Degradation Of Hsp90 Client Proteins Research Articles

EP4 receptor agonist CAY10598 upregulates ROS-dependent Hsp90 cleavage in colorectal cancer cells

Prostaglandin E2 (PGE2) interacts with four specific G protein-coupled receptors, namely EP1, EP2, EP3, and EP4, playing a pivotal role in determining the fate of cells. Our previous findings highlighted that stimulating the EP4 receptor with its agonist, CAY10598, triggers apoptosis in colon cancer HCT116 cells via the production of reactive oxygen species (ROS). This process also reduces the phosphorylation of the oncogenic protein JAK2 and leads to its degradation in these cells. In this study, our goal was to explore the pathways through which CAY10598 leads to JAK2 degradation. We focused on Hsp90, a heat shock protein family member known for its role as a molecular chaperone maintaining the stability of several key proteins including EGFR, MET, Akt, and JAK2. Our results show that CAY10598 decreases the levels of client proteins of Hsp90 in HCT116 cells, an effect reversible by pretreatment with the ROS scavenger N-acetyl cysteine (NAC) or the proteasome inhibitor MG132, indicating that the degradation is likely driven by ROS. Furthermore, we observed that CAY10598 cleaves both α and β isoforms of Hsp90, the process inhibited by NAC. Inhibition of EP4 with the antagonist GW627368x not only prevented the degradation of Hsp90 client proteins but also the cleavage of Hsp90 itself in CAY10598-treated HCT116 cells. Additionally, CAY10598 suppressed the growth of HCT116 cells implanted in mice. Our findings reveal that CAY10598 induces apoptosis in cancer cells by a novel mechanism involving the ROS-dependent cleavage of Hsp90, thereby inhibiting the function of crucial Hsp90 client proteins.

Anti-Cancer Properties of Ginkgolic Acids in Human Nasopharyngeal Carcinoma CNE-2Z Cells via Inhibition of Heat Shock Protein 90.

Ginkgo biloba L. has been used in traditional Chinese medicine (TCM) for thousands of years. However, the anti-cancer properties of ginkgolic acids (GAS) isolated from G. biloba have not been investigated in human nasopharyngeal carcinoma cells. In this study, GAS exhibited an inhibitory effect on the ATPase activity of heat shock protein 90 (Hsp90) and anti-proliferative activities against four human cancer cell lines, with IC50 values ranging from 14.91 to 23.81 μg·mL−1. In vivo experiments confirmed that GAS inhibited tumor growth in CNE-2Z cell-xenografted nude mice with low hepatotoxicity. We further demonstrated that GAS suppressed migration and invasion and induced the apoptosis of CNE-2Z cells by inducing the degradation of Hsp90 client proteins (MMP-2, MMP-9, Her-2, c-Raf, Akt, and Bcl-2). Together, GAS are new Hsp90 inhibitors by binding to Hsp90 (hydrogen bond and hydrophobic interaction). Thus, GAS from G. biloba might represent promising Hsp90 inhibitors for the development of anti-nasopharyngeal carcinoma agents.

Structure-Activity Relationships of Benzothiazole-Based Hsp90 C-Terminal-Domain Inhibitors.

Heat shock protein 90 (Hsp90) is a chaperone responsible for the maturation of many cancer-related proteins, and is therefore an important target for the design of new anticancer agents. Several Hsp90 N-terminal domain inhibitors have been evaluated in clinical trials, but none have been approved as cancer therapies. This is partly due to induction of the heat shock response, which can be avoided using Hsp90 C-terminal-domain (CTD) inhibition. Several structural features have been shown to be useful in the design of Hsp90 CTD inhibitors, including an aromatic ring, a cationic center and the benzothiazole moiety. This study established a previously unknown link between these structural motifs. Using ligand-based design methodologies and structure-based pharmacophore models, a library of 29 benzothiazole-based Hsp90 CTD inhibitors was prepared, and their antiproliferative activities were evaluated in MCF-7 breast cancer cells. Several showed low-micromolar IC50, with the most potent being compounds 5g and 9i (IC50, 2.8 ± 0.1, 3.9 ± 0.1 μM, respectively). Based on these results, a ligand-based structure–activity relationship model was built, and molecular dynamics simulation was performed to elaborate the binding mode of compound 9i. Moreover, compound 9i showed degradation of Hsp90 client proteins and no induction of the heat shock response.

