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The computational simulations for electronic properties of cadmium (Cd) coordinated L-alanine NDI ligand (H2-l-ala NDI) based complex are the focus of this research. For the first time, the Cd-NDI complex (monomer) has been produced using water as the solvent; this is a new approach to synthesizing the Cd-NDI complex that has not been reported yet. Along with crystallography and Hirsch field analysis, CAM-B3LYP/LANL2DZ and B3LYP/LANL2MB basis sets were used, and in-depth characterisation of the Cd-NDI complex by following DFT and TD-DFT hypothetical simulations. Hyperpolarizabilities, frontier molecular orbitals (FMOs), the density of states (DOS), dipole moment (µ), electron density distribution map (EDDM), transition density matrix (TDM), molecular electrostatic potential (MEP), electron-hole analysis (EHA), and electrical conductivity (σ) have all been studied regarding the Cd-NDI complex. The vibrational frequencies and types of interaction are studied using infrared (IR) and non-covalent interaction (NCI) analysis with iso-surface. In comparison to the Cd-NDI complex with 2.61, 2.42 eV Eg (using CAM-B3LYP/LANL2DZ and B3LYP/LANL2MB basis sets, respectively) and 376 nm λmax, (in case of B3LYP/LANL2MB λmax is higher), H2-l-ala NDI have 3.387 eV Eg and 375 nm λmax, metal-ligand coordination in complex dramatically altered charge transfer properties, such as narrowing band gap (Eg). Based on the electronic properties analysis of Cd-NDI complex, it is predicted that the Cd-NDI complex will have a spectacular (nonlinear optical) NLO response. The Cd-NDI complex is discovered to be advantageous for the creation of future nanoscale devices due to the harmony between the Cd metal and H2-l-ala NDI, in addition to their influences on NLO characteristics.
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In this study, to evaluate the effect of different biocontrol agents (BCAs) on the soil bacterial community, we investigated the effects of Bacillus amyloliquefaciens, synthetic bacterial community (Aspergillus niger:Bacillus subtilis:Bacillus licheniformis:Streptomyces microflavus = 3:3:3:1, SynCom), and BCAs combined with lime-nitrogen on soil bacterial community by utilizing 16S rRNA sequencing technology. The sequencing shows that BCAs application can improve the value of Shannon and Sobs index of bacterial community during tobacco rosette and vigorous growing period. With the growth of tobacco, the effect of BCAs on the composition and difference of soil bacterial community structure becomes more and more obvious. In terms of average relative richness, the top six phyla of soil bacterial community are Proteobacteria, Actinobacteria, Chloroflexi, Acidobacteria, Gemmatimonadetes, and Bacteroidetes. Bacillus amyloliquefaciens application can increase the relative richness of Proteobacteria and Bacteroidetes. And the combination between BCAs and lime-nitrogen can increase the relative richness of Gemmatimonadetes and Bacteroidetes. The SynCom also can increase the relative richness of Bacteroidetes, whereas it decreases the relative richness of Acidobacteria. Proteobacteria, Acidobacteria, Gemmatimonadetes, and Bacteroidetes showing an extremely significant correlation with pH and exchangeable magnesium (EMg). BCAs application can improve the tobacco yield, effective leaves, and reducing sugar content that also has extremely significant positive correlation with pH and EMg. In conclusion, the results of our field experiments clearly show that BCAs application can significantly affect the soil pH and EMg by changing most of the dominant soil bacterial species. The richness of Bacteroidetes can serve as an indicator of the changes in soil pH and EMg caused by BCAs application.
