RESUMO
Microscopy of Lewy bodies in Parkinson's disease (PD) suggests they are not solely filamentous deposits of α-synuclein (αS) but also contain vesicles and other membranous material. We previously reported the existence of native αS tetramers/multimers and described engineered mutations of the αS KTKEGV repeat motifs that abrogate the multimers. The resultant excess monomers accumulate in lipid membrane-rich inclusions associated with neurotoxicity exceeding that of natural familial PD mutants, such as E46K. Here, we use the αS "3K" (E35K+E46K+E61K) engineered mutation to probe the mechanisms of reported small-molecule modifiers of αS biochemistry and then identify compounds via a medium-throughput automated screen. αS 3K, which forms round, vesicle-rich inclusions in cultured neurons and causes a PD-like, l-DOPA-responsive motor phenotype in transgenic mice, was fused to YFP, and fluorescent inclusions were quantified. Live-cell microscopy revealed the highly dynamic nature of the αS inclusions: for example, their rapid clearance by certain known modulators of αS toxicity, including tacrolimus (FK506), isradipine, nilotinib, nortriptyline, and trifluoperazine. Our automated 3K cellular screen identified inhibitors of stearoyl-CoA desaturase (SCD) that robustly prevent the αS inclusions, reduce αS 3K neurotoxicity, and prevent abnormal phosphorylation and insolubility of αS E46K. SCD inhibition restores the E46K αS multimer:monomer ratio in human neurons, and it actually increases this ratio for overexpressed wild-type αS. In accord, conditioning 3K cells in saturated fatty acids rescued, whereas unsaturated fatty acids worsened, the αS phenotypes. Our cellular screen allows probing the mechanisms of synucleinopathy and refining drug candidates, including SCD inhibitors and other lipid modulators.
Assuntos
Corpos de Inclusão/efeitos dos fármacos , Lipídeos/análise , Mutação , Neuroblastoma/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/farmacologia , Estearoil-CoA Dessaturase/antagonistas & inibidores , alfa-Sinucleína/química , Animais , Linhagem Celular , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Estearoil-CoA Dessaturase/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMO
Efforts to develop more effective therapies for acute leukemia may benefit from high-throughput screening systems that reflect the complex physiology of the disease, including leukemia stem cells (LSCs) and supportive interactions with the bone marrow microenvironment. The therapeutic targeting of LSCs is challenging because LSCs are highly similar to normal hematopoietic stem and progenitor cells (HSPCs) and are protected by stromal cells in vivo. We screened 14,718 compounds in a leukemia-stroma co-culture system for inhibition of cobblestone formation, a cellular behavior associated with stem-cell function. Among those compounds that inhibited malignant cells but spared HSPCs was the cholesterol-lowering drug lovastatin. Lovastatin showed anti-LSC activity in vitro and in an in vivo bone marrow transplantation model. Mechanistic studies demonstrated that the effect was on target, via inhibition of HMG-CoA reductase. These results illustrate the power of merging physiologically relevant models with high-throughput screening.
Assuntos
Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Leucemia , Células-Tronco Neoplásicas/efeitos dos fármacos , Linhagem Celular Tumoral , Células-Tronco Hematopoéticas , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Lovastatina/farmacologia , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/fisiologiaRESUMO
RAD54, an important homologous recombination protein, is a member of the SWI2/SNF2 family of ATPase-dependent DNA translocases. In vitro, RAD54 stimulates RAD51-mediated DNA strand exchange and promotes branch migration of Holliday junctions. It is thought that an ATPase-dependent DNA translocation is required for both of these RAD54 activities. Here we identified, by high-throughput screening, a specific RAD54 inhibitor, streptonigrin (SN), and used it to investigate the mechanisms of RAD54 activities. We found that SN specifically targets the RAD54 ATPase, but not DNA binding, through direct interaction with RAD54 and generation of reactive oxygen species. Consistent with the dependence of branch migration (BM) on the ATPase-dependent DNA translocation of RAD54, SN inhibited RAD54 BM. Surprisingly, the ability of RAD54 to stimulate RAD51 DNA strand exchange was not significantly affected by SN, indicating a relatively smaller role of RAD54 DNA translocation in this process. Thus, the use of SN enabled us to identify important differences in the effect of the RAD54 ATPase and DNA translocation on two major activities of RAD54, BM of Holliday junctions and stimulation of DNA pairing.
