RESUMEN
In utero mammalian development relies on the establishment of the maternal-fetal exchange interface, which ensures transportation of nutrients and gases between the mother and the fetus. This exchange interface is established via development of multinucleated syncytiotrophoblast cells (SynTs) during placentation. In mice, SynTs develop via differentiation of the trophoblast stem cell-like progenitor cells (TSPCs) of the placenta primordium, and in humans, SynTs are developed via differentiation of villous cytotrophoblast (CTB) progenitors. Despite the critical need in pregnancy progression, conserved signaling mechanisms that ensure SynT development are poorly understood. Herein, we show that atypical protein kinase C iota (PKCλ/ι) plays an essential role in establishing the SynT differentiation program in trophoblast progenitors. Loss of PKCλ/ι in the mouse TSPCs abrogates SynT development, leading to embryonic death at approximately embryonic day 9.0 (E9.0). We also show that PKCλ/ι-mediated priming of trophoblast progenitors for SynT differentiation is a conserved event during human placentation. PKCλ/ι is selectively expressed in the first-trimester CTBs of a developing human placenta. Furthermore, loss of PKCλ/ι in CTB-derived human trophoblast stem cells (human TSCs) impairs their SynT differentiation potential both in vitro and after transplantation in immunocompromised mice. Our mechanistic analyses indicate that PKCλ/ι signaling maintains expression of GCM1, GATA2, and PPARγ, which are key transcription factors to instigate SynT differentiation programs in both mouse and human trophoblast progenitors. Our study uncovers a conserved molecular mechanism, in which PKCλ/ι signaling regulates establishment of the maternal-fetal exchange surface by promoting trophoblast progenitor-to-SynT transition during placentation.
Asunto(s)
Diferenciación Celular/fisiología , Isoenzimas/metabolismo , Intercambio Materno-Fetal/fisiología , Placenta/metabolismo , Proteína Quinasa C/metabolismo , Trofoblastos/fisiología , Animales , Proteínas de Unión al ADN/metabolismo , Femenino , Factor de Transcripción GATA2/metabolismo , Humanos , Isoenzimas/genética , Masculino , Ratones , Ratones Noqueados , Modelos Animales , PPAR gamma/metabolismo , Placenta/citología , Placentación/fisiología , Embarazo , Proteína Quinasa C/genética , Transducción de Señal , Células Madre/citología , Factores de Transcripción/metabolismo , Trofoblastos/citologíaRESUMEN
Early pregnancy loss affects â¼15% of all implantation-confirmed human conceptions. However, evolutionarily conserved molecular mechanisms that regulate self-renewal of trophoblast progenitors and their association with early pregnancy loss are poorly understood. Here, we provide evidence that transcription factor TEAD4 ensures survival of postimplantation mouse and human embryos by controlling self-renewal and stemness of trophoblast progenitors within the placenta primordium. In an early postimplantation mouse embryo, TEAD4 is selectively expressed in trophoblast stem cell-like progenitor cells (TSPCs), and loss of Tead4 in postimplantation mouse TSPCs impairs their self-renewal, leading to embryonic lethality before embryonic day 9.0, a developmental stage equivalent to the first trimester of human gestation. Both TEAD4 and its cofactor, yes-associated protein 1 (YAP1), are specifically expressed in cytotrophoblast (CTB) progenitors of a first-trimester human placenta. We also show that a subset of unexplained recurrent pregnancy losses (idiopathic RPLs) is associated with impaired TEAD4 expression in CTB progenitors. Furthermore, by establishing idiopathic RPL patient-specific human trophoblast stem cells (RPL-TSCs), we show that loss of TEAD4 is associated with defective self-renewal in RPL-TSCs and rescue of TEAD4 expression restores their self-renewal ability. Unbiased genomics studies revealed that TEAD4 directly regulates expression of key cell cycle genes in both mouse and human TSCs and establishes a conserved transcriptional program. Our findings show that TEAD4, an effector of the Hippo signaling pathway, is essential for the establishment of pregnancy in a postimplantation mammalian embryo and indicate that impairment of the Hippo signaling pathway could be a molecular cause for early human pregnancy loss.
