RESUMEN
Various preparations of follicle-stimulating hormone (FSH) are commercially available; however, they differ in glycoforms composition and purity owing to their respective sources. Additional chemical/physical changes can also be introduced during manufacturing and can impact their biological activity (biopotency), which is routinely assessed using an in vivo bioassay (Steelman-Pohley). This study aimed to determine whether an in vitro bioassay could assess biopotency by distinguishing between r-hFSH chemical/physical variants with similar ability to the in vivo bioassay. The specific activity (units of biological activity per mg of product) of variants of r-hFSH generated through enrichment (acidic/basic), stress (oxidative/acidic pH) and enzymatic treatment (desialylation and desialylation/degalactosylation) was compared using the in vivo and in vitro bioassays. The in vitro bioassay reliably detected potential chemical/physical modifications in r-hFSH variants that may impact biopotency. Overall, the methods demonstrated a comparable ability to detect changes in specific activities due to chemical/physical differences in r-hFSH variants. These data indicate that the in vitro bioassay is suitable to replace the in vivo bioassay.
Asunto(s)
Hormona Folículo Estimulante Humana , Hormona Folículo Estimulante , Bioensayo/métodos , Técnicas In VitroRESUMEN
Interferon beta (IFNß) is a well-known cytokine, belonging to the type I family, that exerts antiviral, immunomodulatory, and antiproliferative activity. It has been reported that the artificially deamidated form of recombinant IFNß-1a at Asn25 position shows an increased biological activity. As a deepening of the previous study, the molecular mechanism underlying this biological effect was investigated in this work by combining experimental and computational techniques. Specifically, the binding to IFNAR1 and IFNAR2 receptors and the canonical pathway of artificially deamidated IFNß-1a molecule were analyzed in comparison to the native form. As a result, a change in receptor affinity of deamidated IFNß-1a with respect to the native form was observed, and to better explore this molecular interaction, molecular dynamics simulations were carried out. Results confirmed, as previously hypothesized, that the N25D mutation can locally change the interaction network of the mutated residue but also that this effect can be propagated throughout the molecule. In fact, many residues not involved in the interaction with IFNAR1 in the native form participate to the recognition in the deamidated molecule, enhancing the binding to IFNAR1 receptor and consequently an increase of signaling cascade activation. In particular, a higher STAT1 phosphorylation and interferon-stimulated gene expression was observed under deamidated IFNß-1a cell treatment. In conclusion, this study increases the scientific knowledge of deamidated IFNß-1a, deciphering its molecular mechanism, and opens new perspectives to novel therapeutic strategies.
Asunto(s)
Antivirales , Interferón beta , Antivirales/metabolismo , Factores Inmunológicos , Interferón beta-1a , Interferón beta/metabolismo , Interferones , Transducción de SeñalRESUMEN
Cytokinins are adenine-based phytohormones that regulate key processes in plants, such as cell division and differentiation, root and shoot growth, apical dominance, branching, and seed germination. In preliminary studies, they have also shown protective activities against human neurodegenerative diseases. To extend knowledge of the protection (protective activity) they offer, we investigated activities of natural cytokinins against salsolinol (SAL)-induced toxicity (a Parkinson's disease model) and glutamate (Glu)-induced death of neuron-like dopaminergic SH-SY5Y cells. We found that kinetin-3-glucoside, cis-zeatin riboside, and N6-isopentenyladenosine were active in the SAL-induced PD model. In addition, trans-, cis-zeatin, and kinetin along with the iron chelator deferoxamine (DFO) and the necroptosis inhibitor necrostatin 1 (NEC-1) significantly reduced cell death rates in the Glu-induced model. Lactate dehydrogenase assays revealed that the cytokinins provided lower neuroprotective activity than DFO and NEC-1. Moreover, they reduced apoptotic caspase-3/7 activities less strongly than DFO. However, the cytokinins had very similar effects to DFO and NEC-1 on superoxide radical production. Overall, they showed protective activity in the SAL-induced model of parkinsonian neuronal cell death and Glu-induced model of oxidative damage mainly by reduction of oxidative stress.
