RESUMEN
The quantitative analysis of cell surface antigens has attracted increasing attention due to the antigenic variation recognition that can facilitate early diagnoses. This paper presents a novel methodology based on the optical "cell-tearing" and the especially proposed "dilution regulations" to detect variations in cell surface antigens. The cell attaches to the corresponding antibody-coated slide surface. Then, the cell-binding firmness between a single cell and the functionalized surface is assayed by optically tearing using gradually reduced laser powers incorporated with serial antibody dilutions. Groups B and B3 of red blood cells (RBCs) were selected as the experiment subject. The results indicate that a higher dilution called for lower power to tear off the cell binding. According to the proposed relative-quantitative analysis theory, antigenic variation can be intuitively estimated by comparing the maximum allowable dilution folds. The estimation result shows good consistency with the finding in the literature. This study suggests a novel methodology for examining the variation in cell surface antigens, expected to be widely capable with potential sensor applications not only in biochemistry and biophysics, but also in the micro-/nano- engineering field.
Asunto(s)
Antígenos de Superficie , Pinzas Ópticas , Anticuerpos , Eritrocitos , Rayos LáserRESUMEN
Pharmacologic intervention to affect the membrane lipid homeostasis of lipid rafts is a potent therapeutic strategy for cancer. Here we showed that gallic acid (GA) caused the complex formation of inactive Ras-related C3 botulinum toxin substrate 1 (Rac1)-phospho (p)-casein kinase 2 α (CK2α) (Tyr 255) in human tongue squamous carcinoma (TSC) cells, which disturbed the lipid raft membrane-targeting of phosphatidylinositol 3-kinase (PI3K)-Rac1-protein kinase B (Akt) signal molecules by inducing the association of p110α-free p85α with unphosphorylated phosphatase tensin homolog deleted on chromosome 10 (PTEN) in lipid rafts. The effects on induction of inactive Rac1-p-CK2α (Tyr 255) complex formation and attenuation of p-Akt (Ser 473), GTP-Rac1, glucose transporter-1 (GLUT-1) lipid raft membrane-targeting, and cell invasive activity by GA were counteracted either by CK2α short hairpin RNA or cellular-Src (c-Src) inhibitor PP1. PP1 treatment, GLUT-1 or constitutively active Rac1 ectopic-expression blocked GA-induced decreases in cellular glucose, sphingolipid and cholesterol of lipid raft membranes, p85α-p110α-GTP-Rac1 complexes, glucosylceramide synthase activity and increase in ceramide and p110α-free p85α-PTEN complex levels of lipid raft membranes, which reversed the inhibition on matrix metalloproteinase (MMP)-2/-9-mediated cell invasion induced by GA. Using transient ectopic expression of nuclear factor-kappa B (NF-κB) p65, MMP-2/-9 promoter-driven luciferase, and NF-κB-dependent luciferase reporter genes and NF-κB specific inhibitors or Rac1 specific inhibitor NSC23766, we confirmed that an attenuation of Rac1 activity by GA confers inhibition of NF-κB-mediated MMP-2/-9 expression and cell invasion. In conclusion, GA-induced c-Src activation is a key inductive event for the formation of inactive Rac1-p-CK2α (Tyr 255) complexes, which disturbed lipid raft compartment of PI3K and PTEN molecules by impairing Akt-regulated GLUT-1-mediated sphingolipid synthesis, and finally resulting in inhibition of TSC cell invasion.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Microdominios de Membrana/metabolismo , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal , Esfingolípidos/biosíntesis , Neoplasias de la Lengua/metabolismo , Familia-src Quinasas/metabolismo , Carcinoma de Células Escamosas/patología , Compartimento Celular , Línea Celular Tumoral , Regulación hacia Abajo/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Epítopos/metabolismo , Ácido Gálico/farmacología , Transportador de Glucosa de Tipo 1/metabolismo , Homeostasis/efectos de los fármacos , Humanos , Metaloproteinasas de la Matriz/metabolismo , Microdominios de Membrana/efectos de los fármacos , Modelos Biológicos , FN-kappa B/metabolismo , Invasividad Neoplásica , Proteínas Proto-Oncogénicas c-akt/metabolismo , Neoplasias de la Lengua/patología , Proteína de Unión al GTP rac1/metabolismoRESUMEN
