TRPA1 activation and Hsp90 inhibition synergistically downregulate macrophage activation and inflammatory responses in vitro.

BACKGROUND: Transient receptor potential ankyrin 1 (TRPA1) channels are known to be actively involved in various pathophysiological conditions, including neuronal inflammation, neuropathic pain, and various immunological responses. Heat shock protein 90 (Hsp90), a cytoplasmic molecular chaperone, is well-reported for various cellular and physiological processes. Hsp90 inhibition by various molecules has garnered importance for its therapeutic significance in the downregulation of inflammation and are proposed as anti-cancer drugs. However, the possible role of TRPA1 in the Hsp90-associated modulation of immune responses remains scanty. RESULTS: Here, we have investigated the role of TRPA1 in regulating the anti-inflammatory effect of Hsp90 inhibition via 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) in lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA) stimulation in RAW 264.7, a mouse macrophage cell lines and PMA differentiated THP-1, a human monocytic cell line similar to macrophages. Activation of TRPA1 with Allyl isothiocyanate (AITC) is observed to execute an anti-inflammatory role via augmenting Hsp90 inhibition-mediated anti-inflammatory responses towards LPS or PMA stimulation in macrophages, whereas inhibition of TRPA1 by 1,2,3,6-Tetrahydro-1,3-dimethyl-N-[4-(1-methylethyl)phenyl]-2,6-dioxo-7 H-purine-7-acetamide,2-(1,3-Dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7 H-purin-7-yl)-N-(4-isopropylphenyl)acetamide (HC-030031) downregulates these developments. LPS or PMA-induced macrophage activation was found to be regulated by TRPA1. The same was confirmed by studying the levels of activation markers (major histocompatibility complex II (MHCII), cluster of differentiation (CD) 80 (CD80), and CD86, pro-inflammatory cytokines (tumor necrosis factor (TNF) and interleukin 6 (IL-6)), NO (nitric oxide) production, differential expression of mitogen-activated protein kinase (MAPK) signaling pathways (p-p38 MAPK, phospho-extracellular signal-regulated kinase 1/2 (p-ERK 1/2), and phosphor-stress-activated protein kinase/c-Jun N-terminal kinase (p-SAPK/JNK)), and induction of apoptosis. Additionally, TRPA1 has been found to be an important contributor to intracellular calcium levels toward Hsp90 inhibition in LPS or PMA-stimulated macrophages. CONCLUSION: This study indicates a significant role of TRPA1 in Hsp90 inhibition-mediated anti-inflammatory developments in LPS or PMA-stimulated macrophages. Activation of TRPA1 and inhibition of Hsp90 has synergistic roles towards regulating inflammatory responses associated with macrophages. The role of TRPA1 in Hsp90 inhibition-mediated modulation of macrophage responses may provide insights towards designing future novel therapeutic approaches to regulate various inflammatory responses.

The HSP90 inhibitor 17-AAG induced calcium-mediated apoptosis in filarial parasites.

The available antifilarial medications are effective only against the larval stage of the filarial parasite. As a result, there is a pressing need for an adulticidal drug. The development of drugs requires the identification of molecular targets that are critical for parasite life. In this study, we observed the effect of 17-N-allyl-17-demethoxygeldanamycin on the survival of adult filarial parasites. The 17-N-allyl-17-demethoxygeldanamycin (17-AAG) is a derivative of geldanamycin (GA), which is an inhibitor of heat shock protein (HSP)90. It is less toxic as compared to geldanamycin. The motility and viability of the adult filarial parasite Setaria cervi were decreased on exposure to 17-AAG at 2.5 and 5.0 µM/ml concentrations. The 17-AAG treated parasites showed induction of oxidative stress as evidenced by decreased activity of various antioxidant enzymes like glutathione s-transferase, glutathione reductase, thioredoxin reductase, and an increase in ROS production in comparison to control. Oxidative stress may lead to altered calcium homeostasis. Indeed, in 17-AAG treated worms, there was a rise in calcium in the cytosol and mitochondria, as well as a decrease in the ER. We also observed enhanced activity of phospholipase C in the treated parasite, suggesting the opening of calcium channels located on the ER membrane. ER stress is marked by a reduced level of protein disulfide isomerase. Further, 17-AAG treated worms showed an increase in apoptotic marker enzyme activities like calpain, cyt-c, and caspase-3. The 2D-gel electrophoresis technique showed 142 protein spots in the control and 112 spots in the 17-AAG treated parasite. Thus, 17-AAG induced oxidative stress, and altered calcium, and proteostasis of parasites, which led to apoptosis.

