monorden and Breast-Neoplasms

Drug-induced resistance, or tolerance, is an emerging yet poorly understood failure of anticancer therapy. The interplay between drug-tolerant cancer cells and innate immunity within the tumor, the consequence on tumor growth, and therapeutic strategies to address these challenges remain undescribed. Here, we elucidate the role of taxane-induced resistance on natural killer (NK) cell tumor immunity in triple-negative breast cancer (TNBC) and the design of spatiotemporally controlled nanomedicines, which boost therapeutic efficacy and invigorate "disabled" NK cells. Drug tolerance limited NK cell immune surveillance via drug-induced depletion of the NK-activating ligand receptor axis, NK group 2 member D, and MHC class I polypeptide-related sequence A, B. Systems biology supported by empirical evidence revealed the heat shock protein 90 (Hsp90) simultaneously controls immune surveillance and persistence of drug-treated tumor cells. On the basis of this evidence, we engineered a "chimeric" nanotherapeutic tool comprising taxanes and a cholesterol-tethered Hsp90 inhibitor, radicicol, which targets the tumor, reduces tolerance, and optimally reprimes NK cells via prolonged induction of NK-activating ligand receptors via temporal control of drug release

Topics: Animals; Antineoplastic Agents, Immunological; Breast Neoplasms; Cell Line, Tumor; Cholesterol; Docetaxel; Drug Delivery Systems; Drug Liberation; Drug Resistance, Neoplasm; Female; HSP90 Heat-Shock Proteins; Humans; Killer Cells, Natural; Macrolides; Mice, Inbred BALB C; Molecular Targeted Therapy; Nanoparticles; Triple Negative Breast Neoplasms; Tumor Microenvironment

Previously, we reported the Hsp90 inhibitory activity of radamide, an open chain amide chimera of geldanamycin and radicicol. Attempts to further expand upon structure-activity relationships for this class of Hsp90 inhibitors led to the preparation of a series of radamide analogues focused on differing tether lengths and quinone mimics. In addition, the cup-shaped conformation adopted by the two natural products when bound to the Hsp90 N-terminal ATP binding pocket suggests that conformationally biased compounds may demonstrate improved binding and inhibition. The preparation and evaluation of radamide analogues with cis/trans alpha,beta-unsaturated amides yielded compounds that exhibit improved antiproliferative activity. In addition, several analogues demonstrated the ability to induce degradation of Hsp90-dependent oncogenic signaling proteins in vitro, a hallmark of Hsp90 N-terminal inhibition.

Topics: Acetanilides; Adenosine Triphosphatases; Antineoplastic Agents; Benzoates; Benzoquinones; Binding Sites; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Drug Design; Female; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Lactones; Macrolides; Molecular Structure; Receptor, ErbB-2; Structure-Activity Relationship

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.

Topics: Adenine; Anisoles; Antineoplastic Agents; Benzoquinones; Breast Neoplasms; Cell Line, Tumor; Dimerization; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Macrolides; Peptide Fragments

Topics: Animals; Antineoplastic Agents; Benzoquinones; Breast Neoplasms; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Macrolides; Mice; Mice, Inbred Strains; Small Molecule Libraries; Structure-Activity Relationship; Xenograft Model Antitumor Assays

In an effort to discover small molecule inhibitors of Hsp90, we have screened over 500 EtOAc extracts of Sonoran desert plant-associated fungi using a two-stage strategy consisting of a primary cell-based heat shock induction assay (HSIA) followed by a secondary biochemical luciferase refolding assay (LRA). Bioassay-guided fractionation of extracts active in these assays derived from Chaetomium chiversii and Paraphaeosphaeria quadriseptata furnished the Hsp90 inhibitors radicicol (1) and monocillin I (2), respectively. In SAR studies, 1, 2, and their analogues, 3-16, were evaluated in these assays, and the antiproliferative activity of compounds active in both assays was determined using the breast cancer cell line MCF-7. Radicicol and monocillin I were also evaluated in a solid-phase competition assay for their ability to bind Hsp90 and to deplete cellular levels of two known Hsp90 client proteins with relevance to breast cancer, estrogen receptor (ER), and the type 1 insulin-like growth factor receptor (IGF-1R). Some inferences on SAR were made considering the crystal structure of the N-terminus of yeast Hsp90 bound to 1 and the observed biological activities of 1-16. Isolation of radicicol and monocillin I in this study provides evidence that we have developed an effective strategy for discovering natural product-based Hsp90 inhibitors with potential anticancer activity.

