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Introduction: Cerebral cavernous malformations (CCMs) are pathologic lesions comprised of clusters of thin-walled capillaries characterized by abnormal proliferation, angiogenesis, and bleeding secondary to somatic or germline mutations in endothelial cells. CCMs can cause headaches, seizures and/or neurological defects. There is a clinical need to develop better tools to detect CCMs and follow their progression in conjunction with the current use of neuroimaging techniques. Here we present data supporting the utility of LOX-1 (lectin-type oxidized LDL receptor 1), a 50 kDa transmembrane protein implicated in endothelial cell dysfunction and ischemia, as a putative biomarker for CCM. Methods: CCM urine samples (n = 23) were collected from pediatric CCM patients. Matched healthy controls (n = 24) were collected from pediatric patients with either Chiari I malformation or fatty filum terminale, and otherwise normal findings. All samples were collected with patient/family consent and institutional review board approval.Samples were analyzed with Olink Proteomic Proximity Extension Assay (PEA). Differences in expression for 2,925 unique proteins were quantified between healthy control urine samples and CCM urine samples. The results were normalized, validated, and analyzed for demographic bias. In addition to urine samples, CCM tissue from patients was harvested and used to create primary cell lines for in vitro analysis of LOX-1 expression, in addition to immunofluorescence of lesional tissue excised at surgery. Results: ANOVA analysis of the CCM urine samples showed a statistically significant increase in LOX-1 compared to the control samples, with CCM patients exhibiting a > 5-fold increase in urinary expression. Corroborating these elevated levels of circulating marker, analysis of source tissue from surgically resected CCMs revealed that LOX-1 is increased in both CCM patient cavernoma primary cell lines and operative specimens. Conclusion: LOX-1 is involved with pathways implicated in CCM pathogenesis and our data here reveals that LOX-1 expression is significantly elevated in CCM patients as compared to matched healthy control individuals, including both source tissue from surgically excised CCMs and in analysis of samples collected from outside of the central nervous system, particularly urine. This proof-of-principle data suggests that LOX-1 may have potential utility as a target for CCM treatment and supports further investigation related to its potential mechanistic impact on CCM pathogenesis.
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Proprotein convertase subtilisin/kexin type 9 (PCSK9) is one of the major regulators of low-density lipoprotein receptor (LDLR). Information on role and regulation of PCSK9 in lung is very limited. Our study focuses on understanding the role and regulation of PCSK9 in the lung. PCSK9 levels are higher in Bronchoalveolar lavage fluid (BALF) of smokers with or without chronic obstructive pulmonary diseases (COPD) compared to BALF of nonsmokers. PCSK9-stimulated cells induce proinflammatory cytokines and activation of MAPKp38. PCSK9 transcripts are highly expressed in healthy individuals compared to COPD, pulmonary fibrosis or pulmonary systemic sclerosis. Cigarette smoke extract reduce PCSK9 levels in undifferentiated pulmonary bronchial epithelial cells (PBEC) but induce in differentiated PBEC. PCSK9 inhibition affect biological pathways, induces lipid peroxidation, and higher level of apoptosis in response to staurosporine. Our results suggest that higher levels of PCSK9 in BALF acts as an inflammatory marker. Furthermore, extracellular and intracellular PCSK9 play different roles.
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Inflamación , Proproteína Convertasa 9 , Humanos , Proproteína Convertasa 9/metabolismo , Proproteína Convertasa 9/genética , Inflamación/metabolismo , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/patología , Enfermedad Pulmonar Obstructiva Crónica/genética , Muerte Celular , Metabolismo de los Lípidos , Masculino , Apoptosis , Células Epiteliales/metabolismo , FemeninoRESUMEN
Intracranial vascular malformations manifest on a continuum ranging from predominantly arterial to predominantly venous in pathology. Cerebral cavernous malformations (CCMs) are capillary malformations that exist at the midpoint of this continuum. The axon guidance factor Ephrin B2 and its receptor EphB4 are critical regulators of vasculogenesis in the developing central nervous system. Ephrin B2/EphB4 dysregulation has been implicated in the pathogenesis of arterial-derived arteriovenous malformations and vein-based vein of Galen malformations. Increasing evidence supports the hypothesis that aberrant Ephrin B2/EphB4 signaling may contribute to developing vascular malformations, but their role in CCMs remains largely uncharacterized. Evidence of Ephrin dysregulation in CCMs would be important to establish a common link in the pathogenic spectrum of EphrinB2/Ephb4 dysregulation. By studying patient-derived primary CCM endothelial cells (CCMECs), we established that CCMECs are functionally distinct from healthy endothelial cell controls; CCMECs demonstrated altered patterns of migration, motility, and impaired tube formation. In addition to the altered phenotype, the CCMECs also displayed an increased ratio of EphrinB2/EphB4 compared to the healthy endothelial control cells. Furthermore, whole exome sequencing identified mutations in both EphrinB2 and EphB4 in the CCMECs. These findings identify functional alterations in the EphrinB2/EphB4 ratio as a feature linking pathophysiology across the spectrum of arterial, capillary, and venous structural malformations in the central nervous system while revealing a putative therapeutic target.
