leupeptins and tanespimycin

Porcine Circovirus Type 2 (PCV2) is a pathogen that has the ability to cause devastating disease manifestations in pig populations with major economic implications. Our previous research found that Hsp90 is required for PCV2 production in PK-15 and 3D4/31 cells. The aim of this study was to evaluate the effect of Hsp90 inhibitor regulating PCV2 replication and to explore its underlying mechanism. In PK-15 and 3D4/31 cells treated with 17-AAG after viral adsorption, replication of PCV2 was attenuated as assessed by quantitating the expression of viral protein. Following NF-κB activation it was observed that 24hpi with PCV2 was significantly inhibited in the presence of 17-AAG. The expression of Hsp90 associated client proteins in PCV2-infected cells were also reduced in the presence of 17-AAG. However, treatment with MG-132 failed to rescue 17-AAG mediated reduction of PCV2 production in host cells. Thus, Hsp90 regulates PCV2 by modulating cellular signaling proteins. These results highlight the importance of cellular proteins during PCV2 infection and the possibility of targeting cellular chaperones for developing new anti-rotaviral strategies.

Topics: Animals; Benzoquinones; Cell Line; Cell Survival; Circoviridae Infections; Circovirus; Host-Pathogen Interactions; HSP90 Heat-Shock Proteins; Lactams, Macrocyclic; Leupeptins; NF-kappa B; Swine; Swine Diseases; Virus Replication

Osteosarcoma (OS) is the most malignant primary bone tumor in children and adolescents with limited treatment options and poor prognosis. Recently, aberrant expression of Runx2 has been found in OS, thereby contributing to the development, and progression of OS. However, the upstream signaling molecules that regulate its expression in OS remain largely unknown. In the present study, we first confirmed that the inhibition of HSP90 with 17-AAG caused significant apoptosis of OS cells via a caspase-3-dependent mechanism, and that inhibition or knockdown of HSP90 by 17-AAG or siRNAs significantly suppressed mRNA and protein expression of Runx2. Furthermore, we provided evidence that Runx2 was transcriptionally regulated by HSP90 when using MG132 and CHX chase assay. We also demonstrated that β-catenin was overexpressed in OS tissue, and that knockdown of β-catenin induced pronounced apoptosis of OS cells in the presence or absence of 17-AAG. Interestingly, this phenomenon was accompanied with a significant reduction of Runx2 and Cyclin D1 expression, indicating an essential role of Runx2/Cyclin D1 in 17-AAG-induced cells apoptosis. Moreover, we demonstrated that the apoptosis of OS cells induced by 17-AAG did require the involvement of the AKT/GSK-3β/β-catenin signaling pathway by using pharmacological inhibitor GSK-3β (LiCl) or siGSK-3β. Our findings reveal a novel mechanism that Runx2 is transcriptionally regulated by HSP90 via the AKT/GSK-3β/β-catenin signaling pathway, and by which leads to apoptosis of OS cells.

Topics: Benzoquinones; beta Catenin; Bone Neoplasms; Cell Line, Tumor; Cell Survival; Core Binding Factor Alpha 1 Subunit; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3 beta; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Leupeptins; Osteosarcoma; Proto-Oncogene Proteins c-akt; Signal Transduction; Transcription, Genetic

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

Hsp90 is an important driver of stabilization and activation of several oncogenic proteins in many key pathways in oncogenesis, including HER2. The present study demonstrated that synuclein gamma (SNCG) prevents the protein degradation and protects the function of HER2 in the condition when the function of Hsp90 is blocked. Disruption of Hsp90 resulted in a significant degradation of HER2 and the loss of activity. However, SNCG completely recovered Hsp90 disruption-mediated losses of HER2 and the function. SNCG bound to HER2 in the presence and absence of Hsp90. Specifically, the C-terminal (Gln106-Asp127) of SNCG bound to the loop connecting αC helix and β4 sheet of the kinase domain of HER2. SNCG renders resistance to 17-AAG-induced tumor suppression in tumor xenograft. Crossing SNCG transgenic mice with HER2 mice stimulated HER2-induced tumor growth and rendered resistance to Hsp90 disruption. The present study indicates that SNCG protects Hsp90 client protein of HER2, and renders resistance to Hsp90 disruption.

