RESUMEN
Many factors regulate cancer cell apoptosis, among which Survivin has a strong anti-apoptotic effect and PHLPP is a tumor suppressor gene that can induce significant apoptosis. However, the relationship between PHLPP and Survivin in gallbladder carcinoma (GBC) has not been reported. This study found that PHLPP expression is decreased and Survivin expression is increased in GBC tissues and cell lines. Their expression levels showed an inverse relationship and were associated with poor prognosis of GBC patients. Loss of PHLPP can increase the level of phosphorylated Survivin and induce the nuclear export of Survivin, which thus inhibit cell apoptosis and promote cell proliferation in GBC cells. The process that PHLPP regulates Survivin phosphorylation and intracellular localization is involved in AKT activity. Re-overexpression of PHLPP in GBC cells can decrease AKT phosphorylation level. Reduced expression of PHLPP in GBC is associated with high expression of miR-495. Increasing PHLPP expression or inhibiting miR-495 expression can induce apoptosis and suppress tumor growth in GBC xenograft model in nude mice. The results revealed the role and mechanism of PHLPP and Survivin in GBC cells and proposed strategies for gene therapies targeting the miR-495 / PHLPP / AKT / Survivin regulatory pathway.
Asunto(s)
Neoplasias de la Vesícula Biliar/terapia , Terapia Genética/métodos , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Proteínas Nucleares/administración & dosificación , Proteínas Nucleares/genética , Fosfoproteínas Fosfatasas/administración & dosificación , Fosfoproteínas Fosfatasas/genética , Transporte Activo de Núcleo Celular , Adulto , Anciano , Animales , Apoptosis/genética , Proliferación Celular/genética , Femenino , Neoplasias de la Vesícula Biliar/enzimología , Neoplasias de la Vesícula Biliar/genética , Neoplasias de la Vesícula Biliar/patología , Humanos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Fosforilación , Pronóstico , Distribución Aleatoria , Survivin , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Recently, we found that sMEK1 effectively regulates pro-apoptotic activity when combined with a traditional chemotherapeutic drug. Therefore, combinational therapeutic strategies targeting critical molecular and cellular mechanisms are urgently required. In this present work, we evaluated whether sMEK1 enhanced the pro-apoptotic activity of chemotherapeutic drugs in ovarian carcinoma cells. Combined with a chemotherapeutic drug, sMEK1 showed an additive effect on the suppression of ovarian cancer cell growth by inducing cell cycle arrest and apoptosis and regulating related gene expression levels or protein activities. In addition, the phosphoinositide-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway was strongly inhibited by the combined treatment, showing de-repression of the tuberous sclerosis complex (TSC) and suppression of ras homolog enriched in the brain (Rheb) and mTOR and raptor in aggressive ovarian carcinoma cells and mouse xenograft models. Treatment with sMEK1 and paclitaxel reduced phosphorylation of ribosomal S6 kinase (S6K) and 4E-binding protein (4E-BP), two critical downstream targets of the mTOR-signaling pathway. Furthermore, both sMEK1 and paclitaxel significantly inhibited the expression of signaling components downstream of S6K/4E-BP, such as hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF), both in vitro and in vivo. Therefore, our data suggest that the combination of sMEK1 and paclitaxel is a promising and effective targeted therapy for chemotherapy-resistant or recurrent ovarian cancers.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Subunidad alfa del Factor 1 Inducible por Hipoxia/biosíntesis , Neoplasias Glandulares y Epiteliales/tratamiento farmacológico , Neoplasias Glandulares y Epiteliales/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Apoptosis/efectos de los fármacos , Carcinoma Epitelial de Ovario , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular , Movimiento Celular/efectos de los fármacos , Femenino , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Complejos Multiproteicos/metabolismo , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Glandulares y Epiteliales/patología , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Paclitaxel/administración & dosificación , Fosfoproteínas Fosfatasas/administración & dosificación , Fosfoproteínas/metabolismo , Fosforilación , Proteínas Quinasas S6 Ribosómicas/metabolismo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