The Heat Shock Protein 90 (HSP90) Is Required for the IL-33-Induced Cytokine Production in Mast Cells (MCs).

The alarmin interleukin-33 (IL-33) is released upon cell stress and damage in peripheral tissues. The receptor for IL-33 is the Toll/Interleukin-1 receptor (TIR)-family member T1/ST2 (the IL-33R), which is highly and constitutively expressed on MCs. The sensing of IL-33 by MCs induces the MyD88-TAK1-IKK2-dependent activation of p65/RelA and MAP-kinases, which mediate the production of pro-inflammatory cytokines and amplify FcεRI-mediated MC-effector functions and the resulting allergic reactions. Therefore, the investigation of IL-33-induced signaling is of interest for developing therapeutic interventions effective against allergic reactions. Importantly, beside the release of IL-33, heat shock proteins (HSPs) are upregulated during allergic reactions. This maintains the biological functions of signaling molecules and/or cytokines but unfortunately also strengthens the severity of inflammatory reactions. Here, we demonstrate that HSP90 does not support the IL-33-induced and MyD88-TAK1-IKK2-dependent activation of p65/RelA and of mitogen-activated protein (MAP)-kinases. We found that HSP90 acts downstream of these signaling pathways, mediates the stability of produced cytokine mRNAs, and therefore facilitates the resulting cytokine production. These data show that IL-33 enables MCs to perform an effective cytokine production by the upregulation of HSP90. Consequently, HSP90 might be an attractive therapeutic target for blocking IL-33-mediated inflammatory reactions.

Synergistic Action between PfHsp90 Inhibitor and PfRad51 Inhibitor Induces Elevated DNA Damage Sensitivity in the Malaria Parasite.

The DNA recombinase Rad51 from the human malaria parasite Plasmodium falciparum has emerged as a potential drug target due to its central role in the homologous recombination (HR)-mediated double-strand break (DSB) repair pathway. Inhibition of the ATPase and strand exchange activity of P. falciparum Rad51 (PfRad51) by a small-molecule inhibitor, B02 [3-(phenylmethyl)-2-[(1E)-2-(3-pyridinyl)ethenyl]-4(3H)-quinazolinone], renders the parasite more sensitive to genotoxic agents. Here, we investigated whether the inhibition of the molecular chaperone PfHsp90 potentiates the antimalarial action of B02. We found that the PfHsp90 inhibitor 17-AAG [17-(allylamino)-17-demethoxygeldanamycin] exhibits strong synergism with B02 in both drug-sensitive (strain 3D7) and multidrug-resistant (strain Dd2) P. falciparum parasites. 17-AAG causes a greater than 200-fold decrease in the half-maximal inhibitory concentration (IC50) of B02 in 3D7 parasites. Our results provide mechanistic insights into such profound synergism between 17-AAG and B02. We report that PfHsp90 physically interacts with PfRad51 and promotes the UV irradiation-induced DNA repair activity of PfRad51 by controlling its stability. We find that 17-AAG reduces PfRad51 protein levels by accelerating proteasomal degradation. Consequently, PfHsp90 inhibition renders the parasites more susceptible to the potent DNA-damaging agent methyl methanesulfonate (MMS) in a dose-dependent manner. Thus, our study provides a rationale for targeting PfHsp90 along with the recombinase PfRad51 for controlling malaria propagation.

Cytosolic and Mitochondrial Hsp90 in Cytokinesis, Mitochondrial DNA Replication, and Drug Action in Trypanosoma brucei.

