RESUMO
The splicing of pre-mRNA is a critical process in normal cells and is deregulated in cancer. Compounds that modulate this process have recently been shown to target a specific vulnerability in tumors. We have developed a novel cell-based assay that specifically activates luciferase in cells exposed to SF3B1 targeted compounds, such as sudemycin D6. This assay was used to screen a combined collection of approved drugs and bioactive compounds. This screening approach identified several active hits, the most potent of which were CGP-74514A and aminopurvalanol A, both have been reported to be cyclin-dependent kinases (CDKs) inhibitors. We found that these compounds, and their analogs, show significant cdc2-like kinase (CLK) inhibition and clear structure-activity relationships (SAR) at CLKs. We prepared a set of analogs and were able to 'dial out' the CDK activity and simultaneously developed CLK inhibitors with low nanomolar activity. Thus, we have demonstrated the utility of our exon-skipping assay and identified new molecules that exhibit potency and selectivity for CLK, as well as some structurally related dual CLK/CDK inhibitors.
Assuntos
Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , 2-Aminopurina/análogos & derivados , 2-Aminopurina/química , 2-Aminopurina/metabolismo , Adenina/análogos & derivados , Adenina/química , Adenina/metabolismo , Sítios de Ligação , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Éxons , Genes Reporter , Ensaios de Triagem em Larga Escala , Humanos , Concentração Inibidora 50 , Luciferases/genética , Simulação de Dinâmica Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína , Splicing de RNA , Relação Estrutura-AtividadeRESUMO
UNLABELLED: A major goal in HIV eradication research is characterizing the reservoir cells that harbor HIV in the presence of antiretroviral therapy (ART), which reseed viremia after treatment is stopped. In general, it is assumed that the reservoir consists of CD4(+) T cells that express no viral proteins. However, recent findings suggest that this may be an overly simplistic view and that the cells that contribute to the reservoir may be a diverse population that includes both CD4(+) and CD4(-) cells. In this study, we directly infected resting CD4(+) T cells and used fluorescence-activated cell sorting (FACS) and fiber-optic array scanning technology (FAST) to identify and image cells expressing HIV Gag. We found that Gag expression from integrated proviruses occurred in resting cells that lacked surface CD4, likely resulting from Nef- and Env-mediated receptor internalization. We also extended our approach to detect cells expressing HIV proteins in patients suppressed on ART. We found evidence that rare Gag(+) cells persist during ART and that these cells are often negative for CD4. We propose that these double-negative α/ß T cells that express HIV protein may be a component of the long-lived reservoir. IMPORTANCE: A reservoir of infected cells persists in HIV-infected patients during antiretroviral therapy (ART) that leads to rebound of virus if treatment is stopped. In this study, we used flow cytometry and cell imaging to characterize protein expression in HIV-infected resting cells. HIV Gag protein can be directly detected in infected resting cells and occurs with simultaneous loss of CD4, consistent with the expression of additional viral proteins, such as Env and Nef. Gag(+) CD4(-) cells can also be detected in suppressed patients, suggesting that a subset of infected cells express proteins during ART. Understanding the regulation of viral protein expression during ART will be key to designing effective strategies to eradicate HIV reservoirs.
