Expression Of Hsp90 Client Proteins Research Articles

The HSP90 inhibitor HVH-2930 exhibits potent efficacy against trastuzumab-resistant HER2-positive breast cancer.

Rationale: Resistance to targeted therapies like trastuzumab remains a critical challenge for HER2-positive breast cancer patients. Despite the progress of several N-terminal HSP90 inhibitors in clinical trials, none have achieved approval for clinical use, primarily due to issues such as induction of the heat shock response (HSR), off-target effects, and unfavorable toxicity profiles. We sought to examine the effects of HVH-2930, a novel C-terminal HSP90 inhibitor, in overcoming trastuzumab resistance. Methods: The effect of HVH-2930 on trastuzumab-sensitive and -resistant cell lines in vitro was evaluated in terms of cell viability, expression of HSP90 client proteins, and impact on cancer stem cells. An in vivo model with trastuzumab-resistant JIMT-1 cells was used to examine the efficacy and toxicity of HVH-2930. Results: HVH-2930 was rationally designed to fit into the ATP-binding pocket interface cavity of the hHSP90 homodimer in the C-terminal domain of HSP90, stabilizing its open conformation and hindering ATP binding. HVH-2930 induces apoptosis without inducing the HSR but by specifically suppressing the HER2 signaling pathway. This occurs with the downregulation of HER2/p95HER2 and disruption of HER2 family member heterodimerization. Attenuation of cancer stem cell (CSC)-like properties was associated with the downregulation of stemness factors such as ALDH1, CD44, Nanog and Oct4. Furthermore, HVH-2930 administration inhibited angiogenesis and tumor growth in trastuzumab-resistant xenograft mice. A synergistic effect was observed when combining HVH-2930 and paclitaxel in JIMT-1 xenografts. Conclusion: Our findings highlight the potent efficacy of HVH-2930 in overcoming trastuzumab resistance in HER2-positive breast cancer. Further investigation is warranted to fully establish its therapeutic potential.

Transferrin Modified GSH Sensitive Hyaluronic Acid Derivative Micelle to Deliver HSP90 Inhibitors to Enhance the Therapeutic Efficacy of Brain Cancers.

Simple SummaryHeat shock protein 90 (HSP90) is a key element of a multi-chaperone complex involved in the stabilizing of many client proteins, oncoproteins, which play essential roles in tumorigenesis. As the result, HSP90 has been taken as a promising target for anticancer therapies. AUY922 has good antitumor activity by inhibiting the ATPase activity of HSP90, while it has certain limitations, including poor water solubility and lack of selectivity, which have incited the development of a novel targeted nanoformulation. In this study, we have successfully synthesized and characterized a GSH-sensitive micelle that can encapsulate hydrophobic AUY922 in its core region to enhance its therapeutic efficacy against brain cancers. All in vitro and in vivo experimental results showed nanoformulated AUY922 has a better therapeutic efficacy against brain cancer in comparison to the free AUY922.Herein, GSH-sensitive hyaluronic acid-poly(lactic-co-glycolic acid) (HA-SS-PLGA) was synthesized. Surface modification of PLGA with hyaluronic acid produced a highly stable micelle at physiological pH while a micelle was destabilized at a higher GSH level. Fluorescence microscopy results showed that rhodamine-encapsulated micelle was taken up by brain cancer cells, while competitive inhibition was observed in the presence of free HA and free transferrin. In vitro cytotoxicity results revealed that transferrin-targeted nanoformulated AUY922 (TF-NP-AUY922) shows higher cytotoxicity than either free AUY922 or non-targeted AUY922-loaded micelles (NP-AUY922). In comparison to the control groups, free AUY922, TF-NP-AUY922 or NP-AUY922 treatment revealed the upregulation of HSP70, while the expression of HSP90 client proteins was simultaneously depleted. In addition, the treatment group induced caspase-dependent PARP cleavage and the upregulation of p53 expression, which plays a key role in apoptosis of brain cancer cells. In vivo and ex vivo biodistribution studies showed that cypate-loaded micelle was taken up and accumulated in the tumor regions. Furthermore, in vivo therapeutic efficacy studies revealed that the AUY922-loaded micelle significantly suppressed tumor growth in comparison to the free AUY922, or control groups using tumor-bearing NOD-SCID mice. Moreover, biochemical index and histological analysis revealed synthesized micelle does not show any significant cytotoxicity to the selected major organs. Overall, a synthesized micelle is the best carrier for AUY922 to enhance the therapeutic efficiency of brain cancer.

