ABSTRACT
PURPOSE: The prognosis of patients with multiple myeloma (MM) is still dismal despite recent improvements achieved by introducing new therapeutic agents. However, there remains an urgent need for progress in myeloma drug development. We here show that novel marine-derived compounds can exert potent anti-myeloma activity. EXPERIMENTAL DESIGN: Nine marine-derived compounds were applied at low nM concentrations (0.1-100 nM) to MM cell lines (OPM-2, NCI-H929, U266, RPMI-8226), to primary human myeloma cells and to peripheral blood mononuclear cells. Apoptosis was determined by flow cytometry. In addition, eGFP-transgenic MM cell lines growing with mesenchymal cells from bone marrow were used to visualize tumors by fluorescence stereomicroscopy. Anti-myelomaactivities were studied in vitro in 3D spheroids and in vivo in myeloma xenografts on chicken embryos. Tumor size was analyzed by measuring GFP content with a GFP ELISA. Anti-angiogenic activities of compounds were tested in an in vivo gelatin sponge assay with conditioned media from primary bone marrow-derived endothelial cells. RESULTS: We identified a subset of marine compounds with strong anti-myeloma activity in vitro and in vivo. Moreover, some of the compounds inhibited myeloma-related angiogenesis in the in vivo gelatin sponge assay. They merit further drug development to improve treatment options for MM.
Subject(s)
Aquatic Organisms/chemistry , Biological Products/pharmacology , Multiple Myeloma/drug therapy , Angiogenesis Inhibitors/pharmacology , Animals , Biological Products/chemistry , Cell Line, Tumor , Chick Embryo , Drug Screening Assays, Antitumor , Heterografts , Humans , Multiple Myeloma/blood supply , Multiple Myeloma/pathology , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/pathology , Spheroids, Cellular , TransfectionABSTRACT
We have investigated the target and mechanism of action of a new family of cytotoxic small molecules of marine origin. PM050489 and its dechlorinated analogue PM060184 inhibit the growth of relevant cancer cell lines at subnanomolar concentrations. We found that they are highly potent microtubule inhibitors that impair mitosis with a distinct molecular mechanism. They bind with nanomolar affinity to unassembled αß-tubulin dimers, and PM050489 binding is inhibited by known Vinca domain ligands. NMR TR-NOESY data indicated that a hydroxyl-containing analogue, PM060327, binds in an extended conformation, and STD results define its binding epitopes. Distinctly from vinblastine, these ligands only weakly induce tubulin self-association, in a manner more reminiscent of isohomohalichondrin B than of eribulin. PM050489, possibly acting like a hinge at the association interface between tubulin heterodimers, reshapes Mg(2+)-induced 42 S tubulin double rings into smaller 19 S single rings made of 7 ± 1 αß-tubulin dimers. PM060184-resistant mutants of Aspergillus nidulans map to ß-tubulin Asn100, suggesting a new binding site different from that of vinblastine at the associating ß-tubulin end. Inhibition of assembly dynamics by a few ligand molecules at the microtubule plus end would explain the antitumor activity of these compounds, of which PM060184 is undergoing clinical trials.
Subject(s)
Antineoplastic Agents/pharmacology , Polyketides/pharmacology , Pyrones/pharmacology , Tubulin Modulators/pharmacology , Animals , Antineoplastic Agents/chemistry , Cell Line, Tumor , Humans , Mitosis/drug effects , Models, Molecular , Neoplasms/drug therapy , Neoplasms/metabolism , Polyketides/chemistry , Porifera/chemistry , Pyrones/chemistry , Tubulin/metabolism , Tubulin Modulators/chemistryABSTRACT
This study: (i) investigated the in vitro cytotoxicity and mode of action of lurbinectedin (PM01183) and Zalypsis® (PM00104) compared with trabectedin in cell lines deficient in specific mechanisms of repair, (ii) evaluated their in vivo antitumor activity against a series of murine tumors and human xenografts. The antiproliferative activity, the DNA damage and the cell cycle perturbations induced by the three compounds on tumor lines were very similar. Nucleotide Excision Repair (NER) deficient cells were approximately fourfold more resistant to trabectedin, lurbinectedin and Zalypsis®. Cells deficient in non-homologous end joining (NHEJ), MRN complex and translesion synthesis (TLS) were slightly more sensitive to the three compounds (approximately fivefold) while cells deficient in homologous recombination (HR) were markedly more sensitive (150-200-fold). All three compounds showed a good antitumor activity in several in vivo models. Lurbinectedin and trabectedin had a similar pattern of antitumor activity in murine tumors and in xenografts, whereas Zalypsis® appeared to have a distinct spectrum of activity. The fact that no relationship whatsoever was found between the in vitro cytotoxic potency and the in vivo antitumor activity, suggests that in addition to direct cytotoxic mechanisms other host-mediated effects are involved in the in vivo pharmacological effects.