Assuntos
Antirretrovirais/uso terapêutico , Antígenos CD4/análise , Antígenos CD8/análise , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Subpopulações de Linfócitos T/virologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/biossíntese , Citometria de Fluxo , Humanos , Imagem Óptica , Subpopulações de Linfócitos T/químicaRESUMO
Rapid tumor growth can establish metabolically stressed microenvironments that activate 5'-AMP-activated protein kinase (AMPK), a ubiquitous regulator of ATP homeostasis. Previously, we investigated the importance of AMPK for the growth of experimental tumors prepared from HRAS-transformed mouse embryo fibroblasts and for primary brain tumor development in a rat model of neurocarcinogenesis. Here, we used triple-negative human breast cancer cells in which AMPK activity had been knocked down to investigate the contribution of AMPK to experimental tumor growth and core glucose metabolism. We found that AMPK supports the growth of fast-growing orthotopic tumors prepared from MDA-MB-231 and DU4475 breast cancer cells but had no effect on the proliferation or survival of these cells in culture. We used in vitro and in vivo metabolic profiling with [(13)C]glucose tracers to investigate the contribution of AMPK to core glucose metabolism in MDA-MB-231 cells, which have a Warburg metabolic phenotype; these experiments indicated that AMPK supports tumor glucose metabolism in part through positive regulation of glycolysis and the nonoxidative pentose phosphate cycle. We also found that AMPK activity in the MDA-MB-231 tumors could systemically perturb glucose homeostasis in sensitive normal tissues (liver and pancreas). Overall, our findings suggest that the contribution of AMPK to the growth of aggressive experimental tumors has a critical microenvironmental component that involves specific regulation of core glucose metabolism.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Neoplasias da Mama/enzimologia , Proteínas de Neoplasias/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Glucose/genética , Glucose/metabolismo , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Via de Pentose Fosfato/genética , RatosRESUMO
Combinatorial methods enable the synthesis of chemical libraries on scales of millions to billions of compounds, but the ability to efficiently screen and sequence such large libraries has remained a major bottleneck for molecular discovery. We developed a novel technology for screening and sequencing libraries of synthetic molecules of up to a billion compounds in size. This platform utilizes the fiber-optic array scanning technology (FAST) to screen bead-based libraries of synthetic compounds at a rate of 5 million compounds per minute (â¼83â¯000 Hz). This ultra-high-throughput screening platform has been used to screen libraries of synthetic "self-readable" non-natural polymers that can be sequenced at the femtomole scale by chemical fragmentation and high-resolution mass spectrometry. The versatility and throughput of the platform were demonstrated by screening two libraries of non-natural polyamide polymers with sizes of 1.77M and 1B compounds against the protein targets K-Ras, asialoglycoprotein receptor 1 (ASGPR), IL-6, IL-6 receptor (IL-6R), and TNFα. Hits with low nanomolar binding affinities were found against all targets, including competitive inhibitors of K-Ras binding to Raf and functionally active uptake ligands for ASGPR facilitating intracellular delivery of a nonglycan ligand.
RESUMO
Angiogenesis is one of the major processes controlling growth and metastasis of tumors. Angiogenesis inhibitors have been targeted for the treatment of various cancers for more than 2 decades. We have developed a novel class of steroidal compounds aimed at blocking the angiogenic process in cancerous tissues. Our lead compound, SR16388, is a potent antiangiogenic agent with binding affinity to estrogen receptor-α (ER-α) and -ß (ER-ß) at the nanomolar range. This compound inhibited the proliferation of human microvascular endothelial cells (HMVEC) and various types of human cancer cells in vitro. SR16388 inhibited embryonic angiogenesis as measured in the chick chorioallantoic membrane (CAM) assay. The blood vessel density in the CAM was greatly reduced after the embryos were treated with 3 µg/CAM of SR16388 for 24 h. SR16388 at a dose of 2 µM prevented tube formation in Matrigel after HMVEC cells were treated for 8 h. In a modified Boyden chamber assay, SR16388 inhibited the migration of HMVECs by 80% at 500 nM. Using a novel in vivo Fibrin Z-chamber model, we demonstrated that SR16388 at a single daily oral dose of 3 mg/kg for 12 days significantly inhibited the granulation tissue (GT) thickness and the microvessel density of the GT as compared to control. More importantly, SR16388 down-regulated the pro-angiogenic transcription factors, hypoxia inducible factor 1α (HIF-1α) and signal transducer and activator of transcription 3 (STAT3) in non-small cell lung cancer (NSCLC) cells. Together, these effects of SR16388 can lead to the reduction of vascularization and tumor growth in vivo.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Estradiol/análogos & derivados , Neoplasias/tratamento farmacológico , Esteroides/uso terapêutico , Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Galinhas , Regulação para Baixo/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Estradiol/química , Estradiol/farmacologia , Estradiol/uso terapêutico , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Fibrina/metabolismo , Fase G1/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Camundongos Nus , Microvasos/citologia , Neoplasias/irrigação sanguínea , Neoplasias/patologia , Neovascularização Patológica/tratamento farmacológico , Fosforilação/efeitos dos fármacos , Ratos , Ratos Endogâmicos F344 , Fator de Transcrição STAT3/metabolismo , Esteroides/química , Esteroides/farmacologiaRESUMO