The specialized cholesterol/sphingolipid-rich membrane domains termed lipid rafts are highly dynamic in the cancer cells, which rapidly assemble effector molecules to form a sorting platform essential for oncogenic signaling transduction in response to extra- or intracellular stimuli. Density-based membrane flotation, subcellular fractionation, cell surface biotinylation, and co-immunoprecipitation analyses of bichalcone analog ((E)-1-(4-Hydroxy-3-((4-(4-((E)-3-(pyridin-3-yl)acryloyl)phenyl)piperazin-1-yl)methyl)phenyl)-3-(pyridin-3-yl)prop-2-en-1-one (TSWU-BR4)-treated cancer cells showed dissociation between GRP78 and p85α conferring the recruitment of PTEN to lipid raft membranes associated with p85α. Ectopic expression of GRP78 could overcome induction of lipid raft membrane-associated p85α-unphosphorylated PTEN complex formation and suppression of GRP78PI3KAktGTP-Rac1-mediated and GRP78-regulated PERKNrf2 antioxidant pathway and cancer cell invasion by TSWU-BR4. Using specific inducer, inhibitor, or short hairpin RNA for ASM demonstrated that induction of the lipid raft membrane localization and activation of ASM by TSWU-BR4 is responsible for perturbing homeostasis of cholesterol and ceramide levels in the lipid raft and ER membranes, leading to alteration of GRP78 membrane trafficking and subsequently inducing p85α-unphosphorylated PTEN complex formation, causing disruption of GRP78PI3KAktGTP-Rac1-mediated signal and ER membrane-associated GRP78-regulated oxidative stress balance, thus inhibiting cancer cell invasion. The involvement of the enrichment of ceramide to lipid raft membranes in inhibition of NF-κB-mediated MMP-2 expression was confirmed through attenuation of NF-κB activation using C2-ceramide, NF-κB specific inhibitors, ectopic expression of NF-κB p65, MMP-2 promoter-driven luciferase, and NF-κB-dependent reporter genes. In conclusion, localization of ASM in the lipid raft membranes by TSWU-BR4 is a key event for initiating formation of ceramide-enriched lipid raft membrane platforms, which causes delocalization of GRP78 from the lipid raft and ER membranes to the cytosol and formation of p85α-unphosphorylated PTEN complexes to attenuate the GRP78-regulated oxidative stress balance and GRP78p85αAktGTP-Rac1NF-κBMMP-2-mediated cancer cell invasion.
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Chalconas/farmacología , Proteínas de Choque Térmico/metabolismo , Homeostasis , Lípidos de la Membrana/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Línea Celular Tumoral , Membrana Celular/metabolismo , Ceramidas/metabolismo , Chalconas/química , Colesterol/metabolismo , Fosfatidilinositol 3-Quinasa Clase Ia/metabolismo , Chaperón BiP del Retículo Endoplásmico , Homeostasis/efectos de los fármacos , Humanos , Metaloproteinasa 2 de la Matriz/metabolismo , Microdominios de Membrana/efectos de los fármacos , Microdominios de Membrana/metabolismo , Modelos Biológicos , FN-kappa B/metabolismo , Invasividad Neoplásica , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Esfingomielina FosfodiesterasaRESUMEN
Citrate is a key intermediate of the tricarboxylic acid cycle and acts as an allosteric signal to regulate the production of cellular ATP. An elevated cytosolic citrate concentration inhibits growth in several types of human cancer cells; however, the underlying mechanism by which citrate induces the growth arrest of cancer cells remains unclear. The results of this study showed that treatment of human pharyngeal squamous carcinoma (PSC) cells with a growth-suppressive concentration of citrate caused cell cycle arrest at the G2/M phase. A coimmunoprecipitation study demonstrated that citrate-induced cell cycle arrest in the G2/M phase was associated with stabilizing the formation of cyclin B1-phospho (p)-cyclin-dependent kinase 1 (CDK1) (Thr 161) complexes. The citrate-induced increased levels of cyclin B1 and G2/M phase arrest were suppressed by the caspase-3 inhibitor Ac-DEVD-CMK and caspase-3 cleavage of mutant p21 (D112N). Ectopic expression of the constitutively active form of protein kinase B (Akt1) could overcome the induction of p21 cleavage, cyclin B1-p-CDK1 (Thr 161) complexes, and G2/M phase arrest by citrate. p85α-phosphatase and tensin homolog deleted from chromosome 10 (PTEN) complex-mediated inactivation of Akt was required for citrate-induced G2/M phase cell cycle arrest because PTEN short hairpin RNA or a PTEN inhibitor (SF1670) blocked the suppression of Akt Ser 473 phosphorylation and the induction of cyclin B1-p-CDK1 (Thr 161) complexes and G2/M phase arrest by citrate. In conclusion, citrate induces G2/M phase arrest in PSC cells by inducing the formation of p85α-PTEN complexes to attenuate Akt-mediated signaling, thereby causing the formation of cyclin B1-p-CDK1 (Thr 161) complexes.