Assuntos
Antibióticos Antineoplásicos/farmacologia , DNA Helicases/antagonistas & inibidores , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/antagonistas & inibidores , Enzimas Reparadoras do DNA/metabolismo , DNA Fúngico/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Estreptonigrina/farmacologia , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , DNA Cruciforme/genética , DNA Cruciforme/metabolismo , DNA Fúngico/genética , Recombinação Homóloga/efeitos dos fármacos , Recombinação Homóloga/fisiologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Conventional assays evaluating antitumor activity of immune effector cells have limitations that preclude their high-throughput application. We adapted the recently developed Compartment-Specific Bioluminescence Imaging (CS-BLI) technique to perform high-throughput quantification of innate antitumor activity and to show how pharmacologic agents (eg, lenalidomide, pomalidomide, bortezomib, and dexamethasone) and autologous BM stromal cells modulate that activity. CS-BLI-based screening allowed us to identify agents that enhance or inhibit innate antitumor cytotoxicity. Specifically, we identified compounds that stimulate immune effector cells against some tumor targets but suppressed their activity against other tumor cells. CS-BLI offers rapid, simplified, and specific evaluation of multiple conditions, including drug treatments and/or cocultures with stromal cells and highlights that immunomodulatory pharmacologic responses can be heterogeneous across different types of tumor cells. This study provides a framework to identify novel immunomodulatory agents and to prioritize compounds for clinical development on the basis of their effect on antitumor immunity.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Imunidade Inata/fisiologia , Medições Luminescentes/métodos , Neoplasias/diagnóstico , Neoplasias/imunologia , Animais , Antígeno CD56/metabolismo , Compartimento Celular/genética , Compartimento Celular/imunologia , Compartimento Celular/fisiologia , Sobrevivência Celular , Diagnóstico por Imagem/instrumentação , Diagnóstico por Imagem/métodos , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Imunoterapia/métodos , Células K562 , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Medições Luminescentes/instrumentação , Neoplasias/patologia , Neoplasias/terapia , Especificidade por Substrato/imunologia , Células Tumorais CultivadasRESUMO
BACKGROUND: Alzheimer's Disease (AD) affects millions globally, but therapy development is lagging. New experimental systems that monitor neuronal functions in conditions approximating the AD brain may be beneficial for identifying new therapeutic strategies. METHODS: We expose cultured neurons to aqueous-soluble human brain extract from 43 individuals across a spectrum of AD pathology. Multi-electrode arrays (MEAs) and live-cell imaging were used to assess neuronal firing and neurite integrity (NI), respectively, following treatments of rat cortical neurons (MEA) and human iPSC-derived neurons (iN) with human brain extracts. RESULTS: We observe associations between spontaneous activity and Aß42:40 levels, between neurite integrity and oligomeric Aß, and between neurite integrity and tau levels present in the brain extracts. However, these associations with Aß and tau do not fully account for the effects observed. Proteomic profiling of the brain extracts revealed additional candidates correlated with neuronal structure and activity. Neurotoxicity in MEA and NI assays was associated with proteins implicated in lysosomal storage disorders, while neuroprotection was associated with proteins of the WAVE regulatory complex controlling actin cytoskeleton dynamics. Elevated ganglioside GM2 activator (GM2A) associates with reductions in both NI and MEA activity, and cell-derived GM2A alone is sufficient to induce a loss of neurite integrity and a reduction in neuronal firing. CONCLUSIONS: The techniques and data herein introduce a system for modeling neuronal vulnerability in response to factors in the human brain and provide insights into proteins potentially contributing to AD pathogenesis.
Assuntos
Doença de Alzheimer , Neuritos , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Encéfalo/metabolismo , Gangliosídeo G(M2)/metabolismo , Gangliosídeos/metabolismo , Humanos , Neuritos/metabolismo , Neuritos/patologia , Neurônios/metabolismo , Proteínas/metabolismo , Proteômica , Ratos , Proteínas tau/metabolismoRESUMO
Cell cycle regulators, such as cyclin-dependent kinases (CDKs), are appealing targets for multiple myeloma (MM) therapy given the increased proliferative rates of tumour cells in advanced versus early stages of MM. We hypothesized that a multi-targeted CDK inhibitor with a different spectrum of activity compared to existing CDK inhibitors could trigger distinct molecular sequelae with therapeutic implications for MM. We therefore studied the small molecule heterocyclic compound NVP-LCQ195/AT9311 (LCQ195), which inhibits CDK1, CDK2 and CDK5, as well as CDK3 and CDK9. LCQ195 induced cell cycle arrest and eventual apoptotic cell death of MM cells, even at sub-µmol/l concentrations, spared non-malignant cells, and overcame the protection conferred to MM cells by stroma or cytokines of the bone marrow milieu. In MM cells, LCQ195 triggered decreased amplitude of transcriptional signatures associated with oncogenesis, drug resistance and stem cell renewal, including signatures of activation of key transcription factors for MM cells e.g. myc, HIF-1α, IRF4. Bortezomib-treated MM patients whose tumours had high baseline expression of genes suppressed by LCQ195 had significantly shorter progression-free and overall survival than those with low levels of these transcripts in their MM cells. These observations provide insight into the biological relevance of multi-targeted CDK inhibition in MM.
