RESUMEN
The progression of normal cells to a tumorigenic and metastatic state involves the accumulation of mutations in multiple key signaling proteins, encoded by oncogenes and tumor suppressor genes. Recently, members of the TRP channel family have been included in the oncogenic and tumor suppressor protein family. TRPM1, TRPM8, and TRPV6 are considered to be tumor suppressors and oncogenes in localized melanoma and prostate cancer, respectively. Herein, we focus our attention on the antioncogenic properties of TRPV1. Changes in TRPV1 expression occur during the development of transitional cell carcinoma (TCC) of human bladder. A progressive decrease in TRPV1 expression as the TCC stage increases triggers the development of a more aggressive gene phenotype and invasiveness. Finally, downregulation of TRPV1 represents a negative prognostic factor in TCC patients. The knowledge of the mechanism controlling TRPV1 expression might improve the diagnosis and new therapeutic strategies in bladder cancer.
RESUMEN
Malignant transformation of cells resulting from enhanced proliferation and aberrant differentiation is often accompanied by changes in transient receptor potential vanilloid (TRPV) channels expression. In gliomas, recent evidence indicates that TRPV type 2 (TRPV2) negatively controls glioma cell survival and proliferation. In addition, cannabinoids, the ligands of both cannabinoid and TRPV2 receptors, promote glioblastoma stem-like cells (GSCs) differentiation and inhibit gliomagenesis. Herein, we provide evidence on the expression of TRPV2 in human GSCs and that GSCs differentiation reduces nestin and progressively increases both the glial fibrillary acidic protein (GFAP) and TRPV2 expression. Therefore, we evaluated the role of TRPV2 cation channel in GSC lines differentiation. Treatment of GSC lines with the TRPV antagonist Ruthenium Red, with ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid or knockdown of TRPV2 gene during differentiation, decreases GFAP and class III beta-tubulin (ß(III)-tubulin) expression; conversely, phorbol-12-myristate-13-acetate stimulates GSCs proliferation, reduces TRPV2 expression and partially reverts astroglial differentiation. In addition, forced TRPV2 expression in GSC lines by stable TRPV2 transfection increases GFAP and ß(III)-tubulin expression and parallelly reduces proliferation. Finally, TRPV2 overexpression inhibits GSCs proliferation in a xenograft mouse model, as shown by reduced tumor diameter and mitotic index, and promotes the differentiation of GSCs toward a more mature glial phenotype. Overall, our results demonstrate that TRPV2 promotes in vitro and in vivo GSCs differentiation and inhibits their proliferation. Better understanding of the molecular mechanisms that regulate the balance between proliferation and differentiation of GSCs would lead to more specific and efficacious pharmacological approaches.
Asunto(s)
Diferenciación Celular/genética , Glioblastoma/genética , Glioblastoma/metabolismo , Células Madre Neoplásicas/citología , Células Madre Neoplásicas/metabolismo , Canales Catiónicos TRPV/genética , Animales , Calcio/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Expresión Génica , Humanos , Ratones , Ratones Desnudos , Ésteres del Forbol/farmacologíaRESUMEN
T helper (T(H) )-17 lymphocytes are characterized by the expression of many regulatory cytokines, including IL-17A and IL-22, but at present no clinical data are available on the expression of these cytokines in peripheral blood mononuclear cells (PBMC) from allergic asthmatic and rhinitic (AR) children. The aim of this study was to investigate a possible relationship between IL-22 and IL-17A mRNAs and clinical parameters in seroatopic, AR, and asthmatic children. The study, conducted during the pollen season, included 18 healthy and 18 allergic (n=9 asthmatic and n=9 rhinitic) children. Serum total and specific IgE, eosinophil count, and skin prick test were performed; in addition, IL-22 and IL-17A mRNA levels were detected in PBMC from healthy and allergic subjects by quantitative real-time PCR. Despite the absence of the mRNA for the IL-17A cytokine, IL-22 expression was found in PBMC from asthmatic patients, with increased IL-22 mRNA levels in patients with chronic severe respect to those with moderate asthma. A positive correlation between IL-22 mRNA and serum total IgE levels was found in asthmatic children. In addition, higher IL-22 and IL-17A mRNA levels were detected in both AR and asymptomatic seroatopic children, compared to healthy individuals, and a correlation between IL-22 and IL17A mRNA and serum total IgE levels was demonstrated. Moreover, the mRNA level of retinoic acid-related orphan receptor C, the T(H) 17 transcription factor, was found to be increased in AR but not in asthmatic patients. This study provides the first evidence that IL-22 mRNA might be expressed in chronic severe asthmatic and AR children. The expression of IL-22 and IL-17A mRNAs in asymptomatic monosensitized seroatopic children suggests a role of these cytokines in the early events involved in the development of these allergic diseases.
