RESUMO
BACKGROUND: Development of cancer therapeutics partially depends upon selection of appropriate animal models. Therefore, improvements to model selection are beneficial. RESULTS: Forty-nine human tumor xenografts at in vivo passages 1, 4 and 10 were subjected to cDNA microarray analysis yielding a dataset of 823 Affymetrix HG-U133 Plus 2.0 arrays. To illustrate mining strategies supporting therapeutic studies, transcript expression was determined: 1) relative to other models, 2) with successive in vivo passage, and 3) during the in vitro to in vivo transition. Ranking models according to relative transcript expression in vivo has the potential to improve initial model selection. For example, combining p53 tumor expression data with mutational status could guide selection of tumors for therapeutic studies of agents where p53 status purportedly affects efficacy (e.g., MK-1775). The utility of monitoring changes in gene expression with extended in vivo tumor passages was illustrated by focused studies of drug resistance mediators and receptor tyrosine kinases. Noteworthy observations included a significant decline in HCT-15 colon xenograft ABCB1 transporter expression and increased expression of the kinase KIT in A549 with serial passage. These trends predict sensitivity to agents such as paclitaxel (ABCB1 substrate) and imatinib (c-KIT inhibitor) would be altered with extended passage. Given that gene expression results indicated some models undergo profound changes with in vivo passage, a general metric of stability was generated so models could be ranked accordingly. Lastly, changes occurring during transition from in vitro to in vivo growth may have important consequences for therapeutic studies since targets identified in vitro could be over- or under-represented when tumor cells adapt to in vivo growth. A comprehensive list of mouse transcripts capable of cross-hybridizing with human probe sets on the HG-U133 Plus 2.0 array was generated. Removal of the murine artifacts followed by pairwise analysis of in vitro cells with respective passage 1 xenografts and GO analysis illustrates the complex interplay that each model has with the host microenvironment. CONCLUSIONS: This study provides strategies to aid selection of xenograft models for therapeutic studies. These data highlight the dynamic nature of xenograft models and emphasize the importance of maintaining passage consistency throughout experiments.
Assuntos
Perfilação da Expressão Gênica , Neoplasias/genética , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Análise por Conglomerados , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Paclitaxel/uso terapêutico , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Prostaglandina E Subtipo EP2/genética , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Transplante Heterólogo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The response to extracellular stimuli often alters the phosphorylation state of plasma membrane- associated proteins. In this regard, generation of a comprehensive membrane phosphoproteome can significantly enhance signal transduction and drug mechanism studies. However, analysis of this subproteome is regarded as technically challenging, given the low abundance and insolubility of integral membrane proteins, combined with difficulties in isolating, ionizing and fragmenting phosphopeptides. In this article, we highlight recent advances in membrane and phosphoprotein enrichment techniques resulting in improved identification of these elusive peptides. We also describe the use of alternative fragmentation techniques, and assess their current and future value to the field of membrane phosphoproteomics.
Assuntos
Membrana Celular/metabolismo , Fosfoproteínas/metabolismo , Proteômica/métodos , Sequência de Aminoácidos , Animais , Humanos , Dados de Sequência Molecular , Fosfopeptídeos/química , Fosfopeptídeos/isolamento & purificação , Fosfopeptídeos/metabolismo , Fosfoproteínas/química , Fosfoproteínas/isolamento & purificação , Fosforilação , SolubilidadeRESUMO
The indenoisoquinoline NSC724998 is a novel topoisomerase I (Top1) inhibitor entering Phase I clinical trials at the National Cancer Institute, USA. In this study, 2-D PAGE analysis was performed on nuclear lysates prepared from HCT-116 and A375 cells treated with 1 microM NSC724998 for 24 h and the differentially regulated spots identified by LC-MS/MS. One-hundred fourteen protein spot differentials were identified, 66 from A375 cells and 48 from HCT-116 cells. Proteins related to apoptosis changed specifically in A375 cells, whereas proteins involved in the ubiquitin-proteasome system were highly enriched in treated HCT-116 cells. Importantly, 12 differentially expressed proteins (ETFA, HCC1, HNRCL, KAP1, NPM, NUCL, PRDX1, PRP19, PSB6, RAE1L, RU2A, and SFRS9) were common to both cell lines. Western blotting and immunocytochemistry confirmed significant nuclear upregulation of both the proteasome subunit PSB6 and the transcriptional repressor KAP1. Interestingly, increased KAP1 polypeptide was accompanied by enhanced phosphorylation at Ser824. Similar to gammaH2AX, KAP1 phosphorylation was consistently enhanced in a panel of 12 cell lines and in A375 xenografts following NSC 724998 treatment. In summary, these data enhance our understanding of protein dynamics in the nucleus following DNA damage and provide an alternate marker (pKAP1) with potential for monitoring clinical responses to Top1 poisons.
