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Microorganisms are shown to actively partition their intracellular resources, such as proteins, for growth optimization. Recent experiments have begun to reveal molecular components unpinning the partition; however, quantitatively, it remains unclear how individual parts orchestrate to yield precise resource allocation that is both robust and dynamic. Here, we developed a coarse-grained mathematical framework that centers on guanosine pentaphosphate (ppGpp)-mediated regulation and used it to systematically uncover the design principles of proteome allocation in Escherichia coli. Our results showed that the cellular ability of resource partition lies in an ultrasensitive, negative feedback-controlling topology with the ultrasensitivity arising from zero-order amino acid kinetics and the negative feedback from ppGpp-controlled ribosome synthesis. In addition, together with the time-scale separation between slow ribosome kinetics and fast turnovers of ppGpp and amino acids, the network topology confers the organism an optimization mechanism that mimics sliding mode control, a nonlinear optimization strategy that is widely used in man-made systems. We further showed that such a controlling mechanism is robust against parameter variations and molecular fluctuations and is also efficient for biomass production over time. This work elucidates the fundamental controlling mechanism of E. coli proteome allocation, thereby providing insights into quantitative microbial physiology as well as the design of synthetic gene networks.
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Tricalcium phosphate (TCP) has gained attention due to its interconnected porous structures which promote fibrovascular invasion and bony replacement. Moreover, when gelatin is added and crosslinked with genipin (GGT), TCP exhibits robust biocompatibility and stability, making it an excellent bone substitute. In this study, we incorporated emodin and lumbrokinase (LK) into GGT to develop an antibacterial biomaterial. Emodin, derived from various plants, possesses antibacterial and anti-inflammatory properties. LK comprises proteolytic enzymes extracted from the earthworm Lumbricus rubellus and exhibits fibrinolytic activity, enabling it to dissolve biofilms. Additionally, LK stimulates osteoblast activity while inhibiting osteoclast differentiation. GGT was combined with emodin and lumbrokinase to produce the GGTELK composite. The biomedical effects of GGTELK were assessed through in vitro assays and an ex vivo bone defect model. The GGTELK composite demonstrated antibacterial properties, inhibiting the growth of S. aureus and reducing biofilm formation. Moreover, it exhibited anti-inflammatory effects by reducing the secretion of IL-6 in both in vivo cell experiments and the ex vivo model. Therefore, the GGTELK composite, with its stability, efficient degradation, biocompatibility, and anti-inflammatory function, is expected to serve as an ideal bone substitute.
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Achieving cellular uptake is a central challenge for novel antibiotics targeting Gram-negative bacterial pathogens. One strategy is to hijack the bacterial iron transport system by siderophore-antibiotic conjugates that are actively imported into the cell. This was realized with the MECAM-ampicillin conjugate LP-600 we recently reported that was highly active against E. coli. In the present study, we investigate a paradoxical regrowth of E. coli upon treatment of LP-600 at concentrations 16-32 times above the minimum inhibitory concentration (MIC). The phenomenon, coined "Eagle-effect" in other systems, was not due to resistance formation, and it occurred for the siderophore conjugate but not for free ampicillin. To investigate the molecular imprint of the Eagle effect, a combined transcriptome and untargeted metabolome analysis was conducted. LP-600 induced the expression of genes involved in iron acquisition, SOS response, and the e14 prophage upon regrowth conditions. The Eagle effect was diminished in the presence of sulbactam, which we ascribe to a putative synergistic antibiotic action but not to ß-lactamase inhibition. The study highlights the relevance of the Eagle effect for siderophore conjugates. Through the first systematic -omics investigations, it also demonstrates that the Eagle effect manifests not only in a paradoxical growth but also in unique gene expression and metabolite profiles.