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Hemangioma Cavernoso del Sistema Nervioso Central , Receptor EphB2 , Receptor EphB4 , Humanos , Receptor EphB4/genética , Receptor EphB2/genética , Hemangioma Cavernoso del Sistema Nervioso Central/genética , Células Endoteliales/patología , Cultivo Primario de Células , Secuenciación del Exoma , Masculino , Femenino , Preescolar , Niño , AdolescenteRESUMEN
Urinary biomarkers can diagnose and monitor pathophysiologic conditions in the central nervous system (CNS). However, focus is often on single diseases, with limited data on discriminatory capability of this approach in a general setting. Here, we demonstrate that different classes of CNS disease exhibit distinct biomarker patterns, evidence of disease-specific "fingerprinting." Urine from 218 patients with pathology-confirmed tumors or cerebrovascular disease, controls (n = 33) were collected. ELISA and/or bead-based multiplexing quantified levels of 21 putative urinary biomarkers. Analysis identified biomarkers capable of distinguishing each disease from controls and other diseases. Mann-Whitney U tests identified biomarkers with differential expression between disease types and controls (P ≤ 0.001). Subsequent receiver-operating characteristic (ROC) analyses revealed distinguishing biomarkers with high sensitivity and specificity. Areas under the curve (AUCs) ranged 0.8563-1.000 (P values ≤ 0.0003), sensitivities ranged 80.00-100.00%, and specificities ranged 80.95-100.00%. These data demonstrate proof-of-principle evidence that disease-specific urinary biomarker signatures exist. In contrast to non-specific responses to ischemia or injury, these results suggest that urinary biomarkers accurately reflect unique biological processes distinct to different diseases. This work can be used to generate disease-specific panels for enhancing diagnosis, assisting less-invasive follow-up and herald utility by revealing putative disease-specific therapeutic targets.
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Enfermedades del Sistema Nervioso Central , Humanos , Biomarcadores , Curva ROC , Sistema Nervioso CentralRESUMEN
We have synthesized an oxetane derivative of the benzimidazole compound mebendazole (OBD9) with enhanced solubility and strong anticancer activity in multiple types of cancer cells, especially colorectal cancer. In this report, we provide evidence that OBD9 suppresses colorectal cancer growth by interfering with the Wnt signaling pathway, a main driver of cell growth in colorectal cancer. Specifically, we find that OBD9 induces autophagic degradation of TNIK (traf2 and Nck-interacting kinase), which promotes T-cell factor-4 (TCF4)/beta-catenin-mediated gene expression. Thus, OBD9 as a TNIK inhibitor blocks Wnt/beta-catenin signaling at the final step of transcriptional activation. We suggest that OBD9 provides a potential novel autophagy-mediated, Wnt-damping therapeutic strategy for the treatment of colorectal cancer.
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Neoplasias del Colon , Neoplasias Colorrectales , Humanos , Vía de Señalización Wnt , beta Catenina/metabolismo , Línea Celular Tumoral , Neoplasias del Colon/tratamiento farmacológico , Neoplasias Colorrectales/tratamiento farmacológicoRESUMEN
The transcript encoding Antizyme Inhibitor 1 (AZIN1) is frequently edited in various cancers, and this editing is associated with enhanced tumor aggressiveness. After comparison of wild-type AZIN1 (wtAZIN1) and edited AZIN1 (edAZIN1, which contains a Ser367Gly substitution), we report differential binding of edAZIN1 to a small set of proteins; specifically, edAZIN1 binds to alpha-smooth muscle actin (ACTA2), gamma actin 1 (ACTG1), and myosin9, whereas wtAZIN1 does not. This binding enables nuclear translocation of edAZIN1. In contrast to overexpression of edAZIN1 and, to a lesser extent, (editable) wtAZIN1, overexpression of an uneditable AZIN1 allele does not promote a cellular phenotype associated with increased tumorigenicity. In patients, both editing and nuclear localization of AZIN1 are common and are associated with tumor aggressiveness, i.e., a higher Gleason score, higher genomic instability, and a shorter progression-free survival time. In conclusion, the data indicate that binding of edAZIN1 to the actin/myosin9 complex supports its nuclear translocation, leading to enhanced cellular aggressiveness, and is associated with worse prostate cancer outcomes.
