The Hsp90 inhibitor SNX-2112 induces apoptosis of human hepatocellular carcinoma cells: the role of ER stress.

Heat shock protein 90 (Hsp90) has been predicted to be involved in hepatocellular carcinoma (HCC) therapy; however, the mechanisms of action remain elusive. SNX-2112 is an Hsp90 inhibitor showing broad antitumor activity. Here we aim to determine the role of the endoplasmic reticulum (ER) stress in SNX-2112-induced apoptosis in HCC cells. In general, three HCC cells (i.e., HepG2, Huh7, and SK-Hep1) were used in our experiments. The cell viability was determined by the CCK-8 assay. The apoptosis was analyzed using flow cytometry, laser scanning confocal microscopy (LSM) and Western blotting. The efficacy and mechanisms of action of SNX-2112 were also evaluated in a mouse xenograft model. We found that SNX-2112 showed stronger inhibition on cell growth than 17-AAG, a classical Hsp90 inhibitor. SNX-2112 treatment led to the caspase-dependent apoptosis. Interestingly, SNX-2112 decreased the expression levels of the ER chaperone proteins calnexin and immunoglobulin binding protein (BiP). It also inhibited all three ER stress sensors, namely, inositol-requiring gene 1 (IRE1), PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF-6) in vitro and/or in vivo. However, the ER stress inducer tunicamycin strongly enhanced SNX-2112-induced apoptosis, whereas the IRE1 knockdown did not. Taken together, we for the first time indicated the possible apoptotic pathways of SNX-2112 in HCC cells, raising the possibility that the induction of ER stress might be favorable for SNX-2112-induced apoptosis.

P-glycoprotein (P-gp)-mediated efflux limits intestinal absorption of the Hsp90 inhibitor SNX-2112 in rats.

1. The promising anticancer agent SNX-2112 (a novel Hsp90 inhibitor) is poorly bioavailable after oral administration. Here, we aim to determine the role of P-glycoprotein (P-gp) in the intestinal absorption of SNX-2112. 2. We found that SNX-2112 significantly stimulated P-gp ATPase activity in in vitro ATPase assay with a small EC50 (the half-maximal effective concentration) value of 0.32 µM. 3. In the single-pass perfused rat intestine model, absorption of SNX-2112 was not favored in the small intestine with a [Formula: see text] (the wall permeability) value of 0.38-0.64. By contrast, the compound was well absorbed in the colon with a [Formula: see text] value of 1.19. The P-gp inhibitors cyclosporine and elacridar (i.e. GF120918A) markedly enhanced SNX-2112 absorption in all four intestinal segments (i.e. duodenum, jejunum, ileum and colon) and the fold change ranged from 3.1 to 14.1. Pharmacokinetic study revealed that cyclosporine increased the systemic exposure of SNX-2112 by a 2.5-fold after oral administration. 4. This is the first report that P-gp-mediated efflux is a limiting factor for intestinal absorption of SNX-2112 in rats.

Identification of circRNA-miRNA-mRNA network regulated by Hsp90 in human melanoma A375 cells.

BACKGROUND: Melanoma is the deadliest form of skin cancer. Heat shock protein 90 (Hsp90) is highly expressed in human melanoma. Hsp90 inhibitors can suppress the growth of human melanoma A375 cells; however, the underlying mechanism remains unclear. METHODS: A375 cells were treated with SNX-2112, an Hsp90 inhibitor, for 48 h, and whole-transcriptome sequencing was performed. RESULTS: A total of 2,528 differentially expressed genes were identified, including 895 upregulated and 1,633 downregulated genes. Pathway enrichment analyses of differentially expressed mRNAs identified the extracellular matrix (ECM)-receptor interaction pathway as the most significantly enriched pathway. The ECM receptor family mainly comprises integrins (ITGs) and collagens (COLs), wherein ITGs function as the major cell receptors for COLs. 19 upregulated miRNAs were found to interact with 6 downregulated ITG genes and 8 upregulated miRNAs were found to interact with 3 downregulated COL genes. 9 differentially expressed circRNAs in SNX-2112-treated A375 cells were identified as targets of the ITG- and COL-related miRNAs. Based on the differentially expressed circRNAs, miRNAs, and mRNAs, ITGs- and COL-based circRNA-miRNA-mRNA regulatory networks were mapped, revealing a novel regulatory mechanism of Hsp90-regulated melanoma. CONCLUSION: Targeting the ITG-COL network is a promising approach to the treatment of melanoma.