Asunto(s)
Autorrenovación de las Células/genética , Proteínas de Unión al ADN/genética , Desarrollo Embrionario/genética , Proteínas Musculares/genética , Factores de Transcripción/genética , Trofoblastos/citología , Trofoblastos/metabolismo , Aborto Habitual/etiología , Aborto Habitual/metabolismo , Aborto Espontáneo/etiología , Aborto Espontáneo/metabolismo , Animales , Biomarcadores , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Implantación del Embrión , Femenino , Técnica del Anticuerpo Fluorescente , Regulación del Desarrollo de la Expresión Génica , Humanos , Inmunohistoquímica , Ratones , Proteínas Musculares/metabolismo , Placenta/metabolismo , Embarazo , Factores de Transcripción de Dominio TEA , Factores de Transcripción/metabolismoRESUMEN
Early mammalian development is crucially dependent on the establishment of oxidative energy metabolism within the trophectoderm (TE) lineage. Unlike the inner cell mass, TE cells enhance ATP production via mitochondrial oxidative phosphorylation (OXPHOS) and this metabolic preference is essential for blastocyst maturation. However, molecular mechanisms that regulate establishment of oxidative energy metabolism in TE cells are incompletely understood. Here, we show that conserved transcription factor TEAD4, which is essential for pre-implantation mammalian development, regulates this process by promoting mitochondrial transcription. In developing mouse TE and TE-derived trophoblast stem cells (TSCs), TEAD4 localizes to mitochondria, binds to mitochondrial DNA (mtDNA) and facilitates its transcription by recruiting mitochondrial RNA polymerase (POLRMT). Loss of TEAD4 impairs recruitment of POLRMT, resulting in reduced expression of mtDNA-encoded electron transport chain components, thereby inhibiting oxidative energy metabolism. Our studies identify a novel TEAD4-dependent molecular mechanism that regulates energy metabolism in the TE lineage to ensure mammalian development.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Desarrollo Embrionario/genética , Metabolismo Energético , Mamíferos/embriología , Mamíferos/genética , Mitocondrias/genética , Proteínas Musculares/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Animales , Blastocisto/citología , Blastocisto/metabolismo , Blastocisto/ultraestructura , ADN Mitocondrial/genética , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Ectodermo/citología , Transporte de Electrón , Metabolismo Energético/genética , Ratones , Mitocondrias/ultraestructura , Modelos Biológicos , Proteínas Musculares/deficiencia , Proteínas Musculares/genética , Oxidación-Reducción , Células Madre/citología , Células Madre/metabolismo , Factores de Transcripción de Dominio TEA , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Trofoblastos/citologíaRESUMEN
A successful pregnancy is critically dependent upon proper placental development and function. During human placentation, villous cytotrophoblast (CTB) progenitors differentiate to form syncytiotrophoblasts (SynTBs), which provide the exchange surface between the mother and fetus and secrete hormones to ensure proper progression of pregnancy. However, epigenetic mechanisms that regulate SynTB differentiation from CTB progenitors are incompletely understood. Here, we show that lysine-specific demethylase 1 (LSD1; also known as KDM1A), a histone demethylase, is essential to this process. LSD1 is expressed both in CTB progenitors and differentiated SynTBs in first-trimester placental villi; accordingly, expression in SynTBs is maintained throughout gestation. Impairment of LSD1 function in trophoblast progenitors inhibits induction of endogenous retrovirally encoded genes SYNCYTIN1/endogenous retrovirus group W member 1, envelope (ERVW1) and SYNCYTIN2/endogenous retrovirus group FRD member 1, envelope (ERVFRD1), encoding fusogenic proteins critical to human trophoblast syncytialization. Loss of LSD1 also impairs induction of chorionic gonadotropin α (CGA) and chorionic gonadotropin ß (CGB) genes, which encode α and ß subunits of human chorionic gonadotrophin (hCG), a hormone essential to modulate maternal physiology during pregnancy. Mechanistic analyses at the endogenous ERVW1, CGA, and CGB loci revealed a regulatory axis in which LSD1 induces demethylation of repressive histone H3 lysine 9 dimethylation (H3K9Me2) and interacts with transcription factor GATA2 to promote RNA polymerase II (RNA-POL-II) recruitment and activate gene transcription. Our study reveals a novel LSD1-GATA2 axis, which regulates human trophoblast syncytialization.
Asunto(s)
Diferenciación Celular/genética , Factor de Transcripción GATA2/genética , Histona Demetilasas/genética , Trofoblastos/metabolismo , Vellosidades Coriónicas/crecimiento & desarrollo , Vellosidades Coriónicas/metabolismo , Epigénesis Genética/genética , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Productos del Gen env/genética , Humanos , Relaciones Madre-Hijo , Placentación/genética , Embarazo , Proteínas Gestacionales/genética , ARN Polimerasa II/genética , Transducción de Señal/genéticaRESUMEN
GATA transcription factors are implicated in establishing cell fate during mammalian development. In early mammalian embryos, GATA3 is selectively expressed in the extraembryonic trophoblast lineage and regulates gene expression to promote trophoblast fate. However, trophoblast-specific GATA3 function is dispensable for early mammalian development. Here, using dual conditional knockout mice, we show that genetic redundancy of Gata3 with paralog Gata2 in trophoblast progenitors ensures the successful progression of both pre- and postimplantation mammalian development. Stage-specific gene deletion in trophoblasts reveals that loss of both GATA genes, but not either alone, leads to embryonic lethality prior to the onset of their expression within the embryo proper. Using ChIP-seq and RNA-seq analyses, we define the global targets of GATA2/GATA3 and show that they directly regulate a large number of common genes to orchestrate stem versus differentiated trophoblast fate. In trophoblast progenitors, GATA factors directly regulate BMP4, Nodal and Wnt signaling components that promote embryonic-extraembryonic signaling cross-talk, which is essential for the development of the embryo proper. Our study provides genetic evidence that impairment of trophoblast-specific GATA2/GATA3 function could lead to early pregnancy failure.