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Ácido Cítrico/farmacología , Fosfatidilinositol 3-Quinasa Clase Ia/metabolismo , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Fosfohidrolasa PTEN/metabolismo , Neoplasias Faríngeas/metabolismo , Proteína Quinasa CDC2/genética , Proteína Quinasa CDC2/metabolismo , Carcinoma de Células Escamosas/genética , Línea Celular Tumoral , Ciclina B1/metabolismo , Humanos , Modelos Biológicos , Complejos Multiproteicos/metabolismo , Neoplasias Faríngeas/genética , Fosforilación , Transducción de SeñalRESUMEN
Selective pharmacologic targeting of cell cycle regulators is a potent anti-cancer therapeutic strategy. Here, we show that caspase-3-mediated p21 cleavage involves p53 independent of triptolide (TPL)-induced S phase arrest in human type 1 nasopharyngeal carcinoma (NPC) cells. Coimmunoprecipitation studies demonstrated that TPL causes S phase cell cycle arrest by suppressing the formation of cyclin A-phosphor (p)-cyclin-dependent kinas 2 (CDK2) (Thr 39) complexes. Ectopic expression of constitutively active protein kinase B1 (Akt1) blocks the induction of S phase arrest and the suppression of cyclin A expression and CDK2 Thr 39 phosphorylation by TPL. Expression of the phosphomimetic mutant CDK2 (T39E) rescues the cells from TPL-induced S phase arrest, whereas phosphorylation-deficient CDK2 (T39A) expression regulates cell growth with significant S phase arrest and enhances TPL-triggered S phase arrest. Treatment with TPL induces an increase in the formation of complexes between unphosphorylated phosphatase and tensin homolog deleted from chromosome 10 (PTEN) and p85α in the plasma membrane. Decreased microRNA (miR)-144 expression and increased PTEN expression after TPL treatment were demonstrated, and TPL-enhanced p85α-PTEN complexes and inhibitory effects on Akt (Ser 473) phosphorylation and S phase arrest were suppressed by ectopic PTEN short hairpin RNA or miR-144 expression. Knockdown of endogenous miR-144 by miR-144 Trap upregulated PTEN expression and accordingly enhanced p85α-PTEN complex formation and S phase arrest. Collectively, the effect of TPL on S phase arrest in human NPC cells is likely to enhance the p85α-PTEN interaction in the plasma membrane by suppressing miR-144 expression, resulting in the attenuation of cyclin A-p-CDK2 (Thr 39) complex formation via Akt inactivation.
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Fosfatidilinositol 3-Quinasa Clase Ia/metabolismo , Diterpenos/farmacología , Regulación hacia Abajo/efectos de los fármacos , MicroARNs/genética , Neoplasias Nasofaríngeas/patología , Fosfohidrolasa PTEN/metabolismo , Fenantrenos/farmacología , Puntos de Control de la Fase S del Ciclo Celular/efectos de los fármacos , Caspasa 3/metabolismo , Línea Celular Tumoral , Quinasa 2 Dependiente de la Ciclina/química , Quinasa 2 Dependiente de la Ciclina/metabolismo , Compuestos Epoxi/farmacología , Humanos , Fosforilación/efectos de los fármacos , Proteolisis/efectos de los fármacos , Treonina/metabolismo , Quinasas p21 Activadas/metabolismoRESUMEN
Caloric restriction activates sirtuin 1 (SIRT1) and induces a variety of metabolic effects that are beneficial for preventing age-related disease. The present study screened a commercially available used drug library to develop small molecule activators of SIRT1 as therapeutics for treatment of metabolic disorders. Using an in vitro fluorescence assay, the cancer therapeutic camptothecin increased SIRT1 enzymatic activity by 5.5-fold, indicating it to be a potent SIRT1 activator. Camptothecin also elevated the nicotinamide adenine dinucleotide (NAD)+/NADH ratio and increased SIRT1 protein levels in differentiated C2C12 myogenic cells. Treatment of C2C12 myotubes with camptothecin increased phosphorylation of AMP-dependent kinase (AMPK) and acetyl-coenzyme A carboxylase, caused nuclear translocation and deacetylation of forkhead box O1 (FoxO1), increased transcription and protein expression of adipose triglyceride lipase (ATGL), decreased the amount of intracellular oil droplets, and significantly increased ß-oxidation of fatty acids. These in vitro data were confirmed in vivo as camptothecin treatment of C57BL/6J mice reduced fat and plasma triglyceride levels. All of the above camptothecin-induced alterations were attenuated by the SIRT1-specific inhibitor nicotinamide and/or 6-[4-(2-piperidin-1-ylethoxy) phenyl]-3-pyridin-4-ylpyrazolo [1,5-a]pyrimidin (compound C). Thus, camptothecin activation of SIRT1 promotes lipid catabolism through AMPK/FoxO1/ATGL signaling.