RESUMEN
The probability of acute kidney injury (AKI) is higher in septic diabetic patients, which is associated with, among other factors, proximal tubular cell (PTC) injury induced by the hypoxic/hyperglycemic/inflammatory microenvironment that surrounds PTCs in these patients. Here, we exposed human PTCs (HK-2 cells) to 1% O2/25 mM glucose/inflammatory cytokines with the aim of studying the role of prostaglandin uptake transporter (PGT) and dipeptidyl peptidase-4 (DPP-4, a target of anti-hyperglycemic agents) as pharmacological targets to prevent AKI in septic diabetic patients. Our model reproduced two pathologically relevant mechanisms: (i) pro-inflammatory PTC activation, as demonstrated by the increased secretion of chemokines IL-8 and MCP-1 and the enhanced expression of DPP-4, intercellular leukocyte adhesion molecule-1 and cyclo-oxygenase-2 (COX-2), the latter resulting in a PGT-dependent increase in intracellular prostaglandin E2 (iPGE2); and (ii) epithelial monolayer injury and the consequent disturbance of paracellular permeability, which was related to cell detachment from collagen IV and the alteration of the cell cytoskeleton. Most of these changes were prevented by the antagonism of PGE2 receptors or the inhibition of COX-2, PGT or DPP-4, and further studies suggested that a COX-2/iPGE2/DPP-4 pathway mediates the pathogenic effects of the hypoxic/hyperglycemic/inflammatory conditions on PTCs. Therefore, inhibitors of PGT or DPP-4 ought to undergo testing as a novel therapeutic avenue to prevent proximal tubular damage in diabetic patients at risk of AKI.
Asunto(s)
Lesión Renal Aguda , Diabetes Mellitus , Inhibidores de la Dipeptidil-Peptidasa IV , Humanos , Ciclooxigenasa 2/metabolismo , Diabetes Mellitus/tratamiento farmacológico , Lesión Renal Aguda/tratamiento farmacológico , Lesión Renal Aguda/etiología , Lesión Renal Aguda/prevención & control , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas , Prostaglandinas , Inhibidores de la Dipeptidil-Peptidasa IV/farmacología , Inhibidores de la Dipeptidil-Peptidasa IV/uso terapéutico , Dipeptidil Peptidasa 4RESUMEN
Among the extracellular vesicles, apoptotic bodies (ABs) are only formed during the apoptosis and perform a relevant role in the pathogenesis of different diseases. Recently, it has been demonstrated that ABs from human renal proximal tubular HK-2 cells, either induced by cisplatin or by UV light, can lead to further apoptotic death in naïve HK-2 cells. Thus, the aim of this work was to carry out a non-targeted metabolomic approach to study if the apoptotic stimulus (cisplatin or UV light) affects in a different way the metabolites involved in the propagation of apoptosis. Both ABs and their extracellular fluid were analyzed using a reverse-phase liquid chromatography-mass spectrometry setup. Principal components analysis showed a tight clustering of each experimental group and partial least square discriminant analysis was used to assess the metabolic differences existing between these groups. Considering the variable importance in the projection values, molecular features were selected and some of them could be identified either unequivocally or tentatively. The resulting pathways indicated that there are significant, stimulus-specific differences in metabolites abundancies that may propagate apoptosis to healthy proximal tubular cells; thus, we hypothesize that the share in apoptosis of these metabolites might vary depending on the apoptotic stimulus.
Asunto(s)
Cisplatino , Vesículas Extracelulares , Humanos , Cisplatino/farmacología , Rayos Ultravioleta , Metabolómica/métodos , ApoptosisRESUMEN
We report the design of a new model based on a small neutral 8-aryl-3-formylBODIPY and its suitability to develop privileged highly bright and photostable fluorescent probes for selective and, more importantly, covalent staining of mitochondria.