Subject(s)
Carbolines/pharmacology , DNA Damage/drug effects , DNA Repair/drug effects , Dioxoles/pharmacology , Heterocyclic Compounds, 4 or More Rings/pharmacology , Neoplasms/drug therapy , Tetrahydroisoquinolines/pharmacology , Alkaloids/pharmacology , Animals , Antineoplastic Agents, Alkylating/pharmacology , Apoptosis/drug effects , Cell Cycle/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Chickens , Flow Cytometry , Humans , In Vitro Techniques , Mice , Mice, Inbred C57BL , Mice, Nude , Molecular Structure , Neoplasms/pathology , Trabectedin , Xenograft Model Antitumor AssaysABSTRACT
This paper reports on the syntheses and spectrometric characterisation of eleven novel ent-kaurane diterpenoids, including a complete set of (1)H, (13)C NMR and crystallographic data for two novel ent-kaurane diepoxides. Moreover, the antineoplastic cytotoxicity for kaurenoic acid and the majority of ent-kaurane derivatives were assessed in vitro against a panel of fourteen cancer cell lines, of which allylic alcohols were shown to be the most active compounds. The good in vitro antimalarial activity and the higher selectivity index values observed for some ent-kaurane epoxides against the chloroquine-resistant W2 clone of Plasmodium falciparum indicate that this class of natural products may provide new hits for the development of antimalarial drugs.
Subject(s)
Antimalarials/pharmacology , Antineoplastic Agents/pharmacology , Diterpenes, Kaurane/pharmacology , Plasmodium falciparum/drug effects , Antimalarials/chemical synthesis , Antimalarials/chemistry , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Death/drug effects , Cell Proliferation/drug effects , Diterpenes, Kaurane/chemical synthesis , Diterpenes, Kaurane/chemistry , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Models, Molecular , Molecular Structure , Parasitic Sensitivity Tests , Structure-Activity RelationshipABSTRACT
Several pinacol derivatives of podophyllotoxins bearing different side chains and functions at C-7 were synthesized through reductive cross-coupling of podophyllotoxone and several aldehydes and ketones. While possessing a hydroxylated chain at C-7, the compounds retained their respective hydroxyl group with either the 7α (podo) or 7ß (epipodo) configuration. Along with pinacols, some C-7 alkylidene and C-7 alkyl derivatives were also prepared. Cytotoxicities against neoplastic cells followed by cell cycle arrest and cellular microtubule disruption were evaluated and mechanistically characterized through tubulin polymerization inhibition and assays of binding to the colchicine site. Compounds of the epipodopinacol (7ß-OH) series behaved similarly to podophyllotoxin in all the assays and proved to be the most potent inhibitors. Significantly, 7α-isopropyl-7-deoxypodophyllotoxin (20), without any hydroxyl function, appeared as a promising lead compound for a novel type of tubulin polymerization inhibitors. Experimental results were in overall agreement with modeling and docking studies performed on representative compounds of each series.
Subject(s)
Antimitotic Agents/chemical synthesis , Antineoplastic Agents/chemical synthesis , Podophyllotoxin/analogs & derivatives , Podophyllotoxin/chemical synthesis , Antimitotic Agents/pharmacology , Antineoplastic Agents/pharmacology , Binding, Competitive , Cell Cycle/drug effects , Cell Line, Tumor , Cell Proliferation , Drug Screening Assays, Antitumor , Humans , Microtubules/drug effects , Microtubules/ultrastructure , Models, Molecular , Podophyllotoxin/pharmacology , Stereoisomerism , Tubulin Modulators/chemical synthesis , Tubulin Modulators/pharmacologyABSTRACT
Irvalec is a marine-derived antitumor agent currently undergoing phase II clinical trials. In vitro, Irvalec induces a rapid loss of membrane integrity in tumor cells, accompanied of a significant Ca(2+) influx, perturbations of membrane conductivity, severe swelling and the formation of giant membranous vesicles. All these effects are not observed in Irvalec-resistant cells, or are significantly delayed by pretreating the cells with Zn(2+). Using fluorescent derivatives of Irvalec it was demonstrated that the compound rapidly interacts with the plasma membrane of tumor cells promoting lipid bilayer restructuration. Also, FRET experiments demonstrated that Irvalec molecules localize in the cell membrane close enough to each other as to suggest that the compound could self-organize, forming supramolecular structures that likely trigger cell death by necrosis through the disruption of membrane integrity.
