RESUMO
Ovarian cancer is the leading cause of death among gynecological malignancies. It is detected at late stages when the disease is spread through the abdominal cavity in a condition known as peritoneal carcinomatosis. Thus, there is an urgent need to develop novel therapeutic interventions to target advanced stages of ovarian cancer. Mammary serine protease inhibitor (Maspin) represents an important metastasis suppressor initially identified in breast cancer. Herein we have generated a sequence-specific zinc finger artificial transcription factor (ATF) to up-regulate the Maspin promoter in aggressive ovarian cancer cell lines and to interrogate the therapeutic potential of Maspin in ovarian cancer. We found that although Maspin was expressed in some primary ovarian tumors, the promoter was epigenetically silenced in cell lines derived from ascites. Transduction of the ATF in MOVCAR 5009 cells derived from ascitic cultures of a TgMISIIR-TAg mouse model of ovarian cancer resulted in tumor cell growth inhibition, impaired cell invasion, and severe disruption of actin cytoskeleton. Systemic delivery of lipid-protamine-RNA nanoparticles encapsulating a chemically modified ATF mRNA resulted in inhibition of ovarian cancer cell growth in nude mice accompanied with Maspin re-expression in the treated tumors. Gene expression microarrays of ATF-transduced cells revealed an exceptional specificity for the Maspin promoter. These analyses identified novel targets co-regulated with Maspin in human short-term cultures derived from ascites, such as TSPAN12, that could mediate the anti-metastatic phenotype of the ATF. Our work outlined the first targeted, non-viral delivery of ATFs into tumors with potential clinical applications for metastatic ovarian cancers.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias Experimentais/metabolismo , Neoplasias Ovarianas/metabolismo , Fatores de Transcrição/biossíntese , Dedos de Zinco , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Camundongos , Camundongos Transgênicos , Invasividade Neoplásica , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/terapia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/farmacologia , Serpinas/biossíntese , Tetraspaninas/genética , Tetraspaninas/metabolismo , Fatores de Transcrição/genética , TranscriptomaRESUMO
Human equilibrative nucleoside transporter 1 (hENT1) is an important determinant for nucleoside analog based chemotherapy success. Preliminary data suggest hENT1 regulation by PPARs. Using A2780 cells, we investigated the role of PPARs on hENT1 expression and activity. PPARα and PPARγ agonists, Wy14,643 and RGZ, increased hENT1 expression, but only PPARα activation or overexpression resulted in higher hENT1 transport activity. On the other hand, promoter analysis showed two putative PPRE in hENT1 promoter and luciferase-coupled promoter constructs were generated and analyzed. Our results suggest that PPARα-but not PPARγ-mediated expression regulation of hENT1 is PPRE-dependent. In conclusion, PPARα and PPARγ activation modulate hENT1 expression.
Assuntos
Transportador Equilibrativo 1 de Nucleosídeo/genética , Regulação da Expressão Gênica , PPAR alfa/metabolismo , PPAR gama/metabolismo , Linhagem Celular , Humanos , PPAR alfa/agonistas , PPAR gama/agonistas , Regiões Promotoras Genéticas , Pirimidinas/farmacologia , Transfecção , Regulação para CimaRESUMO
Equilibrative nucleoside transporters (ENTs) are facilitative transporters broadly selective for pyrimidine and purine nucleosides and are essential for the modulation of nucleoside concentration and nucleoside analog availability. Resistance to nucleoside-derived drugs strongly correlates with a deficiency of ENT1 expression in several tumor cells. Thus, it is crucial to understand the mechanisms by which this transporter is modulated. Using a mouse myeloid leukemic cell line as a model, we investigated whether stress-activated kinases regulate ENT1 expression and function. JNK activation, but not p38 MAPK results in rapid loss of mENT1 function, mRNA expression and promoter activity. c-Jun but not the mutant c-Jun Ser63/73Ala, decreased mENT1 promoter activity. Moreover cJun bound to an AP-1 site identified at -1196 of the promoter, suggesting a specific role for this transcription factor in mENT1 regulation. We propose that activation of JNK-cJun pathway negatively regulates mENT1 and suggest that this mechanism might contribute to the development of nucleoside analog-derived drug resistance.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Transportador Equilibrativo 1 de Nucleosídeo/genética , Regulação da Expressão Gênica , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Nucleosídeos/metabolismo , Animais , Antineoplásicos/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Regulação para Baixo , Humanos , Camundongos , Regiões Promotoras GenéticasRESUMO
