Suppression of heat- and polyglutamine-induced cytotoxicity by nonsteroidal anti-inflammatory drugs.

We have shown that sodium salicylate activates the heat shock promoter and induces the expression of heat shock proteins (hsps), with a concomitant increase in the thermotolerance of cells. To determine whether these effects are generally displayed by nonsteroidal anti-inflammatory drugs (NSAIDs), we examined the effects of a cyclooxygenase inhibitor, indomethacin, and a lipoxygenase inhibitor, nordihydroguaiaretic acid. Both inhibitors up-regulated the hsp promoter at 37 degrees C through the activation of heat shock factors, and increased cellular levels of hsps in mammalian cells, although the degree of the expression of hsps and thermotolerance of cells differed depending on the drugs. Furthermore, NSAIDs such as sodium salicylate and indomethacin suppressed the protein aggregation and apoptosis caused by an expanded polyglutamine tract in a cellular model of polyglutamine disease. These findings suggest that NSAIDs generally induce the expression of hsps in mammalian cells and may be used for the protection of cells against deleterious stressors and neurodegenerative diseases.     

NF-kappaB p65 regulates nuclear translocation of Ku70 via degradation of heat shock cognate protein 70 in pancreatic acinar AR42J cells

Ku proteins such as Ku70 and Ku80 play key roles in multiple nuclear processes. Nuclear translocation of Ku70 is independent of Ku80 translocation and mediated by nuclear localization signal (NLS) receptors including importin-alpha. In the present study using pancreatic acinar AR42J cells, heat shock cognate protein 70 (Hsc70) was identified as the protein associated with NLS of Ku70. Interaction of Ku70 with importin-alpha and nuclear translocation of Ku70 was suppressed by overexpression of Hsc70, but enhanced by downregulation of Hsc70. The results suggest that the formation of Ku70 complex with Hsc70 prevents NLS of Ku70 from access of importin-alpha and inhibits nuclear translocation of Ku70. Since NF-kappaB p65 activation induced the decrease of Hsc70 level, the interaction of Ku70 with importin-alpha and nuclear translocation of Ku70 increased upon the activation of NF-kappaB p65. NF-kappaB p65 induced cell proliferation through decrease of Hsc70 levels and increase of nuclear translocation of Ku70. In the cells treated with cerulein as a physiological stimulus to activate NF-kappaB p65, nuclear translocation of Ku70 increased through NF-kappaB p65-mediated decrease of Hsc70 level. The results suggest that the involvement of NF-kappaB p65 in nuclear translocation of Ku70 may be mediated by Hsc70 degradation, which may play a key role in cell proliferation of pancreatic acinar AR42J cells.     

MAPK (ERK2) kinase--a key target for NSAIDs-induced inhibition of gastric cancer cell proliferation and growth.

Limited clinical and experimental studies indicate that nonsteroidal anti-inflammatory drugs (NSAIDs) may inhibit gastric cancer growth. However, the mechanisms involved are not completely understood and cannot be explained by COX-2 inhibition alone. MAPK signaling pathway is essential for cell proliferation, but the effect of NSAIDs on MAPK activity and phosphorylation in gastric cancer has never been studied. Since increased and unregulated cell proliferation and reduced cell apoptosis are important features of cancer growth, we studied whether NS-398, a selective COX-2 inhibitor and/ or indomethacin (IND), a non-selective NSAID: 1) inhibit gastric cancer cell proliferation, 2) whether this inhibition is mediated via MAPK (ERK2), and 3) whether NSAIDs enhance apoptosis in gastric cancer cells. Human gastric epithelial cells (MKN28) derived from gastric tubular adenocarcinoma were cultured and treated with either vehicle, IND (0.25-0.5mM) or NS-398 (50-100 microM) for 6, 16, 24 and 48h. Studies: 1) Cellular proliferation was determined by 3H-thymidine uptake. 2) MAPK activity was measured by incorporation of radiolabeled phosphate into myelin basic protein. 3) Apoptosis was evaluated using TUNEL assay. IND and NS-398 significantly inhibited the proliferation of MKN28 cells at 24h by 3.5 - 5 fold (p<0.002) and at 48h by 2.5 - 10 fold (p<0.02). Both NSAIDs also significantly inhibited ERK2 activity: IND >53% inhibition, NS-398, 100 microM >72% inhibition; all p<0.05. Both IND and NS-398 significantly increased apoptotic index. In conclusion, IND and NS-398 significantly inhibit proliferation and growth of human gastric cancer cell line MKN28. This effect is mediated by NSAID-induced inhibition of MAPK (ERK2) kinase signaling pathway, essential for cell proliferation. NSAIDs also increase apoptosis in MKN28 cells. In addition to inhibiting cyclooxygenase, NSAIDs inhibit phosphorylating enzymes--kinases essential for signaling cell proliferation.     

