Proteasome inhibitor-I enhances tunicamycin-induced chemosensitization of prostate cancer cells through regulation of NF-κB and CHOP expression

Although endoplasmic reticulum (ER) stress induction by some anticancer drugs can lead to apoptotic death of cancer cells, combination therapy with other chemicals would be much more efficient. It has been reported that proteasome inhibitors could induce cancer cell death through ER-stress. Our study, however, showed a differential mechanism of proteasome inhibitor-I (Pro-I)-induced cell death. Pro-I significantly enhanced apoptotic death of PC3 prostate cancer cells pretreated with tunicamycin (TM) while other signaling inhibitors against p38, mitogen activated kinase (MEK) and phosphatidyl-inositol 3-kinase (PI3K) did not, as evidenced by cell proliferation and cell cycle analyses. NF-κB inhibition by Pro-I, without direct effect on ER-stress, was found to be responsible for the TM-induced chemosensitization of PC3 cells. Moreover, TM-induced/enhancer-binding protein (C/EBP) homologous protein (CHOP) expression was enhanced by Pro-I without change in GRP78 expression. CHOP knockdown by siRNA also showed a significant decrease in Pro-I chemosensitization. All these data suggest that although TM could induce both NF-κB activation and CHOP expression through ER-stress, both NF-κB inhibition and increased CHOP level by Pro-I are required for enhanced chemosensitization of PC3 prostate cancer cells. Thus, our study might contribute to the identification of anticancer targets against prostate cancer cells.     

Beneficial Effect of Astragalosides on Stroke Condition Using PC12 Cells under Oxygen Glucose Deprivation and Reperfusion

Astragalosides (AST) are reported to be neuroprotective in focal cerebral ischemic models in vivo. In this study, the direct effect of AST against oxygen and glucose deprivation (OGD) including neuronal injury and the underlying mechanisms in vitro were investigated. 5 h OGD followed by 24 h of reperfusion [adding back oxygen and glucose (OGD-R)] was used to induce in vitro ischemia reperfusion injury in differentiated rat pheochromocytoma PC12 cells. AST (1, 100, and 200 µg/mL) were added to the culture after 5 h of the OGD ischemic insult and was present during the reoxygenation phases. A key finding was that OGD-R decreased cell viability, increased lactate dehydrogenase, increased reactive oxygen species, apoptosis, autophagy, functional impairment of mitochondria, and endoplasmic reticulum stress in PC12 cells, all of which AST treatment significantly reduced. In addition, AST attenuated OGD-R-induced cell loss through P38 MAPK activation a neuroprotective effect blunted by SB203580, a specific inhibitor of P38 MAPK. Our data suggest that both apoptosis and autophagy are important characteristics of OGD-R-induced PC12 death and that treating PC12 cells with AST blocked OGD-R-induced apoptosis and autophagy by suppressing intracellular oxidative stress, functional impairment of mitochondria, and endoplasmic reticulum stress. Our data provide identification of AST that can concomitantly inhibit multiple cells death pathways following OGD injuries in neural cells.     

Proteomic analysis of lipopolysaccharide-induced apoptosis in PC12 cells

We employed rat pheochromocytoma PC12 cells as our model system to identify cellular proteins that accompany Escherichia coli lipopolysaccharide (LPS)-induced apoptosis, based on a proteomic approach. Cell viability tests revealed that na?ve PC12 cells underwent cell death in a dose-dependent manner after treatment with LPS. Flow cytometric analysis confirmed that apoptosis was primarily responsible for the observed cell death. Two-dimensional electrophoresis in conjunction with N-terminal sequencing, immunoblot, matrix-assisted laser desorption/ionization-time of flight analysis or computer matching with protein databases further revealed that the LPS-induced apoptosis is accompanied by an augmented level of calreticulin, calcium binding protein 50, endoplasmic reticulum protein 60 (ERP60), heat shock protein 60 (HSP60) or HSP90, and a reduced level of amphoterin, cytochrome c oxidase polypeptide VIa-liver or ERP29. These proteins are associated with endoplasmic reticulum, mitochondria or cell membrane, and are with known or potential roles in apoptosis. Their identification therefore provides an impetus for further delineation of the cellular and molecular basis of apoptotic cell death and sepsis based on proteomic profiling of PC12 cells.     