Peroxisome proliferator activated receptors (PPARs, alpha, beta/delta, gamma) control lipid homeostasis and differentiation in various tissues and tumor cells. PPARbeta and PPARgamma increase oligodendrocyte maturation in glial mixed populations and spinal cord oligodendrocytes, respectively, and PPARbeta is known to modulate the activity of other PPARs. To assess a possible interaction between PPARs in glial cell differentiation we used the undifferentiated C6 glioma cell line as model. These cells express all three PPARs, but only PPARgamma shows transcriptional activity in agonist-based reporter gene assay. Agonist-activated PPARgamma up-regulates oligodendrocyte markers, down-regulates an astrocyte marker, and increases alkyl-dihydroxyacetone phosphate synthase, enzyme involved in the synthesis of myelin-rich plasmalogens. Similar effects are induced in PPARgamma overexpressing cells, which in addition show PPARbeta up-regulation. PPARbeta or PPARalpha agonists show no effect. Nevertheless, PPARbeta overexpression up-regulates PPARgamma and commits C6 cells to oligodendrocytes; effect that is abrogated by a PPARgamma antagonist or PPARgamma interference RNA. Moreover, PPARbeta overexpression also induces PPARalpha and its target genes, including acyl-CoA oxidase, enzyme involved in very long chain fatty acid recycling, and in the synthesis of myelin components such as docosahexaenoic acid. These results indicate for the first time, that PPARs concertedly cooperate in C6 glioma cell differentiation to oligodendrocytes. Further, they suggest that active PPARbeta might be essential for increasing oligodendrocyte distinctive markers and enzymes required for myelin synthesis in C6 glioma cells through up-regulation of PPARgamma and PPARalpha.
Assuntos
Diferenciação Celular , Linhagem da Célula , Glioma/metabolismo , Metabolismo dos Lipídeos , Bainha de Mielina/metabolismo , Oligodendroglia/metabolismo , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Receptor Cross-Talk , Alquil e Aril Transferases/biossíntese , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Indução Enzimática , Glioma/enzimologia , Glioma/patologia , Metabolismo dos Lipídeos/genética , Camundongos , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/enzimologia , Oligodendroglia/patologia , PPAR alfa/metabolismo , PPAR gama/metabolismo , PPAR beta/metabolismo , Receptores Ativados por Proliferador de Peroxissomo/agonistas , Receptores Ativados por Proliferador de Peroxissomo/genética , Pirimidinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Rosiglitazona , Tiazóis/farmacologia , Tiazolidinedionas/farmacologia , Fatores de Tempo , Transcrição Gênica , TransfecçãoRESUMO
Acute myeloid leukemia (AML) has a high mortality rate despite chemotherapy and transplantation. Both CXCR4/SDF-1 and VLA-4/VCAM1 axes are involved in leukemia protection but little is known about the role of CCL2/CCR2 in AML biology and protection against chemotherapy. We measured CCR2 expression in AML cell lines and primary AML cells by flow cytometry (FCM), real time PCR (RT-PCR) and western blot (WB). CCL2 production was quantified by solid phase ELISA in peripheral blood (PB) and bone marrow (BM) serum. We measured chemotaxis in a transwell system with different concentrations of CCL2/CCR2 blockers; cell cycle with BrDU and propidium iodide and proliferation with yellow tetrazolium MTT. We determined synergy in in vitro cell apoptosis combining chemotherapy and CCL2/CCR2 blockade. Finally, we performed chemoprotection studies in an in vivo mouse model. Of 35 patients, 23 (65%) expressed CCR2 by FCM in PB. Two cell lines expressed high levels of CCR2 (THP-1 and murine AML). RT-PCR and WB confirmed CCR2 production. CCL2 solid phase ELISA showed significantly lower levels of CCL2 in PB and BM compared to normal controls. Chemotaxis experiments confirmed a dose-dependent migration in AML primary cells expressing CCR2 and THP-1 cells. A significant inhibition of transmigration was seen after CCL2/CCR2 blockade. Proliferation of CCR2+ AML cell lines was slightly increased (1.4-fold) after axis stimulation. We observed a non-significant increase in phase S THP-1 cells exposed to CCL2 and a concomitant decrease of cells in G1. The chemotherapy studies did not show a protective effect of CCL2 on cytarabine-induced apoptosis or synergy with chemotherapy after CCL2/CCR2 blockade both in vitro and in vivo. In conclusion, CCL2/CCR2 axis is expressed in the majority of monocytoid AML blasts. The axis is involved in cell trafficking and proliferation but no in vitro and in vivo chemotherapy protective effect was seen.