Asunto(s)
Factor de Transcripción GATA2/fisiología , Factor de Transcripción GATA3/fisiología , Placenta/fisiología , Células Madre/citología , Trofoblastos/citología , Animales , Diferenciación Celular , Linaje de la Célula , Implantación del Embrión , Desarrollo Embrionario , Femenino , Eliminación de Gen , Humanos , Ratones , Ratones Noqueados , Embarazo , Preñez , Análisis de Secuencia de ARNRESUMEN
Cell fate decisions are fundamental to the development of multicellular organisms. In mammals the first cell fate decision involves segregation of the pluripotent inner cell mass and the trophectoderm, a process regulated by cell polarity proteins, HIPPO signaling and lineage-specific transcription factors such as CDX2. However, the regulatory mechanisms that operate upstream to specify the trophectoderm lineage have not been established. Here we report that transcription factor AP-2γ (TFAP2C) functions as a novel upstream regulator of Cdx2 expression and position-dependent HIPPO signaling in mice. Loss- and gain-of-function studies and promoter analysis revealed that TFAP2C binding to an intronic enhancer is required for activation of Cdx2 expression during early development. During the 8-cell to morula transition TFAP2C potentiates cell polarity to suppress HIPPO signaling in the outside blastomeres. TFAP2C depletion triggered downregulation of PARD6B, loss of apical cell polarity, disorganization of F-actin, and activation of HIPPO signaling in the outside blastomeres. Rescue experiments using Pard6b mRNA restored cell polarity but only partially corrected position-dependent HIPPO signaling, suggesting that TFAP2C negatively regulates HIPPO signaling via multiple pathways. Several genes involved in regulation of the actin cytoskeleton (including Rock1, Rock2) were downregulated in TFAP2C-depleted embryos. Inhibition of ROCK1 and ROCK2 activity during the 8-cell to morula transition phenocopied TFAP2C knockdown, triggering a loss of position-dependent HIPPO signaling and decrease in Cdx2 expression. Altogether, these results demonstrate that TFAP2C facilitates trophectoderm lineage specification by functioning as a key regulator of Cdx2 transcription, cell polarity and position-dependent HIPPO signaling.
Asunto(s)
Diferenciación Celular/fisiología , Linaje de la Célula/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas de Homeodominio/metabolismo , Transducción de Señal/fisiología , Factor de Transcripción AP-2/metabolismo , Factores de Transcripción/metabolismo , Trofoblastos/fisiología , Amidas/farmacología , Análisis de Varianza , Animales , Factor de Transcripción CDX2 , Polaridad Celular/fisiología , Inmunoprecipitación de Cromatina , Regulación del Desarrollo de la Expresión Génica/genética , Vía de Señalización Hippo , Luciferasas , Ratones , Microscopía Fluorescente , Proteínas Serina-Treonina Quinasas/metabolismo , Piridinas/farmacología , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/genética , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/metabolismoRESUMEN
Umbilical cord blood (UCB) engraftment is in part limited by graft cell dose, generally one log less than that of bone marrow (BM)/peripheral blood (PB) cell grafts. Strategies toward increasing hematopoietic stem/progenitor cell (HSPC) homing to BM have been assessed to improve UCB engraftment. Despite recent progress, a complete understanding of how HSPC homing and engraftment are regulated is still elusive. We provide evidence that blocking erythropoietin (EPO)-EPO receptor (R) signaling promotes homing to BM and early engraftment of UCB CD34+ cells. A significant population of UCB CD34+ HSPC expresses cell surface EPOR. Exposure of UCB CD34+ HSPC to EPO inhibits their migration and enhances erythroid differentiation. This migratory inhibitory effect was reversed by depleting EPOR expression on HSPC. Moreover, systemic reduction in EPO levels by hyperbaric oxygen (HBO) used in a preclinical mouse model and in a pilot clinical trial promoted homing of transplanted UCB CD34+ HSPC to BM. Such a systemic reduction of EPO in the host enhanced myeloid differentiation and improved BM homing of UCB CD34+ cells, an effect that was overcome with exogenous EPO administration. Of clinical relevance, HBO therapy before human UCB transplantation was well-tolerated and resulted in transient reduction in EPO with encouraging engraftment rates and kinetics. Our studies indicate that systemic reduction of EPO levels in the host or blocking EPO-EPOR signaling may be an effective strategy to improve BM homing and engraftment after allogeneic UCB transplantation. This clinical trial was registered at www.ClinicalTrials.gov (#NCT02099266).