Assuntos
Antineoplásicos/farmacologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Mieloma Múltiplo/patologia , Apoptose/efeitos dos fármacos , Ácidos Borônicos/uso terapêutico , Bortezomib , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Quinases Ciclina-Dependentes/metabolismo , Citocinas/antagonistas & inibidores , Citocinas/farmacologia , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Combinação de Medicamentos , Interações Medicamentosas , Ensaios de Seleção de Medicamentos Antitumorais , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/enzimologia , Mieloma Múltiplo/genética , Pirazinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Células Estromais/fisiologia , Análise de Sobrevida , Transcrição Gênica , Resultado do Tratamento , Células Tumorais CultivadasRESUMO
BACKGROUND: Isothiocyanates, a family of phytochemicals found in cruciferous vegetables, have cytotoxic effects against several types of tumor cells. Multiple myeloma is a fatal disease characterized by clonal proliferation of plasma cells in the bone marrow. The growing body of preclinical information on the anti-cancer activity of isothiocyanates led us to investigate their anti-myeloma properties. DESIGN AND METHODS: We evaluated the anti-myeloma activity of the isothiocyanates, sulforaphane and phenethyl isothiocyanate, on a panel of human myeloma cell lines as well as primary myeloma tumor cells. Cell viability, apoptosis, cell cycle alterations and cell proliferation were then analyzed in vitro and in a xenograft mouse model in vivo. The molecular sequelae of isothiocyanate treatment in multiple myeloma cells were evaluated by multiplex analyses using bead arrays and western blotting. RESULTS: We observed that sulforaphane and phenylethyl isothiocyanate have activity against myeloma cell lines and patients' myeloma cells both in vitro and in vivo using a myeloma xenograft mouse model. Isothiocyanates induced apoptotic death of myeloma cells; depletion of mitochondrial membrane potential; cleavage of PARP and caspases-3 and -9; as well as down-regulation of anti-apoptotic proteins including Mcl-1, X-IAP, c-IAP and survivin. Isothiocyanates induced G(2)/M cell cycle arrest accompanied by mitotic phosphorylation of histone H3. Multiplex analysis of phosphorylation of diverse components of signaling cascades revealed changes in MAPK activation; increased phosphorylation of c-jun and HSP27; as well as changes in the phosphorylation of Akt, and GSK3α/ß and p53. Isothiocyanates suppressed proliferation of myeloma cells alone and when co-cultured with HS-5 stromal cells. Sulforaphane and phenylethyl isothiocyanate enhanced the in vitro anti-myeloma activity of several conventional and novel therapies used in multiple myeloma. CONCLUSIONS: Our study shows that isothiocyanates have potent anti-myeloma activities and may enhance the activity of other anti-multiple myeloma agents. These results indicate that isothiocyanates may have therapeutic potential in multiple myeloma and provide the preclinical framework for future clinical studies of isothiocyanates in multiple myeloma.
Assuntos
Antineoplásicos/farmacologia , Isotiocianatos/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Tiocianatos/farmacologia , Animais , Antineoplásicos/uso terapêutico , Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Humanos , Isotiocianatos/uso terapêutico , Isotiocianatos/toxicidade , Camundongos , Camundongos SCID , Mieloma Múltiplo/metabolismo , Células Estromais/efeitos dos fármacos , Sulfóxidos , Tiocianatos/uso terapêutico , Tiocianatos/toxicidade , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Multi-electrode arrays (MEAs) are being more widely used by researchers as an instrument platform for monitoring prolonged, non-destructive recordings of spontaneously firing neurons in vitro for applications in modeling Alzheimer's, Parkinson's, schizophrenia, and many other diseases of the human CNS. With the more widespread use of these instruments, there is a need to examine the prior art of studies utilizing MEAs and delineate best practices for data acquisition and analysis to avoid errors in interpretation of the resultant data. Using a dataset of recordings from primary rat (Rattus norvegicus) cortical cultures, methods and statistical power for discerning changes in neuronal activity on the array level are examined. Further, a method for unsupervised spike sorting is implemented, allowing for the resolution of action potential incidents down to the single neuron level. Following implementation of spike sorting, the dynamics of firing frequency across populations of individual neurons and networks are examined longitudinally. Finally, the ability to detect a frequency independent phenotype, the change in action potential amplitude, is demonstrated through the use of pore-forming neurotoxin treatments. Taken together, this study provides guidance and tools for users wishing to incorporate multi-well MEA usage into their studies.