Topics: Animals; Benzoquinones; Breast Neoplasms; Cell Line, Tumor; gamma-Synuclein; HSP90 Heat-Shock Proteins; Humans; Immunoprecipitation; Lactams, Macrocyclic; Leupeptins; Mice; Mice, Nude; Mice, Transgenic; Receptor, ErbB-2

The receptor tyrosine kinase AXL is overexpressed in many cancer types including thyroid carcinomas and has well established roles in tumor formation and progression. Proper folding, maturation, and activity of several oncogenic receptor tyrosine kinases require HSP90 chaperoning. HSP90 inhibition by the antibiotic geldanamycin or its derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) causes destabilization of its client proteins. Here we show that AXL is a novel client protein of HSP90. 17-AAG induced a time- and dose-dependent down-regulation of endogenous or ectopically expressed AXL protein, thereby inhibiting AXL-mediated signaling and biological activity. 17-AAG-induced AXL down-regulation specifically affected fully glycosylated mature receptor present on cell membrane. By using biotin and [(35)S]methionine labeling, we showed that 17-AAG caused depletion of membrane-localized AXL by mediating its degradation in the intracellular compartment, thus restricting its exposure on the cell surface. 17-AAG induced AXL polyubiquitination and subsequent proteasomal degradation; under basal conditions, AXL co-immunoprecipitated with HSP90. Upon 17-AAG treatment, AXL associated with the co-chaperone HSP70 and the ubiquitin E3 ligase carboxyl terminus of HSC70-interacting protein (CHIP). Overexpression of CHIP, but not of the inactive mutant CHIP K30A, induced accumulation of AXL polyubiquitinated species upon 17-AAG treatment. The sensitivity of AXL to 17-AAG required its intracellular domain because an AXL intracellular domain-deleted mutant was insensitive to the compound. Active AXL and kinase-dead AXL were similarly sensitive to 17-AAG, implying that 17-AAG sensitivity does not require receptor phosphorylation. Overall our data elucidate the molecular basis of AXL down-regulation by HSP90 inhibitors and suggest that HSP90 inhibition in anticancer therapy can exert its effect through inhibition of multiple kinases including AXL.

Topics: Aniline Compounds; Antineoplastic Agents; Axl Receptor Tyrosine Kinase; Benzoquinones; Catalytic Domain; Cell Membrane; Glycosylation; HeLa Cells; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Leupeptins; Nitriles; Proteasome Inhibitors; Protein Binding; Protein Isoforms; Protein Kinase Inhibitors; Protein Stability; Protein Transport; Proteolysis; Proto-Oncogene Proteins; Quinolines; Receptor Protein-Tyrosine Kinases; Signal Transduction; Ubiquitin-Protein Ligases; Ubiquitination

GABARAPL1 belongs to the small family of GABARAP proteins (including GABARAP, GABARAPL1 and GABARAPL2/GATE-16), one of the two subfamilies of the yeast Atg8 orthologue. GABARAPL1 is involved in the intracellular transport of receptors, via an interaction with tubulin and GABA(A) or kappa opioid receptors, and also participates in autophagy and cell proliferation. In the present study, we identify the HSP90 protein as a novel interaction partner for GABARAPL1 using GST pull-down, mass spectrometry and coimmunoprecipitation experiments. GABARAPL1 and HSP90 partially colocalize in MCF-7 breast cancer cells overexpressed Dsred-GABARAPL1 and in rat brain. Moreover, treatment of MCF-7 cells overexpressed FLAG-GABARAPL1-6HIS with the HSP90 inhibitor 17-AAG promotes the GABARAPL1 degradation, a process that is blocked by proteasome inhibitors such as MG132, bortezomib and lactacystin. Accordingly, we demonstrate that HSP90 interacts and protects GABARAPL1 from its degradation by the proteasome.

Topics: Adaptor Proteins, Signal Transducing; Animals; Benzoquinones; Blotting, Western; Cell Line; Cell Line, Tumor; Cysteine Proteinase Inhibitors; HSP90 Heat-Shock Proteins; Humans; Immunoprecipitation; Lactams, Macrocyclic; Leupeptins; Mass Spectrometry; Microscopy, Confocal; Microtubule-Associated Proteins; Rats

Small ubiquitin-related modifiers 1, 2 and 3 (SUMO-1, -2, -3), members of the ubiquitin-like protein family, can be conjugated to various cellular proteins. Conjugates of SUMO-2 and SUMO-3 (SUMO-2/3) accumulate in cells exposed to various stress stimuli or to MG132 treatment. Although the proteins modified by SUMO-2/3 during heat shock or under MG132 treatment have been identified, the significance of this modification remains unclear. Our data show that the inhibition of translation by puromycin or cycloheximide blocks both the heat shock and MG132 induced accumulation of SUMO-2/3 conjugates in HEK 293T and U2OS cells. However, the heat shock induced accumulation of SUMO-2/3 conjugates was restored by proteasome inhibition, which suggests that the inhibition of translation did not abolish SUMOylation itself. Furthermore, we show that some of the proteins truncated due to the treatment by low concentration of puromycin are SUMOylated in HEK 293T cells. We suggest that the SUMO-2/3 conjugates accumulating under the heat shock or MG132 treatment result largely from new protein synthesis and that portion of them is incorrectly folded.