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Neoplasias de la Próstata , Edición de ARN , Masculino , Humanos , Actinas/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Neoplasias de la Próstata/genéticaRESUMEN
Akt kinase regulates several cellular processes, among them growth, proliferation and survival, and has been correlated to neoplastic disease. We report here crosstalk between several Akt regulatory phosphatases that controls the level of the activated form (phosphorylated) of Akt and affects tumor cell aggressiveness. In prostate cancer cell lines, we observed that transient transfection of PTEN decreased the endogenous level of PHLPPs and in contrast, the transient transfection of PHLPPs decreased the endogenous level of PTEN. Furthermore, silencing of PTEN by siRNA resulted in increased PHLPP levels. This phenomenon was not seen in non-transformed cells or in prostate stem cells. This crosstalk promoted cancer cell invasion and was controlled by epigenetically regulated processes where activation of miRs (miR-190 and miR214), the polycomb group of proteins and DNA methylation were involved. The purinergic P2X4 receptor, which has been shown to have a role in wound healing, was identified to be the mediator of this crosstalk. We also studied prostate stem cells and found this crosstalk in the TGFß1-activated epithelial-mesenchymal transition (EMT). The crosstalk seemed to be a natural part of EMT. In summary, we identify a crosstalk between Akt phosphatases which is not present in non-transformed prostate cells but occurs in cancer cells and stem cells transformed by TGFß-1. This crosstalk is important for cellular invasion. BACKGROUND: Phosphatases regulate the Akt oncogene. RESULTS: Crosstalk between Akt phosphatases in prostate cancer cells and in TGF-ß1 activated stem cells but not in non-transformed cells. CONCLUSION: This back-up mechanism facilitates invasive migration of prostate stem and cancer cells. SIGNIFICANCE: Characterization of Akt regulation may lead to a better understanding of tumor development and to novel strategies for treatment.
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Proteínas Nucleares/fisiología , Fosfohidrolasa PTEN/fisiología , Fosfoproteínas Fosfatasas/fisiología , Células Madre/metabolismo , Línea Celular Tumoral , Epigénesis Genética , Transición Epitelial-Mesenquimal/fisiología , Humanos , Invasividad Neoplásica/fisiopatología , Proteínas Nucleares/metabolismo , Fosfohidrolasa PTEN/antagonistas & inhibidores , Fosfohidrolasa PTEN/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , ARN Interferente Pequeño/farmacología , Receptores Purinérgicos P2X4/fisiología , Transfección , Factor de Crecimiento Transformador beta1RESUMEN
Endometriosis is an estrogen-dependent inflammatory disease that affects approximately 10% of women. Debilitating pelvic or abdominal pain is one of its major clinical features. Current animal models of endometriosis-associated pain require surgery either to implant tissue or to remove the ovaries. Moreover, existing models do not induce spontaneous pain, which is the primary symptom of patients with chronic pain, including endometriosis. A lack of models that accurately recapitulate the disease phenotype must contribute to the high failure rate of clinical trials for analgesic drugs directed at chronic pain, including those for endometriosis. We set out to establish a murine model of endometriosis-associated pain. Endometriosis was induced nonsurgically by injecting a dissociated uterine horn into a recipient mouse. The induced lesions exhibited histological features that resemble human lesions along with an increase in proinflammatory cytokines and recruitment of immune cells. We also observed the presence of calcitonin gene-related peptide-, TRPA1-, and TRPV1-expressing nerve fibers in the lesions. This model induced mechanical allodynia, spontaneous abdominal pain, and changes in thermal selection behavior that indicate discomfort. These behavioral changes were reduced by drugs used clinically for endometriosis, specifically letrozole (aromatase inhibitor) and danazol (androgen). Endometriosis also induced neuronal changes as evidenced by activation of the NF-κB signaling pathway in TRPA1- and TRPV1-expressing dorsal root ganglion neurons. In conclusion, we have established a model of endometriosis-associated pain that responds to clinically active drugs and can, therefore, be used to identify novel therapies.