Abstract 2317: Prostanoid EP4 receptor induces cleavage of HSP90 via ROS generation in human colon cancer cells

Abstract Prostaglandin E2 (PGE2) has been reported to play critical roles in cell fate decision by interacting with four types of G protein-coupled membrane receptors such as EP1, EP2, EP3 and EP4. We previously reported that EP4 stimulation by treatment with its agonist CAY10598 induced apoptosis via reactive oxygen species (ROS) formation in colon cancer HCT116 cells. Moreover, treatment with CAY10598 diminished the phosphorylation of JAK2 and induced degradation of this oncoprotein, leading to the attenuation of STAT3 activation in HCT116 cells. In the present study, we attempted to delineate the molecular mechanisms underlying the degradation of JAK2 by activation of EP4. HSP90, a member of the heat shock protein family, is a molecular chaperone that supports stability of client proteins, such as, EGFR, MET, Akt and JAK2. HSP90-mediated stabilization/activation of these client proteins contributes to the acquisition of cancer cell hallmarks, including proliferation, survival, angiogenesis and invasion. It has been recently reported that the chaperoning function of HSP90 may be disrupted by post-translational modification induced by oxidative stress. Treatment of human colon cancer HCT116 cells with CAY10598 down-regulated expression of HSP90 client proteins in a concentration- and time-dependent manner and the down-regulation was restored by pretreatment with ROS scavenger N-acetyl cysteine (NAC) or proteasome inhibitor MG132. However, cotreatment with cycloheximide, protein synthesis inhibitor, accelerated the CAY10598-induced degradation of HSP90 client proteins. These data suggest that CAY10598-induced HSP90 client protein degradation may be caused by ROS generation. In renal carcinoma Caki cells, CAY10598 also down-regulated expression of HSP90 client proteins, suggesting that EP4 stimulation may regulate HSP90 activity in colon cancer cells as well as renal cancer cells. We found that HSP90α was cleaved to 40 or 55 kDa, while HSP90β cleaved to 25 kDa by CAY10598 treatment and the cleavage of HSP90α and β was blocked by NAC treatment. Furthermore, EP4 inhibition by treatment with antagonist GW627368x attenuated not only degradation of HSP90 client proteins but also cleavage of HSP90 in CAY105998-treated HCT116 cells. In conclusions, EP4 agonist CAY10598 induces degradation of HSP90 client proteins via ROS-dependent HSP90 cleavage, leading to apoptosis in HCT116 cells. This is a novel mechanism by which EP4 activation induces apoptosis of cancer cells that involved inhibiting HSP90 function. Citation Format: In Gyeong Chae, Mi-hee Yu, Kyung-Soo Chun. Prostanoid EP4 receptor induces cleavage of HSP90 via ROS generation in human colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2317. doi:10.1158/1538-7445.AM2017-2317

Antileukemic Scalarane Sesterterpenoids and Meroditerpenoid from Carteriospongia (Phyllospongia) sp., Induce Apoptosis via Dual Inhibitory Effects on Topoisomerase II and Hsp90.

Two new scalarane sesterterpenoids, 12β-(3′β-hydroxybutanoyloxy)-20,24-dimethyl-24-oxo-scalara-16-en-25-al (1) and 12β-(3′β-hydroxypentanoyloxy)-20,24-dimethyl-24-oxo-scalara-16-en-25-al (2), along with one known tetraprenyltoluquinol-related metabolite (3), were isolated from the sponge Carteriospongia sp. In leukemia Molt 4 cells, 1 at 0.0625 μg/mL (125 nM) triggered mitochondrial membrane potential (MMP) disruption and apoptosis showing more potent effect than 2 and 3. The isolates inhibited topoisomerase IIα expression. The apoptotic-inducing effect of 3 was supported by the in vivo experiment through suppressing the volume of xenograft tumor growth (47.58%) compared with the control. Compound 1 apoptotic mechanism of action in Molt 4 cells was further elucidated through inducing ROS generation, calcium release and ER stress. Using the molecular docking analysis, 1 exhibited more binding affinity to N-terminal ATP-binding pocket of Hsp90 protein than 17-AAG, a standard Hsp90 inhibitor. The expression of Hsp90 client proteins, Akt, p70S6k, NFκB, Raf-1, p-GSK3β, and XIAP, MDM 2 and Rb2, and CDK4 and Cyclin D3, HIF 1 and HSF1 were suppressed by the use of 1. However, the expression of Hsp70, acetylated tubulin, and activated caspase 3 were induced after 1 treatment. Our results suggested that the proapoptotic effect of the isolates is mediated through the inhibition of Hsp90 and topoisomerase activities.

