RESUMO
CCT251236 1, a potent chemical probe, was previously developed from a cell-based phenotypic high-throughput screen (HTS) to discover inhibitors of transcription mediated by HSF1, a transcription factor that supports malignancy. Owing to its activity against models of refractory human ovarian cancer, 1 was progressed into lead optimization. The reduction of P-glycoprotein efflux became a focus of early compound optimization; central ring halogen substitution was demonstrated by matched molecular pair analysis to be an effective strategy to mitigate this liability. Further multiparameter optimization led to the design of the clinical candidate, CCT361814/NXP800 22, a potent and orally bioavailable fluorobisamide, which caused tumor regression in a human ovarian adenocarcinoma xenograft model with on-pathway biomarker modulation and a clean in vitro safety profile. Following its favorable dose prediction to human, 22 has now progressed to phase 1 clinical trial as a potential future treatment for refractory ovarian cancer and other malignancies.
Assuntos
Antineoplásicos , Neoplasias Ovarianas , Humanos , Feminino , Fatores de Transcrição/metabolismo , Neoplasias Ovarianas/patologia , Linhagem Celular Tumoral , Antineoplásicos/farmacologiaRESUMO
BACKGROUND AND AIMS: Changes in single microRNA (miRNA) expression have been associated with chemo-resistance in biliary tract cancers (BTCs). However, a global assessment of the dynamic role of the microRNome has never been performed to identify potential therapeutic targets that are functionally relevant in the BTC cell response to chemotherapy. APPROACH AND RESULTS: High-throughput screening (HTS) of 997 locked nucleic acid miRNA inhibitors was performed in six cholangiocarcinoma cell lines treated with cisplatin and gemcitabine (CG) seeking changes in cell viability. Validation experiments were performed with mirVana probes. MicroRNA and gene expression was assessed by TaqMan assay, RNA-sequencing, and in situ hybridization in four independent cohorts of human BTCs. Knockout of microRNA was achieved by CRISPR-CAS9 in CCLP cells (MIR1249KO) and tested for effects on chemotherapy sensitivity in vitro and in vivo. HTS revealed that MIR1249 inhibition enhanced chemotherapy sensitivity across all cell lines. MIR1249 expression was increased in 41% of cases in human BTCs. In validation experiments, MIR1249 inhibition did not alter cell viability in untreated or dimethyl sulfoxide-treated cells; however, it did increase the CG effect. MIR1249 expression was increased in CD133+ biliary cancer cells freshly isolated from the stem cell niche of human BTCs as well as in CD133+ chemo-resistant CCLP cells. MIR1249 modulated the chemotherapy-induced enrichment of CD133+ cells by controlling their clonal expansion through the Wnt-regulator FZD8. MIR1249KO cells had impaired expansion of the CD133+ subclone and its enrichment after chemotherapy, reduced expression of cancer stem cell markers, and increased chemosensitivity. MIR1249KO xenograft BTC models showed tumor shrinkage after exposure to weekly CG, whereas wild-type models showed only stable disease over treatment. CONCLUSIONS: MIR1249 mediates resistance to CG in BTCs and may be tested as a target for therapeutics.
Assuntos
Neoplasias do Sistema Biliar , Colangiocarcinoma , Cisplatino/farmacologia , Desoxicitidina/análogos & derivados , MicroRNAs , Antineoplásicos/farmacologia , Neoplasias do Sistema Biliar/tratamento farmacológico , Neoplasias do Sistema Biliar/metabolismo , Neoplasias do Sistema Biliar/patologia , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Colangiocarcinoma/tratamento farmacológico , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patologia , Desoxicitidina/farmacologia , Descoberta de Drogas , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Ensaios de Triagem em Larga Escala/métodos , Humanos , MicroRNAs/antagonistas & inibidores , MicroRNAs/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
Deregulation of the cyclin-dependent kinases (CDKs) has been implicated in the pathogenesis of multiple cancer types. Consequently, CDKs have garnered intense interest as therapeutic targets for the treatment of cancer. We describe herein the molecular and cellular effects of CCT068127, a novel inhibitor of CDK2 and CDK9. Optimized from the purine template of seliciclib, CCT068127 exhibits greater potency and selectivity against purified CDK2 and CDK9 and superior antiproliferative activity against human colon cancer and melanoma cell lines. X-ray crystallography studies reveal that hydrogen bonding with the DFG motif of CDK2 is the likely mechanism of greater enzymatic potency. Commensurate with inhibition of CDK activity, CCT068127 treatment results in decreased retinoblastoma protein (RB) phosphorylation, reduced phosphorylation of RNA polymerase II, and induction of cell cycle arrest and apoptosis. The transcriptional signature of CCT068127 shows greatest similarity to other small-molecule CDK and also HDAC inhibitors. CCT068127 caused a dramatic loss in expression of DUSP6 phosphatase, alongside elevated ERK phosphorylation and activation of MAPK pathway target genes. MCL1 protein levels are rapidly decreased by CCT068127 treatment and this associates with synergistic antiproliferative activity after combined treatment with CCT068127 and ABT263, a BCL2 family inhibitor. These findings support the rational combination of this series of CDK2/9 inhibitors and BCL2 family inhibitors for the treatment of human cancer.