Trypanosoma brucei subspecies cause African sleeping sickness in humans, an infection that is commonly fatal if not treated, and available therapies are limited. Previous studies have shown that heat shock protein 90 (Hsp90) inhibitors have potent and vivid activity against bloodstream-form trypanosomes. Hsp90s are phylogenetically conserved and essential catalysts that function at the crux of cell biology, where they ensure the proper folding of proteins and their assembly into multicomponent complexes. To assess the specificity of Hsp90 inhibitors and further define the role of Hsp90s in African trypanosomes, we used RNA interference (RNAi) to knock down cytosolic and mitochondrial Hsp90s (HSP83 and HSP84, respectively). Loss of either protein led to cell death, but the phenotypes were distinctly different. Depletion of cytosolic HSP83 closely mimicked the consequences of chemically depleting Hsp90 activity with inhibitor 17-AAG. In these cells, cytokinesis was severely disrupted, and segregation of the kinetoplast (the massive mitochondrial DNA structure unique to this family of eukaryotic pathogens) was impaired, leading to cells with abnormal kinetoplast DNA (kDNA) structures. Quite differently, knockdown of mitochondrial HSP84 did not impair cytokinesis but halted the initiation of new kDNA synthesis, generating cells without kDNA. These findings highlight the central role of Hsp90s in chaperoning cell cycle regulators in trypanosomes, reveal their unique function in kinetoplast replication, and reinforce their specificity and value as drug targets.

Regulation of ClC-2 Chloride Channel Proteostasis by Molecular Chaperones: Correction of Leukodystrophy-Associated Defect.

The ClC-2 channel plays a critical role in maintaining ion homeostasis in the brain and the testis. Loss-of-function mutations in the ClC-2-encoding human CLCN2 gene are linked to the white matter disease leukodystrophy. Clcn2-deficient mice display neuronal myelin vacuolation and testicular degeneration. Leukodystrophy-causing ClC-2 mutant channels are associated with anomalous proteostasis manifesting enhanced endoplasmic reticulum (ER)-associated degradation. The molecular nature of the ER quality control system for ClC-2 protein remains elusive. In mouse testicular tissues and Leydig cells, we demonstrated that endogenous ClC-2 co-existed in the same protein complex with the molecular chaperones heat shock protein 90ß (Hsp90ß) and heat shock cognate protein (Hsc70), as well as the associated co-chaperones Hsp70/Hsp90 organizing protein (HOP), activator of Hsp90 ATPase homolog 1 (Aha1), and FK506-binding protein 8 (FKBP8). Further biochemical analyses revealed that the Hsp90ß-Hsc70 chaperone/co-chaperone system promoted mouse and human ClC-2 protein biogenesis. FKBP8 additionally facilitated membrane trafficking of ClC-2 channels. Interestingly, treatment with the Hsp90-targeting small molecule 17-allylamino-17-demethoxygeldanamycin (17-AAG) substantially boosted ClC-2 protein expression. Also, 17-AAG effectively increased both total and cell surface protein levels of leukodystrophy-causing loss-of-function ClC-2 mutant channels. Our findings highlight the therapeutic potential of 17-AAG in correcting anomalous ClC-2 proteostasis associated with leukodystrophy.

Heat Shock Protein 90 Chaperones E1A Early Protein of Adenovirus 5 and Is Essential for Replication of the Virus.

Adenovirus infections tend to be mild, but they may pose a serious threat for young and immunocompromised individuals. The treatment is complicated because there are no approved safe and specific drugs for adenovirus infections. Here, we present evidence that 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG), an inhibitor of Hsp90 chaperone, decreases the rate of human adenovirus 5 (HAdV-5) replication in cell cultures by 95%. 17-AAG inhibited the transcription of early and late genes of HAdV-5, replication of viral DNA, and expression of viral proteins. 6 h after infection, Hsp90 inhibition results in a 6.3-fold reduction of the newly synthesized E1A protein level without a decrease in the E1A mRNA level. However, the Hsp90 inhibition does not increase the decay rate of the E1A protein that was constitutively expressed in the cell before exposure to the inhibitor. The co-immunoprecipitation proved that E1A protein interacted with Hsp90. Altogether, the presented results show, for the first time. that Hsp90 chaperones newly synthesized, but not mature, E1A protein. Because E1A serves as a transcriptional co-activator of adenovirus early genes, the anti-adenoviral activity of the Hsp90 inhibitor might be explained by the decreased E1A level.