SNX-2112 Induces Apoptosis and Inhibits Proliferation, Invasion, and Migration of Non-Small Cell Lung Cancer by Downregulating Epithelial-Mesenchymal Transition via the Wnt/ß-Catenin Signaling Pathway.

Lung cancer is the most frequent malignant tumor, and non-small cell lung cancer (NSCLC) is responsible for substantial mortality worldwide. The small molecule SNX-2112 was recently shown to critically effect the proliferation and apoptosis of tumor cells. Nevertheless, the precise mechanism by which SNX-2112 affects NSCLC remains poorly understood. Therefore, we investigated the function of SNX-2112 in NSCLC. We verified that SNX-2112 promoted apoptosis and suppressed the proliferation, invasion, and migration of A549 and H520 NSCLC cells in vitro. We further verified the potential mechanism of SNX-2112 in NSCLC. The changes in the protein levels demonstrated that SNX-2112 inhibited the epithelial-mesenchymal transition (EMT) (increased E-cadherin and decreased N-cadherin and vimentin) and the Wnt/ß-catenin signaling pathway (glycogen synthase kinase (GSK) 3ß and phosphorylated (p)-ß-catenin increased, ß-catenin and p-GSK3ß decreased) in NSCLC cells. These results were verified by rescue experiments using a Wnt/ß-catenin pathway agonist. We also established a tumor xenograft model and confirmed that SNX-2112 reduced tumor growth and proliferation and enhanced necrosis and apoptosis in a NSCLC model in vivo. In conclusion, the current study is the first to discover the mechanism of SNX-2112 in NSCLC. SNX-2112 induced apoptosis and also inhibited the proliferation, invasion, and migration of NSCLC cells by downregulating EMT via the Wnt/ß-catenin signaling pathway.

Complex Crystal Structure Determination and in vitro Anti-non-small Cell Lung Cancer Activity of Hsp90 N Inhibitor SNX-2112.

SNX-2112, as a promising anticancer lead compound targeting heat shock protein 90 (Hsp90), absence of complex crystal structure of Hsp90 N -SNX-2112 hindered further structural optimization and understanding on molecular interaction mechanism. Herein, a high-resolution complex crystal structure of Hsp90 N -SNX-2112 was successfully determined by X-ray diffraction, resolution limit, 2.14 Å, PDB ID 6LTK, and their molecular interaction was analyzed in detail, which suggested that SNX-2112 was well accommodated in the ATP-binding pocket to disable molecular chaperone activity of Hsp90, therefore exhibiting favorable inhibiting activity on three non-small cell lung cancer (NSCLC) cell lines (IC50, 0.50 ± 0.01 µM for A549, 1.14 ± 1.11 µM for H1299, 2.36 ± 0.82 µM for H1975) by inhibited proliferation, induced cell cycle arrest, and aggravated cell apoptosis. SNX-2112 exhibited high affinity and beneficial thermodynamic changes during the binding process with its target Hsp90 N confirmed by thermal shift assay (TSA, ΔTm, and -9.51 ± 1.00°C) and isothermal titration calorimetry (K d , 14.10 ± 1.60 nM). Based on the complex crystal structure and molecular interaction analysis, 32 novel SNX-2112 derivatives were designed, and 25 new ones displayed increased binding force with the target Hsp90 N verified by molecular docking evaluation. The results would provide new references and guides for anti-NSCLC new drug development based on the lead compound SNX-2112.

Identification of the circRNA-miRNA-mRNA regulatory network of Hsp90 inhibitor-induced cell death in colorectal cancer by integrated analysis.