Synergistic cytotoxicity of BIIB021 with triptolide through suppression of PI3K/Akt/mTOR and NF-κB signal pathways in thyroid carcinoma cells.

The effec.t of BIIB021, a novel heat shock protein 90 (hsp90) inhibitor, on survival of thyroid carcinoma cells has not been evaluated. In this study, the impact of BIIB021 alone or in combination with the histone acetyltransferase inhibitor triptolide on survival of thyroid carcinoma cells was identified. In 8505C and TPC-1 thyroid carcinoma cells, BIIB021 caused cell death in conjunction with alterations in expression of hsp90 client proteins. Cotreatment of both BIIB021 and triptolide, compared with treatment of BIIB021 alone, decreased cell viability, and increased the percentage of dead cells and cytotoxic activity. All of the combination index values were lower than 1.0, suggesting synergistic activity of BIIB021 with triptolide in induction of cytotoxicity. In treatment of both BIIB021 and triptolide, compared with treatment of BIIB021 alone, the protein levels of total and phospho-p53, and cleaved caspase-3 were elevated, while those of total Akt, phospho-mTOR, phospho-4EBP1, phospho-S6K, phospho-NF-κB, survivin, X-linked inhibitor of apoptosis protein (xIAP), cellular inhibitor of apoptosis protein (cIAP) and acetyl. histone H4 were reduced. These results suggest that BIIB021 has a cytotoxic activity accompanied by regulation of hsp90 client proteins in thyroid carcinoma cells. Moreover, the synergism between BIIB021 and triptolide in induction of cytotoxicity is associated with the inhibition of PI3K/Akt/mTOR and NF-κB signal pathways, the underexpression of survivin and the activation of DNA damage response in thyroid carcinoma cells.

The heat shock protein 90 inhibitor SNX5422 has a synergistic activity with histone deacetylase inhibitors in induction of death of anaplastic thyroid carcinoma cells.

The influence of the heat shock protein 90 (hsp90) inhibitor SNX5422 alone or in combination with the histone deacetylase (HDAC) inhibitors PXD101, suberoylanilide hydroxamic acid (SAHA), and trichostatin A (TSA) on survival of anaplastic thyroid carcinoma (ATC) cells was investigated. In 8505C and CAL62 cells, SNX5422 caused cell death with concomitant changes in the expression of hsp90 client proteins. After treatment of both SNX5422 and PXD101, SAHA and TSA, compared with treatment of SNX5422 alone, cell viability was diminished, whereas inhibition rate and cytotoxic activity were enhanced. All of the combination index values were lower than 1.0, suggesting the synergism between SNX5422 and PXD101, SAHA and TSA in induction of cell death. In cells treated with both SNX5422 and PXD101, SAHA and TSA, compared with cells treated with SNX5422 alone, the protein levels of Akt, phospho-4EBP1, phospho-S6 K, and survivin were diminished, while those of γH2AX, acetyl. histone H3, acetyl. histone H4, cleaved PARP, and cleaved caspase-3 were enhanced. In conclusion, these results demonstrate that SNX5422 has a cytotoxic activity in conjunction with alterations in the expression of hsp90 client proteins in ATC cells. Moreover, SNX5422 synergizes with HDAC inhibitors in induction of cytotoxicity accompanied by the suppression of PI3K/Akt/mTOR signaling and survivin, and the overexpression of DNA damage-related proteins in ATC cells.

Novel heat shock protein 90 inhibitor NVP-AUY922 synergizes with the histone deacetylase inhibitor PXD101 in induction of death of anaplastic thyroid carcinoma cells.