Assuntos
Microeletrodos , Neurônios , Potenciais de Ação , Animais , Neurônios/fisiologia , RatosRESUMO
This study characterized the preclinical anti-myeloma activity of VE465, a low molecular weight pan-Aurora kinase inhibitor. After 96-h drug exposure, several multiple myeloma (MM) cell lines were more sensitive to VE465 compared to non-malignant cells. The anti-MM activity of VE465 was maintained in the presence of interleukin-6 and, interestingly, enhanced by co-culture with stromal cells. However, primary MM cells were less responsive than cell lines. Combinations with dexamethasone (Dex), doxorubicin (Doxo) and bortezomib showed no antagonism. Our study highlights the potential role of the tumour microenvironment in modulating the activity of this drug class.
Assuntos
Antineoplásicos/farmacologia , Mieloma Múltiplo/patologia , Piperazinas/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Aurora Quinases , Comunicação Celular/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Técnicas de Cocultura , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Células-Tronco Hematopoéticas/citologia , Humanos , Células Tumorais CultivadasRESUMO
B-Raf is an important mediator of cell proliferation and survival signals transduced via the Ras-Raf-MEK-ERK cascade. BRAF mutations have been detected in several tumors, including papillary thyroid carcinoma, but the precise role of B-Raf as a therapeutic target for thyroid carcinoma is still under investigation. We analyzed a panel of 93 specimens and 14 thyroid carcinoma cell lines for the presence of BRAF mutations and activation of the mitogen-activated protein/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway. We also compared the effect of a B-Raf small inhibitory RNA construct and the B-Raf kinase inhibitor AAL881 on both B-Raf wild-type and mutant thyroid carcinoma cell lines. We found a high prevalence of the T1799A (V600E) mutation in papillary and anaplastic carcinoma specimens and cell lines. There was no difference in patient age, B-Raf expression, Ki67 immunostaining, or clinical stage at presentation between wild-type and BRAF(V600E) specimens. Immunodetection of phosphorylated and total forms of MEK and ERK revealed no difference in their phosphorylation between wild-type and BRAF(V600E) patient specimens or cell lines. Furthermore, a small inhibitory RNA construct targeting the expression of both wild-type B-Raf and B-Raf(V600E) induced a comparable reduction of viability in both wild-type and BRAF(V600E) mutant cancer cells. Interestingly, AAL881 inhibited MEK and ERK phosphorylation and induced apoptosis preferentially in BRAF(V600E)-harboring cells than wild-type ones, possibly because of better inhibitory activity against B-Raf(V600E). We conclude that B-Raf is important for the pathophysiology of thyroid carcinomas irrespective of mutational status. Small molecule inhibitors that selectively target B-Raf(V600E) may provide clinical benefit for patients with thyroid cancer.