Nerve growth factor withdrawal-mediated apoptosis in naive and differentiated PC12 cells through p53/caspase-3-dependent and -independent pathways

Programmed cell death is regulated in response to a variety of stimuli, including the tumor suppressor protein p53, that can mediate cell cycle arrest through p21/Waf1 and apoptosis through the Bcl-2/Bax equilibrium and caspases. Neuronal cell apoptosis has been reported to require p53, whereas other data suggest that neuronal cell death may be independent of p53. Comparison of wild type PC12 to a temperature-sensitive PC12 cell line that depresses the normal function of p53 has permitted investigation of the importance of p53 in a variety of cell functions. This study examined the role of p53 in trophic factor withdrawal-mediated apoptosis in both na?ve and differentiated PC12 cells. Our data show that as PC12 cells differentiate they are more poised to undergo apoptosis than their undifferentiated counterparts. Survival assays with XTT (sodium 3'-1-(phenylaminocarbonyl)-3,4-tetrazolium-bis(4-methoxy-6-nitro)benzene sulfonic acid) and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) demonstrated that lack of p53 is initially protective against apoptosis. The window of protection is about 20 h for na?ve and 36 h for differentiated cells. Apoptosis involved caspases 3, 6, and 9. However, caspase 3 activation was absent in cells lacking p53, concomitant with the delayed apoptosis. When the expression of caspase 3 was silenced with interference RNA, wild type PC12 cells revealed a morphology and biochemistry similar to PC12[p53ts] cells, indicating that caspase 3 accounts for the observed delay in apoptosis in p53 dysfunction. These results suggest that p53 is important, but not essential, in factor withdrawal-mediated apoptosis. Parallel pathways of caspase-mediated apoptosis are activated later in the absence of functional p53.     

Dopamine auto-oxidation aggravates non-apoptotic cell death induced by over-expression of human A53T mutant alpha-synuclein in dopaminergic PC12 cells

In this study, we demonstrated that transient transfection and over-expression of human mutant A53T alpha-synuclein (α-syn) could induce expression level- and time-dependent, non-apoptotic cell death in PC12 cells, while wild-type and mutant A30P α-syn could not. The non-apoptotic cell death induced by over-expression of A53T α-syn in PC12 cells was found to be dopamine (DA) related. It could be alleviated by nerve growth factor but not by chemicals that abrogate endoplasmic reticulum stress. Furthermore, PC12 cell death could be alleviated by N -acetyl-cysteine (NAC) as well as by l -cysteine; but not by cell permeable tyrosinase inhibitors. NAC could prevent DA auto-oxidation and tyrosinase-catalyzed DA oxidation, whereas l -cysteine could potently abrogate DA auto-oxidation but could not prevent tyrosinase-catalyzed DA oxidation. Both NAC and l -cysteine could increase the reduced and total GSH levels, and concurrently decrease the oxidized GSH level in PC12 cells. On the other hand, over-expression of human mutant A53T α-syn could decrease the reduced and total GSH levels, and increase the oxidized GSH level in the cells. Taken together, we concluded that auto-oxidation of endogenous DA aggravates non-apoptotic cell death induced by over-expression of human mutant A53T α-syn in PC12 cells.     