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Camptotecina/farmacología , Proteína Forkhead Box O1/metabolismo , Lipasa/metabolismo , Sirtuina 1/metabolismo , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Metabolismo de los Lípidos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Transducción de Señal/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
Iron or oxygen regulates the stability of hypoxia inducible factor-1α (HIF-1α). We investigated whether ferrous glycinate would affect HIF-1α accumulation, aerobic glycolysis and mitochondrial energy metabolism in human A549 lung cancer cells. Incubation of A549 cells with ferrous glycinate decreased the protein levels of HIF-1α, which was abrogated by proteosome inhibitor, or prolyl hydroxylase inhibitor. The addition of ferrous glycinate decreased protein levels of glucose transporter-1, hexokinase-2, and lactate dehydrogenase A, and decreased pyruvate dehydrogenase kinase-1 (PDK-1) and pyruvate dehydrogenase (PDH) phosphorylation in A549 cells. Ferrous glycinate also increased the expression of the mitochondrial transcription factor A (TFAM), and the mitochondrial protein, cytochrome c oxidase (COX-IV). Silencing of HIF-1α expression mimicked the effects of ferrous glycinate on PDK-1, PDH, TFAM and COX-IV in A549 cells. Ferrous glycinate increased mitochondrial membrane potential and ATP production in A549 cells. These results suggest that ferrous glycinate may reverse Warburg effect through down regulating HIF-1α in A549 cells.
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Metabolismo Energético/efectos de los fármacos , Compuestos Ferrosos/farmacología , Glicina/farmacología , Subunidad alfa del Factor 1 Inducible por Hipoxia/biosíntesis , Subunidad alfa del Factor 1 Inducible por Hipoxia/deficiencia , Células A549 , Relación Dosis-Respuesta a Droga , Compuestos Ferrosos/química , Glicina/análogos & derivados , Glicina/química , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Healthcare professionals have a high risk of needlestick and sharps injuries (NSIs), which have a high potential for disease transmission. Ambulatory care follow up is essential, but is usually overlooked. This study aimed to investigate the annual and cumulative (age-, sex-, and subtype-specific) incidences of ambulatory care visits after NSIs. This study was also designed to evaluate the incidences of blood-borne diseases associated with NSIs among Taiwanese health professionals in Taiwan between 2004 and 2010. Data were obtained from the National Health Insurance Research Database, which contains anonymized records representing approximately 99% of the Taiwan population. A total of 4443 nurse healthcare workers (NHCWs) and 3138 non-nurse healthcare workers (NNHCWs), including physicians, medical technologists, and other health professionals were included in this longitudinal study. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. The Mantel-Haenszel method was used to adjust for sex, age, and type of affiliation. Results showed that the annual incidence of ambulatory care visits of NHCWs increased from 0.7% in 2004 to 1.9% in 2010; this incidence was significantly higher than that of NNHCWs (from 0.3% in 2004 to 0.5% in 2010) in any yearly comparison (p < 0.05). The sex-adjusted 7-year cumulative incidence rate was 3.23 (95% CI = 1.23-8.45) in males and 3.92 (95% CI = 2.70-5.69) in females (p < 0.05). The age-adjusted 7-year cumulative incidence rate was 2.74 (95% CI = 1.99-3.77) and 2.14 (95% CI = 1.49-3.07) in subjects ≤ 30 and ≥31 years old, respectively (p < 0.0005). The affiliation-adjusted 7-year cumulative incidence rate was 1.89 (95% CI = 1.21-2.94) in medical centers and 3.33 (95% CI = 2.51-4.41) in nonmedical centers (p < 0.01). In conclusion, NSIs increased steadily from 2004 to 2010 in Taiwan with NHCWs having higher NSIs incidences than NNHCWs. A routine ambulatory care visit after NSIs can prevent blood-borne transmission, especially for NHCWs. Educational programs may be helpful for reducing the incidence of NSIs and increasing ambulatory care visit ratios after NSIs.