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Endometriosis , Preparaciones Farmacéuticas , Animales , Modelos Animales de Enfermedad , Endometriosis/complicaciones , Endometriosis/tratamiento farmacológico , Estrógenos , Femenino , Humanos , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/etiología , RatonesRESUMEN
Extracellular nucleotides are important mediators of cell activation and trigger multiple responses via membrane receptors known as purinergic receptors (P2). P2X receptors are ligand-gated ion channels, activated by extracellular ATP. P2X4 is one of the most sensitive purinergic receptors, that is typically expressed by neurons, microglia, and some epithelial and endothelial cells. P2X4 mediates neuropathic pain via brain-derived neurotrophic factor and is also involved in inflammation in response to high ATP release. It is therefore involved in multiple inflammatory pathologies as well as neurodegenerative diseases. We have produced monoclonal antibodies (mAb) directed against this important human P2X4 receptor. Focusing on two mAbs, we showed that they also recognize mouse and rat P2X4. We demonstrated that these mAbs can be used in flow cytometry, immunoprecipitation, and immunohistochemistry, but not in Western blot assays, indicating that they target conformational epitopes. We also characterized the expression of P2X4 receptor on mouse and human peripheral blood lymphocytes (PBL). We showed that P2X4 is expressed at the surface of several leukocyte cell types, with the highest expression level on eosinophils, making them potentially sensitive to adenosine triphosphate (ATP). P2X4 is expressed by leucocytes, in human and mouse, with a significant gender difference, males having higher surface expression levels than females. Our findings reveal that PBL express significant levels of P2X4 receptor, and suggest an important role of this receptor in leukocyte activation by ATP, particularly in P2X4high expressing eosinophils.
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Eosinófilos/inmunología , Eosinófilos/metabolismo , Expresión Génica , Receptores Purinérgicos P2X4/genética , Animales , Astrocitoma/genética , Astrocitoma/metabolismo , Biomarcadores , Línea Celular , Femenino , Glioma/genética , Glioma/metabolismo , Humanos , Inmunofenotipificación , Leucocitos/inmunología , Leucocitos/metabolismo , Leucocitos/patología , Masculino , Ratones , Microglía/inmunología , Microglía/metabolismo , Receptores Purinérgicos P2X4/metabolismoRESUMEN
Juniper (Juniperus communis L.) is a northern coniferous plant generally used as a spice and for nutritional purposes in foods and drinks. It was previously reported that juniper extract (JE) affects p53 activity, cellular stress, and gene expression induced cell death in human neuroblastoma cells. Therefore, the effects of juniper on p53 and Akt signaling was examined further in A549 lung, 22RV1 and DU145 prostate, and HepG2 liver cancer cells using Western blot, confocal microscopy, and MTT analysis. We found that juniper simultaneously decreased cell viability, activated the p53 pathway, and inactivated the PI3K/Akt pathway. The p53 activation was associated with increased nuclear p53 level. Akt was dephosphorylated, and its inactivation was associated with increased levels of PHLPP1 and PHLPP2 phosphatases. Parallel increases of PARP suggest that JE decreased cell viability by activating cell death. In adtion, JE potentiated the effects of gemcitabine and 5-fluorouracil anticancer drugs. Thus, JE can activate cell death in different cancer cell lines through p53 and Akt pathways.
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Muerte Celular/efectos de los fármacos , Citostáticos/farmacología , Juniperus/química , Extractos Vegetales/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Células A549 , Supervivencia Celular/efectos de los fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Fluorouracilo/farmacología , Células Hep G2 , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteína p53 Supresora de Tumor/genética , GemcitabinaRESUMEN
Antizyme inhibitor (AZIN) stimulates cell proliferation by binding to and sequestering the cell cycle suppressor antizyme. Despite the important role of the antizyme-AZIN protein-protein interaction (PPI) in cell cycle regulation, there are no assays for directly measuring the binding of AZIN to antizyme that are amenable to high throughput screening. To address this problem, we developed and validated a novel antizyme-AZIN intramolecular FRET sensor using clover and mRuby2 fluorescent proteins. By introducing alanine mutations in the AZIN protein, we used this sensor to probe the PPI for key residues governing the binding interaction. We found that like many PPIs, the energy of the antizyme-AZIN binding interaction is distributed across many amino acid residues; mutation of individual residues did not have a significant effect on disrupting the PPI. We also examined the interaction between Clover-AZIN and antizyme-mRuby2 in cells. Evidence of a direct interaction between Clover-AZIN and antizyme-mRuby2 was observed within cells, validating the use of this FRET sensor for probing intracellular antizyme-AZIN PPI. In conclusion, we have developed and optimized a FRET sensor which can be adapted for high throughput screening of either in vitro or intracellular activity.