Compromising the constitutive p16INK4a expression sensitizes human neuroblastoma cells to Hsp90 inhibition and promotes premature senescence.

The Hsp90 chaperone has become the attractive pharmacological target to inhibit tumor cell proliferation. However, tumor cells can evolve with mechanisms to overcome Hsp90 inhibition. Using human neuroblastoma, we have investigated one such limitation. Here, we demonstrate that neuroblastoma cells overcome the interference of tumor suppressor p16INK4a in cell proliferation, which is due to its latent interaction with CDK4 and CDK6. Cells also displayed impedance to the pharmacological inhibition of cancer chaperone Hsp90 inhibition with respect to induced cytotoxicity. However, the p16INK4a knockdown has triggered the activation of cyclin-CDK6 axis and enhanced the cell proliferation. These cells are eventually sensitized to Hsp90 inhibition by activating the DNA damage response mediated through p53-p21WAF-1 axis and G1 cell cycle exit. While both CDK4 and CDK6 have exhibited low affinity to p16INK4a , CDK6 has exhibited high affinity to Hsp90. Destabilizing the CDK6 interaction with Hsp90 has prolonged G2/M cell cycle arrest fostering to premature cellular senescence. The senescence driven cells exhibited compromised metastatic potential both in vitro as well as in mice xenografts. Our study unravels that cancer cells can be adapted to the constitutive expression of tumor suppressors to overcome therapeutic interventions. Our findings display potential implication of Hsp90 inhibitors to overcome such adaptations.

HSP90 inhibitor 17AAG attenuates sevoflurane-induced neurotoxicity in rats and human neuroglioma cells via induction of HSP70.

BACKGROUND: 17AAG has been extensively studied for its antitumor effects that protect cells from lethal stress by maintaining protein stability. The role of 17AAG in sevoflurane-induced neuronal injury has never been studied. We aim to investigate the effect of 17AAG on sevoflurane-induced neurotoxicity in vivo and in vitro. METHODS: Sevoflurane-induced hippocampal neuron injury model was established in aged Sprague-Dawley rats. Pretreatment of vehicle or 17AAG was administered prior to sevoflurane inhalation. H4 neuroglioma cells were pretreated with vehicle or 17AAG and exposed to sevoflurane. Apoptosis, oxidative stress, expression of interleukin-6 (IL-6), and activation of the nuclear factor-κB (NF-κB) signaling pathway in H4 cells were examined by Hoechst assay, flow cytometry, Western blot, and immunofluorescent staining. RNA interference against HSPA1A was performed to test the function of HSP70 in neuroprotection. RESULTS: Exogenous 17AAG reduced sevoflurane-induced apoptosis and oxidative stress in rat hippocampal neurons and in H4 cells. In H4 cells, 17AAG suppressed sevoflurane-induced upregulation of IL-6 and activation of NF-κB signaling. 17AAG enhanced sevoflurane-induced upregulation of HSP70 in rat hippocampal neurons and in H4 cells. Conversely, silencing of HSPA1A in H4 cells blocked the cytoprotective effect of 17AAG against sevoflurane-induced apoptosis and oxidative stress, and prevented upregulation of IL-6 and activation of NF-κB signaling. CONCLUSIONS: 17AAG protects against sevoflurane-induced neurotoxicity in vivo and in vitro via HSP70-dependent inhibition of apoptosis, oxidative stress, and pro-inflammatory signaling pathway.

Enzymatic biosynthesis and biological evaluation of novel 17-AAG glucoside as potential anti-cancer agents.