Assuntos
Benzodioxóis/farmacologia , Núcleo Celular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Isoquinolinas/farmacologia , Proteômica , Inibidores da Topoisomerase I/farmacologia , Western Blotting , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida , Dano ao DNA , Eletroforese em Gel Bidimensional , Humanos , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Espectrometria de Massas , Proteínas Repressoras/metabolismo , Proteína 28 com Motivo TripartidoRESUMO
The "Warburg effect," also termed aerobic glycolysis, describes the increased reliance of cancer cells on glycolysis for ATP production, even in the presence of oxygen. Consequently, there is continued interest in inhibitors of glycolysis as cancer therapeutics. One example is dichloroacetate (DCA), a pyruvate mimetic that stimulates oxidative phosphorylation through inhibition of pyruvate dehydrogenase kinase. In this study, the mechanistic basis for DCA anti-cancer activity was re-evaluated in vitro using biochemical, cellular and proteomic approaches. Results demonstrated that DCA is relatively inactive (IC(50) ≥ 17 mM, 48 hr), induces apoptosis only at high concentrations (≥ 25 mM, 48 hr) and is not cancer cell selective. Subsequent 2D-PAGE proteomic analysis confirmed DCA-induced growth suppression without apoptosis induction. Furthermore, DCA depolarizes mitochondria and promotes reactive oxygen species (ROS) generation in all cell types. However, DCA was found to have selective activity against rho(0) cells [mitochondrial DNA (mtDNA) deficient] and to synergize with 2-deoxyglucose in complex IV deficient HCT116 p53(-/-) cells. DCA also synergized in vitro with cisplatin and topotecan, two antineoplastic agents known to damage mitochondrial DNA. These data suggest that in cells "hardwired" to selectively utilize glycolysis for ATP generation (e.g., through mtDNA mutations), the ability of DCA to force oxidative phosphorylation confers selective toxicity. In conclusion, although we provide a mechanism distinct from that reported previously, the ability of DCA to target cell lines with defects in the electron transport chain and to synergize with existing chemotherapeutics supports further preclinical development.
Assuntos
Ácido Dicloroacético/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Transporte de Elétrons , Glicólise , Células HCT116 , Células HL-60 , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteômica , Coelhos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Analogs of the malaria therapeutic, artemisinin, possess in vitro and in vivo anticancer activity. In this study, two dimeric artemisinins (NSC724910 and 735847) were studied to determine their mechanism of action. Dimers were >1,000 fold more active than monomer and treatment was associated with increased reactive oxygen species (ROS) and apoptosis induction. Dimer activity was inhibited by the antioxidant L-NAC, the iron chelator desferroxamine and exogenous hemin. Similarly, induction of heme oxygenase (HMOX) with CoPPIX inhibited activity, whereas inhibition of HMOX with SnPPIX enhanced it. These results emphasize the importance of iron, heme and ROS in activity. Microarray analysis of dimer treated cells identified DNA damage, iron/heme and cysteine/methionine metabolism, antioxidant response, and endoplasmic reticulum (ER) stress as affected pathways. Detection of an ER-stress response was relevant because in malaria, artemisinin inhibits pfATP6, the plasmodium orthologue of mammalian sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPases (SERCA). A comparative study of NSC735847 with thapsigargin, a specific SERCA inhibitor and ER-stress inducer showed similar behavior in terms of transcriptomic changes, induction of endogenous SERCA and ER calcium mobilization. However, thapsigargin had little effect on ROS production, modulated different ER-stress proteins and had greater potency against purified SERCA1. Furthermore, an inactive derivative of NSC735847 that lacked the endoperoxide had identical inhibitory activity against purified SERCA1, suggesting that direct inhibition of SERCA has little inference on overall cytotoxicity. In summary, these data implicate indirect ER-stress induction as a central mechanism of artemisinin dimer activity.
