RESUMEN
BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) has poor survival and treatment options. PDAC cells shift their metabolism towards glycolysis, which fuels the plasma membrane calcium pump (PMCA), thereby preventing Ca2+-dependent cell death. The ATP-generating pyruvate kinase-M2 (PKM2) is oncogenic and overexpressed in PDAC. This study investigated the PKM2-derived ATP supply to the PMCA as a potential therapeutic locus. METHODS: PDAC cell growth, migration and death were assessed by using sulforhodamine-B/tetrazolium-based assays, gap closure assay and poly-ADP ribose polymerase (PARP1) cleavage, respectively. Cellular ATP and metabolism were assessed using luciferase/fluorescent-based assays and the Seahorse XFe96 analyzer, respectively. Cell surface biotinylation identified membrane-associated proteins. Fura-2 imaging was used to assess cytosolic Ca2+ overload and in situ Ca2+ clearance. PKM2 knockdown was achieved using siRNA. RESULTS: The PKM2 inhibitor (shikonin) reduced PDAC cell proliferation, cell migration and induced cell death. This was due to inhibition of glycolysis, ATP depletion, inhibition of PMCA and cytotoxic Ca2+ overload. PKM2 associates with plasma membrane proteins providing a privileged ATP supply to the PMCA. PKM2 knockdown reduced PMCA activity and reduced the sensitivity of shikonin-induced cell death. CONCLUSIONS: Cutting off the PKM2-derived ATP supply to the PMCA represents a novel therapeutic strategy for the treatment of PDAC.
Asunto(s)
Carcinoma Ductal Pancreático/tratamiento farmacológico , Proteínas Portadoras/genética , Proliferación Celular/genética , Proteínas de la Membrana/genética , Neoplasias Pancreáticas/tratamiento farmacológico , Hormonas Tiroideas/genética , Adenosina Trifosfato/metabolismo , Calcio/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Proteínas Portadoras/antagonistas & inhibidores , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Citosol/efectos de los fármacos , Glucólisis/efectos de los fármacos , Humanos , Proteínas de la Membrana/antagonistas & inhibidores , Naftoquinonas/farmacología , Páncreas/efectos de los fármacos , Páncreas/metabolismo , Páncreas/patología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Proteínas de Unión a Hormona TiroideRESUMEN
OBJECTIVE: The role of C-type lectin receptor, a type of pattern recognition receptor, in otitis media with effusion (OME) is unclear. We assayed the levels of expression of C-type lectin receptor mRNA in children with OME and evaluated its relationship to the presence of bacteria, accompanying diseases, and characteristics of exudates. SUBJECTS AND METHODS: The study population consisted of 73 children with OME who had undergone ventilating tube insertion. The levels of expression of Dectin-1, MR1, MR2, DC-SIGN, Syk, Card-9, Bcl-10, Malt-1, Src, Dec-205, Galectin-1, Tim-3, Trem-1, and DAP-12 mRNA in middle ear effusion were determined by real-time PCR. The level of expression of each mRNA was correlated with the presence of bacteria, accompanying diseases, and exudates characteristics. RESULTS: The levels of expression of C-type lectin receptor mRNAs were not associated with bacterial presence or exudates characteristics (p>0.05 each). Levels of expression, however, were significantly higher in patients with sinusitis, adenoid vegetation or adenoiditis, and allergic rhinitis (p<0.05 each). CONCLUSIONS: Levels of expression of C-type lectin receptor mRNA may be associated with the pathogenesis of OME, being significantly higher in patients with than without accompanying sinusitis, adenoid vegetation or adenoiditis, and allergic rhinitis.