Taspase1 is an Ntn-hydrolase overexpressed in primary human cancers, coordinating cancer cell proliferation, invasion, and metastasis. Loss of Taspase1 activity disrupts proliferation of human cancer cells in vitro and in mouse models of glioblastoma. Taspase1 is synthesized as an inactive proenzyme, becoming active upon intramolecular cleavage. The activation process changes the conformation of a long fragment at the C-terminus of the α subunit, for which no full-length structural information exists and whose function is poorly understood. We present a cloning strategy to generate a circularly permuted form of Taspase1 to determine the crystallographic structure of active Taspase1. We discovered that this region forms a long helix and is indispensable for the catalytic activity of Taspase1. Our study highlights the importance of this element for the enzymatic activity of Ntn-hydrolases, suggesting that it could be a potential target for the design of inhibitors with potential to be developed into anticancer therapeutics.
Assuntos
Endopeptidases/química , Endopeptidases/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Difusão Dinâmica da Luz , Endopeptidases/genética , Ativação Enzimática , Humanos , Modelos Moleculares , Domínios Proteicos , Estrutura Secundária de ProteínaRESUMO
Agents that modulate pre-mRNA splicing are of interest in multiple therapeutic areas, including cancer. We report our recent screening results with the application of a cell-based Triple Exon Skipping Luciferase Reporter (TESLR) using a library that is composed of FDA approved drugs, clinical compounds, and mechanistically characterized tool compounds. Confirmatory assays showed that three clinical antitumor therapeutic candidates (milciclib, PF-3758309 and PF-562271) are potent splicing modulators and that these drugs are, in fact, nanomolar inhibitors of multiple kinases involved in the regulation the spliceosome. We also report the identification of new SF3B1 antagonists (sudemycinol C and E) and show that these antagonists can be used to develop a displacement assay for SF3B1 small molecule ligands. These results further support the broad potential for the development of agents that target the spliceosome for the treatment of cancer and other diseases, as well as new avenues for the discovery of new chemotherapeutic agents for a range of diseases.
Assuntos
Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Éxons/efeitos dos fármacos , Precursores de RNA/genética , Splicing de RNA/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Inibidores de Proteínas Quinases/farmacologiaRESUMO
A set of genetically engineered isogenic cell lines is developed to express either folate receptor alpha or mesothelin, and a control cell line negative for both antigens. These cell lines also express fluorescent and bioluminescent reporter transgenes. The cell lines are used to authenticate specificity and function of a T-cell biofactory, a living vector that is developed to express proportionate amounts of engineered proteins upon engaging with disease cells through their specific antigenic biomarkers. The engineered cell lines are also used to assess the cytolytic function and specificity of primary T cells engineered with chimeric antigen receptors; and the specificity of monoclonal antibodies. The strategy described can be used to generate other cell lines to present different disease-specific biomarkers for use as quality control tools.
Assuntos
Engenharia Celular/métodos , Engenharia Genética/métodos , Neoplasias Ovarianas/genética , Receptores de Antígenos Quiméricos/genética , Linfócitos T , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Ovarianas/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismoRESUMO
An artificial cell-signaling pathway is developed that capitalizes on the T-cell's innate extravasation ability and transforms it into a vector (T-cell Biofactory) for synthesizing calibrated amounts of engineered proteins in vivo. The modularity of this pathway enables reprogramming of the T-cell Biofactory to target biomarkers on different disease cells, e.g. cancer, viral infections, autoimmune disorders. It can be expected that the T-cell Biofactory leads to a "living drug" that extravasates to the disease sites, assesses the disease burden, synthesizes the calibrated amount of engineered therapeutic proteins upon stimulation by the diseased cells, and reduces targeting of normal cells.