Asunto(s)
Citocininas/uso terapéutico , Modelos Biológicos , Fármacos Neuroprotectores/uso terapéutico , Enfermedad de Parkinson/tratamiento farmacológico , Reguladores del Crecimiento de las Plantas/farmacología , Acetilcisteína/farmacología , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Muerte Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Citocininas/farmacología , Ácido Glutámico/toxicidad , Humanos , Isoquinolinas/farmacología , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Estrés Oxidativo/efectos de los fármacos , Oxígeno/metabolismo , Enfermedad de Parkinson/patología , Superóxidos/metabolismoRESUMEN
Novel triterpene derivatives were prepared and evaluated in salsolinol (SAL)- and glutamate (Glu)-induced models of neurodegeneration in neuron-like SH-SY5Y cells. Among the tested compounds, betulin triazole 4 bearing a tetraacetyl-ß-d-glucose substituent showed a highly potent neuroprotective effect. Further studies revealed that removal of tetraacetyl-ß-d-glucose part (free triazole derivative 10) resulted in strong neuroprotection in the SAL model at 1 µM, but this derivative suffered from cytotoxicity at higher concentrations. Both compounds modulated oxidative stress and caspase-3,7 activity, but 10 showed a superior effect comparable to the Ac-DEVD-CHO inhibitor. Interestingly, while both 4 and 10 outperformed the positive controls in blocking mitochondrial permeability transition pore opening, only 4 demonstrated potent restoration of the mitochondrial membrane potential (MMP) in the model. Derivatives 4 and 10 also showed neuroprotection in the Glu model, with 10 exhibiting the strongest oxidative stress reducing effect among the tested compounds, while the neuroprotective activity of 4 was probably due recovery of the MMP.
Asunto(s)
Ácido Glutámico/metabolismo , Isoquinolinas/antagonistas & inhibidores , Modelos Biológicos , Fármacos Neuroprotectores/farmacología , Triazoles/farmacología , Triterpenos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Isoquinolinas/farmacología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Estructura Molecular , Fármacos Neuroprotectores/síntesis química , Fármacos Neuroprotectores/química , Estrés Oxidativo/efectos de los fármacos , Relación Estructura-Actividad , Triazoles/química , Triterpenos/síntesis química , Triterpenos/químicaRESUMEN
BACKGROUND/AIMS: Pigment epithelium-derived factor (PEDF) is a potent endogenous inhibitor of angiogenesis, and a promising anticancer agent. We have previously shown that PEDF can be phosphorylated, and that distinct phosphorylations differentially regulate its physiological functions. We also demonstrated that triple phosphomimetic mutant (EEE-PEDF), has significantly increased antiangiogenic activity, and is much more efficient than WT-PEDF in inhibiting neovascularization and tumor growth. The enhanced antiangiogenic effect was associated with a direct ability to facilitate apoptosis of tumor-residing endothelial cells (EC), and subsequently, disruption of intratumoral vascularization. In the present report, we elucidated the molecular mechanism by which EEE-PEDF exerts more profound effects at the cellular level. METHODS: Here we used Western blotting, as well as in vitro binding, proliferation, apoptosis and migration assays to follow the signaling components responsible for the PEDF and EEE-PEDF effects. RESULTS: We found that EEE-PEDF suppresses EC proliferation due to caspase-3-dependent apoptosis, and also inhibits migration of the EC much better than WT-PEDF. Although WT-PEDF and EEE-PEDF did not affect proliferation and did not induce apoptosis of cancer cells, these agents efficiently inhibited cancer cell motility, with EEE-PEDF showing stronger effect. The stronger activity of EEE-PEDF was correlated to a better binding to laminin receptors. Furthermore, the proapoptotic and antimigratory activities of WT-PEDF and EEE-PEDF were found respectively regulated by differential activation of two distinct MAPK pathways, namely JNK and p38. We show that JNK and p38 phosphorylation is much higher in cells treated with EEE-PEDF. JNK leads to apoptosis of ECs, while p38 leads to antimigratory effect in both EC and cancer cells. CONCLUSION: These results reveal the molecular signaling mechanism by which the phosphorylated PEDF exerts its stronger antiangiogenic, antitumor activities.