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Águilas , Sideróforos , Animales , Sideróforos/farmacología , Sideróforos/metabolismo , Escherichia coli , Águilas/metabolismo , Antibacterianos/farmacología , Antibacterianos/metabolismo , Ampicilina/farmacología , Hierro/metabolismoRESUMEN
Our research was designed to evaluate the effect on bone regeneration with 3-dimensional (3D) printed polylactic acid (PLA) and 3D printed polycaprolactone (PCL) scaffolds, determine the more effective option for enhancing bone regeneration, and offer tentative evidence for further research and clinical application. Employing the 3D printing technique, the PLA and PCL scaffolds showed similar morphologies, as confirmed via scanning electron microscopy (SEM). Mechanical strength was significantly higher in the PLA group (63.4 MPa) than in the PCL group (29.1 MPa) (p < 0.01). Average porosity, swelling ratio, and degeneration rate in the PCL scaffold were higher than those in the PLA scaffold. SEM observation after cell coculture showed improved cell attachment and activity in the PCL scaffolds. A functional study revealed the best outcome in the 3D printed PCL-TGF-ß1 scaffold compared with the 3D printed PCL and the 3D printed PCL-Polydopamine (PDA) scaffold (p < 0.001). As confirmed via SEM, the 3D printed PCL- transforming growth factor beta 1 (TGF-ß1) scaffold also exhibited improved cell adhesion after 6 h of cell coculture. The 3D printed PCL scaffold showed better physical properties and biocompatibility than the 3D printed PLA scaffold. Based on the data of TGF-ß1, this study confirms that the 3D printed PCL scaffold may offer stronger osteogenesis.
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The development of novel drugs against Gram-negative bacteria represents an urgent medical need. To overcome their outer cell membrane, we synthesized conjugates of antibiotics and artificial siderophores based on the MECAM core, which are imported by bacterial iron uptake systems. Structures, spin states, and iron binding properties were predicted in silico using density functional theory. The capability of MECAM to function as an effective artificial siderophore in Escherichia coli was proven in microbiological growth recovery and bioanalytical assays. Following a linker optimization focused on transport efficiency, five ß-lactam and one daptomycin conjugates were prepared. The most potent conjugate 27 showed growth inhibition of Gram-positive and Gram-negative multidrug-resistant pathogens at nanomolar concentrations. The uptake pathway of MECAMs was deciphered by knockout mutants and highlighted the relevance of FepA, CirA, and Fiu. Resistance against 27 was mediated by a mutation in the gene encoding ExbB, which is involved in siderophore transport.
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Antibacterianos/farmacología , Benzamidas/química , Benzamidas/farmacología , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Hidroxibenzoatos/química , Hidroxibenzoatos/farmacología , Sideróforos/farmacología , Antibacterianos/síntesis química , Antibacterianos/química , Relación Dosis-Respuesta a Droga , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Sideróforos/síntesis química , Sideróforos/química , Relación Estructura-ActividadRESUMEN
Uneven distribution of pores, lack of connection between holes, low reproducibility, insufficient mechanical strength, and incomplete volatility of organic solvents are some problems associated with traditional tissue engineering methods for bone defect repair. These characteristics reduce the quality and stability of products. This study uses 3D printing (3DP) to fabricate a biocompatible poly(lactic) acid-based scaffold for repairing bone tissue. Hence, three different types of scaffolds are assessed: a freeze-dried polylactic acid (PLA) scaffold constructed using the traditional freeze-extraction method; a 3D-PLA scaffold produced through the 3DP technique; and a 3D-PLA-bone morphogenetic protein-2 (BMP-2) scaffold that is prepared using 3DP technology, with the addition of BMP-2. To enhance biological activity, polydopamine (pDA) is used to graft BMP-2 on the surface of the 3D-PLA-BMP-2 scaffold. Then, the scaffolds are implanted into the bilateral femoral condyles of rabbits, and their ability to repair the bone tissue defects is tested. The results of the experiments reveal that the 3DP scaffolds are more biocompatible than the ones produced through the traditional manufacturing methods because they enhance cell adhesion and differentiation after pDA modification and BMP-2 fixation. In the future, the 3DP products may be applied for the repair of larger bone defects in the clinical setting.