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Atención Ambulatoria/estadística & datos numéricos , Personal de Salud/estadística & datos numéricos , Encuestas Epidemiológicas/estadística & datos numéricos , Lesiones por Pinchazo de Aguja/epidemiología , Adulto , Factores de Edad , Enfermedades Transmisibles/etiología , Demografía , Femenino , Humanos , Incidencia , Masculino , Lesiones por Pinchazo de Aguja/complicaciones , Enfermeras y Enfermeros/estadística & datos numéricos , Factores de Riesgo , Taiwán/epidemiologíaRESUMEN
AIM: To investigate the effects of punicalagin, a polyphenol isolated from Punica granatum, on human U87MG glioma cells in vitro. METHODS: The viability of human U87MG glioma cells was evaluated using MTT assay. Cell cycle was detected with flow cytometry analysis. The levels of Bcl-2, cleaved caspase-9, cleaved poly(ADP-ribose) polymerase (PARP), phosphor-AMPK and phosphor-p27 at Thr198 were measured using immunoblot analyses. Caspase-3 activity was determined with spectrophotometer. To determine autophagy, LC3 cleavage and punctate patterns were examined. RESULTS: Punicalagin (1-30 µg/mL) dose-dependently inhibited the cell viability in association with increased cyclin E level and decreased cyclin B and cyclin A levels. The treatment also induced apoptosis as shown by the cleavage of PARP, activation of caspase-9, and increase of caspase-3 activity in the cells. However, pretreatment of the cells with the pan-caspase inhibitor z-DEVD-fmk (50 µmol/L) did not completely prevent the cell death. On the other hand, punicalagin treatment increased LC3-II cleavage and caused GFP-LC3-II-stained punctate pattern in the cells. Suppressing autophagy of cells with chloroquine (1-10 µmol/L) dose-dependently alleviated the cell death caused by punicalagin. Punicalagin (1-30 µg/mL) also increased the levels phosphor-AMPK and phosphor-p27 at Thr198 in the cells, which were correlated with the induction of autophagic cell death. CONCLUSION: Punicalagin induces human U87MG glioma cell death through both apoptotic and autophagic pathways.
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Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Glioma/tratamiento farmacológico , Taninos Hidrolizables/farmacología , Proteínas Quinasas Activadas por AMP/metabolismo , Antineoplásicos Fitogénicos/administración & dosificación , Antineoplásicos Fitogénicos/aislamiento & purificación , Antineoplásicos Fitogénicos/farmacología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Glioma/patología , Humanos , Taninos Hidrolizables/administración & dosificación , Taninos Hidrolizables/aislamiento & purificación , Lythraceae/química , FosforilaciónRESUMEN
BACKGROUND: A shift in glucose metabolism from oxidative phosphorylation to anaerobic glycolysis is the biochemical hallmark of malignant cancer cells. METHODS: In the present study, we demonstrated that Nodal stimulated the expression of glycolytic enzymes and decreased reliance on mitochondrial oxidative phosphorylation in human glioma cancer cells. The shift in glucose metabolism was mediated by induction of the hypoxia-inducible factor (HIF). RESULTS: Nodal protein expression was shown to be correlated with expression levels of glucose transporter (Glut)-1, hexokinase (HK)-II, pyruvate dehydrogenase kinase (PDK)-1, the phosphorylation level of pyruvate dehydrogenase (PDH), glucose uptake, and lactate accumulation in human glioma cells. These effects were inversely correlated with mitochondrial oxygen consumption and ATP production. Knockdown of Nodal expression with specific small hairpin RNA reduced Glut-1, HK-II, and PDK-1 expressions and PDH phosphorylation. Nodal knockdown also reduced glucose uptake and lactate generation, which in turn increased mitochondrial membrane potential (Ψ), O2 utilization, and ATP synthesis. The ectopic expression of Nodal in low-expressing Nodal glioma cells resulted in the opposite results compared with those of Nodal knockdown glioma cells. Treatment of cells with recombinant Nodal increased HIF-1 expression, and this effect was regulated at the transcriptional level. Blockage of the Nodal receptor by a pharmacological inhibitor or Nodal knockdown in U87MG cells decreased HIF-1α expression. Furthermore, HIF-1α knockdown in U87MG cells decreased Glut-1, HK-II, and PDK-1 expressions and PDH phosphorylation, which were similar to results in Nodal knockdown cells. CONCLUSION: Taken together, these results suggest that Nodal affects energy metabolism through HIF-1α.
