Pharmacological inhibition of Hsp90 is an exciting option for cancer therapy. The clinical efficacy of Hsp90 inhibitors is, however, less than expected. Binding of the co-chaperone p23 to Hsp90 and ...induced overexpression of anti-apoptotic proteins Hsp70 and Hsp27 are thought to contribute to this outcome. Herein, we report that the natural product gedunin may provide a new alternative to inactivate the Hsp90 machine. We show that gedunin directly binds to p23 and inactivates it, without overexpression of Hsp27 and relatively modest induction of Hsp70. Using molecular docking and mutational analysis, we mapped the gedunin-binding site on p23. Functional analysis shows that gedunin inhibits the p23 chaperoning activity, blocks its cellular interaction with Hsp90, and interferes with p23-mediated gene regulation. Cell treatment with gedunin leads to cancer cell death by apoptosis through inactivation of p23 and activation of caspase 7, which cleaves p23 at the C terminus. These results provide important insight into the molecular mechanism of action of this promising lead compound.
Background: The Hsp90 chaperoning machine is an exciting therapeutic target for cancer treatment.
Results: We report that the natural product gedunin inactivates the Hsp90 co-chaperone p23 in vitro and in vivo. The lethal effect of gedunin-p23 complex on cancer cells is amplified by caspase-7-mediated cleavage of the co-chaperone, leading to apoptotic cell death.
Conclusions: Gelduin binds directly to p23 leading to inactivation of the Hsp90 machine and selective destabilization of steroid receptors.
Significance: Gedunin is a promising compound to develop anti-cancer therapeutics.
Heat shock protein 90 (Hsp90) and its co-chaperones promote cancer, and targeting Hsp90 holds promise for cancer treatment. Most of the efforts to harness this potential have focused on targeting the ...Hsp90 N-terminus ATP binding site. Although newer-generation inhibitors have shown improved efficacy in aggressive cancers, induction of the cellular heat shock response (HSR) by these inhibitors is thought to limit their clinical efficacy. Therefore, Hsp90 inhibitors with novel mechanisms of action and that do not trigger the HSR would be advantageous. Here, we investigated the mechanism by which capsaicin inhibits Hsp90. Through mutagenesis, chemical modifications, and proteomic studies, we show that capsaicin binds to the N-terminus of Hsp90 and inhibits its ATPase activity. Consequently, capsaicin and its analogs inhibit Hsp90 ATPase-dependent progesterone receptor reconstitution in vitro. Capsaicin did not induce the HSR, instead, it promoted the degradation of Hsp70 through the lysosome-autophagy pathway. Remarkably, capsaicin did not induce degradation of the constitutively expressed cognate Hsc70, indicating selectivity for Hsp70. Combined treatments of capsaicin and the Hsp90 inhibitor 17-AAG improved the anti-tumor efficacy of 17-AAG in cell culture and tridimensional tumor spheroid growth assays using breast and prostate cancer models. Consistent with this, in silico docking studies revealed that capsaicin binding to the ATP binding site of Hsp90 was distinct from classical N-terminus Hsp90 inhibitors, indicating a novel mechanism of action. Collectively, these findings support the use of capsaicin as a chemical scaffold to develop novel Hsp90 N-terminus inhibitors as well as its ability to be a potential cancer co-therapeutic.