Assuntos
Antineoplásicos/farmacologia , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 9 Dependente de Ciclina/metabolismo , Melanoma/metabolismo , Purinas/farmacologia , Compostos de Anilina/farmacologia , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quinase 2 Dependente de Ciclina/genética , Quinase 9 Dependente de Ciclina/genética , Células HCT116 , Células HT29 , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Purinas/química , Proteína do Retinoblastoma/metabolismo , Sulfonamidas/farmacologiaRESUMO
Phenotypic screens, which focus on measuring and quantifying discrete cellular changes rather than affinity for individual recombinant proteins, have recently attracted renewed interest as an efficient strategy for drug discovery. In this article, we describe the discovery of a new chemical probe, bisamide (CCT251236), identified using an unbiased phenotypic screen to detect inhibitors of the HSF1 stress pathway. The chemical probe is orally bioavailable and displays efficacy in a human ovarian carcinoma xenograft model. By developing cell-based SAR and using chemical proteomics, we identified pirin as a high affinity molecular target, which was confirmed by SPR and crystallography.
Assuntos
Amidas/química , Proteínas de Transporte/química , Proteínas de Ligação a DNA/química , Proteínas Nucleares/química , Quinolinas/química , Fatores de Transcrição/química , Administração Oral , Amidas/administração & dosagem , Amidas/farmacologia , Disponibilidade Biológica , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Dioxigenases , Descoberta de Drogas , Fatores de Transcrição de Choque Térmico , Ligantes , Espectroscopia de Prótons por Ressonância Magnética , Quinolinas/administração & dosagem , Quinolinas/farmacologia , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Heat shock factor 1 (HSF1) is a transcription factor that plays key roles in cancer, including providing a mechanism for cell survival under proteotoxic stress. Therefore, inhibition of the HSF1-stress pathway represents an exciting new opportunity in cancer treatment. We employed an unbiased phenotypic screen to discover inhibitors of the HSF1-stress pathway. Using this approach we identified an initial hit (1) based on a 4,6-pyrimidine scaffold (2.00 µM). Optimisation of cellular SAR led to an inhibitor with improved potency (25, 15 nM) in the HSF1 phenotypic assay. The 4,6-pyrimidine 25 was also shown to have high potency against the CDK9 enzyme (3 nM).
RESUMO
PURPOSE: Deregulated phosphatidylinositol 3-kinase pathway signaling through AGC kinases including AKT, p70S6 kinase, PKA, SGK and Rho kinase is a key driver of multiple cancers. The simultaneous inhibition of multiple AGC kinases may increase antitumor activity and minimize clinical resistance compared with a single pathway component. EXPERIMENTAL DESIGN: We investigated the detailed pharmacology and antitumor activity of the novel clinical drug candidate AT13148, an oral ATP-competitive multi-AGC kinase inhibitor. Gene expression microarray studies were undertaken to characterize the molecular mechanisms of action of AT13148. RESULTS: AT13148 caused substantial blockade of AKT, p70S6K, PKA, ROCK, and SGK substrate phosphorylation and induced apoptosis in a concentration and time-dependent manner in cancer cells with clinically relevant genetic defects in vitro and in vivo. Antitumor efficacy in HER2-positive, PIK3CA-mutant BT474 breast, PTEN-deficient PC3 human prostate cancer, and PTEN-deficient MES-SA uterine tumor xenografts was shown. We show for the first time that induction of AKT phosphorylation at serine 473 by AT13148, as reported for other ATP-competitive inhibitors of AKT, is not a therapeutically relevant reactivation step. Gene expression studies showed that AT13148 has a predominant effect on apoptosis genes, whereas the selective AKT inhibitor CCT128930 modulates cell-cycle genes. Induction of upstream regulators including IRS2 and PIK3IP1 as a result of compensatory feedback loops was observed. CONCLUSIONS: The clinical candidate AT13148 is a novel oral multi-AGC kinase inhibitor with potent pharmacodynamic and antitumor activity, which shows a distinct mechanism of action from other AKT inhibitors. AT13148 will now be assessed in a first-in-human phase I trial.