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Proteínas Portadoras/química , Transferencia Resonante de Energía de Fluorescencia/métodos , Proteínas/química , Secuencia de Aminoácidos , Animales , Fenómenos Biofísicos , Proteínas Portadoras/metabolismo , Humanos , Ornitina Descarboxilasa/metabolismo , Unión Proteica/fisiología , Proteínas/metabolismoRESUMEN
Clusterin (CLU) is a chaperone-like protein with multiple functions. sCLU is frequently upregulated in prostate tumor cells after chemo- or radiotherapy and after surgical or pharmacological castration. Moreover, CLU has been documented to modulate the cellular homolog of murine thymoma virus akt8 oncogene (AKT) activity. Here, we investigated how CLU overexpression influences phosphatidylinositol 3'-kinase (PI3K)/AKT signaling in human normal and cancer epithelial prostate cells. Human prostate cells stably transfected with CLU were broadly profiled by reverse phase protein array (RPPA), with particular emphasis on the PI3K/AKT pathway. The effect of CLU overexpression on normal and cancer cell motility was also tested. Our results clearly indicate that CLU overexpression enhances phosphorylation of AKT restricted to isoform 2. Mechanistically, this can be explained by the finding that the phosphatase PH domain leucine-rich repeat-containing protein phosphatase 1 (PHLPP1), known to dephosphorylate AKT2 at S474, is markedly downregulated by CLU, whereas miR-190, a negative regulator of PHLPP1, is upregulated. Moreover, we found that phosphatase and tensin homolog (PTEN) was heavily phosphorylated at the inhibitory site S380, contributing to the hyperactivation of AKT signaling. By keeping AKT2 phosphorylation high, CLU dramatically enhances the migratory behavior of prostate epithelial cell lines with different migratory and invasive phenotypes, namely prostate normal epithelial 1A (PNT1A) and prostatic carcinoma 3 (PC3) cells. Altogether, our results unravel for the first time a circuit by which CLU can switch a low migration phenotype toward a high migration phenotype, through miR-190-dependent downmodulation of PHLPP1 expression and, in turn, stabilization of AKT2 phosphorylation.
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Clusterina/metabolismo , Proteínas Nucleares/metabolismo , Fosfohidrolasa PTEN/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Células 3T3 , Animales , Línea Celular Tumoral , Movimiento Celular/fisiología , Clusterina/genética , Células Epiteliales/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Masculino , Ratones , MicroARNs/genética , Células PC-3 , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Próstata/patología , Proteínas Proto-Oncogénicas c-akt/genética , Transducción de Señal/genéticaRESUMEN
Tight junctions (TJ) act as hubs for intracellular signaling pathways controlling epithelial cell fate and function. Deregulation of TJ is a hallmark of epithelial-mesenchymal transition (EMT), which contributes to carcinoma progression and metastasis. However, the signaling mechanisms linking TJ to the induction of EMT are not understood. Here, we identify a TJ-based signalosome, which controls AKT signaling and EMT in breast cancer. The coxsackie and adenovirus receptor (CXADR), a TJ protein with an essential yet uncharacterized role in organogenesis and tissue homeostasis, was identified as a key component of the signalosome. CXADR regulated the stability and function of the phosphatases and AKT inhibitors PTEN and PHLPP2. Loss of CXADR led to hyperactivation of AKT and sensitized cells to TGFß1-induced EMT. Conversely, restoration of CXADR stabilized PHLPP2 and PTEN, inhibited AKT, and promoted epithelial differentiation. Loss of CXADR in luminal A breast cancer correlated with loss of PHLPP2 and PTEN and poor prognosis. These results show that CXADR promotes the formation of an AKT-inhibitory signalosome at TJ and regulates epithelial-mesenchymal plasticity in breast cancer cells. Moreover, loss of CXADR might be used as a prognostic marker in luminal breast cancer. SIGNIFICANCE: The tight junction protein CXADR controls epithelial-mesenchymal plasticity in breast cancer by stabilizing the AKT regulators PTEN and PHLPP2.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/1/47/F1.large.jpg.