Cumulative evidence suggests that the heat shock protein 90 (Hsp90) co-chaperone UNC-45 myosin chaperone A (UNC45A) contributes to tumorigenesis and that its expression in cancer cells correlates ...with proliferation and metastasis of solid tumors. However, the molecular mechanism by which UNC45A regulates cancer cell proliferation remains largely unknown. Here, using siRNA-mediated gene silencing and various human cells, we report that UNC45A is essential for breast cancer cell growth, but is dispensable for normal cell proliferation. Immunofluorescence microscopy, along with gene microarray and RT-quantitative PCR analyses, revealed that UNC45A localizes to the cancer cell nucleus, where it up-regulates the transcriptional activity of the glucocorticoid receptor and thereby promotes expression of the mitotic kinase NIMA-related kinase 7 (NEK7). We observed that UNC45A-deficient cancer cells exhibit extensive pericentrosomal material disorganization, as well as defects in centrosomal separation and mitotic chromosome alignment. Consequently, these cells stalled in metaphase and cytokinesis and ultimately underwent mitotic catastrophe, phenotypes that were rescued by heterologous NEK7 expression. Our results identify a key role for the co-chaperone UNC45A in cell proliferation and provide insight into the regulatory mechanism. We propose that UNC45A represents a promising new therapeutic target to inhibit cancer cell growth in solid tumor types.
Hsp90 and its co-chaperones are known to be important for cancer cell survival. The N-terminal inhibitors of Hsp90 that are in ongoing clinical trials as antitumor agents have unfortunately shown ...disappointing efficacies in the clinic. Thus, novel inhibitors of the Hsp90 machine with a different mechanism of action are urgently needed. We report here the development of a novel high-throughput screening assay platform to identify small-molecule inhibitors of Hsp90 and its co-chaperones. This assay quantitatively measures the ability of Hsp90 and its co-chaperones to refold/protect the progesterone receptor, a physiological client of Hsp90, in a 96-well plate format. We screened the National Institutes of Health clinical collection drug library and identified capsaicin as a hit molecule. Capsaicin is a Food and Drug Administration-approved drug for topical use in pain management. Cell survival assays showed that capsaicin selectively kills cancer cells and destabilizes several Hsp90 client proteins. Thus, our data may explain the seemingly pleotropic effect of capsaicin.
Abstract
Pharmacological inhibition of Hsp90 is an exciting option for cancer therapy. The clinical efficacy of Hsp90 inhibitors is, however, less than expected. Binding of the co-chaperone p23 to ...Hsp90, and induced overexpression of anti-apoptotic proteins, Hsp70 and Hsp27, are thought to contribute to this outcome. Herein, we report that the natural product, gedunin, may provide a new alternative to inactivate the Hsp90 machine. We show that gedunin directly binds to p23 and inactivates it, without overexpression of Hsp27 and relatively modest induction of Hsp70. Using molecular docking and mutational analysis, we mapped the gedunin-binding site on p23. Functional analysis shows that gedunin inhibits p23 chaperoning activity, blocks its cellular interaction with Hsp90 and interferes with p23-mediated gene regulation. Cell treatment with gedunin leads to cancer cell death by apoptosis through inactivation of p23 and activation of caspase 7, which cleaves p23 at the C-terminus. These results provide important insight into the molecular mechanism of action of this promising lead compound.
Citation Format: Chaitanya A. Patwardhan, Laura B. Peterson, Brian Blagg, Ahmed Chadli. Gedunin inactivates the co-chaperone p23 causing cancer cell death by apoptosis. abstract. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3197. doi:10.1158/1538-7445.AM2014-3197
•Comparison of different phase interaction models have been performed.•Inclusion of wall lubrication force showed void peak near the wall.•Behavior of boiling parameter models has been studied.•CFD ...model has been developed on the basis of model comparison.•CFD model is used for pressure drop prediction in long vertical tube boiling.
In this article, Eulerian two fluid model is used for prediction of high pressure subcooled nucleate flow boiling in vertical tubes. Predictions of different turbulence models, phase interaction models have been compared with previous experimental data. Axial and radial profiles of vapor fraction and liquid temperature have been used for comparison of these models. Bubble departure diameter models and bubble induced turbulence models show the significant effect on the vapor fraction prediction. It was found that use of Kocamustafaogullari and Ishii models for bubble departure diameter and nucleation site density are suitable for predicting vapor distribution and liquid temperature for high pressure boiling. Models of interfacial heat transfer and bubble departure frequency developed for low pressure or atmospheric conditions showed a good applicability for high pressure conditions also. Inclusion of wall lubrication force showed the void fraction peak near the wall. Simulated pressure drop predictions for high pressure flow boiling in a 13m long vertical tube shows the error of 2–10% with experimental data. This indicates that computational fluid dynamics (CFD) is the promising technique for pressure drop prediction.
