Metal-enriched HSP90 nanoinhibitor overcomes heat resistance in hyperthermic intraperitoneal chemotherapy used for peritoneal metastases.

Clinical hyperthermic intraperitoneal chemotherapy (HIPEC) is regarded as a potential treatment that can prolong survival of patients with peritoneal metastases after cytoreductive surgery. However, treated tumor cells are prone to becoming heat resistant to HIPEC therapy through high expression of heat shock proteins (HSPs). Here, a carrier-free bifunctional nanoinhibitor was developed for HIPEC therapy in the management of peritoneal metastases. Self-assembly of the nanoinhibitor was formed by mixing Mn ion and epigallocatechin gallate (EGCG) in a controllable manner. Such nanoinhibitor directly inhibited HSP90 and impaired the HSP90 chaperone cycle by reduced intracellular ATP level. Additionally, heat and Mn ion synergistically induced oxidative stress and expression of caspase 1, which activated GSDMD by proteolysis and caused pyroptosis in tumor cells, triggering immunogenic inflammatory cell death and induced maturation of dendritic cells through the release of tumor antigens. This strategy to inhibit heat resistance in HIPEC presented an unprecedented paradigm for converting "cold" tumors into "hot" ones, thus significantly eradicating disseminated tumors located deep in the abdominal cavity and stimulating immune response in peritoneal metastases of a mouse model. Collectively, the nanoinhibitor effectively induced pyroptosis of colon tumor cells under heat conditions by inhibiting heat stress resistance and increasing oxidative stress, which may provide a new strategy for treatment of colorectal peritoneal metastases.

Discovery of three-dimensional bicyclo[3.3.1]nonanols as novel heat shock protein 90 inhibitors.

We developed an efficient synthetic method for constructing bicyclo[3.3.1]nonane, an sp3-carbon-rich three-dimensional scaffold consisting of two fused six-membered rings. Among the bicyclo[3.3.1]nonanols synthesized, several bicyclo[3.3.1]nonane derivatives were found to inhibit gene transcription by hypoxia-inducible factor-1 (HIF-1). The structure-activity relationship study revealed that the number of hydrophobic functional groups and a carboxylic acid moiety in the bicyclo[3.3.1]nonanols are important for inhibitory activities of both gene transcription by HIF-1 and cell growth. Bicyclo[3.3.1]nonanols fluctuated the amounts of client proteins of heat shock protein (HSP) 90 without inducing a heat shock response in cells and specifically inhibited the ATPase activity of HSP90. These results indicate that bicyclo[3.3.1]nonanols are novel HSP90 inhibitors with a different mechanism of action from conventional HSP90 inhibitors.

Synergistic effect of Hsp90 inhibitor ginkgolic acids C15꞉1 combined with paclitaxel on nasopharyngeal carcinoma. / Hsp90æŠ‘åˆ¶å‰‚ç™½æžœé ¸ä¸Žç´«æ‰é†‡çš„ååŒæŠ—é¼»å’½ç™Œä½œç”¨.

