Pristimerin induces apoptosis by targeting the proteasome in prostate cancer cells

Pristimerin is a natural product derived from the Celastraceae and Hippocrateaceae families that were used as folk medicines for anti inflammation in ancient times. Although it has been shown that pristimerin induces apoptosis in breast cancer cells, the involved mechanism of action is unknown. The purpose of the current study is to investigate the primary target of pristimerin in human cancer cells, using prostate cancer cells as a working model. Nucleophilic susceptibility and in silico docking studies show that C6 of pristimerin is highly susceptible towards a nucleophilic attack by the hydroxyl group of N-terminal threonine of the proteasomal chymotrypsin subunit. Consistently, pristimerin potently inhibits the chymotrypsin-like activity of a purified rabbit 20S proteasome (IC50 2.2 micromol/L) and human prostate cancer 26S proteasome (IC50 3.0 micromol/L). The accumulation of ubiquitinated proteins and three proteasome target proteins, Bax, p27 and I kappa B-alpha, in androgen receptor (AR)-negative PC-3 prostate cancer cells supports the conclusion that proteasome inhibition by pristimerin is physiologically functional. This observed proteasome inhibition subsequently led to the induction of apoptotic cell death in a dose- and kinetic-dependent manner. Furthermore, in AR-positive, androgen-dependent LNCaP and AR-positive, androgen-independent C4-2B prostate cancer cells, proteasome inhibition by pristimerin results in suppression of AR protein prior to apoptosis. Our data demonstrate, for the first time, that the proteasome is a primary target of pristimerin in prostate cancer cells and inhibition of the proteasomal chymotrypsin-like activity by pristimerin is responsible for its cancer cell death-inducing property.     

APP and APLP1 are degraded through autophagy in response to proteasome inhibition in neuronal cells

Amyloid beta (Aβ) precursor protein (APP) is a key protein in the pathogenesis of Alzheimer’s disease (AD). Both APP and its paralogue APLP1 (amyloid beta precursor-like protein 1) have multiple functions in cell adhesion and proliferation. Previously it was thought that autophagy is a novel beta-amyloid peptide (Aβ)-generating pathway activated in AD. However, the protein proteolysis of APLP1 is still largely unknown. The present study shows that APLP1 is rapidly degraded in neuronal cells in response to stresses, such as proteasome inhibition. Activation of the endoplasmic reticulum (ER) stress by proteasome inhibitors induces autophagy, causing reduction of mature APLP1/APP. Blocking autophagy or JNK stress kinase rescues the protein expression for both APP and APLP1. Therefore, our results suggest that APP/APLP1 is degraded through autophagy and the APLP1 proteolysis is mainly mediated by autophagy-lysosome pathway.     

Dynamic analysis of apoptosis in primary cortical neurons by fixed- and real-time cytofluorometry

We describe here a cytofluorometric technology for the characterization of decision, execution, and degradation steps of neuronal apoptosis. Multiparametric flow cytometry was developed and combined to detailed fluorescence microscopy observations to establish the chronology and hierarchy of death-related events: neuron morphological changes, mitochondrial transmembrane potential (DeltaPsi(m)) collapse, caspase-3 and -9 activation, phosphatidyl-serine exposure, nuclear dismantling and final plasma membrane permeabilization. Moreover, we developed a reliable real-time flow cytometric monitoring of DeltaPsi(m) and plasma membrane integrity in response to neurotoxic insults including MPTP treatment. Taking advantage of recently developed specific fluorescent probes and a third generation pan-caspase inhibitor, this integrated approach will be pertinent to study the cell biology of neuronal apoptosis and to characterize new neuro-toxic/protective molecules.     

