thapsigargin and benzyloxycarbonylleucyl-leucyl-leucine-aldehyde

Topics: Animals; Bortezomib; Cell Line, Tumor; Drug Resistance, Multiple; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Leupeptins; Mice; Thapsigargin; Tunicamycin; Vincristine

Endoplasmic reticulum (ER) stress causes neuronal dysfunction followed by cell death and is recognized as a feature of many neurodegenerative diseases. Using a phenotypic screen, we recently identified benzodiazepinone derivatives that reduce ER stress-mediated apoptosis in a rat neuronal progenitor cell line (CSM14.1). Herein we describe how structure-activity relationship (SAR) studies around these screening hits led to compounds that display robust cytoprotective activity against thapsigargin-induced ER stress in SH-SY5Y and H4 human neuronal cell lines. We demonstrate that the most potent of these derivatives, compound 4hh, inhibits the activation of p38 MAP kinase (p38) and c-Jun N-terminal kinase (JNK), protein kinases that are downstream signal effectors of the unfolded protein response (UPR). Compound 4hh specifically protects against thapsigargin-induced cell death and displays no protection against other insults known to induce cellular stress or activate p38. However, compound 4hh provides moderate inhibition of p38 activity stimulated by compounds that disrupt calcium homeostasis. Our data indicate that probe compound 4hh is a valuable small molecule tool that can be used to investigate the effects of ER stress on human neurons. This approach may provide the basis for the future development of therapeutics for the treatment of neurodegenerative diseases.

Topics: Animals; Benzodiazepinones; Calcium; Cell Death; Cell Line; Dose-Response Relationship, Drug; Endoplasmic Reticulum Stress; Enzyme Inhibitors; Homeostasis; Humans; Imidazoles; Ionomycin; Leupeptins; MAP Kinase Signaling System; Neurons; Oleanolic Acid; Rats; Structure-Activity Relationship; Thapsigargin

Genome-wide transcript profiling to elucidate responses to HSP90 inhibition revealed strong induction of HSPA6 in MCF-7 cells treated with 17-AAG. Time- and dose dependent induction of HSPA6 (confirmed by qPCR and Western Blots) occurred also upon treatment with Radicicol, another HSP90 inhibitor. HSPA6 was not detectable in untreated cells or cells treated with toxins that do not inhibit HSP90, or upon applying oxidative stress. Thus, HSPA6 induction is not a general response to cytotoxic insults. Modulation of HSPA6 levels by siRNA-mediated inhibition or recombinant expression did not influence 17-AAG mediated cell death. HSPA6 induction as a consequence of HSP90 inhibition occurs in various (but not all) cell lines and may be a more specific marker for HSP90 inhibition than induction of other HSP70 proteins.

Topics: Amino Acid Sequence; Benzoquinones; Blotting, Western; Brefeldin A; Cell Line, Tumor; Dose-Response Relationship, Drug; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Hot Temperature; HSP70 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Leupeptins; Macrolides; MCF-7 Cells; Molecular Sequence Data; RNA Interference; Sequence Homology, Amino Acid; Thapsigargin; Time Factors; Transcriptional Activation

Recently, cation transport regulator homolog 1 (Chac1) has been identified as a novel pro-apoptotic factor in cells under endoplasmic reticulum (ER) stress. Of the three major ER stress sensors, it is suggested that ATF4 participates in the transcriptional regulation of Chac1 gene expression. The precise characterization of the Chac1 promoter, however, has not yet been elucidated. In this study, we detected the induction of Chac1 mRNA expression using DNA array analysis and RT-PCR of thapsigargin (Tg)-inducible genes in Neuro2a cells. Chac1 mRNA expression was also induced immediately following treatment with tunicamycin (Tm) and brefeldin A. Characterization of the mouse Chac1 promoter activity using a luciferase reporter assay revealed that the CREB/ATF element and amino acid response element in the mouse Chac1 promoter are functional and respond to Tm stimulation and ATF4 overexpression. Mutations in either element in the Chac1 promoter did not inhibit the responsiveness of this promoter to Tm and ATF4; however, mutations in both of these elements dramatically decreased the basal activity and response to ER stress stimuli. In addition to the transcriptional regulation, we found that Chac1 protein expression was only detected in the presence of MG132, a proteasome inhibitor, even though mouse Chac1 gene was transiently overexpressed in Neuro2a cells. Taken together, we are the first to demonstrate the transcriptional and post-translational regulation of Chac1 expression in a neuronal cell line.