Asunto(s)
Compuestos de Boro/química , Colorantes Fluorescentes/química , Mitocondrias/química , Imagen Óptica , Compuestos de Bencilo/química , Compuestos de Bencilo/metabolismo , Compuestos de Boro/síntesis química , Colorantes Fluorescentes/síntesis química , Humanos , Mitocondrias/metabolismo , Estructura Molecular , Células PC-3 , Procesos FotoquímicosRESUMEN
Proximal tubular cells (PTC) are particularly vulnerable to hypoxia-induced apoptosis, a relevant factor for kidney disease. We hypothesized here that PTC death under hypoxia is mediated by cyclo-oxygenase (COX-2)-dependent production of prostaglandin E2 (PGE2), which was confirmed in human proximal tubular HK-2 cells because hypoxia (1% O2)-induced apoptosis (i) was prevented by a COX-2 inhibitor and by antagonists of prostaglandin (EP) receptors and (ii) was associated to an increase in intracellular PGE2 (iPGE2) due to hypoxia-inducible factor-1α-dependent transcriptional up-regulation of COX-2. Apoptosis was also prevented by inhibitors of the prostaglandin uptake transporter PGT, which indicated that iPGE2 contributes to hypoxia-induced apoptosis (on the contrary, hypoxia/reoxygenation-induced PTC death was exclusively due to extracellular PGE2). Thus, iPGE2 is a new actor in the pathogenesis of hypoxia-induced tubular injury and PGT might be a new therapeutic target for the prevention of hypoxia-dependent lesions in renal diseases.
Asunto(s)
Muerte Celular , Hipoxia de la Célula , Dinoprostona/metabolismo , Túbulos Renales Proximales/metabolismo , Humanos , Túbulos Renales Proximales/patologíaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Cisplatin's toxicity in renal tubular epithelial cells limits the therapeutic efficacy of this antineoplastic drug. In cultured human proximal tubular HK-2 cells (PTC) a prostaglandin uptake transporter (PGT)-dependent increase in intracellular prostaglandin E2 (iPGE2) mediates cisplatin's toxicity (i.e. increased cell death and loss of cell proliferation) so that it is prevented by PGT inhibitors. Here we found in cisplatin-treated PTC that 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a PGT inhibitor, prevented cisplatin's toxicity but not the increase in iPGE2. Because expression of retinoic acid receptor-ß (RAR-ß) is dependent on iPGE2 and because RAR-ß is a regulator of cell survival and proliferation, we hypothesized that RAR-ß might mediate the protective effect of DIDS against cisplatin's toxicity in PTC. Our results confirmed this hypothesis because: i) protection of PTC by DIDS was abolished by RAR-ß antagonist LE-135; ii) DIDS increased the expression of RAR-ß in PTC and prevented its decrease in cisplatin-treated PTC but not in cisplatin-treated human cervical adenocarcinoma HeLa cells in which DIDS failed to prevent cisplatin's toxicity; iii) while RAR-ß expression decreased in cisplatin-treated PTC, RAR-ß over-expression prevented cisplatin's toxicity. RAR-ß agonist CH55 or RAR pan-agonist all-trans retinoic acid did not prevent cisplatin's toxicity, which suggests that RAR-ß does not protect PTC through activation of gene transcription. In conclusion, RAR-ß might be a new player in cisplatin-induced proximal tubular injury and the preservation of its expression in proximal tubules through treatment with DIDS might represent a novel strategy in the prevention of cisplatin's nephrotoxicity without compromising cisplatin's chemotherapeutic effect on cancer cells.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Cisplatino/efectos adversos , Dinoprostona/genética , Receptores de Ácido Retinoico/genética , Neoplasias del Cuello Uterino/tratamiento farmacológico , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Adenocarcinoma/genética , Adenocarcinoma/patología , Antineoplásicos/efectos adversos , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Chalconas/farmacología , Cisplatino/farmacología , Dibenzazepinas/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Túbulos Renales Proximales/efectos de los fármacos , Sustancias Protectoras , Transducción de Señal/efectos de los fármacos , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/patologíaRESUMEN
Renal hypoxia and loss of proximal tubular cells (PTC) are relevant in diabetic nephropathy. Hypoxia inhibits hypoxia-inducible factor-1α (HIF-1α) degradation, which leads to cellular adaptive responses through HIF-1-dependent activation of gene hypoxia-responsive elements (HRE). However, the diabetic microenvironment represses the HIF-1/HRE response in PTC. Here we studied the mechanism and consequences of impaired HIF-1α regulation in human proximal tubular HK-2 cells incubated in hyperglycemia. Inhibition at different levels of the canonical pathway of HIF-1α degradation did not activate the HIF-1/HRE response under hyperglycemia, except when proteasome was inhibited. Further studies suggested that hyperglycemia disrupts the interaction of HIF-1α with Hsp90, a known cause of proteasomal degradation of HIF-1α. Impaired HIF-1α regulation in cells exposed to hyperglycemic, hypoxic diabetic-like milieu led to diminished production of vascular endothelial growth factor-A and inhibition of cell migration (responses respectively involved in tubular protection and repair). These effects, as well as impaired HIF-1α regulation, were reproduced in normoglycemia in HK-2 cells incubated with microparticles released by HK-2 cells exposed to diabetic-like milieu. In summary, these results highlight the role of proteasome-dependent mechanisms of HIF-1α degradation on diabetes-induced HK-2 cells dysfunction and suggest that cell-derived microparticles may mediate negative effects of the diabetic milieu on PTC.
