RESUMEN
Non-small-cell lung cancer (NSCLC) is a leading cause of cancer-related death worldwide. NSCLC patients with overexpressed or mutated epidermal growth factor receptor (EGFR) related to disease progression are treated with EGFR-tyrosine kinase inhibitors (EGFR-TKIs). Acquired drug resistance after TKI treatments has been a major focus for development of NSCLC therapies. This study aimed to establish afatinib-resistant cell lines from which afatinib resistance-associated genes are identified and the underlying mechanisms of multiple-TKI resistance in NSCLC can be further investigated. Nude mice bearing subcutaneous NSCLC HCC827 tumors were administered with afatinib at different dose intensities (5-100 mg/kg). We established three HCC827 sublines resistant to afatinib (IC50 > 1 µM) with cross-resistance to gefitinib (IC50 > 5 µM). cDNA microarray revealed several of these sublines shared 27 up- and 13 down-regulated genes. The mRNA expression of selective novel genes - such as transmembrane 4 L six family member 19 (TM4SF19), suppressor of cytokine signaling 2 (SOCS2), and quinolinate phosphoribosyltransferase (QPRT) - are responsive to afatinib treatments only at high concentrations. Furthermore, c-MET amplification and activations of a subset of tyrosine kinase receptors were observed in all three resistant cells. PHA665752, a c-MET inhibitor, remarkably increased the sensitivity of these resistant cells to afatinib (IC50 = 12-123 nM). We established afatinib-resistant lung cancer cell lines and here report genes associated with afatinib resistance in human NSCLC. These cell lines and the identified genes serve as useful investigational tools, prognostic biomarkers of TKI therapies, and promising molecule targets for development of human NSCLC therapeutics.
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Afatinib/farmacología , Antineoplásicos/farmacología , Biomarcadores de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Resistencia a Antineoplásicos/genética , Neoplasias Pulmonares/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Animales , Biomarcadores de Tumor/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Ratones Desnudos , Análisis de Secuencia por Matrices de Oligonucleótidos , Transcriptoma , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Geranium wilfordii is one of the major species used as Herba Geranii (lao-guan-cao) in China, it is commonly used solely or in polyherbal formulations for treatment of joint pain resulted from rheumatoid arthritis (RA) and gout. This herb is used to validate a target-based drug screening platform called Herbochip® and evaluate anti-inflammatory effects of Geranium wilfordii ethanolic extract (GWE) using tumor necrosis factor-alpha (TNF-α) as a drug target together with subsequent in vitro and in vivo assays. METHODS: A microarray-based drug screening platform was constructed by arraying HPLC fractions of herbal extracts onto a surface-activated polystyrene slide (Herbochip®). Using TNF-α as a molecular probe, fractions of 82 selected herbal extracts, including GWE, were then screened to identify plant extracts containing TNF-α-binding agents. Cytotoxicity of GWE and modulatory effects of GWE on TNF-α expression were evaluated by cell-based assays using TNF-α sensitive murine fibrosarcoma L929 cells as an in vitro model. RESULTS: The in vivo anti-inflammatory effects of GWE were further assessed by animal models including carrageenan-induced hind paw edema in rats and xylene-induced ear edema in mice, in comparison with aspirin. The hybridization data obtained by Herbochip® analysis showed unambiguous signals which confirmed TNF-α binding activity in 46 herbal extracts including GWE. In L929 cells GWE showed significant inhibitory effect on TNF-α expression with negligible cytotoxicity. GWE also significantly inhibited formation of carrageenan-induced hind paw edema and xylene-induced ear edema in animal models, indicating that it indeed possessed anti-inflammatory activity. CONCLUSION: We have thus validated effectiveness of the Herbochip® drug screening platform using TNF-α as a molecular target. Subsequent experiments on GWE lead us to conclude that the anti-RA activity of GWE can be attributed to inhibitory effect of GWE on the key inflammatory factor, TNF-α. Our results contribute towards validation of the traditional use of GWE in the treatment of RA and other inflammatory joint disorders.