Asunto(s)
Proteínas del Ojo/farmacología , Factores de Crecimiento Nervioso/farmacología , Serpinas/farmacología , Animales , Apoptosis/efectos de los fármacos , Bovinos , Línea Celular , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Lipasa/química , Lipasa/genética , Lipasa/metabolismo , Mutagénesis , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/metabolismo , Fosforilación/efectos de los fármacos , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , Receptores de Laminina/química , Receptores de Laminina/genética , Receptores de Laminina/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Serpinas/genética , Serpinas/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Alzheimer's disease (AD) is a progressive neurodegenerative disorder affecting tens of million people. Currently marketed drugs have limited therapeutic efficacy and only slowing down the neurodegenerative process. Interestingly, it has been suggested that biometal cations in the amyloid beta (Aß) aggregate deposits contribute to neurotoxicity and degenerative changes in AD. Thus, chelation therapy could represent novel mode of therapeutic intervention. Here we describe the features of chelators with therapeutically relevant mechanism of action. We have found that the tested compounds effectively reduce the toxicity of exogenous Aß and suppress its endogenous production as well as decrease oxidative stress. Cholyl hydrazones were found to be the most active compounds. In summary, our data show that cation complexation, together with improving transport efficacy may represent basis for eventual treatment strategy in AD.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Quelantes/farmacología , Terapia por Quelación , Inhibidores de la Colinesterasa/farmacología , Metales/farmacología , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/antagonistas & inhibidores , Péptidos beta-Amiloides/metabolismo , Cationes/química , Cationes/farmacología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Quelantes/síntesis química , Quelantes/química , Inhibidores de la Colinesterasa/síntesis química , Inhibidores de la Colinesterasa/química , Relación Dosis-Respuesta a Droga , Humanos , Metales/química , Estructura Molecular , Agregado de Proteínas/efectos de los fármacos , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Human type I interferons are a family of pleiotropic cytokines with antiviral, anti-proliferative and immunomodulatory activities. They signal through the same cell surface receptors IFNAR1 and IFNAR2 yet evoking markedly different physiological effects. One differentiating factor of interferon-beta (IFN-ß) from other type I interferons is the presence of theAsn-Gly-Arg (NGR) sequence motif, which, upon deamidation, converts to Asp-Gly-Arg (DGR) and iso-Asp-Gly-Arg (iso-DGR) motifs. In other proteins, the NGR and iso-DGR motifs are reported as CD13- and αvß3, αvß5, αvß6, αvß8 and α5ß1 integrin-binding motifs, respectively. The scope of this study was to perform exploratory surface plasmon resonance (SPR) experiments to assess the binding properties of a deamidated IFN-ß variant to integrins. For this purpose, integrin αvß3 was selected as a reference model within the iso-DGR- integrin binding members. The obtained results show that deamidated IFN-ß binds integrin αvß3 with nanomolar affinity and that the response was dependent on the deamidation extent. Based on these results, it can be expected that deamidated IFN-ß also binds to other integrin family members that are able to bind to the iso-DGR binding motif. The novel binding properties could help elucidate specific IFN-ß attributes that under physiological conditions may be modulated by the deamidation.
Asunto(s)
Amidas/metabolismo , Integrina alfaVbeta3/metabolismo , Interferón beta/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Unión Proteica , Resonancia por Plasmón de SuperficieRESUMEN
The present study aims to focus on the bioprospecting of marine macroalgae of Turbinaria species, plenteous biomass of the world ocean. Three types of solvents, i.e., H2 O, MeOH/H2 O (80:20, v/v) and hexane/i-PrOH (50:50, v/v), were used for extraction. Both the biological activity and the pattern of present chemicals were characterized. For the cell proliferation assay, the human embryonic kidney 293 cells, cervix/breast/pancreatic adenocarcinoma, and osteosarcoma cells were used. For the antioxidant activity determination, both intracellular assay with human embryonic kidney and cervix adenocarcinoma cells, as well as the biochemical DPPH test, were employed. To complete the information about macroalgae composition, organic compounds were characterized by the liquid chromatography coupled with high resolution tandem mass spectrometry. Attention was concentrated mainly on the lipidomic profile characterization. In spite the fact that any significant antiproliferative effect was not observed for cancer cells, both the Turbinaria species were shown to be good protectors against the oxidative stress of the non-cancer cells. Most of the antioxidants were determined in the hexane/i-PrOH extract. As regards the lipids identified, most of them belonged to the triacylglycerols followed by sphingomyelins, diacylglycerols, and polar (lyso)phospholipids. Additionally to fatty acids with 14, 16 and 18 carbons, also those with odd carbon numbers were frequently present.