Assuntos
Antineoplásicos/farmacologia , Artemisininas/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Heme Oxigenase-1/metabolismo , Heme/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacologia , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Artemisia/química , Biomarcadores/metabolismo , Western Blotting , Cálcio/metabolismo , Ciclo Celular/efeitos dos fármacos , Dimerização , Retículo Endoplasmático/metabolismo , Inibidores Enzimáticos/farmacologia , Perfilação da Expressão Gênica , Humanos , Lisina/análogos & derivados , Lisina/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/antagonistas & inibidores , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Tapsigargina/farmacologiaRESUMO
BACKGROUND: The nucleoside analog, ARC (NSC 188491) is a recently characterized transcriptional inhibitor that selectively kills cancer cells and has the ability to perturb angiogenesis in vitro. In this study, the mechanism of action of ARC was further investigated by comparing in vitro and in vivo activity with other anti-neoplastic purines. METHODS: Structure-based homology searches were used to identify those compounds with similarity to ARC. Comparator compounds were then evaluated alongside ARC in the context of viability, cell cycle and apoptosis assays to establish any similarities. Following this, biological overlap was explored in detail using gene-expression analysis and kinase inhibition assays. RESULTS: Results demonstrated that sangivamycin, an extensively characterized pro-apoptotic nucleoside isolated from Streptomyces, had identical activity to ARC in terms of 1) cytotoxicity assays, 2) ability to induce a G2/M block, 3) inhibitory effects on RNA/DNA/protein synthesis, 4) transcriptomic response to treatment, 5) inhibition of protein kinase C, 6) inhibition of positive transcription elongation factor b (P-TEFb), 7) inhibition of VEGF secretion, and 8) activity within hollow fiber assays. Extending ARC activity to PKC inhibition provides a molecular basis for ARC cancer selectivity and anti-angiogenic effects. Furthermore, functional overlap between ARC and sangivamycin suggests that development of ARC may benefit from a retrospective of previous sangivamycin clinical trials. However, ARC was found to be inactive in several xenograft models, likely a consequence of rapid serum clearance. CONCLUSION: Overall, these data expand on the biological properties of ARC but suggest additional studies are required before it can be considered a clinical trials candidate.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Nucleosídeos/farmacologia , Fator B de Elongação Transcricional Positiva/antagonistas & inibidores , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Nucleosídeos de Pirimidina/farmacologia , Pirimidinas/farmacologia , Animais , Antibióticos Antineoplásicos/química , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , DNA de Neoplasias/biossíntese , Ensaios de Seleção de Medicamentos Antitumorais , Células HL-60 , Humanos , Camundongos , Proteínas de Neoplasias/biossíntese , Nucleosídeos/química , Fosforilação , Inibidores de Proteínas Quinases/química , Nucleosídeos de Pirimidina/química , Pirimidinas/química , RNA Polimerase II/metabolismo , RNA Neoplásico/biossíntese , Relação Estrutura-Atividade , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Antibodies can be conjugated to effector molecules to derive targeted therapeutics with properties such as cell-specific cytotoxicity. The murine anti-CD22 antibody RFB4 linked to a member of the ribonuclease A superfamily, Onconase (Onc), becomes a potential drug candidate for non-Hodgkin's lymphoma. Onc is currently in Phase III clinical trials for unresectable malignant mesothelioma but conjugation to RFB4 considerably enhances its specificity for CD22+ lymphomas. RFB4-targeted Onc is effective in preclinical models, causes little non-specific toxicities in mice, and has favorable formulation properties. Derivatization and conjugation of RFB4 and Onc have been optimized.