RESUMO
We have synthesized a histone deacetylase inhibitor, NVP-LAQ824, a cinnamic hydroxamic acid, that inhibited in vitro enzymatic activities and transcriptionally activated the p21 promoter in reporter gene assays. NVP-LAQ824 selectively inhibited growth of cancer cell lines at submicromolar levels after 48-72 h of exposure, whereas higher concentrations and longer exposure times were required to retard the growth of normal dermal human fibroblasts. Flow cytometry studies revealed that both tumor and normal cells arrested in the G(2)-M phase of the cell cycle after compound treatment. However, an increased sub-G(1) population at 48 h (reminiscent of apoptotic cells) was observed only in the cancer cell line. Annexin V staining data supported our hypothesis that NVP-LAQ824 induced apoptosis in tumor and transformed cells but not in normal cells. Western blotting experiments showed an increased histone H3 and H4 acetylation level in NVP-LAQ824-treated cancer cells, suggesting that the likely in vivo target of NVP-LAQ824 was histone deacetylase(s). Finally, NVP-LAQ824 exhibited antitumor effects in a xenograft animal model. Together, our data indicated that the activity of NVP-LAQ824 was consistent with its intended mechanism of action. This novel histone deacetylase inhibitor is currently in clinical trials as an anticancer agent.
Assuntos
Antineoplásicos/toxicidade , Neoplasias do Colo/tratamento farmacológico , Inibidores de Histona Desacetilases , Ácidos Hidroxâmicos/toxicidade , Animais , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Linhagem Celular Tumoral , Neoplasias do Colo/patologia , Inibidores Enzimáticos/toxicidade , Fluoruracila/uso terapêutico , Histona Desacetilases/isolamento & purificação , Humanos , Cinética , Masculino , Camundongos , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Transplante HeterólogoRESUMO
Here we describe a new approach for tumor targeting in which augmented concentrations of Fe(II) in cancer cells and/or the tumor microenvironment triggers drug release from an Fe(II)-reactive prodrug conjugate. The 1,2,4-trioxolane scaffold developed to enable this approach can in principle be applied to a broad range of cancer therapeutics and is illustrated here with Fe(II)-targeted forms of a microtubule toxin and a duocarmycin-class DNA-alkylating agent. We show that the intrinsic reactivity/toxicity of the duocarmycin analog is masked in the conjugated form and this greatly reduced toxicity in mice. This in turn permitted elevated dosing levels, leading to higher systemic exposure and a significantly improved response in tumor xenograft models. Overall our results suggest that Fe(II)-dependent drug delivery via trioxolane conjugates could have significant utility in expanding the therapeutic index of a range of clinical and preclinical stage cancer chemotherapeutics.
Assuntos
Antineoplásicos/farmacologia , Compostos Ferrosos/farmacologia , Indóis/farmacologia , Pró-Fármacos/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Sistemas de Liberação de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Duocarmicinas , Feminino , Compostos Ferrosos/química , Humanos , Indóis/síntese química , Indóis/química , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Estrutura Molecular , Pró-Fármacos/síntese química , Pró-Fármacos/química , Pirróis/síntese química , Pirróis/química , Pirróis/farmacologia , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
BACKGROUND AND AIMS: Recognition of abnormal glycosylation in virtually every cancer type has raised great interest in exploration of the tumor glycome for biomarker discovery. Identifying glycan markers of circulating tumor cells (CTCs) represents a new development in tumor biomarker discovery. The aim of this study was to establish an experimental approach to enable rapid screening of CTCs for glycan marker identification and characterization. METHODS: We applied carbohydrate microarrays and a high-speed fiber-optic array scanning technology (FAST scan) to explore potential glycan markers of breast CTCs (bCTCs) and targeting antibodies. An anti-tumor monoclonal antibody, HAE3-C1 (C1), was identified as a key immunological probe in this study. RESULTS: In our carbohydrate microarray analysis, C1 was found to be highly specific for an O-glycan cryptic epitope, gp(C1). Using FAST-scan technology, we established a procedure to quantify expression levels of gp(C1) in tumor cells. In blood samples from five stage IV metastatic breast cancer patients, the gp(C1) positive CTCs were detected in all subjects; â¼40% of bCTCs were strongly gp(C1) positive. Interestingly, CTCs from a triple-negative breast cancer patient with multiple sites of metastasis were predominantly gp(C1) positive (92.5%, 37/40 CTCs). CONCLUSIONS: Together we present here a practical approach to examine rare cell expression of glycan markers. Using this approach, we identified an O-core glyco-determinant gp(C1) as a potential immunological target of bCTCs. Given its bCTC-expression profile, this target warrants an extended investigation in a larger cohort of breast cancer patients.