Asunto(s)
Antiinflamatorios/uso terapéutico , Artritis Reumatoide/tratamiento farmacológico , Medicamentos Herbarios Chinos/farmacología , Geranium/química , Inflamación/tratamiento farmacológico , Fitoterapia , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Antiinflamatorios/análisis , Antiinflamatorios/farmacología , Artritis Reumatoide/metabolismo , Carragenina/uso terapéutico , Línea Celular , China , Medicamentos Herbarios Chinos/química , Medicamentos Herbarios Chinos/uso terapéutico , Edema/tratamiento farmacológico , Edema/metabolismo , Inflamación/inducido químicamente , Inflamación/metabolismo , Masculino , Ratones Endogámicos ICR , Análisis por Micromatrices/métodos , Ratas Sprague-Dawley , XilenosRESUMEN
In order to develop more effective therapeutic vaccines against cancers with high-risk human papillomavirus (HPV) infection, it is crucial to enhance the immunogenicity, eliminate the oncogenicity of oncoproteins, and take a combination of E7- and E6-containing vaccines. It has been shown recently that PE(ΔIII)-E7-KDEL3 (E7), a fusion protein containing the HPV16 oncoprotein E7 and the translocation domain of Pseudomonas aeruginosa exotoxin A, is effective against TC-1 tumor cells inoculated in mice, therefore, we engineered PE(ΔIII)-E6-CRL-KDEL3 (E6), the de-oncogenic versions of the E7 and E6 fusion proteins [i.e. PE(ΔIII)-E7(d)-KDEL3, E7(d), and PE(ΔIII)-E6(d)-CRL-KDEL3, E6(d)] and tested the immunoefficacies of these fusion proteins as mono- and bivalent vaccines. Results indicated that the E7(d) get higher immunogenicity than its wild type and the E6 fusion proteins augmented the immunogenicity and antitumor effects of their E7 counterparts. Furthermore, the bivalent vaccine system E7(d) plus E6(d), in the presence of cisplatin, showed the best tumoristatic and tumoricidal effects against established tumors in vivo. Therefore, it can be concluded that this novel therapeutic vaccine system, upon further optimization, may shed new light on clinical management of HPV-related carcinomas.
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Cisplatino/uso terapéutico , Proteínas de Unión al ADN/uso terapéutico , Proteínas de Fusión Oncogénica/uso terapéutico , Proteínas Oncogénicas Virales/uso terapéutico , Proteínas E7 de Papillomavirus/uso terapéutico , Vacunas contra Papillomavirus/uso terapéutico , ADP Ribosa Transferasas/uso terapéutico , Animales , Antineoplásicos/uso terapéutico , Toxinas Bacterianas/uso terapéutico , Vacunas contra el Cáncer/inmunología , Línea Celular Tumoral , Sinergismo Farmacológico , Exotoxinas/uso terapéutico , Ratones , Proteínas de Fusión Oncogénica/inmunología , Vacunas contra Papillomavirus/inmunología , Factores de Virulencia/uso terapéutico , Exotoxina A de Pseudomonas aeruginosaRESUMEN
A survey of sequence databases revealed 10 instances of subgroup IIB1 mitochondrial ribosomal introns with 1 to 33 additional nucleotides inserted between the 5' exon and the consensus sequence at the intron 5' end. These 10 introns depart further from the IIB1 consensus in their predicted domain VI structure: In contrast to its basal helix and distal GNRA terminal loop, the middle part of domain VI is highly variable and lacks the bulging A that serves as the branchpoint in lariat formation. In vitro experiments using two closely related IIB1 members inserted at the same ribosomal RNA site in the basidiomycete fungi Grifola frondosa and Pycnoporellus fulgens revealed that both ribozymes are capable of efficient self-splicing. However, whereas the Grifola intron was excised predominantly as a lariat, the Pycnoporellus intron, which possesses six additional nucleotides at the 5' end, yielded only linear products, consistent with its predicted domain VI structure. Strikingly, all of the introns with 5' terminal insertions lack the EBS2 exon-binding site. Moreover, several of them are part of the small subset of group II introns that encode potentially functional homing endonucleases of the LAGLIDADG family rather than reverse transcriptases. Such coincidences suggest causal relationships between the shift to DNA-based mobility, the loss of one of the two ribozyme sites for binding the 5' exon, and the exclusive use of hydrolysis to initiate splicing.