A novel 17-allylamino-17-demethoxygeldanamycin (17-AAG) glucoside (1) was obtained from in vitro enzymatic glycosylation using a UDP-glycosyltransferase (YjiC). The water-solubility of compound 1 was approximately 10.5 times higher than that of the substrate, 17-AAG. Compound 1 showed potential anti-proliferative activities against five human cancer cell lines, with IC50 values ranging from 5.26 to 28.52 µM. Further studies also indicated that compound 1 could inhibit the growth of CNE-2Z cells by inducing the degradation of Hsp90 client proteins (Akt, c-Raf, Bcl-2, and HIF-1α). In addition, compound 1 showed greater potential anti-tumor efficacy than 17-AAG in nude mice xenografted with CNE-2Z cells. Therefore, we suggest that in vitro enzymatic glycosylation is a powerful approach for the structural optimization of 17-AAG.

17-Allylamino-Demethoxygeldanamycin Ameliorate Microthrombosis Via HSP90/RIP3/NLRP3 Pathway After Subarachnoid Hemorrhage in Rats.

BACKGROUND: Subarachnoid hemorrhage (SAH) is a severe and emergent cerebrovascular disease, the prognosis of which usually very poor. Microthrombi formation highlighted with inflammation occurs early after SAH. As the main cause of DCI, microthrombosis associated with the prognosis of SAH. The aim of this study was to show HSP90 inhibitor 17-AAG effect on microthrombosis after SAH in rats. METHODS: Ninety-five SD rats were used for the experiment. For time course study, the rats were randomly divided into five groups: sham group and SAH group with different time point (1d, 2d, 3d, 5d). Endovascular perforation method was conducted for SAH model. Neurological score, SAH grade, and mortality were measured after SAH. The samples of the left hemisphere brain were collected. The expression of HSP90 was detected by Western blot. The microthrombosis after SAH in rats' brain was detected by immunohistochemistry. For mechanism study, rats were randomly divided into three groups: sham, SAH + vehicle, and SAH +17-AAG (n = 6/group). 17-AAG was given by intraperitoneal injection (80 mg/kg) 1 h after SAH. Neurological function were measured at 24 h after SAH. The expression of RIP3, NLRP3, ASC, and IL-1ß was measured by Western blot. Microthrombosis was detected by immunohistochemistry. RESULTS: Our results showed that the HSP90 protein level increased and peaked at 2 days after SAH. Microthrombosis caused by SAH was increased in 1 day and peaked at 2 days after SAH. Administration HSP90 specific inhibitor 17-AAG reduced expression of RIP3, NLRP3, ASC, and IL-1ß, reduced microthrombosis after SAH, and improved neurobehavior when compared to vehicle group. CONCLUSIONS: 17-AAG can ameliorate microthrombosis via HSP90/RIP3/NLRP3 pathway and improve neurobehavior after SAH.

HSP90 Inhibition and Modulation of the Proteome: Therapeutical Implications for Idiopathic Pulmonary Fibrosis (IPF).

Idiopathic Pulmonary fibrosis (IPF) is a catastrophic disease with poor outcomes and limited pharmacological approaches. Heat shock protein 90 (HSP90) has been recently involved in the wound-healing pathological response that leads to collagen deposition in patients with IPF and its inhibition represents an exciting drug target against the development of pulmonary fibrosis. Under physiological conditions, HSP90 guarantees proteostasis through the refolding of damaged proteins and the degradation of irreversibly damaged ones. Additionally, its inhibition, by specific HSP90 inhibitors (e.g., 17 AAG, 17 DAG, and AUY-922) has proven beneficial in different preclinical models of human disease. HSP90 inhibition modulates a complex subset of kinases and interferes with intracellular signaling pathways and proteome regulation. In this review, we evaluated the current evidence and rationale for the use of HSP90 inhibitors in the treatment of pulmonary fibrosis, discussed the intracellular pathways involved, described the limitations of the current understanding and provided insights for future research.

Influenza A virus neuraminidase protein interacts with Hsp90, to stabilize itself and enhance cell survival.