Assuntos
Anticorpos Monoclonais/imunologia , Biomarcadores Tumorais/sangue , Neoplasias da Mama/patologia , Células Neoplásicas Circulantes/imunologia , Polissacarídeos/imunologia , Adulto , Linhagem Celular Tumoral , Feminino , Glicosilação , Humanos , Pessoa de Meia-Idade , Projetos PilotoRESUMO
The spliceosome has recently emerged as a new target for cancer chemotherapy and novel antitumor spliceosome targeted agents are under development. Here, we describe two types of novel pharmacodynamic assays that facilitate drug discovery and development of this intriguing class of innovative therapeutics; the first assay is useful for preclinical optimization of small-molecule agents that target the SF3B1 spliceosomal protein in animals, the second assay is an ex vivo validated, gel-based assay for the measurement of drug exposure in human leukocytes. The first assay utilizes a highly specific bioluminescent splicing reporter, based on the skipping of exons 4-11 of a Luc-MDM2 construct, which specifically yields active luciferase when treated with small-molecule spliceosome modulators. We demonstrate that this reporter can be used to monitor alternative splicing in whole cells in vitro. We describe here that cell lines carrying the reporter can be used in vivo for the efficient pharmacodynamic analysis of agents during drug optimization and development. We also demonstrate dose- and time-dependent on-target activity of sudemycin D6 (SD6), which leads to dramatic tumor regression. The second assay relies on the treatment of freshly drawn human blood with SD6 ex vivo treatment. Changes in alternative splicing are determined by RT-PCR using genes previously identified in in vitro experiments. The Luc-MDM2 alternative splicing bioluminescent reporter and the splicing changes observed in human leukocytes should allow for the more facile translation of novel splicing modulators into clinical application.
RESUMO
Inhibitors of histone deacetylase (HDAC) have been shown to induce terminal differentiation of human tumor cell lines and to have antitumor effects in vivo. We have prepared analogues of suberoylanilide hydroxamic acid (SAHA) and trichostatin A and have evaluated them in a human HDAC enzyme inhibition assay, a p21(waf1) (p21) promoter assay, and in monolayer growth inhibition assays. One compound, 4-(dimethylamino)-N-[7-(hydroxyamino)-7-oxoheptyl]-benzamide, was found to affect the growth of a panel of eight human tumor cell lines differentially.
Assuntos
Antineoplásicos/síntese química , Benzamidas/síntese química , Inibidores Enzimáticos/síntese química , Inibidores de Histona Desacetilases , Ácidos Hidroxâmicos/síntese química , Hidroxilaminas/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Benzamidas/química , Benzamidas/farmacologia , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/farmacologia , Hidroxilaminas/química , Hidroxilaminas/farmacologia , Modelos Moleculares , Regiões Promotoras Genéticas , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
A series of N-hydroxy-3-phenyl-2-propenamides were prepared as novel inhibitors of human histone deacetylase (HDAC). These compounds were potent enzyme inhibitors, having IC(50)s < 400 nM in a partially purified enzyme assay. However, potency in cell growth inhibition assays ranged over 2 orders of magnitude in two human carcinoma cell lines. Selected compounds having cellular IC(50) < 750 nM were tested for maximum tolerated dose (MTD) and for efficacy in the HCT116 human colon tumor xenograft assay. Four compounds having an MTD > or = 100 mg/kg were selected for dose-response studies in the HCT116 xenograft model. One compound, 9 (NVP-LAQ824), had significant dose-related activity in the HCT116 colon and A549 lung tumor models, high MTD, and low gross toxicity. On the basis, in part, of these properties, 9 has entered human clinical trials in 2002.