Asunto(s)
Metabolismo Energético , Glioma/metabolismo , Glioma/patología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Mitocondrias/metabolismo , Proteína Nodal/metabolismo , Adenosina Trifosfato/metabolismo , Western Blotting , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Glucosa/metabolismo , Glucólisis , Humanos , Hipoxia/metabolismo , Hipoxia/patología , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Ácido Láctico/metabolismo , Luciferasas/metabolismo , Potencial de la Membrana Mitocondrial , Proteína Nodal/antagonistas & inhibidores , Proteína Nodal/genética , Consumo de Oxígeno , ARN Interferente Pequeño/genética , Células Tumorales CultivadasRESUMEN
Monacolin K is the secondary metabolite isolated from Monascus spp. It is the natural form of lovastatin, which is clinically used to reduce the synthesis of cholesterol by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A reductase. In the present study, monacolin K increased protein expression of SIRT1 and phosphorylation level of AMP-activated protein kinase (AMPK) in HepG2 cells. Through activation of SIRT1/AMPK pathway, monacolin K increased phosphorylation of acetyl CoA carboxylase and caused nuclear translocation of forkhead box O1. The western blotting results showed that monacolin K increased expression of adipose triglyceride lipase but decreased abundances of fatty acid synthase (FAS) and sterol regulatory element-binding protein 1 (SREBP1). Monacolin K also decreased the intracellular accumulation of lipids as demonstrated by Oil Red O staining. In addition, the immunostaining showed that monacolin K prevented the nuclear translocation of SREBP1, indicating the association with down-regulation of FAS. All the demonstrated effects of monacolin K were counteracted by nicotinamide or compound C, the inhibitors of SIRT1 or AMPK. In summary, monacolin K reduces the lipid content through SIRT1/AMPK pathway in HepG2 cells, which promotes catabolism and inhibits anabolism of lipid.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Anticolesterolemiantes/farmacología , Metabolismo de los Lípidos/fisiología , Lovastatina/farmacología , Transducción de Señal/fisiología , Sirtuina 1/metabolismo , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Relación Dosis-Respuesta a Droga , Células Hep G2 , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: Musculoskeletal disorders (MSDs) represent the leading causes of occupational injuries among nursing staff. This population-based study was designed to assess the incidence and age-specific incidence of MSDs among a Taiwanese nurse cohort compared with non-nurses. DESCRIPTION: Data from the Taiwan National Health Insurance Research Database were used to identify MSDs in the study population. A total of 3914 nurses with a diagnosis of MSD were included, together with 11,744 non-nurses as a comparison group. The comparison subjects were randomly selected at a ratio of 3:1 relative to the nurse population and were matched by gender and age. The incidence of MSDs was calculated for the study group, with nurse-to-reference risk ratios presented as odds ratios with 95% confidence intervals (CIs). During the period 2004-2010, 3004 MSDs occurred among the nurses (76.24%) and 7779 (65.79%) in the non-nurses. The annual incidence of MSDs for the nurses increased from 28.35% in 2006 to 33.65% in 2010. The nurse-to-reference risk ratio was 1.27 (95% CI 1.19-1.35) in 2004 and 1.46 (1.37-1.55) in 2010. Herniation of intervertebral disc, lumbago, rotator cuff syndrome, medial epicondylitis, trigger finger and carpal tunnel syndrome were the most common problems. CONCLUSIONS: Nurses are at higher risk of MSDs and the trend is increasing. Incorrect work-related posture/movement, psychological issues and the rolling shift system may be the major causes of MSDs among nurses in Taiwan.