Neuraminidase protein (NA) of influenza A virus (IAV) is popularly known for its sialidase function to assist in the release of progeny virus. However, involvement of NA in other stages of the IAV life cycle also indicates its multifunctional nature and necessity to interact with other host proteins. Here, we report a host protein-heat shock protein 90 (Hsp90), as a novel interacting partner of IAV NA. A classical yeast two-hybrid screen was conducted to identify a new host interacting partner for NA and the interaction was further validated by coimmunoprecipitation from cells, transiently expressing both proteins and also from IAV-infected cells. Confocal imaging showed that both proteins colocalized in the cytoplasm in transfected host cells. Interestingly, increased levels of NA in the presence of Hsp90 was observed, which tends to decrease if adenosine triphosphatase activity of Hsp90 is inhibited using 17-N-allylamino-17-demethoxygeldanamycin (17AAG). This establishes viral NA as a client protein of host chaperone Hsp90 contributing toward NA's stability via the NA-Hsp90 interaction. This is the first report showing the interaction of NA with Hsp90 and its role in stabilizing viral NA thus preventing it from degradation. Enhanced cell survival in the presence of this interaction was also observed, thus suggesting the requirement of stable viral NA, post-IAV infection, for efficient virus production in infected mammalian cells.

HSP90 inhibitor 17-AAG prevents apoptosis of cardiomyocytes via miR-93-dependent mitigation of endoplasmic reticulum stress.

Heart failure accounts for substantial morbidity and mortality worldwide. Accumulating evidence suggests that aberrant cardiac cell death caused by endoplasmic reticulum stress (ERS) is often associated with structural or functional cardiac abnormalities that lead to insufficient cardiac output. The detailed molecular mechanism underlying the pathological death of cardiomyocytes still remains poorly understood. We found that 17-AAG (tanespimycin), an HSP90 (heat shock protein 90) inhibitor often used to kill cancer cells, could potently inhibit tunicamycin-induced ERS and the downstream nuclear factor kappa B activity in neonatal rat cardiomyocytes, leading to diminished apoptotic signaling and thus enhanced cell survival. Interestingly, the antiapoptotic effect of 17-AAG on cardiomyocytes required normal expression of miR-93, an oncogenic microRNA known to promote cell survival and growth. Our study implicated a new pharmacological role of 17-AAG in supporting the miR-93-associated oncogenic signaling to prevent the pathological death of cardiomyocytes. The results opened opportunities for exploring new strategies in the development of therapeutic agents.

17-allylamino-17-demethoxygeldanamycin impeded chemotherapy through antioxidant activation via reducing reactive oxygen species-induced cell death.

Hyperthermia enhances the anticancer effects of thymidylate synthase (TYMS) inhibitors (raltitrexed, RTX) and improves the precise biochemical mechanisms partially through enhancement of intracellular drug absorption. Recent research focuses on the potential anticancer drug target Heat Shock Protein 90 (HSP90), which could increase the sensitivity of cancer cells to TYMS inhibitors; however, with different HSP90 inhibitors, several research studies finally showed a poor efficacy in preclinical or clinical research. Here, we showed that 17-allylamino-17-demethoxygeldanamycin (17-AAG, HSP90 inhibitor) affects the efficacy of chemotherapy through antioxidant activation-induced resistance. In this study, we found that RTX, alone or in combination with hyperthermia, triggers reactive oxygen species (ROS) exposure and thus induces cell death. Also, the addition of hyperthermia showed more ROS exposure and function. The pharmacologic inhibition of HSP90 reversed the effects of chemotherapeutical treatments, while the overexpression of HSP90 showed no relation with these effects, which demonstrated that dysregulation of HSP90 might have a significant impact on chemotherapeutic treatments. The addition of 17-AAG increased the activation of antioxidant with increased antioxidant enzymes, thus affecting the RTX efficacy.

HSP90 as a novel therapeutic target for posterior capsule opacification.