Assuntos
Antineoplásicos/administração & dosagem , Neoplasias , Fosfatidilinositol 3-Quinase/metabolismo , Inibidores de Proteínas Quinases/administração & dosagem , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Pirimidinas/administração & dosagem , Pirróis/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Human cancers often contain genetic alterations that disable G1/S checkpoint control and loss of this checkpoint is thought to critically contribute to cancer generation by permitting inappropriate proliferation and distorting fate-driven cell cycle exit. The identification of cell permeable small molecules that activate the G1/S checkpoint may therefore represent a broadly applicable and clinically effective strategy for the treatment of cancer. Here we describe the identification of several novel small molecules that trigger G1/S checkpoint activation and characterise the mechanism of action for one, CCT020312, in detail. Transcriptional profiling by cDNA microarray combined with reverse genetics revealed phosphorylation of the eukaryotic initiation factor 2-alpha (EIF2A) through the eukaryotic translation initiation factor 2-alpha kinase 3 (EIF2AK3/PERK) as the mechanism of action of this compound. While EIF2AK3/PERK activation classically follows endoplasmic reticulum (ER) stress signalling that sets off a range of different cellular responses, CCT020312 does not trigger these other cellular responses but instead selectively elicits EIF2AK3/PERK signalling. Phosphorylation of EIF2A by EIF2A kinases is a known means to block protein translation and hence restriction point transit in G1, but further supports apoptosis in specific contexts. Significantly, EIF2AK3/PERK signalling has previously been linked to the resistance of cancer cells to multiple anticancer chemotherapeutic agents, including drugs that target the ubiquitin/proteasome pathway and taxanes. Consistent with such findings CCT020312 sensitizes cancer cells with defective taxane-induced EIF2A phosphorylation to paclitaxel treatment. Our work therefore identifies CCT020312 as a novel small molecule chemical tool for the selective activation of EIF2A-mediated translation control with utility for proof-of-concept applications in EIF2A-centered therapeutic approaches, and as a chemical starting point for pathway selective agent development. We demonstrate that consistent with its mode of action CCT020312 is capable of delivering potent, and EIF2AK3 selective, proliferation control and can act as a sensitizer to chemotherapy-associated stresses as elicited by taxanes.
Assuntos
Ativadores de Enzimas/farmacologia , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , eIF-2 Quinase/metabolismo , Animais , Análise por Conglomerados , Ciclina D1/metabolismo , DNA Complementar/genética , Avaliação Pré-Clínica de Medicamentos , Interações Medicamentosas , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Ativadores de Enzimas/química , Fator de Iniciação 2 em Eucariotos/metabolismo , Humanos , Camundongos , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Paclitaxel/farmacologia , Fosforilação/efeitos dos fármacos , Proteína do Retinoblastoma/metabolismo , Transcriptoma/efeitos dos fármacos , Transcriptoma/genéticaRESUMO
Histone deacetylase (HDAC) inhibitors are currently approved for cutaneous T-cell lymphoma and are in mid-late stage trials for other cancers. The HDAC inhibitors LAQ824 and SAHA increase phosphocholine (PC) levels in human colon cancer cells and tumor xenografts as observed by magnetic resonance spectroscopy (MRS). In this study, we show that belinostat, an HDAC inhibitor with an alternative chemical scaffold, also caused a rise in cellular PC content that was detectable by (1)H and (31)P MRS in prostate and colon carcinoma cells. In addition, (1)H MRS showed an increase in branched chain amino acid and alanine concentrations. (13)C-choline labeling indicated that the rise in PC resulted from increased de novo synthesis and correlated with an induction of choline kinase α expression. Furthermore, metabolic labeling experiments with (13)C-glucose showed that differential glucose routing favored alanine formation at the expense of lactate production. Additional analysis revealed increases in the choline/water and phosphomonoester (including PC)/total phosphate ratios in vivo. Together, our findings provide mechanistic insights into the impact of HDAC inhibition on cancer cell metabolism and highlight PC as a candidate noninvasive imaging biomarker for monitoring the action of HDAC inhibitors.