Asunto(s)
Trasplante de Células Madre de Sangre del Cordón Umbilical , Eritropoyetina/metabolismo , ADP-Ribosil Ciclasa 1/metabolismo , Adolescente , Adulto , Anciano , Animales , Antígenos CD34/metabolismo , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Quimiocina CXCL12/farmacología , Quimerismo , Estudios de Cohortes , Trasplante de Células Madre de Sangre del Cordón Umbilical/efectos adversos , Femenino , Enfermedad Injerto contra Huésped/etiología , Humanos , Oxigenoterapia Hiperbárica , Masculino , Ratones , Persona de Mediana Edad , Células Mieloides/citología , Células Mieloides/efectos de los fármacos , Células Mieloides/metabolismo , Receptores de Eritropoyetina/metabolismo , Análisis de Supervivencia , Acondicionamiento Pretrasplante , Resultado del Tratamiento , Adulto JovenRESUMEN
Pluripotent stem cells (PSCs) contain functionally immature mitochondria and rely upon high rates of glycolysis for their energy requirements. Thus, altered mitochondrial function and promotion of aerobic glycolysis are key to maintain and induce pluripotency. However, signaling mechanisms that regulate mitochondrial function and reprogram metabolic preferences in self-renewing versus differentiated PSC populations are poorly understood. Here, using murine embryonic stem cells (ESCs) as a model system, we demonstrate that atypical protein kinase C isoform, PKC lambda/iota (PKCλ/ι), is a key regulator of mitochondrial function in ESCs. Depletion of PKCλ/ι in ESCs maintains their pluripotent state as evident from germline offsprings. Interestingly, loss of PKCλ/ι in ESCs leads to impairment in mitochondrial maturation, organization, and a metabolic shift toward glycolysis under differentiating condition. Our mechanistic analyses indicate that a PKCλ/ι-hypoxia-inducible factor 1α-PGC1α axis regulates mitochondrial respiration and balances pluripotency in ESCs. We propose that PKCλ/ι could be a crucial regulator of mitochondrial function and energy metabolism in stem cells and other cellular contexts.
Asunto(s)
Diferenciación Celular/fisiología , Células Madre Embrionarias/metabolismo , Metabolismo Energético/fisiología , Isoenzimas/metabolismo , Mitocondrias/metabolismo , Células Madre Pluripotentes/metabolismo , Proteína Quinasa C/metabolismo , Animales , Glucólisis/fisiología , Humanos , Ratones , Transducción de Señal/fisiologíaRESUMEN
Embryonic stem cell (ESC) pluripotency is orchestrated by distinct signaling pathways that are often targeted to maintain ESC self-renewal or their differentiation to other lineages. We showed earlier that inhibition of PKC signaling maintains pluripotency in mouse ESCs. Therefore, in this study, we investigated the importance of protein kinase C signaling in the context of rat ESC (rESC) pluripotency. Here we show that inhibition of PKC signaling is an efficient strategy to establish and maintain pluripotent rESCs and to facilitate reprogramming of rat embryonic fibroblasts to rat induced pluripotent stem cells. The complete developmental potential of rESCs was confirmed with viable chimeras and germ line transmission. Our molecular analyses indicated that inhibition of a PKCζ-NF-κB-microRNA-21/microRNA-29 regulatory axis contributes to the maintenance of rESC self-renewal. In addition, PKC inhibition maintains ESC-specific epigenetic modifications at the chromatin domains of pluripotency genes and, thereby, maintains their expression. Our results indicate a conserved function of PKC signaling in balancing self-renewal versus differentiation of both mouse and rat ESCs and indicate that targeting PKC signaling might be an efficient strategy to establish ESCs from other mammalian species.
Asunto(s)
Células Madre Embrionarias/enzimología , Células Madre Pluripotentes/enzimología , Proteína Quinasa C-epsilon/metabolismo , Transducción de Señal/fisiología , Animales , Células Madre Embrionarias/citología , Indoles/farmacología , Maleimidas/farmacología , MicroARNs/metabolismo , FN-kappa B/metabolismo , Células Madre Pluripotentes/citología , Proteína Quinasa C-epsilon/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Transducción de Señal/efectos de los fármacosRESUMEN
Methyltransferase-like 3 (METTL3), the catalytic enzyme of methyltransferase complex for m6A methylation of RNA, is essential for mammalian development. However, the importance of METTL3 in human placentation remains largely unexplored. Here, we show that a fine balance of METTL3 function in trophoblast cells is essential for successful human placentation. Both loss-of and gain-in METTL3 functions are associated with adverse human pregnancies. A subset of recurrent pregnancy losses and preterm pregnancies are often associated with loss of METTL3 expression in trophoblast progenitors. In contrast, METTL3 is induced in pregnancies associated with fetal growth restriction (FGR). Our loss of function analyses showed that METTL3 is essential for the maintenance of human TSC self-renewal and their differentiation to extravillous trophoblast cells (EVTs). In contrast, loss of METTL3 in human TSCs promotes syncytiotrophoblast (STB) development. Global analyses of RNA m6A modification and METTL3-RNA interaction in human TSCs showed that METTL3 regulates m6A modifications on the mRNA molecules of critical trophoblast regulators, including GATA2, GATA3, TEAD1, TEAD4, WWTR1, YAP1, TFAP2C and ASCL2, and loss of METTL3 leads to depletion of mRNA molecules of these critical regulators. Importantly, conditional deletion of Mettl3 in trophoblast progenitors of an early post-implantation mouse embryo also leads to arrested self-renewal. Hence, our findings indicate that METLL3 is a conserved epitranscriptomic governor in trophoblast progenitors and ensures successful placentation by regulating their self-renewal and dictating their differentiation fate.
