RESUMEN
Peroxisome proliferator activated receptor gamma (PPARgamma) plays a critical role in controlling immune and inflammatory responses. However, its effect on pulpal inflammation has not been clarified. The purpose of this study was to determine the anti-inflammatory effect of PPARgamma on pulpal inflammation. Human dental pulp cells treated with lipopolysaccharide exhibited elevated levels of matrix metalloproteinase-2 (MMP-2), MMP-9, intracellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1). However, when treated with rosiglitazone (PPARgamma agonist) or adenoviral PPARgamma in same culture system, the expression of ICAM-1 and VCAM-1 was markedly inhibited along with decreased secretion of MMPs. In addition, the coadministration of GW9662 (PPARgamma antagonist) and rosiglitazone blocked the inhibition of MMP-2, MMP-9, ICAM-1, and VCAM-1. These results suggest that PPARgamma decreased the production of MMPs, ICAM-1, and VCAM-1 and might offer a possible attempt of using it as one of anti-inflammatory modulators in a pulpal inflammation.
Asunto(s)
Antiinflamatorios/farmacología , Pulpa Dental/metabolismo , Mediadores de Inflamación/metabolismo , PPAR gamma/fisiología , Pulpitis/metabolismo , Anilidas/farmacología , Western Blotting , Células Cultivadas , Pulpa Dental/citología , Escherichia coli/química , Fibroblastos/metabolismo , Humanos , Molécula 1 de Adhesión Intercelular/biosíntesis , Lipopolisacáridos , Metaloproteinasa 2 de la Matriz/biosíntesis , Metaloproteinasa 9 de la Matriz/biosíntesis , Inhibidores de la Metaloproteinasa de la Matriz , PPAR gamma/agonistas , PPAR gamma/antagonistas & inhibidores , PPAR gamma/farmacología , Proteínas Recombinantes/farmacología , Rosiglitazona , Tiazolidinedionas/farmacología , Molécula 1 de Adhesión Celular Vascular/biosíntesisRESUMEN
The stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) pathway is a well-known senescence-related stress activated protein kinase. Multiple environmental stresses induce programmed cell death, such as apoptosis. Normal human diploid fibroblast (HDF) cells have a limited life span in vitro, halting proliferation after a fixed number of cell divisions. Aged passage HDF showed resistance to oxidative stress involving heat shock proteins (Hsp60) through a mechanism involving the translocation of Hsp60 from the mitochondria to the cytosol. The present study showed that the translocation of Hsp60 from the mitochondria to the cytosol followed by high levels of p-SAPK/JNK activation as a result of oxidative stress was observed in the young cells only. The inhibition of SAPK/JNK activation by SP600125 under oxidative stress almost completely blocked the translocation of Hsp60 in both young and aged cells. This suggests that aged HDF cells are resistant to oxidative stress by blocking the translocation of Hsp60 from the mitochondria to the cytosol followed by SAPK/JNK inhibition. Overall, the mechanism of resistance by oxidative stress in aged cells is induced by blocked of the translocation of Hsp60 followed by SAPK/JNK inactivation.
Asunto(s)
Senescencia Celular/fisiología , Chaperonina 60/metabolismo , Fibroblastos/metabolismo , MAP Quinasa Quinasa 4/metabolismo , Proteínas Mitocondriales/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Estrés Oxidativo/fisiología , Antracenos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , División Celular/efectos de los fármacos , División Celular/fisiología , Células Cultivadas , Senescencia Celular/efectos de los fármacos , Diploidia , Fibroblastos/citología , Humanos , MAP Quinasa Quinasa 4/antagonistas & inhibidores , Masculino , Mitocondrias/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiologíaRESUMEN
Paired box gene 9 (Pax9) and c-myb are transcription factors that regulate the expression of the genes involved in mediating cell proliferation, resistance to apoptosis, and migration. However, the function of Pax9 in oral squamous cell carcinoma (OSCC) is virtually unknown. This study examined the anti-apoptotic roles of Pax9 and c-myb, and clarified interaction between the two genes in KB cells. Inhibition of Pax9 caused the induction of apoptosis with enhanced cleavage of caspase-3 and PARP, accelerated Bax, and reduced Bcl-2 expression. Transducing c-myb cells with adenovirus c-myb (Ad/c-myb) were induced cell growth and inhibited apoptosis, but dominant-negative myb cells (Ad/DN-myb) were not affected. Pax9 was upregulated in the Ad/c-myb cells with simultaneous decrease in the Ad/DN-myb infection. However, c-myb remained unaffected in the Pax9 small interfering RNA (siRNA) transfected cells. Moreover, the Pax9 siRNA transfected cells and Ad/DN-myb infected cells were able to arrest the cell cycle at the G(0) phase. This suggests that Pax9 and c-myb expression in KB cells is essential for cell growth, and survival is enhanced by c-myb. Disrupting the function of c-myb and Pax9 could be a potential target for cancer treatment.
