cct018159 and tanespimycin

Cushing's disease is primarily caused by adrenocorticotropic hormone (ACTH)-producing pituitary adenomas. If excision of the tumor from the pituitary, which is the primary treatment for Cushing's disease, is unsuccessful, further medical therapy is needed to treat the resultant hypercortisolism. Some of the drugs used to treat this condition have shown potential therapeutic benefits, but a more effective treatment should be explored for the treatment of Cushing's disease. In the present study, we determined the effect of heat shock protein 90 inhibitors on ACTH production and cell proliferation of AtT-20 corticotroph tumor cells.. AtT-20 pituitary corticotroph tumor cells were cultured. The expression levels of mouse proopiomelanocortin (POMC) and pituitary tumor transforming gene 1 (PTTG1) mRNA were evaluated using quantitative real-time PCR. Cellular DNA content was analyzed with fluorescence-activated cell sorting (FACS) analysis. The protein levels were determined by Western blot analysis.. Both 17-allylamino-17-demethoxygeldanamycin and CCT018159 decreased POMC mRNA levels in AtT-20 cells and ACTH levels in the culture medium of these cells, suggesting that both drugs suppress ACTH synthesis and secretion in corticotroph tumor cells. Both drugs also decreased cell proliferation and induced apoptosis. FACS analyses revealed that both agents increased the percentage of AtT-20 cells in the G2/M phase. These drugs decreased cell proliferation, presumably due to the induction of cell death and arrest of the cell cycle in AtT-20 cells. Tumor weight in mice xenografted with AtT-20 cells and treated with CCT018159 was lower than in AtT-20-xenografted control mice. CCT018159 also decreased plasma ACTH levels, and POMC and PTTG1 mRNA levels in the tumor cells.. CCT018159 inhibits ACTH production and corticotroph tumor cell proliferation in vitro and in vivo.

Topics: ACTH-Secreting Pituitary Adenoma; Adenoma; Adrenocorticotropic Hormone; Animals; Benzoquinones; Cell Line, Tumor; Cell Proliferation; Heterocyclic Compounds, 2-Ring; HSP90 Heat-Shock Proteins; Lactams, Macrocyclic; Mice; Neoplasm Transplantation; Pro-Opiomelanocortin; Pyrazoles; Real-Time Polymerase Chain Reaction; RNA, Messenger; Securin; Tumor Burden

The HSP90 (heat-shock protein 90) inhibitor 17-AAG (17-allylamino-demethoxygeldanamycin) increases osteoclast formation both in vitro and in vivo, an action that can enhance cancer invasion and growth in the bone microenvironment. The cellular mechanisms through which 17-AAG exerts this action are not understood. Thus we sought to clarify the actions of 17-AAG on osteoclasts and determine whether other HSP90 inhibitors had similar properties. We determined that 17-AAG and the structurally unrelated HSP90 inhibitors CCT018159 and NVP-AUY922 dose-dependently increased RANKL [receptor activator of NF-κB (nuclear factor κB) ligand]-stimulated osteoclastogenesis in mouse bone marrow and pre-osteoclastic RAW264.7 cell cultures. Moreover, 17-AAG also enhanced RANKL- and TNF (tumour necrosis factor)-elicited osteoclastogenesis, but did not affect RANKL-induced osteoclast survival, suggesting that only differentiation mechanisms are targeted. 17-AAG affected the later stages of progenitor maturation (after 3 days of incubation), whereas the osteoclast formation enhancer TGFβ (transforming growth factor β) acted prior to this, suggesting different mechanisms of action. In studies of RANKL-elicited intracellular signalling, 17-AAG treatment did not increase c-Fos or NFAT (nuclear factor of activated T-cells) c1 protein levels nor did 17-AAG increase activity in luciferase-based NF-κB- and NFAT-response assays. In contrast, 17-AAG treatment (and RANKL treatment) increased both MITF (microphthalmia-associated transcription factor) protein levels and MITF-dependent vATPase-d2 (V-type proton ATPase subunit d2) gene promoter activity. These results indicate that HSP90 inhibitors enhance osteoclast differentiation in an NFATc1-independent manner that involves elevated MITF levels and activity.

Topics: Animals; Benzoquinones; Bone Marrow Cells; Cell Differentiation; Cells, Cultured; Heterocyclic Compounds, 2-Ring; HSP90 Heat-Shock Proteins; Isoxazoles; Lactams, Macrocyclic; Mice; Mice, Inbred C57BL; Microphthalmia-Associated Transcription Factor; NF-kappa B; NFATC Transcription Factors; Osteoclasts; Promoter Regions, Genetic; Proto-Oncogene Proteins c-fos; Pyrazoles; Resorcinols; Stem Cells; Transforming Growth Factor beta; Vacuolar Proton-Translocating ATPases

Dying cells release pro-inflammatory molecules, functioning as cytokines to trigger cell/tissue inflammation that is relevant to disease pathology. Heat-shock protein 90 (HSP90) is believed to act as a danger signal for tissue damage once released extracellularly. Potential roles of HSP90 were explored in retinal pigment epithelial (RPE) inflammatory responses to necrosis. Cellular extracts can trigger ARPE-19 cell inflammatory responses, producing cytokines that lead to an increase in ARPE-19 cell monolayer permeability. Addition of recombinant HSP90β mimics the induction of chemokines IL-8 and MCP-1 in cultured RPE cells, suggesting that released HSP90 can incite RPE cell sterile inflammatory responses. Consistent with this, classical HSP90 inhibitors were shown to substantially reduce necrosis-induced cytokine production and permeability increases in ARPE-19 cells. Moreover, a cell-impermeable inhibitor, 17-N,N-dimethylaminoethylamino-17-demethoxy-geldanamycin-N-oxide, also efficiently inhibited necrosis-induced cytokine production and TNF-α/IL-1β-induced increase in ARPE-19 cell permeability in vitro and endotoxin-induced development of uveitis in vivo, suggesting that HSP90 can contribute to necrosis-induced RPE inflammatory responses. Collectively, our data identify HSP90 as a pro-inflammatory molecule in RPE cell sterile inflammatory responses.