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Intrones/genética , Mutagénesis Insercional/fisiología , ARN Ribosómico/genética , ARN/genética , Secuencia de Bases , Basidiomycota/genética , Basidiomycota/metabolismo , Grifola/genética , Datos de Secuencia Molecular , Mutagénesis Insercional/genética , Conformación de Ácido Nucleico , Nucleótidos/genética , Pycnoporus/genética , Precursores del ARN/química , Precursores del ARN/genética , Empalme del ARN , ARN Catalítico/química , ARN Catalítico/genética , ARN Catalítico/metabolismo , ARN de Hongos/química , ARN de Hongos/genética , ARN Mitocondrial , ARN Ribosómico/químicaRESUMEN
Mitochondrial dysfunction is a hallmark of amyloid ß-peptide (Aß)-induced neurodegeneration of Alzheimer's disease (AD). This study investigated whether mtDNA T8993G mutation-induced complex V inhibition, clinically associated with neurological muscle weakness, ataxia, and retinitis pigmentosa (NARP), is a potential risk factor for AD and the pathological link for long-term exposure of Aß-induced mitochondrial toxicity and apoptosis in NARP cybrids. Using noninvasive fluorescence probe-coupled laser scanning imaging microscopy and NARP cybrids harboring 98% mutant genes along with its parental 143B osteosarcoma cells, we demonstrated that Aß-augmented mitochondrial Ca(2+) (mCa(2+))-independent mitochondrial reactive oxygen species (mROS) formation for a cardiolipin (CL, a major mitochondrial protective phospholipid)-dependent lethal modulation of the mitochondrial permeability transition (MPT). Aß augmented not only the amount but also the propagation rate of mROS-induced mROS formation to significantly depolarize mitochondrial membrane potential (∆Ψ(m)) and reduce mCa(2+) stress. Aß-augmented mROS oxidized and depleted CL, thereby enhances mitochondrial fission and movement retardation, which promoted the NARP-augmented lethal transient-MPT (t-MPT) to switch to its irreversible mode of permanent-MPT (p-MPT). Interestingly, melatonin, a multiple mitochondrial protector, markedly reduced Aß-augmented mROS formation and therefore significantly reduced mROS-mediated depolarization of ∆Ψ(m), fission of mitochondria and retardation of mitochondrial movement to stabilize CL and hence the MPT. In the presence of melatonin, Aß-promoted p-MPT was reversed to a protective t-MPT, which preserved ∆Ψ(m) and lowered elevated mCa(2+) to sublethal levels for an enhanced mCa(2+)-dependent O(2) consumption. Thus, melatonin may potentially rescue AD patients associated with NARP symptoms.
Asunto(s)
Péptidos beta-Amiloides/farmacología , Cardiolipinas/metabolismo , Melatonina/uso terapéutico , Proteínas de Transporte de Membrana Mitocondrial/efectos de los fármacos , Calcio/metabolismo , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/metabolismo , Miopatías Mitocondriales/tratamiento farmacológico , Poro de Transición de la Permeabilidad Mitocondrial , Especies Reactivas de Oxígeno/metabolismo , Retinitis Pigmentosa/tratamiento farmacológicoRESUMEN
Treatment with geldanamycin (GA) leads to an increase in [Ca2+]c and the production of reactive oxygen species (ROS) in rat brain tumor 9L RBT cells. GA-exerted calcium signaling was blocked by BAPTA/AM and EGTA. The effect of GA on [Ca2+]c was significantly reduced in the presence of thapsigargin (TG) and ruthenium red (RR). GA-induced GRP78 expression is significantly decreased in the presence of BAPTA/AM, EGTA and RR, suggesting that the calcium influx from the extracellular space and intracellular calcium store oscillations are contributed to by the calcium mobilization and GRP78 expression induced by GA. The induced GRP78 expression is sensitive to added U73122 and Ro-31-8425, pinpointing the involvement of phospholipase C (PLC) and protein kinase C (PKC) in GA-induced endoplasmic reticulum (ER) stress. The antioxidants N-acetylcysteine (NAC), BAPTA/AM, EGTA and H7 also have significant inhibitory effects on ROS generation. Finally, neither H7 nor NAC was able to affect the calcium response elicited by GA. Our results suggest that the causal signaling cascade during GA-inducted GRP78 expression occurs via a pathway that connects PLC to cytoplasmic calcium increase, PKC activation and, then, finally, ROS generation. Our data provides new insights into the influence of GA on ER stress response in 9L RBT cells.
Asunto(s)
Antibióticos Antineoplásicos/toxicidad , Benzoquinonas/toxicidad , Calcio/metabolismo , Expresión Génica/efectos de los fármacos , Proteínas de Choque Térmico/metabolismo , Lactamas Macrocíclicas/toxicidad , Especies Reactivas de Oxígeno/metabolismo , Animales , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Quelantes/farmacología , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Estrenos/farmacología , Proteínas de Choque Térmico/genética , Indoles/farmacología , Maleimidas/farmacología , Proteína Quinasa C/metabolismo , Pirrolidinonas/farmacología , Ratas , Fosfolipasas de Tipo C/antagonistas & inhibidores , Fosfolipasas de Tipo C/metabolismoRESUMEN
In our previous research, Cordyceps militaris (CM) had a hypoglycemic effect in normal rats. In this study we wanted to elucidate whether CM also had an effect on diabetic rats. Twelve rats with streptozotocin-induced diabetes were separated randomly into 2 groups. First, aqueous extracts of CM 10 mg/kg (CM group) or saline (control group) was fed to the rats; then the plasma glucose levels were assayed. Second, the signaling proteins IRS-1 and GLUT-4 collected from the muscle were detected. Finally, another 2 groups of rats were injected with atropine 0.1 mg/kg intraperitoneally just before the CM/saline feeding, and the assays mentioned above were repeated. Blood glucose decreased 7.2% in the CM group but only 1.5% in the control group (P < 0.05). The IRS-1 signal was 2.9-fold higher than actin in the CM group but only 0.8-fold higher in the control group (P < 0.005). In GLUT-4 signal, the difference was 1.7- vs. 0.6-fold, respectively, compared with actin (P < 0.05). However, atropine injection made CM-induced hypoglycemia or elevation of IRS-1 and GLUT-4 not significant. In conclusion, CM had a hypoglycemic effect in diabetic rats and atropine blocked it. Therefore, the cholinergic activation also was considered to be involved in the hypoglycemic effect of CM in rats with streptozotocin-induced diabetes.