Topics: Benzoquinones; Cycloheximide; Heat-Shock Response; HEK293 Cells; HeLa Cells; Humans; Lactams, Macrocyclic; Leupeptins; Models, Biological; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Biosynthesis; Protein Synthesis Inhibitors; Puromycin; Small Ubiquitin-Related Modifier Proteins; Sumoylation; Ubiquitins

Histone deacetylase 6 (HDAC6) inhibition, recently, has been shown to promote the acetylation of heat-shock protein 90 (Hsp90) and disrupt its chaperone function. Her-2 oncoprotein is identified as a client protein of Hsp90. Therefore, in this study we examined the effect of carbamazepine, which could inhibit HDAC on Hsp90 acetylation and Her-2 stability. The results of this study demonstrate that while carbamazepine had no effect on the Her-2 mRNA level, it induced Her-2 protein degradation via the proteasome pathway by disrupting the chaperone function of Hsp90 in SK-BR-3 cells. Mechanistically, carbamazepine could enhance the acetylation of α-tubulin, indicating its inhibitory effect on HDAC6. Functionally, carbamazepine could synergize with trastuzumab or geldanamycin to promote Her-2 degradation and inhibit breast cancer cell proliferation. Thus, this study has potential clinical implications by providing a promising strategy to overcome the development of resistance against trastuzumab therapy for breast cancer.

Topics: Acetylation; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Benzoquinones; Breast Neoplasms; Carbamazepine; Cell Line, Tumor; Cell Proliferation; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Drug Synergism; Female; Histone Deacetylase 6; Histone Deacetylase Inhibitors; Histone Deacetylases; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Leupeptins; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Protein Stability; Receptor, ErbB-2; RNA, Messenger; Time Factors; Trastuzumab; Tubulin

T cell response initiated by engagement of T cell receptor (TCR) is dependent on signal transduction events composed of protein kinases and adaptor proteins. However, the molecular mechanism for gene expression of these proteins is not entirely understood. Here we identified Heat Shock Protein 90 (HSP90) as an essential regulator for gene expression of Linker for activation of T cells (LAT) in primarily activated human T cells. Primarily activated T cells continuously synthesized LAT protein and treatment of cells with 17-AAG, a pharmacological inhibitor of HSP90, decreased LAT protein level following reduction of LAT mRNA. Furthermore, promoter activity of LAT gene was dramatically inhibited by 17-AAG. These results reveal a novel role of HSP90 as a positive regulator for expression of LAT gene in activated T cells.

Topics: Adaptor Proteins, Signal Transducing; Benzoquinones; Cell Membrane; Down-Regulation; Enzyme Inhibitors; Gene Expression Regulation; HSP90 Heat-Shock Proteins; Humans; Jurkat Cells; Lactams, Macrocyclic; Leupeptins; Lymphocyte Activation; Membrane Proteins; Promoter Regions, Genetic; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Transport; RNA, Messenger; T-Lymphocytes

Clusterin is a stress-activated, cytoprotective chaperone that confers broad-spectrum treatment resistance in cancer. However, the molecular mechanisms mediating CLU transcription following anticancer treatment stress remain incompletely defined. We report that Y-box binding protein-1 (YB-1) directly binds to CLU promoter regions to transcriptionally regulate clusterin expression. In response to endoplasmic reticulum stress inducers, including paclitaxel, YB-1 is translocated to the nucleus to transactivate clusterin. Furthermore, higher levels of activated YB-1 and clusterin are seen in taxane-resistant, compared with parental, prostate cancer cells. Knockdown of either YB-1 or clusterin sensitized prostate cancer cells to paclitaxel, whereas their overexpression increased resistance to taxane. Clusterin overexpression rescued cells from increased paclitaxel-induced apoptosis following YB-1 knockdown; in contrast, however, YB-1 overexpression did not rescue cells from increased paclitaxel-induced apoptosis following clusterin knockdown. Collectively, these data indicate that YB-1 transactivation of clusterin in response to stress is a critical mediator of paclitaxel resistance in prostate cancer.