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Desarrollo Óseo/efectos de los fármacos , Proteína Morfogenética Ósea 2/farmacología , Regeneración Ósea/efectos de los fármacos , Poliésteres/química , Impresión Tridimensional , Andamios del Tejido/química , Fosfatasa Alcalina/metabolismo , Animales , Femenino , Implantación de Prótesis , Conejos , Tomografía Computarizada por Rayos XRESUMEN
BACKGROUND: Brain amyloid deposition is one of the main pathological characteristics of Alzheimer's disease (AD). Soluble oligomers formed during the process that causes ß-amyloid (Aß) to aggregate into plaques are considered to have major neurotoxicity. Currently, drug development for the treatment of Alzheimer's disease has encountered serious difficulties. Our newly proposed solution is to accelerate the aggregation of Aß to reduce the amount of cytotoxic Aß oligomers in brain tissue. This strategy differs from the existing strategy of reducing the total Aß content and the number of amyloid plaques. METHOD: In this study, we screened a small library and found that a flavonoid compound (ZGM1) promoted the aggregation of ß-amyloid (Aß). We further verified the binding of ZGM1 to Aß42 using a microscale thermophoresis (MST) assay. Subsequently, we used dot blotting (DB), transmission electron microscopy (TEM), and thioflavin T fluorescence (ThT) measurements to study the aggregation of Aß under the influence of ZGM1. By using cell experiments, we determined whether ZGM1 can inhibit the cytotoxicity of Aß. Finally, we studied the protective effects of ZGM1 on cognitive function in APPswe/PS1 mice via behavioral experiments and measured the number of plaques in the mouse brain by thioflavin staining. RESULTS: ZGM1 can bind with Aß directly and mediate a new Aß assembly process to form reticular aggregates and reduce the amount of Aß oligomers. Animal experiments showed that ZGM1 can significantly improve cognitive dysfunction and that Aß plaque deposition in the brain tissue of mice in the drug-administered group was significantly increased. CONCLUSION: Our research suggests that promoting Aß aggregation is a promising treatment method for AD and deserves further investigation.
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Encéfalo/efectos de los fármacos , Disfunción Cognitiva/tratamiento farmacológico , Flavonoides/farmacología , Placa Amiloide/tratamiento farmacológico , Agregado de Proteínas/efectos de los fármacos , Precursor de Proteína beta-Amiloide/genética , Animales , Encéfalo/metabolismo , Encéfalo/patología , Disfunción Cognitiva/metabolismo , Disfunción Cognitiva/patología , Modelos Animales de Enfermedad , Flavonoides/uso terapéutico , Ratones , Ratones Transgénicos , Placa Amiloide/metabolismo , Placa Amiloide/patología , Presenilina-1/genética , Resultado del TratamientoRESUMEN
In the original publication of this article [1], there is an error in Fig. 4A.
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Bone scaffold surface characterization is important for improving cell adhesion, migration, and differentiation. In this study, bone morphogenetic protein-2 (BMP-2) was immobilized to the surface of the gelatin/hydroxyapatite composite using avidin-biotin binding system to produce a bone-tissue engineering scaffold. Firstly, hydroxyapatite particles reacted with hexamethylene diisocyanate and then the terminal group was converted into a primary amine group. Avidin was then immobilized on the surfaces of hydroxyapatite particles using N-ethyl-N'-(3-(dimethylamino)propyl) carbodiimide and N-hydroxysuccinimide as coupling agents. Gelatin was blended with avidin-modified hydroxyapatite and pure hydroxyapatite to obtain gelain/hydroxyapatite composite. The composite was then cross-linked with glutaraldehyde. Finally, biotin-conjugated BMP-2 was immobilized on the surface of the composite via avidin-biotin binding. In vitro study indicated that BMP-2-immobilized composite film had a higher ALP activity than that composite film without BMP-2. The composite scaffolds were then implanted into rabbit skulls to check bone-tissue regeneration. Ultrasound and micro-CT scans demonstrated that neovascularization and new bone formation in the BMP-2-immobilized composite scaffolds were higher than those in composite scaffolds without BMP-2. Histological evaluation result was similar to that of the micro-CT. Therefore, the surface immobilization of BMP-2 could effectively improve osteogenesis in the gelatin/hydroxyapatite composite scaffold.