Asunto(s)
Carcinoma de Células Escamosas/genética , Neoplasias de la Boca/genética , Factor de Transcripción PAX9/genética , Proteínas Proto-Oncogénicas c-myb/genética , Adenoviridae/genética , Adenoviridae/metabolismo , Apoptosis , Carcinoma de Células Escamosas/metabolismo , Ciclo Celular , Línea Celular Tumoral , Supervivencia Celular , Humanos , Células KB , Neoplasias de la Boca/metabolismo , Factor de Transcripción PAX9/metabolismo , Proteínas Proto-Oncogénicas c-myb/metabolismo , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Fase de Descanso del Ciclo Celular , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Since an attenuated response to stress is a characteristic of senescence, a cellular senescence model was used to examine the mechanism of resistance against oxidative stress using human diploid fibroblasts (HDF). With increasing passage, the HDF showed increased production of reactive oxygen species (ROS). Late passage HDF were resistant to the lethal effects of oxidative stress, showing less cleavage of pro-caspase-3 and PARP than those of early ones. Since heat shock proteins (Hsps) are not only cytoprotective but also interfere with the apoptotic cascade, the expression patterns of Hsps during cellular senescence were next examined. Oxidative stress induced a decrease in the mitochondrial Hsp60 levels with a concomitant increase in the cytosolic Hsp60 levels in the early passage HDF, but not in late ones. To show that the resistance to oxidative stress is a specific effect of Hsp60, the levels of Hsp60 were knocked down by siRNA. As expected the Hsp60 knock-down cells were more resistant to oxidative stress. These findings show that Hsp60 is a key player in the resistance mechanism against oxidative stress and aging.
Asunto(s)
Apoptosis , Chaperonina 60/metabolismo , Diploidia , Estrés Oxidativo , Apoptosis/efectos de los fármacos , Forma de la Célula , Células Cultivadas , Senescencia Celular/efectos de los fármacos , Chaperonina 60/genética , Fibroblastos , Humanos , Peróxido de Hidrógeno/toxicidad , Masculino , Mitocondrias/metabolismo , ARN Interferente Pequeño/genética , Especies Reactivas de Oxígeno/metabolismo , Factores de TiempoRESUMEN
Terrein is a bioactive fungal metabolite whose anti-inflammatory properties are virtually unknown. The purpose of this study was to determine the effects of terrein on lipopolysaccharide (LPS)-induced expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in human dental pulp cells and to determine the mechanism of the observed effects. The LPS-induced expression of ICAM-1 and VCAM-1 was inhibited by terrein in both a time- and dose-dependent manner. LPS-stimulated translocation of nuclear factor kappa B (NF-kappaB) into the nucleus, which was blocked by inhibitors of amino kinase terminal (AKT, LY294002), extracellular signal regulated kinase 1/2 (ERK 1/2, PD98059), p38 (SB203580), and c-jun NH2-terminal kinase (JNK, SP600125) or terrein. In addition, these inhibitors and terrein also reduced the level of ICAM-1 and VCAM-1 expression in LPS-induced inflammation of pulp cells. Terrein suppressed NF-kappaB activation by blocking the activation of Akt. These results strongly suggest the potential role of terrein as an anti-inflammatory modulator in pulpal inflammation.
Asunto(s)
Antiinflamatorios no Esteroideos/uso terapéutico , Ciclopentanos/uso terapéutico , Pulpa Dental/metabolismo , Pulpitis/tratamiento farmacológico , Transporte Activo de Núcleo Celular/efectos de los fármacos , Ciclopentanos/farmacología , Activación Enzimática/efectos de los fármacos , Humanos , Molécula 1 de Adhesión Intercelular/biosíntesis , Lipopolisacáridos/farmacología , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Pulpitis/metabolismo , Molécula 1 de Adhesión Celular Vascular/biosíntesisRESUMEN
The recent demonstration of aberrant expression of the c-myb proto-oncogene in various cancers suggests that c-myb plays an important role in the development of cancer. On this basis, it has been proposed that ablation of c-myb function might be an effective approach for therapy of c-myb dependent malignancies. We previously used a dominant negative c-myb (DN-myb) construct to induce apoptosis in K562 cells. In this study, DN-myb was expressed in an adenovirus-mediated gene delivery system and introduced into head and neck squamous cell carcinoma cells (HNSCC) in vitro and in vivo to examine its tumor suppressive function and its potential in HNSCC gene therapy. Over expression of DN-myb in HNSCC cells inhibited in vitro cell proliferation, expression of growth factors such as IGF-I, -II, IGF-1R, and VEGF, inhibited Akt/PKB pathway activation, and enhanced induction of apoptosis. Similarly, in vivo administration of DN-myb retarded tumor-growth. Our results support a role for DN-myb in inducing apoptosis and tumor suppression, and, furthermore, suggest that DN-myb gene therapy might provide a powerful tool for treatment of c-myb dependent malignancies such as HNSCC.