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Neoplasias de la Mama/patología , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus/metabolismo , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Uniones Estrechas/patología , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Diferenciación Celular , Movimiento Celular , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus/genética , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus/fisiología , Femenino , Perfilación de la Expresión Génica , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/metabolismo , Pronóstico , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Tasa de Supervivencia , Uniones Estrechas/genética , Uniones Estrechas/metabolismo , Células Tumorales CultivadasRESUMEN
Toluene diisocyanate (TDI) is a reactive chemical used in manufacturing plastics. TDI exposure adversely affects workers' health, causing occupational asthma, but individuals differ in susceptibility. We recently suggested a role for signalling mediated by the enzyme autotaxin (ATX) and its product, lysophosphatidic acid (LPA), in TDI toxicity. Here we genotyped 118 TDI-exposed workers for six single-nucleotide polymorphisms (SNPs) in genes encoding proteins implicated in ATX-LPA signalling: purinergic receptor P2X7 (P2RX7), CC motif chemokine ligand 2 (CCL2), interleukin 1ß (IL1B), and caveolin 1 (CAV1). Two P2RX7 SNPs (rs208294 and rs2230911) significantly modified the associations between a biomarker of TDI exposure (urinary 2,4-toluene diamine) and plasma LPA; two IL1B SNPs (rs16944 and rs1143634) did not. CAV1 rs3807989 modified the associations, but the effect was not statistically significant (pâ¯=â¯0.05-0.09). In vitro, TDI-exposed bronchial epithelial cells (16HBE14o-) rapidly released ATX and IL-1ß. P2X7 inhibitors attenuated both responses, but confocal microscopy showed non-overlapping localizations of ATX and IL-1ß, and down-regulation of CAV1 inhibited the ATX response but not the IL-1ß response. This study indicates that P2X7 is pivotal for TDI-induced ATX-LPA signalling, which was modified by genetic variation in P2RX7. Furthermore, our data suggest that the TDI-induced ATX and IL-1ß responses occur independently.
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Lisofosfolípidos/metabolismo , Hidrolasas Diéster Fosfóricas/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , 2,4-Diisocianato de Tolueno/toxicidad , Adolescente , Adulto , Biomarcadores , Caveolina 1/efectos de los fármacos , Caveolina 1/genética , Línea Celular , Industria Química , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Exposición Profesional/efectos adversos , Hidrolasas Diéster Fosfóricas/genética , Polimorfismo de Nucleótido Simple/genética , ARN Interferente Pequeño/farmacología , Receptores Purinérgicos P2X7/efectos de los fármacos , Receptores Purinérgicos P2X7/genética , Transducción de Señal/genética , Adulto JovenRESUMEN
Hepatocellular carcinoma (HCC) is rated as the fifth most common malignancy and third in cancer-related deaths worldwide. Statins, HMG-CoA reductase inhibitors, are potent cholesterol-lowering drugs, and recent epidemiologic evidence suggests that statins prevent aggressive HCC development. Previous experiments revealed that statins downregulate phosphorylated Akt (pAkt). Here, it is demonstrated that atorvastatin decreases nuclear pAkt levels in pancreatic and lung cancer cell lines within minutes, and this rapid effect is mediated by the purinergic P2X receptors. Akt is upregulated by hepatitis viruses and has oncogenic activity in HCC; therefore, we tested the possibility that the P2X-Akt pathway is important for the anticipated anticancer effects of statins in hepatocytes. Atorvastatin decreased hepatitis B virus X protein- and insulin-induced pAkt and pGsk3ß (Ser9) levels. Furthermore, Akt-induced lipogenesis was counteracted by atorvastatin, and these statin-induced effects were dependent on P2X receptors. Statin also decreased proliferation and invasiveness of hepatocytes. These data provide mechanistic evidence for a P2X receptor-dependent signaling pathway by which statins decrease pAkt, its downstream phosphorylation target pGsk3ß, and lipogenesis in hepatocytes.Implications: The Akt pathway is deregulated and may act as a driver in HCC development; the P2X-Akt signaling pathway may have a role in anticancer effects of statins. Mol Cancer Res; 15(6); 714-22. ©2017 AACR.