The deregulated kinase activity of p210-BCR/ABL oncoproteins, hallmark of chronic myelogenous leukaemia (CML), induces and sustains the leukaemic phenotype, and contributes to disease progression. Imatinib mesylate, a BCR/ABL kinase inhibitor, is effective in most of chronic phase CML patients. However, a significant percentage of CML patients develop resistance to imatinib and/or still progresses to blast crisis, a disease stage that is often refractory to imatinib therapy. Furthermore, there is compelling evidence indicating that the CML leukaemia stem cell is also resistant to imatinib. Thus, there is still a need for new drugs that, if combined with imatinib, will decrease the rate of relapse, fully overcome imatinib resistance and prevent blastic transformation of CML. We recently reported that the activity of the tumour suppressor protein phosphatase 2A (PP2A) is markedly inhibited in blast crisis CML patient cells and that molecular or pharmacologic re-activation of PP2A phosphatase led to growth suppression, enhanced apoptosis, impaired clonogenic potential and decreased in vivo leukaemogenesis of imatinib-sensitive and -resistant (T315I included) CML-BC patient cells and/or BCR/ABL+ myeloid progenitor cell lines. Thus, the combination of PP2A phosphatase-activating and BCR/ABL kinase-inhibiting drugs may represent a powerful therapeutic strategy for blast crisis CML patients.
Asunto(s)
Antineoplásicos/administración & dosificación , Crisis Blástica/tratamiento farmacológico , Fosfoproteínas Fosfatasas/administración & dosificación , Piperazinas/administración & dosificación , Pirimidinas/administración & dosificación , Transducción de Señal/efectos de los fármacos , Animales , Protocolos de Quimioterapia Combinada Antineoplásica , Benzamidas , Resistencia a Antineoplásicos/fisiología , Genes abl/efectos de los fármacos , Humanos , Mesilato de Imatinib , Fosfoproteínas Fosfatasas/metabolismo , Proteína Fosfatasa 2 , Proteínas Proto-Oncogénicas c-bcr/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Glycogen-targeting subunits of protein phosphatase-1 (PP-1) are scaffolding proteins that facilitate the regulation of key enzymes of glycogen metabolism by PP-1. In the current study, we have tested the effects of hepatic expression of GMDeltaC, a truncated version of the muscle-targeting subunit isoform, in rats rendered insulin-deficient via injection of a single moderate dose of streptozotocin (STZ). Three key findings emerged. First, GMDeltaC expression in liver was sufficient to fully normalize blood glucose levels (from 335 +/- 31 mg/dl prior to viral injection to 109 +/- 28 mg/dl 6 days after injection) and liver glycogen content in STZ-injected rats. Second, this normalization occurred despite very low levels of liver glucokinase expression in the insulin-deficient STZ-injected rats. Finally, the hyperphagia induced by STZ injection was completely reversed by GMDeltaC expression in liver. In contrast to these findings with GMDeltaC, overexpression of another targeting subunit, GL, in STZ-injected rats caused a large increase in liver glycogen stores but only a transient decrease in food intake and blood glucose levels. The surprising demonstration of a glucose-lowering effect of GMDeltaC in the background of depressed hepatic glucokinase expression suggests that controlled stimulation of liver glycogen storage may be an effective mechanism for improving glucose homeostasis, even when normal pathways of glucose disposal are impaired.
