17-(dimethylaminoethylamino)-17-demethoxygeldanamycin and Lung-Neoplasms

We have previously identified receptor tyrosine kinase-like orphan receptor 1 (ROR1) as a direct transcriptional target of TTF-1/NKX2-1, a lineage-survival oncogene in lung adenocarcinoma. ROR1 sustains prosurvival signaling from multiple receptor tyrosine kinases including epidermal growth factor receptor, MET, and insulin-like growth factor 1 receptor in part by maintaining the caveolae structure as a scaffold protein of cavin-1 and caveolin-1. In this study, a high throughput screening of the natural product library containing 2560 compounds was undertaken using a cell-based FluoPPI assay detecting ROR1-cavin-1 interaction. As a result, geldanamycin (GA), a known inhibitor of heat shock protein 90 (HSP90), was identified as a potential inhibitor of ROR1. Geldanamycin, as well as two GA derivatives tested in the clinic, 17-allylamino-17-demethoxygeldanamycin (17-AAG) and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), decreased ROR1 protein expression. We found that ROR1 physically interacted with HSP90α, but not with other HSP90 paralogs, HSP90β or GRP94. Geldanamycin in turn destabilized and degraded ROR1 protein in a dose- and time-dependent manner through the ubiquitin/proteasome pathway, resulting in a significant suppression of cell proliferation in lung adenocarcinoma cell lines, for which the kinase domain of ROR1, but not its kinase activity or N-glycosylation, was required. Our findings indicate that HSP90 is required to sustain expression of ROR1 crucial for lung adenosarcoma survival, suggesting that inhibition of HSP90 could be a promising therapeutic strategy in ROR1-positive lung adenocarcinoma.

Topics: Adenocarcinoma of Lung; Antibiotics, Antineoplastic; Benzoquinones; Cell Line, Tumor; Cell Proliferation; Drug Screening Assays, Antitumor; Gene Knockdown Techniques; High-Throughput Screening Assays; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Lung Neoplasms; Proteasome Endopeptidase Complex; Proteolysis; Receptor Tyrosine Kinase-like Orphan Receptors; RNA-Binding Proteins

Up-regulation of heat shock protein 90 (HSP90) gene takes place in lung cancer cells. Therefore, targeting HSP90 in lung cancer may be promising step in lung cancer therapy. The present study aimed to evaluate the efficiency of implantable 17-dimethylaminoethylamino-17-demethoxy geldanamycin (17-DMAG)-loaded Poly(caprolactone)-poly(ethylene glycol) (PCL/PEG) nanofibers to increase the anti-cancer effects via inhibition of HSP90 expression and telomerase activity. For this purpose, 17-DMAG-loaded PCL/PEG nanofibers were successfully fabricated via electrospinning and characterized using FE-SEM and FTIR. Colorimetric MTT assay was used to determine the drug cytotoxicity. Also, the expression levels of HSP90 mRNA in the A549 cells treated with the nanofibers were assessed using Quantitative Real-Time PCR. The effect of free 17-DMAG and 17-DMAG-loaded PCL/PEG nanofiber treatment on telomerase activity was monitored by TRAP assay. MTT assay confirmed that loading of 17-DMAG into PCL/PEG nanofiber enhanced dramatically cytotoxicity in the lung cancer cells. This finding was associated with reduction of HSP90 mRNA expression and telomerase activity in the cells seeded on 17-DMAG-loaded PCL/PEG nanofibers in relative to free 17-DMAG. In conclusion, the findings demonstrated that 17-DMAG-loaded PCL/PEG nanofibers are more effectual than free 17-DMAG against A549 lung cancer cells via modulation of Hsp90 expression and inhibition of telomerase activity. Hence, the implantable 17-DMAG-loaded nanofibrous scaffolds might be an excellent tool for efficiently killing of the lung residual cancer cells and avoid the local cancer recurrence.