Assuntos
Adenocarcinoma Folicular/genética , Carcinoma Papilar/genética , Isoquinolinas/farmacologia , Mutação/genética , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias da Glândula Tireoide/genética , Adenocarcinoma Folicular/patologia , Adenocarcinoma Folicular/prevenção & controle , Adulto , Idoso , Apoptose/efeitos dos fármacos , Carcinoma/genética , Carcinoma/patologia , Carcinoma/prevenção & controle , Carcinoma Papilar/patologia , Carcinoma Papilar/prevenção & controle , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/prevenção & controle , Proliferação de Células/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Neoplasias da Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/prevenção & controle , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Increased expression of the Tribbles pseudokinase 1 gene (TRIB1) is associated with lower plasma levels of LDL cholesterol and triglycerides, higher levels of HDL cholesterol and decreased risk of coronary artery disease and myocardial infarction. We identified a class of tricyclic glycal core-based compounds that upregulate TRIB1 expression in human HepG2 cells and phenocopy the effects of genetic TRIB1 overexpression as they inhibit expression of triglyceride synthesis genes and ApoB secretion in cells. In addition to predicted effects related to downregulation of VLDL assembly and secretion these compounds also have unexpected effects as they upregulate expression of LDLR and stimulate LDL uptake. This activity profile is unique and favorably differs from profiles produced by statins or other lipoprotein targeting therapies. BRD8518, the initial lead compound from the tricyclic glycal class, exhibited stereochemically dependent activity and the potency far exceeding previously described benzofuran BRD0418. Gene expression profiling of cells treated with BRD8518 demonstrated the anticipated changes in lipid metabolic genes and revealed a broad stimulation of early response genes. Consistently, we found that BRD8518 activity is MEK1/2 dependent and the treatment of HepG2 cells with BRD8518 stimulates ERK1/2 phosphorylation. In agreement with down-regulation of genes controlling triglyceride synthesis and assembly of lipoprotein particles, the mass spectrometry analysis of cell extracts showed reduced rate of incorporation of stable isotope labeled glycerol into triglycerides in BRD8518 treated cells. Furthermore, we describe medicinal chemistry efforts that led to identification of BRD8518 analogs with enhanced potency and pharmacokinetic properties suitable for in vivo studies.
RESUMO
CONTEXT: The Bcl-2 family of proteins regulates apoptosis in various models and may represent a promising therapeutic target in human malignancies. OBJECTIVE/METHODS: We evaluated the sensitivity of thyroid carcinoma cell lines (two papillary, one follicular, two anaplastic, three medullary) in vitro to BH3I-1 and BH3I-2', two cell-permeable inhibitors of the Bcl-2 homology (BH)-3 domain-mediated interaction between proapoptotic and antiapoptotic Bcl-2 family members. The thyroid carcinoma cell line FRO was stably transfected with cDNA for Bcl-2 or constitutively active Akt and evaluated for sensitivity to BH3-domain inhibition. RESULTS: BH3-domain inhibition disrupted the mitochondrial membrane potential in thyroid carcinoma cells, induced caspase-dependent apoptosis, and potently sensitized them to sublethal concentrations of doxorubicin and the proteasome inhibitor bortezomib (Velcade). Overexpression of constitutively active Akt suppressed BH3I-1-induced cell death. Bcl-2-overexpressing FRO cells were more resistant to conventional chemotherapeutic agents (such as doxorubicin) but significantly more sensitive to BH3I-1 than control cells and were found to overexpress caspase-9, caspase-8, Bmf, Bok, and Bik transcripts and express less A1, BRaf, and FLIP transcripts. CONCLUSIONS: Bcl-2 expression protects thyroid carcinomas against chemotherapy-induced apoptosis. Nevertheless, overexpression of Bcl-2 may result in "oncogene addiction" of the cancer cell, which can be exploited by using BH3-domain inhibitors alone or in combination with other agents, including conventional chemotherapeutics (such as doxorubicin) or novel targeted therapies (such as the proteasome inhibitor bortezomib), for the treatment of aggressive thyroid cancer, including the medullary and anaplastic types.
Assuntos
Carcinoma Medular/genética , Carcinoma/genética , Genes bcl-2/genética , Neoplasias da Glândula Tireoide/genética , Antibióticos Antineoplásicos/farmacologia , Antineoplásicos/farmacologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Western Blotting , Ácidos Borônicos/farmacologia , Bortezomib , Linhagem Celular Tumoral , Colorimetria , Doxorrubicina/farmacologia , Humanos , Potenciais da Membrana/fisiologia , Proteína Oncogênica v-akt/genética , Inibidores de Proteases/farmacologia , Pirazinas/farmacologia , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Sais de Tetrazólio , Tiazóis , Transcrição GênicaRESUMO