Asunto(s)
Antioxidantes/farmacología , Productos Biológicos/química , Bioprospección/métodos , Proliferación Celular/efectos de los fármacos , Microalgas/química , Sustancias Protectoras/farmacología , Células Cultivadas , Cromatografía Líquida de Alta Presión , Humanos , Lípidos/análisis , Lípidos/química , Metaboloma , Metabolómica , Microalgas/metabolismo , Espectrometría de Masas en TándemRESUMEN
The MYC family of transcription factors consists of three well characterized members, c-MYC, L-MYC, and MYCN, deregulated in the majority of human cancers. In neuronal tumors such as neuroblastoma, MYCN is frequently activated by gene amplification, and reducing its expression by RNA interference has been shown to promote growth arrest and apoptosis of tumor cells. From a clinical perspective, RNA interference is not yet a viable option, and small molecule inhibitors of transcription factors are difficult to develop. We therefore planned to identify, at the global level, the genes interacting functionally with MYCN required to promote fitness of tumor cells facing oncogenic stress. To find genes whose inactivation is synthetically lethal to MYCN, we implemented a genome-wide approach in which we carried out a drop-out shRNA screen using a whole genome library that was delivered into isogenic neuroblastoma cell lines expressing or not expressing MYCN. After the screen, we selected for in-depth analysis four shRNAs targeting AHCY, BLM, PKMYT1, and CKS1B. These genes were chosen because they are directly regulated by MYC proteins, associated with poor prognosis of neuroblastoma patients, and inhibited by small molecule compounds. Mechanistically, we found that BLM and PKMYT1 are required to limit oncogenic stress and promote stabilization of the MYCN protein. Cocktails of small molecule inhibitors of CKS1B, AHCY, BLM, and PKMYT1 profoundly affected the growth of all neuroblastoma cell lines but selectively caused death of MYCN-amplified cells. Our findings suggest that drugging the MYCN network is a promising avenue for the treatment of high risk, neuroblastic cancers.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Proteínas Nucleares/metabolismo , Proteínas Oncogénicas/metabolismo , Apoptosis , Diferenciación Celular , Línea Celular Tumoral , Separación Celular , Citometría de Flujo , Genes myc , Estudio de Asociación del Genoma Completo , Células HEK293 , Humanos , Lentivirus/genética , Proteínas de la Membrana/metabolismo , Proteína Proto-Oncogénica N-Myc , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/metabolismo , Pronóstico , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , ARN Interferente Pequeño/metabolismo , RecQ Helicasas/metabolismoRESUMEN
Annexin A1 (ANXA1) is the first characterized member of the annexins superfamily. It binds the cellular membrane phospholipids in Ca(2+) regulated manner. Annexin A1 has been found in several tissues and many physiological roles as hormones secretion, vesiculation, inflammatory response, apoptosis and differentiation have been shown. Its subcellular localization and binding with many partner proteins are altered accordingly with its physiological role. The Annexin A1 membrane localization is crucial for binding to receptors, suggesting a paracrine and juxtacrine extracellular action. Annexin A1 is subjected to several post-translational modifications. In particular the protein is phosphorylated on several residues both on the N-terminal functional domain and on the C-terminus core. Different kinases have been identified as responsible for the phosphorylation status of selective residues. The specific change in the phosphorylation status on the different sites alters ANXA1 localization, binding properties and functions. This review shows the physiological relevance of the ANXA1 phosphorylation leading to the conclusion that numerous and different roles of Annexin A1 could be associated with different phosphorylations to alter not only intracellular localization and bindings to its partners but also the extracellular receptor interactions.