RESUMEN
Multidrug resistance-associated protein 1 (MRP1) reduces intracellular anticancer drug accumulation either by co transporting them with glutathione (GSH) or extruding drug-GSH conjugates outside of the cell. Thus, MRP1 confers multidrug resistance (MDR) and worsen successful chemotherapeutic treatment against cancer. Although the exact mechanism of MRP1 involved in MDR remains unknown, the elevated level of intracellular GSH is considered as a key factor responsible for MDR in cancer. Hence the quest for non-toxic molecules that are able to deplete intracellular GSH has profound importance to subdue MDR. The present preclinical study depicts the resistance reversal potentiality of an iron complex; viz. Ferrous N-(2-hydroxy acetophenone) glycinate (FeNG) developed by us in doxorubicin resistant Ehrlich ascites carcinoma (EAC/Dox) cells. FeNG potentiate cytotoxic effect of doxorubicin on EAC/Dox cells ex vivo and also increases the survivability EAC/Dox bearing Swiss albino mice in vivo as well. Moreover, in vivo administration of FeNG significantly depletes intracellular GSH with ensuant increase in doxorubicin concentration in EAC/Dox cells without alternation of MRP1 expression. In addition, intra-peritoneal (i.p.) application of FeNG in normal or EAC/Dox bearing mice does not cause any systemic toxicity in preliminary trials in mouse Ehrlich ascites carcinoma model. Therefore, the present report provides evidence that FeNG may be a promising new resistance modifying agent against drug resistant cancers.
Asunto(s)
Acetofenonas/metabolismo , Antineoplásicos/uso terapéutico , Carcinoma de Ehrlich/tratamiento farmacológico , Doxorrubicina/uso terapéutico , Resistencia a Antineoplásicos , Glutatión/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Acetofenonas/química , Animales , Resistencia a Múltiples Medicamentos , Femenino , Humanos , Hierro , Ratones , Estructura Molecular , Distribución TisularRESUMEN
During mouse preimplantation development, the generation of the inner cell mass (ICM) and trophoblast lineages comprises upregulation of Nanog expression in the ICM and its silencing in the trophoblast. However, the underlying epigenetic mechanisms that differentially regulate Nanog in the first cell lineages are poorly understood. Here, we report that BRG1 (Brahma-related gene 1) cooperates with histone deacetylase 1 (HDAC1) to regulate Nanog expression. BRG1 depletion in preimplantation embryos and Cdx2-inducible embryonic stem cells (ESCs) revealed that BRG1 is necessary for Nanog silencing in the trophoblast lineage. Conversely, in undifferentiated ESCs, loss of BRG1 augmented Nanog expression. Analysis of histone H3 within the Nanog proximal enhancer revealed that H3 lysine 9/14 (H3K9/14) acetylation increased in BRG1-depleted embryos and ESCs. Biochemical studies demonstrated that HDAC1 was present in BRG1-BAF155 complexes and BRG1-HDAC1 interactions were enriched in the trophoblast lineage. HDAC1 inhibition triggered an increase in H3K9/14 acetylation and a corresponding rise in Nanog mRNA and protein, phenocopying BRG1 knockdown embryos and ESCs. Lastly, nucleosome-mapping experiments revealed that BRG1 is indispensable for nucleosome remodeling at the Nanog enhancer during trophoblast development. In summary, our data suggest that BRG1 governs Nanog expression via a dual mechanism involving histone deacetylation and nucleosome remodeling.
Asunto(s)
Blastocisto/metabolismo , ADN Helicasas/metabolismo , Células Madre Embrionarias/citología , Histona Desacetilasa 1/metabolismo , Proteínas de Homeodominio/biosíntesis , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Acetilación , Animales , Masa Celular Interna del Blastocisto/citología , Masa Celular Interna del Blastocisto/metabolismo , ADN Helicasas/genética , Embrión de Mamíferos , Regulación del Desarrollo de la Expresión Génica , Histona Desacetilasa 1/antagonistas & inhibidores , Histonas/metabolismo , Ratones , Complejos Multiproteicos/metabolismo , Proteína Homeótica Nanog , Proteínas Nucleares/genética , Procesamiento Proteico-Postraduccional/genética , Interferencia de ARN , ARN Mensajero/genética , ARN Interferente Pequeño , Factores de Transcripción/genética , Transcripción Genética/genética , Activación Transcripcional/genética , Trofoblastos/citologíaRESUMEN
Drug resistance is a problem that hinders the numerous successes of chemotherapeutic intervention of cancer and continues to be a major obstacle for cures. Till date, several attempts have been made to develop suitable multidrug resistance (MDR) reversing agents. But, throughout the clinical development of MDR reversing agents, patients repeatedly suffer from toxicities. So far, some anticancer activity of Schiff bases which are the condensation products of carbonyl compounds and primary amines and their metal complexes has been described. But, overcoming multidrug resistance, by the use of such small molecules still remain unexplored. Under this backdrop, in search of less toxic and more effective MDR reversing agents our laboratory has developed the different metal chelates of Schiff base N-(2-hydroxy acetophenone)glycinate (NG) which is structurally similar to azatyrosine [L-ß-(5-hydroxy-2-pyridyl)-alanine] that inhibits tumor formation by deactivating the c-Raf-1 kinase and c-Ha-ras signalling pathway. A decade-long research proposes possible strategies to overcome MDR by exploiting the chemical nature of such metal chelates. In this review we have catalogued the success of metal chelates of NG to overcome MDR in cancer. The review depict that the problem of MDR can be circumvent by synchronized activation of immunogenic cell death pathways that utilize the components of a host's immune system to kill cancer cells in combination with other conventional strategies. The current wealth of preclinical information promises better understanding of the cellular processes underlying MDR reversing activity of metal derivatives of NG and thus exposes several cellular targets for rational designing of new generation of Schiff base metal chelates as MDR reversing agents.