Assuntos
Quimiocina CCL2/metabolismo , Resistencia a Medicamentos Antineoplásicos , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/tratamento farmacológico , Receptores CCR2/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose , Medula Óssea/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Ensaio de Unidades Formadoras de Colônias , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Promielocítica Aguda/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Monócitos/citologia , Células U937 , Adulto JovemRESUMO
The aberrant epigenetic silencing of tumor suppressor genes (TSGs) plays a major role during carcinogenesis and regaining these dormant functions by engineering of sequence-specific epigenome editing tools offers a unique opportunity for targeted therapies. However, effectively normalizing the expression and regaining tumor suppressive functions of silenced TSGs by artificial transcription factors (ATFs) still remains a major challenge. Herein we describe novel combinatorial strategies for the potent reactivation of two class II TSGs, MASPIN and REPRIMO, in cell lines with varying epigenetic states, using the CRISPR/dCas9 associated system linked to a panel of effector domains (VP64, p300, VPR and SAM complex), as well as with protein-based ATFs, Zinc Fingers and TALEs. We found that co-delivery of multiple effector domains using a combination of CRISPR/dCas9 and TALEs or SAM complex maximized activation in highly methylated promoters. In particular, CRISPR/dCas9 VPR with SAM upregulated MASPIN mRNA (22,145-fold change) in H157 lung cancer cells, with accompanying re-expression of MASPIN protein, which led to a concomitant inhibition of cell proliferation and induction of apoptotic cell death. Consistently, CRISPR/dCas9 VP64 with SAM upregulated REPRIMO (680-fold change), which led to phenotypic reprogramming in AGS gastric cancer cells. Altogether, our results outlined novel sequence-specific, combinatorial epigenome editing approaches to reactivate highly methylated TSGs as a promising therapy for cancer and other diseases.
Assuntos
Sistemas CRISPR-Cas/genética , Proteínas de Homeodomínio/genética , Neoplasias Pulmonares/metabolismo , RNA Longo não Codificante/genética , Proteínas Repressoras/genética , Motivo Estéril alfa/genética , Neoplasias Gástricas/metabolismo , Dedos de Zinco/genética , Apoptose , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Metilação de DNA , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Neoplasias Pulmonares/genética , Serpinas/genética , Serpinas/metabolismo , Neoplasias Gástricas/genética , Ativação Transcricional , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
In order to treat osteoporosis and other bone mass disorders it is necessary to understand the regulatory processes that control the cell fate decisions responsible for going from bone precursor cells to bone tissue. Many processes interact to regulate cell division, differentiation and apoptosis. There are models for these basic processes, but not for their interactions. In this work we use the theory of switched systems, reuse and composition of validated models to describe the cell fate decisions leading to bone and fat formation. We describe the differentiation of osteo-adipo progenitor cells by composing its model with differentiation stimuli. We use the activation of the Wnt pathway as stimulus to osteoblast lineage, including regulation of cell division and apoptosis. This model is our first step to simulate physiological responses in silico to treatments for bone mass disorders.
