bay-11-7082 and parthenolide

In mature erythrocytes, glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) yield NADPH, a crucial cofactor of the enzyme glutathione reductase (GR) converting glutathione disulfide (GSSG) into its reduced state (GSH). GSH is essential for detoxification processes in and survival of erythrocytes. We explored whether the anti-inflammatory compounds Bay 11-7082, parthenolide and dimethyl fumarate (DMF) were able to completely deplete a common target (GSH), and to impair the function of upstream enzymes of GSH recycling and replenishment. Treatment of erythrocytes with Bay 11-7082, parthenolide or DMF led to concentration-dependent eryptosis resulting from complete depletion of GSH. GSH depletion was due to strong inhibition of G6PDH activity. Bay 11-7082 and DMF, but not parthenolide, were able to inhibit the GR activity. This approach "Inhibitors, Detection of their common target that is completely depleted or inactivated when pharmacologically relevant concentrations of each single inhibitor are applied, Subsequent functional analysis of upstream enzymes for this target" (IDS), can be applied to a broad range of inhibitors and cell types according to the selected target. The specific G6PDH inhibitory effect of these compounds may be exploited for the treatment of human diseases with high NADPH and GSH consumption rates, including malaria, trypanosomiasis, cancer or obesity.

Topics: Dimethyl Fumarate; Eryptosis; Erythrocytes; Glucosephosphate Dehydrogenase; Humans; Nitriles; Sesquiterpenes; Sulfones

The effect of lipopolysaccharide (LPS) on valproic acid (VPA)-induced cell death was examined by using mouse RAW 264.7 macrophage cells. LPS inhibited the activation of caspase 3 and poly (ADP-ribose) polymerase and prevented VPA-induced apoptosis. LPS inhibited VPA-induced p53 activation and pifithrin-α as a p53 inhibitor as well as LPS prevented VPA-induced apoptosis. LPS abolished the increase of Bax/Bcl-2 ratio, which is a critical indicator of p53-mediated mitochondrial damage, in response to VPA. The nuclear factor (NF)-κB inhibitors, Bay 11-7082 and parthenolide, abolished the preventive action of LPS on VPA-induced apoptosis. A series of toll-like receptor ligands, Pam3CSK4, poly I:C, and CpG DNA as well as LPS prevented VPA-induced apoptosis. Taken together, LPS was suggested to prevent VPA-induced apoptosis via activation of anti-apoptotic NF-κB and inhibition of pro-apoptotic p53 activation. The detailed inhibitory mechanism of VPA-induced apoptosis by LPS is discussed.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Cell Line; Dose-Response Relationship, Drug; Immunoblotting; Lipopeptides; Lipopolysaccharides; Macrophages; Mice; NF-kappa B; Nitriles; Oligodeoxyribonucleotides; Poly I-C; Proto-Oncogene Proteins c-bcl-2; Sesquiterpenes; Sulfones; Toll-Like Receptor 9; Toll-Like Receptors; Tumor Suppressor Protein p53; Valproic Acid

Erythrocytes may enter eryptosis, a suicidal death characterized by cell shrinkage and phosphatidylserine exposure at the erythrocyte outer membrane. Susceptibility to eryptosis is enhanced in aged erythrocytes and stimulated by NFκB-inhibitors Bay 11-7082 and parthenolide. Here we explored whether expression of NFκB and susceptibility to inhibitor-induced eryptosis is sensitive to erythrocyte age.. Human erythrocytes were separated into five fractions, based on age-associated characteristics cell density and volume. NFκB compared to ß-actin protein abundance was estimated by Western blotting and cell volume from forward scatter. Phosphatidylserine exposure was identified using annexin-V binding.. NFκB was most abundant in young erythrocytes but virtually absent in aged erythrocytes. A 24h or 48h exposure to Ringer resulted in spontaneous decrease of forward scatter and increase of annexin V binding, effects more pronounced in aged than in young erythrocytes. Both, Bay 11-7082 (20 µM) and parthenolide (100 µM) triggered eryptosis, effects again most pronounced in aged erythrocytes.. NFκB protein abundance is lowest and spontaneous eryptosis as well as susceptibility to Bay 11-7082 and parthenolide highest in aged erythrocytes. Thus, inhibition of NFκB signalling alone is not responsible for the stimulation of eryptosis by parthenolide or Bay 11-7082.