PURPOSE: To evaluate the potential of proteasome inhibitors, a novel class of antitumor agents, for the treatment of retinoblastoma. The proteasome inhibitor bortezomib (PS-341, Velcade; Millennium Pharmaceuticals, Cambridge, MA), approved by the US Food and Drug Administration for the treatment of multiple myeloma, is being studied for the treatment of several other malignancies. Among other effects, it inactivates the transcription factor nuclear factor-kappaB (NF-kappaB) by blocking the degradation of its inhibitor, IkappaB. NF-kappaB, which is constitutively active in human retinoblastoma cells and promotes their survival, represents a therapeutic target for patients with this malignancy. METHODS: The authors evaluated the effect of bortezomib on the retinoblastoma cell lines Y79 and WERI-Rb1 in vitro using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry with propidium iodide, gene expression profiling, RT-PCR, and immunoblotting. RESULTS: Bortezomib induced caspase-dependent apoptosis in both retinoblastoma cell lines at clinically achievable concentrations. Bortezomib upregulated heat-shock proteins, other stress-response proteins, proapoptotic molecules, cell-cycle regulators, transcription factors, cytokines, and several proteasome subunits and solute carrier proteins, whereas it downregulated antiapoptotic and adhesion molecules. Bortezomib also induced cleavage of caspases, Bid and poly(ADP-ribose) polymerase (PARP), and sensitized retinoblastoma cells to doxorubicin. CONCLUSIONS: Bortezomib induces a stress response and triggers caspase-dependent apoptosis in human retinoblastoma cells at clinically achievable concentrations. This study provides insight into the molecular mechanism(s) of the antitumor activity of bortezomib and a basis for future preclinical studies leading to clinical trials of bortezomib, alone or in combination with conventional chemotherapy, to improve patient outcomes in retinoblastoma.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Ácidos Borônicos/farmacologia , Inibidores de Proteases/farmacologia , Pirazinas/farmacologia , Neoplasias da Retina/patologia , Retinoblastoma/patologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Bortezomib , Caspases/metabolismo , Proliferação de Células/efeitos dos fármacos , Citometria de Fluxo , Formazans , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Immunoblotting , Indicadores e Reagentes , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias da Retina/genética , Neoplasias da Retina/metabolismo , Retinoblastoma/genética , Retinoblastoma/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sais de Tetrazólio , Células Tumorais Cultivadas , Regulação para CimaRESUMO
Alzheimer's disease (AD) induces memory and cognitive impairment in the absence of motor and sensory deficits during its early and middle course. A major unresolved question is the basis for this selective neuronal vulnerability. Aß, which plays a central role in AD pathogenesis, is generated throughout the brain, yet some regions outside of the limbic and cerebral cortices are relatively spared from Aß plaque deposition and synapse loss. Here, we examine neurons derived from iPSCs of patients harboring an amyloid precursor protein mutation to quantify AD-relevant phenotypes following directed differentiation to rostral fates of the brain (vulnerable) and caudal fates (relatively spared) in AD. We find that both the generation of Aß and the responsiveness of TAU to Aß are affected by neuronal cell type, with rostral neurons being more sensitive than caudal neurons. Thus, cell-autonomous factors may in part dictate the pattern of selective regional vulnerability in human neurons in AD.
Assuntos
Doença de Alzheimer/genética , Peptídeos beta-Amiloides/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios/metabolismo , Proteínas tau/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Camundongos , Neurônios/patologia , Fenótipo , Proteínas tau/metabolismoRESUMO
CONTEXT: The epidermal growth factor receptor (EGFR), a transmembrane tyrosine kinase (TK) receptor that mediates proliferation and survival signaling, is expressed in a wide variety of normal and neoplastic tissues. EGFR inhibitors have produced objective responses in patients with non-small-cell lung carcinomas harboring activating EGFR TK domain somatic mutations. OBJECTIVE AND METHODS: Because the EGFR pathway has been reported to be important for the pathophysiology of thyroid carcinoma, we investigated the expression and mutational status of EGFR in 14 thyroid carcinoma cell lines as well as its functional role by evaluating their in vitro sensitivity to AEE788, a new dual-family EGFR/ErbB2 and vascular endothelial growth factor receptor TK inhibitor. We also evaluated the mutational status, mRNA and protein expression, as well as phosphorylation status of EGFR in a panel of thyroid carcinoma specimens. RESULTS: EGFR expression and phosphorylation in the thyroid carcinoma cell lines and tissue specimens were present but not stronger than in noncancerous thyroid tissue. EGFR TK domain mutations were detected in two of 62 histological specimens (3.2%) but not in cell lines. All thyroid carcinoma cell lines were significantly less sensitive (IC(50) at least 25-fold higher) in vitro to AEE788 than a primary culture of EGFR-mutant lung carcinoma cells. CONCLUSIONS: Thyroid carcinoma cells overall are poorly responsive to clinically relevant concentrations of AEE788 in vitro. The presence of EGFR-activating TK domain mutations may identify a small minority of thyroid cancer patients that may benefit from EGFR inhibitors, but additional preclinical evidence of efficacy is needed.