OBJECTIVES: Nasopharyngeal cracinoma is a kind of head and neck malignant tumor with high incidence and high mortality. Due to the characteristics of local recurrence, distant metastasis, and drug resistance, the survival rate of patients after treatment is not high. Paclitaxel (PTX) is used as a chemotherapy drug in treating nasopharyngeal carcinoma, but nasopharyngeal carcinoma cells are easy to develop resistance to PTX. Inhibition of heat shock protein 90 (Hsp90) can overcome common signal redundancy and resistance in many cancers. This study aims to investigate the anti-tumor effect of ginkgolic acids C15꞉1 (C15:1) combined with PTX on nasopharyngeal carcinoma CNE-2Z cells and the mechanisms. METHODS: This experiment was divided into a control group (without drug), a C15:1 group (10, 30, 50, 70 µmol/L), a PTX group (5, 10, 20, 40 nmol/L), and a combination group. CNE-2Z cells were treated with the corresponding drugs in each group. The proliferation of CNE-2Z cells was evaluated by methyl thiazolyl tetrazolium (MTT). Wound-healing assay and transwell chamber assay were used to determine the migration of CNE-2Z cells. Transwell chamber was applied to the impact of CNE-2Z cell invasion. Annexin V-FITC/PI staining was used to observe the effect on apoptosis of CNE-2Z cells. The changes of proteins involved in cell invasion, migration, and apoptosis after the combination of C15꞉1 and PTX treatment were analyzed by Western blotting. RESULTS: Compared with the control group, the C15꞉1 group and the PTX group could inhibit the proliferation of CNE-2Z cells (all P<0.05). The cell survival rates of the C15꞉1 50 µmol/L combined with 5, 10, 20, or 40 nmol/L PTX group were lower than those of the single PTX group (all P<0.05), the combination index (CI) value was less than 1, suggesting that the combined treatment group had a synergistic effect. Compared with the 50 µmol/L C15꞉1 group and the 10 nmol/L PTX group, the combination group significantly inhibited the invasion and migration of CNE-2Z cells (all P<0.05). The results of Western blotting demonstrated that the combination group could significantly down-regulate Hsp90 client protein matrix metalloproteinase (MMP)-2 and MMP-9. The results of double staining showed that compared with the 50 µmol/L C15꞉1 group and the 10 nmol/L PTX group, the apoptosis ratio of CNE-2Z cells in the combination group was higher (both P<0.05). The results of Western blotting suggested that the combination group could decrease the Hsp90 client proteins [Akt and B-cell lymphoma-2 (Bcl-2)] and increase the Bcl-2-associated X protein (Bax). CONCLUSIONS: The combination of C15꞉1 and PTX has a synergistic effect which can inhibit cell proliferation, invasion, and migration, and induce cell apoptosis. This effect may be related to the inhibition of Hsp90 activity by C15꞉1.

Pimitespib is effective on cecal GIST in a mouse model of familial GISTs with KIT-Asp820Tyr mutation through KIT signaling inhibition.

Three families with multiple gastrointestinal stromal tumors (GISTs) caused by a germline Asp820Tyr mutation at exon 17 of the c-kit gene (KIT-Asp820Tyr) have been reported. We previously generated a knock-in mouse model of the family, and the mice with KIT-Asp818Tyr corresponding to human KIT-Asp820Tyr showed a cecal tumor equivalent to human GIST. In the model mice, we reported that tyrosine kinase inhibitor, imatinib, could stabilize but not decrease the cecal tumor volume. In this report, we examined whether a heat shock protein 90 inhibitor, pimitespib (TAS-116), has an inhibitory effect on phosphorylation of KIT-Asp818Tyr and can decrease the cecal tumor volume in the model mice. First, we showed that pimitespib inhibited KIT phosphorylation both dose- and time-dependently in KIT-Asp818Tyr transfected murine Ba/F3 cells. Then, four 1-week courses of pimitespib were orally administered to heterozygous (KIT-Asp818Tyr/+) model mice. Each course consisted of once-daily administration for consecutive 5 days followed by 2 days-off. Cecal tumors were dissected, and tumor volume was histologically analyzed, Ki-67 labeling index was immunohistochemically examined, and apoptotic figures were counted. Compared to the vehicle treated mice, pimitespib administered mice showed statistically significantly smaller cecal tumor volume, lower Ki-67 labeling index, and higher number of apoptotic figures in 10 high power fields (P = 0.0344, P = 0.0019 and P = 0.0269, respectively). Western blotting revealed that activation of KIT signaling molecules was strongly inhibited in the tumor tissues of pimitespib-administered mice compared to control mice. Thus, pimitespib seemed to inhibit in vivo tumor progression effectively in the model mice. These results suggest that the progression of multiple GISTs in patients with germline KIT-Asp820Tyr might be controllable by pimitespib.

The Effect of Heat Shock Protein 90 Inhibitor on Pain in Cancer Patients: A Systematic Review and Meta-Analysis.