Topics: Animals; Antineoplastic Agents; Ascomycota; Breast Neoplasms; Desert Climate; HSP90 Heat-Shock Proteins; Humans; Insulin-Like Growth Factor I; Lactones; Luciferases; Macrolides; Molecular Structure; Plants; Rabbits; Receptors, Estrogen; Structure-Activity Relationship; Tumor Cells, Cultured

We report on the discovery of benzo- and pyridino- thiazolothiopurines as potent heat shock protein 90 inhibitors. The benzothiazole moiety is exceptionally sensitive to substitutions on the aromatic ring with a 7'-substituent essential for activity. Some of these compounds exhibit low nanomolar inhibition activity in a Her-2 degradation assay (28-150 nM), good aqueous solubility, and oral bioavailability profiles in mice. In vivo efficacy experiments demonstrate that compounds of this class inhibit tumor growth in an N87 human colon cancer xenograft model via oral administration as shown with compound 37 (8-(7-chlorobenzothiazol-2-ylsulfanyl)-9-(2-cyclopropylamino-ethyl)-9H- purin-6-ylamine).

Topics: Animals; Binding Sites; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Female; HSP90 Heat-Shock Proteins; Humans; Mice; Mice, Nude; Molecular Structure; Purines; Stereoisomerism; Structure-Activity Relationship; Sulfhydryl Compounds; Time Factors; Xenograft Model Antitumor Assays

The modulation of DNA topology by topoisomerase II plays a crucial role during chromosome condensation and segregation in mitosis and has thus become a highly attractive target for chemotherapeutic drugs. However, these drugs are highly toxic, and so new approaches are required. One such strategy is to target topoisomerase II-interacting proteins. Here we report the identification of potential topoisomerase II-associated proteins using immunoprecipitation, followed by 1-D and 2-D gel electrophoresis and MALDI-TOF mass spectrometry. A total of 23 proteins were identified and, of these, 17 were further validated as topoisomerase IIalpha-associated proteins by coimmunoprecipitation and Western blot. Six of the interacting proteins were cellular chaperones, including 3 members of the heat shock protein-90 (Hsp90) family, and so the effect of Hsp90 modulation on the antitumor activity of topoisomerase II drugs was tested using the sulforhodamine B assay, clonogenic assays and a xenograft model. The Hsp90 inhibitors geldanamycin, 17-AAG (17-allylamino-17-demethoxygeldanamycin) and radicicol significantly enhanced the activity of the topoisomerase II poisons etoposide and mitoxantrone in vitro and in vivo. Thus, our method of identifying topoisomerase II-interacting proteins appears to be effective, and at least 1 novel topoisomerase IIalpha-associated protein, Hsp90, may represent a valid drug target in the context of topoisomerase II-directed chemotherapy.

Topics: Adenocarcinoma; Animals; Benzoquinones; Breast Neoplasms; Carcinoma, Adenosquamous; Colonic Neoplasms; DNA Topoisomerases, Type II; Electrophoresis, Gel, Two-Dimensional; Enzyme Inhibitors; Female; HSP90 Heat-Shock Proteins; Humans; Immunoprecipitation; Lactams, Macrocyclic; Lactones; Macrolides; Mice; Mice, Nude; Molecular Chaperones; Neoplasms; Protein Binding; Quinones; Rifabutin; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Transplantation, Heterologous; Tumor Cells, Cultured

Molecular chaperones and co-chaperones, such as heat-shock proteins (Hsp's), play a pivotal role in the adequate folding and the stability of steroid hormone receptors. As shown by immunofluorescence staining and immunoblot analysis, the Hsp90 inhibitor radicicol induced a rapid (within hours) depletion of estrogen receptor-alpha (ER) in MCF-7 and IBEP-2 breast carcinoma cells. Inhibition of proteasomes (MG-132, LLnL) or of protein synthesis (cycloheximide), which both suppressed E(2)-induced downregulation of ER, failed to modify ER degradation caused by radicicol. On the other hand, partial antiestrogens, such as hydroxytamoxifen (a triphenylethylene) and LY 117,018 (a benzothiophene) stabilized ER, making it immune to radicicol-induced degradation. Furthermore, radicicol did not interfere with ER upregulation induced by hydroxytamoxifen. Thus, the current study points to possible variation in the mechanism/pathway of ER breakdown. Besides, the protective effect of partial antiestrogens suggests that ER stability is only compromized by Hsp90 disruption when the receptor is in its native, unliganded form.