Asunto(s)
Glucemia/efectos de los fármacos , Colinérgicos/farmacología , Fibras Colinérgicas/efectos de los fármacos , Cordyceps/química , Diabetes Mellitus Experimental/tratamiento farmacológico , Hipoglucemiantes/farmacología , Animales , Atropina/antagonistas & inhibidores , Fraccionamiento Químico , Colinérgicos/química , Hipoglucemiantes/química , Masculino , Distribución Aleatoria , Ratas , Ratas Wistar , AguaRESUMEN
Background: Cancer multidrug resistance (MDR) is an important factor that severely affects the chemotherapeutic efficacy. Among various methods to bypass MDR, usage of cytokines, such as tumor necrosis factor alpha (TNFα) is attractive, which exerts antitumor effects of immunotherapeutic response and apoptotic/proinflammatory pathways. Nevertheless, the challenges remain how to implement targeted delivery of TNFα to reduce toxicity and manifest the involved signaling mechanism that subdues MDR. Methods: We synthesized a multifunctional nanosytem, in which TNFα covalently bound to doxorubicin (Dox)-loaded pH-responsive mesoporous silica nanoparticles (MSN) through bi-functional polyethylene glycol (TNFα-PEG-MSN-Hydrazone-Dox) as a robust design to overcome MDR. Results: The salient features of this nanoplatform are: 1) by judicious tailoring of TNFα concentration conjugated on MSN, we observed it could lead to a contrary effect of either proliferation or suppression of tumor growth; 2) the MSN-TNFα at higher concentration serves multiple functions, besides tumor targeting and inducer of apoptosis through extrinsic pathway, it inhibits the expression level of p-glycoprotein (P-gp), a cell membrane protein that functions as a drug efflux pump; 3) the enormous surface area of MSN provides for TNFα functionalization, and the nanochannels accommodate chemotherapeutics, Dox; 4) targeted intracellular release of Dox through the pH-dependent cleavage of hydrazone bonds induces apoptosis by the specific intrinsic pathway; and 5) TNFα-PEG-MSN-Hydrazone-Dox (MSN-Dox-TNFα) could infiltrate deep into the 3D spheroid tumor model through disintegration of tight junction proteins. When administered intratumorally in a Dox-resistant mouse tumor model, MSN-Dox-TNFα exhibited a synergistic therapeutic effect through the collective performances of TNFα and Dox. Conclusion: We hereby develop and demonstrate a multifunctional MSN-Dox-TNFα system with concentration-tailored TNFα that can abrogate the drug resistance mechanism, and significantly inhibit the tumor growth through both intrinsic and extrinsic apoptosis pathways, thus making it a highly potential nanomedicine translated in the treatment of MDR tumors.
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Nanopartículas , Neoplasias , Ratones , Animales , Citocinas , Factor de Necrosis Tumoral alfa , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Doxorrubicina , Apoptosis , Resistencia a Múltiples Medicamentos , Nanopartículas/química , Proliferación Celular , Hidrazonas/farmacología , Hidrazonas/uso terapéutico , Dióxido de Silicio/química , Resistencia a Antineoplásicos , PorosidadRESUMEN
BACKGROUND: There are few studies that have examined the potential of RNA inference (RNAi) to increase protein production in the baculovirus expression vector system (BEVS). Spodoptera frugiperda (fall armyworm) (Sf)-caspase-1-repressed stable cells exhibit resistance to apoptosis and enhancement of recombinant protein production. However, the mechanism of recombinant protein augmentation in baculovirus-infected Caspase-repressed insect cells has not been elucidated. RESULTS: In the current study, we utilized RNAi-mediated Sf-caspase-1-repressed stable cells to clarify how the resistance to apoptosis can enhance both intracellular (firefly luciferase) and extracellular (secreted alkaline phosphatase [SEAP]) recombinant protein production in BEVS. Since the expression of molecular chaperones is strongly associated with the maximal production of exogenous proteins in BEVS, the differential expression of molecular chaperones in baculovirus-infected stable cells was also analyzed in this study. CONCLUSION: The data indicated that the retention of expression of molecular chaperones in baculovirus-infected Sf-caspase-1-repressed stable cells give the higher recombinant protein accumulation.