Topics: A549 Cells; Benzoquinones; Cell Proliferation; Dose-Response Relationship, Drug; Drug Carriers; Drug Delivery Systems; Drug Implants; Gene Expression; Growth Inhibitors; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Lung Neoplasms; Nanofibers

Small-cell lung cancer (SCLC) accounts for nearly 15% of lung cancer cases and exhibits aggressive clinical behavior characterized by rapid growth and metastatic spread to multiple organs. About 70% of patients with SCLC present with extensive disease and distant metastases at diagnosis. HSP90 is a 90-kDa molecular chaperone whose association is required for the stability and function of its numerous "client proteins." Here, we assessed the therapeutic potential of the HSP90 inhibitor 17-DMAG in SCLC. Notably, 17-DMAG hindered the viability of human SCLC cell lines-regardless of their chemosensitivity-via the decreased expression of client proteins, including the proto-oncogene c-Raf (also known as RAF1). In an in vivo imaging model of SCLC multiple-organ metastasis with the human SCLC cell line SBC-5, treatment with 17-DMAG remarkably inhibited the formation of metastatic sites in the liver, but was ineffective in hindering the progression of bone lesions. The latter was likely the result of activation of osteoclasts. IGF-1, which is supposed to be rich in bone environment, preserved c-Raf expression and maintained viability of SBC-5 cells treated with 17-DMAG. Furthermore, the combined use of a bisphosphonate with 17-DMAG significantly attenuated the progression of metastases in both the liver and the bone. These findings suggest that therapeutic effects of HSP90 inhibitors may be organ-specific and should be carefully monitored in SCLC clinical trials.

Topics: Animals; Benzoquinones; Bone Neoplasms; Cell Line, Tumor; Cell Proliferation; Diphosphonates; HSP90 Heat-Shock Proteins; Humans; Imidazoles; Lactams, Macrocyclic; Liver Neoplasms; Lung Neoplasms; Male; Mice; Organ Specificity; Proto-Oncogene Mas; Proto-Oncogene Proteins c-raf; Small Cell Lung Carcinoma; Zoledronic Acid

Because anaplastic lymphoma kinase (ALK) is dependent on Hsp90 for protein stability, Hsp90 inhibitors are effective in controlling growth of lung cancer cells with ALK rearrangement. We investigated the mechanism of acquired resistance to 17-(Dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), a geldanamycin analogue Hsp90 inhibitor, in H3122 and H2228 non-small cell lung cancer cell lines with ALK rearrangement.. Resistant cell lines (H3122/DR-1, H3122/DR-2 and H2228/DR) were established by repeated exposure to increasing concentrations of 17-DMAG. Mechanisms for resistance by either NAD(P)H/quinone oxidoreductase 1 (NQO1), previously known as a factor related to 17-DMAG resistance, or P-glycoprotein (P-gp; ABCB1/MDR1) were queried using RT-PCR, western blot analysis, chemical inhibitors, the MTT cell proliferation/survival assay, and cellular efflux of rhodamine 123.. The resistant cells showed no cross-resistance to AUY922 or ALK inhibitors, suggesting that ALK dependency persists in cells with acquired resistance to 17-DMAG. Although expression of NQO1 was decreased in H3122/DR-1 and H3122/DR-2, NQO1 inhibition by dicumarol did not affect the response of parental cells (H2228 and H3122) to 17-DMAG. Interestingly, all resistant cells showed the induction of P-gp at the protein and RNA levels, which was associated with an increased efflux of the P-gp substrate rhodamine 123 (Rho123). Transfection with siRNA directed against P-gp or treatment with verapamil, an inhibitor of P-gp, restored the sensitivity to the drug in all cells with acquired resistance to 17-DMAG. Furthermore, we also observed that the growth-inhibitory effect of 17-DMAG was decreased in A549/PR and H460/PR cells generated to over-express P-gp by long-term exposure to paclitaxel, and these cells recovered their sensitivity to 17-DMAG through the inhibition of P-gp.. P-gp over-expression is a possible mechanism of acquired resistance to 17-DMAG in cells with ALK rearrangement.