Posterior capsule opacification (PCO) is a common complication of cataract surgery, resulting from a combination of proliferation, migration, epithelial-mesenchymal transition (EMT) of residual capsular epithelial cells and fibrosis of myofibroblasts. HSP90 is known to regulate the proteostasis of cells under pathophysiological conditions. The role of HSP90 in PCO formation, however, is not clear. To do this, the lens epithelial cell lines and an ex vivo cultured rat capsular bag model were used to study the role of HSP90 in PCO formation. The expression of protein and mRNA was measured by immunoblotting and quantitative RT-PCR, and cell apoptosis was measured by TUNEL(TdT-mediated dUTP nick-end labeling). The cell proliferation was measured by cell viability assays. The results showed that 17-AAG (Tanespimycin), an inhibitor of HSP90, suppresses the proliferation of immortalized lens epithelial cell lines HLE-B3, SRA01/04, and mLEC, with IC50 values of 0.27, 0.27, and 0.49 µM, respectively. In an ex vivo cultured rat capsular model, the capsular residual epithelial cells resisted the stress of the capsulorhexis surgery and took 3-6 days to completely overlay the capsular posterior wall. During this process, heat shock factor 1 and its downstream targets HSP90, HSP25, αB-crystallin, and HSP40 were upregulated. Treatment with 17-AAG inhibited the viability of capsular residual epithelial cells and induced the cells apoptosis, characterized by increases in ROS levels, apoptotic DNA injury, and the activation of caspases 9 and 3. HSP90 participated in regulating both EGF receptor (EGFR) and TGF receptor (TGFR) signaling pathways. HSP90 was found to interact with the EGFR, such that inhibition of HSP90 by 17-AAG destabilized the EGFR protein and suppressed p-ERK1/2 and p-AKT levels. 17-AAG also inhibited the TGF-ß-induced phosphorylation of SMAD2/3 and ERK1/2 and the decrease in E-cadherin and ZO-1 expression. Accordingly, these data suggest that the induction of HSP90 protects capsular residual epithelial cells against capsulorhexis-induced stress and participates in regulating the processes of proliferation, EMT and migration of rat capsular residual epithelial cells, at least partly, through the EGFR and TGFR signaling pathways. Treatment with 17-AAG suppresses PCO formation and is therefore a potential therapeutic candidate for PCO prevention.

Heat-shock protein 90 (Hsp90) promotes opioid-induced anti-nociception by an ERK mitogen-activated protein kinase (MAPK) mechanism in mouse brain.

Recent advances in developing opioid treatments for pain with reduced side effects have focused on the signaling cascades of the µ-opioid receptor (MOR). However, few such signaling targets have been identified for exploitation. To address this need, we explored the role of heat-shock protein 90 (Hsp90) in opioid-induced MOR signaling and pain, which has only been studied in four previous articles. First, in four cell models of MOR signaling, we found that Hsp90 inhibition for 24 h with the inhibitor 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) had different effects on protein expression and opioid signaling in each line, suggesting that cell models may not be reliable for predicting pharmacology with this protein. We thus developed an in vivo model using CD-1 mice with an intracerebroventricular injection of 17-AAG for 24 h. We found that Hsp90 inhibition strongly blocked morphine-induced anti-nociception in models of post-surgical and HIV neuropathic pain but only slightly blocked anti-nociception in a naive tail-flick model, while enhancing morphine-induced precipitated withdrawal. Seeking a mechanism for these changes, we found that Hsp90 inhibition blocks ERK MAPK activation in the periaqueductal gray and caudal brain stem. We tested these signaling changes by inhibiting ERK in the above-mentioned pain models and found that ERK inhibition could account for all of the changes in anti-nociception induced by Hsp90 inhibition. Taken together, these findings suggest that Hsp90 promotes opioid-induced anti-nociception by an ERK mechanism in mouse brain and that Hsp90 could be a future target for improving the therapeutic index of opioid drugs.

Transcriptional regulation of Runx2 by HSP90 controls osteosarcoma apoptosis via the AKT/GSK-3ß/ß-catenin signaling.

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 ß-catenin was overexpressed in OS tissue, and that knockdown of ß-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-3ß/ß-catenin signaling pathway by using pharmacological inhibitor GSK-3ß (LiCl) or siGSK-3ß. Our findings reveal a novel mechanism that Runx2 is transcriptionally regulated by HSP90 via the AKT/GSK-3ß/ß-catenin signaling pathway, and by which leads to apoptosis of OS cells.