Subject(s)
Antineoplastic Agents/pharmacology , Cell Death/drug effects , Depsipeptides/pharmacology , Neoplasms/pathology , Cell Line, Tumor , Cell Membrane/drug effects , Cell Membrane Permeability/drug effects , HumansABSTRACT
BACKGROUND: Several human pathologies, including neoplasia and ischemic cardiovascular diseases, course with an unbalance between oxygen supply and demand (hypoxia). Cells within hypoxic regions respond with the induction of a specific genetic program, under the control of the Hypoxia Inducible Factor (HIF), that mediates their adaptation to the lack of oxygen. The activity of HIF is mainly regulated by the EGL-nine homolog (EGLN) enzymes that hydroxylate the alpha subunit of this transcription factor in an oxygen-dependent reaction. Hydroxylated HIF is then recognized and ubiquitinilated by the product of the tumor suppressor gene, pVHL, leading to its proteosomal degradation. Under hypoxia, the hydroxylation of HIF by the EGLNs is compromised due to the lack of oxygen, which is a reaction cosubstrate. Thus, HIF escapes degradation and drives the transcription of its target genes. Since the progression of the aforementioned pathologies might be influenced by activation of HIF-target genes, development of small molecules with the ability to interfere with the HIF-regulatory machinery is of great interest. RESULTS: Herein we describe a yeast three-hybrid system that reconstitutes mammalian HIF regulation by the EGLNs and VHL. In this system, yeast growth, under specific nutrient restrictions, is driven by the interaction between the beta domain of VHL and a hydroxyproline-containing HIFalpha peptide. In turn, this interaction is strictly dependent on EGLN activity that hydroxylates the HIFalpha peptide. Importantly, this system accurately preserves the specificity of the hydroxylation reaction toward specific substrates. We propose that this system, in combination with a matched control, can be used as a simple and inexpensive assay to identify molecules that specifically modulate EGLN activity. As a proof of principle we show that two known EGLN inhibitors, dimethyloxaloylglycine (DMOG) and 6-chlor-3-hydroxychinolin-2-carbonic acid-N-carboxymethylamide (S956711), have a profound and specific effect on the yeast HIF/EGLN/VHL system. CONCLUSION: The system described in this work accurately reconstitutes HIF regulation while preserving EGLN substrate specificity. Thus, it is a valuable tool to study HIF regulation, and particularly EGLN biochemistry, in a cellular context. In addition, we demonstrate that this system can be used to identify specific inhibitors of the EGLN enzymes.
Subject(s)
Hypoxia-Inducible Factor 1/metabolism , Hypoxia/metabolism , Mammals/metabolism , Saccharomyces cerevisiae/metabolism , Two-Hybrid System Techniques/standards , Animals , Enzyme Inhibitors/pharmacology , Gene Expression Regulation/physiology , Hydroxylation/drug effects , Hypoxia/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Hypoxia-Inducible Factor-Proline Dioxygenases , Oxidation-Reduction , Oxygen/metabolism , Procollagen-Proline Dioxygenase/metabolism , Transcriptional Activation/drug effects , Transcriptional Activation/genetics , Ubiquitination , Von Hippel-Lindau Tumor Suppressor Protein/genetics , Von Hippel-Lindau Tumor Suppressor Protein/metabolismABSTRACT
Chemical investigation of the hybrid soft coral Sinularia maxima x Sinularia polydactyla yielded four new cembranolide diterpenes (1-4), the norcembranoid 5-episinuleptolide (5), and two known sesquiterpenes. The structures and configurations of the new compounds were determined by spectroscopic techniques and comparison of NMR data with those of related metabolites. Compound 3 shows strong cytotoxicity on the breast cancer SK-BR3 cell line and cervix cancer HeLa and HeLa-Apl cell lines with GI(50) values of 0.039, 0.48, and 0.56 microM, respectively.