Asunto(s)
Nefropatías Diabéticas/metabolismo , Células Epiteliales/metabolismo , Glucosa/farmacología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Túbulos Renales Proximales/metabolismo , Proteolisis/efectos de los fármacos , Hipoxia de la Célula , Línea Celular , Nefropatías Diabéticas/patología , Células Epiteliales/patología , Glucosa/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Túbulos Renales Proximales/patología , Complejo de la Endopetidasa Proteasomal/metabolismoRESUMEN
Bodily attractiveness is an important component of mate value. Musculature-a crucial component of men's bodily attractiveness-provides women with probabilistic information regarding a potential mate's quality. Overall musculature is comprised of several muscle groups, each of which varies in information value; different muscles should be weighted differently by attractiveness-assessment adaptations as a result. In the current study, women and men ( N = 1,742) reported size preferences for 14 major muscle groups. Women's reported preferences provided only partial support for our hypotheses that women will prefer muscles that most reliably differentiate between potential mates to be larger; men tended to prefer larger upper-body muscles. We discuss possible interpretations of these mixed findings. Ultimately, our findings suggest that attractiveness-assessment adaptations are sensitive to the information contained within specific muscle groups and they highlight the potential for additional research on the nuances of bodily attractiveness assessment.
Asunto(s)
Belleza , Composición Corporal/fisiología , Hombres , Músculo Esquelético , Aptitud Física/psicología , Parejas Sexuales , Adolescente , Adulto , Conducta de Elección/fisiología , Femenino , Humanos , Masculino , Adulto JovenRESUMEN
The therapeutic efficacy of the antineoplastic drug cisplatin is limited by its nephrotoxicity, which affects particularly to proximal tubular cells (PTC). Cisplatin-induced cytotoxicity appears to be multifactorial and involves inflammation, oxidative stress as well as apoptosis. We have recently shown that the cyclo-oxygenase-2 (COX-2)/intracellular prostaglandin E2 (iPGE2)/EP receptor pathway mediates the apoptotic effect of cisplatin on human proximal tubular HK-2 cells. Here, we studied the effects on HK-2 cells of apoptotic bodies (ABs) generated after treatment of HK-2 cells with cisplatin. We found that ABs inhibited cell growth, induced apoptosis and increased COX-2 expression and iPGE2 in ABs-recipient HK-2 cells. Inhibition of the COX-2/iPGE2/EP receptor pathway in these cells prevented the effects of ABs without interfering with their internalization. Interestingly, 2nd generation ABs (i.e. ABs released by cells undergoing apoptosis upon treatment with ABs) did not trigger apoptosis in naïve HK-2 cells, and stimulated cell proliferation through the COX-2/iPGE2/EP receptor pathway. These results suggest that ABs, through iPGE2-dependent mechanisms, might have a relevant role in the natural history of cisplatin-induced acute kidney failure because they contribute first to the propagation of the noxious effects of cisplatin to non-injured PTC and then to the promotion of the proliferative tubular response required for proximal tubule repair. Since iPGE2 also mediates both cisplatin-induced HK-2 cell apoptosis, intervention in the COX-2/iPGE2/EP receptor pathway might provide us with new therapeutic avenues in patients with cisplatin-induced acute kidney injury.