CDC37 As a Novel Target for the Treatment of NPM1-ALK Expressing Anaplastic Large Cell Lymphomas

Anaplastic large cell lymphoma (ALCL) is an aggressive CD30+ T-cell lymphoma that accounts for 2-8% and 10-15% of non-Hodgkin lymphomas in adults and children, respectively. The currently used standard therapy for anaplastic lymphoma kinase (ALK, a member of the insulin receptor superfamily) positive ALCL has limited effectiveness, resulting in a substantial percentage of cases with poor outcomes, either failing to enter remission or relapse within a few months after starting treatment. Thus, there is a clear unmet clinical need for developing novel, effective and safer therapeutic strategies for ALCL. Nucleophosmin 1 (NPM1) is a nucleolar phosphoprotein, which functions as a molecular chaperone for proteins and nucleic acids. Approximately 50% of ALCL cases are positive for the NPM1-ALK fusion chimera generated by the t(2;5) chromosomal translocation. The oligomerization domain of NPM1 in the fusion protein NPM1-ALK mediates the ligand-independent dimerization of chimeric protein, which results in constitutive activation of the chimeric tyrosine kinase activity leading to downstream signaling pathways responsible for the oncogenicity. As NPM1-ALK is an established heat shock protein 90 (HSP90) client protein, we hypothesized that disruption of the interaction between master chaperone HSP90 and co-chaperone Cdc37 would be a viable druggable target. Celastrol is a natural compound extracted from the root bark of Chinese medicinal plant Tripterygium wilfordi. Anticancer properties of celastrol are attributed to its action on the interaction of HSP90 and its co-chaperone Cdc37. Hsp90 is an ATP-dependent master molecular chaperone. Cdc37 is a Hsp90 co-chaperone and disruption of Hsp90-Cdc37 interaction will result in the degradation of Hsp90 client proteins. Celastrol disrupts the interaction of Hsp90 and Cdc37 through inhibition of Hsp90 ATPase activity without blockage of ATP binding site as observed with Hsp90 inhibitors. We used NPM1-ALK endogenously expressing Karpas299, SUDHL-1 and ectopically expressing Ba/F3-FG-NPM1-ALK cell lines in our study. Treatment with celastrol (0.1-1 µM) significantly induced apoptosis dose dependently in ALCL cells. Apoptosis was associated with activated caspase 3 and poly (ADP-ribose) polymerase cleavage. We also observed upregulation of proapoptotic protein BAD and downregulation of antiapoptotic proteins survivin and MCL1. Celastrol treatment inhibited NPM1-ALK mediated signaling and downstream signaling effectors STAT3, AKT, and ERK1/2 by degradation of the NPM1-ALK fusion protein. Cell survival was evaluated by clonogenic survival assay and results were clearly indicative that the clonogenic survival is diminished in celastrol treated samples compared to controls. Our immunoprecipitation studies clearly demonstrated that celastrol treatment disrupted the interaction between Hsp90 and Cdc37 and induced degradation of the NPM1-ALK fusion protein. Altogether, targeting Cdc37 using celastrol is a novel therapeutic approach to induce apoptosis in ALCL cells expressing NPM1-ALK and warrants developing future therapeutic intervention strategies. DisclosuresGanguly:Amgen: Other: Advisory Board; Seattle Genetics: Speakers Bureau.

C0818, a novel curcumin derivative, induces ROS-dependent cytotoxicity in human hepatocellular carcinoma cells in vitro via disruption of Hsp90 function.