Suppression of endoplasmic reticulum stress-induced caspase activation and cell death by the overexpression of Bcl-xL or Bcl-2

Continuous endoplasmic reticulum (ER) stress, such as the accumulation of unfolded proteins, results in cell death and relates to the pathogenesis of some neurodegenerative diseases. Treatment of brefeldin A, an inhibitor of transport between the ER and Golgi complex, induced cell death during 24 h, which accompanied activation of caspase-2, caspase-3 and caspase-9, starting at 12 h and increasing time-dependently up to 28 h. Caspase-2 was expressed and activated in not only mitochondria and cytosol, but also in the microsomal fraction containing ER and Golgi. Of note is that overexpression of Bcl-x(L) or Bcl-2 in PC12 cells markedly suppressed brefeldin A-induced activation of caspases and resulting cell death. Delivery of anti-Bcl-2 antibody into the Bcl-2-overexpressed cells again recovered apoptosis. While the brefeldin A-treatment induced the phosphorylation of both c-Jun N-terminal kinase (JNK) and p38 MAPK, overexpression of Bcl-x(L) or Bcl-2 reduced the prolonged phosphorylation of JNK, but not of p38 MAPK. Pretreatment with a JNK inhibitor, SP600125, suppressed the brefeldin A-induced caspase-2 activation and cell death significantly. Thus, our results suggest that protective effects of Bcl-x(L) and Bcl-2 against brefeldin A-induced cell death appear to be dependent on the regulation of JNK activation.     

Inhibitors of Trypsin-Like Serine Proteases Inhibit Processing of the Caspase Nedd-2 and Protect PC12 Cells and Sympathetic Neurons from Death Evoked by Withdrawal of Trophic Support

Abstract: Rat pheochromocytoma (PC12) cells and sympathetic neurons undergo apoptotic cell death upon withdrawal of trophic support. We have shown previously that selective cysteine aspartase (caspase) inhibitors protect PC12 cells and sympathetic neurons from such death, and that the caspase Nedd-2 is required for this type of death to occur. We now show that 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) and N ^α- p -tosyl- l -lysine chloromethyl ketone (TLCK), agents that inhibit another class of proteases, the trypsin-like serine proteases, also suppress cell death in this paradigm. The site of action of these agents is upstream of the caspases, because the CPP32-like and Nedd-2-cleaving activities that are induced upon withdrawal of trophic support in PC12 cells are inhibited when AEBSF and TLCK are applied to the cells. Both agents inhibit thymidine incorporation in PC12 cells at concentrations similar to those that promote survival, raising the possibility that they may promote survival in neuronal cells through inhibition of aberrant activation of cell cycle components.     

Piceatannol attenuates hydrogen-peroxide- and peroxynitrite-induced apoptosis of PC12 cells by blocking down-regulation of Bcl-XL and activation of JNK

There is mounting evidence implicating the accumulation of intracellular reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the pathogenesis of neurodegenerative disorders, including Alzheimer's disease. Recently, considerable attention has been focused on identifying naturally occurring antioxidants that are able to reduce excess ROS and RNS, thereby protecting against oxidative stress and neuron death. The present study investigated the possible protective effects of piceatannol (trans-3,4,3',5'-tetrahydroxystilbene), which is present in grapes and other foods, on hydrogen-peroxide- and peroxynitrite-induced oxidative cell death. PC12 rat pheochromocytoma (PC12) cells treated with hydrogen peroxide or SIN-1 (a peroxynitrite-generating compound) exhibited apoptotic death, as determined by nucleus condensation and cleavage of poly(ADP-ribose)polymerase (PARP). Piceatannol treatment attenuated hydrogen-peroxide- and peroxynitrite-induced cytotoxicity, apoptotic features, PARP cleavage and intracellular ROS and RNS accumulation. Treatment of PC12 cells with hydrogen peroxide or SIN-1 led to down-regulation of Bcl-X(L) and activation of caspase-3 and -8, which were also inhibited by piceatannol treatment. Hydrogen peroxide or SIN-1 treatment induced phosphorylation of the c-Jun-N-terminal kinase (JNK), which was inhibited by piceatannol treatment. Moreover, SP600125 (a JNK inhibitor) significantly inhibited hydrogen-peroxide- and peroxynitrite-induced PC12 cell death, revealing inactivation of the JNK pathway as a possible molecular mechanism for the protective effects of piceatannol against hydrogen-peroxide- and peroxynitrite-induced apoptosis of PC12 cells. Collectively, these findings suggest that the protective effect of piceatannol against hydrogen-peroxide- and peroxynitrite-induced apoptosis of PC12 cells is associated with blocking the activation of JNK and the down-regulation of Bcl-XL.     