Asunto(s)
Caspasas/metabolismo , Chaperonas Moleculares/metabolismo , Animales , Baculoviridae/genética , Caspasas/química , Caspasas/genética , Chaperonas Moleculares/genética , Plásmidos/genética , Plásmidos/metabolismo , Interferencia de ARN , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Células Sf9 , Spodoptera , TransfecciónRESUMEN
Mitochondrial dynamics including morphological fission and mitochondrial movement are essential to normal mitochondrial and cellular physiology. This study investigated how mtDNA T8993G (NARP)-induced inhibition of mitochondrial complex V altered mitochondrial dynamics in association with a protective mitochondrial phospholipid, cardiolipin (CL), as a potential therapeutic target. NARP cybrids harboring 98% of mtDNA T8993G genes and its parental osteosarcoma 143B cells were studied for comparison, and protection provided by melatonin, a potent mitochondrial protector, was explored. We demonstrate for the first time that NARP mutation significantly enhances apoptotic death as a result of three distinct lethal mitochondrial apoptotic insults including oxidative, Ca(2+), and lipid stress. In addition, NARP significantly augmented pathological depletion of CL. NARP-augmented depletion of CL results in enhanced retardation of mitochondrial movement and fission and later swelling of mitochondria during all insults. These results suggest that CL is a common and crucial pathological target for mitochondrial apoptotic insults. Furthermore, CL possibly plays a central role in regulating mitochondrial dynamics that are associated with NARP-augmented mitochondrial pathologies. Intriguingly, melatonin, by differentially preserving CL during various stresses (oxidation > Ca(2+) > lipid), rescues differentially CL-altered mitochondrial dynamics and cell death (oxidation > Ca(2+) > lipid). Thus, melatonin, in addition to being a mitochondrial antioxidant to antagonize mitochondrial oxidative stress, a mitochondrial permeability transition modulator to antagonize mitochondrial Ca(2+) stress, may stabilize directly CL to prevent its oxidization and/or depletion and, therefore, exerts great potential in rescuing CL-dependent mitochondrial dynamics-associated mitochondrial pathologies for treatment of NARP-induced pathologies and diseases.
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Proteína C-Reactiva/genética , Cardiolipinas/metabolismo , ADN Mitocondrial/genética , Melatonina/metabolismo , Mitocondrias/metabolismo , Mutación , Proteínas del Tejido Nervioso/genética , Análisis de Varianza , Ácido Araquidónico/farmacología , Proteína C-Reactiva/metabolismo , Línea Celular Tumoral , Movimiento Celular/fisiología , Supervivencia Celular , Células Cultivadas , Ingeniería Genética , Técnicas Genéticas , Humanos , Proteínas del Tejido Nervioso/metabolismo , Estrés Oxidativo/fisiología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
7,7''-Dimethoxyagastisflavone (DMGF), a biflavonoid isolated from the needles of Taxus × media cv. Hicksii, was evaluated for its antiproliferative and antineoplastic effects in three human cancer cell lines. Interestingly, DMGF caused cell death via different pathways in different cancer cells. DMGF induced apoptosis, activated caspase-3 activity and changed the mitochondrial membrane potential in HT-29 human colon cancer cells. However, the apoptotic pathway is not the major pathway involved in DMGF-induced cell death in A549 human lung cancer cells and HepG2 human hepatoma cells. Treatment with 3-MA, an inhibitor of autophagy, significantly decreased DMGF-induced cell death in HepG2 and A549 cells, but did not affect DMGF-induced cell death in HT-29 cells. Following DMGF treatment, the HepG2 cells increased expression of LC3B-II, a marker used to monitor autophagy in cells. Thus, DMGF induced apoptotic cell death in HT-29 cells, triggered both apoptotic and autophagic death in A549 cells and induced autophagic cell death in HepG2 cells.
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Antineoplásicos Fitogénicos/farmacología , Apoptosis , Autofagia , Biflavonoides/farmacología , Alcaloides/aislamiento & purificación , Antineoplásicos Fitogénicos/aislamiento & purificación , Biflavonoides/aislamiento & purificación , Biomarcadores de Tumor/metabolismo , Caspasa 3/metabolismo , Supervivencia Celular , Cromatografía Líquida de Alta Presión , Activación Enzimática , Células HT29 , Células Hep G2 , Humanos , Concentración 50 Inhibidora , Potencial de la Membrana Mitocondrial , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Taxoides/aislamiento & purificación , Taxus/químicaRESUMEN
Previous studies have shown that Cordyceps militaris (CM) has a hypoglycemic effect, but the actual mechanism remains unclear. This study explored the hypoglycemic mechanism of aqueous extracts of CM in normal Wistar rats. First, the optimal dose of CM for lowering plasma glucose and insulin secretion was tested. Further, atropine and hemicholinium-3 (HC-3) were injected and a western blot was used to investigate insulin signaling. It was found that 10 mg/kg CM extracts had a stronger hypoglycemic effect than a higher dose (100 mg/kg); therefore, a dose of 10 mg/kg was used in subsequent experiments. In normal rats, CM extracts decreased plasma glucose by 21.0% and induced additional insulin secretion by 54.5% after 30 min. When atropine or HC-3 was injected, CM induced a hypoglycemic effect, but the enhancement of insulin secretion was blocked. By western blotting, significant increases in the insulin receptor substrate 1 (IRS-1) and glucose transporter 4 (GLUT-4) were observed after CM feeding. However, the elevation of these signaling proteins was abolished by atropine or HC-3. Taken together, these findings indicate that CM can lower plasma glucose via the stimulation of insulin secretion and cholinergic activation involved in the hypoglycemic mechanism of normal Wistar rats.