Asunto(s)
Resistencia a Múltiples Medicamentos/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Bases de Schiff/farmacología , Bases de Schiff/uso terapéutico , Animales , Quelantes/metabolismo , Complejos de Coordinación/metabolismo , HumanosRESUMEN
Anticancer drugs induce apoptosis to cancer cells and also exhibit undesired toxicity to normal cells. Therefore development of novel agents triggering apoptosis and have low toxicity towards normal cells is most important. Hydroxamic acids suppress tumour cell growth through apoptosis but the underlying mechanism is poorly understood. Herein, we describe the apoptotic potential of a dibasic hydroxamic acid derivative, viz., oxayl bis (N-phenyl) hydroxamic acid (OBPHA), which induces apoptosis through generation of both ROS and NO in doxorubicin resistant T-lymphoblastic leukemia, CEM/ADR5000 cells. Present study discloses that OBPHA selectively kills cancerous cells irrespective of their drug resistant phenotype. We also determined the crystal structure of OBPHA to understand the structural requirements for apoptosis; the study reveals that the presence of substituted hydroxamic acid groups (-CO-NH-OH) favours the generation of NO possibly through auto degeneration. Along with the induction of caspase 3 mediated intrinsic apoptosis; OBPHA also activates p53 dependent signalling cascade and downregulates HDAC3 expression in a time dependent manner possibly due to increased ROS and NO production and simultaneous decrease in cellular GSH level. Thus ROS and NO mediated downstream signalling are essential for the anticancer effect of OBPHA. Therefore OBPHA, having a structurally relevant pharmacophore provides important insight into the development of new ROS and RNS generating chemicals inducing p53 dependent apoptosis.
Asunto(s)
Antineoplásicos/farmacología , Bencenoacetamidas/farmacología , Ácidos Hidroxámicos/farmacología , Oxalatos/farmacología , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Glutatión/metabolismo , Humanos , Leucemia/tratamiento farmacológico , Leucemia/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/fisiología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismoRESUMEN
Multidrug resistance (MDR) remains a significant problem for effective cancer chemotherapy. In spite of considerable advances in drug discovery, most of the cancer cases still stay incurable because of resistance to chemotherapy. We synthesized a novel, Mn (II) complex (chelate), viz., manganese N-(2-hydroxy acetophenone) glycinate (MnNG) that exhibits considerable efficacy to overcome drug resistant cancer. The antiproliferative activity of MnNG was studied on doxorubicin resistant and sensitive human T lymphoblastic leukemia cells (CEM/ADR 5000 and CCRF/CEM). MnNG induced apoptosis significantly in CEM/ADR 5000 cells probably through generation of reactive oxygen species. Moreover, intraperitoneal (i.p.) application of MnNG at non-toxic doses caused significant increase in the life-span of Swiss albino mice bearing sensitive and doxorubicin resistant subline of Ehrlich ascites carcinoma cells.
Asunto(s)
Glicina/análogos & derivados , Glicina/farmacología , Manganeso/farmacología , Neoplasias/tratamiento farmacológico , Compuestos Organometálicos/farmacología , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Células de la Médula Ósea/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Doxorrubicina , Resistencia a Múltiples Medicamentos , Femenino , Humanos , Manganeso/química , Ratones , Bazo/citologíaRESUMEN
Multidrug resistance (MDR) in cancer, a major obstacle to successful application of cancer chemotherapy, is often characterized by over-expression of multidrug resistance-related proteins such as MRP1, P-gp or elevated glutathione (GSH) level. Efflux of drugs by functional P-gp, MRP1 and elevated GSH level can confer resistance to apoptosis induced by a range of different stimuli. Therefore, it is necessary to develop new cell death inducers with relatively lower toxicity toward non-malignant cells that can overcome MDR by induction of apoptotic or non-apoptotic cell death pathways. Herein we report the synthesis and spectroscopic characterization of a GSH depleting, redox active Schiff's base, viz., potassium-N-(2-hydroxy-3-methoxy-benzaldehyde)-alaninate (PHMBA). Cytotoxic potential of PHMBA has been studied in doxorubicin-resistant and -sensitive T lymphoblastic leukemia cells and Ehrlich ascites carcinoma (EAC) cells. PHMBA kills both the cell types irrespective of their drug-resistance phenotype following apoptotic/necrotic pathways. Moreover, PHMBA-induced cell death is associated with oxidative stress mediated mitochondrial pathway as the H(2)O(2) inhibitor PEG-Catalase abrogated PHMBA-induced apoptosis/necrosis. PHMBA induces anti-tumor activity in both doxorubicin-sensitive and -resistant EAC-tumor-bearing Swiss albino mice. The non-toxicity of PHMBA was also confirmed through cytotoxicity studies on normal cell lines like PBMC, NIH3T3 and Chang Liver. To summarise, our data provide compelling rationale for future clinical use of this redox active Schiff's base in treatment of cancer patients irrespective of their drug-resistance status.