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Anexina A1/metabolismo , Aminoácidos/metabolismo , Animales , Membrana Celular/metabolismo , Humanos , Modelos Biológicos , Fosforilación , Transporte de ProteínasRESUMEN
This review will address the current understanding of the relationship between hyperprolactinemia and antipsychotic drugs. Hyperprolactinemia is a frequent but often neglected side effect of typical, but also of many atypical antipsychotics. Release of PRL from lactotrope cells is influenced by several factors, such as stress, physical and sexual activity and food assumption. PRL secretion is regulated by hypothalamic-pituitary portal system and its homeostasis is the result of a complex balance between stimulating and inhibitory factors, both endogeneous and esogeneous. The main physiological control mechanism of secretion is played by the inhibitory action of dopamine. Conversely, among stimulation factors, serotonin is probably the main modulator of PRL release. An high number of drugs may cause PRL increase too, such as drugs that reduce dopaminergic functions at SNC level, or drugs with an antagonistic action towards dopaminergic receptors and those increasing serotonergic neurotransmission. Hyperprolactinemia is one of the most frequent endocrine pathologies of the hypothalamic-pituitary axis. Antipsychotics (AP) are the most common cause of druginduced hyperprolactinemia. Not all AP have the same impact on inducing hyperprolactinemia. In this review we will focus on the subdivision of AP in 'PRL-raising' (stimulators) and 'PRL-sparing' (sparers) and on their differences in inducing hyperprolactinemia. Finally we evaluated different complications in patients with antipsychotics induced hyperprolactinemia that may cause not only short-term side effects but also important systemic long-term effects. At the end of the review we finally report the possible options of treatment considering however that at present there are no ideal therapies or evaluations, and decisions have to be made on a case by case basis.
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Antipsicóticos/efectos adversos , Hiperprolactinemia/inducido químicamente , Prolactina/sangre , Humanos , Hiperprolactinemia/sangre , Prolactina/efectos de los fármacos , Trastornos Psicóticos/tratamiento farmacológico , Factores de RiesgoRESUMEN
Pigment epithelium-derived factor (PEDF) is a potent endogenous inhibitor of angiogenesis and a promising anticancer agent. We have previously shown that PEDF can be phosphorylated and that distinct phosphorylations differentially regulate its physiological functions. We also demonstrated that triple phosphomimetic mutant (EEE-PEDF), has significantly increased antiangiogenic activity and is much more efficient than WT-PEDF in inhibiting neovascularization and tumor growth. The enhanced antiangiogenic effect was associated with a direct ability to facilitate apoptosis of tumor-residing endothelial cells (ECs), and subsequently, disruption of intratumoral vascularization. In the present report, we elucidated the molecular mechanism by which EEE-PEDF exerts more profound effects at the cellular level. We found that EEE-PEDF suppresses EC proliferation due to caspase-3-dependent apoptosis and also inhibits migration of the EC much better than WT-PEDF. Although WT-PEDF and EEE-PEDF did not affect proliferation and did not induce apoptosis of cancer cells, these agents efficiently inhibited cancer cell motility, with EEE-PEDF showing a stronger effect. The stronger activity of EEE-PEDF was correlated with a better binding to laminin receptors. Furthermore, the proapoptotic and antimigratory activities of WT-PEDF and EEE-PEDF were found regulated by differential activation of two distinct MAPK pathways, namely JNK and p38, respectively. We show that JNK and p38 phosphorylation is much higher in cells treated with EEE-PEDF. JNK leads to apoptosis of ECs, whereas p38 leads to anti-migratory effect in both EC and cancer cells. These results reveal the molecular signaling mechanism by which the phosphorylated PEDF exerts its stronger antiangiogenic, antitumor activities.