Topics: Activating Transcription Factor 4; Animals; Base Sequence; Blotting, Western; Cell Line, Tumor; Connexins; Cysteine Proteinase Inhibitors; Endoplasmic Reticulum Stress; gamma-Glutamylcyclotransferase; Gene Expression Regulation; Intracellular Signaling Peptides and Proteins; Leupeptins; Mice; Molecular Sequence Data; Mutation; Neuroblastoma; Promoter Regions, Genetic; Protein Biosynthesis; Reverse Transcriptase Polymerase Chain Reaction; Thapsigargin; Transcription, Genetic; Tunicamycin

Environmental stresses that disrupt protein homeostasis induce phosphorylation of eIF2, triggering repression of global protein synthesis coincident with preferential translation of ATF4, a transcriptional activator of the integrated stress response (ISR). Depending on the extent of protein disruption, ATF4 may not be able to restore proteostatic control and instead switches to a terminal outcome that features elevated expression of the transcription factor CHOP (GADD153/DDIT3). The focus of this study is to define the mechanisms by which CHOP directs gene regulatory networks that determine cell fate. We find that in response to proteasome inhibition, CHOP enhances the expression of a collection of genes encoding transcription regulators, including ATF5, which is preferentially translated during eIF2 phosphorylation. Transcriptional expression of ATF5 is directly induced by both CHOP and ATF4. Knockdown of ATF5 increases cell survival in response to proteasome inhibition, supporting the idea that both ATF5 and CHOP have proapoptotic functions. Transcriptome analysis of ATF5-dependent genes reveals targets involved in apoptosis, including NOXA, which is important for inducing cell death during proteasome inhibition. This study suggests that the ISR features a feedforward loop of stress-induced transcriptional regulators, each subject to transcriptional and translational control, which can switch cell fate toward apoptosis.

Topics: Activating Transcription Factors; Animals; Apoptosis; Apoptosis Regulatory Proteins; Cell Survival; Cells, Cultured; Eukaryotic Initiation Factor-2; Feedback, Physiological; Gene Expression Regulation; Gene Knockout Techniques; Gene Regulatory Networks; Homeostasis; Leupeptins; Mice; Phosphorylation; Promoter Regions, Genetic; Proteasome Inhibitors; Protein Biosynthesis; Protein Processing, Post-Translational; Proteolysis; Proto-Oncogene Proteins c-bcl-2; Response Elements; Stress, Physiological; Thapsigargin; Transcription Factor CHOP; Transcriptional Activation; Transcriptome

Expression of clusterin (CLU) closely correlates with the regulation of apoptosis in cancer. Although endoplasmic reticulum (ER) stress-induced upregulation and retrotranslocation of cytoplasmic CLU (presecretory (psCLU) and secreted (sCLU) forms) has been linked to its anti-apoptotic properties, mechanisms mediating these processes remain undefined. Here, we show using human prostate cancer cells that GRP78 (Bip) associates with CLU under ER stress conditions to facilitate its retrotranslocation and redistribution to the mitochondria. Many ER stress inducers, including thapsigargin, MG132 or paclitaxel, increased expression levels of GRP78 and CLU, as well as post-translationally modified hypoglycosylated CLU forms. ER stress increased association between GRP78 and CLU, which led to increased cytoplasmic CLU levels, while reducing sCLU levels secreted into the culture media. GRP78 stabilized CLU protein and its hypoglycosylated forms, in particular after paclitaxel treatment. Moreover, subcellular fractionation and confocal microscopy with CLUGFP indicated that GRP78 increased stress-induced CLU retrotranslocation from the ER with co-localized redistribution to the mitochondria, thereby reducing stress-induced apoptosis by cooperatively stabilizing mitochondrial membrane integrity. GRP78 silencing reduced CLU protein, but not mRNA levels, and enhanced paclitaxel-induced cell apoptosis. Taken together, these findings reveal novel dynamic interactions between GRP78 and CLU under ER stress conditions that govern CLU trafficking and redistribution to the mitochondria, elucidating how GRP78 and CLU cooperatively promote survival during treatment stress in prostate cancer.

