RESUMEN
Lipid emulsions are the primary source of calories and fatty acids that are used to provide essential energy and nutrients to patients suffering from severe intestinal failure and critical illness. However, their use has been linked to adverse effects on patient outcomes, notably affecting immune defenses and inflammatory responses. ClinOleic is a lipid emulsion containing a mixture of olive oil and soybean oil (80:20). The effect of ClinOleic on the differentiation of M1 macrophages remains unclear. In this study, we isolated human monocytes and added ClinOleic to differentiation culture media to investigate whether it affects monocyte polarization into M1 macrophages and macrophage functions, such as reactive oxygen species (ROS) production and phagocytosis. ROS production was stimulated by live S. aureus and detected with L-012, a chemiluminescence emission agent. Phagocytic capacity was assayed using pHrodo™ Green S. aureus Bioparticles® Conjugate. We found that M1 cell morphology, surface markers (CD80 and CD86), and M1-associated cytokines (TNF-α and IL-6) did not significantly change upon incubation with ClinOleic during M1 polarization. However, S. aureus-triggered ROS production was significantly lower in M1 macrophages differentiated with ClinOleic than in those not treated with ClinOleic. The inhibitory effect of ClinOleic on macrophage function also appeared in the phagocytosis assay. Taken together, these findings reveal that ClinOleic has a limited impact on the M1 differentiation phenotype but obviously reduces ROS production and phagocytosis.
RESUMEN
Autophagy is a potential target for the treatment of triple negative breast cancer (TNBC). Because of a lack of targeted therapies for TNBC, it is vital to find optimal agents that avoid chemoresistance and metastasis. Flavopereirine has anti-proliferation ability in cancer cells, but whether it regulates autophagy in breast cancer cells remains unclear. A Premo™ Tandem Autophagy Sensor Kit was used to image the stage at which flavopereirine affects autophagy by confocal microscopy. A plasmid that constitutively expresses p-AKT and siRNA targeting p38 mitogen-activated protein kinase (MAPK) was used to confirm the related signaling pathways by Western blot. We found that flavopereirine induced microtubule-associated protein 1 light chain 3 (LC3)-II accumulation in a dose- and time-dependent manner in MDA-MB-231 cells. Confocal florescent images showed that flavopereirine blocked autophagosome fusion with lysosomes. Western blotting showed that flavopereirine directly suppressed p-AKT levels and mammalian target of rapamycin (mTOR) translation. Recovery of AKT phosphorylation decreased the level of p-p38 MAPK and LC3-II, but not mTOR. Moreover, flavopereirine-induced LC3-II accumulation was partially reduced in MDA-MB-231 cells that were transfected with p38 MAPK siRNA. Overall, flavopereirine blocked autophagy via LC3-II accumulation in autophagosomes, which was mediated by the AKT/p38 MAPK signaling pathway.
Asunto(s)
Autofagia/efectos de los fármacos , Neoplasias de la Mama/metabolismo , Carbolinas/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Femenino , HumanosRESUMEN
Breast cancer, which is the most frequently diagnosed cancer, is quite heterogeneous. For breast cancer subtypes lacking targeted therapies, it is vitally essential to find novel agents that prevent chemoresistance and metastatic relapse. Flavopereirine is a ß-carboline alkaloid that has antiplasmodial activity, and its antiproliferative effect in different cancers remains unclear. The effect of flavopereirine on cell cycle arrest and apoptosis signaling in breast cancer cells was analyzed by flow cytometry. An inhibitor and siRNA were used to confirm the related signaling pathways by Western blot analysis. We found that flavopereirine caused G0/G1 phase arrest in MCF-7â¯cells and S phase arrest in MDA-MB-231â¯cells. MDA-MB-231â¯cells were more sensitive to flavopereirine-induced apoptosis. Furthermore, we found that flavopereirine-induced apoptosis was partially reduced in MDA-MB-231â¯cells treated with an extracellular regulated kinase (ERK) inhibitor and p38 mitogen-activated protein kinase (MAPK) siRNA. Moreover, p38 siRNA treatment simultaneously reduced phosphorylated ERK expression levels. Conversely, the recovered phosphorylation of AKT decreased the levels of p-ERK and p-p38 MAPK. Overall, flavopereirine induces cell cycle arrest and the AKT/p38 MAPK/ERK signaling pathway, which contribute to flavopereirine-induced apoptosis in MDA-MB-231â¯cells.