Heat shock protein 90 (Hsp90) is the most common molecular chaperone that controls the maturation of many oncoproteins critical in tumor development. Hsp90 has been considered as a promising target for cancer treatment, but the clinical significance of Hsp90 and the mechanisms of Hsp90 regulating the tumor-promoting effects in hepatocellular carcinoma (HCC) remain obscure. Previous studies have shown that curcumin, a polyphenol derived from the plant turmeric (Curcuma longa), inhibits tumor growth, which may provide an effective alternative therapy for HCC. Compared to curcumin, a novel derivative of curcumin, 3,5-(E)-Bis(3-methoxy-4-hydroxybenzal)-4-piperidinone hydrochloride (C0818) that is more potent in Hsp90 inhibition and antitumor activity. In this study, we investigated the effect of C0818 on HCC cells in vitro and its relation to Hsp90 inhibition. We showed that C0818 concentration-dependently inhibited the proliferation, the colony formation and induced apoptosis in HepG2 and Sk-Hep-1 cells. C0818 concentration-dependently inhibited DNA synthesis and induced G2/M phase arrest in HepG2 and Sk-Hep-1 cells. We further demonstrated that C0818induced ROS- and caspase-dependent apoptosis in HCC cells through the mitochondrial-mediated pathway. C0818 induced the degradation of Hsp90 client proteins as RAS, C-Raf, P-C-Raf, Erk, P-ERK, MEK, P-MEK, Akt and P-Akt, which led to subsequent inhibition of the RAS/RAF/MEK/ERK and PI3K/AKT pathways. We revealed that C0818 could inhibit the binding of Hsp90 with its clients without affecting their transcription, which subsequently induced the degradation of Hsp90 clients by the proteasome rather than the lysosome. These results are of potential importance for elucidating a novel Hsp90 inhibitor targeting HCC.

Gephyromycin C, a novel small-molecule inhibitor of heat shock protein Hsp90, induces G2/M cell cycle arrest and apoptosis in PC3 cells in vitro

Gephyromycin C (GC), a natural compound isolated from a marine-derived actinomycete Streptomyces sp. SS13I, which exerts anti-proliferative effect on PC3 cells. However, its underlying mechanism of the anti-cancer effect remains unknown. The results of SRB assays showed that GC inhibited the proliferation of PC3 cells with an IC50 value of 1.79 ± 0.28 μM. GC also induced G2/M cell cycle arrest which was accompanied by declining levels of cyclin proteins. Possible mechanisms were investigated and it was found that GC bound to Hsp90 and caused the degradation of Hsp90 client proteins (AKT, CHK1, P53, CDK4, Raf-b, and Raf-1). The fluorescent polarization assay with FITC-labeled geldanamycin (FITC-GA) demonstrated that GC was able to compete with FITC-GA in binding to wild type Hsp90 with an IC50 of 2.15 μM. Results of a docking study also suggested that GC interacted with the N-terminal domain of Hsp90. Our results showed that GC could bind to Hsp90, which resulted in down-regulation of Hsp90 client proteins and G2/M arrest in PC3 cells. Since the antitumor effects of this kind of angucycline via targeting Hsp90 has not been reported before, our results indicate that GC is a novel inhibitor of Hsp90 from marine resources and worthy of further study.

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.

DCZ5248, a novel dual inhibitor of Hsp90 and autophagy, exerts antitumor activity against colon cancer.

Hsp90 is a potential therapeutic target for tumor, as it maintains the stability of a variety of proteins related to tumor development and progression. Autophagy is a self-degradation process to maintain cellular homeostasis and autophagy inhibitors can suppress tumor growth. In this study, we identified DCZ5248, a triazine derivative, was a dual inhibitor of both Hsp90 and late-autophagy with potent antitumor activity against colon cancer cells in vitro and in vivo. We showed that DCZ5248 (0.1-10 μM) induced dose-dependent degradation of Hsp90 client proteins (AKT, CDK4, CDK6 and RAF-1) in HCT 116 colon cancer cells through a proteasome-dependent pathway. Meanwhile, DCZ5248 (0.3 μM) induced cytoplasmic vacuole formation, LC3 II conversion, p62 protein upregulation, and inhibited autophagy at the late stage in the colon cancer cell lines tested. We further revealed that the inhibition of autophagy was achieved by impairing lysosomal functions through induction of lysosomal acidification and attenuation of lysosomal cathepsin activity. The modulation of autophagy by DCZ5248 was independent of Hsp90 inhibition as the autophagy inhibition was not blocked by Hsp90 knockdown. Importantly, inhibition of both Hsp90 function and autophagy by DCZ5248 induced G1-phase cell cycle arrest, apoptosis, and exerted potent antitumor activity against colon cancer cells both in vitro and in vivo. These findings demonstrate that DCZ5248 is a novel dual inhibitor of Hsp90 and autophagy with potential for colon cancer therapy.