The influence of the novel heat shock protein 90 (hsp90) inhibitor NVP-AUY922 (AUY922) on anaplastic thyroid carcinoma (ATC) cells has not been investigated. The effect of AUY922 alone or in combination with the histone deacetylase inhibitor PXD101 on survival of ATC cells was evaluated. In ATC cells, AUY922, compared with 17-allylamino-17-demethoxygeldanamycin, herbimycin A and radicicol, potently lead to growth inhibition and cell death with cleavage of caspase-3 and poly (ADP-ribose) polymerase (PARP) and concomitant changes in expression of hsp90 client proteins. After treatment of both AUY922 and PXD101, compared with treatment of AUY922 or PXD101 alone, the percentage of nonviable cells, annexin V-stained cells, and cytotoxic activity increased. All of the combination index values were lower than 1.0, suggesting the synergism between AUY922 and PXD101 in induction of cell death. In cells treated with both AUY922 and PXD101, compared with cells treated with AUY922 alone, the protein levels of phospho-Akt, cellular inhibitor of apoptosis protein, X-linked inhibitor of apoptosis protein, survivin, ataxia telangiectasia mutated (ATM), and ATM and Rad-3 related (ATR) decreased, whereas those of acetyl. histone H3, acetyl. histone H4, phospho-histone H2A.X, cleaved DFF45, and cleaved PARP increased. Our results demonstrate that AUY922 potently induces cytotoxicity with concomitant modulation of hsp90 client proteins in ATC cells. Moreover, AUY922 has a synergistic activity with PXD101 in induction of cytotoxicity in conjunction with the inactivation of PI3K/Akt signaling and survivin and the activation of DNA damage response in ATC cells.

Influence of 17AAG on proliferation and invasion of gastric cancer cell and its mechanism

To investigate the effect of 17-allylamino-demethoxygeldanamycin (17AAG) on the proliferative and invasive ability of gastric cancer cells and associated mechanism. The proliferative ability was tested by MTT method and the cell cycle was detected by flow cytometry(FCM) when 17AAG was used to treat gastric cancer cell SGC7901. Apoptosis was detected by FCM and PI-Annexin V double staining. The invasive ability was tested by transwell method. Expression of HSP90, HSP70, c-met and AKT was detected by Western blot. The growth of SGC7901 cells was inhibited after the administration of 17AAG, and the inhibitation was dose- and time-dependent. The cell cycle was blocked at the G0/G1 phase. The apoptotic ratio in 17AAG group was much higher than that in blank group and DMSO group (P<0.01). The cellular invasive ability decreased significantly (P<0.01). The expression of HSP70 was elevated by 17AAG, and the expression of c-met and AKT was down-regulated, but no change of HSP90 was observed. 17AAG can inhibit the proliferative and invasive ability of SGC7901 cells, and induces apoptosis through down-regulating the expression of HSP90 client proteins instead of the target HSP90 itself.

Anti-tumor activity of WK88-1, a novel geldanamycin derivative, in gefitinib-resistant non-small cell lung cancers with Met amplification.

Although epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) have been introduced for the treatment of non-small cell lung cancer (NSCLC), the emergence of secondary T790M mutation in EGFR or amplification of the Met proto-oncogene restrain the clinical success of EGFR-TKIs. Since heat shock protein-90 (Hsp90) stabilizes various oncoproteins including EGFR and c-Met, the inhibition of Hsp90 activity appears as a rational strategy to develop anticancer drugs. Despite preclinical efficacy of geldanamycin-anasamycin (GA)-derivatives containing benzoquinone moiety as Hsp90 inhibitors, the hepatotoxicity of these GA-derivatives restricts their therapeutic benefit. We have prepared WK-88 series of GA-derivatives, which lack the benzoquinone moiety. In this study, we have examined the anticancer effects of WK88-1 in Met-amplified- and gefitinib-resistant (HCC827GR) NSCLC cells and its parental HCC827 cells. Treatment with WK88-1 reduced the cell viability in both HCC827 and HCC827GR cells, which was associated with marked decrease in the constitutive expression of Hsp90 client proteins, such as EGFR, ErbB2, ErbB3, Met and Akt. Moreover, WK88-1 attenuated phosphorylation of these Hsp90 client proteins and reduced the anchorage-independent growth of HCC827GR cells. Administration of WK88-1 did not cause hepatotoxicity in animals and significantly reduced the growth of HCC827GR cells xenograft tumors in nude mice. Our study provides evidence that ErbB3 might be a client for Hsp90 in Met-amplified NSCLCs. In conclusion, we demonstrate that inhibition of Hsp90 dampens the activation of EGFR- or c-Met-mediated survival of Met-amplified NSCLCs and that WK88-1 as a Hsp90 inhibitor alleviates gefitinib resistance in HCC827GR cells.