Topics: Apoptosis; Cell Line, Tumor; Clusterin; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Heat-Shock Proteins; Humans; Leupeptins; Male; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Membranes; Paclitaxel; Prostatic Neoplasms; Protein Transport; RNA Interference; RNA, Messenger; RNA, Small Interfering; Thapsigargin; Up-Regulation

The transcription factor CHOP/GADD153 is induced during the unfolded protein response and is related to the induction of ER stress-mediated apoptosis. However, how CHOP is organized between the pro-survival and pro-apoptotic roles of ER stress remains largely undefined. In this study, we identified the apoptosis regulating protein suppressed by CHOP. We found that treatment of Caki cells with CHOP-inducing drugs including withaferin A, thapsigargin, brefeldin A, and silybin led to a strong reduction in cFLIP(L) protein levels together with a concomitant increase in the CHOP protein. Interestingly, Wit A down-regulated cFLIP(L) expression via both suppressing mRNA transcription and increasing cFLIPL protein instability. We also found that forced expression of CHOP dose-dependently led to a decrease of cFLIP(L) protein expression but did not alter cFLIP(L) mRNA levels. Additionally, we observed that siRNA-mediated CHOP silencing recovered the cFLIP(L) expression decreased by CHOP-inducing agents in Caki cells. Finally, we showed that CHOP facilitates ubiquitin/proteasome-mediated cFLIP(L) degradation, leading to down-regulation of cFLIP(L). Finally, cFLIP(L) over-expression reduced cell death induced by treatment with brefeldin A, thapsigargin, and silybin. Taken together, our results provide novel evidence that cFLIP(L) is a CHOP control target and that CHOP-induced down-regulation of cFLIP(L) is due to activation of the ubiquitin/proteasome pathways.

Topics: Apoptosis; Blotting, Western; Brefeldin A; CASP8 and FADD-Like Apoptosis Regulating Protein; Cell Line, Tumor; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Genetic Vectors; Humans; Immunoprecipitation; Leupeptins; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Stability; Proteolysis; RNA, Messenger; RNA, Small Interfering; Silybin; Silymarin; Thapsigargin; Transcription Factor CHOP; Transcription, Genetic; Transfection; Ubiquitin; Ubiquitination; Unfolded Protein Response; Withanolides

The BH3-only proteins of the Bcl-2 family are known to mediate mitochondrial dysfunction during apoptosis. However, the identity of the critical BH3-only proteins and the mechanism of their action following treatment by diverse apoptotic stimuli remain to be fully resolved. We therefore used RNAi to screen the entire Bcl-2 family for their involvement in three major apoptotic pathways in HeLa cells. We found that Bcl-xL and Mcl-1 are major inhibitors of apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL), endoplasmic reticulum (ER) stress, and proteasome inhibition. Among the 10 BH3-only proteins, Bid and Noxa were found to be critically involved in TRAIL-induced apoptosis, in which Noxa participates by constitutively binding to Mcl-1. Bim and Noxa were found to be necessary for ER stress-induced apoptosis, in which Noxa assisted Bim function by sequestering Mcl-1 and binding to Bcl-xL. As a critical BH3-only protein, Noxa was strongly upregulated and became associated with both Mcl-1 and Bcl-xL during apoptosis induced by proteasome inhibition. In addition, we found that Noxa became 'Mcl-1 free' following treatment by ER stress and proteasome inhibition, but not after TRAIL treatment. These results defined the critical Bcl-2 network during apoptosis and suggested that Noxa participated in triggering mitochondrial dysfunction in multiple apoptotic pathways through distinct mechanisms.