Asunto(s)
Diabetes Mellitus Experimental/terapia , Hiperglucemia/terapia , Hiperfagia/terapia , Hígado/metabolismo , Fosfoproteínas Fosfatasas/farmacología , Adenoviridae/genética , Animales , Glucemia/análisis , Glucemia/efectos de los fármacos , Terapia Genética/métodos , Glucoquinasa/sangre , Glucógeno/metabolismo , Hiperglucemia/inducido químicamente , Hiperfagia/inducido químicamente , Hígado/efectos de los fármacos , Masculino , Fosfoproteínas Fosfatasas/administración & dosificación , Fosfoproteínas Fosfatasas/genética , Proteína Fosfatasa 1 , Subunidades de Proteína/administración & dosificación , Subunidades de Proteína/genética , Subunidades de Proteína/farmacología , Ratas , Ratas Wistar , EstreptozocinaRESUMEN
Microinjection of bacterially expressed human cdc25A protein into Xenopus prophase oocytes provokes the activation of p34cdc2 kinase and the tyrosine dephosphorylation of p34cdc2 in the presence or absence of protein synthesis. The level of p34cdc2 kinase activity then drops in parallel with the degradation of cyclin B2 and finally increases again to stabilize at a high level. Cdc25 microinjection induces the assembly of a metaphase I spindle which is abnormally located in the deep cytoplasm. Moreover, oocytes arrest at the metaphase I stage and do not reach metaphase II even 10 h after cdc25 microinjection. The extended metaphase I period observed in cdc25-injected oocytes results from an equilibrium between degradation of cyclins and synthesis of new cyclins. This is in contrast with progesterone-stimulated oocytes where cyclin degradation is turned off when oocytes enter metaphase II. During metaphase I, the reactivation of MPF activity can be disrupted in two different ways: 1) cycloheximide, an inhibitor of protein synthesis, by preventing the synthesis of new cyclins, provokes the disappearance of MPF kinase activity and the reformation of a nucleus; 2) when the cAMP level is increased during the metaphase I period in cdc25-injected oocytes, MPF kinase activity drops following a rephosphorylation of tyrosine 15 of p34cdc2, while the cyclin turn-over remains unaffected. Moreover, increasing the cAMP level in prophase oocytes totally prevents the action of cdc25. Our results indicate that in Xenopus oocytes, the PKA pathway negatively regulates the activation of MPF and the activity of p34cdc2/cyclin B complex through tyrosine phosphorylation of p34cdc2 during metaphase I.
Asunto(s)
Proteínas de Ciclo Celular/administración & dosificación , Proteínas de Ciclo Celular/farmacología , Factor Promotor de Maduración/metabolismo , Meiosis/fisiología , Metafase/fisiología , Oocitos/citología , Fosfoproteínas Fosfatasas/administración & dosificación , Fosfoproteínas Fosfatasas/farmacología , Fosfoproteínas Fosfatasas/fisiología , Xenopus laevis/genética , Animales , Proteína Quinasa CDC2/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Ciclinas/metabolismo , Cicloheximida/farmacología , Femenino , Microinyecciones , Fosforilación , Profase/efectos de los fármacos , Precursores de Proteínas/metabolismo , Inhibidores de la Síntesis de la Proteína/metabolismo , Huso Acromático/genética , Factores de Tiempo , Fosfatasas cdc25RESUMEN
Enhancement of cardiac L-type Ca2+ channel activity by norepinephrine via phosphorylation by protein kinase A (PKA) underlines the positive inotropic effect of this transmitter and is a classical example of an ion channel modulation. However, it is not clear whether the channel protein itself (and which subunit) is a substrate for PKA. We have expressed various combinations of the cardiac Ca2+ channel subunits in Xenopus oocytes by injecting subunit mR-NAs. Expression of beta or alpha 2/delta + beta subunits potentiated the native (endogenous) Ca2+ channel currents in the oocyte (similar to T or N but not L-type). This potentiated endogenous current was enhanced by intracellular injection of cAMP or of the catalytic subunit of PKA, and this effect was reversed by the injection of a PKA inhibitor suggesting the presence of basal phosphatase activity. When a cardiac channel of alpha 1 + beta, alpha 1 + alpha 2/delta or alpha 1 + alpha 2/delta + beta composition was expressed at levels high enough that the contribution of the endogenous current became negligible, cAMP and PKA failed to increase the Ca2+ channel current, whereas PKA inhibitors and the catalytic subunit of protein phosphatase 1 reduced the amplitude of the current. Reduction of the current by PKA inhibitors was observed regardless of the presence of the beta subunit, suggesting a major role for the alpha 1 subunit in this process. These results suggest that, like in the heart, when expressed in Xenopus oocytes, the cardiac L-type Ca2+ channels are phosphorylated in basal state and dephosphorylation reduces their activity. However, unlike the situation in the heart, the activity of the channel cannot be enhanced by PKA-catalyzed phosphorylation, suggesting that the channel is already fully phosphorylated in its basal state.