Topics: Aging; Apoptosis; Cells, Cultured; Erythrocytes; Humans; NF-kappa B; Nitriles; Sesquiterpenes; Sulfones; Time Factors

Identification of novel target pathways in glioblastoma (GBM) remains critical due to poor prognosis, inefficient therapies and recurrence associated with these tumors. In this work, we evaluated the role of nuclear-factor-kappa-B (NFκB) in the growth of GBM cells, and the potential of NFκB inhibitors as antiglioma agents. NFκB pathway was found overstimulated in GBM cell lines and in tumor specimens compared to normal astrocytes and healthy brain tissues, respectively. Treatment of a panel of established GBM cell lines (U138MG, U87, U373 and C6) with pharmacological NFκB inhibitors (BAY117082, parthenolide, MG132, curcumin and arsenic trioxide) and NFκB-p65 siRNA markedly decreased the viability of GBMs as compared to inhibitors of other signaling pathways such as MAPKs (ERK, JNK and p38), PKC, EGFR and PI3K/Akt. In addition, NFκB inhibitors presented a low toxicity to normal astrocytes, indicating selectivity to cancerous cells. In GBMs, mitochondrial dysfunction (membrane depolarization, bcl-xL downregulation and cytochrome c release) and arrest in the G2/M phase were observed at the early steps of NFκB inhibitors treatment. These events preceded sub-G1 detection, apoptotic body formation and caspase-3 activation. Also, NFκB was found overstimulated in cisplatin-resistant C6 cells, and treatment of GBMs with NFκB inhibitors overcame cisplatin resistance besides potentiating the effects of the chemotherapeutics, cisplatin and doxorubicin. These findings support NFκB as a potential target to cell death induction in GBMs, and that the NFκB inhibitors may be considered for in vivo testing on animal models and possibly on GBM therapy.

Topics: Animals; Antineoplastic Agents; Apoptosis; Arsenic Trioxide; Arsenicals; Astrocytes; Brain Neoplasms; Cell Cycle; Cell Death; Cell Line, Tumor; Cisplatin; Curcumin; Doxorubicin; Drug Synergism; Glioblastoma; Humans; Leupeptins; Molecular Targeted Therapy; NF-kappa B; Nitriles; Oxides; Rats; Sesquiterpenes; Signal Transduction; Sulfones

The preclinical compounds Bay 11-7082 and parthenolide trigger apoptosis, an effect contributing to their antiinflammatory action. The substances interfere with the activation and nuclear translocation of nuclear factor NFκB, by inhibiting NFκB directly (parthenolide) or by interfering with the inactivation of the NFκB inhibitory protein IκB-α (Bay 11-7082). Beyond that, the substances may be effective in part by nongenomic effects. Similar to apoptosis of nucleated cells, erythrocytes may undergo apoptosis-like cell death (eryptosis) characterized by cell membrane scrambling with phosphatidylserine exposure, and cell shrinkage. Thus, erythrocytes allow the study of nongenomic mechanisms contributing to suicidal cell death, e.g. Ca(2+) leakage or glutathione depletion. The present study utilized Western blotting to search for NFκB and IκB-α expression in erythrocytes, FACS analysis to determine cytosolic Ca(2+) (Fluo3 fluorescence), phosphatidylserine exposure (annexin V binding), and cell volume (forward scatter), as well as an enzymatic method to determine glutathione levels. As a result, both NFκB and IκB-α are expressed in erythrocytes. Targeting the NFκB pathway by Bay 11-7082 (IC(50) ≈ 10 μM) and parthenolide (IC(50) ≈ 30 μM) triggered suicidal erythrocyte death as shown by annexin V binding and decrease of forward scatter. Bay 11-7082 treatment further increased intracellular Ca(2+) and led to depletion of reduced glutathione. The effects of Bay 11-7082 and parthenolide on annexin V binding could be fully reversed by the antioxidant N-acetylcysteine. In conclusion, the pharmacological inhibitors of NFκB, Bay 11-7082 and parthenolide, interfere with the survival of erythrocytes involving mechanisms other than disruption of NFκB-dependent gene expression.