Non-steroidal anti-inflammatory drugs stimulate secretion of non-amyloidogenic precursor protein

Chronic inflammatory processes are associated with the pathophysiology of Alzheimer's disease (AD), and it has been proposed that treatment with non-steroidal anti-inflammatory drugs (NSAIDs) reduces the risk for AD. Here we report that various NSAIDs, such as the cyclooxygenase inhibitors, nimesulide, ibuprofen and indomethacin, as well as thalidomide (Thal) and its non-teratogenic analogue, supidimide, significantly stimulated the secretion of the non-amyloidogenic alpha-secretase form of the soluble amyloid precursor protein (sAPP alpha) into the conditioned media of SH-SY5Y neuroblastoma and PC12 cells. These NSAIDs markedly reduced the levels of the cellular APP holoprotein, further accelerating non-amyloidogenic processes. sAPP alpha release, induced by nimesulide and Thal, was modulated by inhibitors of protein kinase C and Erk mitogen-activated protein (MAP) kinase. Furthermore, in results complementary to the inhibitor studies, we show for the first time that NSAIDs can activate the Erk MAP kinase signaling cascade, thus identifying a novel pharmacology mechanism of NSAIDs. Our findings suggest that NSAIDs and Thal might prove useful to favor non-amyloidogenic APP processing by enhancing alpha-secretase activity, thereby reducing the formation of amyloidogenic derivatives, and therefore are of potential therapeutic value in AD.     

Inhibition of angiogenesis by nonsteroidal anti-inflammatory drugs: insight into mechanisms and implications for cancer growth and ulcer healing

Angiogenesis, the formation of new capillary blood vessels, is essential not only for the growth and metastasis of solid tumors, but also for wound and ulcer healing, because without the restoration of blood flow, oxygen and nutrients cannot be delivered to the healing site. Nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin, indomethacin and ibuprofen are the most widely used drugs for pain, arthritis, cardiovascular diseases and, more recently, the prevention of colon cancer and Alzheimer disease. However, NSAIDs produce gastroduodenal ulcers in about 25% of users (often with bleeding and/or perforations) and delay ulcer healing, presumably by blocking prostaglandin synthesis from cyclooxygenase (COX)-1 and COX-2 (ref. 10). The hypothesis that the gastrointestinal side effects of NSAIDs result from inhibition of COX-1, but not COX-2 (ref. 11), prompted the development of NSAIDs that selectively inhibit only COX-2 (such as celecoxib and rofecoxib). Our study demonstrates that both selective and nonselective NSAIDs inhibit angiogenesis through direct effects on endothelial cells. We also show that this action involves inhibition of mitogen-activated protein (MAP) kinase (ERK2) activity, interference with ERK nuclear translocation, is independent of protein kinase C and has prostaglandin-dependent and prostaglandin-independent components. Finally, we show that both COX-1 and COX-2 are important for the regulation of angiogenesis. These findings challenge the premise that selective COX-2 inhibitors will not affect the gastrointestinal tract and ulcer/wound healing.     