Assuntos
Modelos Biológicos , Osteoblastos/citologia , Osteoblastos/fisiologia , Adipócitos/citologia , Adipócitos/fisiologia , Adipogenia , Apoptose , Diferenciação Celular , Divisão Celular , Linhagem da Célula , Condrogênese , Feminino , Redes Reguladoras de Genes , Humanos , Masculino , Osteogênese , Osteoporose/patologia , Osteoporose/terapia , Biologia de Sistemas , Via de Sinalização WntRESUMO
BACKGROUND: Despite a high response rate to chemotherapy, the majority of patients with acute myeloid leukemia (AML) are destined to relapse due to residual disease in the bone marrow (BM). The tumor microenvironment is increasingly being recognized as a critical factor in mediating cancer cell survival and drug resistance. In this study, we propose to identify mechanisms involved in the chemoprotection conferred by the BM stroma to leukemia cells. METHODS: Using a leukemia mouse model and a human leukemia cell line, we studied the interaction of leukemia cells with the BM microenvironment. We evaluated in vivo and in vitro leukemia cell chemoprotection to different cytotoxic agents mediated by the BM stroma. Leukemia cell apoptosis was assessed by flow cytometry and western blotting. The activity of the equilibrative nucleoside transporter 1 (ENT1), responsible for cytarabine cell incorporation, was investigated by measuring transport and intracellular accumulation of (3)H-adenosine. RESULTS: Leukemia cell mobilization from the bone marrow into peripheral blood in vivo using a CXCR4 inhibitor induced chemo-sensitization of leukemia cells to cytarabine, which translated into a prolonged survival advantage in our mouse leukemia model. In vitro, the BM stromal cells secreted a soluble factor that mediated significant chemoprotection to leukemia cells from cytarabine induced apoptosis. Furthermore, the BM stromal cell supernatant induced a 50% reduction of the ENT1 activity in leukemia cells, reducing the incorporation of cytarabine. No protection was observed when radiation or other cytotoxic agents such as etoposide, cisplatin and 5-fluorouracil were used. CONCLUSION: The BM stroma secretes a soluble factor that significantly protects leukemia cells from cytarabine-induced apoptosis and blocks ENT1 activity. Strategies that modify the chemo-protective effects mediated by the BM microenvironment may enhance the benefit of conventional chemotherapy for patients with AML.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Citarabina/farmacologia , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Animais , Antimetabólitos Antineoplásicos/uso terapêutico , Apoptose/fisiologia , Células da Medula Óssea/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citarabina/uso terapêutico , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Células Tumorais CultivadasRESUMO
The genetic basis for the Ara-C resistance of CCRF-CEM Ara-C/8C leukemia cells was investigated. DNA sequencing revealed that these cells expressed an equilibrative nucleoside transporter 1 (ENT1) with a single missense mutation resulting in glycine to arginine replacement (G24R). To test the importance of this residue, additional G24 mutants were created and examined for [3H]-uridine and [3H]-Ara-C uptake. Both a G24E and G24A mutant showed reduced ENT1-dependent activity. An EGFP-tagged G24R ENT1 displayed plasma membrane localization even though it was unable to bind [3H]-NBMPR, an ENT1-specific inhibitor. These results define G24 as critical amino acid for ENT1 nucleoside uptake and suggest that mutations in TM1 may provide a mechanism for Ara-C resistance in CCRF-CEM Ara-C/8C cells.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Citarabina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Transportador Equilibrativo 1 de Nucleosídeo/genética , Leucemia de Células T/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Glicina/genética , Glicina/metabolismo , Humanos , Dados de Sequência Molecular , Mutação Puntual , Estrutura Terciária de Proteína/genética , RNA Mensageiro/biossíntese , Tioinosina/análogos & derivados , Tioinosina/metabolismoRESUMO
Philadelphia chromosome-positive chronic myelogenus leukemia (CML) is widely treated with imatinib mesylate (imatinib), a potent inhibitor of the Bcr-Abl tyrosine kinase. However, resistance to this compound remains a concern. Current treatment approaches include combinations of imatinib with nucleoside analogs such as gemcitabine, which requires equilibrative nucleoside transporters (ENTs) for uptake, to overcome this resistance. Here we report that imatinib treatment decreased ENT1-dependent activity and mRNA expression. Although, imatinib-resistant cells showed decreased levels of both ENT1 and ENT2 activity and expression, these cells remained sensitive to gemcitabine, suggesting that nucleoside analogs can be used as adjunctive therapy.