Assuntos
Anticorpos Monoclonais/imunologia , Biologia Molecular/métodos , Ribonucleases/biossíntese , Ribonucleases/imunologia , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Antineoplásicos/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Dissulfetos/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Citometria de Fluxo , Imunofluorescência , Humanos , Camundongos , RNA de Transferência/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Ribonucleases/isolamento & purificação , SuccinimidasRESUMO
PURPOSE: Activities distinct from inhibition of Bcr/abl have led to adaphostin (NSC 680410) being described as "a drug in search of a mechanism." In this study, proteomic analysis of adaphostin-treated myeloid leukemia cell lines was used to further elucidate a mechanism of action. EXPERIMENTAL DESIGN: HL60 and K562 cells treated with adaphostin for 6, 12, or 24 h were analyzed using two-dimensional PAGE. Differentially expressed spots were excised, digested with trypsin, and analyzed by liquid chromatography-tandem mass spectrometry. The contribution of the redox-active hydroquinone group in adaphostin was also examined by carrying out proteomic analysis of HL60 cells treated with a simple hydroquinone (1,4-dihydroxybenzene) or H(2)O(2). RESULTS: Analysis of adaphostin-treated cells identified 49 differentially expressed proteins, the majority being implicated in the response to oxidative stress (e.g., CALM, ERP29, GSTP1, PDIA1) or induction of apoptosis (e.g., LAMA, FLNA, TPR, GDIS). Interestingly, modulation of these proteins was almost fully prevented by inclusion of an antioxidant, N-acetylcysteine. Validation of the proteomic data confirmed GSTP1 as an adaphostin resistance gene. Subsequent analysis of HL60 cells treated with 1,4-dihydroxybenzene or H(2)O(2) showed similar increases in intracellular peroxides and an almost identical proteomic profiles to that of adaphostin treatment. Western blotting of a panel of cell lines identified Cu/Zn superoxide dismutase (SOD) as correlating with adaphostin resistance. The role of SOD as a second adaphostin resistance gene was confirmed by demonstrating that inhibition of SOD using diethyldithiocarbamate increased adaphostin sensitivity, whereas transfection of SOD I attenuated toxicity. Importantly, treatment with 1,4-dihydroxybenzene or H(2)O(2) replicated adaphostin-induced Bcr/abl polypeptide degradation, suggesting that kinase inhibition is a ROS-dependent phenomenon. CONCLUSION: Adaphostin should be classified as a redox-active-substituted dihydroquinone.
Assuntos
Adamantano/análogos & derivados , Antineoplásicos/farmacologia , Expressão Gênica/efeitos dos fármacos , Hidroquinonas/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Adamantano/classificação , Adamantano/farmacologia , Antineoplásicos/classificação , Western Blotting , Eletroforese em Gel Bidimensional , Perfilação da Expressão Gênica , Células HL-60 , Humanos , Hidroquinonas/classificação , Oxidantes/classificação , Oxidantes/farmacologia , ProteômicaRESUMO
Here we report that B16F10 murine melanoma cells mimic endothelial cell behavior and the angiogenic process in vitro and in vivo. Cord formation in vitro by tumor cells is stimulated by hypoxia and vascular endothelial growth factor (VEGF) and inhibited by antibodies against VEGF and the VEGF KDR receptor (VEGF receptor 2). We define regulation of tumor cell-derived vascular space formation by these vasoactive compounds as "vasocrine" stimulation. ICRF 159 (Razoxane; NSC 129943) prevents tumor cell but not endothelial cell cord formation in vitro, and the antiangiogenic drug TNP-470 (NSC 642492) inhibits endothelial but not tumor cell cord formation in vitro. Both drugs inhibit formation of blood-filled vascular spaces in vivo. These results bear on the anticipated action of ICRF 159 in human clinical trials and novel strategies for targeting tumor blood supplies.