Topics: Apoptosis; Apoptosis Regulatory Proteins; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Bcl-2-Like Protein 11; bcl-Associated Death Protein; bcl-X Protein; BH3 Interacting Domain Death Agonist Protein; Endoplasmic Reticulum; HeLa Cells; Humans; Leupeptins; Membrane Proteins; Myeloid Cell Leukemia Sequence 1 Protein; Proteasome Inhibitors; Protein Binding; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; RNA Interference; Stress, Physiological; Thapsigargin; TNF-Related Apoptosis-Inducing Ligand

To explore the apoptotic effect of ubiquitin-proteasome inhibitor (PI) MG-132 on human tongue squamous cell carcinoma cell line (Tca-8113 cell) through endoplasmic reticulum stress.. Tca-8113 cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, the exponential cells were divided into 5 groups.The cell culture medium was added to 1640 (negative control group), thapsigargin 5 μmol/L (positive control group), and 10,20,30 μmol/L (MG-132 group). After 24 h, the following experiments were carried out: morphological change of apoptotic cells was observed by Hoechst 33258 fluorescent staining under fluorescent microscope, apoptotic rate of cells was determined with annexin V-FITC/PI double staining by flow cytometry, the GRP78 mRNA level was determined by RT-PCR, the expression of caspase-12 protein was evaluated by Western blot, the human ubiquitin-protein ligase E3 concentration was detected by ELISA. The data was analyzed using SPSS16.0 software package.. Typical morphological change of apoptosis in Tca-8113 cells was observed 24 hours after treating with 10.0, 20.0, 30.0 μmol/L MG-132 and positive control group; The apoptotic rate of MG-132 groups gradually increased with MG-132 concentration; The GRP78 mRNA level was up-regulated; The expression of caspase-12 increased was demonstrated by Western blot; The expression of the human ubiquitin-protein ligase E3 in MG-132 groups was 28.75 ± 2.28, 18.16 ± 0.65 and 8.85 ± 0.72.. MG-132 can induce apoptosis of Tca-8113 cells through endoplasmic reticulum stress; MG-132 can inhibit the expression of human ubiquitin ligase E3.

Topics: Animals; Apoptosis; Carcinoma, Squamous Cell; Cell Line; Endoplasmic Reticulum Chaperone BiP; Heat-Shock Proteins; Humans; Leupeptins; Thapsigargin; Tongue Neoplasms

The aim of this study was to investigate the apoptotic effect of a proteasome inhibitor MG-132 on Tca-8113, a cell line of human tongue squamous cell carcinoma. Tca-8113 cells were treated with 10, 20, and 30μM of MG-132, or 5μM thapsigargin. Apoptosis rate was determined with annexin V/propidium iodide double staining. Expression of E3ubiquitin-protein ligase was determined by ELISA, and Grp78 and caspase-12 mRNA, and Grp78 and caspase-12 protein was assessed by RT-PCR and Western blot, respectively. Apoptosis was observed 18h after MG-132 treatment. The apoptotic rate in the 10, 20, and 30μM MG-132 group was 13.5, 19.6 and 34.7%, respectively, which was higher than in the thapsigargin (8.5%, P<0.01) or control group (0.5%, P<0.01). The expression of E3 ubiquitin-protein ligase in the 10, 20, and 30μM MG-132 group was 28.75±2.28, 18.16±0.65, 8.85±0.72, respectively, which was lower than in the thapsigargin (38.96±0.33, P<0.05 or 0.01) or control (40.88±4.52, P<0.05 or 0.01) group. The levels of Grp78 and capase-12 mRNA, Grp78 and caspase-12 protein in the MG-132 groups were higher than in the control group (P<0.01). In conclusion, MG-132 induces apoptosis in Tca-8113 cells in a concentration-dependent manner. The MG-132-induced apoptosis may involve downregulation of E3 ubiquitin ligase, and upregulation of Grp78 and caspase-12.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Squamous Cell; Caspase 12; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Endoplasmic Reticulum Chaperone BiP; Heat-Shock Proteins; Humans; Leupeptins; RNA, Messenger; Thapsigargin; Tongue Neoplasms; Ubiquitin-Protein Ligases