Asunto(s)
Asma/diagnóstico , Interleucinas/metabolismo , Leucocitos Mononucleares/metabolismo , Rinitis Alérgica Perenne/diagnóstico , Rinitis Alérgica Estacional/diagnóstico , Asma/inmunología , Asma/fisiopatología , Biomarcadores/metabolismo , Niño , Progresión de la Enfermedad , Femenino , Humanos , Inmunoglobulina E/sangre , Interleucina-17/genética , Interleucina-17/inmunología , Interleucina-17/metabolismo , Interleucinas/genética , Interleucinas/inmunología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/patología , Masculino , Rinitis Alérgica Perenne/inmunología , Rinitis Alérgica Perenne/fisiopatología , Rinitis Alérgica Estacional/inmunología , Rinitis Alérgica Estacional/fisiopatología , Balance Th1 - Th2 , Interleucina-22RESUMEN
Capsaicin (CPS) has been found to exhibit either tumor promoting or suppressing effects, many of which are mediated by the specific transient receptor potential vanilloid type-1 (TRPV1). Herein, we provide evidence that CPS treatment induced a more aggressive gene phenotype and invasiveness in 5637 cells-lacking TRPV1 receptor. CPS treatment of 5637 cells induced upregulation of pro-angiogenetic (angiopoietin 1, angiopoietin 2 and vascular endothelial growth factor), pro-invasive and pro-metastatic genes (MMP1, MMP9, TIMP1, TIMP3, granzyme A (GZMA), NM23A and S100A) with a downregulation of apoptotic genes (Fas/CD95 and tumor necrosis factor receptor superfamily member 1A). CPS increased the invasiveness of 5637 cells by triggering IGF (insulin-like growth factor)-1 release, GZMA and MMP9 activation, α-tubulin disassembly and cytoskeleton degradation. Finally, in order to evaluate the relationship between the lack of TRPV1 expression and increased CPS-induced invasiveness, we transfected 5637 cells with the TRPV1 complementary DNA (cDNA) sequence. We found that TRPV1-expressing cells show CPS-mediated calcium level increase, growth inhibition and apoptosis. Moreover, CPS-induced migration and MMP9 activation were reverted, suggesting an inhibitory role played by TRPV1 in urothelial cancer cell invasion and metastasis.
Asunto(s)
Capsaicina/farmacología , Expresión Génica/efectos de los fármacos , Metaloproteinasa 9 de la Matriz/metabolismo , Canales Catiónicos TRPV/genética , Neoplasias Urológicas/patología , Apoptosis/efectos de los fármacos , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Western Blotting , Calcio/metabolismo , Carcinoma Papilar/genética , Carcinoma Papilar/patología , Adhesión Celular/efectos de los fármacos , Línea Celular , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Humanos , Linfocitos Nulos , Metaloproteinasa 9 de la Matriz/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Fármacos del Sistema Sensorial/farmacología , Transducción de Señal , Canales Catiónicos TRPV/metabolismo , Neoplasias Urológicas/genética , Urotelio/metabolismoRESUMEN
AIMS: To evaluate the expression of transient receptor potential vanilloid type-1 channel protein (TRPV1) in normal and neoplastic urothelial tissues and to correlate TRPV1 expression with clinicopathological parameters and disease-specific survival. METHODS AND RESULTS: TRPV1 expression was analysed in normal and neoplastic urothelial samples at both mRNA and protein levels by quantitative real time polymerase chain reaction (qPCR) and immunohistochemistry, respectively. TRPV1 downregulation was found in urothelial cancer (UC) specimens, which correlated with tumour progression. Moreover, TRPV1 mRNA levels were associated with clinicopathological parameters to assess the role of TRPV1 downregulation as a negative prognostic factor for survival. Kaplan-Meier survival analysis demonstrated a significantly shorter survival in patients showing TRPV1 mRNA downregulation. Multivariate Cox regression analysis indicated further that TRPV1 mRNA expression retained its significance as an independent risk factor. CONCLUSIONS: The progression of UC of human bladder is associated with a marked decrease in TRPV1 expression, with a progressive loss in high-grade muscle invasive UC. Downregulation of TRPV1 mRNA expression may represent an independent negative prognostic factor for bladder cancer patients.