Ganetespib, a Novel Hsp90 Inhibitor in Patients With KRAS Mutated and Wild Type, Refractory Metastatic Colorectal Cancer

BackgroundHeat shock protein 90 (Hsp90) is a cellular chaperone that is required for the maturation and stability of a variety of proteins that play key roles in colon cancer initiation and progression. The primary objective of the current study was to define the safety and efficacy of ganetespib, a novel, selective small-molecule Hsp90 inhibitor, in patients with refractory metastatic colorectal cancer. Patients and MethodsThe study was a single-arm, Simon 2-stage, phase II trial for patients with chemotherapy-refractory, metastatic colorectal cancer. Patients received ganetespib 200 mg/m2 intravenously. Tumor tissue was collected before treatment and 48 hours after treatment for changes in expression of Hsp90 client proteins and other potential pharmacodynamics markers. V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS), v-Raf murine sarcoma viral oncogene homolog B, and phosphatidylinositol-4, 5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) mutational status was also determined. ResultsSeventeen patients were treated (median age, 58; range, 44-79 years). No patients demonstrated objective regression of disease. Two patients had stable disease of 6.8 and 5.1 months duration. Serious adverse events that were potentially attributable to ganetespib included diarrhea (12%, n = 2), fatigue (17%, n = 3), and increased aspartate aminotransferase/alanine aminotransferase (12%, n = 2) and alkaline phosphatase (6%, n = 1) levels. Of the 17 evaluable patients, 9 (53%) including patients with stable disease as best response, had KRAS-mutant tumors. ConclusionIn this first phase II investigation of an Hsp90 inhibitor in colorectal cancer, ganetespib as a single agent did not demonstrate activity in chemotherapy-refractory metastatic colorectal cancer. However, on the basis of the drug's promising preclinical combination data and the relatively mild toxicity profile, further clinical investigation of this agent in combination with standard cytotoxic agents is planned.

Profiling Hsp90 differential expression and the molecular effects of the Hsp90 inhibitor IPI-504 in high-grade glioma models.

Retaspimycin hydrochloride (IPI-504), an Hsp90 (heat shock protein 90) inhibitor, has shown activity in multiple preclinical cancer models, such as lung, breast and ovarian cancers. However, its biological effects in gliomas and normal brain derived cellular populations remain unknown. In this study, we profiled the expression pattern of Hsp90α/β mRNA in stable glioma cell lines, multiple glioma-derived primary cultures and human neural stem/progenitor cells. The effects of IPI-504 on cell proliferation, apoptosis, motility and expression of Hsp90 client proteins were evaluated in glioma cell lines. In vivo activity of IPI-504 was investigated in subcutaneous glioma xenografts. Our results showed Hsp90α and Hsp90β expression levels to be patient-specific, higher in high-grade glioma-derived primary cells than in low-grade glioma-derived primary cells, and strongly correlated with CD133 expression and differentiation status of cells. Hsp90 inhibition by IPI-504 induced apoptosis, blocked migration and invasion, and significantly decreased epidermal growth factor receptor levels, mitogen-activated protein kinase and/or Akt activities, and secretion of vascular endothelial growth factor in glioma cell lines. In vivo study showed that IPI-504 could mildly attenuate tumor growth in immunocompromised mice. These findings suggest that targeting Hsp90 by IPI-504 has the potential to become an active therapeutic strategy in gliomas in a selective group of patients, but further research into combination therapies is still needed.