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/patología , Carbolinas/farmacología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Células MCF-7 , Mitocondrias/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Neutrophil extracellular traps (NETs) are net-like chromatin fibers that can trap and kill microorganisms. Although several anti-inflammatory effects of intravenous anesthetics have been reported, it has not been investigated whether intravenous anesthetics influence NET formation. AIMS: To compare the effects of four intravenous anesthetics (propofol, thiamylal sodium, midazolam, and ketamine) on phorbol myristate acetate (PMA)-induced NET formation and analyze the associated signaling pathways. MATERIALS AND METHODS: PMA-stimulated NETs formed in the absence or presence of intravenous anesthetics were stained with SYTOX Green and then quantified. Inhibitors were applied to investigate the related mechanism, which was confirmed by western blotting, and ROS were detected. KEY FINDINGS: The neutrophils incubated with propofol showed the lowest degree of NET formation compared with those incubated with the other intravenous anesthetics. Propofol significantly reduced the level of myeloperoxidase (MPO)-derived HOCl but not that of superoxide. Aminopyrine, an MPO inhibitor, markedly decreased the number of PMA-induced NETs, indicating the involvement of HOCl in the inhibitory effect of propofol on NET formation. According to western blotting results, the level of p-ERK was reduced by propofol during PMA-induced NET formation. The ERK inhibitor PD98059 decreased NET formation but did not inhibit PMA-induced HOCl generation, and aminopyrine did not reduce ERK phosphorylation. SIGNIFICANCE: Through this study, we define a new anti-inflammatory effect of intravenous anesthetics. Of the four intravenous anesthetics tested, propofol was the most potent inhibitor of NET formation. Moreover, propofol resulted in a decrease in PMA-induced NET formation by two independent mechanisms: inhibition of HOCl and p-ERK.
Asunto(s)
Trampas Extracelulares/efectos de los fármacos , Propofol/farmacología , Anestésicos Intravenosos/metabolismo , Anestésicos Intravenosos/farmacología , Cromatina/efectos de los fármacos , Voluntarios Sanos , Humanos , Ácido Hipocloroso/metabolismo , Ketamina/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Midazolam/farmacología , Neutrófilos/fisiología , Propofol/metabolismo , Especies Reactivas de Oxígeno , Transducción de Señal , Acetato de Tetradecanoilforbol/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Tiamilal/farmacologíaRESUMEN
Anesthetics have immunomodulatory effects, but the use of different assay systems has contributed to inconsistent results in the literature. IL-1ß and reactive oxygen species (ROS) secreted by phagocytes are important factors that protect against Staphylococcus aureus infection. In this study, the effects of four intravenous anesthetics (propofol, thiamylal sodium, midazolam, and ketamine) on IL-1ß secretion, ROS, and bacterial survival in S. aureus-infected RAW264.7 cells were evaluated. S. aureus-infected RAW264.7 cells with or without intravenous anesthetic treatment were established as the experimental model. Cell supernatants were subjected to ELISAs to measure secreted IL-1ß. Cell pellets were subjected to qPCR and western blot analyses to analyze IL-1ß mRNA and protein levels. Luminol chemiluminescence assays were used to detect ROS, and bacterial survival was determined by counting the colony forming units at the beginning and end of the infection. Compared with the levels after treatment with the other intravenous anesthetics, secreted IL-1ß levels were lowest in the supernatant of S. aureus-infected RAW264.7 cell cultures after propofol treatment, but propofol did not decrease IL-1ß mRNA or protein expression. However, thiamylal sodium and midazolam decreased IL-1ß mRNA and protein expression in a dose-dependent manner. Additionally, propofol substantially decreased S. aureus-stimulated ROS and phagocytosis. Bacterial survival was strongly increased by propofol treatment. Of the four intravenous anesthetics, propofol was the most potent inhibitor of IL-1ß secretion and ROS level in S. aureus-infected RAW264.7 cells; moreover, propofol resulted in an increase in bacterial survival by inhibiting ROS and phagocytosis.