DNA fragmentation is one of the most characteristic features of apoptotic cells and caspase-activated DNase (CAD) is considered to be a major nuclease responsible for DNA fragmentation. CAD forms a complex with its inhibitor (ICAD), which is also required for the functional folding of CAD, leading to CAD stabilization in cells. In this paper, we investigated the involvement of the ubiquitin-proteasome system in CAD stability. The expression and ubiquitination of CAD was remarkably increased by MG132 treatment in the absence of ICAD. These results suggest that CAD protein may be preferentially degraded by the ubiquitin-proteasome system in the absence of ICAD to maintain protein quality control.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Caspase 3; Chlorides; Deoxyribonucleases; DNA Fragmentation; Fibroblasts; Gene Expression; HeLa Cells; Humans; Leupeptins; Manganese Compounds; Mice; NIH 3T3 Cells; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Rotenone; Staurosporine; Thapsigargin; Transfection; Ubiquitin; Ubiquitination

p53 regulates apoptosis and the cell cycle through actions in the nucleus and cytoplasm. Altering the subcellular localization of p53 can alter its biological function. Therefore, small molecules that change the localization of p53 would be useful chemical probes to understand the influence of subcellular localization on the function of p53. To identify such molecules, a high-content screen for compounds that increased the localization of p53 to the nucleus or cytoplasm was developed, automated, and conducted. With this image-based assay, we identified ellipticine that increased the nuclear localization of GFP-mutant p53 protein but not GFP alone in Saos-2 osteosarcoma cells. In addition, ellipticine increased the nuclear localization of endogenous p53 in HCT116 colon cancer cells with a resultant increase in the transactivation of the p21 promoter. Increased nuclear p53 after ellipticine treatment was not associated with an increase in DNA double stranded breaks, indicating that ellipticine shifts p53 to the nucleus through a mechanism independent of DNA damage. Thus, a chemical biology approach has identified a molecule that shifts the localization of p53 and enhances its nuclear activity.

Topics: Active Transport, Cell Nucleus; Cell Line, Tumor; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; DNA Damage; Drug Evaluation, Preclinical; Ellipticines; Green Fluorescent Proteins; Humans; Leupeptins; Osteosarcoma; Recombinant Fusion Proteins; Thapsigargin; Tumor Suppressor Protein p53

Inactivation of the tumor suppressor p53 by degradation is a mechanism utilized by cells to adapt to endoplasmic reticulum (ER) stress. However, the mechanisms of p53 destabilization by ER stress are not known. We demonstrate here that the E3 ubiquitin-ligase Hdm2 is essential for the nucleocytoplasmic transport and proteasome-dependent degradation of p53 in ER-stressed cells. We also demonstrate that p53 phosphorylation at S315 and S376 is required for its nuclear export and degradation by Hdm2 without interfering with the ubiquitylation process. Furthermore, we show that p53 destabilization in unstressed cells utilizes the cooperative action of Hdm2 and glycogen synthase kinase 3beta, a process that is enhanced in cells exposed to ER stress. In contrast to other stress pathways that stabilize p53, our findings further substantiate a negative role of ER stress in p53 activation with important implications for the function of the tumor suppressor in cells with a dysfunctional ER.