Asunto(s)
Apoptosis/efectos de los fármacos , Resistencia a Antineoplásicos , Mitocondrias/efectos de los fármacos , Neoplasias/patología , Especies Reactivas de Oxígeno/metabolismo , Bases de Schiff/farmacología , Animales , Calcio/metabolismo , Calpaína/metabolismo , Caspasa 3/metabolismo , Línea Celular Tumoral , Citometría de Flujo , Glutatión/metabolismo , Espectroscopía de Resonancia Magnética , Ratones , Mitocondrias/enzimología , Mitocondrias/metabolismo , Necrosis , Neoplasias/enzimología , Neoplasias/metabolismo , Espectrofotometría Ultravioleta , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
TAMs, present in the tumor microenvironment, play an immunosuppressive role, leading to tumor progression and metastasis. Recently, numerous attempts have been made to switch immunosuppressive TAMs into an immunostimulatory type. Previously, we showed that a copper chelate, viz., copper N-(2-hydroxy acetophenone) glycinate [CuNG], can reprogram TAMs toward the proimmunogenic type to mount an antitumor immune response, but the underlying molecular mechanisms of skewing TAMs toward the proimmunogenic type remain elusive. Herein, we tried to explore the signaling mechanisms responsible for the reprogramming of TAMs. We observed that CuNG-induced ROS generation triggers activation of two MAPKs, i.e., p38MAPK and ERK1/2, and also causes up-regulation of intracellular glutathione. Furthermore, activation of p38 MAPK up-regulated the initial IL-12 production and the activation of ERK1/2 in tandem with GSH, found responsible for IFN-γ production by TAMs. This IFN-γ, in turn, prolonged IL-12 production and down-regulated TGF-ß production and thus, plays the decisive role in CuNG-mediated reprogramming of regulatory cytokine production by TAMs. Our work highlights that ROS-mediated activation of MAPKs can convert suppressive macrophages toward the proimmunogenic type. Thus, the present study opens the possibility of targeting TAMs by the use of redox-active compounds for designing a novel, therapeutic strategy against cancer.
Asunto(s)
Glicina/análogos & derivados , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Macrófagos/inmunología , Quinasas de Proteína Quinasa Activadas por Mitógenos/inmunología , Neoplasias/inmunología , Compuestos Organometálicos/farmacología , Especies Reactivas de Oxígeno/inmunología , Microambiente Tumoral/inmunología , Animales , Línea Celular Tumoral , Quelantes/farmacología , Activación Enzimática/efectos de los fármacos , Activación Enzimática/inmunología , Femenino , Glicina/farmacología , Interferón gamma/genética , Interferón gamma/metabolismo , Interleucina-12/inmunología , Interleucina-12/metabolismo , Sistema de Señalización de MAP Quinasas/inmunología , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Neoplasias/metabolismo , Neoplasias/terapia , Especies Reactivas de Oxígeno/metabolismo , Factor de Crecimiento Transformador beta/inmunología , Factor de Crecimiento Transformador beta/metabolismo , Microambiente Tumoral/efectos de los fármacosRESUMEN
Multidrug resistance (MDR) mediated by the over expression of drug efflux protein P-glycoprotein (P-gp) is one of the major impediments to successful treatment of cancer. P-gp acts as an energy-dependent drug efflux pump and reduces the intracellular concentration of structurally unrelated drugs inside the cells. Therefore, there is an urgent need for development of new molecules that are less toxic to normal cell and preferentially effective against drug resistant malignant cells. In this preclinical study we report the apoptotic potential of copper N-(2-hydroxyacetophenone) glycinate (CuNG) on doxorubicin resistant T lymphoblastic leukaemia cells (CEM/ADR5000). To evaluate the cytotoxic effect of CuNG, we used different normal cell lines (NIH 3T3, Chang liver and human PBMC) and cancerous cell lines (CEM/ADR5000, parental sensitive CCRF-CEM, SiHa and 3LL) and conclude that CuNG preferentially kills cancerous cells, especially both leukemic cell types irrespective of their MDR status, while leaving normal cell totally unaffected. Moreover, CuNG involves reactive oxygen species (ROS) for induction of apoptosis in CEM/ADR5000 cells through the intrinsic apoptotic pathway. This is substantiated by our observation that antioxidant N-acetyle-cysteine (NAC) and PEG catalase could completely block ROS generation and, subsequently, abrogates CuNG induced apoptosis. On the other hand, uncomplexed ligand N-(2-hydroxyacetophenone) glycinate (NG) fails to generate a significant amount of ROS and concomitant induction of apoptosis in CEM/ADR5000 cells. Therefore, CuNG induces drug resistant leukemia cells to undergo apoptosis and proves to be a molecule having therapeutic potential to overcome MDR in cancer.