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Materiales Biocompatibles/uso terapéutico , Proteína Morfogenética Ósea 2/uso terapéutico , Regeneración Ósea/efectos de los fármacos , Durapatita/uso terapéutico , Gelatina/uso terapéutico , Proteínas Inmovilizadas/uso terapéutico , Animales , Avidina/uso terapéutico , Osteogénesis/efectos de los fármacos , Conejos , Cráneo/cirugía , Andamios del Tejido/químicaRESUMEN
BACKGROUND: Interleukin-11 (IL-11), a dominant IL-6 family cytokine, is involved in tumorigenesis, tumor progression and differentiation in colon cancer cells. IL-11 signaling has been recently identified as a potential therapeutic target in colon cancer. Bazedoxifene, a third- generation selective estrogen modulator approved by the Food and Drug Administration (FDA), is a novel inhibitor of IL-11/GP130 signaling discovered by docking modeling. METHODS: In this study, the inhibition efficacy of bazedoxifene in colon cancer cells and its potential mechanism were investigated in vitro and in vivo by using MTT cell viability assay, BrdU cell proliferation assay, colony formation assay, wound-healing/cell migration assay, immunofluorescence, western blot assay and the mouse xenograft tumor model. RESULTS: Bazedoxifene inhibits phosphorylation of signal transducer and activator of transcription 3 (p-STAT3) and its nuclear translocation induced by IL-11 in colon cancer cells. It also inhibits p-STAT3 induced by IL-6 and IL-11 but not by OSM or STAT1 phosphorylation induced by INF-γ in human colon cancer cells. In addition, bazedoxifene can significantly inhibit phosphorylation of AKT and STAT3 downstream targets. Furthermore, bazedoxifene alone or together with oxaliplatin can significantly induce apoptosis, inhibit cell viability, cell colony formation and cell migration in colon cancer cells. Knock-down of IL-11R can reduce the sensitivity of colon cancer cells to bazedoxifene. IL-11 can reduce the efficacy of oxaliplatin-mediated inhibition of cell viability. Consistent with in vitro findings, bazedoxifene alone also attenuated HCT-15 xenograft tumor burden and reduced p-STAT3, p-AKT and p-ERK in vivo. Its combination with oxaliplatin attenuated DLD-1 xenograft tumor burden and reduced p-STAT3 in vivo. CONCLUSIONS: Taken together, these results support bazedoxifene as a novel and effective therapeutic agent targeting IL-11/GP130 signaling for human colorectal cancer therapy.
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Neoplasias del Colon/tratamiento farmacológico , Receptor gp130 de Citocinas/metabolismo , Indoles/uso terapéutico , Moduladores Selectivos de los Receptores de Estrógeno/uso terapéutico , Línea Celular Tumoral , Humanos , Indoles/farmacología , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , TransfecciónRESUMEN
Oral cancer is the eighth most common type of cancer among men worldwide, with an age-standardized rate of 6.3 per 100,000, and is the fourth leading cause of cancer-associated mortality among men in Taiwan. Cisplatin and 5-fluorouracil (5-FU) are two of the most frequently utilized chemotherapy drugs for the treatment of oral cancer. Although oral cancer patients initially benefit from chemotherapy with these drugs, they may develop resistance to them, which worsens their prognosis and reduces survival rates. It has been reported that increased levels of epidermal growth factor receptor (EGFR) and multidrug resistance-associated protein 2 (MRP2) induce drug resistance in numerous types of human cancer. Therefore, the present study employed lentivirus vector-mediated RNA interference (RNAi) in order to target the genes encoding EGFR and MRP2 in the oral squamous cell carcinoma cell line OC2. It was observed that RNAi-mediated downregulation of EGFR or MRP2 increased the sensitivity to 5-FU and cisplatin in OC2 cells. Downregulation of EGFR resulted in significant suppression of OC2 tumor growth following 5-FU administration. However, simultaneous downregulation of the two genes did not further suppress the tumor growth, indicating that MRP2 does not have a significant role in the chemosensitivity of EGFR-downregulated cells to 5-FU. In contrast, downregulation of MRP2 was demonstrated to significantly enhance the therapeutic effects of cisplatin in EGFR-downregulated OC2 tumors. The observation that the expression of MRP2 was positively correlated with the level of cisplatin resistance in cells suggests that RNAi-mediated downregulation of MRP2 may be applicable as a therapeutic approach toward reversing MRP2-dependent cisplatin resistance in oral cancer.
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Innate immune responses are important for pathogen elimination and adaptive immune response activation. However, excess inflammation may contribute to immunopathology and disease progression (e.g. inflammation-associated hepatocellular carcinoma). Immune modulation resulting from pattern recognition receptor-induced responses is a potential strategy for controlling immunopathology and related diseases. This study demonstrates that the mycotoxin patulin suppresses Toll-like receptor- and RIG-I/MAVS-dependent cytokine production through GSH depletion, mitochondrial dysfunction, the activation of p62-associated mitophagy, and p62-TRAF6 interaction. Blockade of autophagy restored the immunosuppressive activity of patulin, and pharmacological activation of p62-dependent mitophagy directly reduced RIG-I-like receptor-dependent inflammatory cytokine production. These results demonstrated that p62-dependent mitophagy has an immunosuppressive role to innate immune response and might serve as a potential immunomodulatory target for inflammation-associated diseases.