Heat shock protein 90 (Hsp90) is a molecular chaperone that plays an essential role in tumor growth. Numerous Hsp90 inhibitors have been discovered and tested in preclinical and clinical trials. Recently, several preclinical studies have demonstrated that Hsp90 inhibitors could modulate pain sensitization. However, no studies have evaluated the impact of Hsp90 inhibitors on pain in the patients. This study aims to summarize the pain events reported in clinical trials assessing Hsp90 inhibitors and to determine the effect of Hsp90 inhibitors on pain in patients. We searched PubMed, EBSCOhost, and clinicaltrials.gov for Hsp90 inhibitor clinical trials. The pain-related adverse events were summarized. Meta-analysis was performed using the data reported in randomized controlled trials. We identified 90 clinical trials that reported pain as an adverse effect, including 5 randomized controlled trials. The most common types of pain reported in all trials included headache, abdominal pain, and back pain. The meta-analysis showed that Hsp90 inhibitors increased the risk of abdominal pain significantly and appeared to increase the risk for back pain. In conclusion, Hsp90 inhibitor treatment could potentially increase the risk of pain. However, the meta-analysis demonstrated only moderate evidence for the connection between Hsp90 inhibitor and pain.

HSP90 inhibitors in the context of heat shock and the unfolded protein response: effects on a primary canine pulmonary adenocarcinoma cell line.

BACKGROUND: Agents targeting HSP90 and GRP94 are seldom tested in stressed contexts such as heat shock (HS) or the unfolded protein response (UPR). Tumor stress often activates HSPs and the UPR as pro-survival mechanisms. This begs the question of stress effects on chemotherapeutic efficacy, particularly with drugs targeting chaperones such as HSP90 or GRP94. We tested the utility of several HSP90 inhibitors, including PU-H71 (targeting GRP94), on a primary canine lung cancer line under HS/UPR stress compared to control conditions. METHODS: We cultured canine bronchoalveolar adenocarcinoma cells that showed high endogenous HSP90 and GRP94 expression; these levels substantially increased upon HS or UPR induction. We treated cells with HSP90 inhibitors 17-DMAG, 17-AAG or PU-H71 under standard conditions, HS or UPR. Cell viability/survival was assayed. Antibody arrays measured intracellular signalling and apoptosis profiles. RESULTS: HS and UPR had varying effects on cells treated with different HSP90 inhibitors; in particular, HS and UPR promoted resistance to inhibitors in short-term assays, but combinations of UPR stress and PU-H571 showed potent cytotoxic activity in longer-term assays. Array data indicated altered signalling pathways, with apoptotic and pro-survival implications. UPR induction + dual targeting of HSP90 and GRP94 swayed the balance toward apoptosis. CONCLUSION: Cellular stresses, endemic to tumors, or interventionally inducible, can deflect or enhance chemo-efficacy, particularly with chaperone-targeting drugs. Stress is likely not held accountable when testing new pharmacologics or assessing currently-used drugs. A better understanding of stress impacts on drug activities should be critical in improving therapeutic targeting and in discerning mechanisms of drug resistance.

Targeting of multiple oncogenic signaling pathways by Hsp90 inhibitor alone or in combination with berberine for treatment of colorectal cancer.

There is a wide range of drugs and combinations under investigation and/or approved over the last decade to treat colorectal cancer (CRC), but the 5-year survival rate remains poor at stages II-IV. Therefore, new, more-efficient drugs still need to be developed that will hopefully be included in first-line therapy or overcome resistance when it appears, as part of second- or third-line treatments in the near future. In this study, we revealed that heat shock protein 90 (Hsp90) inhibitors have high therapeutic potential in CRC according to combinative analysis of NCBI's Gene Expression Omnibus (GEO) repository and chemical genomic database of Connectivity Map (CMap). We found that second generation Hsp90 inhibitor, NVP-AUY922, significantly downregulated the activities of a broad spectrum of kinases involved in regulating cell growth arrest and death of NVP-AUY922-sensitive CRC cells. To overcome NVP-AUY922-induced upregulation of survivin expression which causes drug insensitivity, we found that combining berberine (BBR), a herbal medicine with potency in inhibiting survivin expression, with NVP-AUY922 resulted in synergistic antiproliferative effects for NVP-AUY922-sensitive and -insensitive CRC cells. Furthermore, we demonstrated that treatment of NVP-AUY922-insensitive CRC cells with the combination of NVP-AUY922 and BBR caused cell growth arrest through inhibiting CDK4 expression and induction of microRNA-296-5p (miR-296-5p)-mediated suppression of Pin1-ß-catenin-cyclin D1 signaling pathway. Finally, we found that the expression level of Hsp90 in tumor tissues of CRC was positively correlated with CDK4 and Pin1 expression levels. Taken together, these results indicate that combination of NVP-AUY922 and BBR therapy can inhibit multiple oncogenic signaling pathways of CRC.