Topics: Animals; Anti-Inflammatory Agents; Benzoquinones; Cell Line; Chemokine CCL2; Disease Models, Animal; Dose-Response Relationship, Drug; Extracellular Signal-Regulated MAP Kinases; Heterocyclic Compounds, 2-Ring; HSP90 Heat-Shock Proteins; Humans; Inflammation Mediators; Interleukin-1beta; Interleukin-8; JNK Mitogen-Activated Protein Kinases; Lactams, Macrocyclic; Lipopolysaccharides; Male; Necrosis; Permeability; Protein Kinase Inhibitors; Pyrazoles; Rats; Rats, Inbred Lew; Retinal Pigment Epithelium; Signal Transduction; Time Factors; Tumor Necrosis Factor-alpha; Uveitis

Molecular chaperone heat shock protein 90 (Hsp90) inhibitors are promising targeted cancer therapeutic drugs, with the advantage that they deplete multiple oncogenic client proteins and modulate all the classical hallmarks of cancer. They are now in clinical trial and show potential for activity in melanoma and other malignancies. Here we explore the metabolic response to Hsp90 inhibition in human melanoma cells using magnetic resonance spectroscopy. We show that, concomitant with growth inhibition and re-differentiation, Hsp90 inhibition in human melanoma cells is associated with increased glycerophosphocholine content. This was seen with both the clinical geldanamycin-based Hsp90 drug 17-AAG and the structurally dissimilar Hsp90 inhibitor CCT018159. The effect was noted in both BRAF mutant SKMEL28 and BRAF wildtype CHL-1 melanoma cells. Elevated content of the -CH2+CH3 fatty acyl chains and cytoplasmic mobile lipid droplets was also observed in 17-AAG-treated SKMEL28 cells. Importantly, the phospholipase A2 inhibitor bromoenol lactone prevented the rise in glycerophosphocholine seen with 17-AAG, suggesting a role for phospholipase A2 activation in the Hsp90 inhibitor-induced metabolic response. Our findings provide a basis for using metabolic changes as non-invasive indicators of Hsp90 inhibition and potentially as biomarkers of anticancer activity with Hsp90 drugs in malignant melanoma and possibly in other cancers.

Topics: Antineoplastic Agents; Benzoquinones; Biomarkers, Pharmacological; Glycerylphosphorylcholine; Heterocyclic Compounds, 2-Ring; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Lipid Metabolism; Magnetic Resonance Spectroscopy; Melanoma; Mutation; Naphthalenes; Phospholipase A2 Inhibitors; Proto-Oncogene Proteins B-raf; Pyrazoles; Pyrones; Skin Neoplasms

High-throughput screening of chemical libraries and the subsequent rapid progress of hit compounds through an iterative developmental test cascade are essential parts of modern molecular mechanism-based drug discovery. These processes depend on the use of efficient assay technologies and equipment. Enzyme-linked immunosorbent assays have historically been carried out in 96-well microtitre plates. Improvements in reagents and assay technologies mean that solid-phase immunoassays can be adapted for higher throughput to play an important role in modern drug discovery. The molecular chaperone heat-shock protein (Hsp) 90 is an important anticancer drug target because it maintains the conformation, stability, and function of many important oncogenic client proteins, including those involved with signal transduction, cell proliferation, survival, differentiation, motility angiogenesis, and metastasis. Using the standard inhibitors of the adenosine triphosphatase (ATPase) activity of Hsp90, geldanamycin (GA) and 17-allylamino-17- demethoxygeldanamycin (17AAG), novel solid-phase immunoassays have been validated using a time-resolved fluorescence (TRF) end point. Their utility for confirming the mechanism of action of Hsp90 inhibition in secondary cell-based assays has been shown and applied to the novel Hsp90 inhibitor CCT018159. Adaptation of these assays for later studies using human tumour xenografts and samples obtained from a Phase 1 trial of 17AAG is also described. Finally, comparison is made between the use and applicability of this type of immunoassay and other techniques such as western blotting, immunohistochemistry, and flow cytometry analysis.

Topics: Animals; Benzoquinones; Blotting, Western; Carrier Proteins; Cell Extracts; Dose-Response Relationship, Drug; Drug Design; eIF-2 Kinase; Enzyme Inhibitors; HCT116 Cells; Heterocyclic Compounds, 2-Ring; HSP70 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; HT29 Cells; Humans; Immunoassay; Intracellular Signaling Peptides and Proteins; Lactams, Macrocyclic; Lymphocytes; Mice; Neoplasm Transplantation; Proto-Oncogene Proteins c-raf; Pyrazoles; Quinones; Rifabutin; Technology, Pharmaceutical; Transplantation, Heterologous