Nuclear translocation of stress protein Hsc70 during S phase in rat C6 glioma cells

The expression and the nuclear translocation of the constitutive heat shock protein 70 (Hsc70) were determined during the cell cycle in synchronized rat astrocytomic C6 glioma cells. Cells were first shifted to the GO by serum starvation. Twelve hours after a subsequent growth stimulation by transfer to 20% newborn calf serum, about 50% of the cells entered S phase. Western blot analysis with different monoclonal antibodies showed that only the constitutively expressed and moderately stress-activated Hsc70 is induced during serum stimulation. Maximal cellular Hsc70 content (170% of the control) was observed in early to mid S phase followed by a drastic decline while cells pass through G2/M (20% of the control). Hsp70, the major heat-inducible heat shock protein in C6 cells, is not detected in either asynchronously proliferating, serum-starved or in serum-stimulated C6 cells. Analysis of the nuclear and cytoplasmic protein fractions showed a significant increase of Hsc70 translocation into the nucleus during early S phase. These results indicate a role for Hsc70 but not for Hsp70 in the process of S phase entry and/or progression in C6 cells under physiological conditions.     

Physiological and molecular assessment of altered expression of Hsc70-1 in Arabidopsis. Evidence for pleiotropic consequences

Hsp70s function as molecular chaperones. The protective chaperone activities of hsp70 help to confer tolerance to heat, glucose deprivation, and drought. Overexpression of hsp70s in many organisms correlates with enhanced thermotolerance, altered growth, and development. To better understand the roles of hsp70 proteins in Arabidopsis, the molecular and physiological consequences of altered expression of the major heat shock cognate, Hsc70-1, were analyzed. Extensive efforts to achieve underexpression of Hsc70-1 mRNA using a full-length antisense cDNA resulted in no viable transgenic plants, suggesting that reduced expression is lethal. Constitutive overexpression of Hsc70-1 also appeared to be deleterious to viability, growth, and development because fewer transformants were recovered, and most were dwarfed with altered root systems. Despite being dwarfed, the overexpression plants progressed normally through four selected developmental stages. Heat treatment revealed that Hsc70-1 overexpression plants were more tolerant to heat shock (44 degrees C for 10 min). The elevated basal levels of HSC70-1 in transgenic plants led to delayed heat shock response of several heat shock genes. The data in this study suggest that tight regulation of Hsc70-1 expression is critical for the viability of Arabidopsis and that the functions of HSC70-1 contribute to optimum growth, development, thermotolerance, and regulation of the heat shock response.     

Mitochondrial Ca2+ overload underlies Abeta oligomers neurotoxicity providing an unexpected mechanism of neuroprotection by NSAIDs

Dysregulation of intracellular Ca(2+) homeostasis may underlie amyloid beta peptide (Abeta) toxicity in Alzheimer's Disease (AD) but the mechanism is unknown. In search for this mechanism we found that Abeta(1-42) oligomers, the assembly state correlating best with cognitive decline in AD, but not Abeta fibrils, induce a massive entry of Ca(2+) in neurons and promote mitochondrial Ca(2+) overload as shown by bioluminescence imaging of targeted aequorin in individual neurons. Abeta oligomers induce also mitochondrial permeability transition, cytochrome c release, apoptosis and cell death. Mitochondrial depolarization prevents mitochondrial Ca(2+) overload, cytochrome c release and cell death. In addition, we found that a series of non-steroidal anti-inflammatory drugs (NSAIDs) including salicylate, sulindac sulfide, indomethacin, ibuprofen and R-flurbiprofen depolarize mitochondria and inhibit mitochondrial Ca(2+) overload, cytochrome c release and cell death induced by Abeta oligomers. Our results indicate that i) mitochondrial Ca(2+) overload underlies the neurotoxicity induced by Abeta oligomers and ii) inhibition of mitochondrial Ca(2+) overload provides a novel mechanism of neuroprotection by NSAIDs against Abeta oligomers and AD.     