Asunto(s)
Apoptosis/efectos de los fármacos , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Glicina/análogos & derivados , Compuestos Organometálicos/farmacología , Animales , Antibióticos Antineoplásicos/farmacología , Western Blotting , Ciclo Celular/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cobre/química , Citocromos c/metabolismo , Relación Dosis-Respuesta a Droga , Doxorrubicina/farmacología , Citometría de Flujo , Glutatión/metabolismo , Glicina/farmacología , Humanos , Ratones , Células 3T3 NIH , Compuestos Organometálicos/química , Oxidación-Reducción/efectos de los fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Especies Reactivas de Oxígeno/metabolismo , Factores de TiempoRESUMEN
Multidrug resistance (MDR) mediated by the over expression of drug efflux protein P-glycoprotein (P-gp) is one of the major impediments to successful treatment of cancer. P-gp acts as an energy-dependent drug efflux pump and reduces the intracellular concentration of structurally unrelated drugs inside the cells. Therefore, there is an urgent need for development of new compound that are less toxic and effective against drug resistance in cancer. Preclinical studies have shown that quinoline derivatives possess anticancer activities. Here, we report the antitumor potential of quinoline derivative, 2-(2-Methyl-quinolin-4ylamino)-N-phenyl acetamide (S4). To evaluate the cytotoxic potential of S4, we used four different cell lines (Hela, HCT-116, CCRF-CEM, and CEM/ADR 5000) in vitro, and showed that S4 kills doxorubicin resistant T lymphoblastic leukemia cell, CEM/ADR 5000 in a concentration dependent manner while others remains unaffected. Moreover, S4 induces apoptosis in CEM/ADR 5000 cells through generation reactive oxygen species (ROS). This is substantiated by the fact that the antioxidant N-acetyle-cysteine (NAC) completely blocks ROS generation and, subsequently, abrogates S4 induced apoptosis. Furthermore, in vivo treatment with S4 significantly increases the life span of swiss albino mice bearing sensitive and doxorubicin resistant subline of Ehrlich ascites carcinoma. In addition, intraperitoneal application of S4 in mice does not show any systemic toxicity at concentrations that in preliminary trials in a mice Ehrlich ascites carcinoma model. Therefore, present report provides evidence that S4, a quinoline derivative, may be a promising new therapeutic agent against drug resistant cancers.
Asunto(s)
Acetanilidas/farmacología , Aminoquinolinas/farmacología , Antineoplásicos/farmacología , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Neoplasias/tratamiento farmacológico , Acetanilidas/administración & dosificación , Acetanilidas/efectos adversos , Acetanilidas/uso terapéutico , Aminoquinolinas/administración & dosificación , Aminoquinolinas/efectos adversos , Aminoquinolinas/uso terapéutico , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/efectos adversos , Antineoplásicos/uso terapéutico , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Carcinoma de Ehrlich/tratamiento farmacológico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Doxorrubicina/farmacología , Femenino , Humanos , Masculino , Ratones , Neoplasias/metabolismo , Estrés Oxidativo/efectos de los fármacos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Análisis de Supervivencia , Pruebas de Toxicidad CrónicaRESUMEN
Multiple drug resistance (MDR) remains a major clinical challenge for cancer treatment. P-glycoprotein is the major contributor and they exceed their role in the chemotherapy resistance of most of the malignancies. Attempts in several preclinical and clinical studies to reverse the MDR phenomenon by using MDR modulators have not yet generated promising results. In the present study, a co-ordination complex of zinc viz., Zn N-(2-hydroxyacetophenone)glycinate (ZnNG) has been synthesized, characterized and its antitumour activity was tested in vitro against drug sensitive and resistant human T-lymphoblastic leukemic cell lines (CCRF/CEM and CEM/ADR5000 respectively) and in vivo against Ehrlich ascites carcinoma (EAC) implanted in female Swiss albino mice. To evaluate the cytotoxic potential of ZnNG, we used sensitive CCRF/CEM and drug resistant CEM/ADR 5000 cell lines in vitro. Moreover, ZnNG also has the potential ability to reverse the multidrug resistance phenotype in drug resistant CEM/ADR 5000 cell line and induces apoptosis in combination with vinblastine. ZnNG remarkably increases the life span of Swiss albino mice bearing sensitive and doxorubicin resistant subline of EAC in presence and in absence of doxorubicin. In addition, intraperitoneal application of ZnNG in mice does not show any systemic toxicity in preliminary trials in normal mice. To conclude, a novel metal chelate of zinc viz., ZnNG, may be a promising therapeutic agent against sensitive as well as drug resistant cancers.