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Atorvastatina/farmacología , Hepatocitos/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores Purinérgicos P2X/metabolismo , Proliferación Celular/efectos de los fármacos , Células Hep G2 , Hepatocitos/metabolismo , Humanos , Insulina/metabolismo , Insulina/farmacología , Fosforilación/efectos de los fármacos , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Proteínas Reguladoras y Accesorias ViralesRESUMEN
Diisocyanates are industrial chemicals which have a wide range of applications in developed and developing countries. They are notorious lung toxicants and respiratory sensitizers. However, the mechanisms behind their adverse effects are not adequately characterized. Autotaxin (ATX) is an enzyme producing lysophosphatidic acid (LPA), and the ATX-LPA axis has been implicated in lung related inflammatory conditions and diseases, including allergic asthma, but not to toxicity of environmental low-molecular-weight chemicals. We investigated effects of toluene diisocyanate (TDI) on ATX induction in human lung epithelial cell models, and we correlated LPA-levels in plasma to biomarkers of TDI exposure in urine collected from workers exposed to <5ppb (parts per billion). Information on workers' symptoms was collected through interviews. One nanomolar TDI robustly induced ATX release within 10min in vitro. A P2X7- and P2X4-dependent microvesicle formation was implicated in a rapid ATX release and a subsequent protein synthesis. Co-localization between purinergic receptors and ATX was documented by immunofluorescence and confocal microscopy. The release was modulated by monocyte chemoattractant protein-1 (MCP-1) and by extracellular ATP. In workers, we found a dose-response relationship between TDI exposure biomarkers in urine and LPA levels in plasma. Among symptomatic workers reporting "sneezing", the LPA levels were higher than among non-symptomatic workers. This is the first report indicating induction of the ATX-LPA axis by an environmental low-molecular-weight chemical, and our data suggest a role for the ATX-LPA axis in TDI toxicity.
Asunto(s)
Enfermedades Pulmonares/inducido químicamente , Pulmón/efectos de los fármacos , Lisofosfolípidos/metabolismo , Enfermedades Profesionales/inducido químicamente , Hidrolasas Diéster Fosfóricas/biosíntesis , 2,4-Diisocianato de Tolueno/efectos adversos , Adulto , Biomarcadores/sangre , Biomarcadores/orina , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Inducción Enzimática , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Femenino , Humanos , Pulmón/enzimología , Pulmón/fisiopatología , Enfermedades Pulmonares/diagnóstico , Enfermedades Pulmonares/metabolismo , Lisofosfolípidos/sangre , Lisofosfolípidos/orina , Masculino , Persona de Mediana Edad , Enfermedades Profesionales/diagnóstico , Enfermedades Profesionales/metabolismo , Exposición Profesional/efectos adversos , Interferencia de ARN , Receptores Purinérgicos P2X4/efectos de los fármacos , Receptores Purinérgicos P2X4/metabolismo , Receptores Purinérgicos P2X7/efectos de los fármacos , Receptores Purinérgicos P2X7/metabolismo , Medición de Riesgo , Transducción de Señal/efectos de los fármacos , Suecia , Factores de Tiempo , Transfección , Regulación hacia Arriba , Adulto JovenRESUMEN
Akt kinase controls cell survival, proliferation, and invasive growth and is a critical factor for cancer development. Here we describe a cross-talk between phosphatases that may preserve levels of activated/phosphorylated Akt and confer aggressive growth of cancer cells. In prostatic cancer cells, but not in non-transformed cells or in prostate stem cells, we found that the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) overexpression down-regulated PH domain and leucine-rich repeat phosphatase (PHLPP) and that PHLPP overexpression down-regulated PTEN. We also show that silencing PTEN by siRNA increased the levels of PHLPPs. This cross-talk facilitated invasive migration and was mediated by epigenetic alterations, including activation of miR-190, miR-214, polycomb group of proteins, as well as DNA methylation. A role for the purinergic receptor P2X4, previously associated with wound healing, was indicated. We also show that TGF-ß1 induced cross-talk concomitant with epithelial-mesenchymal transition in stem cells. The cross-talk emerged as an integrated part of epithelial-mesenchymal transition. We conclude that cross-talk between PTEN and PHLPPs is silenced in normal prostate cells but activated in TGF-ß1 transformed prostate stem and cancer cells and facilitates invasive growth.