Topics: Aniline Compounds; Annexin A5; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Calcium; Cell Size; Erythrocytes; Glutathione; Humans; I-kappa B Kinase; NF-kappa B; Nitriles; Phosphatidylserines; Protein Binding; Sesquiterpenes; Signal Transduction; Sulfones; Xanthenes

In this study, 2-aminophenoxazine-3-one (Phx-3) exhibited a potent cell growth inhibitory effect with apoptotic features in a dose-dependent manner in various cancer cell lines tested. Comparison of the expression profiles of endoplasmic reticulum (ER) stress-related genes in U266 multiple myeloma cells after treatment with Phx-3 and the ER stress inducers tunicamycin (TNM) and thapsigargin (TPG) indicated that although TNM and TPG potently induced pro-apoptotic transcription factor CHOP (GADD153) within 8 h of treatment, Phx-3 induced almost no CHOP within 48 h of treatment in U266 cells. However, murine embryonic fibroblast (MEF) cells and other cancer cell lines (e.g. A549 lung cancer cells and HL-60 acute leukemia cells) exhibited up-regulation of CHOP after treatment with Phx-3. The potency of CHOP induction in response to Phx-3 appeared to be partially correlated with the cytotoxic sensitivity of Phx-3 among various cell lines tested. MEF cells derived from CHOP knockout mice were more resistant to Phx-3 than wild-type MEF cells. Since Phx-3 has been shown to induce activation of NF-κB, a transcription factor functioning as a repressor of CHOP, we further treated U266 cells with a combination of Phx-3 and NF-κB inhibitors (e.g. BAY11-7082 or parthenolide). This enhanced cytotoxicity along with up-modulation of CHOP in U266 cells. These data suggest that ER stress-mediated CHOP induction by Phx-3 is involved in the cytotoxic effect. Regulation of CHOP expression appears to be a potent therapeutic target for cancer treatment.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Proliferation; Endoplasmic Reticulum; Gene Expression Profiling; HL-60 Cells; Humans; Mice; Mice, Knockout; NF-kappa B; Nitriles; Oxazines; Sesquiterpenes; Sulfones; Thapsigargin; Transcription Factor CHOP; Tunicamycin; Up-Regulation

Activation of the inflammasome generates the pro-inflammatory cytokines interleukin-1 beta and -18, which are important mediators of inflammation. Abnormal activation of the inflammasome leads to many inflammatory diseases, including gout, silicosis, neurodegeneration, and genetically inherited periodic fever syndromes. Therefore, identification of small molecule inhibitors that target the inflammasome is an important step toward developing effective therapeutics for the treatment of inflammation. Here, we show that the herbal NF-kappaB inhibitory compound parthenolide inhibits the activity of multiple inflammasomes in macrophages by directly inhibiting the protease activity of caspase-1. Additional investigations of other NF-kappaB inhibitors revealed that the synthetic I kappaB kinase-beta inhibitor Bay 11-7082 and structurally related vinyl sulfone compounds selectively inhibit NLRP3 inflammasome activity in macrophages independent of their inhibitory effect on NF-kappaB activity. In vitro assays of the effect of parthenolide and Bay 11-7082 on the ATPase activity of NLRP3 demonstrated that both compounds inhibit the ATPase activity of NLRP3, suggesting that the inhibitory effect of these compounds on inflammasome activity could be mediated in part through their effect on the ATPase activity of NLRP3. Our results thus elucidate the molecular mechanism for the therapeutic anti-inflammatory activity of parthenolide and identify vinyl sulfones as a new class of potential therapeutics that target the NLRP3 inflammasome.

Topics: Animals; Anti-Inflammatory Agents; Bone Marrow Cells; Caspase 1; Cell Death; Humans; Immunoblotting; Inflammation; L-Lactate Dehydrogenase; Macrophages; Mice; NF-kappa B; Nitriles; Sesquiterpenes; Sulfones

Chlamydia trachomatis infections represent the leading cause of bacterial sexually-transmitted disease in the United States and can cause serious tissue damage leading to infertility and ectopic pregnancies in women. Inflammation and hence the innate immune response to chlamydial infection contributes significantly to tissue damage, particularly by secreting proinflammatory cytokines such as interleukin (IL)-1beta from monocytes, macrophages and dendritic cells. Here we demonstrate that C. trachomatis or Chlamydia muridarum infection of a monocytic cell line leads to caspase-1 activation and IL-1beta secretion through a process requiring the NLRP3 inflammasome. Thus, secretion of IL-1beta decreased significantly when cells were depleted of NLRP3 or treated with the anti-inflammatory inhibitors parthenolide or Bay 11-7082, which inhibit inflammasomes and the transcription factor NF-kappaB. As for other infections causing NRLP3 inflammasome assembly, caspase-1 activation in monocytes is triggered by potassium efflux and reactive oxygen species production. However, anti-oxidants inhibited IL-1beta secretion only partially. Atypically for a bacterial infection, caspase-1 activation during chlamydial infection also involves partially the spleen tyrosine kinase (Syk), which is usually associated with a pathogen recognition receptor for fungal pathogens. Secretion of IL-1beta during infection by many bacteria requires both microbial products from the pathogen and an exogenous danger signal, but chlamydial infection provides both the pathogen-associated molecular patterns and danger signals necessary for IL-1beta synthesis and its secretion from human monocytes. Use of inhibitors that target the inflammasome in animals should therefore dampen inflammation during chlamydial infection.