Amide-tethered quinoline-resorcinol conjugates as a new class of HSP90 inhibitors suppressing the growth of prostate cancer cells

The study is focused on the design and synthesis of amide tethered quinoline-resorcinol hybrid constructs as a new class of HSP90 inhibitor. In-vitro studies of the synthetic compounds led to the identification of compound 11, which possesses potent cell growth inhibitory effects against HCT116, Hep3B and PC-3 cell lines, exerted through HSP90 inhibition. Compound 11 triggers degradation of HSP90 client proteins along with concomitant induction of HSP70, demonstrates apoptosis inducing ability and causes G2M phase cell cycle arrest in PC-3 cells. Molecular modeling was used to dock compound 11 into the HSP90 active site and key interactions with the amino acid residues of the HSP90 chaperone protein were determined.

Class 1 HDAC and HDAC6 inhibition inversely regulates CD38 induction in myeloma cells via interferon-α and ATRA.

Class 1 HDAC and HDAC6 inhibition inversely regulates CD38 induction in myeloma cells via interferon-α and ATRA.

C1206, a novel curcumin derivative, potently inhibits Hsp90 and human chronic myeloid leukemia cells in vitro.

4-(4-Pyridinyl methylene) curcumin (C1206) is a new derivative of curcumin that is more active than curcumin in inhibition of heat shock protein 90 (Hsp90) and antitumor action. In this study we investigated the relationship between C1206-induced inhibition of Hsp90 and its anti-leukemic effects. The fluorescence quenching experiments showed that C1206 seemed to bind the middle dimerization domain of Hsp90. The interaction between C1206 and Hsp90 was driven mainly by electrostatic interaction. In in vitro enzyme activity assay, C1206 dose-dependently inhibited Hsp90 ATPase activity with an IC50 value of 4.17 μmol/L. In both imatinib-sensitive K562 chronic myeloid leukemia cells and imatinib-resistant K562/G01 chronic myeloid leukemia cells, C1206 (0.4-3.2 μmol/L) dose-dependently caused the degradation of Hsp90 client proteins and downstream proteins (AKT, MEK, ERK, C-RAF, P-AKT, P-MEK and P-ERK). Furthermore, C1206 (0.4-3.2 μmol/L) dose-dependently induced apoptosis of K562 and K562/G01 cells through triggering mitochondrial pathway. Consistent with this result, C1206 inhibited the proliferation of K562 and K562/G01 cells with IC50 values of 1.10 and 0.60 μmol/L, respectively. These results suggest that C1206 is a novel Hsp90 inhibitor and a promising therapeutic agent for chronic myeloid leukemia.

Liposomal honokiol induced lysosomal degradation of Hsp90 client proteins and protective autophagy in both gefitinib-sensitive and gefitinib-resistant NSCLC cells.