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Inmunidad Innata/efectos de los fármacos , Mitofagia/efectos de los fármacos , Micotoxinas/farmacología , Patulina/farmacología , Proteína Sequestosoma-1/inmunología , Animales , Células HEK293 , Humanos , Ratones , Mitofagia/inmunología , Células RAW 264.7RESUMEN
Oral squamous cell carcinoma (OSCC) constitutes >90% of oral cancers and is the sixth most common malignancy among males worldwide and the fourth leading cause of death due to cancer among males in Taiwan. However, most patients do not receive a diagnosis of OSCC until the late stages, which have a lower survival rate. The use of molecular marker analysis to identify early-stage OSCC would permit optimal timing for treatments and consequently prolong survival. The aim of this study was to identify biomarkers of OSCC using the Illumina GoldenGate Methylation Cancer Panel, which comprised a total of 1,505 CpG sites covering 807 genes. Samples of buccal mucosa resected from 40 OSCC patients and normal tissue samples obtained from 15 patients (normal mucosa from OSCC patients or from patients undergoing surgery unrelated to OSCC) were analyzed. Fms-related tyrosine kinase 4 (FLT4) methylation exhibited a perfect specificity for detecting OSCC, with an area under the receiver operating characteristic curve of 0.91 for both all-stage and early-stage OSCC. Methylation of 7 genes (ASCL1, FGF3, FLT4, GAS7, KDR, TERT, and TFPI2) constitutes the top-20 panels for detecting OSCC. The top-20 panels for detecting early-stage OSCC contain 8 genes: ADCYAP1, EPHA7, FLT4, GSTM2, KDR, MT1A, NPY, and TFPI2. FLT4 RNA expression and methylation level were validated using RT-PCR and a pyrosequencing methylation assay. The median level of FLT4 expression was 2.14-fold for normal relative to OSCC tissue samples (P < 0.0001). Among the 8 pyrosequenced FLT4 CpG sites, methylation level was much higher in the OSCC samples. In conclusion, methylation statuses of selected genes, and especially FLT4, KDR, and TFPI2, might be of great potential as biomarkers for early detection of buccal OSCC.
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Biomarcadores de Tumor/metabolismo , Carcinoma de Células Escamosas/metabolismo , Metilación de ADN , Neoplasias de la Boca/metabolismo , Adulto , Islas de CpG , Glicoproteínas/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: The dysregulation of transforming growth factor-ß (TGF-ß) signaling plays a crucial role in ovarian carcinogenesis and in maintaining cancer stem cell properties. Classified as a member of the ATP-binding cassette (ABC) family, ABCA1 was previously identified by methylated DNA immunoprecipitation microarray (mDIP-Chip) to be methylated in ovarian cancer cell lines, A2780 and CP70. By microarray, it was also found to be upregulated in immortalized ovarian surface epithelial (IOSE) cells following TGF-ß treatment. Thus, we hypothesized that ABCA1 may be involved in ovarian cancer and its initiation. RESULTS: We first compared the expression level of ABCA1 in IOSE cells and a panel of ovarian cancer cell lines and found that ABCA1 was expressed in HeyC2, SKOV3, MCP3, and MCP2 ovarian cancer cell lines but downregulated in A2780 and CP70 ovarian cancer cell lines. The reduced expression of ABCA1 in A2780 and CP70 cells was associated with promoter hypermethylation, as demonstrated by bisulfite pyro-sequencing. We also found that knockdown of ABCA1 increased the cholesterol level and promoted cell growth in vitro and in vivo. Further analysis of ABCA1 methylation in 76 ovarian cancer patient samples demonstrated that patients with higher ABCA1 methylation are associated with high stage (P = 0.0131) and grade (P = 0.0137). Kaplan-Meier analysis also found that patients with higher levels of methylation of ABCA1 have shorter overall survival (P = 0.019). Furthermore, tissue microarray using 55 ovarian cancer patient samples revealed that patients with a lower level of ABCA1 expression are associated with shorter progress-free survival (P = 0.038). CONCLUSIONS: ABCA1 may be a tumor suppressor and is hypermethylated in a subset of ovarian cancer patients. Hypermethylation of ABCA1 is associated with poor prognosis in these patients.