Colorectal cancer (CRC) is a global disease with high incidence and mortality rate. Hsp90 inhibitors induce cell death in various cancers, including CRC. However, the underlying mechanisms need to be clarified further. In this study, Caco-2 cells were treated with 0.25 µM SNX-2112, an Hsp90 inhibitor, for 48 h; subsequently, whole-transcriptome sequencing was performed. At the mRNA level in SNX-2112-treated Caco-2 cells, 1588 genes were upregulated, and 433 genes were downregulated. Six genes were found to be associated with necroptosis and apoptosis, and these 6 upregulated genes were validated by RT-qPCR. Hundred and six miRNAs were upregulated, and 48 miRNAs were downregulated in SNX-2112-treated Caco-2 cells. Eleven downregulated miRNAs were found to interact with the 6 upregulated genes. Moreover, 676 circRNAs were upregulated, and 291 circRNAs were downregulated in SNX-2112-treated Caco-2 cells. Among them, 126 circRNAs were found to be the target of the 11 downregulated miRNAs. The circRNA-miRNA-mRNA regulatory network of Hsp90 inhibitor-induced cell death in colorectal cancer was constructed. This regulatory network extends the underlying mechanism of Hsp90 and improves our understanding of Hsp90 inhibitors as potential targeted therapeutic agents.

Suppression of Esophageal Cancer Stem-like Cells by SNX-2112 Is Enhanced by STAT3 Silencing.

Many studies have demonstrated that cancer stem cells (CSCs) or tumor-initiating cells (TICs) are responsible for tumor cell proliferation, chemotherapy resistance, metastasis, and relapse in various cancers. We, and others, have previously shown that the signal transducer and activator of transcription 3 (STAT3) signaling pathway is responsible for CSCs and TICs growth. Recent reports have indicated that the heat shock protein 90 (Hsp90) is also essential for the survival of CSCs and TICs. SNX-2112 is an Hsp90 inhibitor. However, it remains unclear whether proliferation of esophageal cancer stem-like cells (ECSLCs) is suppressed by SNX-2112 with knockdown of STAT3 (shSTAT3). Here, we explored the association between SNX-2112 with shSTAT3 and the suppression of ECSLCs growth. We found that the expression level of both STAT3 and p-STAT3 was higher in clinical esophageal cancer tissue than in the adjacent normal tissue, using western blot and qPCR analysis. Furthermore, differential expression analysis demonstrated that STAT3 was overexpressed in clinical specimens. We demonstrated that SNX-2112 inhibited cancer cell proliferation, decreased ABCB1 and ABCG2 gene expression levels and reduced the colony formation capacity of ECSLCs, which was enhanced by STAT3 silencing. Flow cytometry analysis revealed that the combination of SNX-2112 and shSTAT3 significantly induced apoptosis and cell cycle arrest at G2/M phase in ECSLCs. Levels of proliferation pathway proteins, including p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) which were also client proteins of Hsp90, were also reduced. In addition, SNX-2112 with shSTAT3 inhibited the proliferation of ECSLCs in vivo. Finally, STAT3 overexpression eliminated the apoptotic and antiproliferative effects of SNX-2112 on ECSLCs. Hence, these results provide a rationale for the therapeutic potential of the combination of SNX-2112 with shSTAT3 in esophageal cancer, and may indicate new targets for clinical intervention in human cancer.

Antitumorigenic Effect of Hsp90 Inhibitor SNX-2112 on Tongue Squamous Cell Carcinoma is Enhanced by Low-Intensity Ultrasound.