Honokiol (HK), a natural chemical isolated from Mangnolia officinalis, has shown antitumorigenic activities when used to treat a variety of tumor cell lines. The mechanism of honokiol activity when used to treat gefitinib-sensitive and gefitinib-resistant non-small cell lung cancer (NSCLC) requires elucidation. Here, the presence of liposomal honokiol (LHK) induced apoptotic and antitumor activities in four xenograft models generated using NSCLC cell lines such as HCC827 (gefitinib-sensitive) and H1975 (gefitinib-resistant). Mechanistic studies revealed that LHK inhibited the Akt and Erk1/2, both EGFR signaling cascades effectors, by promoting degradation of HSP90 client proteins (HCP), including wild-type or mutant EGFR, Akt and C-Raf. Molecular biology assays showed that LHK induced HCP degradation through a lysosomal pathway, rather than the canonical proteasome protein degradation pathway. As a result of misfolded protein accumulation, LHK induced endoplasmic reticulum (ER) stress and autophagy. Inhibition of ER stress (with 4-phenylbutyrate) or autophagy (with small interfering RNA) reduced LHK-induced HCP degradations. Additionally, LHK induced autophagy showed a protective role for cancer cell as inhibition of autophagy invitro and invivo by autophagosome degradation inhibitors could promote the anticancer activity of LHK. LHK has been approved by the China Food and Drug Administration for first-in-human clinical trials in NSCLC. The current study will guide the design of future LHK clinical trials.

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

Abstract 2022: Hsp90 inhibitor (NVP-AUY922) enhances anti-cancer effect of Bcl-2 inhibitor (ABT-737) in small cell lung cancer

Abstract Background: Bcl-2 family is a group of apoptosis regulators that plays an anti-apoptotic role for cell survival and is known to contribute to induce chemotherapy resistance in small cell lung cancer (SCLC). ABT-737 is an anti-cancer drug that induces apoptosis by selectively blocking the activities of Bcl-2 and Bcl-xL, but not Mcl-1. Consequently, the efficacy of ABT-737 is largely restricted in the presence of Mcl-1. Heat-shock-protein 90 (Hsp90) is highly expressed in most tumors and Hsp90 inhibitors induce the proteasomal degradation of Hsp90 client proteins. In addition, Hsp90 inhibitor can reduce Mcl-1 expression, a down-stream of Akt and Erk pathway. Furthermore, Hsp90 inhibitor inhibits activities of NF-κB by degradation of IKK. Thus, we show that downregulation of Mcl-1 and NF-κB by Hsp90 inhibitor can lead to synergistic pro-apoptotic effects with ABT-737. Materials and methods: The proliferative activity, apoptotic activities, and expression of apoptotic proteins were assessed in SCLC cell lines after treatment with ABT-737, NVP-AUY922, or both drugs. The synergy effects of ABT-737 and NVP-AUY922 were analyzed by cell viabilities with different concentrations in SCLC cell lines, and the combination index values were < 1. In addition, the synergy effects of the drugs were showed with xenograft model with human SLCL cell line in vivo. Results: Here, we show that NVP-AUY922, an Hsp90 inhibitor, can potentiate the pro-apoptotic effects of ABT-737 not only by reducing the levels of Akt but also by inhibiting pNF-κB, both of which are proteins regulating apoptosis signaling. Western blot analysis revealed that proteins associated with apoptosis such as PARP, Caspase 3 and 7 were more upregulated in SCLC cells exposed to the drug combination than in cells exposed to NVP-AUY922 or ABT-737 alone. In addition, Annexin V & dead cell assay showed dual inhibition of the Hsp90 and Bcl-2 signaling pathways more increased apoptotic cells and dead cells in SCLC. In our result, ABT-737 induced apoptosis by blocking Bcl-2 activation, and NVP-AUY922 blocked the levels of Hsp90 client proteins, Akt and pErk, ultimately leading to decreased level of Mcl-1. In addition, NVP-AUY922 induced degradation of IKK, and increased IκB-α inhibited activation of NF-κB. And this combination treatment showed higher BIM and BID expression, pro-apoptotic proteins, than single treatment. Furthermore, synergy effects of combination were verified in xenograft model with human SCLC cell line. Conclusions: Consequently, NVP-AUY922 synergizes with ABT-737 to induce apoptosis by reducing activities of Mcl-1 and NF-κB in SCLC. This study suggests that adopting an appropriate combination of drugs can lead to better outcomes compared with monotherapy in SCLC. Citation Format: Hannah Yang, Kang-Seo Park, Junyoung Choi, Sang-We Kim Kim, Dae Ho Lee. Hsp90 inhibitor (NVP-AUY922) enhances anti-cancer effect of Bcl-2 inhibitor (ABT-737) in small cell lung cancer [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 2022. doi:10.1158/1538-7445.AM2017-2022

Non-benzoquinone geldanamycin analogs trigger various forms of death in human breast cancer cells.