Asunto(s)
Canales Catiónicos TRPV/metabolismo , Neoplasias de la Vejiga Urinaria/patología , Vejiga Urinaria/patología , Adulto , Anciano , Anciano de 80 o más Años , Progresión de la Enfermedad , Femenino , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Estadificación de Neoplasias , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Canales Catiónicos TRPV/genética , Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/mortalidad , Urotelio/metabolismo , Urotelio/patologíaRESUMEN
The aim of this study was to investigate the expression and function of the transient receptor potential vanilloid 2 (TRPV2) in human glioma cells. By Real-Time-PCR and western blot analysis, we found that TRPV2 messenger RNA (mRNA) and protein were expressed in benign astrocyte tissues, and its expression progressively declined in high-grade glioma tissues as histological grade increased (n = 49 cases), and in U87MG cells and in MZC, FCL and FSL primary glioma cells. To investigate the function of TRPV2 in glioma, small RNA interfering was used to silence TRPV2 expression in U87MG cells. As evaluated by RT-Profiler PCR array, siTRPV2-U87MG transfected cells displayed a marked downregulation of Fas and procaspase-8 mRNA expression, associated with upregulation of cyclin E1, cyclin-dependent kinase 2, E2F1 transcriptor factor 1, V-raf-1 murine leukemia viral oncogene homolog 1 and Bcl-2-associated X protein (Bcl-X(L)) mRNA expression. TRPV2 silencing increased U87MG cell proliferation as shown by the increased percentage of cells incorporating 5-bromo-2-deoxyuridine expressing beta(III)-tubulin and rescued glioma cells to Fas-induced apoptosis. These events were dependent on extracellular signal-regulated kinase (ERK) activation: indeed inhibition of ERK activation in siTRPV2-U87MG transfected cells by treatment with PD98059, a specific mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor, reduced Bcl-X(L) protein levels, promoted Fas expression, and restored Akt/protein kinase B pathway activation leading to reduced U87MG cell survival and proliferation, and increased sensitivity to Fas-induced apoptosis. In addition, transfection of TRPV2 in MZC glioma cells, by inducing Fas overexpression, resulted in a reduced viability and an increased spontaneous and Fas-induced apoptosis. Overall, our findings indicate that TRPV2 negatively controls glioma cell survival and proliferation, as well as resistance to Fas-induced apoptotic cell death in an ERK-dependent manner.
Asunto(s)
Apoptosis , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Glioma/patología , Canales Catiónicos TRPV/fisiología , Receptor fas/fisiología , Línea Celular Tumoral , Proliferación Celular , Flavonoides/farmacología , Glioma/tratamiento farmacológico , Humanos , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/análisis , Canales Catiónicos TRPV/análisis , Canales Catiónicos TRPV/antagonistas & inhibidores , Canales Catiónicos TRPV/genética , Proteína bcl-X/análisisRESUMEN
Herein, we provide evidence on the expression of transient receptor potential vanilloid type 1 (TRPV1) on human urothelial cancer (UC) cells and its involvement in the apoptosis induced by the selective agonist capsaicin (CPS). We analyzed TRPV1 messenger RNA and protein expression on human UC cell lines demonstrating its progressive decrease in high-grade UC cells. Treatment of RT4 cells with CPS induced cell cycle arrest in G(0)/G(1) phase and apoptosis. These events were associated with rapid co-ordinated transcription of pro-apoptotic genes including Fas/CD95, Bcl-2 and caspase families and ataxia telangiectasia mutated (ATM)/CHK2/p53 DNA damage response pathway. CPS induced Fas/CD95 upregulation, but more importantly Fas/CD95 ligand independent, TRPV1-dependent death receptor clustering and triggering of both extrinsic and intrinsic mitochondrial-dependent pathways. Moreover, we observed that CPS activates ATM kinase that is involved in Ser15, Ser20 and Ser392 p53 phosphorylation as shown by the use of the specific inhibitor KU55933. Notably, ATM activation was also found to control upregulation of Fas/CD95 expression and its co-clustering with TRPV1 as well as RT4 cell growth and apoptosis. Altogether, we describe a novel connection between ATM DNA damage response pathway and Fas/CD95-mediated intrinsic and extrinsic apoptotic pathways triggered by TRPV1 stimulation on UC cells.