p53-independent endoplasmic reticulum stress-mediated cytotoxicity of a Newcastle disease virus strain in tumor cell lines

While Newcastle disease virus (NDV) causes serious infections in birds, it is apparently nonpathogenic in mammalian species, including humans. Previous observations and small-scale clinical trials indicated that NDV exerts oncolytic effects. Isolates of NDV were found to have selective affinity to transformed cells. We previously showed that the attenuated NDV strain MTH-68/H causes apoptotic cell death in cultures of PC12 rat pheochromocytoma cells. The aim of the present study was to extend MTH-68/H cytotoxicity testing with human tumor cell lines and to analyze certain biochemical aspects of its oncolytic effect. MTH-68/H was found to be able to kill a wide range of transformed cells by apoptosis. While caspase-8 and caspase-9 are not involved in MTH-68/H-induced apoptosis, activation of caspase-3 and caspase-12 was detected in virus-infected PC12 cells. A human glioblastoma cell line with repressible expression of the p53 protein did not show any difference in MTH-68/H sensitivity in its p53-expressing and p53-depleted states, indicating that the apoptotic process induced by MTH-68/H does not depend on p53. Apoptosis was accompanied by virus replication in two tumor cell lines tested (PC12 cells and HeLa human cervical cells), and signs of endoplasmic reticulum stress (phosphorylation of protein kinase R-like endoplasmic reticulum kinase and eIF2alpha) were also detected in transformed cells. In contrast, proliferation of nontransformed mouse and rat fibroblast cell lines and human primary fibroblasts was not affected by MTH-68/H treatment. MTH-68/H thus selectively kills tumor cell cultures by inducing endoplasmic reticulum stress leading to p53-independent apoptotic cell death.     

Colchicine-Induced Apoptosis Was Prevented by Glycogen Synthase Kinase-3 Inhibitors in PC12 Cells

The purpose of this study was to examine whether glycogen synthase kinase-3 (GSK-3) is involved in colchicine-induced cell death in PC12 cells by using GSK inhibitors. Colchicine increased apoptotic cell death with morphological changes characterized by cell shrinkage and nuclear condensation or fragmentation. GSK-3 inhibitors such as alsterpaullone, SB216763, and AR-A014418 prevented colchicine-induced cell death and caspase-3 activation. These results suggest that colchicine induces caspase-dependent apoptotic cell death and that GSK-3 activation is involved in cell death in PC12 cells.     

ER stress is the initial response to polyglutamine toxicity in PC12 cells

Persistent endoplasmic reticulum (ER) stress and impairment of the ubiquitin-proteasome system (UPS) cause neuronal cell death. However, the relationship between these two phenomena remains controversial. In our current study, we have utilized an expanded polyglutamine fusion protein (polyQ81) expression system in PC12 cells to further examine the involvement of ER stress and UPS impairment in cell death. The expression of polyQ81-induced ER stress and cell death. PolyQ81 also induced the activation of c-Jun N-terminal kinase (JNK) and caspase-3 and an increase in polyubiquitin immunoreactivity, suggesting UPS impairment. ER stress was induced prior to the accumulation of polyubiquitinated proteins. Low doses of lactacystin had almost similar effects on cell viability and on the activation of JNK and caspase-3 between normal cells and polyQ81-expressing cells. These results suggest that ER stress mediates polyglutamine toxicity prior to UPS impairment during the initial stages of these toxic effects.     