PURPOSE: The novel Hsp90 inhibitor SNX-2112 showed broad antitumor activity. However, it was still necessary to optimize the therapeutic dosage of SNX-2112 applied on tumors to obtain effective therapy with minimal dose to reduce toxicity. We investigated the role of low-intensity US in promoting antitumorigenic effect of low doses of SNX-2112 on tongue squamous cell carcinoma. METHODS: Cell viability was measured using CCK-8 assay or staining with Calcein AM/PI. Relative cumulative levels of SNX-2112 in cells were detected using high-performance liquid chromatography. The production of ROS was analyzed using fluorescence microscope and flow cytometer. Cellular apoptosis was detected using flow cytometer. The expression levels of proteins of the ERS-associated apoptosis signaling pathway were detected using Western blotting analysis. The efficacy and biosafety of SNX-2112 were also investigated in a mouse xenograft model. RESULTS: Low-intensity US combined with SNX-2112 exhibited significant antitumor effect, increased the absorption of SNX-2112 by cells even with a low dose, enhanced ROS generation and apoptosis. The combination regimen also inhibited the protein expression of Hsp90 and triggered apoptosis through endoplasmic reticulum stress (ERS) by enhancing PERK, CHOP and Bax protein levels, while downregulating the level of Bcl-2. Additionally, N-acetyl-L-cysteine (NAC), ROS scavenger, was able to reverse these results. Low-intensity US combined with SNX-2112 significantly inhibited tumor growth, prolonged survival of mice, decreased proliferation and promoted apoptosis with no visible damage or abnormalities in major organs in the mouse xenograft model with tongue squamous cell carcinoma. CONCLUSION: The antitumor effects of SNX-2112 were enhanced by low-intensity US. The most probable mechanism was that US sonoporation induced more SNX-2112 delivery to the cells and enhanced ROS production, triggering the ERS-associated apoptosis signaling pathway. Therefore, low-intensity US may increase the efficiency of conventional chemotherapy and reduce the dosage of SNX-2112 required and its side effects.

SNX-2112 Induces Apoptosis and Autophagy of Nara-H Cells.

BACKGROUND/AIM: Selective heat shock protein 90 (Hsp90) inhibitor SNX-2112 exhibits antitumor activity in multiple cancer cell types. Here, the antitumor activity of SNX-2112 in Nara-H cells was analyzed. MATERIALS AND METHODS: Antitumor activity of SNX-2112 was assessed using a cell proliferation assay. We also examined the signalling pathways involved in SNX-2112-mediated autophagy and apoptosis of Nara-H cells by western blot and morphological analyses. RESULTS: Cell proliferation assays demonstrated that SNX-2112 inhibited Nara-H cell growth. Western blotting revealed that SNX-2112 induced apoptosis and autophagy, inhibited mammalian target of rapamycin (mTOR) phosphorylation, and suppressed the mitogen-activated protein kinase (MAPK) signalling pathway. Morphological analysis confirmed that SNX-2112 induced autophagy and apoptosis. CONCLUSION: SNX-2112 induced autophagy and apoptosis of Nara-H cells by inhibiting mTOR and MAPK pathways. Our results support developing SNX-2112 to treat human soft tissue sarcomas.

Chitosan-functionalised single-walled carbon nanotube-mediated drug delivery of SNX-2112 in cancer cells.

Delivery of amphiphobic drugs (insoluble in both water and oil) has been a great challenge in drug delivery. SNX-2112, a novel inhibitor of Hsp90, is a promising drug candidate for treating various types of cancers; however, the insolubility greatly limits its clinical application. This study aimed to build a new type of drug delivery system using single-walled carbon nanotubes (SWNTs) for controllable release of SNX-2112; chitosan (CHI) was non-covalently added to SWNTs to improve their biocompatibility. SWNTs-CHI demonstrated high drug-loading capability; the release of SNX-2112 was pH triggered and time related. The intracellular reactive oxygen species of SWNTs-CHI increased, compared with that of SWNTs, leading to higher mitogen-activated protein kinase and cell apoptosis. The results of western-blotting, lactate dehydrogenase (LDH) release assay, and cell viability assay analyses indicated that apoptosis-related proteins were abundantly expressed in K562 cells and that the drug delivery system significantly inhibited K562 cells. Thus, SWNT-CHI/SNX-2112 shows great potential as a drug delivery system for cancer therapy.

Phospholipid-stabilized mesoporous carbon nanospheres as versatile carriers for systemic delivery of amphiphobic SNX-2112 (a Hsp90 inhibitor) with enhanced antitumor effect.