Ethanol induces apoptosis in hepatocytes by a pathway involving novel protein kinase C isoforms

Ethanol abuse is one of the major etiologies of cirrhosis. Ethanol has been shown to induce apoptosis via activation of oxidative stress, mitogen-activated protein kinases (MAPK), and tyrosine kinases. However, there is a paucity of data that examine the interplay among these molecules. In the present study we have systematically elucidated the role of novel protein kinase C isoforms (nPKC; PKCdelta and PKCepsilon) in ethanol-induced apoptosis in hepatocytes. Ethanol enhanced membrane translocation of PKCdelta and PKCepsilon, which was associated with the phosphorylation of p38MAPK, p42/44MAPK and JNK1/2, and the nuclear translocation of NF-kappaB and AP-1. This resulted in increased apoptosis in primary rat hepatocytes. Inhibition of both PKCdelta and PKCepsilon resulted in a decreased MAPK activation, decreased nuclear translocation of NF-kappaB and AP-1, and inhibition of apoptosis. In addition, ethanol activated the tyrosine phosphorylation of PKCdelta via tyrosine kinase in hepatocytes. The tyrosine phosphorylated PKCdelta was cleaved by caspase-3 and these fragments were translocated to the nucleus. Inhibition of ethanol-induced oxidative stress blocked the membrane translocation of PKCdelta and PKCepsilon, and the tyrosine phosphorylation of PKCdelta in hepatocytes. Inhibition of oxidative stress, tyrosine kinase or caspase-3 activity caused a decreased nuclear translocation of PKCdelta in response to ethanol, and was associated with less apoptosis. Conclusion: These results provide a newly-described mechanism by which ethanol induces apoptosis via activation of nPKC isoforms in hepatocytes.     

Differential regulation of anti-inflammatory proteins in human rheumatoid synoviocyte MH7A cell by celecoxib and ibuprofen

Non-steroidal anti-inflammatory drugs are known to be the most widely used drugs to exert their anti-inflammatory activities. It was examined protein expression profiles of human rheumatoid fibroblast-like synoviocyte MH7A cells treated with celecoxib, a selective cyclooxygenase-2 inhibitor, or ibuprofen, a non-selective cyclooxygenase inhibitor, using two-dimensional gel electrophoresis for comparison the mechanism of the drugs. Altered expression pattern in response to celecoxib is significantly different from that of ibuprofen treated cells. When MH7A cells were treated with celecoxib, 28 proteins were affected at their expression levels. Among them, heat shock proteins (Hsp60 and 70), glucose regulated proteins (Hsp75 and 78) were observed to be up-regulated by 1 to 30 microM concentrations of celecoxib but those proteins were not affected in ibuprofen treated cells. On the other hand, the expression of 19 proteins was changed by ibuprofen and the expression of apolipoprotein E, RNA binding motif 4, CTP-phosphocholine cytidylyltransferase, and phospholipase A2 inhibitory protein was only altered by ibuprofen. The expressions of 15 proteins were affected by both celecoxib and ibuprofen. Our results showed that celecoxib and ibuprofen, though they are known to act as cyclooxygenase inhibitors, could exert a different mode of acting mechanisms in anti-inflammatory processes. The chemical proteomic approach will be useful for figuring out the mode of actions of drugs.     