Assuntos
Inibidores da Angiogênese/farmacologia , Melanoma Experimental/irrigação sanguínea , Neovascularização Patológica/patologia , Animais , Antineoplásicos/farmacologia , Divisão Celular/fisiologia , Hipóxia Celular/fisiologia , Cicloexanos , Desenho de Fármacos , Fatores de Crescimento Endotelial/biossíntese , Endotélio Vascular/citologia , Endotélio Vascular/fisiologia , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Linfocinas/biossíntese , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , O-(Cloroacetilcarbamoil)fumagilol , Razoxano/farmacologia , Sesquiterpenos/farmacologia , Transdução de Sinais/fisiologia , Fator A de Crescimento do Endotélio Vascular , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Fatores de Crescimento do Endotélio VascularRESUMO
The crystal structure of a post-translationally modified form of eosinophil-derived neurotoxin (EDN) with four extra residues on its N terminus ((-4)EDN) has been solved and refined at atomic resolution (1 A). Two of the extra residues can be placed unambiguously, while the density corresponding to two others is poor. The modified N terminus appears to influence the position of the catalytically important His129, possibly explaining the diminished catalytic activity of this variant. However, (-4)EDN has been shown to be cytotoxic to a Kaposi's sarcoma tumor cell line and other endothelial cell lines. Analysis of the structure and function suggests that the reason for cytotoxicity is most likely due to cellular recognition by the N-terminal extension, since the intrinsic activity of the enzyme is not sufficient for cytotoxicity and the N-terminal extension does not affect the conformation of EDN.
Assuntos
Ribonucleases/química , Ribonucleases/metabolismo , Animais , Sítios de Ligação , Bovinos , Cristalografia por Raios X , Endotélio/efeitos dos fármacos , Endotélio/metabolismo , Endotélio/patologia , Neurotoxina Derivada de Eosinófilo , Imunofluorescência , Humanos , Modelos Moleculares , Ligação Proteica , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína , Ribonuclease Pancreático/química , Ribonucleases/genética , Ribonucleases/farmacologia , Sarcoma de Kaposi/metabolismo , Sarcoma de Kaposi/patologia , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Bleomycin is a clinically used antitumor antibiotic long thought to function therapeutically at the level of DNA cleavage. Recently, it has become clear that bleomycin can also cleave selected members of all major classes of RNA. Using the computer program COMPARE to search the database established by the Anticancer Drug Screening Program of the National Cancer Institute, a possible mechanism-based correlation was found between onconase, an antitumor ribonuclease currently being evaluated in phase III clinical trials, and the chemotherapeutic agent bleomycin. Following these observations, experimentation revealed that bleomycin caused tRNA cleavage and DNA-independent protein synthesis inhibition in rabbit reticulocyte lysate and when microinjected into Xenopus oocytes. The correlation of protein synthesis inhibition to the previously reported site-specific RNA cleavage caused by bleomycin supports the thesis that RNA cleavage may constitute an important element of the mechanism of action of bleomycin.
Assuntos
Antineoplásicos/farmacologia , Bleomicina/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , RNA/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Sistema Livre de Células , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Oócitos , RNA/metabolismo , Coelhos , Reticulócitos , Ribonucleases/metabolismo , Ribonucleases/farmacologia , Software , XenopusRESUMO
This article describes the construction, expression, and purification of RNase single-chain antibody fusion proteins. To construct a fusion protein, the gene for each moiety, the RNase and the binding ligand, is modified separately to contain complementary DNA encoding a 13 amino acid spacer that separates the RNase from the binding moiety. Appropriate restriction enzyme sites for cloning into the vector are also added. The modified DNA is combined and fused using the PCR technique of splicing by overlap extension (1). The resulting DNA construct is expressed in inclusion bodies in BL21(DE3) bacteria that are specifically engineered for the expression of toxic proteins (2). After isolation and purification of the inclusion bodies, the fusion protein is solubilized, denatured, and renatured. The renatured RNase fusion protein mixture is purified to homogeneity by two chromatography steps. The first column, a CM-Sephadex C-50 or a heparin Sepharose column, eliminates the majority of contaminating proteins while the second column, an affinity column (Ni2+-NTA agarose), results in the final purification of the RNase fusion protein.