Assuntos
Biomarcadores Tumorais/análise , Colina Quinase/metabolismo , Neoplasias do Colo/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Fosforilcolina/análise , Fosforilcolina/metabolismo , Neoplasias da Próstata/metabolismo , Animais , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Humanos , Espectroscopia de Ressonância Magnética/métodos , Masculino , Camundongos , Camundongos Nus , Sulfonamidas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Preoperative chemotherapy has demonstrated a survival benefit for patients with potentially resectable esophageal cancer; however, currently it is not possible to predict the benefit of this treatment for an individual patient. This prospective study was designed to correlate gene expression profiles with clinical outcome in this setting. PATIENTS AND METHODS: Eligible patients were deemed to have resectable disease after staging by computed tomography, endoscopic ultrasound, and laparoscopy as indicated and following discussion at the multidisciplinary team meeting. All patients received neoadjuvant platinum and fluoropyrimidine-based chemotherapy; and clinical data were entered prospectively onto a study-specific database. Total RNA was isolated from pretreatment tumor biopsies obtained at baseline endoscopy and analyzed using a cDNA array consisting of 22,000 cDNA clones. RESULTS: Of the patients with adequate follow-up accrued between 2002 and 2005, 35 satisfied the quality control measures for the microarray profiling. Median follow-up was 938 days. Supervised hierarchical clustering of normalized data revealed 165 significantly differentially expressed genes based on overall survival (OS; P < .01) with 2 distinct clusters: a poor outcome group: N = 17 (1 year OS 46.2%) and a good outcome group: N = 18 (1 year OS 100%). Genes identified included those previously associated with esophageal cancer and, interestingly, a group of genes encoding proteins involved in the regulation of the TOLL receptor-signaling pathway. CONCLUSION: This initial study has highlighted groups of tumors with distinct gene expression profiles based on survival and warrants further validation in a larger cohort. This approach may further our understanding of individual tumor biology and thus facilitate the development of tailored treatment.
Assuntos
Adenocarcinoma/mortalidade , Endossonografia/instrumentação , Neoplasias Esofágicas/mortalidade , Perfilação da Expressão Gênica/métodos , Proteínas de Membrana/genética , Adenocarcinoma/genética , Adenocarcinoma/cirurgia , Idoso , Idoso de 80 Anos ou mais , DNA/análise , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/cirurgia , Feminino , Genes Supressores de Tumor , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos , RNA/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estatística como Assunto , Estatísticas não Paramétricas , Análise de SobrevidaRESUMO
Gliomas are primary brain tumors with poor prognosis that exhibit frequent abnormalities in phosphatidylinositol 3-kinase (PI3 kinase) signaling. We investigated the molecular mechanism of action of the isoform-selective class I PI3 kinase and mTOR inhibitor PI-103 in human glioma cells. The potent inhibitory effects of PI-103 on the PI3 kinase pathway were quantified. PI-103 and the mTOR inhibitor rapamycin both inhibited ribosomal protein S6 phosphorylation but there were clear differences in the response of upstream components of the PI3 kinase pathway, such as phosphorylation of Thr(308)-AKT, that were inhibited by PI-103 but not rapamycin. Gene expression profiling identified altered expression of genes encoding regulators of the cell cycle and cholesterol metabolism, and genes modulated by insulin or IGF1 signaling, rapamycin treatment or nutrient starvation. PI-103 decreased expression of positive regulators of G(1)/S phase progression and increased expression of the negative cell cycle regulator p27(kip1). A reversible PI-103-mediated G(1) cell cycle arrest occurred without significant apoptosis, consistent with the altered gene expression detected. PI-103 induced vacuolation and processing of LC-3i to LC-3ii, which are features of an autophagic response. In contrast to PI-103, LY294002 and PI-387 induced apoptosis, indicative of likely off-target effects. PI-103 interacted synergistically or additively with cytotoxic agents used in the treatment of glioma, namely vincristine, BCNU and temozolomide. Compared to individual treatments, the combination of PI-103 with temozolomide significantly improved the response of U87MG human glioma xenografts. Our results support the therapeutic potential for PI3 kinase inhibitors with a PI-103-like profile as therapeutic agents for the treatment of glioma.
