Downregulation of uPAR and Cathepsin B Induces Apoptosis via Regulation of Bcl-2 and Bax and Inhibition of the PI3K/Akt Pathway in Gliomas

Glioma is the most commonly diagnosed primary brain tumor and is characterized by invasive and infiltrative behavior. uPAR and cathepsin B are known to be overexpressed in high-grade gliomas and are strongly correlated with invasive cancer phenotypes.In the present study, we observed that simultaneous downregulation of uPAR and cathepsin B induces upregulation of some pro-apoptotic genes and suppression of anti-apoptotic genes in human glioma cells. uPAR and cathepsin B (pCU)-downregulated cells exhibited decreases in the Bcl-2/Bax ratio and initiated the collapse of mitochondrial membrane potential. We also observed that the broad caspase inhibitor, Z-Asp-2, 6-dichlorobenzoylmethylketone rescued pCU-induced apoptosis in U251 cells but not in 5310 cells. Immunoblot analysis of caspase-9 immunoprecipitates for Apaf-1 showed that uPAR and cathepsin B knockdown activated apoptosome complex formation in U251 cells. Downregulation of uPAR and cathepsin B also retarded nuclear translocation and interfered with DNA binding activity of CREB in both U251 and 5310 cells. Further western blotting analysis demonstrated that downregulation of uPAR and cathepsin B significantly decreased expression of the signaling molecules p-PDGFR-β, p-PI3K and p-Akt. An increase in the number of TUNEL-positive cells, increased Bax expression, and decreased Bcl-2 expression in nude mice brain tumor sections and brain tissue lysates confirm our in vitro results.In conclusion, RNAi-mediated downregulation of uPAR and cathepsin B initiates caspase-dependent mitochondrial apoptosis in U251 cells and caspase-independent mitochondrial apoptosis in 5310 cells. Thus, targeting uPAR and cathepsin B-mediated signaling using siRNA may serve as a novel therapeutic strategy for the treatment of gliomas

Frequent reduced expression of alpha-1B-adrenergic receptor caused by aberrant promoter methylation in gastric cancers

Recent studies have suggested that epigenetic inactivation of tumour-related genes by promoter methylation participates in the development of gastric cancer. We newly identified the frequently aberrant promoter methylation of alpha-1B-adrenergic receptor (ADRA1B) in colorectal cancer by methylation-sensitive representational difference analysis (MS-RDA) and examined the methylation status of the ADRA1B promoter in 34 paired samples of colorectal cancer and surrounding epithelial tissue, and 34 paired samples of gastric cancer and surrounding epithelial tissue. In colorectal cancers, only four of 34 (11.8%) tumours showed ADRA1B promoter methylation. In contrast, ADRA1B promoter methylation was detected in 24 of 34 (70.6%) gastric cancers and in 14 of 34 (41.2%) surrounding epithelial tissues. The frequency of ADRA1B promoter methylation was higher in gastric epithelial tissues with intestinal metaplasia (41.6%) than in those without intestinal metaplasia (25.0%). Reverse transcription–PCR detected reduced ADRA1B expression in 12 of 18 (66.7%) gastric cancers, and its promoter methylation was detected in 11 of these 12 (91.7%) gastric cancers with reduced ADRA1B expression. Thus, ADRA1B promoter is frequently methylated in gastric cancer. Our results suggest that the ADRA1B gene is an important tumour-related gene frequently involved in the development and progression of gastric cancer

Expression and function of G-protein-coupled receptorsin the male reproductive tract

This review focuses on the expression and function of muscarinic acetylcholine receptors (mAChRs), α1-adrenoceptors and relaxin receptors in the male reproductive tract. The localization and differential expression of mAChR and α1-adrenoceptor subtypes in specific compartments of the efferent ductules, epididymis, vas deferens, seminal vesicle and prostate of various species indicate a role for these receptors in the modulation of luminal fluid composition and smooth muscle contraction, including effects on male fertility. Furthermore, the activation of mAChRs induces transactivation of the epidermal growth factor receptor (EGFR) and the Sertoli cell proliferation. The relaxin receptors are present in the testis, RXFP1 in elongated spermatids and Sertoli cells from rat, and RXFP2 in Leydig and germ cells from rat and human, suggesting a role for these receptors in the spermatogenic process. The localization of both receptors in the apical portion of epithelial cells and smooth muscle layers of the vas deferens suggests an involvement of these receptors in the contraction and regulation of secretion.Esta revisão enfatiza a expressão e a função dos receptores muscarínicos, adrenoceptores α1 e receptores para relaxina no sistema reprodutor masculino. A expressão dos receptores muscarínicos e adrenoceptores α1 em compartimentos específicos de dúctulos eferentes, epidídimo, ductos deferentes, vesícula seminal e próstata de várias espécies indica o envolvimento destes receptores na modulação da composição do fluido luminal e na contração do músculo liso, incluindo efeitos na fertilidade masculina. Além disso, a ativação dos receptores muscarínicos leva à transativação do receptor para o fator crescimento epidermal e proliferação das células de Sertoli. Os receptores para relaxina estão presentes no testículo, RXFP1 nas espermátides alongadas e células de Sertoli de rato e RXFP2 nas células de Leydig e germinativas de ratos e humano, sugerindo o envolvimento destes receptores no processo espermatogênico. A localização de ambos os receptores na porção apical das células epiteliais e no músculo liso dos ductos deferentes de rato sugere um papel na contração e na regulação da secreção.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de FarmacologiaUNIFESP, EPM, Depto. de FarmacologiaSciEL

Overexpression of the alpha1B-adrenergic receptor causes apoptotic neurodegeneration: multiple system atrophy.

Progress toward elucidating the function of α(1B)-adrenergic receptors (α(1B)ARs) in the central nervous system has been constrained by a lack of agonists and antagonists with adequate α(1B)-specificity. We have obviated this constraint by generating transgenic mice engineered to overexpress either wild-type or constitutively active α(1B)ARs in tissues that normally express the receptor, including the brain. All transgenic lines showed granulovacular neurodegeneration, beginning in α(1B)-expressing domains of the brain and progressing with age to encompass all areas. The degeneration was apoptotic and did not occur in non-transgenic mice. Correspondingly, transgenic mice showed an age-progressive hindlimb disorder that was parkinsonian-like, as demonstrated by rescue of the dysfunction by 3, 4-dihydroxyphenylalanine and considerable dopaminergic-neuronal degeneration in the substantia nigra. Transgenic mice also had a grand mal seizure disorder accompanied by a corresponding dysplasia and neurodegeneration of the cerebral cortex. Both behavioral phenotypes (locomotor impairment and seizure) could be partially rescued with the α1AR antagonist terazosin, indicating that α1AR signaling participated directly in the pathology. Our results indicate that overstimulation of α(1B)AR leads to apoptotic neurodegeneration with a corresponding multiple system atrophy indicative of Shy-Drager syndrome, a disease whose etiology is unknown.</p