Topics: Anaplastic Lymphoma Kinase; ATP Binding Cassette Transporter, Subfamily B; Benzoquinones; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dicumarol; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Lactams, Macrocyclic; Lung Neoplasms; NAD(P)H Dehydrogenase (Quinone); Paclitaxel; Receptor Protein-Tyrosine Kinases

Alectinib is a new generation ALK inhibitor with activity against the gatekeeper L1196M mutation that showed remarkable activity in a phase I/II study with echinoderm microtubule associated protein-like 4 (EML4)--anaplastic lymphoma kinase (ALK) non-small cell lung cancer (NSCLC) patients. However, alectinib resistance may eventually develop. Here, we found that EGFR ligands and HGF, a ligand of the MET receptor, activate EGFR and MET, respectively, as alternative pathways, and thereby induce resistance to alectinib. Additionally, the heat shock protein 90 (Hsp90) inhibitor suppressed protein expression of ALK, MET, EGFR, and AKT, and thereby induced apoptosis in EML4-ALK NSCLC cells, even in the presence of EGFR ligands or HGF. These results suggest that Hsp90 inhibitors may overcome ligand-triggered resistance to new generation ALK inhibitors and may result in more successful treatment of NSCLC patients with EML4-ALK.

Topics: Benzoquinones; Blotting, Western; Carbazoles; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Hepatocyte Growth Factor; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Ligands; Lung Neoplasms; Mutation; Oncogene Proteins, Fusion; Phosphorylation; Piperidines; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-met; Transforming Growth Factor alpha; Triazoles

Lung cancer remains the most common cause of cancer-related death worldwide. This malignancy is a complex disease, and it is important to identify potential biological targets, the blockade of which would affect multiple downstream signaling cascades. A growing number of reports recognize novel therapeutic targets in the protein homeostasis network responsible for generating and protecting the protein fold. The heat shock protein 90 (Hsp90) is an essential molecular chaperon involved in the posttranslational folding and stability of proteins. It is required for conformational maturation of multiple oncogenic kinases that drive signal transduction and proliferation of cancer cells. However, in the case of unfolded protein accumulation endoplasmic reticulum (ER) stress is induced and several response pathways such as proteasome functions are activated. The ubiquitin-proteasome system orchestrates the turnover of innumerable cellular proteins. Here, we suggest that the therapeutic efficacy of Hsp90 inhibition may be augmented by coadministering proteasome inhibitor on human non-small-cell lung cancer (NSCLC) cell lines. Indeed, we showed that coadministration of the Hsp90 inhibitor 17-demethoxygeldanamycin (17-DMAG) and proteasome inhibitor (velcade) induced ER stress evidenced by increased unfolded protein response markers. The consequences were evident in multiple aspects of the NSCLC phenotype: reduced viability and cell count, increased apoptotic cell death, and most profoundly, synergistically decreased cell motility. Our findings provide proof-of-concept that targeting ER homeostasis is therapeutically beneficial in NSCLC cell lines.

Topics: Antineoplastic Agents; Apoptosis; Benzoquinones; Biomarkers, Tumor; Boronic Acids; Bortezomib; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Drug Synergism; Endoplasmic Reticulum Stress; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Lung Neoplasms; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Pyrazines; Unfolded Protein Response

Up-regulation of hsp90 gene expression occurs in numerous cancers such as lung cancer. D,L-lactic-co-glycolic acid-poly ethylene glycol-17-dimethylaminoethylamino-17-demethoxy geldanamycin (PLGA-PEG-17DMAG) complexes and free 17-DMAG may inhibit the expression. The purpose of this study was to examine whether nanocapsulating 17DMAG improves the anti cancer effect over free 17DMAG in the A549 lung cancer cell line.. Cells were grown in RPMI 1640 supplemented with 10% FBS. Capsulation of 17DMAG is conducted through double emulsion, then the amount of loaded drug was calculated. Other properties of this copolymer were characterized by Fourier transform infrared spectroscopy and H nuclear magnetic resonance spectroscopy. Assessment of drug cytotoxicity on the grown of lung cancer cell line was carried out through MTT assay. After treatment, RNA was extracted and cDNA was synthesized. In order to assess the amount of hsp90 gene expression, real-time PCR was performed.. In regard to the amount of the drug load, IC50 was significant decreased in nanocapsulated(NC) 17DMAG in comparison with free 17DMAG. This was confirmed through decrease of HSP90 gene expression by real-time PCR.. The results demonstrated that PLGA-PEG-17DMAG complexes can be more effective than free 17DMAG in down-regulating of hsp90 expression by enhancing uptake by cells. Therefore, PLGA-PEG could be a superior carrier for this kind of hydrophobic agent.