Inhibition of neuronal apoptosis by the cyclin-dependent kinase inhibitor GW8510: identification of 3' substituted indolones as a scaffold for the development of neuroprotective drugs

Increasing evidence suggests that neuronal apoptosis is triggered by the inappropriate activation of cyclin-dependent kinases leading to an abortive re-entry of neurons into the cell cycle. Pharmacological inhibitors of cell-cycle progression may therefore have value in the treatment of neurodegenerative diseases in humans. GW8510 is a 3' substituted indolone that was developed recently as an inhibitor of cyclin-dependent kinase 2 (CDK2). We found that GW8510 inhibits the death of cerebellar granule neurons caused by switching them from high potassium (HK) medium to low potassium (LK) medium. Although GW8510 inhibits CDK2 and other CDKs when tested in in vitro biochemical assays, when used on cultured neurons it only inhibits CDK5, a cytoplasmic CDK that is not associated with cell-cycle progression. Treatment of cultured HEK293T cells with GW8510 does not inhibit cell-cycle progression, consistent with its inability to inhibit mitotic CDKs in intact cells. Neuroprotection by GW8510 is independent of Akt and MEK-ERK signaling. Furthermore, GW8510 does not block the LK-induced activation of Gsk3beta and, while inhibiting c-jun phosphorylation, does not inhibit the increase in c-jun expression observed in apoptotic neurons. We also examined the effectiveness of other 3' substituted indolone compounds to protect against neuronal apoptosis. We found that like GW8510, the VEGF Receptor 2 Kinase Inhibitors [3-(1H-pyrrol-2-ylmethylene)-1,3-dihydroindol-2-one], {(Z)-3-[2,4-Dimethyl-3-(ethoxycarbonyl)pyrrol-5-yl)methylidenyl]indol-2-one} and [(Z)-5-Bromo-3-(4,5,6,6-tetrahydro-1H-indol-2-ylmethylene)-1,3-dihydroindol-2-one], the Src family kinase inhibitor SU6656 and a commercially available inactive structural analog of an RNA-dependent protein kinase inhibitor 5-Chloro-3-(3,5-dichloro-4-hydroxybenzylidene)-1,3-dihydro-indol-2-one, are all neuroprotective when tested on LK-treated neurons. Along with our recent identification of the c-Raf inhibitor GW5074 (also a 3' substituted indolone) as a neuroprotective compound, our findings identify the 3' substituted indolone as a core structure for the designing of neuroprotective drugs that may be used to treat neurodegenerative diseases in humans.     

Reactive oxygen species induced by proteasome inhibition in neuronal cells mediate mitochondrial dysfunction and a caspase-independent cell death

While increasing evidence shows that proteasome inhibition triggers oxidative damage, mitochondrial dysfunction and death in neuronal cells, the regulatory relationship among these events is unclear. Using mouse neuronal cells we show that the cytotoxicity induced by mild (0.25 μM) and potent (5.0 μM) doses of the proteasome inhibitor, N-Benzyloxycarbonyl-Ile-Glu (O-t-butyl)-Ala-leucinal, (PSI) involved a dose-dependent increase in caspase activation, overproduction of reactive oxygen species (ROS) and a mitochondrial dysfunction manifested by the translocation of the proapoptotic protein, Bax, from the cytoplasm to the mitochondria, membrane depolarization and the release of cytochrome c and the apoptosis inducing factor (AIF) from mitochondria to the cytoplasm and nucleus, respectively. Whereas caspase or Bax inhibition failed to prevent mitochondrial membrane depolarization and neuronal cell death, pretreatments with the antioxidant N-acetyl-l-cysteine (NAC) or overexpression of the antiapoptotic protein Bcl-xL abrogated these events in cells exposed to mild levels of PSI. These findings implicated ROS as a mediator of PSI-induced cytotoxicity. However, depletions in glutathione and Bcl-xL with potent proteasome inhibition exacerbated this response whereupon survival required the cooperative protection of NAC with Bcl-xL overexpression. Collectively, ROS induced by proteasome inhibition mediates a mitochondrial dysfunction in neuronal cells that culminates in death through caspase- and Bax-independent mechanisms. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Curcumin induces apoptosis in human neuroblastoma cells via inhibition of AKT and Foxo3a nuclear translocation