Assuntos
Neoplasias Encefálicas/metabolismo , Inibidores Enzimáticos/farmacologia , Glioma/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Transdução de Sinais/efeitos dos fármacos , Animais , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Ciclo Celular/fisiologia , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Cromonas/farmacologia , Cromonas/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Furanos/farmacologia , Furanos/uso terapêutico , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Glioma/tratamento farmacológico , Glioma/patologia , Humanos , Camundongos , Morfolinas/farmacologia , Morfolinas/uso terapêutico , Transplante de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Fosfatidilinositol 3-Quinases/metabolismo , Piridinas/farmacologia , Piridinas/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêuticoRESUMO
Compounds within the 2-(4-aminophenyl)benzothiazole class represent extremely potent and selective experimental antitumour agents. The lysylamide prodrug of 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole is undergoing phase I clinical evaluation. Extensive studies to elucidate mechanisms underlying the stark selectivity demonstrated potent cytosolic AhR ligand binding and cytochrome P450 1A1-catalysed bioactivation. Two human derived breast cell lines, initially exquisitely sensitive to this class of agent (GI50 < 5 nM) have been derived displaying acquired resistance to 2-(4-amino-3-methylphenyl)benzothiazole (DF 203; GI50 > 50 microM). Cross resistance to 2-(4-amino-3-iodophenyl)benzothiazole and 2-(4-amino-3-cyanophenyl)benzothiazole is observed (GI50 > 30 microM) as is > 100-fold reduced sensitivity of the two variant lines to 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203). In contrast, cell lines possessing acquired resistance to DF 203 (203R) retain sensitivity to benzo[a]pyrene and doxorubicin. Examination of DF 203-treated cells by confocal microscopy and HPLC analyses of nutrient media concur revealing diminished depletion of DF 203 from medium and impaired intracellular DF 203 retention. In contrast to cytosolic arylhydrocarbon (AhR) receptors of wild type cells, AhR appears constitutively localised within nuclei of 203R cells; consequently, DF 203 fails to drive transcription of cyp1a1. DF 203- and 5F 203-derived DNA adducts fall significantly in 203R cells. Reduced number and intensity of gamma H2AX foci report protection against DF 203-evoked DNA double strand breaks. In conclusion, aberrant AhR signalling underlies at least in part acquired resistance to DF 203. Intriguingly, comparisons of gene transcription profiles between sensitive and resistant paired lines reveal > 5-fold up-regulation of cyp1b1 expression, a protein implicated in resistance to therapeutic agents.
Assuntos
Compostos de Anilina/farmacologia , Antineoplásicos/farmacologia , Benzotiazóis/farmacologia , Neoplasias da Mama/tratamento farmacológico , Compostos de Anilina/química , Compostos de Anilina/metabolismo , Hidrocarboneto de Aril Hidroxilases , Benzotiazóis/química , Benzotiazóis/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Citocromo P-450 CYP1B1 , Sistema Enzimático do Citocromo P-450/análise , Adutos de DNA/análise , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Dibenzodioxinas Policloradas/farmacologia , Receptores de Hidrocarboneto Arílico/análise , Receptores de Hidrocarboneto Arílico/efeitos dos fármacosRESUMO
The cyclin-dependent kinase (CDK) inhibitor seliciclib (R-roscovitine, CYC202) shows promising antitumor activity in preclinical models and is currently undergoing phase II clinical trials. Inhibition of the CDKs by seliciclib could contribute to cell cycle arrest and apoptosis seen with the drug. However, it is common for drugs to exert multiple effects on gene expression and biochemical pathways. To further our understanding of the molecular pharmacology of seliciclib, we employed cDNA microarrays to determine changes in gene expression profiles induced by the drug in HT29 human colon cancer cells. Concentrations of seliciclib were used that inhibited RB phosphorylation and cell proliferation. An increase in the mRNA expression for CJUN and EGR1 was confirmed by Western blotting, consistent with activation of the ERK1/2 MAPK pathway by seliciclib. Transcripts of key genes required for the progression through mitosis showed markedly reduced expression, including Aurora-A/B (AURK-A/B), Polo-like kinase (PLK), cyclin B2 (CCNB2), WEE1 and CDC25C. Reduced expression of these mitotic genes was also seen at the protein level. siRNA-mediated depletion of Aurora-A protein led to an arrest of cells in the G(2)/M phase, consistent with the effects of seliciclib treatment. Inhibition of mitotic entry following seliciclib treatment was indicated by a reduction of histone H3 phosphorylation, which is catalyzed by Aurora-B, and by decreased expression of mitotic markers, including phospho-protein phosphatase 1 alpha. The results indicate a potential mechanism through which seliciclib prevents entry into mitosis. Gene expression profiling has generated hypotheses that led to an increase in our knowledge of the cellular effects of seliciclib and could provide potential pharmacodynamic or response biomarkers for use in animal models and clinical trials.