RESUMO
The Notch pathway plays a key role in the development and is increasingly recognized for its importance in cancer. We demonstrated previously the overexpression of Notch-1 and its ligands in gliomas and showed that their knockdown inhibits glioma cell proliferation and survival. To elucidate the mechanisms downstream of Notch-1 in glioma cells, we performed microarray profiling of glioma cells transfected with Notch-1 small interfering RNA. Notable among downregulated transcripts was the epidermal growth factor receptor (EGFR), known to be overexpressed or amplified in gliomas and prominent in other cancers as well. Further studies confirmed that Notch-1 inhibition decreased EGFR messenger RNA (mRNA) and EGFR protein in glioma and other cell lines. Transfection with Notch-1 increased EGFR expression. Additionally, we found a significant correlation in levels of EGFR and Notch-1 mRNA in primary high-grade human gliomas. Subsequent experiments showed that p53, an activator of the EGFR promoter, is regulated by Notch-1. Experiments with p53-positive and -null cell lines confirmed that p53 partially mediates the effects of Notch-1 on EGFR expression. These results show for the first time that Notch-1 upregulates EGFR expression and also demonstrate Notch-1 regulation of p53 in gliomas. These observations have significant implications for understanding the mechanisms of Notch in cancer and development.
Assuntos
Receptores ErbB/genética , Regulação da Expressão Gênica , Glioma/genética , Receptor Notch1/fisiologia , Transcrição Gênica , Proteína Supressora de Tumor p53/genética , Biópsia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Genes Reporter , Genes p53 , Glioma/patologia , Humanos , Luciferases/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Receptor Notch1/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
The aim of this work was to study the effect of a sustained activation of AMP-activated protein kinase (AMPK) on liver cell survival. AMPK activation was achieved by incubating FTO2B cells with AICA-riboside, which is transformed into ZMP, an AMP analogue, or by adenoviral transfection of hepatocytes with a constitutively active form of AMPK. Prolonged AMPK activation triggered apoptosis and activated c-Jun N-terminal kinase (JNK) and caspase-3. Experiments with iodotubercidin, dicoumarol and z-VAD-fmk, which inhibited AMPK, JNK and caspase activation, respectively, supported the notion that prolonged AMPK activation in liver cells induces apoptosis through an activation pathway that involves JNK and caspase-3.
Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Apoptose/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fígado/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Aminoimidazol Carboxamida/metabolismo , Animais , Linhagem Celular , Ativação Enzimática , Proteínas Quinases JNK Ativadas por Mitógeno , Fígado/citologia , Fígado/enzimologia , Proteínas Recombinantes/metabolismo , Ribonucleotídeos/metabolismo , TransfecçãoRESUMO
Metformin is an anti-diabetic drug that increases glucose utilization in insulin-sensitive tissues. The effect is in part attributable to a stimulation of AMP-activated protein kinase (AMPK). The present study demonstrates that metformin (0.5-2mM) also dose-dependently activates AMPK in insulin-producing MIN6 cells and in primary rat beta-cells, leading to increased phosphorylation of acetyl coA carboxylase (ACC). The maximal effect was reached within 12h and sustained up to 48h. After 24h exposure to metformin (0.5-1mM), rat beta-cells exhibited a reduced secretory and synthetic responsiveness to 10mM glucose, which was also the case following 24h culture with the AMPK-activator 5-amino-imidazole-4-carboxamide riboside (AICAR; 1mM). Longer metformin exposure (>24h) resulted in a progressive increase in apoptotic beta-cells as was also reported for AICAR; metformin-induced apoptosis was reduced by compound C, an AMPK-inhibitor. As with AICAR, metformin activated c-Jun-N-terminal kinase (JNK) and caspase-3 prior to the appearance of apoptosis. It is concluded that metformin-induced AMPK-activation in beta-cells reduces their glucose responsiveness and may, following sustained exposure, result in apoptosis.
Assuntos
Apoptose , Glucose/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Metformina/farmacologia , Complexos Multienzimáticos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Ativadas por AMP , Animais , Ativação Enzimática/efeitos dos fármacos , Feminino , Hipoglicemiantes/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/enzimologia , Proteínas Quinases JNK Ativadas por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Ratos , Ratos WistarRESUMO
Although diacylglycerol kinase α (DGKα) has been linked to several signaling pathways related to cancer cell biology, it has been neglected as a target for cancer therapy. The attenuation of DGKα activity via DGKα-targeting siRNA and small-molecule inhibitors R59022 and R59949 induced caspase-mediated apoptosis in glioblastoma cells and in other cancers, but lacked toxicity in noncancerous cells. We determined that mTOR and hypoxia-inducible factor-1α (HIF-1α) are key targets of DGKα inhibition, in addition to its regulation of other oncogenes. DGKα regulates mTOR transcription via a unique pathway involving cyclic AMP. Finally, we showed the efficacy of DGKα inhibition with short hairpin RNA or a small-molecule agent in glioblastoma and melanoma xenograft treatment models, with growth delay and decreased vascularity. This study establishes DGKα as a central signaling hub and a promising therapeutic target in the treatment of cancer.