Increased expression of heat shock protein 90 in keratinocytes and mast cells in patients with psoriasis.

BACKGROUND: Psoriasis is a chronic inflammatory skin disease and various stress factors mediate inflammation. Heat shock protein (HSP) 90 plays an important role in cell survival; cytokine signaling, such as interleukin-17 receptor signaling; and immune responses. OBJECTIVE: We sought to elucidate protein expression and distribution of HSP90 in psoriasis. METHODS: HSP90 expression and its cellular source were analyzed on normal-appearing, nonlesional, lesional, and ustekinumab-treated psoriatic skin using immunohistochemistry and double immunofluorescence. RESULTS: HSP90α, the inducible isoform of HSP90, was significantly up-regulated in epidermal keratinocytes and mast cells of lesional skin and down-regulated after ustekinumab therapy. LIMITATIONS: There was a limited sample size. CONCLUSIONS: HSP90 from keratinocytes and mast cells is a key regulator of psoriatic inflammation and HSP90 inhibitors may represent a novel therapeutic approach to the disease.

HSP90 C-terminal domain inhibition promotes VDAC1 oligomerization via decreasing K274 mono-ubiquitination in Hepatocellular Carcinoma.

Voltage-dependent anion-selective channel protein 1 (VDAC1) is the most abundant protein in the mitochondrial outer membrane and plays a crucial role in the control of hepatocellular carcinoma (HCC) progress. Our previous research found that cytosolic molecular chaperone heat shock protein 90 (Hsp90) interacted with VDAC1, but the effect of the C-terminal and N-terminal domains of Hsp90 on the formation of VDAC1 oligomers is unclear. In this study, we focused on the effect of the C-terminal domain of Hsp90 on VDAC1 oligomerization, ubiquitination, and VDAC1 channel activity. We found that Hsp90 C-terminal domain inhibitor Novobiocin promoted VDAC1 oligomerization, release of cytochrome c, and activated mitochondrial apoptosis pathway. Atomic coarse particle modeling simulation revealed C-terminal domain of Hsp90α stabilized VDAC1 monomers. The purified VDAC1 was reconstituted into a planar lipid bilayer, and electrophysiology experiments of patch clamp showed that the Hsp90 C-terminal inhibitor Novobiocin increased VDAC1 channel conductance via promoting VDAC1 oligomerization. The mitochondrial ubiquitination proteomics results showed that VDAC1 K274 mono-ubiquitination was significantly decreased upon Novobiocin treatment. Site-directed mutation of VDAC1 (K274R) weakened Hsp90α-VDAC1 interaction and increased VDAC1 oligomerization. Taken together, our results reveal that Hsp90 C-terminal domain inhibition promotes VDAC1 oligomerization and VDAC1 channel conductance by decreasing VDAC1 K274 mono- ubiquitination, which provides a new perspective for mitochondria-targeted therapy of HCC.

Preferential radiosensitization to glioblastoma cancer stem cell-like cells by a Hsp90 inhibitor, N-vinylpyrrolidone-AUY922.