Abstract

Neuroblastoma (NB) is one of the most frequent extracranial solid tumors in children. It accounts for 8–10% of all childhood cancer deaths, and there is a need for development of new drugs for its treatment. Curcumin (diferuloylmethane), a major active component of turmeric (Curcuma longa), has been shown to exert anti-tumor activity on NB, but the specific mechanism by which curcumin inhibits cancer cells proliferation remains unclear. In the present study, we investigated the anti-proliferative effect of curcumin in human LAN5 NB cells. Curcumin treatment causes a rapid increase in reactive oxygen species and a decrease in the mitochondrial membrane potential—events leading to apoptosis activation. Furthermore, curcumin induces decrease in haet shock protein (Hsp)60 and hexokinase II mitochondrial protein levels and increase in the pro-apoptotic protein, bcl-2 associated death promoter (BAD). Moreover, we demonstrate that curcumin modulates anti-tumor activity through modulation of phosphatase and tensin homolog deleted on chromosome 10 and consequential inhibition of the survival Akt cell-signaling pathway. Inhibition of Akt causes its translocation into the cytoplasm and import of Foxo3a into the nucleus where it activates the expression of p27, Bim, and Fas-L pro-apoptotic genes. Together, these results take evidence for considering curcumin as a potential therapeutic agent for patients with NB.     

A comparative study of proteasomal inhibition and apoptosis induced in N27 mesencephalic cells by dopamine and MG132

Dopamine (DA) and its metabolites have been implicated in the pathogenesis of Parkinson's disease. DA can produce reactive-oxygen species and DA-derived quinones such as aminochrome can induce proteasomal inhibition. We therefore examined the ability of DA and MG132 to induce apoptosis and proteasomal inhibition in N27 rat dopaminergic cells. DA (0-500 micromol/L, 0-24 h) and MG132 (0-5 micromol/L, 0-24 h) treated N27 cells resulted in time- and concentration-dependent apoptosis. To better define DA and MG132-induced apoptosis, the activation of initiator caspases 2 and caspase 9 and the executioner caspase 3 was investigated. Activation of caspase 2, caspase 9, and caspase 3 occurred early and prior to cell death. In addition, N-acetylcysteine (NAC) blocked DA but not MG132-induced apoptosis and mitochondrial membrane potential loss. NAC can react with both reactive-oxygen and quinoid metabolites and its inhibitory activity suggests a role for reactive species in DA-induced apoptosis. Proteasomal inhibition was detected after DA treatment in N27 cells which occurred prior to cell death and was abrogated by NAC. Our results implicate DA-derived reactive species in proteasomal inhibition and caspase-dependent apoptosis in N27 cells. The ability of endogenous DA-derived metabolites to induce proteasomal inhibition and apoptosis may contribute to the selective loss of dopaminergic neurons in Parkinson's disease.     

Transport of methotrexate in L1210 cells: Mechanism for inhibition by p-chloromercuriphenylsulfonate and N-ethylmaleimide

Methotrexate transport in L1210 cells is highly sensitive to inhibition by p-chloromercuriphenylsulfonate (CMPS) and, to a lesser extent, by N-ethylmaleimide. A 50% reduction in the methotrexate influx rate occurred upon exposure of cells to 3 μM CMPS or 175 μM N-ethylmaleimide, while complete inhibition was achieved at higher levels of these agents. Dithiothreitol reversed the inhibition by CMPS, suggesting that a sulfhydryl residue is involved. This residue is apparently not located at the substrate binding site of the transport protein, since methotrexate failed to protect the system from inactivation by either CMPS or N-ethylmaleimide, and the transport protein retained the ability to bind substrate (at 4°C) after exposure to these inhibitors (at 37°C). Methotrexate efflux was also inhibited by CMPS (50% at 4 μM), indicating that both the uptake and efflux of methotrexate in L1210 cells occur via the same transport system. High concentrations of CMPS (greater than 20 μM) increased the efflux rate, apparently by damaging the cell membrane and allowing the passive diffusion of methotrexate out of the cell.     