BackgroundHsp90 proteins are important therapeutic targets for many anti-cancer drugs in clinical trials. Geldanamycin (GA) was identified as the first natural inhibitor of Hsp90, increasing evidence suggests that GA was not a good choice for clinical trials. In this study, we investigated two new non-benzoquinone geldanamycin analogs of Hsp90 inhibitors, DHQ3 and 17-demethoxy-reblastatin (17-DR), to explore the molecular mechanisms of their anti-cancer activity in vivo and vitro.MethodsMTT and colony formation assays were used to measure cell viability. Flow cytometry, DAPI staining, ATP assay, electron microscopy, western blots, siRNAs transfection and immunofluorescence were used to determine the molecular mechanism of DHQ3- or 17-DR-induced different forms of death in human breast cancer MDA-MB-231 cells. Malachite green reagent was used to measure ATPase activity of the analogs.ResultsDHQ3 and 17-DR presented efficiently inhibitory effect in MDA-MB-231 cell lines, and DHQ3 induced necroptosis by activation of the RIP1-RIP3-MLKL necroptosis cascade. And DHQ3-induced cell death was inhibited by a necroptosis inhibitor, necrostatin-1 (Nec-1), but not by a caspase inhibitor z-VAD-fmk. On the other hand, 17-DR induced apoptosis in MDA-MB-231 cells, indicating a caspase-dependent killing mechanism. We further demonstrated that down-regulation of RIP1 and RIP3 by siRNA protected against DHQ3 but not 17-DR induced cell death. These results were confirmed by electron microscopy. DHQ3 and 17-DR induced the degradation of Hsp90 client proteins, and they showed strong antitumor effects in MDA-MB-231 cell-xenografted nude mice.ConclusionsThese findings supported that DHQ3 and 17-DR induce different forms of death in some cancer cell line via activation of different pathways. All of the results provided evidence for its anti-tumorigentic action with low hepatotoxicity in vivo, making them promising anti-breast cancer agents.

Structure–activity relationship of Garcinia xanthones analogues: Potent Hsp90 inhibitors with cytotoxicity and antiangiogenesis activity

Hsp90 has long been recognized as an attractive and crucial molecular target for cancer therapy. Gambogic acid (GA), the main active compound of Gamboge hanburyi, has been reported as a natural inhibitor of Hsp90. Here, we present the structure–activity relationship of Garcinia xanthones analogues as Hsp90 inhibitors and identify that compound 25, with a simplified skeleton, had an improved inhibitory effect toward Hsp90. Compound 25 inhibited the ATPase activity of Hsp90 with an IC50 value of 3.68±0.18μM. It also exhibited potent antiproliferative activities in some solid tumor cells. In SK-BR-3 cells with high Hsp90 expression, compound 25 induced the degradation of Hsp90 client proteins including Akt and Erk1/2 without causing the heat shock response. Additionally, compound 25 inhibited angiogenesis in HUVEC cells through Hsp90 regulation of the HIF-1α pathway. These results demonstrate that compound 25 as an Hsp90 inhibitor with a new structure could be further studied for the development of tumor therapy.

Novel 1,6-naphthyridin-2(1H)-ones as potential anticancer agents targeting Hsp90

Hsp90 is an ATP-dependent chaperone known to be overexpressed in many cancers. This way, Hsp90 is an important target for drug discovery. Novobiocin, an aminocoumarin antibiotic, was reported to inhibit Hsp90 targeting C-terminal domain, and showed anti-proliferative properties, leading to the development of new and more active compounds. Consequently, a new set of novobiocin analogs derived from 1,6-naphthyridin-2(1H)-one scaffold was designed, synthesized and evaluated against two breast cancer cell lines. Subsequently, cell cycle progression and apoptosis were conducted on best candidates, finally Western Blot analysis was performed to measure their ability to induce degradation of Hsp90 client proteins.