The present study examined the radiosensitization induced by a heat shock protein 90 inhibitor, N-vinylpyrrolidone (NVP)-AUY922, in CD133-positive cells in a hypoxic area of T98G spheroids. CD133-positive cells that are induced in the hypoxic microenvironment of spheroids have previously been reported to exhibit cancer stem cell-like properties. The present study used CD133-positive cells from a glioblastoma cell line (T98G) as cancer stem cell-like cells. CD133-positive and negative cells were sorted from T98G spheroids using fluorescence-activated cell sorting and used for colony formation assay. Colony formation assay results indicated that NVP-AUY922 enhanced radiosensitivity more strongly in CD133-positive cells compared with CD133-negative cells. This result showed that NVP-AUY922 was a preferential radiosensitization candidate targeting glioblastoma cancer stem cells. The mechanisms underlying radiosensitization by NVP-AUY922 are discussed in relation to the properties of cancer stem cells. Overall, HIF-1α inhibition by NVP-AUY922 may induce higher sensitization of cancer stem cells to radiation.

Studies on Preformulation and Formulation of JIN-001 Liquisolid Tablet with Enhanced Solubility.

This study aimed to develop a heat shock protein 90 (Hsp90) inhibitor liquisolid tablet with improved solubility to overcome low bioavailability issues. As an active pharmaceutical ingredient (API), JIN-001, a novel Hsp90 inhibitor, was reported to have substantial in vitro antiproliferative and in vivo antitumor activity; however, JIN-001 was a crystalline solid with very low solubility in an aqueous solution, and therefore, Capryol 90, which has excellent solubilization ability, was selected as an optimal liquid vehicle based on solubility studies. JIN-001 liquisolid (JLS) powder was successfully prepared by dissolving JIN-001 in Capryol 90 and mixing colloidal silicon dioxide (CSD) used as an oil adsorption agent. The prepared JLS was confirmed to be amorphous. Based on the result of the solubility test of JLS, compared to JIN-001, the solubility of the former was significantly improved in all solvents regardless of pH. JLS tablets were prepared through wet granulation using JIN-001 and stable excipients based on the compatibility test. The developed JLS tablet significantly increased the drug release rate in all tested solutions; however, the liquisolid method had no significant effect on bioavailability in the pharmacokinetics study in beagle dogs. In conclusion, the liquisolid system influenced the solubility and dissolution rate of JIN-001.

Novel C-terminal heat shock protein 90 inhibitors target breast cancer stem cells and block migration, self-renewal, and epithelial-mesenchymal transition.

In patients with triple-negative breast cancer (TNBC), evidence suggests that tumor-initiating cells (TIC) have stem cell-like properties, leading to invasion and metastasis. HSP90 plays a critical role in the conformational maintenance of many client proteins in TIC development. Therefore, we hypothesize that the novel C-terminal HSP90 inhibitors KU711 and KU758 can target TIC and represent a promising strategy for overcoming metastasis. Human breast cancer cells (MDA-MB-468LN, MDA-MB-231) treated with the HSP90 inhibitors KU711, KU758, and 17-AAG showed a 50-80% decrease in TIC markers CD44 and aldehyde dehydrogenase (P < 0.01) as assessed by flow cytometry. A decrease in sphere formation, which was used to assess self-renewal, was observed after the treatment of TNBC cells starting at 2.5 µm KU711 and 0.31 µm KU758. KU compounds also blocked the invasion and migration of TNBC cells in a dose-dependent manner. The knockdown of HSP90 clients was observed without any change in prosurvival HSP70 levels. In vivo, in a murine orthotopic breast cancer model, treatment with KU758 and KU711 yielded an approximately twofold and a fourfold reduction in tumor volumes versus control, respectively, without demonstrated toxicity. In conclusion, C-terminal HSP90 inhibitors are potent novel therapeutics against TNBC in vitro and in vivo as they target TICs and block invasion, EMT transition, and self-renewal.

17­AAG synergizes with Belinostat to exhibit a negative effect on the proliferation and invasion of MDA­MB­231 breast cancer cells.