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/fisiologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Perfilação da Expressão Gênica , Mitose/efeitos dos fármacos , Purinas/farmacologia , Proliferação de Células , Células HT29 , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , RoscovitinaRESUMO
The Aurora family of serine/threonine kinases is important for the regulation of centrosome maturation, chromosome segregation, and cytokinesis during mitosis. Overexpression of Aurora kinases in mammalian cells leads to genetic instability and transformation. Increased levels of Aurora kinases have also been linked to a broad range of human tumors. Here, we describe the properties of CCT129202, a representative of a structurally novel series of imidazopyridine small-molecule inhibitors of Aurora kinase activity. This compound showed high selectivity for the Aurora kinases over a panel of other kinases tested and inhibits proliferation in multiple cultured human tumor cell lines. CCT129202 causes the accumulation of human tumor cells with >or=4N DNA content, leading to apoptosis. CCT120202-treated human tumor cells showed a delay in mitosis, abrogation of nocodazole-induced mitotic arrest, and spindle defects. Growth of HCT116 xenografts in nude mice was inhibited after i.p. administration of CCT129202. We show that p21, the cyclin-dependent kinase inhibitor, is induced by CCT129202. Up-regulation of p21 by CCT129202 in HCT116 cells led to Rb hypophosphorylation and E2F inhibition, contributing to a decrease in thymidine kinase 1 transcription. This has facilitated the use of 3'-deoxy-3'[(18)F]fluorothymidine-positron emission tomography to measure noninvasively the biological activity of the Aurora kinase inhibitor CCT129202 in vivo.
Assuntos
Inibidores Enzimáticos/farmacologia , Imidazóis/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Piridinas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Aurora Quinases , Linhagem Celular Tumoral , Regulação para Baixo , Inibidores Enzimáticos/farmacocinética , Ensaio de Imunoadsorção Enzimática , Feminino , Histonas/metabolismo , Humanos , Camundongos , Microscopia de Fluorescência , Mitose/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Proteína do Retinoblastoma/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Carcinogens induce complex transcriptional responses in cells that may hold key mechanistic information. Benzo(a)pyrene (BaP) modulation of transcription may occur through the activation of the aryl hydrocarbon receptor (AHR) or through responses to DNA damage. To characterize further the expression profiles induced by BaP in HepG2 and MCF-7 cells obtained in our previous study, they were compared to those induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which activates AHR but does not bind to DNA, and anti-benzo(a)pyrene- trans-7,8-dihydrodiol-9,10-epoxide (BPDE), which binds directly to DNA but does not activate AHR. A total of 22 genes had altered expression in MCF-7 cells after both BaP and TCDD exposure, and a total of 29 genes had altered expression in HepG2 cells. In both cell lines, xenobiotic metabolism was upregulated through induction of NQO1, MGST1, and CYP1B1. A total of 78 expression changes were induced by both BaP and BPDE in MCF-7 cells, and a total of 29 expression changes were induced by both BaP and BPDE in HepG2 cells. These genes were predominantly involved in cell cycle regulation, apoptosis, and DNA repair. BaP and BPDE caused the repression of histone genes in both cell lines, suggesting that regulation of these genes is an important component of the DNA damage response. Interestingly, overlap of the BPDE and TCDD gene expression profiles was also observed. Furthermore, some genes were modulated by BaP but not by TCDD or BPDE, including induction of CRY1 and MAK, which may represent novel signaling pathways that are independent of both AHR activation and DNA damage. Promoter analysis identified candidate genes for direct transcriptional regulation by either AHR or p53. These analyses have further dissected and characterized the complex cellular response to BaP.