Quiescence Promotes Latent HIV Infection and Resistance to Reactivation from Latency with Histone Deacetylase Inhibitors.

Human immunodeficiency virus type 1 (HIV-1) establishes transcriptionally silent latent infections in resting memory T cells and hematopoietic stem and progenitor cells (HSPCs), which allows the virus to persist in infected individuals despite antiretroviral therapy. Developing in vitro models of HIV-1 latency that recapitulate the characteristics of latently infected cells in vivo is crucial to identifying and developing effective latency-reversing therapies. HSPCs exist in a quiescent state in vivo, and quiescence is correlated with latent infections in T cells. However, current models for culturing HSPCs and for infecting T cells in vitro require that the cells be maintained in an actively proliferating state. Here we describe a novel culture system in which primary human HSPCs cultured under hypothermic conditions are maintained in a quiescent state. We show that these quiescent HSPCs are susceptible to predominantly latent infection with HIV-1, while actively proliferating and differentiating HSPCs obtain predominantly active infections. Furthermore, we demonstrate that the most primitive quiescent HSPCs are more resistant to spontaneous reactivation from latency than more differentiated HSPCs and that quiescent HSPCs are resistant to reactivation by histone deacetylase inhibitors or P-TEFb activation but are susceptible to reactivation by protein kinase C (PKC) agonists. We also demonstrate that inhibition of HSP90, a known regulator of HIV transcription, recapitulates the quiescence and latency phenotypes of hypothermia, suggesting that hypothermia and HSP90 inhibition may regulate these processes by similar mechanisms. In summary, these studies describe a novel model for studying HIV-1 latency in human primary cells maintained in a quiescent state.IMPORTANCE Human immunodeficiency virus type 1 (HIV-1) establishes a persistent infection for which there remains no feasible cure. Current approaches are unable to clear the virus despite decades of therapy due to the existence of latent reservoirs of integrated HIV-1, which can reactivate and contribute to viral rebound following treatment interruption. Previous clinical attempts to reactivate the latent reservoirs in an individual so that they can be eliminated by the immune response or viral cytopathic effect have failed, indicating the need for a better understanding of the processes regulating HIV-1 latency. Here we characterize a novel in vitro model of HIV-1 latency in primary hematopoietic stem and progenitor cells isolated from human cord blood that may better recapitulate the behavior of latently infected cells in vivo This model can be used to study mechanisms regulating latency and potential therapeutic approaches to reactivate latent infections in quiescent cells.

In vitro interactions between 17-AAG and azoles against Exophiala dermatitidis.

BACKGROUND: Exophiala dermatitidis causes a variety of illnesses in humans which are always refractory to available treatment modalities. Hsp90 governs crucial stress responses, cell wall repair mechanisms and antifungal resistance in pathogenic fungi. Thus, targeting Hsp90 with specific inhibitors holds considerable promise as combination strategy. OBJECTIVES: To investigate the antifungal effect of 17-AAG alone or combined with azoles against E. dermatitidis. METHODS: In vitro interactions of 17-AAG, a Hsp90 inhibitor, and azoles including itraconazole, voriconazole and posaconazole against E. dermatitidis were evaluated via broth microdilution chequerboard technique, adapted from the CLSI M38-A2 method. A total of 18 clinical strains were studied. Candida parapsilosis (ATCC22019) was included to ensure quality control. RESULTS AND CONCLUSIONS: 17-AAG alone exhibited minimal antifungal activity against all tested isolates. However, synergistic effects between 17-AAG and posaconazole, itraconazole or voriconazole were observed against 15 (83.3%), 12 (66.7%) and 1 (5.6%) isolates of E. dermatitidis, respectively. The effective working ranges of 17-AAG in synergistic combinations were mostly within 2-8 µg/mL. No antagonism was observed. In conclusion, harnessing fungal Hsp90 with 17-AAG might prove a potential antifungal regimen for E. dermatitidis infections. However, due to the host toxicity of 17-AAG, more efforts are needed to develop fungal specific Hsp90 inhibitors.