Synthesis of Butein Analogues and their Anti-proliferative Activity Against Gefitinib-resistant Non-small Cell Lung Cancer (NSCLC) through Hsp90 Inhibition

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer representing 85% of lung cancer patients. Despite several EGFR-targeted drugs have been developed in the treatment of NSCLC, the clinical efficacy of these EGFR-targeted therapies is being challenged by the occurrence of drug resistance. In this regard, Hsp90 represents great promise as a therapeutic target of cancerous diseases due to its role in modulating and stabilizing numerous oncogenic proteins. Accordingly, inhibition of single Hsp90 protein simultaneously disables multiple signaling networks so as to overcome drug resistance in cancer. In this study, we synthesized a series of 11 butein analogues and evaluated their biological activities against gefitinibresistant NSCLC cells (H1975). Our study indicated that analogue 1h inhibited the proliferation of H1975 cells, down-regulated the expression of Hsp90 client proteins, including EGFR, Met, Her2, Akt and Cdk4, and upregulated the expression of Hsp70. The result suggested that compound 1h disrupted Hsp90 chaperoning function and could serve a potential lead compound to overcome the drug resistance in cancer chemotherapy.

Synthesis of Flavokawain Analogues and their Anti-neoplastic Effects on Drug-resistant Cancer Cells Through Hsp90 Inhibition

Hsp90 is an ubiquitous molecular chaperone protein, which plays an important role in regulating maturation and stabilization of many oncogenic proteins. Due to its potential to simultaneously disable multiple signaling pathways, Hsp90 represents great promise as a therapeutic target of cancer. In this study, we synthesized flavokawain analogues and evaluated their biological activities against drug-resistant cancer cells. The study indicated that compound 1i impaired the growth of gefitinib-resistant non-small cell lung cancer (H1975), down-regulated the expression of Hsp90 client proteins including EGFR, Her2, Met, Akt and Cdk4, and upregulated the expression of Hsp70. The result strongly suggested that compound 1i inhibited the proliferation of cancer cells through Hsp90 inhibition. Overall, compound 1i could serve as a potential lead compound to overcome the drug resistance in cancer chemotherapy.

HSP27 Expression Levels are Associated with the Sensitivity of Hepatocellular Carcinoma Cells to 17-Allylamino-17-Demethoxygeldanamycin

AIMS, MATERIALS & METHODS: As heat-shock proteins are associated with tumor proliferation, differentiation, invasion and metastasis, we investigated whether targeting Hsp90 with the geldanamycin derivative 17-allylamino-17-demethoxygeldanamycin (17AAG) can inhibit the viability of hepatocellular carcinoma cell lines with various levels of metastatic potential. In addition, we investigated whether the use of Hsp27-siRNA can decrease resistance to 17AAG. Although 17AAG upregulated the expression of heat-shock proteins, it did not affect the expression of Hsp90 client proteins in normal hepatocytes. Hsp90 inhibition by 17AAG degraded its client proteins in both low- and high-metastatic potential cell lines. siRNA inhibited Hsp27 expression in cell lines and improved the sensitivity of 17AAG. 17AAG inhibited the viability of hepatocellular carcinoma cells by degrading Hsp90 client proteins. The sensitivity of cells to 17AAG is associated with the level of Hsp27 expression.

Radiosensitizing effect of the novel Hsp90 inhibitor NVP-AUY922 in human tumour cell lines silenced for Hsp90α

Hsp90 inhibitors can enhance the tumour sensitivity to ionising radiation (IR). However, Hsp90 inhibition leads to the up-regulation of anti-apoptotic Hsp90 and Hsp70, which might diminish the radiosensitizing effects of the inhibitors. Therefore, inhibition of the up-regulation of Hsp90 by siRNA might be a promising strategy to enhance drug-mediated radiosensitization. The expression of Hsp90α was silenced in A549 and GaMG tumour cell lines by siRNA treatment. Pre-silenced for Hsp90α cells were treated with NVP-AUY922, a novel Hsp90 inhibitor, for 24 h and then irradiated. Radiation response was determined by colony-forming ability. The expression of several marker proteins was analysed by Western blot. DNA damage and repair were assessed by histone γH2AX measurements. We found that transfection with siRNA against Hsp90α reduced Hsp90α at mRNA and protein levels. Pre-silencing of Hsp90α reduced NVP-AUY922-mediated up-regulation of Hsp90α but it did not increase drug-mediated radiosensitization in both tumour cell lines. As revealed by Western blot, pre-silencing of Hsp90α followed by NVP-AUY922 did not change the expression of Hsp90 client proteins (Akt, Raf-1, Cdk1 and Cdk4) compared with drug treatment alone, suggesting unchanged chaperone function in transfected cells. Pre-silencing of Hsp90α followed by Hsp90 inhibition did not enhance the radiosensitizing effect of NVP-AUY922 in both tested tumour cell lines. Future work will be done on stable transfection with shRNA against Hsp90α or simultaneous silencing of both Hsp90 isoforms, Hsp90α and Hsp90β, in order to optimize tumour cell killing.