Assuntos
Anticorpos/genética , Imunoconjugados/genética , Proteínas Recombinantes de Fusão/genética , Ribonucleases/genética , Animais , Cromatografia/métodos , Humanos , Imunoconjugados/metabolismo , Neoplasias/imunologia , Neoplasias/terapia , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
Targeting CD22 on human B-cells with a monoclonal antibody conjugated to a cytotoxic RNAse causes potent and specific killing of the lymphoma cells in vitro. This translates to anti-tumor effects in human lymphoma models in SCID mice. RNA damage caused by RNAses could be an important alternative to standard DNA-damaging chemotherapeutics. A second generation construct with an improved recombinant cytotoxic RNAse is described. Targeted RNAses may overcome problems of toxicity and immunogenicity associated with plant or bacterial toxin-containing immunoconjugates.
Assuntos
Moléculas de Adesão Celular , Imunotoxinas/uso terapêutico , Lectinas/antagonistas & inibidores , Linfoma de Células B/tratamento farmacológico , Ribonucleases/uso terapêutico , Animais , Anticorpos Monoclonais/uso terapêutico , Antígenos CD , Antígenos de Diferenciação de Linfócitos B , Humanos , Camundongos , Camundongos SCID , Lectina 2 Semelhante a Ig de Ligação ao Ácido SiálicoRESUMO
We previously reported a potent small molecule Mer tyrosine kinase inhibitor UNC1062. However, its poor PK properties prevented further assessment in vivo. We report here the sequential modification of UNC1062 to address DMPK properties and yield a new potent and highly orally bioavailable Mer inhibitor, 11, capable of inhibiting Mer phosphorylation in vivo, following oral dosing as demonstrated by pharmaco-dynamic (PD) studies examining phospho-Mer in leukemic blasts from mouse bone marrow. Kinome profiling versus more than 300 kinases in vitro and cellular selectivity assessments demonstrate that 11 has similar subnanomolar activity against Flt3, an additional important target in acute myelogenous leukemia (AML), with pharmacologically useful selectivity versus other kinases examined.
Assuntos
Adenina/análogos & derivados , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Adenina/administração & dosagem , Adenina/farmacocinética , Adenina/farmacologia , Administração Oral , Animais , Disponibilidade Biológica , Linhagem Celular Tumoral/efeitos dos fármacos , Técnicas de Química Sintética , Humanos , Concentração Inibidora 50 , Leucemia de Células B/tratamento farmacológico , Leucemia de Células B/metabolismo , Leucemia de Células B/patologia , Camundongos SCID , Terapia de Alvo Molecular , Piperazinas/administração & dosagem , Piperazinas/farmacocinética , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de Xenoenxerto , c-Mer Tirosina Quinase , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
Oxyphenisatin (3,3-bis(4-hydroxyphenyl)-1H-indol-2-one) and several structurally related molecules have been shown to have in vitro and in vivo antiproliferative activity. This study aims to confirm and extend mechanistic studies by focusing on oxyphenisatin acetate (OXY, NSC 59687), the pro-drug of oxyphenisatin. Results confirm that OXY inhibits the growth of the breast cancer cell lines MCF7, T47D, HS578T, and MDA-MB-468. This effect is associated with selective inhibition of translation accompanied by rapid phosphorylation of the nutrient sensing eukaryotic translation initiation factor 2α (eIF2α) kinases, GCN2 and PERK. This effect was paralleled by activation of AMP-activated protein kinase (AMPK) combined with reduced phosphorylation of the mammalian target of rapamycin (mTOR) substrates p70S6K and 4E-BP1. Microarray analysis highlighted activation of pathways involved in apoptosis induction, autophagy, RNA/protein metabolism, starvation responses, and solute transport. Pathway inhibitor combination studies suggested a role for AMPK/mTOR signaling, de novo transcription and translation, reactive oxygen species (ROS)/glutathione metabolism, calcium homeostasis and plasma membrane Na(+) /K(+) /Ca(2+) transport in activity. Further examination confirmed that OXY treatment was associated with autophagy, mitochondrial dysfunction, and ROS generation. Additionally, treatment was associated with activation of both intrinsic and extrinsic apoptotic pathways. In the estrogen receptor (ER) positive MCF7 and T47D cells, OXY induced TNFα expression and TNFR1 degradation, indicating autocrine receptor-mediated apoptosis in these lines. Lastly, in an MCF7 xenograft model, OXY delivered intraperitoneally inhibited tumor growth, accompanied by phosphorylation of eIF2α and degradation of TNFR1. These data suggest that OXY induces a multifaceted cell starvation response, which ultimately induces programmed cell death.
Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Neoplasias da Mama/patologia , Mitocôndrias/efeitos dos fármacos , Acetato de Oxifenisatina/farmacologia , Animais , Comunicação Autócrina/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Proliferação de Células/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Humanos , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Nus , Mitocôndrias/fisiologia , Proteínas de Neoplasias/metabolismo , Fosforilação/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Análise Serial de Tecidos/métodos , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/fisiologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Differential (18)O/(16)O stable isotopic labeling that relies on post-digestion (18)O exchange is a simple and efficient method for the relative quantitation of proteins in complex mixtures. This method incorporates two (18)O atoms onto the C-termini of proteolytic peptides resulting in a 4 Da mass-tag difference between (18)O- and (16)O-labeled peptides. This allows for wide-range relative quantitation of proteins in complex mixtures using shotgun proteomics. Because of minimal sample consumption and unrestricted peptide tagging, the post-digestion (18)O exchange is suitable for labeling of low-abundance membrane proteins enriched from cancer cell lines or clinical specimens, including tissues and body fluids. This chapter describes a protocol that applies post-digestion (18)O labeling to elucidate putative endogenous tumor hypoxia markers in the plasma membrane fraction enriched from a hypoxia-adapted malignant melanoma cell line. Plasma membrane proteins from hypoxic and normoxic cells were differentially tagged using (18)O/(16)O stable isotopic labeling. The initial tryptic digestion and solubilization of membrane proteins were carried out in a buffer containing 60 % methanol followed by post-digestion (18)O exchange/labeling in buffered 20 % methanol. The differentially labeled peptides were mixed in a 1:1 ratio and fractionated using off-line strong cation exchange (SCX) liquid chromatography followed by on-line reversed-phase nano-flow RPLC-MS identification and quantitation of peptides/proteins in respective SCX fractions. The present protocol illustrates the utility of (18)O/(16)O stable isotope labeling in the context of quantitative shotgun proteomics that provides a basis for the discovery of hypoxia-induced membrane protein markers in malignant melanoma cell lines.
Assuntos
Biomarcadores Tumorais/análise , Marcação por Isótopo , Melanoma/metabolismo , Isótopos de Oxigênio/química , Proteínas/análise , Tripsina/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Cromatografia Líquida , Humanos , Espectrometria de Massas , Proteínas/química , Proteômica/métodosRESUMO
PURPOSE: To establish whether NSC80467, a novel fused naphthquinone imidazolium, has a similar spectrum of activity to the well-characterized "survivin suppressant" YM155 and to extend mechanistic studies for this structural class of agent. METHODS: NSC80467 and YM155 were analyzed in parallel using assays measuring viability, survivin suppression, inhibition of DNA/RNA/protein synthesis and the cellular response to DNA damage. RESULTS: GI(50) values generated for both compounds in the NCI-60 screen yielded a correlation coefficient of 0.748, suggesting significant concordance. Both agents were also shown to inhibit protein expression of survivin [BIRC5]. COMPARE analysis identified DNA damaging agents chromomycin A3 and bisantrene HCl and one DNA-directed inhibitor of transcription, actinomycin D, as correlating with the activity of NSC80467 and YM155. Furthermore, both agents were shown to preferentially inhibit DNA, over RNA and protein synthesis. Thus, the ability of NSC80467 and YM155 to induce a DNA damage response was examined further. Treatment of PC3 cells with either agent resulted in dose-dependent induction of γH2AX and pKAP1, two markers of DNA damage. The concentrations of agent required to stimulate γH2AX were considerably lower than those required to inhibit survivin, implicating DNA damage as an initiating event. The DNA damage response was then confirmed in a panel of cell lines treated with NSC80467 or YM155, suggesting that γH2AX and pKAP1 have potential as response biomarkers. CONCLUSIONS: These data provide the first evidence that NSC80467 and YM155 are DNA damaging agents where suppression of survivin is a secondary event, likely a consequence of transcriptional repression.
