leupeptins and monorden

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

Intrinsic stress response pathways are frequently mobilized within tumor cells. The mediators of these adaptive mechanisms and how they contribute to carcinogenesis remain poorly understood. A striking example is heat shock factor 1 (HSF1), master transcriptional regulator of the heat shock response. Surprisingly, we found that loss of the tumor suppressor gene neurofibromatosis type 1 (Nf1) increased HSF1 levels and triggered its activation in mouse embryonic fibroblasts. As a consequence, Nf1-/- cells acquired tolerance to proteotoxic stress. This activation of HSF1 depended on dysregulated MAPK signaling. HSF1, in turn, supported MAPK signaling. In mice, Hsf1 deficiency impeded NF1-associated carcinogenesis by attenuating oncogenic RAS/MAPK signaling. In cell lines from human malignant peripheral nerve sheath tumors (MPNSTs) driven by NF1 loss, HSF1 was overexpressed and activated, which was required for tumor cell viability. In surgical resections of human MPNSTs, HSF1 was overexpressed, translocated to the nucleus, and phosphorylated. These findings reveal a surprising biological consequence of NF1 deficiency: activation of HSF1 and ensuing addiction to this master regulator of the heat shock response. The loss of NF1 function engages an evolutionarily conserved cellular survival mechanism that ultimately impairs survival of the whole organism by facilitating carcinogenesis.

Topics: Active Transport, Cell Nucleus; Animals; Cell Line, Tumor; Cell Transformation, Neoplastic; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Genes, Neurofibromatosis 1; Heat Shock Transcription Factors; Hot Temperature; Humans; Leupeptins; Macrolides; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Neoplasm Proteins; Nerve Sheath Neoplasms; Neurofibromin 1; NIH 3T3 Cells; Phosphorylation; Protein Processing, Post-Translational; RNA, Messenger; RNA, Neoplasm; RNA, Small Interfering; Transcription Factors; Withanolides

The underlying cause of amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disorder, remains unknown. However, there is strong evidence that one pathophysiological mechanism, toxic protein misfolding and/or aggregation, may trigger motor neuron dysfunction and loss. Since the clinical and pathological features of sporadic and familial ALS are indistinguishable, all forms of the disease may be better understood and ultimately treated by studying pathogenesis and therapy in models expressing mutant forms of SOD1. We developed a cellular model in which cell death depended on the expression of G93A-SOD1, a mutant form of superoxide dismutase found in familial ALS patients that produces toxic protein aggregates. This cellular model was optimized for high throughput screening to identify protective compounds from a >50,000 member chemical library. Three novel chemical scaffolds were selected for further study following screen implementation, counter-screening and secondary testing, including studies with purchased analogs. All three scaffolds blocked SOD1 aggregation in high content screening assays and data on the optimization and further characterization of these compounds will be reported separately. These data suggest that optimization of these chemicals scaffolds may produce therapeutic candidates for ALS patients.

Topics: Amyotrophic Lateral Sclerosis; Animals; Benzoquinones; Cell Death; Cytoprotection; Drug Design; Drug Evaluation, Preclinical; High-Throughput Screening Assays; Humans; Lactams, Macrocyclic; Leupeptins; Macrolides; Mutant Proteins; PC12 Cells; Rats; Recombinant Fusion Proteins; Small Molecule Libraries; Superoxide Dismutase

Since many Hsp90 client proteins are key players in tumour pathways, the ubiquitylation and subsequent degradation of Hsp90-substrates as a consequence of pharmacologically inhibiting Hsp90 represents an innovative approach for cancer therapy. We therefore identified Hsp90-binding proteins which accumulated as ubiquityl-tagged aggregates in the detergent insoluble fraction of HeLa cells as a consequence of simultaneously inhibiting Hsp90 and the proteasome. 2-DE followed by nanoLC-MS/MS of trypsinised protein spots provided the Hsp90-dependent ubiquitylated proteome which was finally annotated and functionally classified. The overall picture thus obtained emphasised the well-established role of Hsp90 in stabilising proteins involved in gene transcription and signal transduction. It also provided a novel Hsp90-related link to metabolic pathways as the inhibition of Hsp90 caused the ubiquitylation of a significant amount of metabolic enzymes. These findings serve to support cumulating indications which attribute Hsp90 to diverse stabilising functions beyond signal transduction and gene transcription.

Topics: Electrophoresis, Gel, Two-Dimensional; HeLa Cells; HSP90 Heat-Shock Proteins; Humans; Leupeptins; Macrolides; Protein Binding; Proteome; Proteomics; Solubility; Ubiquitin

Molecular chaperones and co-chaperones, such as heat-shock proteins (Hsp's), play a pivotal role in the adequate folding and the stability of steroid hormone receptors. As shown by immunofluorescence staining and immunoblot analysis, the Hsp90 inhibitor radicicol induced a rapid (within hours) depletion of estrogen receptor-alpha (ER) in MCF-7 and IBEP-2 breast carcinoma cells. Inhibition of proteasomes (MG-132, LLnL) or of protein synthesis (cycloheximide), which both suppressed E(2)-induced downregulation of ER, failed to modify ER degradation caused by radicicol. On the other hand, partial antiestrogens, such as hydroxytamoxifen (a triphenylethylene) and LY 117,018 (a benzothiophene) stabilized ER, making it immune to radicicol-induced degradation. Furthermore, radicicol did not interfere with ER upregulation induced by hydroxytamoxifen. Thus, the current study points to possible variation in the mechanism/pathway of ER breakdown. Besides, the protective effect of partial antiestrogens suggests that ER stability is only compromized by Hsp90 disruption when the receptor is in its native, unliganded form.

Topics: Breast Neoplasms; Cycloheximide; Enzyme Inhibitors; Estrogen Antagonists; Estrogen Receptor alpha; Female; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; HSP90 Heat-Shock Proteins; Humans; Immunoenzyme Techniques; Lactones; Leupeptins; Ligands; Macrolides; Molecular Chaperones; Proteasome Inhibitors; Protein Synthesis Inhibitors; Protein-Tyrosine Kinases; Pyrrolidines; Signal Transduction; Tamoxifen; Thiophenes; Tumor Cells, Cultured