Subject(s)
Anthozoa/chemistry , Antineoplastic Agents/isolation & purification , Antineoplastic Agents/pharmacology , Diterpenes/isolation & purification , Diterpenes/pharmacology , Animals , Antineoplastic Agents/chemistry , Diterpenes/chemistry , Drug Screening Assays, Antitumor , Female , Guam , HeLa Cells , Humans , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Sesquiterpenes/chemistry , Sesquiterpenes/isolation & purification , Sesquiterpenes/pharmacology , Tumor Cells, CulturedABSTRACT
The marine natural product thiocoraline A displayed approximately equal cytotoxic activity at nanomolar concentrations in a panel of 12 human cancer cell lines. X-ray diffraction analyses of orthorhombic crystals of this DNA-binding drug revealed arrays of docked pairs of staple-shaped molecules in which one pendent hydroxyquinoline chromophore from each cysteine-rich molecule appears intercalated between the two chromophores of a facing molecule. This arrangement is in contrast to the proposed mode of binding to DNA that shows the two drug chromophores clamping two stacked base pairs, in agreement with the nearest-neighbor exclusion principle. Proof of DNA sequence recognition was obtained from both classical DNase I footprinting experiments and determination of the melting temperatures of several custom-designed fluorescently labeled oligonucleotides. A rationale for the DNA-binding behavior was gained when models of thiocoraline clamping a central step embedded in several octanucleotides were built and studied by means of unrestrained molecular dynamics simulations in aqueous solution.
Subject(s)
Antineoplastic Agents/pharmacology , DNA/metabolism , Depsipeptides/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Base Sequence , Cell Line, Tumor , Crystallography, X-Ray , DNA Footprinting , Depsipeptides/chemistry , Depsipeptides/metabolism , Humans , StereoisomerismABSTRACT
Aplidin (plitidepsin) is an antitumoral agent that induces apoptosis via Rac1-JNK activation. A proteomic approach using 2D-DIGE technology found 52 cytosolic and 39 membrane proteins differentially expressed in wild-type and Aplidin-resistant HeLa cells, of which 39 and 27 were identified by MALDI-TOF mass spectrometry and database interrogation. A number of proteins involved in apoptosis pathways were found to be deregulated. Alterations in Rab geranylgeranyltransferase, protein disulfide isomerase (PDI), cystathionine gamma-lyase, ezrin, and cyclophilin A (CypA) were confirmed by immunoblotting. Moreover, the role of PDI and CypA in Aplidin resistance was functionally confirmed by using the inhibitor bacitracin and overexpression, respectively. These deregulated proteins are candidates to mediate, at least partially, Aplidin action and might provide a route to the cells to escape the induction of apoptosis by this drug.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis Regulatory Proteins/analysis , Depsipeptides/pharmacology , Drug Resistance, Neoplasm , Neoplasm Proteins/analysis , Neoplasms/chemistry , Proteomics , Blotting, Western , Cell Membrane/chemistry , Cytosol/chemistry , Echocardiography, Doppler , HeLa Cells , Humans , Peptides, CyclicABSTRACT
We report that Aplidin, a novel antitumor agent of marine origin presently undergoing Phase II clinical trials, induced growth arrest and apoptosis in human MDA-MB-231 breast cancer cells at nanomolar concentrations. Aplidin induced a specific cellular stress response program, including sustained activation of the epidermal growth factor receptor (EGFR), the non-receptor protein-tyrosine kinase Src, and the serine/threonine kinases JNK and p38 MAPK. Aplidin-induced apoptosis was only partially blocked by the general caspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone and was also sensitive to AG1478 (an EGFR inhibitor), PP2 (an Src inhibitor), and SB203580 (an inhibitor of JNK and p38 MAPK) in MDA-MB-231 cells. Supporting a role for EGFR in Aplidin action, EGFR-deficient mouse embryo fibroblasts underwent apoptosis upon treatment more slowly than wild-type EGFR fibroblasts and also showed delayed JNK and reduced p38 MAPK activation. N-Acetylcysteine and ebselen (but not other antioxidants such as diphenyleneiodonium, Tiron, catalase, ascorbic acid, and vitamin E) reduced EGFR activation by Aplidin. N-Acetylcysteine and PP2 also partially inhibited JNK and p38 MAPK activation. The intracellular level of GSH affected Aplidin action; pretreatment of cells with GSH or N-acetylcysteine inhibited, whereas GSH depletion caused, hyperinduction of EGFR, Src, JNK, and p38 MAPK. Remarkably, Aplidin also induced apoptosis and activated EGFR, JNK, and p38 MAPK in two cell lines (A-498 and ACHN) derived from human renal cancer, a neoplasia that is highly refractory to chemotherapy. These data provide a molecular basis for the anticancer activity of Aplidin.