Asunto(s)
Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Cisplatino/toxicidad , Vesículas Extracelulares/metabolismo , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/patología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Ciclooxigenasa 2/química , Ciclooxigenasa 2/metabolismo , Inhibidores de la Ciclooxigenasa 2/farmacología , Dinoprostona/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Humanos , Túbulos Renales Proximales/citología , Receptores de Prostaglandina E/antagonistas & inhibidores , Receptores de Prostaglandina E/metabolismo , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Cyclooxygenase (COX)-derived prostaglandin E2 (PGE2 ) affects many mechanisms that have been shown to play roles in carcinogenesis. Recently, we found that, in androgen-independent prostate cancer PC3 cells, PGE2 acts through an intracrine mechanism by which its uptake by the prostaglandin transporter (PGT) results in increased intracellular PGE2 (iPGE2 ), leading to enhanced cell proliferation, migration, invasion, angiogenesis, and loss of cell adhesion to collagen I. These iPGE2 -mediated effects were dependent on hypoxia-inducible factor 1-α (HIF-1α), whose expression increased upon epidermal growth factor receptor (EGFR) transactivation by a subset of intracellular PGE2 receptors. Here, we aimed to study the role of COX in PGE2 protumoral effects in PC3 cells and found that the effects were prevented by inhibition of COX-2, which highlights its crucial role amplifying the levels of iPGE2 . Treatment with exogenous PGE2 determined a transcriptional increase in COX-2 expression, which was abolished by genetic or pharmacologic inhibition of PGT. PGE2 -induced increase in COX-2 expression and, thereby, in transcriptional increase in HIF-1α expression was due to EGFR activation, leading to the activation of Phosphoinositide 3-kinase/Akt, Extracellular signal -regulated kinases 1/2, p38 and Mitogen- and stress-activated protein kinase-1 (PI3K/Akt, Erk1/2, p38 and MSK-1). Collectively, the data suggest that EGFR-dependent COX-2 upregulation by a novel positive feedback loop triggered by iPGE2 underlies the intracrine pro-tumoral effects of PGE2 in PC3 cells. Therefore, this feedback loop may be relevant in prostate cancer for the maintenance of PGE2 -dependent cancer cell growth through amplifying the activity of the COX-2 pathway.
Asunto(s)
Ciclooxigenasa 2/metabolismo , Dinoprostona/metabolismo , Línea Celular Tumoral , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Colágeno Tipo I/metabolismo , Receptores ErbB/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Masculino , Neovascularización Patológica/metabolismo , Células PC-3 , Fosfatidilinositol 3-Quinasas/metabolismo , Neoplasias de la Próstata/metabolismo , Transducción de Señal/fisiología , Activación Transcripcional/fisiología , Regulación hacia Arriba/fisiologíaRESUMEN
Prostaglandin E2 (PGE2 ) increases cell proliferation and stimulates migratory and angiogenic abilities in prostate cancer cells. However, the effects of PGE2 on non-transformed prostate epithelial cells are unknown, despite the fact that PGE2 overproduction has been found in benign hyperplastic prostates. In the present work we studied the effects of PGE2 in immortalized, non-malignant prostate epithelial RWPE-1 cells and found that PGE2 increased cell proliferation, cell migration, and production of vascular endothelial growth factor-A, and activated in vitro angiogenesis. These actions involved a non-canonic intracrine mechanism in which the actual effector was intracellular PGE2 (iPGE2 ) instead of extracellular PGE2 : inhibition of the prostaglandin uptake transporter (PGT) or antagonism of EP receptors prevented the effects of PGE2 , which indicated that PGE2 activity depended on its carrier-mediated translocation from the outside to the inside of cells and that EP receptors located intracellularly (iEP) mediated the effects of PGE2 . iPGE2 acted through transactivation of epidermal growth factor-receptor (EGFR) by iEP, leading to increased expression and activity of hypoxia-inducible factor-1α (HIF-1α). Interestingly, iPGE2 also mediates the effects of PGE2 on prostate cancer PC3 cells through the axis iPGE2 -iEP receptors-EGFR-HIF-1α. Thus, this axis might be responsible for the growth-stimulating effects of PGE2 on prostate epithelial cells, thereby contributing to prostate proliferative diseases associated with chronic inflammation. Since this PGT-dependent non-canonic intracrine mechanism of PGE2 action operates in both benign and malignant prostate epithelial cells, PGT inhibitors should be tested as a novel therapeutic modality to treat prostate proliferative disease.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Dinoprostona/farmacología , Células Epiteliales/efectos de los fármacos , Neovascularización Patológica/tratamiento farmacológico , Línea Celular Tumoral/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Epiteliales/metabolismo , Receptores ErbB/metabolismo , Humanos , Masculino , Neovascularización Patológica/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
The role and reliable quantification of intracellular hydrogen peroxide during cancer therapy constitutes an unexplored and fascinating application. In this work, we report the fabrication of vertically aligned copper nanowires (v-CuNWs) using electrosynthesis on templates, and their application as a cut-and-paste exclusive and flexible electrochemical transducer. This easily adaptable electrodic platform is demonstrated for a fast, simple and free-enzyme selective quantification of intracellular hydrogen peroxide in Cisplatin-treated human renal HK-2 cells. The v-CuNWs sensor was compared with an HRP-enzyme-based biosensor showing excellent correlation and indicates the good selectivity and analytical performance of the v-CuNWs. This sensing approach opens novel avenues for monitoring cell death processes and shows the potential of H2O2 as a cellular damage biomarker, with a clear potency for further developments for in vitro diagnosis and its implication in cancer therapy.
