Inhibition of homologous recombination repair in irradiated tumor cells pretreated with Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin

In order to investigate the mechanism of radio-sensitization by an Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG), we studied repair of DNA double strand breaks (DSBs) in irradiated human cells pre-treated with 17-AAG. DSBs are thought to be the critical target for radiation-induced cell death. Two human tumor cell lines DU145 and SQ-5 which showed clear radio-sensitization by 17-AAG revealed a significant inhibition of DSB repair, while normal human cells which did not show radio-sensitization by the drug indicated no change in the DSB repair kinetics with 17-AAG. We further demonstrated that BRCA2 was a novel client protein for Hsp90, and 17-AAG caused the degradation of BRCA2 and in turn altered the behavior of Rad51, a critical protein for homologous recombination (HR) pathway of DSB repair. Our data demonstrate for the first time that 17-AAG inhibits the HR repair process and could provide a new therapeutic strategy to selectively result in higher tumor cell killing.     

Protection of murine neural progenitor cells by the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin in the low nanomolar concentration range

Stem cell-based approaches provide hope as a potential therapy for neurodegenerative diseases and stroke. One of the major scientific hurdles for stem cell therapy is the poor survival rate of the newly formed or transplanted neural stem cells. In this study, we found that low-dose treatment with the Heat shock protein 90 (Hsp90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG), a heavily investigated anti-cancer drug, prevented neural progenitor cells from either naturally-occurring or stress-induced apoptosis, although it induced apoptosis at higher doses. This stress adaptation effect mediated by low-dose 17-AAG is accompanied by activation of multiple cell survival pathways, including the stress response pathway (induction of Hsp70), the MAPK pathway, and the PI3K/Akt pathway. When administered in vivo, 17-AAG led to Akt and glycogen synthase kinase 3β phosphorylation, and more 5-bromo-2'-deoxyuridine positive cells in the mouse brain. These findings could have profound implications in stem cell therapy for neurodegenerative diseases and stroke.     

Medulloblastoma sensitivity to 17-allylamino-17-demethoxygeldanamycin requires MEK/ERKM

ERBB2 increases the sensitivity of breast cancer cells to the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). This has been attributed to the disruption of ERBB3/ERBB2 heterodimers that maintain a crucial cell survival signal via phosphatidylinositol 3-kinase/AKT. ERBB2 confers a poor clinical outcome in medulloblastoma, the most common malignant pediatric brain tumor. Here, we show that medulloblastoma cell sensitivity to 17-AAG is directly related to ERBB2 expression level. Furthermore, overexpression of exogenous ERBB2 in these cells induces spontaneous homodimerization, further enhancing cell sensitivity to 17-AAG. In contrast to breast cancer cells, this increased sensitivity to 17-AAG does not result from cell dependence on AKT1 activity. Rather, we show that 17-AAG generates a dose- and time-dependent increase in MEK/ERK signaling that is required for the drug to inhibit the proliferation of medulloblastoma cells and that ERBB2 sensitizes medulloblastoma cells to 17-AAG by up-regulating basal MEK/ERK signaling. We further show that down-regulation of MEK1 activity markedly reduces the sensitivity of medulloblastoma, breast, and ovarian cancer cells to 17-AAG, whereas expression of a constitutively active MEK1 potentiates the activity of 17-AAG against these cells. Therefore, intact MEK/ERK signaling may be required for optimal 17AAG activity against a variety of tumor cell types. These data identify a new mechanism by which 17-AAG inhibits the proliferation of cancer cells. Defining the precise mode of action of these agents within specific tumor cell types will be crucial if this class of drugs is to be efficiently developed in the clinic.     

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.     

Low dose Hsp90 inhibitor 17AAG protects neural progenitor cells from ischemia induced death

Stress adaptation effect provides cell protection against ischemia induced apoptosis. Whether this mechanism prevents other types of cell death in stroke is not well studied. This is an important question for regenerative medicine to treat stroke since other types of cell death such as necrosis are also prominent in the stroke brain apart from apoptosis. We report here that treatment with 17-N-Allylamino-17-demethoxygeldanamycin (17AAG), an Hsp90 inhibitor, protected neural progenitor cells (NPCs) against oxygen glucose deprivation (OGD) induced cell death in a dose dependent fashion. Cell death assays indicated that 17AAG not only ameliorated apoptosis, but also necrosis mediated by OGD. This NPC protection was confirmed by exposing cells to oxidative stress, a major stress signal prevalent in the stroke brain. Mechanistic studies demonstrated that 17AAG activated PI3K/Akt and MAPK cell protective pathways. More interestingly, these two pathways were activated in vivo by 17AAG and 17AAG treatment reduced infarct volume in a middle cerebral artery occlusion (MCAO) stroke model. These data suggest that 17AAG protects cells against major cell death pathways and thus might be used as a pharmacological conditioning agent for regenerative medicine for stroke.     