Selective inhibition of carbonic anhydrase-IX by sulphonamide derivatives induces pH and reactive oxygen species-mediated apoptosis in cervical cancer HeLa cells

Selective inhibition with sulphonamides of carbonic anhydrase (CA) IX reduces cell proliferation and induces apoptosis in human cancer cells. The effect on CA IX expression of seven previously synthesised sulphonamide inhibitors, with high affinity for CA IX, as well as their effect on the proliferation/apoptosis of cancer/normal cell lines was investigated. Two normal and three human cancer cell lines were used. Treatment resulted in dose- and time-dependent inhibition of the growth of various cancer cell lines. One compound showed remarkably high toxicity towards CA IX-positive HeLa cells. The mechanisms of apoptosis induction were determined with Annexin-V and AO/EB staining, cleaved caspases (caspase-3, caspase-8, caspase-9) and cleaved PARP activation, reactive oxygen species production (ROS), mitochondrial membrane potential (MMP), intracellular pH (pHi), extracellular pH (pHe), lactate level and cell cycle analysis. The autophagy induction mechanisms were also investigated. The modulation of apoptotic and autophagic genes (Bax, Bcl-2, caspase-3, caspase-8, caspase-9, caspase-12, Beclin and LC3) was measured using real time PCR. The positive staining using γ-H2AX and AO/EB dye, showed increased cleaved caspase-3, caspase-8, caspase-9, increased ROS production, MMP and enhanced mRNA expression of apoptotic genes, suggesting that anticancer effects are also exerted through its apoptosis-inducing properties. Our results show that such sulphonamides might have the potential as new leads for detailed investigations against CA IX-positive cervical cancers.     

Blockade by NS-7, a neuroprotective compound, of both L-type and P/Q-type Ca2+ channels involving depolarization-stimulated nitric oxide synthase activity in primary neuronal culture

The effect of 4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy)pyrimidine hydrochloride (NS-7), a neuroprotective compound, on Ca2+ channels involving the activation of nitric oxide synthase (NOS) was investigated in primary neuronal culture. The NOS activity was estimated from the cyclic GMP formation. The KCl (25 mM)-stimulated cyclic GMP formation was totally abolished by a combined treatment with nifedipine and omega-agatoxin IVA (omega-Aga), whereas spontaneous cyclic GMP formation was partially but significantly reduced by nifedipine. In contrast to nifedipine, NS-7 blocked KCl-stimulated cyclic GMP formation without affecting spontaneous cyclic GMP formation. Subsequently, the effects of nifedipine and NS-7 on L-type Ca2+ channels were compared. Nifedipine blocked equally the cyclic GMP formation stimulated by various concentrations of (+/-)-Bay K 8644, whereas NS-7 inhibited the maximal response without affecting the responses induced by low concentrations of (+/-)-Bay K 8644. The effects of NS-7 on L-type and P/Q-type Ca2+ channels involving KCl-stimulated cyclic GMP formation were subsequently examined. NS-7 suppressed the KCl-stimulated cyclic GMP formation measured in the presence of omega-Aga to almost the same extent as that determined in the presence of nifedipine. In contrast, NS-7 had no influence on ionomycin-induced enhancement of cyclic GMP formation. Finally, NS-7 reversed KCl-induced elevation of the intracellular free Ca2+ concentration. These findings suggest that NS-7 inhibits NOS activation in primary neuronal culture by reducing Ca2+ entry through L-type and P/Q-type Ca2+ channels, in which the inhibition is largely dependent on Ca2+ channel activity.     