Non-steroidal anti-inflammatory drugs inhibit nitric oxide-induced apoptosis and dedifferentiation of articular chondrocytes independent of cyclooxygenase activity

Nitric oxide (NO) causes apoptosis and dedifferentiation of articular chondrocytes by the modulation of extracellular signal-regulated kinase (ERK), p38 kinase, and protein kinase C (PKC) alpha and -zeta. In this study, we investigated the effects and mechanisms of non-steroidal anti-inflammatory drugs (NSAIDs), such as indomethacin, ketoprofen, ibuprofen, sulindac sulfide, and flurbiprofen, in NO-induced apoptosis and dedifferentiation of articular chondrocytes. We found that all of the examined NSAIDs inhibited apoptosis and dedifferentiation. NO production in chondrocytes caused activation of ERK-1/2 and p38 kinase, which oppositely regulate apoptosis and dedifferentiation. NO production also caused inhibition of PKCalpha and -zeta independent of and dependent on, respectively, p38 kinase, which is required for apoptosis and dedifferentiation. Among the signaling molecules modulated by NO, NSAIDs blocked NO-induced activation of p38 kinase, potentiated ERK activation, and blocked inhibition of PKCalpha and -zeta. NSAIDs also inhibited some of the apoptotic signaling that is downstream of p38 kinase and PKC, such as NFkappaB activation, p53 accumulation, and caspase-3 activation. The inhibitory effects of NSAIDs on apoptosis and dedifferentiation were independent of the inhibition of cyclooxygenase (COX)-2 and prostaglandin E(2) (PGE(2)) production, as evidenced by the observation that specific inhibition of COX-2 activity and PGE(2) production or exogenous PGE(2) did not affect NO-induced apoptosis and dedifferentiation. Taken together, our results indicate that NSAIDs block NO-induced apoptosis and dedifferentiation of articular chondrocytes by the modulation of ERK, p38 kinase, and PKCalpha and -zeta in a manner independent of their ability to inhibit COX-2 and PGE(2) production.     

Sialyl-glycoconjugates in cholesterol-rich microdomains of P388 cells are the triggers for apoptosis induced by Rana catesbeiana oocyte ribonuclease

SBL/RC-RNase was originally isolated from frog (Rana catesbeiana) oocytes and purified as a novel sialic acid-binding lectin (SBL) that displayed strong anti-cancer activity. SBL was later shown to be identical to a ribonuclease (RC-RNase) from oocytes of the same species. The administration of SBL/RC-RNase induced apoptosis (with nuclear condensation and DNA fragmentation) in mouse leukemia P388 cells but did not kill umbilical vein endothelial or fibroblast cells derived from normal tissues. The cytotoxic activity of SBL/RC-RNase was inhibited by desialylation of P388 cells and/or the co-presence of free bovine submaxillary mucin. FACS analysis showed that SBL/RC-RNase was incorporated into cells after attachment to cholesterol-rich microdomains. Addition of the cholesterol remover methyl-β-cyclodextrin reduced SBL/RC-RNase-induced apoptosis. Apoptosis occurred through the caspase-3 pathway following activation of caspase-8 by SBL/RC-RNase. A heat shock cognate protein (Hsc70) and a heat shock protein (Hsp70) (each 70 kDa) on the cell membrane were shown to bind to SBL/RC-RNase by mass spectrometric and flow cytometric analyses. Quercetin, an inhibitor of Hsc70 and Hsp70, significantly reduced SBL/RC-RNase-induced apoptosis. Taken together, our findings suggest that sialyl-glycoconjugates present in cholesterol-rich microdomains form complexes with Hsc70 or Hsp70 that act as triggers for SBL/RC-RNase to induce apoptosis through a pathway involving the activation of caspase-3 and caspase-8.     

Identification of novel nuclear export and nuclear localization-related signals in human heat shock cognate protein 70