Assuntos
Acetiltransferases/antagonistas & inibidores , Acrilamidas/síntese química , Acrilamidas/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Ácidos Hidroxâmicos/síntese química , Ácidos Hidroxâmicos/farmacologia , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Animais , Peso Corporal/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Histona Acetiltransferases , Humanos , Indicadores e Reagentes , Camundongos , Camundongos Nus , Conformação Molecular , Transplante de NeoplasiasRESUMO
We previously identified metal-responsive transcription factor-1 (MTF-1) as a positive contributor to mouse fibrosarcoma growth through effects on cell survival, proliferation, tumor angiogenesis and extracellular matrix remodeling. In the present study, we investigated MTF-1 protein expression in human tissues by specific immunostaining of both normal and tumor tissue samples. Immunohistochemical (IHC) staining of a human tissue microarray (TMA), using a unique anti-human MTF-1 antibody, indicated constitutive MTF-1 expression in most normal tissues, with liver and testis displaying comparatively high levels of expression. Nevertheless, MTF-1 protein levels were found to be significantly elevated in diverse human tumor types, including breast, lung and cervical carcinomas. IHC analysis of a separate panel of full-size tissue sections of human breast cancers, including tumor and normal adjacent, surrounding tissue, confirmed and extended the results of the TMA analysis. Taken with our previous findings, this new study suggests a role for MTF-1 in human tumor development, growth or spread. Moreover, the study suggests that MTF-1 could be a novel therapeutic target that offers the opportunity to manipulate metal or redox homeostasis in tumor cells.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Neoplasias/metabolismo , Fatores de Transcrição/metabolismo , Mama/química , Mama/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proteínas de Ligação a DNA/genética , Feminino , Humanos , Neoplasias/genética , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Fatores de Transcrição/genética , Fator MTF-1 de TranscriçãoRESUMO
The orphan nuclear receptor estrogen-related receptor alpha (ERRalpha) has been implicated in the development of various human malignancies, including breast, prostate, ovary, and colon cancer. ERRalpha, bound to a co-activator protein (e.g., peroxisome proliferator receptor gamma co-activator-1alpha, PGC-1alpha), regulates cellular energy metabolism by activating transcription of genes involved in various metabolic processes, such as mitochondrial genesis, oxidative phosphorylation, and fatty acid oxidation. Accumulating evidence suggests that ERRalpha is a novel target for solid tumor therapy, conceivably through effects on the regulation of tumor cell energy metabolism associated with energy stress within solid tumor microenvironments. This report describes a novel steroidal antiestrogen (SR16388) that binds selectively to ERRalpha, but not to ERRbeta or ERRgamma, as determined using a time-resolved fluorescence resonance energy transfer assay. SR16388 potently inhibits ERRalpha's transcriptional activity in reporter gene assays, and prevents endogenous PGC-1alpha and ERRalpha from being recruited to the promoters or enhancers of target genes. Representative in vivo results show that SR16388 inhibited the growth of human prostate tumor xenografts in nude mice as a single agent at 30mg/kg given once daily and 100mg/kg given once weekly. In a combination study, SR16388 (10mg/kg, once daily) and paclitaxel (7.5mg/kg, twice weekly) inhibited the growth of prostate tumor xenografts in nude mice by 61% compared to untreated xenograft tumors. SR16388 also inhibited the proliferation of diverse human tumor cell lines after a 24-h exposure to the compound. SR16388 thus has utility both as an experimental antitumor agent and as a chemical probe of ERRalpha biology.