1143 INTRAVESICAL ADMINISTRATION OF HEAT SHOCK PROTEIN 90-SURVIVIN COMPLEX INHIBITOR SUPPRESSES THE GROWTH OF NON-INVASIVE BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Basic Research III1 Apr 20101143 INTRAVESICAL ADMINISTRATION OF HEAT SHOCK PROTEIN 90-SURVIVIN COMPLEX INHIBITOR SUPPRESSES THE GROWTH OF NON-INVASIVE BLADDER CANCER Akihiro Yano, Yasuhisa Fujii, Hitoshi Masuda, Satoru Kawakami, and Kazunori Kihara Akihiro YanoAkihiro Yano More articles by this author , Yasuhisa FujiiYasuhisa Fujii More articles by this author , Hitoshi MasudaHitoshi Masuda More articles by this author , Satoru KawakamiSatoru Kawakami More articles by this author , and Kazunori KiharaKazunori Kihara More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.642AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Heat shock protein (Hsp) 90 is a molecular chaperone that is involved in signaling pathways for cell proliferation and/or survival of cancer cells. Survivin, one of the Hsp90 client proteins, plays important roles in regulation of cellular survival. Survivin is selectively expressed in most solid cancers and is rarely detected in normal differentiated tissues. Recently, shepherdin, a novel anticancer peptidomimetic modeled on the Hsp90-survivin binding interface was generated and shown to destabilize survivin plus additional Hsp90 client proteins, causing massive killing of cancer cells. In the present study, we investigated whether shepherdin can be a cancer-specific molecular targeting agent for non-invasive bladder cancer by intravesical administration using an orthotopic xenograft model. METHODS The effects of shepherdin on bladder cancer cell proliferation and client protein expression were examined by MTS assay and Western blotting, respectively. The effects of shepherdin by intravesical administration on the growth of UM-UC-3 orthotopic xenograft tumors were determined by IVIS real-time in vivo imaging system using UM-UC-3-luciferase cells. RESULTS Treatment of the bladder cancer cell lines with shepherdin resulted in dose-dependent cell killing as compared with untreated or control scrambled peptide-treated cells. UM-UC-3 cells treated with shepherdin exhibited loss of expression of Hsp90 client proteins, including survivin and Akt, and increase in the level of cytochrome c in the cytosol. The scrambled peptide did not modulate client protein expression. In vivo, intravesical administration of shepherdin abolished the growth of UM-UC-3 orthotopic xenograft tumors (P < 0.01). Shepherdin treatment attenuated expression of survivin and Akt compared with the control group, and did not show any toxicity to normal bladder urothelial cells in the histological specimens. CONCLUSIONS This study is the first to show that intravesical administration of Hsp90-, especially survivin-targeting agent can be a potent and promising therapy for non-invasive bladder cancer minimizing damage to normal urothelial cells. Optimal cancer therapy is a fine balance between effective cancer cell killing and at the same time avoiding damage to the surrounding normal tissue. Intravesical administration of molecular targeting agent, which minimizes toxicity to healthy tissues, such as shepherdin may be the treatment of choice for patients with non-invasive bladder cancer in clinical setting. Tokyo, Japan© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e442 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Akihiro Yano More articles by this author Yasuhisa Fujii More articles by this author Hitoshi Masuda More articles by this author Satoru Kawakami More articles by this author Kazunori Kihara More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Structural basis for depletion of heat shock protein 90 client proteins by deguelin.