beta-Amyloid-induced neuronal apoptosis requires c-Jun N-terminal kinase activation

beta-Amyloid (A beta) has been strongly implicated in the pathophysiology of Alzheimer's disease (AD), but the means by which the aggregated form of this molecule induces neuronal death have not been fully defined. Here, we examine the role of the c-Jun N-terminal kinases (JNKs) and of their substrate, c-Jun, in the death of cultured neuronal PC12 cells and sympathetic neurons evoked by exposure to aggregated A beta. The activities of JNK family members increased in neuronal PC12 cells within 2 h of A beta treatment and reached 3--4-fold elevation by 6 h. To test the role of these changes in death caused by A beta, we examined the effects of CEP-1347 (KT7515), an indolocarbazole that selectively blocks JNK activation. Inclusion of CEP-1347 (100--300 nM) in the culture medium effectively blocked the increases in cellular JNK activity caused by A beta and, at similar concentrations, protected both PC12 cells and sympathetic neurons from A beta-evoked-death. Effective protection required addition of CEP-1347 within 2 h of A beta treatment, indicating that the JNK pathway acts relatively proximally and as a trigger in the death mechanism. A dominant-negative c-Jun construct also conferred protection from A beta-evoked death, supporting a model in which JNK activation contributes to death via activation of c-Jun. Finally, CEP-1347 blocked A beta-stimulated activation of caspase-2 and -3, placing these downstream of JNK activation. These observations implicate the JNK pathway as a required element in death evoked by A beta and hence identify it as a potential therapeutic target in AD.     

Phycocyanin protects INS-1E pancreatic beta cells against human islet amyloid polypeptide-induced apoptosis through attenuating oxidative stress and modulating JNK and p38 mitogen-activated protein kinase pathways

It is widely accepted that human islet amyloid polypeptide (hIAPP) aggregation plays an important role in the loss of insulin-producing pancreatic beta cells. hIAPP-induced cytotoxicity is mediated by generation of reactive oxygen species (ROS). Phycocyanin (PC) is a natural compound from blue-green algae that is widely used as food supplement. Currently, little is known about the effects of PC on beta cells with the presence of hIAPP. The aim of this study was to investigate the in vitro protective effects of PC on INS-1E rat insulinoma beta cells against hIAPP-induced cell death, as well as the underlying mechanisms. Our results showed that hIAPP-induced cell death with apoptotic characteristics including growth inhibition, chromatin condensation and DNA fragmentation. However, cytotoxicity of hIAPP was significantly attenuated by co-incubation of the cells with PC. The results of Western blotting showed that activation of caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP) in hIAPP-treated cells was blocked by PC. Moreover, PC significantly prevented the hIAPP-induced overproduction of intracellular ROS and malondialdehyde (MDA), as well as changes in activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) enzymes. Furthermore, hIAPP triggered the activation of mitogen-activated protein kinases (MAPKs), and these effects were effectively suppressed by PC. Taken together, our results suggest that PC protects INS-1E pancreatic beta cells against hIAPP-induced apoptotic cell death through attenuating oxidative stress and modulating c-Jun N-terminal kinase (JNK) and p38 pathways.     

Cyclooxygenase 2 inhibits SAPK activation in neuronal apoptosis

Cyclooxygenase 2 (COX-2) expressed in cultured neuronal PC12 cells under inducible promoter protects cells from trophic withdrawal apoptosis. Stimulation of SAPK is thought to play a significant role in initiation of PC12 cell death. We have therefore examined whether COX-2 expression inhibits trophic withdrawal-mediated activation of SAPK. SAPK activity increased during the first 6h after NGF removal in mock-transfected PC12 cells. COX-2 expression attenuated the increase of SAPK, as detected by Western blot analysis with phosphorylation state specific anti-SAPK antibodies and by SAPK activity assays. We propose that COX-2 attenuated SAPK activation by preventing activation of nNOS, which occurs, as we have shown before, via COX-2-mediated expression of dynein light chain (DLC). Activation of SAPK in neuronal cell death was attenuated by DLC expression. These observations support a role for NO production and SAPK activation in the neuronal death mechanisms.     