Asunto(s)
Apoptosis , Movimiento Celular , Proteínas del Ojo/fisiología , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Proteínas Mutantes/fisiología , Factores de Crecimiento Nervioso/fisiología , Serpinas/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Inhibidores de la Angiogénesis , Animales , Bovinos , Línea Celular Tumoral , Proliferación Celular , Células Cultivadas , Células Endoteliales/citología , Endotelio Vascular/citología , Glioblastoma/patología , Humanos , Imitación Molecular , FosforilaciónRESUMEN
BACKGROUND: It has been shown that chemo-therapeutic agents, such as histone deacetylase inhibitors (HDACi), are able to increase TRAIL-induced apoptosis in many types of cancer. In the present study, we investigated the effects of the novel HDACi LGP1, a new simplified analogue of FR235222, in human leukaemia U937 cells resistant to TRAIL-induced apoptosis. MATERIALS AND METHODS: U937 cells were incubated with TRAIL/LGP1 for 24 h and apoptosis was evaluated using flow cytometric assay and cleavage of caspase-3 and (PARP) by Western blot. Western blot analysis was also used to detect the expression of p21, p27, (NF-kappaB), Bcl-2 and the levels of H4 histone acetylation. Finally, flow cytometry was used to monitor the enhancement of TRAIL-receptor levels. RESULTS: Treatment with LGP1 caused accumulation of acetylated histone H4 and G(1) cycle arrest accompanied by increase of p21. The compound was also able to sensitize U937 cells to TRAIL-induced apoptosis through multiple mechanisms: (i) activation of caspase-3 and cleavage of PARP; (ii) induction of p21 and p27; (iii) cleavage of NF-kappaB and down-regulation of Bcl-2. Finally, LGP1 induced up-regulation of TRAIL-R1 receptor expression. CONCLUSION: These results demonstrate that U937 cells can be effectively killed by a combination treatment of subtoxic doses of LGP1 and TRAIL.
Asunto(s)
Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Inhibidores de Histona Desacetilasas/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Acetilación/efectos de los fármacos , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Ciclo Celular/efectos de los fármacos , Sinergismo Farmacológico , Activación Enzimática , Fase G1/efectos de los fármacos , Histonas/metabolismo , Humanos , Leucemia Promielocítica Aguda/tratamiento farmacológico , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patología , Péptidos Cíclicos , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Células U937RESUMEN
The reduction of Annexin A1 (ANXA1) expression, commonly associated with prostate cancer, could be due to elevated activity of histone deacetylases. We have investigated the mechanisms of apoptotic effects of FR235222 in LNCaP cell line and the role of ANXA1. We showed that treatment with FR235222 induced apoptosis through a caspase-dependent mechanism. FR235222 was able to increase the protein levels of ANXA1 at a transcriptional level. Finally, the inhibition of ANXA1 expression by siRNA leads to a partial reduction of FR235222-induced apoptosis. The results suggest that elevated activity of HDACs is responsible for the reduction of ANXA1, indicating that ANXA1 expression is a contributing factor to the proapoptotic effects in prostate cancer.
Asunto(s)
Adenocarcinoma/patología , Anexina A1/metabolismo , Apoptosis/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Péptidos Cíclicos/farmacología , Neoplasias de la Próstata/patología , Acetilación , Adenocarcinoma/metabolismo , Anexina A1/genética , Western Blotting , Caspasa 3/metabolismo , Línea Celular Tumoral , Histonas/metabolismo , Humanos , Masculino , Neoplasias de la Próstata/metabolismo , ARN Interferente Pequeño/genética , Regulación hacia ArribaRESUMEN
FR235222, a novel histone deacetylase inhibitor (HDACi), at 50nM caused accumulation of acetylated histone H4, inhibition of cell proliferation and G1 cycle arrest accompanied by increase of p21 and down-regulation of cyclin E in human promyelocytic leukaemia U937 cells. The compound was also able to increase the protein and mRNA levels of annexin A1 (ANXA1) without effects on apoptosis. Similar effects were observed in human chronic myelogenous leukaemia K562 cells and human T cell leukaemia Jurkat cells. Cycle arrest and ANXA1 expression, without significant effects on apoptosis, were also induced by different HDACi like suberoylanilide hydroxamic acid (SAHA) and trichostatin-A (TSA). FR235222 at 0.5 microM stimulated apoptosis of all leukaemia cell lines associated to an increased expression of the full-length (37kDa) protein and the appearance of a 33kDa N-terminal cleavage product in both cytosol and membrane. These results suggest that ANXA1 expression may mediate cycle arrest induced by low doses FR235222, whereas apoptosis induced by high doses FR235222 is associated to ANXA1 processing.