The molecular chaperone heat shock protein 90 (Hsp90) participates in preserving the expression and activity of various oncoproteins, including hypoxia-inducible factor 1alpha (HIF-1alpha) and Akt. Deguelin is a rotenoid with antitumor activities. We investigated whether the antitumor activities of deguelin involve the functional inhibition of Hsp90. Human xenograft tumors were generated in mice from H1299 (n = 6 per group) and A549 (n = 4 per group) non-small-cell lung cancer cells, UMSCC38 (n = 5 per group) head and neck cancer cells, MKN45 (n = 5 per group) stomach cancer cells, and PC-3 (n = 3 per group) prostate cancer cells. Tumor-bearing mice were treated with deguelin at 4 or 8 mg/kg or with vehicle (as a control) twice a day by oral gavage for 15-28 days. Protein expression was assessed by western blot analysis. Akt and Hsp90 were assessed by use of adenoviral vectors expressing constitutively active Akt or Hsp90. Binding of deguelin to Hsp90 was examined by docking analysis and by competition binding experiments with ATP-Sepharose beads. The proteasome inhibitor MG132 was used to investigate deguelin's effect on the induction of ubiquitin-mediated proteasomal degradation of HIF-1alpha. All statistical tests were two-sided. Deguelin bound to the ATP-binding pocket of Hsp90 and disrupted Hsp90 function, leading to ubiquitin-mediated degradation of HIF-1alpha. Administration of deguelin to xenograft-bearing mice statistically significantly decreased tumor growth by inducing apoptosis and decreasing the expression of Hsp90 client proteins, without detectable toxic effects. For example, at 15 days after the start of deguelin treatment, the volume of untreated control H1299 xenograft tumors was 798 mm3 and that of xenograft tumors treated with deguelin at 4 mg/kg was 115.9 mm3 (difference = 682.1 mm3, 95% confidence interval = 480.4 to 883.9 mm3; P<.001). The antitumor activities of deguelin appear to involve its binding to the ATP-binding pocket of Hsp90, which suppresses Hsp90 function.

Antileukemic Activity of Shepherdin and Molecular Diversity of Hsp90 Inhibitors

Heat shock protein 90 (Hsp90) is a molecular chaperone that is involved in signaling pathways for cell proliferation, survival, and cellular adaptation. Inhibitors of Hsp90 are being examined as cancer therapeutic agents, but the molecular mechanism of their anticancer activity is still unclear. We investigated Hsp90 as a therapeutic target for acute myeloid leukemia (AML) by use of the Hsp90 inhibitor shepherdin (a novel peptidyl antagonist of the interaction between Hsp90 and survivin, which is a regulator of cell proliferation and cell viability in cancer). We studied protein interactions by molecular dynamics simulations and conducted competition experiments by use of enzyme-linked immunosorbent assay (ELISA). Shepherdin[79-83], a novel variant carrying the survivin sequence from Lys-79 through Gly-83, or its scrambled peptide was made permeable to cells by adding the antennapedia helix III carrier sequence. Apoptosis, Hsp90 client protein expression, and mitochondrial dysfunction were evaluated in AML types (myeloblastic, monocytic, and chronic myelogenous leukemia in blast crisis), patient-derived blasts, and normal mononuclear cells. Effects of shepherdin on tumor growth were evaluated in AML xenograft tumors in mice (n = 6). Organ tissues were examined histologically. Shepherdin[79-83] bound to Hsp90, inhibited formation of the survivin-Hsp90 complex, and competed with ATP binding to Hsp90. Cell-permeable shepherdin[79-83] induced rapid (within 30 minutes) and complete (with concentrations inducing 50% cell death of 24-35 microM) killing of AML types and blasts, but it did not affect normal mononuclear cells. Shepherdin[79-83] made contact with unique residues in the ATP pocket of Hsp90 (Ile-96, Asp-102, and Phe-138), did not increase Hsp70 levels in AML cells, disrupted mitochondrial function within 2 minutes of treatment, and eliminated the expression of Hsp90 client proteins. Shepherdin[79-83] abolished growth of AML xenograft tumors (mean of control group = 1698 mm3 and mean of treated group = 232 mm3; difference = 1466 mm3, 95% confidence interval = 505.8 to 2426; P = .008) without systemic or organ toxicity and inhibited Hsp90 function in vivo. Shepherdin is a novel Hsp90 inhibitor with a unique mechanism of anticancer activity.