Bcl-2 protects against Abeta(25-35)-induced oxidative PC12 cell death by potentiation of antioxidant capacity

A substantial body of data indicates that reactive oxygen intermediates (ROIs) are implicated in pathogenesis of diverse human diseases. Oxidative stress induced by ROIs often causes cell death via apoptosis that is regulated by a plenty of functional genes and their protein products. Bcl-2 is one such protein that blocks apoptosis induced by various death stimuli. In spite of extensive research, the molecular mechanisms underlying antiapoptotic function of Bcl-2 are not fully clarified. In the present work, we have investigated the role of bcl-2 in protecting against beta-amyloid (Abeta)-induced oxidative death in rat pheochromocytoma (PC12) cells. Transfection with the antiapoptotic bcl-2 gene rescued PC12 cells from apoptotic death induced by Abeta. Addition of an NF-kappaB inhibitor, such as pyrrolidine dithiocarbamate or N-tosyl-l-phenylalanine chloromethyl ketone, to the media aggravated Abeta-induced PC12 cell death. PC12 cells overexpressing bcl-2 exhibited higher levels of constitutively activated NF-kappaB compared with vector-transfected controls, which appear to be mediated by the elevated activation of Akt/protein kinase B. The ectopic expression of bcl-2 enhanced both the expression and the activity of catalase, which were attenuated by NF-kappaB blockers. These results suggest that NF-kappaB plays a role in bcl-2-mediated protection against Abeta-induced apoptosis in PC12 cells through augmentation of cellular antioxidant capacity.     

Mitochondria-endoplasmic reticulum choreography: structure and signaling dynamics

Mitochondria and endoplasmic reticulum (ER) have different roles in living cells but they interact both physically and functionally. A key aspect of the mitochondria-ER relationship is the modulation of Ca(2+) signaling during cell activation, which thus affects a variety of physiological processes. We focus here on the molecular aspects that control the dynamics of the organelle-organelle interaction and their relationship with Ca(2+) signals, also discussing the consequences that these phenomena have, not only for cell physiology but also in the control of cell death.     

Involvement of p38 mitogen-activated protein kinase and apoptosis signal-regulating kinase-1 in nitric oxide-induced cell death in PC12 cells

Although nitric oxide (NO) plays key signaling roles in the nervous systems, excess NO leads to cell death. In this study, the involvement of p38 mitogen-activated protein kinase (p38 MAPK) and apoptosis signal-regulating kinase-1 (ASK1) in NO-induced cell death was investigated in PC12 cells. NO donor transiently activated p38 MAPK in the wild type parental PC12 cells, whereas the p38 MAPK activation was abolished in NO-resistant PC12 cells (PC12-NO-R). p38 MAPK inhibitors protected the cells against NO-induced death, whereas the inhibitors were not significantly protective against the cytotoxicity of reactive oxygen species. Stable transfection with dominant negative p38 MAPK mutant reduced NO-induced cell death. Stable transfection with dominant negative mutant of ASK1 attenuated NO-stimulated activation of p38 MAPK and decreased NO-induced cell death. These results suggest that p38 MAPK and its upstream regulator ASK1 are involved in NO-induced PC12 cell death.     

The stressosome,a caspase-8-activating signalling complex assembled in response to cell stress in an ATG5-mediated manner

Stress-induced apoptosis is mediated primarily through the intrinsic pathway that involves caspase-9. We previously reported that in caspase-9-deficient cells, a protein complex containing ATG5 and Fas-associated death domain (FADD) facilitated caspase-8 activation and cell death in response to endoplasmic reticulum (ER) stress. Here, we investigated whether this complex could be activated by other forms of cell stress. We show that diverse stress stimuli, including etoposide, brefeldin A and paclitaxel, as well as heat stress and gamma-irradiation, caused formation of a complex containing ATG5-ATG12, FADD and caspase-8 leading to activation of downstream caspases in caspase-9-deficient cells. We termed this complex the ‘stressosome’. However, in these cells, only ER stress and heat shock led to stressosome-dependent cell death. Using in silico molecular modelling, we propose the structure of the stressosome complex, with FADD acting as an adaptor protein, interacting with pro-caspase-8 through their respective death effector domains (DEDs) and interacting with ATG5-ATG12 through its death domain (DD). This suggests that the complex could be regulated by cellular FADD-like interleukin-1β-converting enzyme–inhibitory protein (cFLIP_L), which was confirmed experimentally. This study provides strong evidence for an alternative mechanism of caspase-8 activation involving the stressosome complex.