RESUMO
Autophagy is an evolutionarily preserved degradation process of cytoplasmic cellular constituents and plays important physiological roles in human health and disease. It has been proposed that autophagy plays an important role both in tumor progression and in promotion of cancer cell death, although the molecular mechanisms responsible for this dual action of autophagy in cancer have not been elucidated. Pancreatic ductal adenocarcinoma is one of the most aggressive human malignancies with 2-3% five-year survival rate. Its poor prognosis has been attributed to the lack of specific symptoms and early detection tools, and its relatively refractory to traditional cytotoxic agents and radiotherapy. Experimental evidence pointed at autophagy as a pancreatic cancer cell mechanism to survive under adverse environmental conditions, or as a defective programmed cell death mechanism that favors pancreatic cancer cell resistance to treatment. Here, we consider several phenotypical alterations that have been related to increase or decrease the autophagic process in pancreatic tumor cells. We specially review autophagy as a cell death mechanism in response to chemotherapeutic drugs.
Assuntos
Autofagia , Carcinoma Ductal Pancreático/patologia , Neoplasias Pancreáticas/patologia , Animais , Autofagia/efeitos dos fármacos , Capecitabina , Linhagem Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Fluoruracila/análogos & derivados , Fluoruracila/uso terapêutico , Humanos , Prognóstico , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/fisiologia , GencitabinaRESUMO
Autophagy has recently elicited significant attention as a mechanism that either protects or promotes cell death, although different autophagy pathways, and the cellular context in which they occur, remain to be elucidated. We report a thorough cellular and biochemical characterization of a novel selective autophagy that works as a protective cell response. This new selective autophagy is activated in pancreatic acinar cells during pancreatitis-induced vesicular transport alteration to sequester and degrade potentially deleterious activated zymogen granules. We have coined the term "zymophagy" to refer to this process. The autophagy-related protein VMP1, the ubiquitin-protease USP9x, and the ubiquitin-binding protein p62 mediate zymophagy. Moreover, VMP1 interacts with USP9x, indicating that there is a close cooperation between the autophagy pathway and the ubiquitin recognition machinery required for selective autophagosome formation. Zymophagy is activated by experimental pancreatitis in genetically engineered mice and cultured pancreatic acinar cells and by acute pancreatitis in humans. Furthermore, zymophagy has pathophysiological relevance by controlling pancreatitis-induced intracellular zymogen activation and helping to prevent cell death. Together, these data reveal a novel selective form of autophagy mediated by the VMP1-USP9x-p62 pathway, as a cellular protective response.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagia , Endopeptidases/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Membrana/metabolismo , Pâncreas Exócrino/metabolismo , Pancreatite Necrosante Aguda/metabolismo , Ubiquitina Tiolesterase/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linhagem Celular Tumoral , Endopeptidases/genética , Ativação Enzimática/genética , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Proteínas de Choque Térmico/genética , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Pancreatite Necrosante Aguda/genética , Ratos , Proteína Sequestossoma-1 , Ubiquitina Tiolesterase/genéticaRESUMO
BACKGROUND/AIM: Autophagy is a degradation process of cytoplasmic cellular constituents. We have described the vacuole membrane protein-1 (VMP1) whose expression triggers autophagy in mammalian cells. The aim of this study was to analyze the role of autophagy in human pancreatic cancer cell death. METHODS/RESULTS: Here we show that gemcitabine, the standard chemotherapy for pancreatic cancer, induced autophagy in PANC-1 and MIAPaCa-2 cells, as evidenced by the accumulation of acidic vesicular organelles, the recruitment of microtubule-associated protein-1 light chain-3, and electron microscopy. In addition, gemcitabine treatment induced early expression of VMP1 in cancer cells. Gemcitabine also induced apoptosis detected by morphology, annexin V-positive cells, and cleavage of caspase-3. Surprisingly, 3-methyladenine, an autophagy inhibitor, decreased apoptosis in gemcitabine-treated cells, showing that autophagy leads to cancer cell apoptotic death. Finally, VMP1 knockdown decreased autophagy and apoptosis in gemcitabine-treated cancer cells. CONCLUSIONS: The VMP1-autophagy pathway promotes apoptosis in pancreatic cancer cells and mediates gemcitabine-induced cytotoxicity. and IAP.
Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Desoxicitidina/análogos & derivados , Proteínas de Membrana/fisiologia , Neoplasias Pancreáticas/patologia , Adenina/análogos & derivados , Adenina/farmacologia , Caspase 3/metabolismo , Linhagem Celular Tumoral , Desoxicitidina/farmacologia , Técnicas de Silenciamento de Genes , Humanos , Proteínas de Membrana/genética , Neoplasias Pancreáticas/metabolismo , Vacúolos/metabolismo , GencitabinaRESUMO
BACKGROUND/AIMS: We have described VMP1 as a new protein which expression triggers autophagy in mammalian cells. Here we show that experimental diabetes activates VMP1 expression and autophagy in pancreas beta cells as a direct response to streptozotocin (STZ). METHODS: Male Wistar rats were treated with 65 mg/kg STZ and pancreas islets from untreated rats were incubated with 1 mM STZ. RESULTS: RT-PCR analysis shows early VMP1 induction after STZ treatment. In situ hybridization reveals VMP1 mRNA in islet beta cells. Electron microscopy shows chromatin aggregation and autophagy morphology that was confirmed by LC3 expression and LC3-VMP1 co-localization. Apoptotic cell death and the reduction of beta cell pool are evident after 24 h treatment, while VMP1 is still expressed in the remaining cells. VMP1-Beclin1 colocalization in pancreas tissue from STZ-treated rats suggests that VMP1-Beclin1 interaction is involved in the autophagic process activation during experimental diabetes. Results were confirmed using pancreas islets, showing VMP1 expression and autophagy in beta cells as a direct effect of STZ treatment. CONCLUSION: Pancreas beta cells trigger VMP1 expression and autophagy during the early cellular events in response to experimental diabetes.
Assuntos
Autofagia , Diabetes Mellitus Experimental/fisiopatologia , Proteínas de Membrana/biossíntese , Animais , Expressão Gênica/efeitos dos fármacos , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/fisiopatologia , Masculino , Ratos , Ratos Wistar , EstreptozocinaRESUMO
Autophagy is a degradation process of cytoplasmic cellular constituents, which serves as a survival mechanism in starving cells, and it is characterized by sequestration of bulk cytoplasm and organelles in double-membrane vesicles called autophagosomes. Autophagy has been linked to a variety of pathological processes such as neurodegenerative diseases and tumorigenesis, which highlights its biological and medical importance. We have previously characterized the vacuole membrane protein 1 (VMP1) gene, which is highly activated in acute pancreatitis, a disease associated with morphological changes resembling autophagy. Here we show that VMP1 expression triggers autophagy in mammalian cells. VMP1 expression induces the formation of ultrastructural features of autophagy and recruitment of the microtubule-associated protein 1 light-chain 3 (LC3), which is inhibited after treatment with the autophagy inhibitor 3-methiladenine. VMP1 is induced by starvation and rapamycin treatments. Its expression is necessary for autophagy, because VMP1 small interfering RNA inhibits autophagosome formation under both autophagic stimuli. VMP1 is a transmembrane protein that co-localizes with LC3, a marker of the autophagosomes. It interacts with Beclin 1, a mammalian autophagy initiator, through the VMP1-Atg domain, which is essential for autophagosome formation. VMP1 endogenous expression co-localizes with LC3 in pancreas tissue undergoing pancreatitis-induced autophagy. Finally, VMP1 stable expression targeted to pancreas acinar cell in transgenic mice induces autophagosome formation. Our results identify VMP1 as a novel autophagy-related membrane protein involved in the initial steps of the mammalian cell autophagic process.