Topics: Benzoquinones; Cell Survival; Dose-Response Relationship, Drug; Drug Carriers; Gene Expression Regulation, Neoplastic; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Lung Neoplasms; Nanoparticles; Real-Time Polymerase Chain Reaction; Tumor Cells, Cultured

In the clinical treatment of patients with non-small cell lung cancer (NSCLC), the primary and acquired resistance of epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) limits its clinical application, this need to explore new strategy or method to overcome this problem. Recently, some literatures have indicated that the antitumor role of heat shock protein 90 (HSP90) inhibitors by a variety of pathways may provide new strategy for resolving this problem. In this study, we examined the effect of 17-DMAG on NSCLC cell lines A549 and H1975 which were primary and acquired resistant to EGFR-TKI respectively, the purpose was to explore its influence on cell proliferation, apoptosis and the expression of EGFR in vitro as well as possible mechanism.. After A549 and H1975 cell lines were treated with different concentrations of 17-DMAG respectively, the inhibitory rate of cell proliferation was measured by MTT assay in 24 h, 48 h and 72 h. We investigated the effect of 17-DMAG on the cell apoptosis with flow cytometry and the expression of HSP90 and EGFR with Western blot after treated with 17-DMAG for 48 h.. After treated with 17-DMAG, the inhibitory rate of different concentrations and time groups was significant (P<0.01), and the effect was in time- and dose-dependent manner; the apoptosis rate of both two cell lines in all treated groups were significantly higher than control group (P<0.01), and the effect was in dose-dependent manner. By Western blot analysis, there was no significant difference between all treated groups and control group for the expression of both HSP90 and EGFR protein in A549 cell line and HSP90 protein in H1975 cell line after exposed to 17-DMAG for 48 h (P>0.05), while the difference was significant for the expression of EGFR protein in H1975 cell line (P<0.01).. 17-DMAG inhibited the proliferation of NSCLC cell lines A549 and H1975 and also induced apoptosis of both cell lines. It down-regulated the expression of mutant EGFR protein while this phenomenon was not observed in EGFR-wild type cell line. This suggested that the mechanism maybe different between A549 and H1975 cell lines with different genetic backgroud. Our study provided new strategy for treatment with NSCLC being resistant to EGFR-TKI.. 背景与目的 表皮生长因子受体酪氨酸激酶抑制剂（epidermal growth factor receptor-tyrosine kinase inhibitor, EGFR-TKI）在非小细胞肺癌（non-small cell lung cancer, NSCLC）患者的临床治疗中产生的原发性及获得性耐药限制了其临床应用，需要探索新的策略或方法来克服这个问题。最近有文献报道认为热休克蛋白90（heat shock protein 90, HSP90）抑制剂能从多种途径和环节发挥抗肿瘤作用，这为解决NSCLC对EGFR-TKI的耐药提供了新的思路。本研究通过观察HSP90抑制剂17-DMAG对EGFR-TKI分别原发性及获得性耐药的NSCLC细胞株A549和H1975的作用，旨在探讨它对细胞增殖、凋亡与EGFR蛋白表达的影响及其可能的机制。方法 以不同浓度的17-DMAG分别作用于A549和H1975细胞株24 h、48 h、72 h，应用四甲基偶氮唑蓝（MTT）比色法检测细胞增殖；作用48 h后，应用流式细胞术PI单染法检测细胞凋亡，并应用Western blot检测细胞HSP90及EGFR蛋白表达水平。结果 17-DMAG在不同药物浓度和作用时间对A549和H1975细胞的增殖抑制率差异均有统计学意义（P<0.01），且呈时间和剂量依赖性；两种细胞不同药物浓度组和空白对照组之间的凋亡率差异均有统计学意义（P<0.01），且呈剂量依赖性； 17-DMAG作用48 h后，A549细胞的EGFR/GADPH和HSP90/GADPH及H1975细胞的HSP90/GADPH在不同药物浓度组和空白对照组之间的灰度比值差异均无统计学意义（P>0.05），而H1975细胞的EGFR/GADPH在不同药物浓度组和空白对照组之间的灰度比值差异均有统计学意义（P<0.01）。结论 17-DMAG对NSCLC细胞株A549和H1975均具有抑制增殖及促进凋亡作用，且它能降低突变型EGFR的蛋白表达水平，而对野生型EGFR的蛋白表达无明显影响。本研究为EGFR-TKI耐药的非小细胞肺癌提供了新的治疗策略。