Breast cancer is one of the most common malignancies that threaten the health of women. Although there are a few chemotherapies for the clinical treatment of breast cancer, these therapies are faced with the problems of drug­resistance and metastasis. Drug combination can help to reduce the adverse side effects of chemotherapies using single drugs, and also help to overcome common drug­resistance during clinical treatment of breast cancer. The present study reported the synergistic effect of the heat shock protein 90 inhibitor 17­AAG and the histone deacetylase 6 inhibitor Belinostat in triple­negative breast cancer (TNBC) MDA­MB­231 cells, by detection of proliferation, apoptosis and cell cycle arrest following treatment with this combination. Subsequently, RNA sequencing (RNA­seq) data was collected and analyzed to investigate the synergistic mechanism of this combination. Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathways revealed by RNA­seq data analysis, a wound­healing assay was used to investigate the effect of this combination on the migration of MDA­MB­231 cells. Compared with treatment with 17­AAG or Belinostat alone, both the viability inhibition and apoptosis rate of MDA­MB­231 cells were significantly enhanced in the combination group. The combination index values were <1 in three concentration groups. Revealed by the RNA­seq data analysis, the most significantly enriched KEGG pathways in the combination group were closely associated with cell migration. Based on these findings, the anti­migration effect of this combination was investigated. It was revealed that the migration of MDA­MB­231 cells was significantly suppressed in the combination group compared with in the groups treated with 17­AAG or Belinostat alone. In terms of specific genes, the mRNA expression levels of TEA domain family proteins were significantly decreased in the combination group, whereas the phosphorylation of YY1 associated protein 1 and modulator of VRAC current 1 was significantly enhanced in the combination group. These alterations may help to explain the anti­migration effect of this combination. Belinostat has already been approved as a treatment for T­cell lymphoma and 17­AAG is undergoing clinical trials. These findings could provide a beneficial reference for the clinical treatment of patients with TNBC.

Treating patients with ALK-positive non-small cell lung cancer: latest evidence and management strategy.

Rearrangements in anaplastic lymphoma kinase (ALK) gene and echinoderm microtubule-associated protein-like 4 (EML4) gene were first described in a small portion of patients with non-small cell lung cancer (NSCLC) in 2007. Fluorescence in situ hybridization is used as the diagnostic test for detecting an EML4-ALK rearrangement. Crizotinib, an ALK inhibitor, is effective in treating advanced ALK-positive NSCLC, and the US Food and Drug Administration approved it for treating ALK-positive NSCLC in 2011. Several mechanisms of acquired resistance to crizotinib have recently been reported. Second-generation ALK inhibitors were designed to overcome these resistance mechanisms. Two of them, ceritinib and alectinib, were approved in 2014 for advanced ALK-positive NSCLC in the US and Japan, respectively. Heat shock protein 90 (Hsp90) inhibitors also showed activity against ALK-positive NSCLC. Here we review the recent development of crizotinib, ceritinib, alectinib and other second-generation ALK inhibitors as well as Hsp90 inhibitors. We also discuss management strategies for advanced ALK-positive NSCLC.

Phase I dose-escalation studies of SNX-5422, an orally bioavailable heat shock protein 90 inhibitor, in patients with refractory solid tumours.

BACKGROUND: Orally administered SNX-5422, a novel, selective prodrug of the Heat shock protein 90 (Hsp90) inhibitor SNX-2112, was investigated in two sequential phase I studies to determine the safety, maximum tolerated doses (MTDs) and pharmacokinetic profile of SNX-5422. METHODS: Using a dose-escalation design, 3-6 adults with advanced solid tumours received SNX-5422 every-other-day (QOD) or once-daily (QD) 3weeks on/1week off or QD continuously, with disease assessments every 8 weeks. Single-dose and steady-state pharmacokinetic parameters of SNX-2112 were determined. RESULTS: In total, 56 patients were enrolled: QOD 3 weeks on/1 week off, n=36; QD 3weeks on/1 week off, n=17; QD continuous, n=3. Doses ranged from 4 to 133mg/m(2) QOD and 50 to 89 mg/m(2) QD. The MTDs were defined as 100mg/m(2) QOD and 67 mg/m(2) QD, respectively, with diarrhoea being dose-limiting on both 3 weeks on/1 week off schedules. Overall, treatment-related adverse events were mainly low grade, including diarrhoea (64%), nausea (39%), fatigue (28%), and vomiting (28%). Reversible grade 1-3 nyctalopia (night blindness) was reported by four patients (dose: 50-89mg/m(2) QD; 100mg/m(2) QOD). Exposure was generally linear, though greater than dose-proportional. Of 32 evaluable patients on QOD dosing, there was one durable complete response (prostate cancer), one confirmed (HER2+breast cancer) and one unconfirmed partial response (adrenal gland cancer). Three patients (QOD schedule) had stable disease for ⩾ 6 months. CONCLUSIONS: The dose and schedule recommended for further study with SNX-5422 is 100mg/m(2) QOD 3 weeks on/1 week off based on improved tolerability and preliminary evidence of clinical activity.

