC3-I linkage to autophagic vacuoles, was evident after 6h of treatment with both 100 and 500 microM dopamine. The mTOR pathway was inhibited by dopamine, probably due to the intracellular redox changes and energy depletion leading to AMPK activation. However, this mechanism is not sufficient to explain the high LC3-II activation caused by dopamine: the LC3-II increase was not reversed by IGF-1, which prevented this effect when caused by the mTOR inhibitor rapamycin. Our results suggest that the aggregation of ubiquitinated non-degraded proteins may be the main cause of LC3-II activation and autophagy. As we have reported previously, cytosolic dopamine may cause damage by autophagy in neuroblastoma cells (and presumably in dopaminergic neurons), which develops to apoptosis and leads to cell degeneration.

LC3-I conversion to LC3-II does not necessarily result in complete autophagy.

Autophagy was induced in human neuroblastoma SH-SY5Y cells by two different procedures: deprivation of fetal serum in culture medium, or treatment with dopamine. 3-methyladenine prevented autophagy in the two procedures. Although it is usually considered that the conversion of soluble LC3-I to lipid bound LC3-II is associated with the formation of autophagosomes, the inhibition of autophagy with 3-methyladenine prevented this transformation in serum-deprived but not in dopamine-treated cells. While the PI3K-mTOR pathway was inhibited by serum deprivation, dopamine increased the phosphorylation of Akt but inhibited mTOR activity in a similar way to rapamycin. Dopamine and rapamycin increased LC3-II levels by a mechanism not prevented by 3-methyladenine. The activation of LC3-I to LC3-II may then be necessary but not sufficient to trigger cell autophagy. Thus, the increase in LC3-II, as the main biochemical parameter for autophagy at present, should be considered with caution.

Anti-apoptotic effect of Mao-B inhibitor PF9601N [N-(2-propynyl)-2-(5-benzyloxy-indolyl) methylamine] is mediated by p53 pathway inhibition in MPP+-treated SH-SY5Y human dopaminergic cells.

PF9601N [N-(2-propynyl) 2-(5-benzyloxyindol) methylamine] is a non-amphetamine type MAO-B inhibitor that has shown neuroprotective properties in vivo using different experimental models of Parkinson's disease. The mechanisms underlying its neuroprotective effects are poorly understood, but appear to be independent of MAO-B inhibition. We have studied its neuroprotective properties using the human SH-SY5Y dopaminergic cell line exposed to 1-methyl-4-phenylpyridinium (MPP(+)), a cellular model of Parkinson's disease. PF9601N pre-treatment significantly reduced MPP(+)-induced cell death and decreased the activation of one of the main executioner caspases, caspase-3. MPP(+) induced stabilization of transcription factor p53, which led to increased levels of this transcription factor, its nuclear translocation and transactivation of p53 response elements. PF9601N prevented this increase, thus reducing its transcriptional activity. Additional results showed that p53 may mediate its pro-apoptotic actions through caspase-2 under our experimental conditions. PUMA-alpha may also contribute to the p53-induced cell death. Since PF9601N significantly reduced MPP(+)-induced caspase-2 activity and PUMA-alpha levels, this reduction may lead to increased cell survival. Thus, PF9601N is a novel molecule with an apparently novel mechanism of action which has a promising potential as a therapeutic agent in the treatment of neurodegenerative diseases.

Dopamine induces TNFalpha and TNF-R1 expression in SH-SY5Y human neuroblastoma cells.

Cytotoxic concentrations of dopamine (100-500 microM DA) induce expression of tumour necrosis factor receptor-1 (TNF-R1) and tumour necrosis factor-alpha (TNFalpha) in SH-SY5Y human neuroblastoma cells. TNFalpha expression is dose-dependent and can also be detected after 6-hydroxydopamine (6-OHDA) or 1-methyl-4-phenylpyridinium iodide (MPP) treatment. The expression of TNF-R1 is also dose-dependent, but was not observed in 6-OHDA or MPP-treatment. Cells not expressing TNF-R1 were insensitive to TNFalpha, whereas those treated with DA showed a further decrease in viability when subsequently treated with TNFalpha. Thus, DA treatment confers sensitivity to TNFalpha. The decrease of cell viability caused by DA was in part prevented by neutralizing TNFalpha with anti-TNFalpha. As TNF-R1 is increased in substantia nigra of Parkinsonian brains, we suggest that nonvesiculated DA might also play a role in inducing TNF-R1 expression and predispose the neuron to the action of cytokines released in a microglia-mediated inflammatory response.

Mechanisms of prodigiosin cytotoxicity in human neuroblastoma cell lines.

Prodigiosin is a bacterial red pigment with cytotoxic properties and potential antitumor activity that has been tested against different cancerous cells. In this study we report the effect and mechanisms of action of prodigiosin against different human neuroblastoma cell lines: SH-SY5Y, LAN-1, IMR-32 (N-type) and SK-N-AS (S-type). We compare the anticancerous effect of prodigiosin with that of cisplatin at different concentrations during 24 h of exposure. Prodigiosin is more potent, with IC50 values lower than 1.5 microM in N-type neuroblastoma cells and around 7 microM in the S-type neuroblastoma cell line. We describe prodigiosin as a proton sequestering agent that destroys the intracellular pH gradient, and propose that its main cytotoxic effect could be related to its action on mitochondria, where it exerts an uncoupling effect on the electronic chain transport of protons to mitochondrial ATP synthase. As a result of this action, ATP production is reduced but without decreasing in oxygen consumption. This mechanism of action differs from those induced by conventional chemotherapeutic drugs, suggesting a possible role for prodigiosin to enhance the effect of antitumor agents in the treatment of neuroblastoma.