Systemic delivery of amphiphobic drugs (insoluble in both water and oil) represents a formidable challenge in drug delivery. This work aimed to engineer a functional mesoporous carbon material to efficiently load SNX-2112, an amphiphobic anticancer agent, and to evaluate its performance in tumor-targeting delivery. Hydrothermal reaction combined with high-temperature activation was used to fabricate glucose-based mesoporous carbon nanospheres (MCNs). SNX-2112-loaded MCNs stabilized by phospholipid (SN-PMCNs) were prepared by the absorption/solvent diffusion/high-pressure homogenization method. The obtained SN-PMCNs were 180nm around in particle size, showing a high drug load (42.7%) and acceptable physical stability. SN-PMCNs demonstrated an enhanced in vitro antitumor effect and increased uptake into cancer cells in comparison with the formulation of SNX-2112 solution (SN-Sol). The in vivo antitumor effect and biodistribution in 4T1 xenograft tumor mice, a breast cancer model, were also significantly improved through SN-PMCNs. It was shown that specific clathrin-dependent and nonspecific caveolae-dependent endocytosis were involved in the cellular trafficking of SN-PMCNs. Glucose transporter-mediated transport, prolonged body residence time and improved biodistribution via EPR effect were the main mechanisms of enhanced antitumor effect. SN-PMCNs have presented excellent tumor targeting properties and should be a promising carrier to address the systemic delivery of SNX-2112.

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.

Exploring the potential of self-assembled mixed micelles in enhancing the stability and oral bioavailability of an acid-labile drug.

Oral delivery of many drugs is plagued with limited solubility and/or poor stability. This paper aimed to explore the performance of polymeric mixed micelles on solubilization, stabilization and bioavailability enhancement with stiripentol as model drug. Stiripentol-loaded mixed micelles were prepared by solvent-diffusion method: rapid dispersion of an ethanol solution containing stiripentol, monomethoxy poly(ethylene glycol)-b-poly(ε-caprolactone) and sodium oleate into water. Stiripentol micelles were characterized by the particle size, entrapment efficiency, in vitro drug release, TEM, DSC and FTIR. The pharmacokinetic profile of stiripentol was determined in rats after oral administration of stiripentol micelles. The obtained stiripentol micelles were 44.2 nm in size with an entrapment efficiency over 90%. It was shown that micelles substantially improved the solubility and gastric stability of stiripentol. The oral absorption of stiripentol was also enhanced to a great extent with a relative bioavailability of 157% and 444% to the commercial formulation (Diacomit®) and in-house suspensions. Mixed micelles assembled by di-block copolymer/sodium oleate exhibited a good potential in the improvement of drug stability and bioavailability. It should be a promising carrier for oral delivery of therapeuticals with solubility and stability issues.

The Hsp90 inhibitor SNX-2112, induces apoptosis in multidrug resistant K562/ADR cells through suppression of Akt/NF-κB and disruption of mitochondria-dependent pathways.

Heat shock protein 90 (Hsp90) serves as an ATP-dependent molecular chaperone for numerous cell signaling proteins, including many oncogenes and clinically validated cancer targets that are involved in cell proliferation and survival. Recent studies have shown that the Hsp90 inhibitor, SNX-2112, effectively inhibits tumor cell growth and angiogenesis in hematological and solid tumors. However, little is known about the effects of SNX-2112 on leukemias that are resistant to chemotherapy, which is emerging as a major clinical problem. In this study, the effects of SNX-2112 on the multidrug-resistant human chronic myeloid leukemia (CML) K562/ADR cell line were investigated. We observed that SNX-2112 exhibited dose- and time-dependent inhibitory activities against K562/ADR cells. These effects included the induction of apoptosis and secondary necrosis in addition to cell cycle arrest at the G1 and G2 phases. Furthermore, SNX-2112-induced apoptosis was predominantly mediated by the mitochondrial pathway, initiated by the release of cytochrome c and the participation of Bcl-2 family proteins. SNX-2112 also induced the activation of the caspase-3, -8 and -9 cascade and the subsequent cleavage of PARP in K562/ADR cells. Moreover, the inactivation of the Akt and NF-κB signaling pathways may be involved in SNX-2112-induced apoptosis. The expression levels of P-glycoprotein (P-gp) and several chaperons related to drug resistance and apoptosis were also shown to be inhibited, including the Grp78 and Hsp90 isoforms, Grp94 and Trap1. Taken together, these results provide a possible molecular mechanism for the anti-cancer effect of SNX-2112 on K562/ADR cells and provide new insights into the future application of SNX-2112 as a therapeutic agent for anti-multidrug-resistant leukemias.