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Enfermedades Musculoesqueléticas/epidemiología , Enfermeras y Enfermeros , Traumatismos Ocupacionales/epidemiología , Vigilancia de la Población , Adulto , Estudios de Cohortes , Femenino , Humanos , Masculino , Enfermedades Musculoesqueléticas/diagnóstico , Traumatismos Ocupacionales/diagnóstico , Vigilancia de la Población/métodos , Factores de Riesgo , Taiwán/epidemiología , Adulto JovenRESUMEN
Fenofibrate, a fibric acid derivative, is known to possess lipid-lowering effects. Although fenofibrate may activate peroxisome proliferator-activated receptor (PPAR)α and regulate the transcription of several genes, the underlying mechanisms are poorly understood. In this study, we demonstrated that incubation of C2C12 myotubes with fenofibrate increased adipose triglyceride lipase (ATGL) expression and suppressed fatty acid synthase (FAS) level, thereby decreasing intracellular triglyceride accumulation when cells were incubated at high-glucose condition. Fenofibrate increased the phosphorylation of AMP-activated protein kinase (AMPK), which subsequently increased fatty acid ß-oxidation. AMPK phosphorylation was reduced by pretreatment with GW9662 (a PPARα inhibitor), suggesting that AMPK may be a downstream effector of PPARα. Pretreatment with compound C (an AMPK inhibitor) or GW9662 blocked fenofibrate-induced ATGL expression and the lipid-lowering effect. Our results suggest that AMPK is as an upstream regulator of ATGL. With further exploration, we demonstrated that fenofibrate stimulated FoxO1 translocation from the cytosol to nuclei by immunefluorescence assay, chromatin immuneprecipitation assay, and reporter assay. Furthermore, oral administration of fenofibrate ameliorated the body weight, visceral fat and serum biochemical indexes in db/db mice. Taken together, our results suggest that the lipid-lowering effect of fenofibrate was achieved by activating PPARα and AMPK signaling pathway that resulted in increasing ATGL expression, lipolysis, and fatty acid ß-oxidation.
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Proteínas Quinasas Activadas por AMP/fisiología , Fenofibrato/farmacología , Factores de Transcripción Forkhead/fisiología , Hipolipemiantes/farmacología , Lipasa/fisiología , Metabolismo de los Lípidos , Fibras Musculares Esqueléticas/metabolismo , PPAR alfa/fisiología , Acetil-CoA Carboxilasa/metabolismo , Animales , Línea Celular , Núcleo Celular/metabolismo , Citosol/metabolismo , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/metabolismo , Lipasa/genética , Masculino , Ratones , Fosforilación , Regiones Promotoras Genéticas , Unión Proteica , Transporte de Proteínas , Transducción de SeñalRESUMEN
Aloe-emodin (AE), extracted from the rhizome of Rheum palmatum, has an anti-proliferative effect on different human cancer cell lines. Nonetheless, the underlying mechanism by which AE inhibits nasopharyngeal carcinoma (NPC) cell invasion is still unclear. The results of this study show that treatment of NPC cells with growth suppressive concentrations of AE caused cell cycle arrest at the S-G(2)/M phase. Coimmunoprecipitation and small interfering RNA (siRNA) studies demonstrated that AE-induced cell cycle arrest in NPC cells was associated with increasing levels of cyclin B1 bound to cyclin-dependent kinase 1. The inhibition of NPC cell invasion by AE was evidenced through the suppression of matrix metalloproteinases-2 (MMP-2) expression. MMP-2 promoter activity and cell invasion were inhibited by p38 mitogen-activated protein kinase (MAPK) siRNA, inhibitor 4-(4-Fluorophenyl)-2-[4-(methylsulfinyl)phenyl]-5-(4-pyridyl)-1H-imidazole (SB203580), and AE, but not by JNK siRNA and inhibitor 1,9-pyrazoloanthrone. Treatment with AE, SB203580, NF-kappaB inhibitors N-p-tosyl-(L)-phenylalanine chloromethyl ketone (TPCK) and pyrrolidine dithiocarbamate (PDTC) or transfection with p38 MAPK siRNA significantly inhibited NF-kappaB transcriptional activity. In addition, TPCK and PDTC treatment inhibited the expression and promoter activity of MMP-2 and thereby significantly inhibited cell invasion activity. The involvement of p38 MAPK activity in NF-kappaB-mediated MMP-2 function was further confirmed through the attenuation of p38 MAPK by SB203580 and NF-kappaB ectopic expression. Collectively, our results indicate that AE inhibits invasion of NPC cells by suppressing the expression of MMP-2 via the p38 MAPK-NF-kappaB signaling pathway.