Assuntos
Carcinoma/tratamento farmacológico , Receptores ErbB/antagonistas & inibidores , Purinas/farmacologia , Neoplasias da Glândula Tireoide/tratamento farmacológico , Adolescente , Adulto , Idoso , Carcinoma/genética , Carcinoma/metabolismo , Linhagem Celular Tumoral , Receptores ErbB/biossíntese , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , NF-kappa B/metabolismo , Proteína Oncogênica v-akt/metabolismo , Fosforilação , Estrutura Terciária de Proteína , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismoRESUMO
CONTEXT: The ubiquitin-proteasome pathway is a major pathway for degradation of intracellular proteins. Proteasome inhibitors constitute a novel class of antitumor agents with preclinical and clinical evidence of activity against hematological malignancies and solid tumors. The proteasome inhibitor bortezomib (PS-341, Velcade) has been approved by the Food and Drug Administration for the treatment of multiple myeloma and is being studied intensely in several other malignancies. Its mechanism of action is complex but appears to include the inhibition of inhibitory-kappaB degradation, which leads to inactivation of the transcriptional factor nuclear factor-kappaB (NF-kappaB). NF-kappaB has been implicated in the pathophysiology of the most aggressive forms of thyroid carcinoma, i.e. medullary and anaplastic. OBJECTIVE AND METHODS: We evaluated the effect of bortezomib on a panel of thyroid carcinoma cell lines, originating from papillary, follicular, anaplastic, and medullary carcinomas. RESULTS: Bortezomib induced apoptosis in medullary and anaplastic cell lines with IC(50) values well within the range of clinically achievable concentrations and much lower than respective IC(50) values for other solid malignancies. Bortezomib inhibited NF-kappaB activity; increased p53, p21, and jun expression; and induced caspase-dependent apoptosis. Sensitivity of thyroid carcinoma cells to bortezomib was partially decreased by overexpression of Bcl-2 or treatment with IGF-I, whereas the combination of bortezomib with chemotherapy (doxorubicin) was synergistic. CONCLUSIONS: These data provide both insights into the molecular mechanisms of antitumor activity of proteasome inhibitors and the rationale for future clinical trials of bortezomib, alone or in combination with conventional chemotherapy, to improve patient outcome in medullary and anaplastic thyroid carcinomas.
Assuntos
Antineoplásicos/farmacologia , Ácidos Borônicos/farmacologia , Carcinoma Medular/tratamento farmacológico , Carcinoma/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Inibidores de Proteassoma , Pirazinas/farmacologia , Neoplasias da Glândula Tireoide/tratamento farmacológico , Apoptose/efeitos dos fármacos , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Bortezomib , Caspases/fisiologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/análise , Doxorrubicina/farmacologia , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-jun/metabolismo , Neoplasias da Glândula Tireoide/patologia , Proteína Supressora de Tumor p53/análiseRESUMO
Histone deacetylases (HDAC) and histone acetyltransferases exert opposing enzymatic activities that modulate the degree of acetylation of histones and other intracellular molecular targets, thereby regulating gene expression, cellular differentiation, and survival. HDAC inhibition results in accumulation of acetylated histones and induces differentiation and/or apoptosis in transformed cells. In this study, we characterized the effect of two HDAC inhibitors, suberoylanilide hydroxamic acid (SAHA) and m-carboxycinnamic acid bis-hydroxamide, on thyroid carcinoma cell lines, including lines originating from anaplastic and medullary carcinomas. In these models, both SAHA and m-carboxycinnamic acid bis-hydroxamide induced growth arrest and caspase-mediated apoptosis and increased p21 protein levels, retinoblastoma hypophosphorylation, BH3-interacting domain death agonist cleavage, Bax up-regulation, down-regulation of Bcl-2, A1, and Bcl-x(L) expression, and cleavage of poly(ADP-ribose) polymerase and caspase-8, -9, -3, -7, and -2. Transfection of Bcl-2 cDNA partially suppressed SAHA-induced cell death. SAHA down-regulated the expression of the apoptosis inhibitors FLIP and cIAP-2 and sensitized tumor cells to cytotoxic chemotherapy and death receptor activation. Our studies provide insight into the tumor type-specific mechanisms of antitumor effects of HDAC inhibitors and a framework for future clinical applications of HDAC inhibitors in patients with thyroid cancer, including histologic subtypes (e.g., anaplastic and medullary thyroid carcinomas) for which limited, if any, therapeutic options are available.