Topics: Animals; Cell Line; Cycloheximide; Doxorubicin; Endoplasmic Reticulum; Fatty Acids, Unsaturated; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Leupeptins; Mice; Phosphorylation; Proteasome Endopeptidase Complex; Protein Synthesis Inhibitors; Protein Transport; Serine; Signal Transduction; Thapsigargin; Tumor Suppressor Protein p53; Tunicamycin; Ubiquitin; Ubiquitin-Protein Ligases

Homer proteins physically link metabotropic glutamate receptors with IP3 receptors located at the endoplasmic reticulum (ER) and thereby modulate receptor-activated calcium signaling. Homer 1a, the short form of constitutively expressed homer 1 proteins, exerts dominant negative activity with respect to homer 1 proteins by interfering with the formation of multiprotein complexes. Homer 1a is an immediate early gene, the expression of which is activated by various stimuli including glutamate receptor activation. The mechanisms underlying activation of homer 1a expression are however, not fully understood. Here, we show that homer 1a expression is induced in neuronal cell cultures under experimental conditions associated with ER dysfunction. Increased homer 1a mRNA levels were found in 2 sets of cultures: in those exposed to thapsigargin, a specific inhibitor of ER Ca2+-ATPase, after a transient depletion of ER calcium stores through exposure to calcium-free medium supplemented with EGTA, and in those exposed to a proteasome inhibitor known to induce ER dysfunction. Thus, homer 1a expression may be activated by impairment of ER functioning just as it is by glutamate receptor activation.

Topics: Animals; Calcium; Carrier Proteins; Cells, Cultured; Chelating Agents; Cysteine Endopeptidases; Egtazic Acid; Endoplasmic Reticulum; Enzyme Inhibitors; Gene Expression Regulation; Homer Scaffolding Proteins; Leupeptins; Multienzyme Complexes; Neurons; Neuropeptides; Proteasome Endopeptidase Complex; Protein Isoforms; Rats; Rats, Wistar; RNA, Messenger; Thapsigargin

Influenza viruses bud from the plasma membrane of virus-infected cells. Although budding is a critical step in virus replication, little is known about the requirements of the budding process. In this report, we have investigated the role of ATP in influenza virus budding by treating influenza virus infected Madin-Darby canine kidney (MDCK) cells with a number of metabolic inhibitors. When WSN virus-infected MDCK cells were exposed to antimycin A, carbonyl cyanide m-chlorophenylhydrazone, carbonyl cyanide p-trifluoromethoxy-phenylhydrazone, or oligomycin for a short time (15 min or 1 h) late in the infectious cycle, the rate of virus budding decreased. This inhibitory effect was reversible upon removal of the inhibitors. The role of ATP hydrolysis was analyzed by treating lysophosphatidylcholine (LPC)-permeabilized live filter-grown virus-infected MDCK cells with nonpermeable ATP analogues from the basal side and assaying virus budding from the apical side. In LPC-permeabilized cells, membrane-impermeable ATP analogues such as adenosine 5'-O-(3-thiotriphosphate) or 5'-adenylylimidodiphosphate caused reduction of virus budding which could be partially restored by adding excess ATP. These data demonstrated that ATP hydrolysis and not just ATP binding was required for virus budding. However, inhibitors of ion channel (ATPases) and protein ubiquitinylation, which also required the ATP as energy source, did not affect influenza virus budding, suggesting that neither ion channel nor protein ubiquitinylation activity was involved in influenza virus budding. On the other hand, treatment with dimethyl sulfoxide (DMSO), which decreases membrane viscosity, reduced the rate of virus budding, demonstrating that the physical state of membrane viscosity and membrane fluidity had an important effect on virus budding. Data presented in the report indicate that influenza virus budding is an active ATP-dependent process and suggest that reduced virus budding by ATP depletion and DMSO treatment may be partly due to decreased membrane viscosity.

Topics: Acetylcysteine; Adenosine Triphosphate; Animals; Calcium-Transporting ATPases; Cell Line; Cell Membrane Permeability; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dogs; Enzyme Inhibitors; Humans; Indoles; Influenza A virus; Kinetics; Leupeptins; Lysophosphatidylcholines; Multienzyme Complexes; Oligopeptides; Ouabain; Proteasome Endopeptidase Complex; Sodium-Potassium-Exchanging ATPase; Sulfones; Thapsigargin; Viscosity