Asunto(s)
FN-kappa B/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Antraquinonas , Proteína Quinasa CDC2/genética , Proteína Quinasa CDC2/metabolismo , Proteína Quinasa CDC2/farmacología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Ciclina B1/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Humanos , Imidazoles/farmacología , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 2 de la Matriz/farmacología , FN-kappa B/genética , FN-kappa B/farmacología , Neoplasias Nasofaríngeas/genética , Piridinas , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
Aloe-emodin (AE), a natural, biologically active compound from the rhizome of Rheum palmatum, has been shown to induce apoptosis in several cancer cell lines in vitro. However, its molecular mechanism of action in the apoptosis induction of human nasopharyngeal carcinoma (NPC) cells has not been explored. This study shows that AE induced G(2)/M phase arrest by increasing levels of cyclin B1 bound to Cdc2, and also caused an increase in apoptosis of NPC cells, which was characterized by morphological changes, nuclear condensation, DNA fragmentation, caspase-3 activation, cleavage of poly (ADP-ribose) polymerase (PARP) and increased sub-G(1) population. Treatment of NPC cells with AE also resulted in a decrease in Bcl-X(L) and an increase in Bax expression. Ectopic expression of Bcl-X(L) but not Bcl-2 or small interfering RNA (siRNA)-mediated attenuation of Bax suppressed AE-induced apoptotic cell death. AE-induced loss of mitochondrial membrane potential (MMP) and increase in cellular Ca(++) content, reactive oxygen species (ROS) and apoptotic cell death were suppressed by the treatment of cyclosporin A (CsA) or caspase-8 inhibitor Z-IETD-FMK. Co-treatment with caspase-9 inhibitor Z-LEHD-FMK could inhibit AE-induced cell death and the activation of caspase-3 and -9. In addition, suppression of caspase-8 with the specific inhibitor Z-IETD-FMK inhibited AE-induced the activation of Bax, the cleavage of Bid, the translocation of tBid to the mitochondria and the release of cytochrome c, apoptosis-inducing factor (AIF) and Endo G from the mitochondria and subsequent apoptosis. Taken together, these results indicate that the caspase-8-mediated activation of the mitochondrial death pathway plays a critical role in AE-induced apoptosis of NPC cells.
Asunto(s)
Antraquinonas/farmacología , Apoptosis/efectos de los fármacos , Caspasa 8/fisiología , Mitocondrias/efectos de los fármacos , Neoplasias Nasofaríngeas/tratamiento farmacológico , Proteína Quinasa CDC2 , Caspasa 3/fisiología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Ciclina B/metabolismo , Ciclina B1/metabolismo , Quinasas Ciclina-Dependientes , Humanos , Mitocondrias/metabolismo , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patología , Especies Reactivas de Oxígeno/metabolismo , Proteína X Asociada a bcl-2/fisiología , Proteína bcl-X/fisiologíaRESUMEN
The human TAO kinase 1 (hTAOK1) is a member of the Ste20 group of kinases with the kinase domain located at the N-terminus. The rat homologue, originally named TAO1, has been demonstrated to be highly expressed in brain. In this study, the human TAO kinase 1 was transfected into human neuroblastoma SH-SY5Y cells and its biological effects on the cell morphology were observed by co-expressing the enhanced green fluorescent protein (EGFP). It was found that after 16 h of transfection the cells had shrunk, and finally became rounded when transfected with wild-type or mutant K57A genes encoding either the kinase domain (residues 1-376) or the full-length molecule (residues 1-1001). Thirty-four hours after transfection, cells floated and apoptotic bodies were observed after nuclear staining with DAPI. On the other hand, the cells that were transfected with the gene encoding the C-terminal regulatory region (residues 377-1001) of hTAOK1, appeared to remain unchanged. In order to know the signaling events involved in the above biological phenomena, caspase-3-like activities of the transfected cells were measured in the absence or presence of JNK inhibitor SP600125, in which caspase-3 and JNK (C-jun-N-terminal kinase) are both known to be critical components of the neuronal apoptosis. The results showed that the apoptotic cells exhibited elevated caspase-3-like activity, which could be reduced by SP600125 to some extent. It is concluded that human TAO kinase 1 induces apoptosis in SH-SY5Y cells and the kinase domain is essential, but its catalytic activity seems to be dispensable in this case.