Assuntos
Benzo(a)pireno/toxicidade , Carcinógenos Ambientais/toxicidade , Adutos de DNA/metabolismo , Dano ao DNA , Regulação da Expressão Gênica/efeitos dos fármacos , Receptores de Hidrocarboneto Arílico/metabolismo , Benzo(a)pireno/metabolismo , Sítios de Ligação , Carcinógenos Ambientais/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citocromo P-450 CYP1A1/genética , Perfilação da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Dibenzodioxinas Policloradas/toxicidade , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Supressora de Tumor p53/metabolismoRESUMO
HM74 and HM74a have been identified as receptors for niacin. HM74a mediates the pharmacological anti-lipolytic effects of niacin in adipocytes by reducing intracellular cyclic AMP (cAMP) and inhibiting release of free fatty acids into the circulation. In macrophages, niacin induces peroxisome proliferator-activated receptor gamma (PPARgamma)-dependent and cAMP-dependent expression of genes mediating reverse cholesterol transport, although via an unidentified receptor. We describe constitutive expression of HM74a mRNA and hypoxia- and IFNgamma-inducible expression of HM74 and HM74a in human monocytic cell lines and primary cells in culture. In U937 cells niacin-induced expression of 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), the most potent endogenous ligand of PPARgamma. Both niacin and the structurally distinct HM74/HM74a ligand acifran-induced nuclear expression of PPARgamma protein and enhanced PPARgamma transcriptional activity. Niacin-induced PPARgamma transcriptional activity was pertussis toxin sensitive and required activity of phospholipase A(2) (EC 3.1.1.4), cyclo-oxygenase (EC 1.14.99.1) and prostaglandin D(2) synthase (EC 5.3.99.2). Niacin also induced PPARgamma transcriptional activity in HM74 and HM74a CHO cell transfectants, although not in vector-only control cells. This was sensitive to pertussis toxin and to inhibition of phoshoplipase A(2) and cyclo-oxygenase activity. Additionally, niacin increased intracellular cAMP in U937 via a pertussis toxin and cyclo-oxygenase-sensitive mechanism. These results indicate that HM74 and HM74a can mediate macrophage responses to niacin via activation of the prostaglandin synthesis pathway and induction and activation of PPARgamma. This suggests a novel mechanism(s) mediating the clinical effects of pharmacological doses of niacin.
Assuntos
Macrófagos/efeitos dos fármacos , Niacina/farmacologia , PPAR gama/genética , Prostaglandinas/biossíntese , Receptores Acoplados a Proteínas G/fisiologia , Receptores Nicotínicos/fisiologia , Ativação Transcricional/efeitos dos fármacos , Western Blotting , Hipóxia Celular , Linhagem Celular Tumoral , AMP Cíclico/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Macrófagos/metabolismo , Niacina/metabolismo , Prostaglandinas/metabolismo , Receptores Acoplados a Proteínas G/biossíntese , Receptores Acoplados a Proteínas G/genética , Receptores Nicotínicos/biossíntese , Receptores Nicotínicos/genética , Transdução de Sinais , Ativação Transcricional/fisiologiaRESUMO
Delta9-Tetrahydrocannabinol (THC) is the active metabolite of cannabis. THC causes cell death in vitro through the activation of complex signal transduction pathways. However, the role that the cannabinoid 1 and 2 receptors (CB1-R and CB2-R) play in this process is less clear. We therefore investigated the role of the CB-Rs in mediating apoptosis in 3 leukemic cell lines and performed microarray and immunoblot analyses to establish further the mechanism of cell death. We developed a novel flow cytometric technique of measuring the expression of functional receptors and used combinations of selective CB1-R and CB2-R antagonists and agonists to determine their individual roles in this process. We have shown that THC is a potent inducer of apoptosis, even at 1 x IC(50) (inhibitory concentration 50%) concentrations and as early as 6 hours after exposure to the drug. These effects were seen in leukemic cell lines (CEM, HEL-92, and HL60) as well as in peripheral blood mononuclear cells. Additionally, THC did not appear to act synergistically with cytotoxic agents such as cisplatin. One of the most intriguing findings was that THC-induced cell death was preceded by significant changes in the expression of genes involved in the mitogen-activated protein kinase (MAPK) signal transduction pathways. Both apoptosis and gene expression changes were altered independent of p53 and the CB-Rs.