Heat shock cognate protein 70 (Hsc70) serves nuclear transport of several proteins as a molecular chaperone. We have recently identified a novel variant of human Hsc70, heat shock cognate protein 54 (Hsc54), that lacks amino acid residues 464-616 in the protein binding and variable domains of Hsc70. In the present study, we examined nucleocytoplasmic localization of Hsc70 and Hsc54 by using green fluorescent protein (GFP) fusions. GFP-Hsc70 is localized in both the cytoplasm and the nucleus at 37 degrees C and accumulated into the nucleolus/nucleus after heat shock, whereas GFP-Hsc54 always remained exclusively in the cytoplasm under these conditions. Mutation studies indicated that 20 amino acid residues of nuclear localization-related signals, which are missing in Hsc54 but are retained in Hsc70, are required for proper nuclear localization of Hsc70. We further found that Hsc54 contains a functional leucine-rich nuclear export signal (NES, (394)LDVTPLSL(401)) which is differently situated from the previously proposed NES in Saccharomyces cerevisiae Ssb1p. The cytoplasmic localization of Hsc54 was impaired by a mutation in NES as well as by a nuclear export inhibitor, leptomycin B, suggesting that Hsc54 is actively exported from the nucleus to the cytoplasm through a CRM1-dependent mechanism. In contrast, the nucleocytoplasmic localization of Hsc70 was not affected by the same mutation of NES or leptomycin B. These results suggest that the nuclear localization-related signal could functionally mask NES leading to prolonged retention of Hsc70 in the nucleus. An additional mechanism for unmasking the NES may regulate nucleocytoplasmic trafficking of Hsc70.     

Identification of Hsc70 as an influenza virus matrix protein (M1) binding factor involved in the virus life cycle

Influenza virus matrix protein 1 (M1) has been shown to play a crucial role in the virus replication, assembly and budding. We identified heat shock cognate protein 70 (Hsc70) as a M1 binding protein by immunoprecipitation and MALDI-TOF MS. The C terminal domain of M1 interacts with Hsc70. We found that Hsc70 does not correlate with the transport of M1 to the nucleus, however, it does inhibit the nuclear export of M1 and NP, thus resulting in the inhibition of viral production. This is the first demonstration that Hsc70 is directly associated with M1 and therefore is required for viral production.     

Salicylic acid-mediated potentiation of Hsp70 induction correlates with reduced apoptosis in tobacco protoplasts.

BACKGROUND: Elevated temperatures jeopardize plant disease resistance, as mediated by salicylic acid (SA). SA potentiates heat-induced expression of the 70-kDa heat shock protein (Hsp70) in tomato cells. In mammalian cells, Hsp70 suppresses apoptosis. We hypothesized that potentiation of heat-induced Hsp70 by SA contributes to a reduction in apoptosis in tobacco protoplasts. METHODS: Tobacco protoplasts (Nicotiana tabacum) were exposed to SA (70 microM) at normal temperatures or in combination with heat shock. Hsp70/Hsc70 accumulation and phosphatidylserine (PS) exposure, DNA fragmentation, as well as loss of mitochondrial membrane potential were quantified by flow cytometry. RESULTS AND CONCLUSIONS: SA at normal temperatures did not influence Hsp70/Hsc70 accumulation, but were found to induce apoptosis. In contrast, SA in combination with HS potentiated heat-induced Hsp70/Hsc70 accumulation in tobacco protoplasts that correlated negatively with apoptosis, illustrated by decreased PS exposure and DNA fragmentation and enhanced mitochondrial membrane potential. We propose that this correlation supports a possible role for apoptosis suppression by Hsp70 under elevated temperatures during pathogen infection.     

A Fas-associated death domain protein-dependent mechanism mediates the apoptotic action of non-steroidal anti-inflammatory drugs in the human leukemic Jurkat cell line

Non-steroidal anti-inflammatory drugs (NSAIDs) are inhibitors of cyclooxygenase-1 and -2 and are useful for prevention and cure of cancers, especially colon and rectal cancers. The NSAIDs indomethacin and sulindac sulfide have been shown to induce apoptosis of colon epithelial cancer cells by a Bax-dependent mechanism that involves mitochondria-mediated activation of a caspase-9-dependent pathway. In this report, we demonstrate that indomethacin and sulindac sulfide induce apoptosis of human leukemic Jurkat cells by a mechanism that requires the Fas-associated Death Domain Protein-mediated activation of a caspase-8-dependent pathway. Therefore, NSAIDs induce apoptosis by different mechanisms depending on the cell type.