Assuntos
Antineoplásicos/farmacologia , Carcinoma/patologia , Cinamatos/farmacologia , Inibidores de Histona Desacetilases , Ácidos Hidroxâmicos/farmacologia , Neoplasias da Glândula Tireoide/patologia , Apoptose , Morte Celular , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células Tumorais Cultivadas , VorinostatRESUMO
PURPOSE: Fas ligand (FasL) and TNF-related apoptosis-inducing ligand (TRAIL)/Apo2L are members of the TNFalpha family that can trigger apoptosis in susceptible cells via respective death receptors (DRs). FasL cross-links its receptor Fas, resulting in recruitment and proteolytic activation of caspase-8, which initiates the downstream apoptotic cascade. TRAIL signals through its receptors DR4 and DR5, which can activate caspase-8 as well. This study was undertaken to investigate the functional status of the FasL and TRAIL apoptotic pathways in retinoblastoma (Rb) cells. METHODS: The human Rb cell lines Y79 and WERI-Rb1 were evaluated for their response to the Fas cross-linking antibody CH11 and recombinant TRAIL, as well as for cell surface presence and mutational status of Fas, DR4, and DR5 by flow cytometry and genomic DNA sequencing, respectively. The expression of caspase-8 and its inhibitor FLIP, as well as their recruitment to the DR signaling complex were studied by immunoblot analysis. RESULTS: Rb cells express Fas, DR4, and DR5 on their surfaces, yet were resistant to DR-mediated apoptosis. This was not due to DR mutations or secretion of the soluble decoy Fas, antiapoptotic NF-kappaB activity, or FLIP overexpression, but to the absence of caspase-8 expression. The demethylating agent 5-aza-2'-deoxycytidine restored caspase-8 expression and sensitivity to DR-mediated apoptosis. CONCLUSIONS: Rb cells are resistant to DR-mediated apoptosis because of a deficiency in caspase-8 expression secondary to epigenetic gene silencing by overmethylation. The data help delineate the apoptotic pathways in Rb cells and suggest that the combination of demethylating agents with DR-activating modalities, such as TRAIL receptor monoclonal antibodies, may benefit patients with retinoblastoma.
Assuntos
Apoptose , Caspases/genética , Inativação Gênica/fisiologia , Receptores do Fator de Necrose Tumoral/metabolismo , Neoplasias da Retina/patologia , Retinoblastoma/patologia , Azacitidina/farmacologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Caspase 8 , Inibidores de Caspase , Sobrevivência Celular , Inibidores Enzimáticos/farmacologia , Proteína Ligante Fas , Citometria de Fluxo , Humanos , Immunoblotting , Peptídeos e Proteínas de Sinalização Intracelular/farmacologia , Glicoproteínas de Membrana/metabolismo , NF-kappa B/antagonistas & inibidores , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Neoplasias da Retina/enzimologia , Retinoblastoma/enzimologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Receptor fas/metabolismoRESUMO
Recent genome wide association studies have linked tribbles pseudokinase 1 (TRIB1) to the risk of coronary artery disease (CAD). Based on the observations that increased expression of TRIB1 reduces secretion of VLDL and is associated with lower plasma levels of LDL cholesterol and triglycerides, higher plasma levels of HDL cholesterol and reduced risk for myocardial infarction, we carried out a high throughput phenotypic screen based on quantitative RT-PCR assay to identify compounds that induce TRIB1 expression in human HepG2 hepatoma cells. In a screen of a collection of diversity-oriented synthesis (DOS)-derived compounds, we identified a series of benzofuran-based compounds that upregulate TRIB1 expression and phenocopy the effects of TRIB1 cDNA overexpression, as they inhibit triglyceride synthesis and apoB secretion in cells. In addition, the compounds downregulate expression of MTTP and APOC3, key components of the lipoprotein assembly pathway. However, CRISPR-Cas9 induced chromosomal disruption of the TRIB1 locus in HepG2 cells, while confirming its regulatory role in lipoprotein metabolism, demonstrated that the effects of benzofurans persist in TRIB1-null cells indicating that TRIB1 is sufficient but not necessary to transmit the effects of the drug. Remarkably, active benzofurans, as well as natural products capable of TRIB1 upregulation, also modulate hepatic cell cholesterol metabolism by elevating the expression of LDLR transcript and LDL receptor protein, while reducing the levels of PCSK9 transcript and secreted PCSK9 protein and stimulating LDL uptake. The effects of benzofurans are not masked by cholesterol depletion and are independent of the SREBP-2 regulatory circuit, indicating that these compounds represent a novel class of chemically tractable small-molecule modulators that shift cellular lipoprotein metabolism in HepG2 cells from lipogenesis to scavenging.