Asunto(s)
Antineoplásicos/química , Cisplatino/química , Cobre/química , Técnicas Electroquímicas/métodos , Peróxido de Hidrógeno/análisis , Nanocables/química , Técnicas Biosensibles/métodos , Línea Celular Tumoral , Supervivencia Celular , Electrodos , Humanos , Espacio Intracelular/química , Neoplasias/terapia , Sensibilidad y Especificidad , TransductoresRESUMEN
Nephrotoxicity, particularly in the proximal tubule, limits the therapeutic efficacy of the antineoplastic drug cisplatin. The signaling mechanisms appear to be multifactorial, involving inflammation, oxidative stress, and caspase. Here we studied the role of intracellular prostaglandin E2 (iPGE2) in cisplatin's cytotoxicity in human proximal tubular HK-2 cells. Cisplatin-induced apoptotic cell death was prevented by inhibitors of the prostaglandin transporter (PGT) or by PGT knock-down or by pharmacologic inhibition of PGE2 EP receptors or cyclo-oxygenase-2 (COX-2). iPGE2 also increased in cisplatin-treated cells, which was probably due to increased expression of COX-2, microsomal PGE2 synthase-1 and PGT, and was prevented by inhibitors of PGT or COX-2. Thus iPGE2, most likely acting through intracellular EP receptors, mediates cisplatin-induced HK-2 cell death. Importantly, the tumoricidal effect of cisplatin on human cervical adenocarcinoma HeLa cells was not affected by a pharmacologic inhibitor of PGT. In conclusion, iPGE2 may play a significant role in the pathogenesis of cisplatin's nephrotoxicity and treatment with PGT inhibitors might represent a novel strategy in its prevention.
Asunto(s)
Apoptosis/efectos de los fármacos , Cisplatino/farmacología , Dinoprostona/metabolismo , Túbulos Renales Proximales/efectos de los fármacos , Antineoplásicos/farmacología , Western Blotting , Línea Celular , Supervivencia Celular/efectos de los fármacos , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Expresión Génica , Humanos , Espacio Intracelular/metabolismo , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/metabolismo , Microscopía Confocal , Transportadores de Anión Orgánico/genética , Transportadores de Anión Orgánico/metabolismo , Interferencia de ARN , Receptores de Prostaglandina E/antagonistas & inhibidores , Receptores de Prostaglandina E/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The nuclear factor κB (NF-κB) is a powerful activator of angiogenesis, invasion and metastasis. Transactivation and nuclear localisation of NF-κB is an index of recurrence in prostate cancer. Vasoactive intestinal peptide (VIP) exerts similar effects in prostate cancer models involving increased expression of vascular endothelial growth factor (VEGF) and cyclooxygenase-2 (COX-2) which are related to NF-κB transactivation. Here we studied differential mechanisms of VIP-induced NF-κB transactivation in non-tumour RWPE-1 and tumour LNCaP and PC3 human prostate epithelial cells. Immunofluorescence studies showed that VIP increases translocation of the p50 subunit of NF-κB1 to the nucleus, an effect that was inhibited by curcumin. The signalling transduction pathways involved are different depending on cell transformation degree. In control cells (RWPE1), the effect is mediated by protein kinase A (PKA) activation and does not implicate extracellular signal-regulated kinase (ERK) or phosphoinositide 3-kinase (PI3-K) pathways whereas the opposite is true in tumour LNCaP and PC3 cells. Exchange protein directly activated by cAMP (EPAC) pathway is involved in transformed cells but not in control cells. Curcumin blocks the activating effect of VIP on COX-2 promoter/prostaglandin E2 (PGE2) production and VEGF expression and secretion. The study incorporates direct observation on COX-2 promoter and suggests that VIP effect on VEGF may be indirectly mediated by PGE2 after being synthesised by COX-2, thus amplifying the initial signal. We show that the signalling involved in VIP effects on VEGF is cAMP/PKA in non-tumour cells and cAMP/EPAC/ERK/PI3K in tumour cells which coincides with pathways mediating p50 nuclear translocation. Thus, VIP appears to use different pathways for NF-κB1 (p50) transactivation in prostate epithelial cells depending on whether they are transformed or not. Transformed cells depend on pro-survival and pro-proliferative signalling pathways involving ERK, PI3-K and cAMP/EPAC which supports the potential therapeutic value of these targets in prostate cancer.