Asunto(s)
Proteínas Nucleares/metabolismo , Fosfohidrolasa PTEN/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Neoplasias de la Próstata/metabolismo , Células Madre/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Secuencia de Bases , Línea Celular Tumoral , Cartilla de ADN , Epigénesis Genética , Humanos , Masculino , Neoplasias de la Próstata/patología , Ratas , Ratas Endogámicas F344 , Reacción en Cadena en Tiempo Real de la Polimerasa , Transcripción GenéticaRESUMEN
Epidemiological studies indicate that statins, cholesterol-lowering drugs, prevent aggressive prostate cancer and other types of cancer. Employing essentially non-prostate cell lines, we previously showed that statins rapidly downregulate nuclear levels of phosphorylated Akt via P2X7, a purinergic receptor recently implicated in invasive growth. Here, we present studies on phosphatase and tensin homolog deleted on chromosome 10 (PTEN)-positive prostatic cells. We document an involvement of EH domain-binding protein 1 (EHBP1), previously associated with aggressive prostate cancer and insulin-stimulated trafficking and cell migration, in P2X7 signaling. We also show that EHBP1 is essential for an anti-invasive effect of atorvastatin. Furthermore, EHBP1 interacted with P-Rex1, a guanine nucleotide exchange factor previously implicated in invasive growth. Mevalonate did not prevent this anti-invasive effect of atorvastatin. These data indicate that atorvastatin modulates invasiveness via P2X7, EHBP1 and P-Rex1. Interestingly, the interaction between EHBP1 and P-Rex1 was not induced by extracellular adenosine triphosphate (ATP), the endogenous P2X7 ligand, and statins counteracted invasiveness stimulated by extracellular ATP. In support of these experimental data, a population-based genetic analysis showed that a loss of function allele in the P2X7 gene (rs3751143) associated with non-aggressive cancer, and the common allele with aggressive cancer. Our data indicate a novel signaling pathway that inhibits invasiveness and that is druggable. Statins may reduce the risk of aggressive prostate cancer via P2X7 and by counteracting invasive effects of extracellular ATP.
Asunto(s)
Adenosina Trifosfato/efectos adversos , Proteínas Portadoras/metabolismo , Ácidos Heptanoicos/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Fosfohidrolasa PTEN/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Pirroles/farmacología , Receptores Purinérgicos P2X7/metabolismo , Atorvastatina , Western Blotting , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Movimiento Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Humanos , Técnicas para Inmunoenzimas , Masculino , Invasividad Neoplásica , Fosfohidrolasa PTEN/genética , Fosforilación/efectos de los fármacos , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Interferente Pequeño/genética , Receptores Purinérgicos P2X7/química , Receptores Purinérgicos P2X7/genética , Transducción de Señal/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
The thiopurine antimetabolites, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are inactive pro-drugs that require intracellular metabolism for activation to cytotoxic metabolites. Thiopurine methyltransferase (TPMT) is one of the most important enzymes in this process metabolizing both 6-MP and 6-TG to different methylated metabolites including methylthioinosine monophosphate (meTIMP) and methylthioguanosine monophosphate (meTGMP), respectively, with different suggested pharmacological and cytotoxic properties. While meTIMP is a potent inhibitor of de novo purine synthesis (DNPS) and significantly contributes to the cytotoxic effects of 6-MP, meTGMP, does not add much to the effects of 6-TG, and the cytotoxicity of 6-TG seems to be more dependent on incorporation of thioguanine nucleotides (TGNs) into DNA rather than inhibition of DNPS. In order to investigate the role of TPMT in metabolism and thus, cytotoxic effects of 6-MP and 6-TG, we knocked down the expression of the gene encoding the TPMT enzyme using specifically designed small interference RNA (siRNA) in human MOLT4 leukemia cells. The knock-down was confirmed at RNA, protein, and enzyme function levels. Apoptosis was determined using annexin V and propidium iodide staining and FACS analysis. The results showed a 34% increase in sensitivity of MOLT4 cells to 1µM 6-TG after treatment with TPMT-targeting siRNA, as compared to cells transfected with non-targeting siRNA, while the sensitivity of the cells toward 6-MP was not affected significantly by down-regulation of the TPMT gene. This differential contribution of the enzyme TPMT to the cytotoxicity of the two thiopurines is probably due to its role in formation of the meTIMP, the cytotoxic methylated metabolite of 6-MP, while in case of 6-TG methylation by TPMT substantially deactivates the drug.