Asunto(s)
Apoptosis/efectos de los fármacos , Capsaicina/farmacología , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Canales Catiónicos TRPV/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Neoplasias de la Vejiga Urinaria/patología , Receptor fas/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada , Western Blotting , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Invasividad Neoplásica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Fármacos del Sistema Sensorial/farmacología , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/metabolismo , Urotelio/efectos de los fármacos , Urotelio/metabolismoRESUMEN
OBJECTIVE: To evaluate the expression of transient receptor potential vanilloid type 2 (TRPV2) in normal human bladder and urothelial carcinoma (UC) tissues. METHODS: Bladder specimens were obtained by transurethral resection or radical cystectomy. TRPV2 mRNA expression in normal human urothelial cells (NHUCs), UC cell lines, and formalin-fixed paraffin-embedded normal (n=6) and cancer bladder tissues (n=58) was evaluated by polymerase chain reaction (PCR) and quantitative real-time PCR (RT-PCR). TRPV2 protein expression was assessed by cytofluorimetric and confocal microscopy analyses in NHUCs and UC cells and by Western blotting and immunohistochemistry in normal and UC tissues. RESULTS: Enhanced TRPV2 mRNA and protein expression was found in high-grade and -stage UC specimens and UC cell lines. Both the full-length TRPV2 (hTRPV2) and a short splice-variant (s-TRPV2) were detected in NHUC and normal bladder specimens, whereas a progressive decline of s-TRPV2 in pTa, pT1, and pT2 stages was observed, up to a complete loss in pT3 and pT4 UC specimens. CONCLUSIONS: Normal human urothelial cells and bladder tissue specimens express TRPV2 at both the mRNA and protein levels. A progressive loss of s-TRPV2 accompanied by a marked increase of hTRPV2 expression was found in high-grade and -stage UC tissues.
Asunto(s)
Carcinoma de Células Transicionales/genética , Carcinoma de Células Transicionales/patología , Canales Catiónicos TRPV/metabolismo , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/patología , Urotelio/metabolismo , Urotelio/patología , Análisis de Varianza , Biomarcadores de Tumor/análisis , Western Blotting , Carcinoma de Células Transicionales/metabolismo , Distribución de Chi-Cuadrado , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Microscopía Confocal , Estadificación de Neoplasias , Pronóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias de la Vejiga Urinaria/metabolismoRESUMEN
Acute promyelocytic leukemia (APL) is a human cancer generated by a chromosomal translocation t(15;17) involving the promyelocytic leukemia (PML) and retinoic acid receptor alpha (RARalpha) genes. The PML/RARalpha oncoprotein expressing blasts show two of the most important biological features of neoplastic progression: block of differentiation, at the promyelocytic state, and increased survival. Although PML/RARalpha interferes with the normal maturation of myeloid precursors to granulocytes, pharmacological doses of retinoic acid are sufficient to restore the differentiation processes. We designed an assay based on the Real-Time reverse transcriptase polymerase chain reaction (RT-PCR) to experimentally follow the differentiation response of leukemic cells even after short-time differentiating treatments. Amplifying CD11b, CD11c, and CD14 mRNAs, as specific markers of differentiation, by the real-time RT-PCR assay we could detect both retinoic acid (RA) and vitamin D3 and human transforming growth factor beta1 (VitD3/TGFbeta1) induced cellular maturation more precociously than the canonical flow-cytofluorimetric assay. Moreover, by amplifying CD14 mRNA it was possible to monitor the ability of PML/RARalpha oncoprotein to block VitD3/TGFbeta1 induced differentiation in U937-PR9 promonocytic inducible model systems.The proposed real-time quantitative RT-PCR approach is a reproducible and highly sensitive assay and can be considered a valid method to study both cellular maturation state and differentiation response.