Asunto(s)
Núcleo Celular/metabolismo , Subunidad p50 de NF-kappa B/metabolismo , Transducción de Señal/efectos de los fármacos , Péptido Intestinal Vasoactivo/farmacología , Línea Celular , Curcumina/farmacología , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Dinoprostona/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Masculino , Fosfatidilinositol 3-Quinasas/metabolismo , Próstata/citología , Próstata/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Receptores de Péptido Intestinal Vasoactivo/metabolismo , Activación Transcripcional/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The pharmacological modulation of hypoxia-inducible factor-1α (HIF-1α) and HIF-1α-regulated vascular endothelial growth factor-A (VEGF-A) in the kidney has therapeutic interest. Although it is assumed that prostaglandin E(2) (PGE(2)) exerts its biological effects from the extracellular medium through activation of EP receptors located at the cell membrane, we have shown in human renal proximal tubular HK-2 cells (and other cell lines) that intracellular PGE(2) regulates the expression of HIF-1α expression and the production of VEGF-A. Here, we have found--through experiments involving EP receptors agonists, EP receptor gene silencing and inhibition of the prostaglandin uptake transporter--that these biological effects of PGE(2) are mediated by intracellular EP(2) receptors. In sharp contrast with cell membrane EP(2), whose activation results in increased production of cAMP, intracellular EP(2) signaling was independent of cAMP. Instead, it involved c-src-dependent transactivation of epidermal growth factor receptor, which led to p38/ERK1/2-dependent activation of mitogen- and stress-activated kinase-1 (MSK-1) and to MSK-1-dependent-histone H3 phosphorylation and transcriptional up-regulation of retinoic acid receptor-ß. Even more important, this signaling pathway was fully reproduced in nuclei isolated from HK-2 cell, which highlights the relevance of nuclear EP receptors in the up-regulation of HIF-1α. These results open the possibility that signal cascades that proceed entirely in the cell nucleus might be responsible for several PGE(2) effects that are assumed to be due to cell membrane EP receptors.
Asunto(s)
Receptores ErbB/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/biosíntesis , Subtipo EP2 de Receptores de Prostaglandina E/metabolismo , Activación Transcripcional/genética , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Núcleo Celular/metabolismo , AMP Cíclico/metabolismo , Activación Enzimática , Células Epiteliales/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Histonas/metabolismo , Humanos , Túbulos Renales Proximales/citología , Fosforilación , Subtipo EP2 de Receptores de Prostaglandina E/genética , Receptores de Ácido Retinoico/biosíntesis , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Transducción de Señal , Regulación hacia Arriba , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The pharmacological modulation of putative renoprotective factors hypoxia-inducible factor-1α (HIF-1α) and HIF-1α-regulated vascular endothelial growth factor-A (VEGF-A) in the kidney has therapeutic interest. In human renal proximal tubular HK2 cells, prostaglandin E2 (PGE2) up-regulates HIF-1α and VEGF-A through epidermal growth factor receptor (EGFR)-dependent up-regulation of retinoic acid receptor-ß (RARß). Here we studied the role of mitogen-activated protein kinases (MAPKs) ERK1/2 and p38 and their target kinase, mitogen- and stress activated kinase-1 (MSK1), in the signaling cascade. Treatment of HK2 cells with PGE2 resulted in increased phosphorylation of EGFR, the three studied kinases and the histone H3 (Ser10) at the RARß gene promoter (the latter has been proposed as a molecular signature of the activated RARß gene promoter). Prevention of the phosphorylation of EGFR, ERK1/2, p38 MAPK or MSK1 is by incubating, respectively, with AG1478, PD98059, SB203580 or H89 allowed to elucidate the precise phosphorylation order in the signaling cascade triggered by PGE2: first, EGFR; then, ERK1/2 and p38 MAPK and, finally, MSK1. Phosphorylation of MSK1 led to that of Ser10 in histone H3 and to activation of RARß gene transcription (and the consequent increase in the expression of HIF-1α and VEGF-A), which was suppressed by H89 or by transfecting cells with a vector encoding for a dominant-negative mutant of MSK1. These results highlight the relevance of MSK1 in the up-regulation of RARß by PGE2. They also may contribute to new therapeutic approaches based upon the pharmacological control of HIF-1α/VEGF-A in the proximal tubule through the modulation of the PGE2/EGFR/MAPK/MSK1/RARß pathway.