•CFD simulations have been carried out on honeycomb shaped orifice plates.•Effect of geometrical parameters on pressure drop and cavitation have been studied.•Threshold flow velocities to avoid ...cavitation have been identified.•Reduction in flow area increases the cavitation possibility.•Increase in plate spacing between two consecutive plates, allows flow uniformity.
Flow zoning devices in nuclear power plants are used to achieve desired pressure drop and flow rates. Cavitation in these devices may cause erosion, flow chocking, noise, vibration, resulting in severe damage. Optimization of flow conditions, geometrical parameters, and prediction of the hydrodynamics of the flow field are necessary to avoid cavitation in these devices. In the present study, Computational Fluid Dynamics (CFD) simulations have been carried out to model flow through honeycomb shaped orifices. The effect of various geometrical parameters such as flow area, the spacing between plates, number of plates, orientation between plates (inline or offset) on pressure drop, and cavitation characteristics has been studied. The multiphase mixture model (water-liquid and water vapor), in conjunction with the Schnerr and Sauer cavitation model, is used to model the flow through orifice plates. Axial distribution profiles and contours of vapor fraction and pressure coefficient have been plotted to analyze the cavitation characteristics for different plate configurations and to get a better understanding of the flow field. Lowest pressure values are observed at the orifice throat and near plate exit. Threshold flow velocities above which flow is subjected to cavitation have been identified for different geometrical configurations. An increase in the number of plates reduces the probability of cavitation. Reduction in the flow area of the orifice plate increases the cavitation potential and pressure drop. However, an increase in plate spacing enhances the pressure recovery and reduces non-uniformities in the flow field.
Flow boiling in long vertical tubes in evaporators or boilers is governed by hydrodynamics and boiling heat transfer processes. Precise modeling of these flow boiling processes is necessary to design ...evaporator/boiler in nuclear/thermal power plants and to avoid complexities such as flow instabilities, critical heat flux etc. The present study involves development of the new methodology to model the full range flow boiling phenomenon which covers all flow boiling regimes in long vertical tubes at high pressure. Computational fluid dynamics (CFD) simulations have been performed on tubes of lengths 7 m, 13.4 m and 23 m at a pressure range of 86–172 bar. The entire tube has been divided into four sections based on the value of the vapor fraction at the end of the section and different modeling strategies are applied to each section. Eulerian-Eulerian two fluid model along with appropriate wall boiling models and phase interaction models are used to model the different sections. It has been found that the developed methodology shows more accuracy in predicting the pressure drop in the boiler tube (1–9% error) than single section approach. For 23 m long tube full range flow boiling simulations, a reduction in two phase flow boiling heat transfer coefficient is observed due to the presence of instability at 20% design heat duty. Axial distribution profiles of vapor fraction, heat flux, heat transfer coefficient, water/steam temperature have been plotted to get insights of flow boiling phenomenon occurring in a 23 m long evaporator tube. CFD approach shows advancement over one dimensional (1-D) approach in the prediction of thermal hydraulic characteristics.