Determination of the heat shock protein 90 inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin in plasma by liquid chromatography-electrospray mass spectrometry

A rapid method was developed for the quantitative determination of the novel heat shock protein 90 inhibitor, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG; NSC707545), in human plasma. Calibration curves were constructed, and were analyzed using a weight factor proportional to the nominal concentration. Sample pretreatment involved a one-step extraction with ethyl acetate of 0.5-ml samples. The analysis was performed in the range of 1-100 ng/ml on a column (75 mm x 2.1 mm internal diameter with 3.5 microm C18 particle size), using 55% methanol in water containing formic acid as the mobile phase. The column effluent was monitored by mass spectrometry with positive electrospray ionization. The values for precision and accuracy were always <8% and <10% relative error, respectively. The method was successfully applied to examine the pharmacokinetics of 17-DMAG in a cancer patient.     

17-AAG, an Hsp90 inhibitor, ameliorates polyglutamine-mediated motor neuron degeneration

Heat-shock protein 90 (Hsp90) functions as part of a multichaperone complex that folds, activates and assembles its client proteins. Androgen receptor (AR), a pathogenic gene product in spinal and bulbar muscular atrophy (SBMA), is one of the Hsp90 client proteins. We examined the therapeutic effects of 17-allylamino-17-demethoxygeldanamycin (17-AAG), a potent Hsp90 inhibitor, and its ability to degrade polyglutamine-expanded mutant AR. Administration of 17-AAG markedly ameliorated motor impairments in the SBMA transgenic mouse model without detectable toxicity, by reducing amounts of monomeric and aggregated mutant AR. The mutant AR showed a higher affinity for Hsp90-p23 and preferentially formed an Hsp90 chaperone complex as compared to wild-type AR; mutant AR was preferentially degraded in the presence of 17-AAG in both cells and transgenic mice as compared to wild-type AR. 17-AAG also mildly induced Hsp70 and Hsp40. 17-AAG would thus provide a new therapeutic approach to SBMA and probably to other related neurodegenerative diseases.     

FK228 inhibits Hsp90 chaperone function in K562 cells via hyperacetylation of Hsp70

Some pan-histone-deacetylase (HDAC) inhibitors have recently been reported to exert their anti-leukemia effect by inhibiting the activity of class IIB HDAC6, which is the deacetylase of Hsp90 and α-tubulin, thereby leading to hyperacetylation of Hsp90, disruption of its chaperone function and apoptosis. In this study, we compared the effect of a class I HDAC inhibitor FK228 with the pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) on the Hsp90 chaperone function of K562 cells. We demonstrated that, although having a weaker inhibitory effect on HDAC6, FK228 mediated a similar disruption of Hsp90 chaperone function compared to SAHA. Unlike SAHA, FK228 did not mediate hyperacetylation of Hsp90, instead the acetylation of Hsp70 was increased and Bcr-Abl was increasingly associated with Hsp70 rather than Hsp90, forming an unstable complex that promotes Bcr-Abl degradation. These results indicated that FK228 may disrupt the function of Hsp90 indirectly through acetylation of Hsp70 and inhibition of its function.     

Treatment with 17-allylamino-17-demethoxygeldanamycin ameliorated symptoms of Bartter syndrome type IV caused by mutated Bsnd in mice

Mutations of BSND, which encodes barttin, cause Bartter syndrome type IV. This disease is characterized by salt and fluid loss, hypokalemia, metabolic alkalosis, and sensorineural hearing impairment. Barttin is the β-subunit of the ClC-K chloride channel, which recruits it to the plasma membranes, and the ClC-K/barttin complex contributes to transepithelial chloride transport in the kidney and inner ear. The retention of mutant forms of barttin in the endoplasmic reticulum (ER) is etiologically linked to Bartter syndrome type IV. Here, we report that treatment with 17-allylamino-17-demethoxygeldanamycin (17-AAG), an Hsp90 inhibitor, enhanced the plasma membrane expression of mutant barttins (R8L and G47R) in Madin–Darby canine kidney cells. Administration of 17-AAG to Bsnd^R8L/R8L knock-in mice elevated the plasma membrane expression of R8L in the kidney and inner ear, thereby mitigating hypokalemia, metabolic alkalosis, and hearing loss. These results suggest that drugs that rescue ER-retained mutant barttin may be useful for treating patients with Bartter syndrome type IV.     