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Radioresistance is still an emerging problem for radiotherapy of oral cancer. Aberrant epigenetic alterations play an important role in cancer development, yet the role of such alterations in radioresistance of oral cancer is not fully explored. Using a methylation microarray, we identified promoter hypermethylation of FHIT (fragile histidine triad) in radioresistant OML1-R cells, established from hypo-fractionated irradiation of parental OML1 radiosensitive oral cancer cells. Further analysis confirmed that transcriptional repression of FHIT was due to promoter hypermethylation, H3K27me3 and overexpression of methyltransferase EZH2 in OML1-R cells. Epigenetic interventions or depletion of EZH2 restored FHIT expression. Ectopic expression of FHIT inhibited tumor growth in both in vitro and in vivo models, while also resensitizing radioresistant cancer cells to irradiation, by restoring Chk2 phosphorylation and G2/M arrest. Clinically, promoter hypermethylation of FHIT inversely correlated with its expression and independently predicted both locoregional control and overall survival in 40 match-paired oral cancer patient samples. Further in vivo therapeutic experiments confirmed that inhibition of DNA methylation significantly resensitized radioresistant oral cancer cell xenograft tumors. These results show that epigenetic silencing of FHIT contributes partially to radioresistance and predicts clinical outcomes in irradiated oral cancer. The radiosensitizing effect of epigenetic interventions warrants further clinical investigation.
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Ácido Anhídrido Hidrolasas/genética , Metilación de ADN , Neoplasias de la Boca/radioterapia , Proteínas de Neoplasias/genética , Tolerancia a Radiación/genética , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Ácido Anhídrido Hidrolasas/metabolismo , Animales , Línea Celular Tumoral , Proteína Potenciadora del Homólogo Zeste 2 , Epigénesis Genética , Femenino , Silenciador del Gen , Células HEK293 , Histonas/metabolismo , Humanos , Estimación de Kaplan-Meier , Masculino , Metilación , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Neoplasias de la Boca/genética , Neoplasias de la Boca/patología , Proteínas de Neoplasias/metabolismo , Evaluación de Resultado en la Atención de Salud/métodos , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Pronóstico , Carga Tumoral/genética , Carga Tumoral/efectos de la radiaciónRESUMEN
BACKGROUND: The treatment of oral squamous cell carcinoma (OSCC) following early detection is associated with good outcomes. Therefore, the survival and prognosis of OSCC patients could be hugely improved by identifying reliable biomarkers for the early diagnosis of the disease. Our previous methylation microarray analysis results have suggested that the gene encoding tissue factor pathway inhibitor-2 (TFPI-2) is a potential clinical predictor as well as a key regulator involved in OSCC malignancy. METHODS: Methylation of the TFPI-2 promoter in oral tissue specimens was evaluated by bisulfite sequencing assay, quantitative methylation-specific PCR, and pyrosequencing assay. The differences in methylation levels among the groups were compared using the Mann-Whitney U test. The area under the receiver operating characteristic curve (AUROC) was used to evaluate the discrimination ability for detecting OSCC. Cellular TFPI-2 expression was analyzed by quantitative reverse-transcription PCR before and after treatment with 5'-aza-2'-deoxycytidine and trichostatin A, to confirm whether TFPI-2 was epigenetically silenced in OSCC cells. We investigated whether TFPI-2 plays a role as a tumor suppressor by establishing TFPI-2-overexpressing OSCC cells and subjecting them to in vitro cellular proliferation, migration, and invasion assays, as well as an in vivo metastasis assay. RESULTS: TFPI-2 was hypermethylated in OSCC tissues versus normal oral tissues (P < 0.0001), with AUROC = 0.91, when using a pyrosequencing assay to quantify the methylation level. TFPI-2 silencing in OSCC was regulated by both DNA methylation and chromatin histone modification. Restoration of TFPI-2 counteracted the invasiveness of OSCC by inhibiting the enzymatic activity of matrix metalloproteinase-2, and consequently interfered with OSCC metastasis in vivo. CONCLUSIONS: Our data suggest strongly that TFPI-2 is a down-regulated tumor suppressor gene in OSCC, probably involving epigenetic silencing mechanisms. The loss of TFPI-2 expression is a key event for oral tumorigenesis, especially in the process of tumor metastasis.