Steroid hormone receptors are ligand-dependent transcription factors that require the ordered assembly of multichaperone complexes for transcriptional activity. Although heat shock protein (Hsp) 90 ...and Hsp70 are key players in this process, multiple Hsp70- and Hsp90-associated cochaperones associate with receptor-chaperone complexes to regulate receptor folding and activation. Small glutamine-rich tetratricopeptide repeat-containing protein alpha (SGTA) was recently characterized as an Hsp70 and Hsp90-associated cochaperone that specifically regulates androgen receptor activity. However, the specificity of SGTA for additional members of the steroid hormone receptor superfamily and the mechanism by which SGTA regulates receptor activity remain unclear. Here we report that SGTA associates with and specifically regulates the androgen, glucocorticoid, and progesterone receptors and has no effect on the mineralocorticoid and estrogen receptors in both yeast and mammalian cell-based reporter assays. In both systems, SGTA knockdown/deletion enhances receptor activity, whereas SGTA overexpression suppresses receptor activity. We demonstrate that SGTA binds directly to Hsp70 and Hsp90 in vitro with similar affinities yet predominately precipitates with Hsp70 from cell lysates, suggesting a role for SGTA in early, Hsp70-mediated folding. Furthermore, SGTA expression completely abrogates the regulation of receptor function by FKBP52 (52-kDa FK506-binding protein), which acts at a later stage of the chaperone cycle. Taken together, our data suggest a role for SGTA at distinct steps in the chaperone-dependent modulation of androgen, glucocorticoid, and progesterone receptor activity.
Cochaperones are important for the folding and activation of steroid hormone receptors.
The androgen receptor-associated cochaperone SGTA binds both Hsp70 and Hsp90 and regulates progesterone and glucocorticoid receptors.
SGTA is a receptor-specific cochaperone that regulates distinct steps in the receptor chaperoning cycle.
SGTA is a relevant factor in diseases that depend on androgens, progestins and/or glucocorticoids.
Pharmacological inhibition of the Hsp90 machinery is an exciting option for cancer therapy. Clinical efficacy of Hsp90 inhibitors is, however, less than expected. Binding of the co-chaperone p23 to ...Hsp90, and induced over-expression of anti-apoptotic proteins, Hsp70 and Hsp27, is thought to contribute to this undesired outcome. We therefore face an urgent need to develop much better inhibitors of the Hsp90 machinery that can effectively kill cancer cells with minimal side effects. The goal of this dissertation is to identify novel inhibitors of Hsp90 chaperoning machinery to efficiently kill cancer cells with minimal side effects on normal cell survival. First, we report that the natural product, gedunin, may provide a new alternative to inactivate the Hsp90 machine. We show that gedunin directly binds to the co-chaperone p23 and inactivates it, without inducing over-expression of Hsp27 and only a relatively modest induction of Hsp70. Using molecular docking and mutational analyses; we mapped the gedunin-binding site on p23. Functional analysis shows that gedunin inhibits p23 chaperoning activity, blocks its cellular interaction with Hsp90 and interferes with p23-mediated gene regulation. Cell treatment with gedunin leads to cancer cell death by apoptosis through inactivation of p23 and activation of caspase 7, which cleaves p23 at the C-terminus. These results provide important insight into the molecular mechanism of action of this promising lead compound. Second, we report the development of a novel semi-high-throughput drug-screening assay to identify small molecule inhibitors of Hsp90 and its co-chaperones. Our assay quantitatively measures the ability of Hsp90 and its co-chaperones to refold the progesterone receptor (PR), a physiological client of Hsp90, in an in vitro assay performed in a 96-well plate format. We tested the NIH clinical collection drug library of 446 compounds and identified capsaicin as a "hit". Our data show that capsaicin targets the Hsp70-Hsp90 chaperone complex in cells and alters Hsp70 multi-chaperone complexes. It induces cellular destabilization of Hsp90-Hsp70 client proteins and causes degradation of the Hsp70 (induced form) but not the Hsc70 (constitutive form) protein through lysosome-autophagy pathway. Cell survival assays showed that capsaicin selectively kills cancer cells by inducing mitophagy. Taken together, our data suggest that capsaicin could be used in combination with Hsp90 inhibitors for cancer treatment. INDEX WORDS: Hsp90, Hsp70, p23, Hsp90 inhibitors, Gedunin, Capsaicin, High-throughput drug screening, NIH clinical collection.
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