4-Amino derivatives of the Hsp90 inhibitor CCT018159

Novel piperazinyl, morpholino and piperidyl derivatives of the pyrazole-based Hsp90 inhibitor CCT018159 are described. Structure-activity relationships have been elucidated by X-ray co-crystal analysis of the new compounds bound to the N-terminal domain of human Hsp90. Key features of the binding mode are essentially identical to the recently reported potent analogue VER-49009. The most potent of the new compounds has a methylsulfonylbenzyl substituent appended to the piperazine nitrogen, possesses an IC50 of less than 600 nM binding against the enzyme and demonstrates low micromolar inhibition of tumour cell proliferation.     

The effects of Th17 cytokines on the inflammatory mediator production and barrier function of ARPE-19 cells

Th17 cells have emerged as a key factor in the pathogenesis of uveitis as well as other autoimmune disorders. They secrete a number of cytokines including IL-17A, IL-17F and IL-22 and until now the effects of these cytokines on resident cells of the eye were not yet clear. The purpose of this study was to investigate the effects of Interleukin (IL)-17A, IL-17F and IL-22 on the production of inflammatory mediators and barrier function of retinal pigment epithelium cells. We showed that ARPE-19 cells, a spontaneously arisen cell line of retinal pigment epithelium (RPE), constitutively expressed IL-17RC and IL-22R, but not IL-17RA. IL-17A significantly enhanced the production of CXCL8, CCL2, CCL20 and IL-6 by these cells. IL-17F had a similar effect on the production of CXCL8, CCL2 and IL-6 by ARPE-19 cells, but did not influence the expression of CCL20. Both IL-17A and IL-17F significantly decreased the transepithelial electrical resistance (TER) of the ARPE-19 monolayer and increased the diffusion rate of fluorescein isothiocyanate (FITC)-dextran. They also disrupted the distribution of the junction proteins zonula occludens (ZO)-1 and occludin at the interface of adjacent cells. IL-22 did not have a detectable effect on the production of the tested inflammatory mediators by ARPE-19 cells, TER of the ARPE-19 monolayer, the diffusion rate of FITC-dextran or the distribution of ZO-1 and occludin. This study demonstrates that IL-17A and IL-17F, but not IL-22, significantly promoted ARPE-19 cells to secrete inflammatory mediators and compromised the ARPE-19 monolayer barrier function in association with a disrupted distribution of ZO-1 and occludin. These results suggest that both IL-17A and IL-17F may play a role in posterior segment inflammation of the eye.     

Oxysterol-induced toxicity in R28 and ARPE-19 cells

Studies have shown an intimate relationship between cholesterol and retinal diseases; we examined the effects of cholesterol oxides on cultured cells. Using the rat retinal precursor cell line R28 and the human RPE cell line ARPE-19, we investigated the potential cytotoxicity of cholesterol oxides. Cultured R28 and ARPE-19 cells were treated with either 25-hydroxycholesterol and 7-ketocholesterol (0–50 µg/ml). Cell viability was determined by the WST-1 colorimetric assay. Production of reactive oxygen intermediate (ROI) was assessed by a fluorescent probe–based assay (2,7-dichlorodihydrofluorescein diacetate [H₂DCFDA]). To detect the presence of apoptosis, DNA fragmentation gel analysis and Hoescht nuclear staining were performed. Both cholesterol oxides tested were toxic in a time- and dose-dependent fashion to the two cell lines used in this study. Treatment of R28 cells with either 25-hydroxycholesterol or 7-ketocholesterol at a concentration of 25 µg/ml resulted in greater than 50% loss of cell viability after 24 h. ARPE-19 cells were slightly less affected, with a loss of cell viability of approximately 20% and 40% after 24 h-exposure of 25-hydroxycholesterol and 7-ketocholesterol, respectively. DNA fragmentation and chromatin condensation demonstrated apoptotic events occurring in 7-ketocholesterol–treated cells. The fluorescent assay for ROI production showed that after an hour of exposure to 7-ketocholesterol, R28 cells responded with increased levels of ROIs, whereas no immediate production of ROIs were detected with treated ARPE-19 cells. These in vitro findings provide evidence that cholesterol oxides can directly damage cultured retinal and RPE cells. The oxysterol-induced oxidative stress in these cells may be a factor in the pathology of retinal degenerative diseases.     