Topics: Antineoplastic Agents; Apoptosis; Benzoquinones; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; ErbB Receptors; Flow Cytometry; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Lung Neoplasms; Protein Kinase Inhibitors; Time Factors

Sensitivity to a tyrosine kinase inhibitor (TKI) is correlated with the presence of somatic mutations that affect the kinase domain of epidermal growth factor receptor (EGFR). Development of resistance to TKI is a major therapeutic problem in non-small cell lung cancer (NSCLC). Aim of this study is to identify agents that can overcome TKI resistance in NSCLC.. We used a carefully selected panel of 12 NSCLC cell lines to address this clinical problem. Initially, the cell lines were treated with a variety of 10 compounds. Cellular proliferation was measured via MTT assay. We then focused on the gefitinib-resistant, EGFR mutant cell lines [H1650: exon 19 and PTEN mutations; and H1975: exons 20 (T790M) and 21 (L858R)] to identify agents that could overcome TKI resistance.. Both 17-DMAG (Hsp90 inhibitor) and belinostat (histone deacetylase inhibitor, HDACi) effectively decreased the growth of almost all NSCLC lines. Also, belinostat markedly decreased the expression of EGFR and phospho-Akt in the cells. Combination of 17-DMAG and belinostat synergistically inhibited in vitro proliferation of these cells. Furthermore, both agents and their combination almost completely prevented TKI-resistant tumor formation (EGFR T790M mutation) in a xenograft model.. These results suggest that the combination of 17-DMAG and belinostat should be examined in a clinical trial for TKI-resistant NSCLC cell.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Benzoquinones; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Neoplasm; Drug Synergism; ErbB Receptors; Gefitinib; Humans; Hydroxamic Acids; Lactams, Macrocyclic; Lung Neoplasms; Mice; Mice, Nude; Mutation; Protein Kinase Inhibitors; PTEN Phosphohydrolase; Quinazolines; Sulfonamides; Xenograft Model Antitumor Assays

The tumor necrosis factor (TNF) and the cellular NF-κB pathway protein IKKβ play important roles in various cellular processes such as cell proliferation, survival, differentiation, and apoptosis. A heat shock protein 90 inhibitor, 17-DMAG, can induce apoptosis of some tumor cells. This study is to determine the combined effects of 17-DMAG and TNF on malignant cells and the related mechanisms.. We have determined effects of 17-DMAG, an Hsp90 inhibitor, and TNF treatments on the small cell lung cancer cell line (MS-1), the adenocarcinoma cell line (A549), the squamous-cell carcinoma cell line (LK-2), and the normal human bronchial epithelium cell line (NuLi-1) by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrozolium bromide assay. To determine if 17-DMAG inhibit the expression of IKKβ in the normal human NuLi-1 cells, and the malignant MS-1, A549, and LK-2 cells, immunoblotting assays and luciferase assays were performed.. It was found that the combined treatments resulted in synergistic killing of malignant cells, which was confirmed by the apoptosis determination using a fluorescence microscopic assay following staining of the drug-treated cells with Hoescht 33258. The immunoblotting results indicated that the synergistic killing due to 17-DMAG and TNF treatments may be related to the decreases in IKKβ levels in the presence of 17-DMAG.. The results suggest that combination of 17-DMAG and TNF treatments might be useful for treating malignancies upon further study in the further.. The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2041198513886824.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Benzoquinones; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Drug Synergism; Gene Expression Regulation, Neoplastic; HSP90 Heat-Shock Proteins; Humans; I-kappa B Kinase; Lactams, Macrocyclic; Lung Neoplasms; NF-kappa B; Signal Transduction; Time Factors; Transcription, Genetic; Transfection; Tumor Necrosis Factor-alpha

Acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), gefitinib and erlotinib, is frequently observed after initiation of TKIs therapy. Non-small-cell lung cancers (NSCLC) with activating EGFR mutations were reported to be sensitive to heat shock protein 90 (Hsp90) inhibitors regardless of the secondary TKI-resistant T790M mutation. We established EGFR-TKI resistant clones for PC-9 cell lines, harboring EGFR exon 19 deletions, with or without the secondary T790M mutation. We examined the anti-proliferative effect of 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), an orally active Hsp90 inhibitor, on the growth of NSCLC cell lines in vitro and in vivo. In MTS assay, the IC(50) values of 17-DMAG for 13 EGFR-mutant cell lines including eight EGFR-TKI resistant cell lines ranged from 0.04 to 0.16 μM while those for seven EGFR-wild type cell lines ranged from 1.6 to 27.4 μM. Western blot analysis revealed that phospho-EGFR, phospho-Akt, phospho-MAPK, cdk4, and cyclin D1 were more readily depleted by 17-DMAG treatment in EGFR-mutant cell lines than in EGFR-wild type cell lines. Cleaved PARP expression confirmed apoptosis in response to 17-DMAG treatment in EGFR-mutant cell lines but not in EGFR-wild type cell lines. In mice xenograft models, 17-DMAG significantly reduced the growth of EGFR-mutant lines irrespective of T790M mutation. These results suggested that 17-DMAG is a potential novel therapeutic agent for NSCLC patients with EGFR mutations with or without EGFR-TKI resistance.

Topics: Animals; Antineoplastic Agents; Apoptosis; Benzoquinones; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Drug Resistance, Neoplasm; ErbB Receptors; Erlotinib Hydrochloride; Gefitinib; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Lung Neoplasms; Mice; Mutation; Quinazolines; Xenograft Model Antitumor Assays

The three major clinically relevant mechanisms of acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in EGFR mutant lung cancer are a second mutation in the EGFR gene (T790M), Met amplification, and increased expression of hepatocyte growth factor (HGF). Heat shock protein90 (Hsp90) is a 90 kDa molecular chaperone for proteins that include EGFR, Met, and echinoderm microtubule-associated proetin-like-4-the anaplastic lymphoma kinase. Here, we determined whether inhibition of Hsp90 could overcome HGF-triggered EGFR-TKI resistance in EGFR mutant lung cancer cells.. The effects of the Hsp90 inhibitor 17-demethoxygeldanamycin (17-DMAG) on the growth of lung cancer cells resistant to the EGFR-TKI were examined in the presence and absence of HGF, and in cells transfected with the HGF gene in vitro and in vivo.. EGFR-TKI erlotinib did not inhibit the growth of HGF-gene transfected Ma-1 (Ma-1/HGF) cells and H1975 cells, containing the EGFR L858R and T790M mutations, respectively. Erlotinib also did not inhibit the growth of PC-9 and Ma-1 cells, with deletions in EGFR exon19, in the presence of HGF. However, 17-DMAG induced apoptosis and markedly inhibited the growth of these cell lines, even in the presence of HGF. This inhibition by 17-DMAG was associated with decreased expression of EGFR and Met in tumor cells. An in vivo model of HGF-triggered erlotinib-resistance, which used Ma-1/HGF cells, showed that 17-DMAG markedly suppressed tumor growth by decreasing angiogenesis and increasing apoptosis.. Hsp90 inhibitors may overcome HGF-triggered resistance to EGFR-TKIs and may result in more successful treatment of patients with EGFR-mutant lung cancers.

Topics: Adenocarcinoma; Animals; Apoptosis; Benzoquinones; Blotting, Western; Drug Resistance, Neoplasm; ErbB Receptors; Female; Hepatocyte Growth Factor; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Lung Neoplasms; Mice; Mutation; Prognosis; Protein Kinase Inhibitors; Tyrosine; Xenograft Model Antitumor Assays

Inhibition of heat shock protein 90 (HSP90) leads to inappropriate processing of proteins involved in cell survival pathways. We found that HSP90 inhibitor, 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (DMAG), is synergistic with radiation for non-small cell lung cancer cell lines, NCI-H460 and A549. To establish the optimal schedule for this combination, cells were radiated before, after, or simultaneously with DMAG, and survival was scored by clonogenic assay. The sequence of DMAG administration was critical for synergy with radiation, and pretreatment for 16 h led to maximal synergy. Similar radiosensitization was observed in isogenic cells in which expression of wild-type p53 was silenced by RNA interference, although p53 loss rendered cells overall less radiosensitive. The mechanistic basis for synergy was studied by Western blotting, cell cycle analysis, alkaline comet assay, and direct measurement of the activities of key base excision repair enzymes. Regardless of schedule of administration, DMAG led to degradation of proteins involved in activation of cell survival pathways after radiation, which did not explain the differences in the schedule of administration observed in clonogenic assays. In addition to previously reported decrease in activation of ATM, pretreatment with DMAG blocked activation of base excision repair machinery and activity of key enzymes, apurinic/apyrimidinic endonuclease, and DNA polymerase-beta. Similarly, pretreatment with specific apurinic/apyrimidinic endonuclease inhibitor, CRT0044876, reproduced the effects of DMAG. Thus, administration of HSP90 inhibitors before radiation is critical for optimizing their use as radiosensitizers.