Topics: Breast Neoplasms; Cycloheximide; Enzyme Inhibitors; Estrogen Antagonists; Estrogen Receptor alpha; Female; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; HSP90 Heat-Shock Proteins; Humans; Immunoenzyme Techniques; Lactones; Leupeptins; Ligands; Macrolides; Molecular Chaperones; Proteasome Inhibitors; Protein Synthesis Inhibitors; Protein-Tyrosine Kinases; Pyrrolidines; Signal Transduction; Tamoxifen; Thiophenes; Tumor Cells, Cultured

A chimera composed of the natural products radicicol and geldanamycin has been prepared through an amide linkage connecting the resorcinol moiety of radicicol to the quinone ring of geldanamycin. The inhibitory activity of these compounds was determined by their ability to inhibit Hsp90's inherent ATPase activity along with degradation of the Hsp90 client protein, HER-2 in MCF-7 breast cancer cells. [reaction: see text]

Topics: Acetanilides; Adenosine Triphosphatases; Antineoplastic Agents; Benzoates; Benzoquinones; Breast Neoplasms; Cell Line, Tumor; Drug Design; Female; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Lactones; Macrolides; Quinones; Receptor, ErbB-2

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Division; Cyclopropanes; Drug Design; HSP90 Heat-Shock Proteins; Humans; Inhibitory Concentration 50; Lactones; Macrolides; Molecular Structure; Protein Binding; Structure-Activity Relationship; Tumor Cells, Cultured

Novel halohydrin and oxime derivatives of radicicol (1) were prepared and evaluated for their v-src tyrosine kinase inhibitory, antiproliferative, and antitumor activities. Some of the resulting derivatives showed significantly improved antitumor activities than those of 1 in vitro as tested in a cell proliferation assay and in vivo using sc-inoculated human breast carcinoma and epidermoid tumor models. Design and synthesis of radicicol-based novel affinity probes are also described.

Topics: Antineoplastic Agents; Breast Neoplasms; Carcinoma, Squamous Cell; Chromatography, High Pressure Liquid; Drug Screening Assays, Antitumor; Enzyme Inhibitors; Female; Humans; Indicators and Reagents; Lactones; Macrolides; Oximes; src-Family Kinases; Structure-Activity Relationship; Tumor Cells, Cultured

The estrogen receptor (ER) is a hormone-dependent transcription factor that belongs to the steroid/thyroid hormone receptor superfamily. Since the ER contributes to development and progression in human breast cancer, a number of studies have explored ways to inactivate this receptor. Previous studies have suggested that the 90-kDa heat shock protein (Hsp90) interacts with the ER, thus stabilizing the receptor in an inactive state. Here, we report that radicicol, an Hsp90-specific inhibitor, repressed estrogen-dependent transactivation of the ER as measured by pS2 gene transcription and a reporter gene encoding an estrogen-responsive element. Furthermore, we showed that radicicol induced rapid degradation of ERalpha, while the amount of ubiquitinated ERalpha was increased. A proteasome inhibitor, LLnL, almost completely abrogated the radicicol-induced decrease in expression level, as well as in transcriptional activity of ERalpha. These results suggest that radicicol disrupts the ER-Hsp90 heterodimeric complex, thereby generating ERalpha that is susceptible to ubiquitin/proteasome-induced degradation.

Topics: Animals; Binding Sites; Blotting, Northern; Blotting, Western; Breast Neoplasms; Cells, Cultured; Chlorocebus aethiops; DNA; Down-Regulation; Enzyme Inhibitors; Female; HSP90 Heat-Shock Proteins; Humans; Lactones; Macrolides; Multienzyme Complexes; Peptide Hydrolases; Protease Inhibitors; Receptors, Estrogen; Regulatory Sequences, Nucleic Acid; Repressor Proteins; Transcription, Genetic; Transcriptional Activation; Ubiquitins

A novel oxime derivative of radicicol, KF58333, binds to the heat shock protein 90 (Hsp90) and destabilizes its associated signaling molecules. These effects play a critical role in the growth inhibition of tumor cells. To further investigate the effects of this agent, it was administered to two human breast cancer cell lines, KPL-1 and KPL-4, both in vitro and in vivo. KF58333 dose-dependently inhibited the growth and vascular endothelial growth factor (VEGF) secretion, concomitantly with a decrease in VEGF mRNA expression, in each cell line. This agent also suppressed the increase of VEGF secretion and expression induced by hypoxia (1% O(2)). Intravenous injections of this agent into nude mice bearing either KPL-1 or KPL-4 xenografts significantly inhibited the tumor growth associated with a decrease in the Ki67 labeling index and microvascular area and an increase in apoptosis and the necrotic area. These findings indicate that the antitumor activity of this radicicol derivative may be partly mediated by decreasing VEGF secretion from tumor cells and inhibiting tumor angiogenesis. To explore the action mechanisms of the anti-angiogenic effect, the expression level of hypoxia-inducible factor (HIF)-1alpha was investigated. KF58333 provided a significant decrease in the HIF-1alpha protein expression under both normoxic and hypoxic conditions. In contrast, the mRNA expression of HIF-1alpha was not decreased by this agent. It is suggested that the post-transcriptional down-regulation of HIF-1alpha expression by this agent may result in a decrease of VEGF expression and tumor angiogenesis.