Asunto(s)
Canales de Calcio/metabolismo , AMP Cíclico/fisiología , Proteínas Musculares/metabolismo , Miocardio/química , Procesamiento Proteico-Postraduccional , Animales , Artefactos , Bario/metabolismo , Calcio/metabolismo , Canales de Calcio/genética , Proteínas Quinasas Dependientes de AMP Cíclico/administración & dosificación , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , ADN Complementario/administración & dosificación , ADN Complementario/genética , Femenino , Microinyecciones , Modelos Biológicos , Proteínas Musculares/genética , Norepinefrina/farmacología , Oocitos , Especificidad de Órganos , Fosfoproteínas Fosfatasas/administración & dosificación , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Proteína Fosfatasa 1 , ARN Mensajero/administración & dosificación , ARN Mensajero/genética , Conejos , Regulación hacia Arriba , Xenopus laevisRESUMEN
Calcineurin, an intracellular protein phosphatase (type 2B), is reported to inhibit L-type (slow) calcium channels and thereby play a key role in channel inactivation. The present study was undertaken to examine effects of calcineurin on slow channel dependent action potentials of 3-days-old embryonic chick ventricle and to assess the role of this enzyme in regulation of developing slow channels. Calcineurin trapped in phosphatidylcholine-liposomes to facilitate its intracellular uptake was found to inhibit maximal upstroke velocity (+Vmax), overshoot and duration of action potentials. At higher doses of calcineurin containing liposomes the preparations ceased to exhibit spontaneous activity but elicited electrically driven action potentials with lower +Vmax and overshoot. These observations show that calcineurin down-modulates the embryonic cardiac slow channels under basal conditions.
Asunto(s)
Proteínas de Unión a Calmodulina/administración & dosificación , Corazón Fetal/efectos de los fármacos , Fosfoproteínas Fosfatasas/administración & dosificación , Potenciales de Acción/efectos de los fármacos , Animales , Calcineurina , Calmodulina/administración & dosificación , Calmodulina/farmacología , Proteínas de Unión a Calmodulina/farmacología , Embrión de Pollo , Relación Dosis-Respuesta a Droga , Combinación de Medicamentos , Ventrículos Cardíacos , Técnicas In Vitro , Liposomas , Fosfatidilcolinas , Fosfoproteínas Fosfatasas/farmacologíaRESUMEN
A protein-tyrosine-phosphatase (PTPase 1B; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48), specific for phosphotyrosyl residues, was microinjected into Xenopus oocytes. This resulted in a 3- to 5-fold increase in PTPase activity over endogenous levels. The PTPase blocked the insulin-stimulated phosphorylation of tyrosyl residues on endogenous proteins, including a protein having a molecular mass in the same range as the beta subunit of the insulin or insulin-like growth factor I receptor. PTPase 1B also blocked the activation of an S6 peptide kinase--i.e., an enzyme recognizing a peptide having the sequence RRLSSLRA found in a segment of ribosomal protein S6 and known to be activated early in response to insulin. On the other hand, the insulin stimulation of an S6 kinase, detected by using 40S ribosomes as substrate, was unaffected even though PTPase 1B partially prevented the phosphorylation of ribosomal protein S6 in vivo. Mono Q chromatography of insulin-treated oocyte extracts revealed two main peaks of S6 kinase activity. Fractions from the first peak displayed S6 peptide kinase activity that was essentially abolished in profiles from PTPase 1B-injected oocytes. Material from the second peak, which was best revealed by using 40S ribosomes as substrate and had comparatively little S6 peptide kinase activity, was minimally affected by PTPase 1B. These observations suggest that at least two distinct "S6 kinases" are involved in ribosomal protein S6 phosphorylation in vivo and that the activation pathways for these enzymes differ in their sensitivity to PTPase 1B.