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Carcinoma de Células Escamosas/genética , Metilación de ADN , Silenciador del Gen , Glicoproteínas/genética , Neoplasias de la Boca/genética , Regiones Promotoras Genéticas , Animales , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Glicoproteínas/metabolismo , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias de la Boca/diagnóstico , Neoplasias de la Boca/patología , PronósticoRESUMEN
Gene therapy mediated by murine leukemia virus (MLV)-based replicating retrovirus vector (RRV) was previously proven to be highly effective in tumor cell killing, resulting in significant suppression of tumor growth in vivo. Recently, we developed a different form of RRV which is derived from another retrovirus, gibbon ape leukemia virus (GALV), as a cancer therapeutic agent. We compared the gene delivery efficiency and antitumor effects in the two types of RRV in experimental hepatocellular carcinoma (HCC). Our results show that both RRVs can efficiently spread throughout entire HCC cell populations in vitro and achieve high transduction efficiency in HCC xenografts in vivo, while GALV RRV, in general, exhibited more rapid replication kinetics in the tumors. In vitro, substantial HCC cell killing was achieved even when initially only 1% of the HCC cells were producing RRVs that express the yeast cytosine deaminase suicide gene, indicating that the high efficiency of gene transfer by replicative spread of RRVs greatly increased suicide gene toxicity. In vivo, GALV RRV-mediated suicide gene therapy efficiently suppressed HCC tumor growth and no detectable RRV signals were observed in extratumoral tissues, showing promise in using GALV RRV as a cancer therapeutic agent.
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Carcinoma Hepatocelular/terapia , Virus de la Leucemia del Gibón/genética , Neoplasias Hepáticas Experimentales/terapia , Virus Oncolíticos/genética , Animales , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Biotransformación , Carcinoma Hepatocelular/patología , Supervivencia Celular/efectos de los fármacos , Citosina Desaminasa/biosíntesis , Citosina Desaminasa/genética , Flucitosina/metabolismo , Flucitosina/farmacología , Flucitosina/uso terapéutico , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/genética , Terapia Genética , Células Hep G2 , Humanos , Virus de la Leucemia del Gibón/enzimología , Virus de la Leucemia del Gibón/fisiología , Neoplasias Hepáticas Experimentales/patología , Ratones , Ratones Desnudos , Viroterapia Oncolítica , Virus Oncolíticos/enzimología , Virus Oncolíticos/fisiología , Profármacos/metabolismo , Profármacos/farmacología , Profármacos/uso terapéutico , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Transducción Genética , Carga Tumoral/efectos de los fármacos , Replicación Viral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Resistance to TGF-beta is frequently observed in ovarian cancer, and disrupted TGF-beta/SMAD4 signaling results in the aberrant expression of downstream target genes in the disease. Our previous study showed that ADAM19, a SMAD4 target gene, is downregulated through epigenetic mechanisms in ovarian cancer with aberrant TGF-beta/SMAD4 signaling. In this study, we investigated the mechanism of downregulation of FBXO32, another SMAD4 target gene, and the clinical significance of the loss of FBXO32 expression in ovarian cancer. Expression of FBXO32 was observed in the normal ovarian surface epithelium, but not in ovarian cancer cell lines. FBXO32 methylation was observed in ovarian cancer cell lines displaying constitutive TGF-beta/SMAD4 signaling, and epigenetic drug treatment restored FBXO32 expression in ovarian cancer cell lines regardless of FBXO32 methylation status, suggesting that epigenetic regulation of this gene in ovarian cancer may be a common event. In advanced-stage ovarian tumors, a significant (29.3%; P<0.05) methylation frequency of FBXO32 was observed and the association between FBXO32 methylation and shorter progression-free survival was significant, as determined by both Kaplan-Meier analysis (P<0.05) and multivariate Cox regression analysis (hazard ratio: 1.003, P<0.05). Reexpression of FBXO32 markedly reduced proliferation of a platinum-resistant ovarian cancer cell line both in vitro and in vivo, due to increased apoptosis of the cells, and resensitized ovarian cancer cells to cisplatin. In conclusion, the novel tumor suppressor FBXO32 is epigenetically silenced in ovarian cancer cell lines with disrupted TGF-beta/SMAD4 signaling, and FBXO32 methylation status predicts survival in patients with ovarian cancer.