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Depsipeptides , ErbB Receptors/metabolism , Glutathione/metabolism , Mitogen-Activated Protein Kinases/metabolism , Peptides, Cyclic/pharmacology , Proto-Oncogene Proteins pp60(c-src)/metabolism , Animals , Breast Neoplasms/metabolism , Cell Division/drug effects , Cell Survival/drug effects , Cells, Cultured , Enzyme Activation , Enzyme Inhibitors/pharmacology , Female , Fibroblasts/cytology , Fibroblasts/drug effects , Fibroblasts/metabolism , Flow Cytometry , Humans , JNK Mitogen-Activated Protein Kinases , Kidney Neoplasms/metabolism , Mice , Phosphorylation , Receptors, Platelet-Derived Growth Factor/metabolism , Tumor Cells, Cultured/drug effects , p38 Mitogen-Activated Protein KinasesABSTRACT
Aplidin, a new antitumoural drug presently in phase II clinical trials, has shown both in vitro and in vivo activity against human cancer cells. Aplidin effectively inhibits cell viability by triggering a canonical apoptotic program resulting in alterations in cell morphology, caspase activation, and chromatin fragmentation. Pro-apoptotic concentrations of Aplidin induce early oxidative stress, which results in a rapid and persistent activation of both JNK and p38 MAPK and a biphasic activation of ERK. Inhibition of JNK and p38 MAPK blocks the apoptotic program induced by Aplidin demonstrating its central role in the integration of the cellular stress induced by the drug. JNK and p38 MAPK activation results in downstream cytochrome c release and activation of caspases -9 and -3 and PARP cleavage, demonstrating the mediation of the mitochondrial apoptotic pathway in this process. We also demonstrate that protein kinase C delta (PKC-delta) mediates the cytotoxic effect of Aplidin and that it is concomitantly processed and activated late in the apoptotic process by a caspase mediated mechanism. Remarkably, cells deficient in PKC-delta show enhanced survival upon drug treatment as compared to its wild type counterpart. PKC-delta thus appears as an important component necessary for full caspase cascade activation and execution of apoptosis, which most probably initiates a positive feedback loop further amplifying the apoptotic process.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Depsipeptides , Isoenzymes/metabolism , Mitochondria/drug effects , Mitogen-Activated Protein Kinases/metabolism , Oxidative Stress , Peptides, Cyclic/pharmacology , Protein Kinase C/metabolism , Blotting, Western , Enzyme Activation , Flow Cytometry , HeLa Cells , Humans , JNK Mitogen-Activated Protein Kinases , Protein Kinase C-delta , Signal Transduction , p38 Mitogen-Activated Protein KinasesABSTRACT
The tau gene encodes a microtubule-associated protein expressed by neuronal and glial cells. Abnormal deposits of Tau protein are characteristic of several neurodegenerative disorders. Additionally, mutations affecting tau pre-mRNA alternative splicing of exon 10 are associated with frontotemporal dementia and Parkinsonism linked to chromosome 17. In rodents, this process is developmentally regulated by thyroid hormone (T3) causing the predominance of exon 10-containing transcripts. Here we demonstrate that musashi-1 (msi-1) gene is induced by T3 during rat brain development and in N2a cells. T3 increases msi-1 mRNA level in an actinomycin D-sensitive, cycloheximide-resistant fashion without affecting its half-life, which suggests a transcriptional effect. Both ectopic Msi-1 expression and T3 treatment increased the proportion of exon 10-containing tau transcripts. Furthermore, antisense msi-1 expression inhibited T3 action. Our results show that msi-1 mediates the posttranscriptional regulation of tau expression by T3.