Assuntos
Antineoplásicos/farmacologia , Desoxicitidina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos , Proteínas de Transporte de Nucleosídeo Equilibrativas/genética , Proteínas de Transporte de Nucleosídeo Equilibrativas/metabolismo , Piperazinas/farmacologia , Pirimidinas/farmacologia , Animais , Antineoplásicos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Benzamidas , Bovinos , Linhagem Celular Tumoral , Desoxicitidina/administração & dosagem , Desoxicitidina/farmacologia , Transportador Equilibrativo 1 de Nucleosídeo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Mesilato de Imatinib , Camundongos , Proteínas de Transporte de Nucleosídeos/genética , Proteínas de Transporte de Nucleosídeos/metabolismo , Piperazinas/administração & dosagem , Pirimidinas/administração & dosagem , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Tempo , GencitabinaRESUMO
Peroxisome proliferator-activated receptors (PPARs) are key transcription factors in the control of lipid homeostasis and cell differentiation, but little is known about their function in oligodendrocytes, the major lipid-synthesizing cells in the central nervous system (CNS). Using the B12 oligodendrocyte-like cell line and rat spinal cord-derived oligodendrocytes, we evaluated the importance of PPARgamma in the maturation process of these cells. B12 cells express all PPAR isoforms (alpha, beta/delta, and gamma), as assessed by RT-PCR, Western-blot, and transactivation assays. B12 cells respond specifically to PPARgamma agonists by arresting cell proliferation and extending cell processes, events that are blocked by the PPARgamma antagonist GW9662. In addition, alkyl-dihydroxyacetone phosphate synthase (ADAPS), a key peroxisomal enzyme involved in the synthesis of myelin-rich lipid plasmalogens, is increased in PPARgamma agonist-treated B12 cells. In contrast with B12 cells, both immature and mature isolated spinal cord oligodendrocytes presented a high and similar expression level of ADAPS, as assessed by immunocytochemistry. However, as in B12 cells, isolated spinal cord oligodendrocytes were also found to respond specifically to PPARgamma agonists with a four-fold increase in the number of mature cells. Our data suggest a relevant role for PPARgamma in oligodendrocyte lipid metabolism and differentiation.
Assuntos
Diferenciação Celular/fisiologia , Metabolismo dos Lipídeos , Oligodendroglia/citologia , Oligodendroglia/fisiologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Alquil e Aril Transferases/efeitos dos fármacos , Alquil e Aril Transferases/metabolismo , Anilidas/farmacologia , Animais , Western Blotting , Neoplasias Encefálicas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Imuno-Histoquímica , Isoformas de Proteínas , Ratos , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Medula Espinal/metabolismo , Fatores de Transcrição/agonistas , Fatores de Transcrição/antagonistas & inibidoresRESUMO
Peroxisome proliferator activated receptors (PPARs) are nuclear receptors that control important genes involved in lipid metabolism. Their role in nerve cells is uncertain, although anomalous myelination of the corpus callosum has been described in the PPARbeta-null mouse, and abnormalities of this tissue have been documented in fetal alcohol syndrome in humans. We report here that ethanol treatment of B12 oligodendrocyte-like cells induces a concentration- and time-dependent decrease in the mRNA and protein levels of PPARbeta, with no effect on PPARalpha or PPARgamma. The effect on PPARbeta is seen as an increase in mRNA degradation, as assessed by run-off assays, due to a significant decrease in PPARbeta mRNA half-life, with no observed changes in intracellular localization. Our results suggest a possible link between PPARbeta function and ethanol-induced abnormal myelination in oligodendrocytes.