Assuntos
Estradiol/análogos & derivados , Antagonistas de Estrogênios/química , Antagonistas de Estrogênios/farmacologia , Receptores de Estrogênio/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Cristalografia por Raios X , Estradiol/química , Estradiol/farmacologia , Antagonistas de Estrogênios/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Receptores de Estrogênio/química , Receptores de Estrogênio/metabolismo , Esteroides/química , Esteroides/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
BACKGROUND: DNA-damaging agents are widely used for the treatment of human malignancies. Agents containing the multifunctional alkylating moiety tetrakis(2-chloroethyl)phosphorodiamidic acid are currently under development as cancer therapeutics. MATERIALS AND METHODS: TLK58747, a phophorodiamidate-based prodrug, was tested in vivo for antitumor efficacy and safety. The in vitro responses of tumor cells to TLK58747 were examined by cytotoxicity assays, cell cycle analysis, immunoblots and microscopy. RESULTS: TLK58747 was efficacious in xenograft models of human breast, pancreas, and prostate cancer, as well as in leukemia and glioma. It caused less bone marrow suppression in rats than did cyclophosphamide. In vitro, TLK58747 inhibited the growth of a wide variety of cancer cells and activated the DNA damage-response pathway, leading to G(2)/M cell cycle arrest and subsequent premature senescence or apoptosis. CONCLUSION: TLK58747 is a promising new alkylating agent with broad antitumor activity and superior safety that warrants further development.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Dano ao DNA , DNA de Neoplasias/efeitos dos fármacos , Compostos Organofosforados/farmacologia , Pró-Fármacos/farmacologia , Animais , Antineoplásicos Alquilantes/toxicidade , Divisão Celular/efeitos dos fármacos , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , DNA de Neoplasias/metabolismo , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Fase G2/efeitos dos fármacos , Células HL-60 , Humanos , Masculino , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Compostos Organofosforados/toxicidade , Pró-Fármacos/toxicidade , Ratos , Ratos Sprague-Dawley , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The GADD45 (growth arrest and DNA damage-inducible) family of genes is involved in the regulation of cell cycle progression and apoptosis. To study signaling pathways affecting GADD45beta expression and to examine systematically in vivo the GADD45beta expression in tissues following various toxic stresses, we created a transgenic mouse by fusing the GADD45beta promoter to firefly luciferase (Gadd45beta-luc). In vivo GADD45beta expression was assessed by measuring the luciferase activity in the Gadd45beta-luc transgenic mouse using a non-invasive imaging system (IVIS Imaging System, Xenogen Corporation). We found that a number of agents that induce oxidative stress, such as sodium arsenite, CCl4, lipopolysaccharide (LPS), or tumor necrosis factor-alpha, are able to induce luciferase expression throughout the entire animal. In liver, spleen, lung, intestine, kidney, and heart, we observed an induction of luciferase activity after LPS treatment, which correlates with an increase of GADD45beta mRNA in these tissues. Processes that induce DNA damage activate the NF-kappaB signaling pathway. Several inhibitors of the NF-kappaB signaling pathway, including dexamethasone, thalidomide, and a proteasome inhibitor, bortezomib, showed inhibitory effects on LPS-induced GADD45beta expression as indicated by a decrease of the luciferase activity. Northern blot analysis confirmed a broad inhibitory effect of bortezomib on LPS-induced GADD45beta mRNA expression in spleen, lung, and intestine. In liver of bortezomib-treated mice, we observed a reverse correlation between the luciferase activity and the GADD45beta mRNA level. We speculate that such a discrepancy could be due to severe liver toxicity caused by bortezomib and LPS co-treatment. MAPK inhibitors had transient and inconsistent effects on LPS-induced luciferase expression. Our data are consistent with the notion that NF-kappaB, but not the MAPK signaling pathways, is involved in the in vivo regulation of GADD45beta expression. Thus, NF-kappaB signaling involves induction of GADD45beta expression, which supports the proposed role of GADD45beta in protecting cells against DNA damaged under various stress conditions.