Asunto(s)
Insulina/farmacología , Oocitos/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Animales , Femenino , Técnicas In Vitro , Cinética , Microinyecciones , Oocitos/efectos de los fármacos , Fosfoproteínas Fosfatasas/administración & dosificación , Fosforilación , Proteínas Quinasas/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Proteínas Tirosina Fosfatasas , Proteínas Quinasas S6 Ribosómicas , Tirosina , Xenopus laevisRESUMEN
Dynamic reorganization of the actin microfilament networks is dependent on the reversible phosphorylation of myosin light chain. To assess the potential role of protein phosphatases in this process in living nonmuscle cells, we have microinjected the purified type-1 and type-2A phosphatases into the cytoplasm of mammalian fibroblasts. Our studies reveal that elevating type-1 phosphatase levels led to the rapid (within 30 min) and fully reversible disassembly of the actin microfilament network as determined by immunofluorescence analysis. In contrast, microinjection of equivalent amounts of the purified type-2A phosphatase had no effect on actin microfilament organization. Metabolic labeling of cells after injection of purified phosphatases was used to analyze changes in protein phosphorylation. Concomitant with the disassembly of the actin microfilaments induced by type-1 phosphatase, there was an extensive dephosphorylation of myosin light chain. No such change was observed when cells were injected with type-2A phosphatase. In addition, after extraction of fibroblasts with Triton X-100, the type-1 phosphatase could be specifically localized by immunofluorescence to a fibrillar network of microfilaments. Furthermore, neutralizing type-1 phosphatase activity in vivo by microinjection of an affinity-purified antibody, prevented the reorganization of actin microfilaments that we had previously described following injection of cAMP-dependent protein kinase. These data support the notion that type 1 and type-2 phosphatases have distinct substrate specificity in living cells, and that type-1 phosphatase plays a predominant role in the dephosphorylation of myosin light chain and thus in the modulation of actin microfilament organization in vivo in intact nonmuscle cells.
Asunto(s)
Citoesqueleto de Actina/ultraestructura , Actinas/metabolismo , Citoesqueleto/ultraestructura , Músculos/enzimología , Subfragmentos de Miosina/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Animales , Línea Celular , Técnica del Anticuerpo Fluorescente , Microinyecciones , Subfragmentos de Miosina/aislamiento & purificación , Fosfoproteínas Fosfatasas/administración & dosificación , Fosfoproteínas Fosfatasas/aislamiento & purificación , Fosforilación , Proteínas Quinasas/aislamiento & purificación , Proteínas Quinasas/metabolismo , ConejosRESUMEN
Homogeneous preparations of a protein phosphatase that is specific for phosphotyrosyl residues (protein tyrosine phosphatase [PTPase] 1B) were isolated from human placenta and microinjected into Xenopus oocytes. This resulted in an increase in activity of up to 10-fold over control levels, as measured in homogenates with use of an artificial substrate (reduced carboxamidomethylated and maleylated lysozyme). Microinjected PTPase was stable for at least 18 h. It is distributed within the oocyte in a manner similar to the endogenous activity and is suggestive of an interaction with cellular structures or molecules located predominantly in the animal hemisphere. The phosphatase markedly retarded (by up to 5 h) maturation induced by insulin. This, in conjunction with the demonstration that PTPase 1B abolished insulin stimulation of an S6 peptide (RRLSSLRA) kinase concomitant with a decrease in the phosphorylation of tyrosyl residues in a protein with the same apparent Mr as the beta subunit of the insulin and insulinlike growth factor 1 receptors (M. F. Cicirelli, N. K. Tonks, C. D. Diltz, E. H. Fischer, and E. G. Krebs, submitted for publication), provides further support for an essential role of protein tyrosine phosphorylation in insulin action. Furthermore, maturation was significantly retarded even when the PTPase was injected 2 to 4 h after exposure of the cells to insulin. PTPase 1B also retarded maturation induced by progesterone and maturation-promoting factor, which presumably do not act through the insulin receptor. These data point to a second site of action of the PTPase in the pathway of meiotic cell division, downstream of the insulin receptor and following the appearance of active maturation-promoting factor.