Hsp90 inhibitor 17-AAG sensitizes Bcl-2 inhibitor (-)-gossypol by suppressing ERK-mediated protective autophagy and Mcl-1 accumulation in hepatocellular carcinoma cells

Natural BH3-memitic (-)-gossypol shows promising antitumor efficacy in several kinds of cancer. However, our previous studies have demonstrated that protective autophagy decreases the drug sensitivities of Bcl-2 inhibitors in hepatocellular carcinoma (HCC) cells. In the present study, we are the first to report that Hsp90 inhibitor 17-AAG enhanced (-)-gossypol-induced apoptosis via suppressing (-)-gossypol-triggered protective autophagy and Mcl-1 accumulation. The suppression effect of 17-AAG on autophagy was mediated by inhibiting ERK-mediated Bcl-2 phosphorylation while was not related to Beclin1 or LC3 protein instability. Meanwhile, 17-AAG downregulated (-)-gossypol-triggered Mcl-1 accumulation by suppressing Mcl-1^Thr163 phosphorylation and promoting protein degradation. Collectively, our study indicates that Hsp90 plays an important role in tumor maintenance and inhibition of Hsp90 may become a new strategy for sensitizing Bcl-2-targeted chemotherapies in HCC cells.     

Synthesis of Hsp90 inhibitor dimers as potential antitumor agents

Structure-based drug design was used to systematically synthesize PU3-dimers. The cytotoxicity of PU3 dimers 6 against breast cancer cell lines was evaluated, and their potency increased as the length of the bridging linker increased. Among the compounds tested, 6e with a C-20 linker was the most potent and exhibited a 20- to 30-fold increase in activity compared with that of the parent compound 5. Western blot analyses of the cell lysates treated with 6c revealed that 6c resulted in the concentration-dependent degradation of the Hsp90 client protein Her2, which is consistent with other Hsp90 inhibitors.     

Proteome-wide changes induced by the Hsp90 inhibitor, geldanamycin in anaplastic large cell lymphoma cells

The molecular chaperone heat shock protein 90 (Hsp90) affects the function of many oncogenic signaling proteins including nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) expressed in anaplastic large cell lymphoma (ALCL). While ALK-positive ALCL cells are sensitive to the Hsp90 inhibitor and the geldanamycin (GA) analog, 17-allylamino-17-demethoxygeldanamycin (17-AAG), the proteomic effects of these drugs on ALK-positive ALCL cells are unpublished. In this study, we investigated the cellular, biologic, and proteomic changes occurring in ALK-positive ALCL cells in response to GA treatment. GA induced G2/M cell cycle arrest and caspase-3-mediated apoptosis. Furthermore, quantitative proteomic changes analyzed by cleavable isotope-coded affinity tag-LC-MS/MS (cICAT-LC-MS/MS) identified 176 differentially expressed proteins. Out of these, 49 were upregulated 1.5-fold or greater and 70 were downregulated 1.5-fold or greater in GA-treated cells. Analysis of biological functions of differentially expressed proteins revealed diverse changes, including induction of proteins involved in the 26S proteasome as well as downregulation of proteins involved in signal transduction and protein and nucleic acid metabolism. Pathway analysis revealed changes in MAPK, WNT, NF-kappaB, TGFbeta, PPAR, and integrin signaling components. Our studies reveal some of the molecular and proteomic consequences of Hsp90 inhibition in ALK-positive ALCL cells and provide novel insights into the mechanisms of its diverse cellular effects.     

A non-toxic Hsp90 inhibitor protects neurons from Abeta-induced toxicity

The molecular chaperones have been implicated in numerous neurodegenerative disorders in which the defining pathology is misfolded proteins and the accumulation of protein aggregates. In Alzheimer's disease, hyperphosphorylation of tau protein results in its dissociation from microtubules and the formation of pathogenic aggregates. An inverse relationship was demonstrated between Hsp90/Hsp70 levels and aggregated tau, suggesting that Hsp90 inhibitors that upregulate these chaperones could provide neuroprotection. We recently identified a small molecule novobiocin analogue, A4 that induces Hsp90 overexpression at low nanomolar concentrations and sought to test its neuroprotective properties. A4 protected neurons against Abeta-induced toxicity at low nanomolar concentrations that paralleled its ability to upregulate Hsp70 expression. A4 exhibited no cytotoxicity in neuronal cells at the highest concentration tested, 10 microM, thus providing a large therapeutic window for neuroprotection. In addition, A4 was transported across BMECs in vitro, suggesting the compound may permeate the blood-brain barrier in vivo. Taken together, these data establish A4, a C-terminal inhibitor of Hsp90, as a potent lead for the development of a novel class of compounds to treat Alzheimer's disease.