Topics: Animals; Apoptosis; Blotting, Western; Breast Neoplasms; Cell Division; DNA-Binding Proteins; Dose-Response Relationship, Drug; Endothelial Growth Factors; Female; Gene Expression Regulation, Neoplastic; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Ki-67 Antigen; Lactones; Lymphokines; Macrolides; Mice; Mice, Nude; Neoplasm Transplantation; Neovascularization, Pathologic; Nuclear Proteins; RNA, Messenger; Time Factors; Transcription Factors; Transplantation, Heterologous; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Radicicol is a novel hsp90 antagonist, distinct from the chemically unrelated benzoquinone ansamycin compounds, geldanamycin and herbimycin. Both geldanamycin and radicicol bind in the aminoterminal nucleotide-binding pocket of hsp90, destabilizing the hsp90 client proteins, many of which are essential for tumor cell growth. We describe here antitumor activity of a novel oxime derivative of radicicol, KF58333. We also investigated the mechanism of antitumor activity of KF58333 in comparison with its oxime isomer KF58332.. Antiproliferative activities were determined in a panel of breast cancer cell lines in vitro. We also examined inhibition of hsp90 function and apoptosis induction in erbB2-overexpressing human breast carcinoma KPL-4 cells in vitro. Direct binding activity to hsp90 was assessed by hsp90-binding assays using geldanamycin or radicicol beads. In animal studies, we investigated plasma concentrations of these compounds after i.v. injection in BALB/c mice and antitumor activity against KPL-4 cells transplanted into nude mice. Inhibition of hsp90 function and induction of apoptosis in vivo were investigated using tumor specimens from drug-treated animals.. KF58333 showed potent antiproliferative activity against all breast cancer cell lines tested in vitro, and was more potent than its stereoisomer KF58332. These results are consistent with the ability of KF58333 to deplete hsp90 client proteins and the induction of apoptosis in KPL-4 cells in vitro. Interestingly, KF58333, but not KF58332, showed significant in vivo antitumor activity accompanied by induction of apoptosis in KPL-4 human breast cancer xenografts. Although the plasma concentrations of these compounds were equivalent, KF58333, but not KF58332, depleted hsp90 client proteins such as erbB2, raf-1 and Akt in the tumor specimen recovered from nude mice.. These results suggest that inhibition of hsp90 function, which causes depletion of hsp90 client proteins in tumor, contributes to the antitumor activity of KF58333, and that the stereochemistry of the oxime moiety is important for the biological activity of radicicol oxime derivatives.

Topics: Animals; Apoptosis; Breast Neoplasms; Enzyme Inhibitors; Female; Gene Expression Regulation; Genes, erbB-2; HSP90 Heat-Shock Proteins; Humans; Injections, Intravenous; Lactones; Macrolides; Mice; Mice, Inbred BALB C; Mice, Nude; Oximes; Stereoisomerism; Structure-Activity Relationship; Tumor Cells, Cultured

The molecular chaperone Hsp90 plays an essential role in the folding and function of important cellular proteins including steroid hormone receptors, protein kinases and proteins controlling the cell cycle and apoptosis. A 15 A deep pocket region in the N-terminal domain of Hsp90 serves as an ATP/ADP-binding site and has also been shown to bind geldanamycin, the only specific inhibitor of Hsp90 function described to date. We now show that radicicol, a macrocyclic antifungal structurally unrelated to geldanamycin, also specifically binds to Hsp90. Moreover, radicicol competes with geldanamycin for binding to the N-terminal domain of the chaperone, expressed either by in vitro translation or as a purified protein, suggesting that radicicol shares the geldanamycin binding site. Radicicol, as does geldanamycin, also inhibits the binding of the accessory protein p23 to Hsp90, and interferes with assembly of the mature progesterone receptor complex. Radicicol does not deplete cells of Hsp90, but rather increases synthesis as well as the steady-state level of this protein, similar to a stress response. Finally, radicicol depletes SKBR3 cells of p185erbB2, Raf-1 and mutant p53, similar to geldanamycin. Radicicol thus represents a structurally unique antibiotic, and the first non-benzoquinone ansamycin, capable of binding to Hsp90 and interfering with its function.

Topics: Animals; Antibiotics, Antineoplastic; Antifungal Agents; Benzoquinones; Binding Sites; Binding, Competitive; Breast Neoplasms; Cell Division; Chickens; Chromatography, Affinity; Female; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Lactones; Macrolides; Molecular Structure; Quinones; Tumor Cells, Cultured