Asunto(s)
Leucemia Promielocítica Aguda/genética , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Receptores de Ácido Retinoico/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/genética , Diferenciación Celular , Línea Celular Tumoral , Colecalciferol/farmacología , Humanos , Leucemia Promielocítica Aguda/patología , Proteína de la Leucemia Promielocítica , Proteínas/farmacología , Receptor alfa de Ácido Retinoico , Tretinoina/farmacologíaRESUMEN
Acute promyelocytic leukemia (APL) is a subtype of myeloid leukemia characterized by the chromosomal translocation t(15:17) that leads to the expression of promyelocytic leukemia/retinoic acid receptor-alpha (PML/ RARalpha) oncofusion protein. The block of differentiation at the promyelocytic stage of the blasts and their increased survival induced by PML/RARalpha are the principal biological features of the disease. Therapies based on pharmacological doses of retinoic acid (RA, 10(-6) M) are able to restore APL cell differentiation in most cases, but not to achieve complete hematological remission because retinoic acid resistance occurs in many patients. In order to elaborate alternative therapeutic approaches, we focused our attention on the use of antisense oligonucleotides as gene-specific drug directed to PML/RARalpha mRNA target. We used antisense molecules containing multiple locked nucleic acid (LNA) modifications. The LNAs are nucleotide analogues that are able to form duplexes with complementary DNA or RNA sequences with highly increased thermal stability and are resistant to 3'-exonuclease degradation in vitro. The DNA/LNA chimeric molecules were designed on the fusion sequence of PML and RARalpha genes to specifically target the oncofusion protein. Cell-free and in vitro experiments using U937-PR9-inducible cell line showed that DNA/LNA oligonucleotides were able to interfere with PML/RARalpha expression more efficiently than the corresponding unmodified DNA oligo. Moreover, the treatment of U937-PR9 cells with these chimeric antisense molecules was able to abrogate the block of differentiation induced by PML/RARalpha oncoprotein. These data suggest a possible application of oligonucleotides containing LNA in an antisense therapeutic strategy for APL.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Leucemia Promielocítica Aguda/tratamiento farmacológico , Oligonucleótidos Antisentido/química , Proteínas Recombinantes de Fusión/química , Translocación Genética , Diferenciación Celular , Humanos , Leucemia Promielocítica Aguda/genética , Liposomas/química , Ácidos Nucleicos/química , Oligonucleótidos/química , Proteínas de Fusión Oncogénica/química , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Transfección , Células U937RESUMEN
Mammalian homologues of Drosophila Seven in Absentia (SIAHs) target for proteasome-mediated degradation several factors involved in cell growth and tumorigenesis. Here we show that SIAH-1/2 binds and targets for proteasome-mediated degradation the putative tumor suppressor and tripartite motif (TRIM) family member PML, leading to the loss of its transcriptional co-activating properties and a reduction in the number of endogenous PML nuclear bodies. Association with PML requires the substrate-binding domain (SBD) of SIAH-1/2 through an interacting surface apparently distinct from those predicted by the structural studies, or shown experimentally to mediate binding to SIAH-associated factors. Within PML, the coiled-coil domain is required for Siah- and proteasome-mediated degradation, and deletions of regions critical for the integrity of this region impair the ability of Siah to trigger PML-RAR degradation. Fusion of the coiled-coil domain to heterologous proteins resulted in the capacity of mSiah-2 to target their degradation. All of the TRIM proteins tested were degraded upon mSiah-2 overexpression. Finally, we show that the fusion protein PML-RAR (that retains the coiled-coil domain), which causes acute promyelocytic leukemias, is also a potential substrate of mSiah-2. As a result of mSiah-2 overexpression and subsequent degradation of the fusion protein, the arrest in hematopoietic differentiation because of expression of PML-RAR is partially rescued. These results identify PML and other TRIMs as new factors post-translationally regulated by SIAH and involve the coiled-coil region of PML and of other SIAH substrates as a novel structural determinant for targeted degradation.