Assuntos
Antineoplásicos/farmacologia , Dano ao DNA , Imidazóis/farmacologia , Naftoquinonas/farmacologia , Antineoplásicos/química , Western Blotting , Linhagem Celular Tumoral , DNA de Neoplasias/biossíntese , DNA de Neoplasias/genética , Relação Dose-Resposta a Droga , Células HCT116 , Células HT29 , Histonas/metabolismo , Humanos , Imidazóis/química , Proteínas Inibidoras de Apoptose/metabolismo , Células K562 , Estrutura Molecular , Naftoquinonas/química , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Biossíntese de Proteínas/efeitos dos fármacos , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Proteínas Repressoras/metabolismo , Survivina , Fatores de Tempo , Proteína 28 com Motivo TripartidoRESUMO
Recently, there has been renewed interest in the role of tumor stem cells (TSCs) in tumorigenesis, chemoresistance, and relapse of malignant tumors including osteosarcoma. The potential exists to improve osteosarcoma treatment through characterization of TSCs and identification of therapeutic targets. Using transcriptome, proteome, immunophenotyping for cell-surface markers, and bioinformatic analyses, heterogeneous expression of previously reported TSC or osteosarcoma markers, such as CD133, nestin, POU5F1 (OCT3/4), NANOG, SOX2, and aldehyde dehydrogenase, among others, was observed in vitro. However, consistently significantly lower CD326, CD24, CD44, and higher ABCG2 expression in TSC-enriched as compared with un-enriched osteosarcoma cultures was observed. In addition, consistently higher CBX3 expression in TSC-enriched osteosarcoma cultures was identified. ABCA5 was identified as a putative biomarker of TSCs and/or osteosarcoma. Lastly, in a high-throughput screen we identified epigenetic (5-azacytidine), anti-microtubule (vincristine), and anti-telomerase (3,11-difluoro-6,8,13-trimethyl- 8H-quino [4,3,2-kl] acridinium methosulfate; RHPS4)-targeted therapeutic agents as candidates for TSC ablation in osteosarcoma.
Assuntos
Transportadores de Cassetes de Ligação de ATP/biossíntese , Biomarcadores Tumorais/biossíntese , Proteínas Cromossômicas não Histona/biossíntese , Neoplasias Femorais/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/biossíntese , Células-Tronco Neoplásicas/metabolismo , Osteossarcoma/metabolismo , Adolescente , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Azacitidina/farmacologia , Azacitidina/uso terapêutico , Linhagem Celular Tumoral , Neoplasias Femorais/tratamento farmacológico , Neoplasias Femorais/patologia , Humanos , Masculino , Células-Tronco Neoplásicas/patologia , Osteossarcoma/tratamento farmacológico , Osteossarcoma/patologia , Vincristina/farmacologia , Vincristina/uso terapêuticoRESUMO
In this study, a Cu(2+) chelate of the novel thiosemicarbazone NSC 689534 was evaluated for in vitro and in vivo anti-cancer activity. Results demonstrated that NSC 689534 activity (low micromolar range) was enhanced four- to fivefold by copper chelation and completely attenuated by iron. Importantly, once formed, the NSC 689534/Cu(2+) complex retained activity in the presence of additional iron or iron-containing biomolecules. NSC 689534/Cu(2+) mediated its effects primarily through the induction of ROS, with depletion of cellular glutathione and protein thiols. Pretreatment of cells with the antioxidant N-acetyl-l-cysteine impaired activity, whereas NSC 689534/Cu(2+) effectively synergized with the glutathione biosynthesis inhibitor buthionine sulfoximine. Microarray analysis of NSC 689534/Cu(2+)-treated cells highlighted activation of pathways involved in oxidative and ER stress/UPR, autophagy, and metal metabolism. Further scrutiny of the role of ER stress and autophagy indicated that NSC 689534/Cu(2+)-induced cell death was ER-stress dependent and autophagy independent. Last, NSC 689534/Cu(2+) was shown to have activity in an HL60 xenograft model. These data suggest that NSC 689534/Cu(2+) is a potent oxidative stress inducer worthy of further preclinical investigation.