Topics: Apoptosis; Benzoquinones; Bridged-Ring Compounds; Cell Line, Tumor; Cell Proliferation; Clusterin; Drug Resistance, Neoplasm; Endoplasmic Reticulum Stress; Gene Expression Regulation, Neoplastic; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Leupeptins; Male; Paclitaxel; Promoter Regions, Genetic; Prostatic Neoplasms; Protein Binding; RNA, Small Interfering; Taxoids; Transcriptional Activation; Y-Box-Binding Protein 1

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease. Here, we report a new and effective strategy for inhibiting HCV replication using an inhibitor of heat-shock protein 90, 17-AAG (17-allylamino-17demethoxygeldanamycin), and a proteasome inhibitor, MG132.. To explore the virological basis of combination therapy, we analysed the effects of 17-AAG and MG132, singly and in combination on HCV replication in an HCV replicon cell system.. In HCV replicon cells, HCV RNA replication was suppressed by 17-AAG in a dose-dependent manner. As shown in the present study, the 50% inhibitory concentration values were 0.82 nM for 17-AAG and 0.21 nM for MG132. Low concentrations of MG132 had strong synergistic inhibitory effects with low toxicity on HCV replicon cells.. The results of this study suggest that the different effects and synergistic actions of 17-AAG and MG132 could provide a new therapeutic approach to HCV infection.

Topics: Benzoquinones; Cell Line, Tumor; Drug Synergism; Drug Therapy, Combination; Hepacivirus; Hepatitis C; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Leupeptins; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Replicon; Transfection; Virus Replication

The mineralocorticoid receptor (MR) plays a crucial role in the regulation of Na(+) balance and blood pressure, as evidenced by gain of function mutations in the MR of hypertensive families. In the kidney, aldosterone binds to the MR, induces its nuclear translocation, and promotes a transcriptional program leading to increased transepithelial Na(+) transport via the epithelial Na(+) channel. In the unliganded state, MR is localized in the cytosol and part of a multiprotein complex, including heat shock protein 90 (Hsp90), which keeps it ligand-binding competent. 17-Allylamino-17-demethoxygeldanamycin (17-AAG) is a benzoquinone ansamycin antibiotic that binds to Hsp90 and alters its function. We investigated whether 17-AAG affects the stability and transcriptional activity of MR and consequently Na(+) reabsorption by renal cells. 17-AAG treatment lead to reduction of MR protein level in epithelial cells in vitro and in vivo, thereby interfering with aldosterone-dependent transcription. Moreover, 17-AAG inhibited aldosterone-induced Na(+) transport, possibly by interfering with MR availability for the ligand. Finally, we identified the ubiquitin-protein ligase, COOH terminus of Hsp70-interacting protein, as a novel partner of the cytosolic MR, which is responsible for its polyubiquitylation and proteasomal degradation in presence of 17-AAG. In conclusion, 17-AAG may represent a novel pharmacological tool to interfere with Na(+) reabsorption and hypertension.

Topics: Aldosterone; Animals; Benzoquinones; Female; HSP70 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Kidney Tubules, Collecting; Lactams, Macrocyclic; Leupeptins; Mice; Mice, Inbred C57BL; Receptors, Mineralocorticoid; Sodium; Transcriptional Activation; Ubiquitin-Protein Ligases

The heat shock protein HSP90 serves as a chaperone for receptor protein kinases, steroid receptors, and other intracellular signaling molecules. Targeting HSP90 with ansamycin antibiotics disrupts the normal processing of clients of the HSP90 complex. The platelet-derived growth factor receptor alpha (PDGFRalpha) is a tyrosine kinase receptor up-regulated and activated in several malignancies. Here we show that the PDGFRalpha forms a complex with HSP90 and the co-chaperone cdc37 in ovarian, glioblastoma, and lung cancer cells. Treatment of cancer cell lines expressing the PDGFRalpha with the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) promotes degradation of the receptor. Likewise, phospho-Akt, a downstream target, is degraded after treatment with 17-AAG. In contrast, PDGFRalpha expression is not affected by 17-AAG in normal human smooth muscle cells or 3T3 fibroblasts. PDGFRalpha degradation by 17-AAG is inhibited by the proteasome inhibitor MG132. High molecular weight, ubiquitinated forms of the receptor are detected in cells treated with 17-AAG and MG132. Degradation of the receptor is also inhibited by a specific neutralizing antibody to the PDGFRalpha but not by a neutralizing antibody to PDGF or by imatinib mesylate (Gleevec). Ultimately, PDGFRalpha-mediated cell proliferation is inhibited by 17-AAG. These results show that 17-AAG promotes PDGFRalpha degradation selectively in transformed cells. Thus, not only mutated tyrosine kinases but also overexpressed receptors in cancer cells can be targeted by 17-AAG.

Topics: 3T3 Cells; Animals; Antibiotics, Antineoplastic; Antineoplastic Agents; Benzamides; Benzoquinones; Cell Line, Tumor; Cell Proliferation; Humans; Imatinib Mesylate; Lactams, Macrocyclic; Leupeptins; Mice; Piperazines; Platelet-Derived Growth Factor; Pyrimidines; Receptor, Platelet-Derived Growth Factor alpha; Signal Transduction