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Glucemia/efectos de los fármacos , Colinérgicos/farmacología , Cordyceps/química , Hipoglucemiantes/farmacología , Insulina/metabolismo , Animales , Atropina/administración & dosificación , Atropina/farmacología , Glucemia/metabolismo , Western Blotting , Fibras Colinérgicas/efectos de los fármacos , Fibras Colinérgicas/metabolismo , Modelos Animales de Enfermedad , Transportador de Glucosa de Tipo 4/metabolismo , Hemicolinio 3/administración & dosificación , Hemicolinio 3/farmacología , Proteínas Sustrato del Receptor de Insulina/metabolismo , Secreción de Insulina , Masculino , Ratas , Ratas WistarRESUMEN
Metastasis to secondary sites remains the leading cause of nasopharyngeal carcinoma (NPC)-associated death. In order to identify the candidate protein(s) responsible for the differential metastatic capacity, the protein expression profiling between NPC cell line CNE-2 and its highly metastatic subclone S-18 were compared by 2-DE. In total, 18 spots were differentially expressed between these two cell lines. Among all, seven proteins were identified with further MS analysis. Western blotting further validated upregulation of HSP27 and ezrin, and downregulation of valosin containing protein and keratin 18 in S-18. Moreover, the knockdown of HSP27 was found to significantly decrease the invasive ability of S-18. On the other hand, overexpression of HSP27 in NP460 cells, which generated little endogenous HSP27 and less invasive, was noted to gain enhanced metastatic capability. Real-time PCR confirmed that the transcriptional levels of NF-κB and MMP9, MMP11 were downregulated after inhibition of HSP27 in S-18, which implicated that HSP27 enhanced the metastatic property of NPC cells probably via the NF-κB-mediated activation of MMPs. The findings in this work provided us a platform for further elucidating the underlying mechanisms of NPC metastasis and demonstrated that HSP27 would be a valid target for anti-cancer drug development.
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Línea Celular Tumoral , Proteínas de Choque Térmico HSP27/metabolismo , Neoplasias Nasofaríngeas/patología , Metástasis de la Neoplasia , Proteínas de Neoplasias/metabolismo , Proteoma/análisis , Carcinoma , Electroforesis en Gel Bidimensional/métodos , Proteínas de Choque Térmico HSP27/química , Proteínas de Choque Térmico HSP27/genética , Humanos , Espectrometría de Masas/métodos , Metaloproteinasas de la Matriz/genética , Metaloproteinasas de la Matriz/metabolismo , FN-kappa B/metabolismo , Carcinoma Nasofaríngeo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteómica/métodos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismoRESUMEN
S100P was recently found to be overexpressed in a variety of cancers and is considered a potential target for cancer therapy, but the functional role or mechanism of action of S100P in colon cancer is not fully understood. In the present study, we knocked down the gene expression of S100P in colon cancer cells using lentivirus-mediated RNA interference. This step resulted in significant inhibition of cancer cell growth, migration and invasion in vitro and tumor growth and liver metastasis in vivo. Moreover, S100P downstream target proteins were identified by proteomic analysis in colon cancer DLD-1 cells with deletion of S100P. Knockdown of S100P led to downregulation of thioredoxin 1 and ß-tubulin and upregulation of Rho guanosine diphosphate (GDP) dissociation inhibitor α (RhoGDIA), all potential therapeutic targets in cancer. Taken together, these findings suggest that S100P plays an important role in colon tumorigenesis and metastasis, and the comprehensive and comparative analyses of proteins associated with S100P could contribute to understanding the downstream signal cascade of S100P, leading to tumorigenesis and metastasis.