Asunto(s)
Dinoprostona/farmacología , Receptores de Ácido Retinoico/genética , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Línea Celular , Receptores ErbB/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Modelos Biológicos , Fosforilación/efectos de los fármacos , Receptores de Ácido Retinoico/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Vasoactive intestinal peptide (VIP) decreases cell proliferation through PI3K signalling and prevents tumour progression in clear renal cell carcinoma (RCC). Here we analyzed the signalling pathways that mediate such VIP effects by using human RCC A498 cells. The effects of treatment with 1 µM VIP and/or specific protein kinase inhibitors such as H89, Wortmannin and PD98059 were studied by cell adhesion assay, ELISA of VEGF165 and ROS production assays. Semiquantitative RT-PCR and western blot were performed to study p53 expression. VIP increased cell adhesion and ROS production, and decreased VEGF165 secretion through PI3K signalling. Moreover, VIP increased nuclear expression of tumour suppressor p53. VIP effects could be blocked by cell incubation with a specific p53 inhibitor, cyclin pifithrin-α hydrobromide (CPFT-αH). In conclusion, this study provides a p53-dependent mechanism by which VIP regulates cell proliferation in RCC development. It supports a potential usefulness of VIP in new therapies of RCC.
Asunto(s)
Carcinoma de Células Renales/genética , Proteína p53 Supresora de Tumor/biosíntesis , Factor A de Crecimiento Endotelial Vascular/genética , Péptido Intestinal Vasoactivo/administración & dosificación , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , AMP Cíclico/metabolismo , Humanos , Fosfatidilinositol 3-Quinasas/genética , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
The pharmacological modulation of renoprotective factor vascular endothelial growth factor-A (VEGF-A) in the proximal tubule has therapeutic interest. In human proximal tubular HK-2 cells, treatment with all-trans retinoic acid or prostaglandin E2 (PGE2) triggers the production of VEGF-A. The pathway involves an initial increase in intracellular PGE2, followed by activation of EP receptors (PGE2 receptors, most likely an intracellular subset) and increase in retinoic acid receptor-ß (RARß) expression. RARß then up-regulates transcription factor hypoxia-inducible factor-1α (HIF-1α), which increases the transcription and production of VEGF-A. Here we studied the role in this pathway of epidermal growth factor receptor (EGFR) transactivation by EP receptors. We found that EGFR inhibitor AG1478 prevented the increase in VEGF-A production induced by PGE2- and all-trans retinoic acid. This effect was due to the inhibition of the transcriptional up-regulation of RARß, which resulted in loss of the RARß-dependent transcriptional up-regulation of HIF-1α. PGE2 and all-trans retinoic acid also increased EGFR phosphorylation and this effect was sensitive to antagonists of EP receptors. The role of intracellular PGE2 was indicated by two facts; i) PGE2-induced EGFR phosphorylation was substantially prevented by inhibitor of prostaglandin uptake transporter bromocresol green and ii) all-trans retinoic acid treatment, which enhanced intracellular but not extracellular PGE2, had lower effect on EGFR phosphorylation upon pre-treatment with cyclooxygenase inhibitor diclofenac. Thus, EGFR transactivation by intracellular PGE2-activated EP receptors results in the sequential activation of RARß and HIF-1α leading to increased production of VEGF-A and it may be a target for the therapeutic modulation of HIF-1α/VEGF-A.