Asunto(s)
Adenoviridae/genética , Apoptosis , Carcinoma de Células Escamosas/terapia , Terapia Genética/métodos , Neoplasias de Cabeza y Cuello/terapia , Proteínas Proto-Oncogénicas c-myb/metabolismo , Animales , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Ciclo Celular , División Celular , Modelos Animales de Enfermedad , Regulación hacia Abajo , Técnicas de Transferencia de Gen , Vectores Genéticos/uso terapéutico , Sustancias de Crecimiento/metabolismo , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Trasplante de Neoplasias , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-myb/genéticaRESUMEN
PTEN/MMAC1 is a tumor suppressor gene that is mutated in a variety of advanced and metastatic cancers. Its major function is likely to be the phosphatase activity that regulates the phosphotidylinositol (PI)3-kinase/Akt pathway. On the other hand, IGF system plays an important role in cell proliferation and cell survival via PI3-kinase/Akt and mitogen-activated protein kinase pathways in many cancer cells. To evaluate effect of PTEN on cell growth and IGF system in gastric cancer, human gastric adenocarcinoma cells (SNU-5 & -216) were transfected with human PTEN cDNA. Those PTEN- transfected gastric cancer cells had a lower proliferation rate than the pcDNA3-transfected cells. PTEN overexpression induced a profound decrease in the IGF-II and IGF-IR expression levels, and downregulation of IGF-II expression by PTEN was mediated through the regulation of the IGF-II promoter. In addition, a PI3-kinase inhibitor, LY294002, induced the downregulation of IGF-II expression. The PTEN-overexpressing SUN-5 and -216 cells were more sensitive to death induced by etoposide and adriamycin that induce DNA damage than the pcDNA3-transfected cells. These findings suggest that PTEN suppresses the cell growth through modulation of IGF system and sensitizing cancer cells to cell death by anticancer drugs.
Asunto(s)
Antineoplásicos/farmacología , Regulación hacia Abajo , Factor II del Crecimiento Similar a la Insulina/genética , Factor II del Crecimiento Similar a la Insulina/metabolismo , Fosfohidrolasa PTEN/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Fosfohidrolasa PTEN/genética , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Neoplasias Gástricas/metabolismoRESUMEN
PURPOSE: The objective of this study is to characterize the micelles of novel block copolymer of poly (p-Dioxanone-co-L-Lactide)-block-Poly (ethylene glycol) (PPDO/PLLA-b-PEG-) and evaluate its ability to induce gene transfection. METHODS: The ability of the block copolymer to self-assemble was determined by viscometery, dye solublization, NMR spectra and dynamic light scattering. The Trypan blue assay for in vitro biocompatibility of the block copolymer was carried out with NIH 3T3, CT-26 and MCF-7 cells, and beta-glactosidase assay was applied to measure the transfection efficiency of the block copolymer on MCF-7 breast cancer cell. RESULTS: Depending on the block lengths and molecular weights, solubility of the polymeric samples in water was varied. Diluted aqueous solution properties of the copolymer were studied. 1,6-Diphenyl-1,3,5-hexatriene solubilization and 1H NMR spectra carried out in CDCl3 and D2O, were used to prove the existence of hydrophobic domains as the core of micelle. Average particle size of 60-165 nm with low polydispersity, and lower negative zeta potential of -3 to -14 mV were observed on the aqueous copolymer dispersion. Copolymer was found with almost no cytotoxic effect and was able to promote the transfection efficiency (about 3-fold) in MCF-7 cells. CONCLUSIONS: The PPDO/PLLA-b-PEG copolymer has ability to assemble into nanoscopic structures in aqueous environment, which enable to enhance gene transfection.
Asunto(s)
Poliésteres/química , Polietilenglicoles/química , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Fenómenos Químicos , Química Física , Colorantes/química , ADN/administración & dosificación , ADN/química , Electroquímica , Humanos , Operón Lac/genética , Luz , Espectroscopía de Resonancia Magnética , Ratones , Micelas , Tamaño de la Partícula , Soluciones Farmacéuticas , Vehículos Farmacéuticos , Dispersión de Radiación , Soluciones , Espectrofotometría Ultravioleta , Transfección , Viscosidad , beta-Galactosidasa/genéticaRESUMEN
The water extract from the root bark of Cortex Mori (CM, Morus alba L.: Sangbaikpi), a mulberry tree, has been known in Chinese traditional medicine to have antiphlogistic, diuretic, and expectorant properties. In this study, the cytotoxicity of CM against tumor cells and its mechanism was examined. CM exhibited cytotoxic activity on K-562, B380 human leukemia cells and B16 mouse melanoma cells at concentrations of > 1 mg/ml. A DNA fragmentation, PARP cleavage, and nuclear condensation assay showed that those cells exposed to CM underwent apoptosis. The water extract of Scutellarie Radix (SR) was used as a negative control and showed no cytotoxicity in those cells. The flow cytometric profiles of the CM-treated cells were also indicative of apoptosis. However, they did not appear to exert the G1 arrest, which is observed in other tubulin inhibitor agents such as vincristine, taxol. The protein-binding test using Biacore and a microtubule assembly-disassembly assay provided evidence showing that CM bound to the tubulins resulting in a marked inhibition of the assembly, but not the disassembly of microtubules. The possible nonspecific effect of the CM extract could be excluded due to the results using SR, which did not affect the assembly process. Overall, the water extract of CM induces apoptosis of tumor cells by inhibiting microtubule assembly.