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Proteínas de Unión al Calcio/genética , Neoplasias del Colon/genética , Proteínas de Neoplasias/genética , Proteómica/métodos , Interferencia de ARN , Animales , Western Blotting , Proteínas de Unión al Calcio/metabolismo , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Regulación hacia Abajo , Electroforesis en Gel Bidimensional , Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Humanos , Lentivirus/genética , Masculino , Ratones , Ratones Desnudos , Metástasis de la Neoplasia , Proteínas de Neoplasias/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Tiorredoxinas/metabolismo , Tubulina (Proteína)/metabolismo , Carga Tumoral/genética , Regulación hacia Arriba , Ensayos Antitumor por Modelo de Xenoinjerto , Inhibidores de la Disociación del Nucleótido Guanina rho-EspecíficoRESUMEN
Dual color fluorescence cross correlation spectroscopy (FCCS) was used to investigate quantitatively the binding kinetics of tumor necrosis factor (TNFα) with TNFα antibody (anti-TNFα) following fluorescent labeling. Through the analysis of the auto correlation curves of fluorescence correlation spectroscopy (FCS), diffusion coefficients of 100.06 ± 4.9 µm(2) s(-1) and 48.96 ± 2.52 µm(2) s(-1) for Alexa488-TNFα and Atto647N-anti-TNFα were obtained. In addition, the calculated hydrodynamic diameters of the Alexa488-TNFα and Atto647N-anti-TNFα were approximately 4.89 ± 0.24 nm and 9.99 ± 0.52 nm, respectively, which agrees with the values of 5.20 ± 1.23 nm and 9.28 ± 0.86 nm for the native TNFα and the anti-TNFα as determined from dynamic light scattering measurements. For the binding kinetics, association (k(on)) and dissociation (k(off)) rate constants were (1.13 ± 0.08) × 10(4) M(-1) s(-1) and (1.53 ± 0.19) × 10(-3) s(-1) while the corresponding dissociation constant (K(d)) at 25 °C was (1.36 ± 0.10) × 10(-7) M. We believe this is the first report on the binding kinetics for TNFα-antibody recognition in the homogeneous phase. Using this technology, we have shown that controlled experiments can be performed to gain insight into molecular mechanisms involved in the immune response.
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Anticuerpos/química , Espectrometría de Fluorescencia/métodos , Factor de Necrosis Tumoral alfa/química , Factor de Necrosis Tumoral alfa/inmunología , Anticuerpos/inmunología , Color , Colorantes Fluorescentes/química , Hidrodinámica , Cinética , Unión Proteica , Factor de Necrosis Tumoral alfa/análisisRESUMEN
BACKGROUND: Eosinophilic granulocytes are important for the human immune system. Many cationic proteins with cytotoxic activities, such as eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN), are released from activated eosinophils. ECP, with low RNase activity, is widely used as a biomarker for asthma. ECP inhibits cell viability and induces apoptosis to cells. However, the specific pathway underlying the mechanisms of ECP-induced cytotoxicity remains unclear. This study investigated ECP-induced apoptosis in bronchial epithelial BEAS-2B cells and elucidated the specific pathway during apoptosis. RESULTS: To address the mechanisms involved in ECP-induced apoptosis in human BEAS-2B cells, investigation was carried out using chromatin condensation, cleavage of poly (ADP-ribose) polymerase (PARP), sub-G1 distribution in cell cycle, annexin V labeling, and general or specific caspase inhibitors. Caspase-8-dependent apoptosis was demonstrated by cleavage of caspase-8 after recombinant ECP treatment, accompanied with elevated level of tumor necrosis factor alpha (TNF-alpha). Moreover, ECP-induced apoptosis was effectively inhibited in the presence of neutralizing anti-TNF-alpha antibody. CONCLUSION: In conclusion, our results have demonstrated that ECP increased TNF-alpha production in BEAS-2B cells and triggered apoptosis by caspase-8 activation through mitochondria-independent pathway.
Asunto(s)
Apoptosis , Proteína Catiónica del Eosinófilo/metabolismo , Eosinófilos/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Anexina A5/metabolismo , Anticuerpos/metabolismo , Caspasa 8/metabolismo , Línea Celular Tumoral , Proteína Catiónica del Eosinófilo/genética , Neurotoxina Derivada del Eosinófilo/metabolismo , Eosinófilos/inmunología , Fase G1 , Humanos , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Helicobacter pylori is a potent carcinogen associated with gastric cancer malignancy. Recently, H. pylori Heat shock protein 60 (HpHSP60) has been reported to promote cancer development by inducing chronic inflammation and promoting tumor cell migration. This study demonstrates a role for HpHSP60 in angiogenesis, a necessary precursor to tumor growth. We showed that HpHSP60 enhanced cell migration and tube formation, but not cell proliferation, in human umbilical vein endothelial cells (HUVECs). HpHSP60 also indirectly promoted HUVEC proliferation when HUVECs were co-cultured with supernatants collected from HpHSP60-treated AGS or THP-1 cells. The angiogenic array showed that HpHSP60 dramatically induced THP-1 cells and HUVECs to produce the chemotactic factors IL-8 and GRO. Inhibition of CXCR2, the receptor for IL-8 and GRO, or downstream PLCbeta2/Ca2+-mediated signaling, significantly abolished HpHSP60-induced tube formation. In contrast, suppression of MAP K or PI3 K signaling did not affect HpHSP60-mediated tubulogenesis. These data suggest that HpHSP60 enhances angiogenesis via CXCR2/PLCbeta2/Ca2+ signal transduction in endothelial cells.