Asunto(s)
Dinoprostona/metabolismo , Receptores ErbB/metabolismo , Túbulos Renales Proximales/metabolismo , Receptores de Prostaglandina E/metabolismo , Receptores de Ácido Retinoico/metabolismo , Activación Transcripcional/fisiología , Regulación hacia Arriba/fisiología , Línea Celular Tumoral , Dinoprostona/genética , Inhibidores Enzimáticos/farmacología , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Queratolíticos/farmacología , Túbulos Renales Proximales/citología , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Quinazolinas/farmacología , Receptores de Prostaglandina E/genética , Receptores de Ácido Retinoico/genética , Activación Transcripcional/efectos de los fármacos , Tretinoina/farmacología , Tirfostinos/farmacología , Regulación hacia Arriba/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
New approaches are needed to the therapy of advanced prostate cancer. This study determined the effect of growth hormone-releasing hormone (GHRH) antagonists, JMR-132 and JV-1-38 on growth of PC3 tumors as well as on angiogenesis and metastasis through the evaluation of various factors that contribute largely to the progression of prostate cancer. Human PC3 androgen-independent prostate cancer cells were injected subcutaneously into nude mice. The treatment with JMR-132 (10 µg/day) or JV-1-38 (20 µg/day) lasted 41 days. We also evaluated the effects of JMR-132 and JV-1-38 on proliferation, cell adhesion and migration in PC-3 cells in vitro. Several techniques (Western blot, reverse transcription polymerase chain reaction, immunohistochemistry, ELISA and zymography) were used to evaluate the expression levels of GHRH receptors and its splice variants, GHRH, vascular endothelial growth factor (VEGF), hypoxia inducible factor (HIF)-1α, metalloproteinases (MMPs) -2 and -9, ß-catenin and E-cadherin. GHRH antagonists suppressed the proliferation of PC-3 cells in vitro and significantly inhibited growth of PC3 tumors. After treatment with these analogues, we found an increase in expression of GHRH receptor accompanied by a decrease of GHRH levels, a reduction in both VEGF and HIF-1α expression and in active forms of MMP-2 and MMP-9, a significant increase in levels of membrane-associated ß-catenin and a significant decline in E-cadherin. These results support that the blockade of GHRH receptors can modulate elements involved in angiogenesis and metastasis. Consequently, GHRH antagonists could be considered as suitable candidates for therapeutic trials in the management of androgen-independent prostate cancer.
Asunto(s)
Antineoplásicos/farmacología , Hormona Liberadora de Hormona del Crecimiento/análogos & derivados , Hormona Liberadora de Hormona del Crecimiento/antagonistas & inhibidores , Metástasis de la Neoplasia/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Neoplasias de la Próstata/tratamiento farmacológico , Sermorelina/análogos & derivados , Animales , Cadherinas/metabolismo , Adhesión Celular , Línea Celular Tumoral , Proliferación Celular , Hormona Liberadora de Hormona del Crecimiento/farmacología , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Desnudos , Invasividad Neoplásica , Neoplasias de la Próstata/patología , Distribución Aleatoria , Receptores de Neuropéptido/metabolismo , Receptores de Hormona Reguladora de Hormona Hipofisaria/metabolismo , Sermorelina/farmacología , Factores de Crecimiento Endotelial Vascular/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , beta Catenina/metabolismoRESUMEN
We have previously found in human renal proximal tubular HK-2 cells that hypoxia- and all-trans retinoic acid-induced hypoxia-inducible factor-1α up-regulation is accompanied by retinoic acid receptor-ß up-regulation. Here we first investigated whether hypoxia-inducible factor-1α expression is dependent on retinoic acid receptor-ß and our results confirmed it since (i) hypoxia-inducible factor-1α-inducing agents hypoxia, hypoxia-mimetic agent desferrioxamine, all-trans retinoic acid and interleukin-1ß increased retinoic acid receptor-ß expression, (ii) hypoxia-inducible factor-1α up-regulation was prevented by retinoic acid receptor-ß antagonist LE-135 or siRNA retinoic acid receptor-ß and (iii) there was direct binding of retinoic acid receptor-ß to the retinoic acid response element in hypoxia-inducible factor-1α promoter upon treatment with all-trans retinoic acid and 16,16-dimethyl-prostaglandin E(2). Since intracellular prostaglandin E(2) mediates hypoxia-inducible factor-1α up-regulation in normoxia in HK-2 cells, we next investigated and confirmed, its role in the up-regulation of retinoic acid receptor-ß in normoxia by hypoxia-inducible factor-1α-inducing agents all-trans retinoic acid, interleukin-1ß and 16,16-dimethyl-prostaglandin E(2) by inhibiting cyclooxygenases, prostaglandin influx transporter or EP receptors. Interestingly, the hypoxia-induced increase in retinoic acid receptor-ß expression and accumulation of hypoxia-inducible factor-1α was also blocked by the inhibitors tested. This is the first time, to our knowledge, that retinoic acid receptor-ß signalling is involved in the control of the expression of transcription factor hypoxia-inducible factor-1α in both normoxia and hypoxia and that retinoic acid receptor-ß expression is found to be strictly regulated by intracellular prostaglandin E(2). Given the relevance of hypoxia-inducible factor-1α in the kidney in terms of tumorigenesis, progressive renal failure, production of erythropoietin and protection in several models of renal disease, our results open new therapeutic opportunities on the control of hypoxia-inducible factor-1α based upon the pharmacological modulation of retinoic acid receptor-ß, either directly or through the control of intracellular prostaglandin E(2) levels/signalling.