Topics: Ataxia Telangiectasia Mutated Proteins; Benzoquinones; Cell Cycle Proteins; Cell Line, Tumor; Cell Survival; DNA Repair; DNA-Binding Proteins; Down-Regulation; Drug Screening Assays, Antitumor; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Lung Neoplasms; Protein Serine-Threonine Kinases; Radiation; Radiation Tolerance; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

The heat shock protein 90 (Hsp90) has a critical role in malignant transformation. Whereas its ability to maintain the functional conformations of mutant and aberrant oncoproteins is established, a transformation-specific regulation of the antiapoptotic phenotype by Hsp90 is poorly understood. By using selective compounds, we have discovered that small-cell lung carcinoma is a distinctive cellular system in which apoptosis is mainly regulated by Hsp90. Unlike the well-characterized antiapoptotic chaperone Hsp70, Hsp90 is not a general inhibitor of apoptosis, but it assumes this role in systems such as small-cell lung carcinoma, in which apoptosis is uniquely dependent on and effected through the intrinsic pathway, without involvement of caspase elements upstream of mitochondria or alternate pathways that are not apoptosome-channeled. These results provide important evidence for a transformation-specific interplay between chaperones in regulating apoptosis in malignant cells.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Small Cell; Cell Line, Tumor; Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; Drug Design; Drug Screening Assays, Antitumor; Gene Expression Regulation, Neoplastic; HSP90 Heat-Shock Proteins; Humans; Lung Neoplasms; Models, Chemical; Phosphatidylinositol 3-Kinases; Time Factors

To describe the preclinical basis for further development of 17-dimethyl aminoethylamino-17-demethoxygeldanamycin hydrochloride (17-DMAG, NSC 707545).. In vitro proliferation assays, and in vivo model studies in metastatic pancreatic carcinoma and subcutaneous xenograft melanoma and small-cell lung carcinoma models.. 17-DMAG emerged from screening studies as a potent geldanamycin analog, with the average concentration inhibiting the growth of the NCI anticancer cell line drug screen by 50% being 0.053 microM. "Head to head" comparison with 17-allylamino-17-demethoxygeldanamycin (17-AAG, NSC 330507) revealed 17-DMAG to possess potent activity against certain cell types, e.g., MDA-MB-231 breast carcinoma and HL60-TB leukemia which were relatively insensitive to 17-AAG. Evidence of oral bioavailability of 17-DMAG in a saline-based formulation prompted more detailed examination of its antitumor efficacy in vivo. 17-DMAG inhibited the growth of the AsPC-1 pancreatic carcinoma xenografts growing as intrahepatic metastases at doses of 6.7-10 mg/kg twice daily for 5 days administered orally under conditions where 17-AAG was without activity. 17-DMAG in an aqueous vehicle at 7.5-15 mg/kg per day for 3 days on days 1-3, 8-10 and 13-17, or 1-5 and 8-12 showed evidence of antitumor activity by the parenteral and oral routes in the MEXF 276 and MEXF 989 melanomas and by the parenteral route in the LXFA 629 and LXFS 650 adenocarcinoma and small-cell carcinoma models. The latter activity was comparable to the historical activity of 17-AAG.. Taken together, the in vivo activity of 17-DMAG supports the further development of this water-soluble and potentially orally administrable geldanamycin congener.

Topics: Animals; Benzoquinones; Carcinoma, Non-Small-Cell Lung; Cell Proliferation; Drug Screening Assays, Antitumor; Lactams, Macrocyclic; Liver Neoplasms; Lung Neoplasms; Male; Melanoma; Mice; Mice, Nude; Quinones; Skin Neoplasms; Solubility; Transplantation, Heterologous; Tumor Cells, Cultured