Endothelial nitric oxide synthase overexpression by neuronal cells in neurodegeneration: a link between inflammation and neuroprotection

The roles of neuronal and inducible nitric oxide synthases in neurones have been extensively investigated; by contrast, the biological significance of endothelial nitric oxide synthase (eNOS) overexpression that occurs in several pathological conditions has not yet been studied. We have started addressing this issue in a cell model of neurodegeneration, i.e. human SKNBE neuroblastoma cells transfected with a mutant form of alsin, a protein causing an early-onset type of amyotrophic lateral sclerosis, ALS2. We found that eNOS, which is endogenously expressed by these cells, was activated by tumour necrosis factor-α (TNF-α), a proinflammatory cytokine that plays important roles in ALS2 and several neurodegenerative diseases. The TNF-α-dependent eNOS activation occurred through generation, by sphingosine-kinase-1, of sphingosine-1-phosphate, stimulation of its membrane receptors and activation of Akt, as determined using small interference RNA and dominant negative constructs specific for the enzymes and receptors. eNOS activation by TNF-α conferred cytoprotection from excitotoxicity and neurotoxic cues such as reactive oxygen species, endoplasmic reticulum stress, DNA damage, and mutated alsin itself. Our results suggest that overexpression of eNOS by neurones is a broad-range protective mechanism activated during damage and establish a link of pathophysiological relevance between this enzyme and inflammation accompanying neurodegenerative diseases. These findings also question the concept that high NO output in the presence of oxidative stress leads always to peroxynitrite formation contributing to neurodegeneration.     

Inhibition of JNK2 and JNK3 by JNK inhibitor IX induces prometaphase arrest-dependent apoptotic cell death in human Jurkat T cells

Exposure of human Jurkat T cells to JNK inhibitor IX (JNKi), targeting JNK2 and JNK3, caused apoptotic DNA fragmentation along with G₂/M arrest, phosphorylation of Bcl-2, Mcl-1, and Bim, Δψm loss, and activation of Bak and caspase cascade. These JNKi-induced apoptotic events were abrogated by Bcl-2 overexpression, whereas G₂/M arrest, cyclin B1 up-regulation, Cdk1 activation, and phosphorylation of Bcl-2 family proteins were sustained. In the concomitant presence of the G₁/S blocking agent aphidicolin and JNKi, the cells underwent G₁/S arrest and failed to induce all apoptotic events. The JNKi-induced phosphorylation of Bcl-2 family proteins and mitochondrial apoptotic events were suppressed by the Cdk1 inhibitor. Immunofluorescence microscopic analysis revealed that mitotic spindle defect and prometaphase arrest were the underlying factors for the G₂/M arrest. These results demonstrate that JNKi-induced mitochondrial apoptosis was caused by microtubule damage-mediated prometaphase arrest, prolonged Cdk1 activation, and phosphorylation of Bcl-2 family proteins in Jurkat T cells.     

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates the expression and the release of tissue plasminogen activator (tPA) in neuronal cells: involvement of tPA in the neuroprotective effect of PACAP

Pituitary adenylate cyclase-activating polypeptide (PACAP) and tissue plasminogen activator (tPA) play important roles in neuronal migration and survival. However, a direct link between the neurotrophic effects of PACAP and tPA has never been investigated. In this study, we show that, in PC12 cells, PACAP induced a 9.85-fold increase in tPA gene expression through activation of the protein kinase A- and protein kinase C-dependent signaling pathways. In immature cerebellar granule neurons (CGN), PACAP stimulated tPA mRNA expression and release of proteolytically active tPA. Immunocytochemical labeling revealed the presence of tPA in the cytoplasm and processes of cultured CGN. The inhibitory effect of PACAP on CGN motility was not affected by the tPA substrate plasminogen or the tPA inhibitor plasminogen activator inhibitor-1. In contrast, plasminogen activator inhibitor-1 significantly reduced the stimulatory effect of PACAP on CGN survival. Altogether, these data indicate that tPA gene expression is activated by PACAP in both tumoral and normal neuronal cells. The present study also demonstrates that PACAP stimulates the release of tPA which promotes CGN survival by a mechanism dependent of its proteolytic activity.