Topics: Carrier Proteins; Caspase 1; Cell Line; Chlamydia muridarum; Chlamydia trachomatis; Enzyme Inhibitors; Humans; Interleukin-1beta; Monocytes; Nitriles; NLR Family, Pyrin Domain-Containing 3 Protein; Sesquiterpenes; Sulfones

Aromatase (Cyp19) is a key enzyme in estrogen biosynthesis and an important target in breast cancer therapy. Within tumor microenvironment, tumor cells stimulate aromatase expression in adipose stromal cells (ASCs), which in turn promotes estrogen-dependent growth of estrogen receptor (ER)-positive tumor cells. However, it is not clear how aromatase transcription and estrogen biosynthesis are regulated in ASCs under a precancerous condition. Here we demonstrate that cell shape change alone is sufficient to induce aromatase expression in primary ASCs from cancer-free individuals. The activation of aromatase transcription is mediated by IkappaB kinase-beta (IKKbeta), a kinase previously known for its cancer-promoting activity in tumor cells. Activation of IKKbeta leads to elevated expression of transcription factor CCAAT/enhancer-binding protein-beta (C/EBPbeta), which binds to and stimulates two breast cancer-associated promoters of the aromatase gene. We also show that shape-induced estrogen production in ASCs can stimulate estrogen-dependent transcription in ER-positive breast tumor cells. We suggest that IKKbeta-dependent aromatase induction due to changes in cellular architecture in adipose tissue may contribute to the breast cancer risks associated with high mammagraphic density and obesity.

Topics: Adipose Tissue; Anti-Inflammatory Agents, Non-Steroidal; Aromatase; Cell Line, Tumor; Cell Shape; Cell Survival; Cells, Cultured; Chromatin Immunoprecipitation; Estrogens; Flow Cytometry; Gene Expression Regulation, Enzymologic; Genetic Vectors; Humans; I-kappa B Kinase; Immunoblotting; Lentivirus; Nitriles; Promoter Regions, Genetic; Receptors, Estrogen; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Sesquiterpenes; Stromal Cells; Sulfones

Nuclear factor-kappaB (NF-kappaB) is a transcription factor with a pivotal role in neuronal homeostasis. Indeed, NF-kappaB trans-activates several antiapoptotic genes in neurons and inhibition of NF-kappaB transcriptional activity triggers neuronal apoptosis. However, the exact mechanisms by which neurons undergo apoptosis in conditions of NF-kappaB inhibition are poorly understood. To further clarify how NF-kappaB operates in neurons, and to gather information on the molecular events occurring during NF-kappaB inhibition-dependent neuronal apoptosis, this study evaluated the effects of recently identified NF-kappaB inhibitors such as parthenolide, SN50, BAY 11-7082 and helenalin on primary cultures of rat cortical neurons. Data show that NF-kappaB was constitutively activated in neurons, and demonstrate for the first time that drug-dependent NF-kappaB inhibition induced rapid mitochondrial release of cytochrome c, caspase-9 and -3 activation, poly(ADP-ribose) polymerase-1 cleavage, membrane blebbing and nuclear fragmentation, without evidence of procaspase-8 and Bid processing. Interestingly, a burst of Akt activation occurred in neurons exposed to NF-kappaB inhibitors. These events were preceded by selective reduction of mRNAs of NF-kappaB-dependent, antiapoptotic Bcl-2 family members such as Bcl-x(L), Bcl-2 and, in particular, A1/Bfl-1. The present study reports a novel, detailed temporal analysis of the molecular events following impairment of NF-kappaB-driven transcription in neurons and demonstrates that inhibition of constitutive neuronal NF-kappaB activity triggers selective activation of the intrinsic apoptotic program.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Apoptosis; Caspases; Cells, Cultured; Cerebral Cortex; Cytochrome c Group; DNA; DNA-Binding Proteins; Homeostasis; Mitochondria; Neurons; NF-kappa B; Nitriles; Organic Chemicals; Peptides; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Protein Binding; Protein Precursors; Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Replication Protein C; Sesquiterpenes; Sesquiterpenes, Guaiane; Sulfones; Transcription, Genetic