Assuntos
Canabinoides/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Dronabinol/toxicidade , Sistema de Sinalização das MAP Quinases/fisiologia , Receptor CB1 de Canabinoide/fisiologia , Receptor CB2 de Canabinoide/fisiologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Regulação da Expressão Gênica/efeitos dos fármacos , Células HL-60 , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Receptor CB1 de Canabinoide/efeitos dos fármacos , Receptor CB2 de Canabinoide/efeitos dos fármacos , Proteína Supressora de Tumor p53/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismoRESUMO
We review in detail how gene expression microarray technology is benefiting all phases of the discovery, development and subsequent use of new cancer therapeutics. Global gene expression profiling is valuable in cancer classification, elucidation of biochemical pathways and the identification of potential targets for novel molecular therapeutics. We exemplify the value in tissue culture and animal models of cancer, as well as in clinical studies. The power of expression profiling alongside gene knockout or knockdown methods such as RNA interference is illustrated. The use of basal or constitutive gene expression profiling to understand and predict drug sensitivity or resistance is described. The ability of expression profiling to define detailed molecular signatures of drug action is emphasised. The approach can identify on-target and off-target effects. It can be used to identify molecular biomarkers for proof of concept studies, pharmacodynamic endpoints and prognostic markers for predicting outcome and patient selection.
Assuntos
Antineoplásicos/uso terapêutico , Desenho de Fármacos , Perfilação da Expressão Gênica/métodos , Neoplasias/tratamento farmacológico , Animais , Dano ao DNA/efeitos dos fármacos , Previsões , Humanos , Modelos Biológicos , Mutação/genética , Neoplasias/genética , FarmacogenéticaRESUMO
Deregulated expression of the Wilms' tumor gene (WT1) has been implicated in the maintenance of a malignant phenotype in leukemias and a wide range of solid tumors through interference with normal signaling in differentiation and apoptotic pathways. Expression of high levels of WT1 is associated with poor prognosis in leukemias and breast cancer. Using real-time (Taqman) reverse transcription-PCR and RNase protection assay, we have shown up-regulation of WT1 expression following cytotoxic treatment of cells exhibiting drug resistance, a phenomenon not seen in sensitive cells. WT1 is subject to alternative splicing involving exon 5 and three amino acids (KTS) at the end of exon 9, producing four major isoforms. Exon 5 splicing was disrupted in all cell lines studied following a cytotoxic insult probably due to increased exon 5 skipping. Disruption of exon 5 splicing may be a proapoptotic signal because specific targeting of WT1 exon 5-containing transcripts using a nuclease-resistant antisense oligonucleotide (ASO) killed HL60 leukemia cells, which were resistant to an ASO targeting all four alternatively spliced transcripts simultaneously. K562 cells were sensitive to both target-specific ASOs. Gene expression profiling following treatment with WT1 exon 5-targeted antisense showed up-regulation of the known WT1 target gene, thrombospondin 1, in HL60 cells, which correlated with cell death. In addition, novel potential WT1 target genes were identified in each cell line. These studies highlight a new layer of complexity in the regulation and function of the WT1 gene product and suggest that antisense directed to WT1 exon 5 might have therapeutic potential.
Assuntos
Processamento Alternativo/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Éxons/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas WT1/genética , Processamento Alternativo/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Desoxirribonucleases/metabolismo , Doxorrubicina/farmacologia , Perfilação da Expressão Gênica , Humanos , Leucemia/genética , Leucemia/metabolismo , Leucemia/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Oligonucleotídeos Antissenso/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Fatores de Tempo , Proteínas WT1/metabolismoRESUMO
Often the use of cytotoxic drugs in cancer therapy results in stable disease rather than regression of the tumor, and this is typically seen as a failure of treatment. We now show that DNA damage is able to induce senescence in tumor cells expressing wild-type p53. We also show that cytotoxics are capable of inducing senescence in tumor tissue in vivo. Our results suggest that p53 and p21 play a central role in the onset of senescence, whereas p16(INK4a) function may be involved in maintaining senescence. Thus, like apoptosis, senescence appears to be a p53-induced cellular response to DNA damage and an important factor in determining treatment outcome.