Irradiation facilitates the inhibitory effect of the heat shock protein 90 inhibitor NVP-BEP800 on the proliferation of malignant glioblastoma cells through attenuation of the upregulation of heat shock protein 70.

The present study aimed to investigate the effect of NVP-BEP800, a novel heat shock protein (Hsp) 90 inhibitor of the 2-aminothieno[2,3-d]pyrimidine class, in combination with radiation on glioblastoma cells. T98G human glioblastoma cells were treated with dimethyl sulfoxide (DMSO), NVP-BEP800, NVP-BEP800 in combination with X-ray irradiation (10 Gy, 20 min), or X-ray irradiation only, and cultured for 40 h. Cell viability was measured upon completion of the treatments. In addition, apoptosis was measured and immunoblot analysis was performed to analyze the expression levels of cellular protein inhibitory κB kinase ß (IKKß). The combined treatment with NVP-BEP800 and X-ray irradiation resulted in the synergistic destruction of malignant cells. Furthermore, NVP-BEP800 significantly induced apoptosis in the human glioblastoma cells. The immunoblot analysis data indicated that NVP-BEP800 markedly reduced the expression level of IKKß. The results also revealed that X-ray irradiation significantly attenuated the increase in the level of Hsp70 in cells treated with NVP-BEP800. Since elevated levels of Hsp70 are associated with drug resistance induced by Hsp90 inhibitors, the effects of X-ray irradiation on Hsp70 levels may be associated with the enhanced effect on cells of the presence of irradiation. The results of the current study suggest that irradiation enhances the inhibitory effect of NVP-BEP800 on the proliferation of malignant glioblastoma cells by downregulating the expression level of cellular signaling protein IKKß and attenuating the upregulation of Hsp70 that is induced by NVP-BEP800.

Autophagy is involved in endogenous and NVP-AUY922-induced KIT degradation in gastrointestinal stromal tumors.

Gastrointestinal stromal tumor (GIST) is a prototype of mutant KIT oncogene-driven tumor. Prolonged tyrosine kinase inhibitor (TKI) treatment may result in a resistant phenotype through acquired secondary KIT mutation. Heat shock protein 90 (HSP90AA1) is a chaperone protein responsible for protein maturation and stability, and KIT is a known client protein of HSP90AA1. Inhibition of HSP90AA1 has been shown to destabilize KIT protein by enhancing its degradation via the proteasome-dependent pathway. In this study, we demonstrated that NVP-AUY922 (AUY922), a new class of HSP90AA1 inhibitor, is effective in inhibiting the growth of GIST cells expressing mutant KIT protein, the imatinib-sensitive GIST882 and imatinib-resistant GIST48 cells. The growth inhibition was accompanied with a sustained reduction of both total and phosphorylated KIT proteins and the induction of apoptosis in both cell lines. Surprisingly, AUY922-induced KIT reduction could be partially reversed by pharmacological inhibition of either autophagy or proteasome degradation pathway. The blockade of autophagy alone led to the accumulation of the KIT protein, highlighting the role of autophagy in endogenous KIT turnover. The involvement of autophagy in endogenous and AUY922-induced KIT protein turnover was further confirmed by the colocalization of KIT with MAP1LC3B-, acridine orange- or SQSTM1-labeled autophagosome, and by the accumulation of KIT in GIST cells by silencing either BECN1 or ATG5 to disrupt autophagosome activity. Therefore, the results not only highlight the potential application of AUY922 for the treatment of KIT-expressing GISTs, but also provide the first evidence for the involvement of autophagy in endogenous and HSP90AA1 inhibitor-induced KIT degradation.