Asunto(s)
Proteínas Bacterianas/metabolismo , Transformación Celular Neoplásica , Endotelio Vascular/microbiología , Helicobacter pylori , Neovascularización Patológica/microbiología , Receptores de Interleucina-8B/metabolismo , Movimiento Celular , Proliferación Celular , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Humanos , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patologíaRESUMEN
BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a common disease in the elderly population and is characterized by airway inflammation. Whether it is a progressive condition resulting from allergic inflammation or a distinct condition involving a pathogen-induced reaction remains unclear. OBJECTIVES: To determine the role of allergic inflammation in the pathogenesis of elderly COPD. METHODS: A total of 63 elderly adults (21 mite-allergic COPD patients, 29 non-allergic COPD patients, and 13 normal controls) were recruited in this study. The serum-specific IgE for mites, level of interleukin-5 (IL-5), IL-8, leptin, adiponectin, regulated upon activation normal T cell expressed and secreted (RANTES), growth-related oncogene-α (GRO-α), vitamin E, and glutathione (GSH) were determined. RESULTS: The serum levels of GRO-α in patients with COPD were higher in comparison to normal controls (105.8 ± 32.7 vs. 7.5 ± 7.5 pg/mL, p= .021). Compared to patients with non-allergic COPD, patients with mite allergies had a higher serum level of IL-8 (63.2 ± 12.6 vs. 35.0 ± 8.2 pg/mL, p= .022). Although both IL-5 and RANTES levels were increased in COPD patients, there were no significant differences between allergic and non-allergic COPD. There were also no differences in serum levels of leptin, adiponectin, vitamin E, and GSH between COPD patients and normal controls. CONCLUSIONS: The increased serum levels of GRO-α indicate that it may have potential as a candidate biomarker for elderly COPD patients. There was no difference of eosinophils-related chemokines in allergic and non-allergic COPD. These results indicated that both adipokines and eosinophil-related chemokines only play trivial roles in the pathogenesis of COPD.
Asunto(s)
Eosinófilos/inmunología , Hipersensibilidad/inmunología , Neutrófilos/inmunología , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Acaridae/inmunología , Anciano de 80 o más Años , Animales , Biomarcadores/sangre , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inmunoglobulina E/sangre , Inflamación/inmunología , Mediadores de Inflamación/sangre , Masculino , Pyroglyphidae/inmunologíaRESUMEN
Boehmeria nivea extract (BNE) is widely used in southern Taiwan as a folk medicine for hepato-protection and hepatitis treatment. In previous studies, we demonstrated that BNE could reduce the supernatant hepatitis B virus (HBV) DNA in HBV-producing HepG2 2.2.15 cells. In the present study, we established an animal model of HBV viremia and used it to validate the efficacy of BNE in vivo. In this animal model, serum HBV DNA and HBsAg were elevated in accordance with tumor growth. To evaluate the anti-HBV activity of BNE, HBV-viremia mice were built up after one subcutaneous inoculation of HepG2 2.2.15 tumor cells in severe combined immunodeficiency mice over 13 days. The levels of serum HBV DNA were elevated around 10(5)-10(6) copies per milliliter. Both oral and intraperitoneal administration of BNE were effective at inhibiting the production of HBsAg and HBV DNA, whereas tumor growth was not affected by all test articles. Intraperitoneal administration of BNE appeared to have greater potential to inhibit serum HBV DNA levels compared with oral administration under the same dosage. Notably, reduced natural killer cell activity was also observed after high dosage of BNE administration, and this correlated with reduced serum HBV DNA. In conclusion, BNE exhibited potential anti-HBV activity in an animal model of HBV viremia.
RESUMEN
Two brain disease-related genes, one coding for the protease inhibitor SERPINI1 which is down-regulated in brain tumors, and the other for the PDCD10 programmed cell death gene which is often mutated in cerebral cavernous malformation, are closely adjacent in a head-to-head configuration and separated by only 851 bp on human chromosome 3q26. The 851-bp intergenic region contains a GC-rich 175-bp minimal bidirectional promoter which is essential for transcriptional activation of the two flanking genes. The oncogenic c-Myc transcription factor was identified to bind to a non-canonical E-box element (5'-CATGCG-3') of the minimal bidirectional promoter to drive both gene expressions. Methylation at the specific C nucleotide within the E-box sequence (5'-CATG(m)CG-3'), however, would severely interfere with the binding of c-Myc to the E-box. These results suggest that c-Myc plays an important role in regulating the coordinated transcription of the PDCD10-SERPINI1 bidirectional gene pair, and is possibly involved in differential expressions of these two neighboring genes in central nervous system diseases such as brain cancer.