CPUY201112, a novel synthetic small-molecule compound and inhibitor of heat shock protein Hsp90, induces p53-mediated apoptosis in MCF-7 cells

Heat-shock protein 90 (Hsp90) is highly expressed in many tumor cells and is associated with the maintenance of malignant phenotypes. Targeting Hsp90 has had therapeutic success in both solid and hematological malignancies, which has inspired more studies to identify new Hsp90 inhibitors with improved clinical efficacy. Using a fragment-based approach and subsequent structural optimization guided by medicinal chemistry principles, we identified the novel compound CPUY201112 as a potent Hsp90 inhibitor. It binds to the ATP-binding pocket of Hsp90 with a kinetic dissociation (Kd) constant of 27 ± 2.3 nM. It also exhibits potent in vitro antiproliferative effects in a range of solid tumor cells. In MCF-7 cells with high Hsp90 expression, CPUY201112 induces the degradation of Hsp90 client proteins including HER-2, Akt, and c-RAF. We prove that treating MCF-7 cells with CPUY201112 results in cell cycle arrest and apoptosis through the wild-type (wt) p53 pathway. CPUY201112 also synergizes with Nutlin-3a to induce cancer cell apoptosis. CPUY201112 significantly inhibited the growth of MCF-7 xenografts in nude mice without apparent body weight loss. These results demonstrate that CPUY201112 is a novel Hsp90 inhibitor with potential use in treating wild-type p53 related cancers.

Hsp90 as a Potential Therapeutic Target in Retinal Disease.

The molecular chaperone heat shock protein 90 (Hsp90) is a pivotal cellular regulator involved in the folding, activation and assembly of a wide range of proteins. Hsp90 has multiple roles in the retina and the use of different Hsp90 inhibitors has been shown to prevent retinal degeneration in models of retinitis pigmentosa and age-related macular degeneration. Hsp90 is also a potential target in uveal melanoma. Mechanistically, Hsp90 inhibition can evoke a dual response in the retina; stimulating a stress response with molecular chaperone expression. Thereby leading to an improvement in visual function and photoreceptor survival; however, prolonged inhibition can also stimulate the degradation of Hsp90 client proteins potentially deleteriously affect vision. Here, we review the multiple roles of Hsp90 in the retina and the therapeutic potential of Hsp90 as a target.

HSP90 inhibition as a means of radiosensitizing resistant, aggressive soft tissue sarcomas

Radiotherapy is an essential part of multi-modal treatment for soft tissue sarcomas. Treatment failure is commonly attributed to radioresistance, but comprehensive analyses of radiosensitivity are not available, and suitable biomarkers or candidates for targeted radiosensitization are scarce. Here, we systematically analyzed the intrinsic radioresistance of a panel of soft tissue sarcoma cell lines, and extracted scores of radioresistance by principal component analysis (PCA). To identify molecular markers of radioresistance, transcriptomic profiling of DNA damage response regulators was performed. The expression levels of HSP90 and its clients ATR, ATM, and NBS1 revealed strong, positive correlations with the PCA-derived radioresistance scores. Their functional involvement was addressed by HSP90 inhibition, which preferentially sensitized radioresistant sarcoma cells and was accompanied by delayed γ-H2AX foci clearance and HSP90 client protein degradation. The induction of apoptosis and necrosis was not significantly enhanced, but increased levels of basal and irradiation-induced senescence upon HSP90 inhibition were detected. Finally, evaluation of our findings in the TCGA soft tissue sarcoma cohort revealed elevated expression levels of HSP90, ATR, ATM, and NBS1 in a relevant subset of cases with particularly poor prognosis, which might preferentially benefit from HSP90 inhibition in combination with radiotherapy in the future.