Assuntos
Nicotiana , Solo , Acidobacteria , Bactérias/genética , Bacteroidetes/genética , Compostos de Cálcio , Magnésio , Nitrogênio/análise , Óxidos , Proteobactérias/genética , RNA Ribossômico 16S/genética , Solo/química , Microbiologia do Solo , Açúcares , Nicotiana/microbiologiaRESUMO
BACKGROUND: We intended to compare and grade the proposed immune treating strategies for non-small cell lung cancer (NSCLC) with negative Programmed Cell Death Ligand 1(PD-L1). METHODS: We compared the efficacy of single immune checkpoint inhibitor (ICI), single ICI plus chemotherapy, and doublet ICIs with chemotherapy alone, as well as single ICI plus radiotherapy with single ICI for negative PD-L1 (<1%) NSCLC patients. Hazard Ratio (HR) and 95% confidence interval (CI) of progression-free survival (PFS) and overall survival (OS) were used as outcomes. RESULTS: We included 23 randomized control trials with 4665 patients. Compared with chemotherapy alone, single ICI, single ICI plus chemotherapy and doublet ICIs all showed a better OS (0.84 [0.71, 0.99] ; 0.77 [0.69, 0.85] ; 0.64 [0.53, 0.77])), while single ICI plus chemotherapy and doublet ICIs showed a better PFS (0.68 [0.61, 0.75] ; 0.69 [0.56, 0.85]). Additionally, single ICI plus radiotherapy obtained a greater pooled PFS (0.49 [0.28-0.87]) than single ICI. CONCLUSIONS: Both single ICI plus chemotherapy and doublet ICIs were probably better treatment decisions than chemotherapy alone for negative PD-L1 NSCLC patients. Also, single ICI plus radiotherapy carved out a new strategy.
Assuntos
Antineoplásicos Imunológicos , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Antineoplásicos Imunológicos/efeitos adversos , Antígeno B7-H1 , Biomarcadores Tumorais/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismoRESUMO
Pulmonary arterial hypertension (PAH) seriously threatens the elder people. Long non-coding RNAs (lncRNAs) are involved in multiple diseases. However, the study of the lncRNAs in the occurrence of PAH is just beginning. For this, we sought to explore the biological function of lncRNA HOXA cluster antisense RNA 3 (HOXA-AS3) in PAH. Hypoxia (HYP) was used to mimic in vitro model of PAH. Gene and protein expressions in cells were detected by q-PCR and Western blotting, respectively. In addition, cell proliferation and viability were tested by CCK-8 and MTT assay. Cell apoptosis was measured by flow cytometry. Wound healing was used to detect cell migration. Furthermore, the connection of HOXA-AS3, miR-675-3p, and phosphodiesterase 5A (PDE5A) was verified by dual-luciferase report assay. HOXA-AS3 and PDE5A were upregulated in human pulmonary artery smooth muscle cells (HPASMCs) in the presence of HYP, while miR-675-3p was downregulated. Moreover, knockdown of HOXA-AS3 suppressed the growth and migration of HPASMCs, but induced the apoptosis. Overexpression of miR-675-3p achieved the same effect. MiR-675-3p inhibitor or overexpression of PDE5A notably reversed the inhibitory effect of HOXA-AS3 knockdown on PAH. Finally, HOXA-AS3 could sponge miR-675-3p, and PDE5A was directly targeted by miR-675-3p. HOXA-AS3 increased the development of PAH via regulation of miR-675-3p/PDE5 axis, which could be the potential biomarker for treatment of PAH.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , MicroRNAs/genética , Hipertensão Arterial Pulmonar/patologia , RNA Antissenso/genética , RNA Longo não Codificante/genética , Apoptose , Biomarcadores/metabolismo , Movimento Celular , Proliferação de Células , Células Cultivadas , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/genética , Humanos , Hipertensão Arterial Pulmonar/genética , Hipertensão Arterial Pulmonar/metabolismoRESUMO
Among the human milk oligosaccharides (HMOs), one of the most abundant oligosaccharides and has great benefits for human health is 3'-sialyllactose (3'-SL). Given its important physiological functions and the lack of cost-effective production processes, we constructed an in vitro multi-enzymatic cofactor recycling system for the biosynthesis of 3'-SL from a low-cost substrate. First, we constructed the biosynthetic pathway and increased the solubility of cytidine monophosphate kinase (CMK) with chaperones. We subsequently identified that ß-galactosidase (lacZ) affects the yield of 3'-SL, and hence with the lacZ gene knocked out, a 3.3-fold increase in the production of 3'-SL was observed. Further, temperature, pH, polyphosphate concentration, and concentration of divalent metal ions for 3'-SL production were optimized. Finally, an efficient biotransformation system was established under the optimized conditions. The maximum production of 3'-SL reached 38.7 mM, and a molar yield of 97.1% from N-acetylneuraminic acid (NeuAc, sialic acid, SA) was obtained. The results demonstrate that the multi-enzymatic cascade biosynthetic pathway with cofactor regeneration holds promise as an industrial strategy for producing 3'-SL.
Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Genética , Oligossacarídeos/biossíntese , Escherichia coli/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Metais/farmacologia , Polifosfatos/farmacologia , Solubilidade , TemperaturaRESUMO
A bronchogenic cyst in the left atrium is rare. We report the case of a 17-year-old male patient who was admitted to the emergency department because of severe chest pain and dyspnea. He was diagnosed using echocardiography and computed tomography, which revealed a huge cardiac tumour in the dome of the left atrium. He was surgically treated with tumour enucleation. The resultant atrial dome defect was reconstructed with a bovine pericardial patch. Pathologic investigation revealed that the tumour was a bronchogenic benign cyst.
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Cisto Broncogênico , Procedimentos Cirúrgicos Cardíacos , Dissecação/métodos , Átrios do Coração , Neoplasias Cardíacas/diagnóstico , Adolescente , Biópsia/métodos , Cisto Broncogênico/diagnóstico , Cisto Broncogênico/patologia , Cisto Broncogênico/fisiopatologia , Cisto Broncogênico/cirurgia , Procedimentos Cirúrgicos Cardíacos/instrumentação , Procedimentos Cirúrgicos Cardíacos/métodos , Dor no Peito/diagnóstico , Dor no Peito/etiologia , Diagnóstico Diferencial , Dispneia/etiologia , Dispneia/cirurgia , Ecocardiografia/métodos , Átrios do Coração/diagnóstico por imagem , Átrios do Coração/patologia , Átrios do Coração/cirurgia , Humanos , Cuidados Intraoperatórios/métodos , Masculino , Tomografia Computadorizada por Raios X/métodos , Resultado do TratamentoRESUMO
PURPOSE: Constitutive NF-κB activation is identified in about 70% of pancreatic ductal adenocarcinoma (PDAC) cases and is required for oncogenic KRAS-induced PDAC development in mouse models. We sought to determine whether targeting IL-1α pathway would inhibit NF-κB activity and thus suppress PDAC cell growth. EXPERIMENTAL DESIGN: We determined whether anakinra, a human IL-1 receptor (rhIL-1R) antagonist, inhibited NF-κB activation. Assays for cell proliferation, migration, and invasion were performed with rhIL-1R antagonist using the human PDAC cell lines AsPc1, Colo357, MiaPaCa-2, and HPNE/K-ras(G12V)/p16sh. In vivo NF-κB activation-dependent tumorigenesis was assayed using an orthotopic nude mouse model (n = 20, 5 per group) treated with a combination of gemcitabine and rhIL-1RA. RESULTS: rhIL-1R antagonist treatment led to a significant decrease in NF-κB activity. PDAC cells treated with rhIL-1R antagonist plus gemcitabine reduced proliferation, migration, and invasion as compared with single gemcitabine treatment. In nude mice, rhIL-1R antagonist plus gemcitabine significantly reduced the tumor burden (gemcitabine plus rhIL-1RA vs. control, P = 0.014). CONCLUSIONS: We found that anakinra, an FDA-approved drug that inhibits IL-1 receptor (IL-1R), when given with or without gemcitabine, can reduce tumor growth by inhibiting IL1α-induced NF-κB activity; this result suggests that it is a useful therapeutic approach for PDAC.
Assuntos
NF-kappa B/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Receptores de Interleucina-1/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Comunicação Autócrina , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Modelos Animais de Doenças , Quimioterapia Combinada , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Proteína Antagonista do Receptor de Interleucina 1/farmacologia , Interleucina-1alfa/metabolismo , Masculino , Camundongos , Neoplasias Pancreáticas/tratamento farmacológico , Receptores de Interleucina-1/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
The anticancer effect of Scutellaria baicalensis extract has recently become a topic of interest. In this study, the anticancer effects and underlying mechanisms of wogonoside, the main constituent of Scutellaria baicalensis, were investigated in a human hepatocellular carcinoma (HCC) cell line in vitro. The effects of wogonoside on the proliferation, cell cycle progression and apoptosis of hepatocellular carcinoma cells were examined. Western blotting was employed to analyze the proteins associated with the biological effects of wogonoside. Wogonoside exerted anti-proliferation properties in vitro. HCC cell growth was attenuated by wogonoside (8 µM) treatment. Cell cycle progression analysis and DNA ladder assay revealed that apoptosis was enhanced in wogonoside-treated cells and that cell cycle arrest occurred in the G2/M phase. It was also demonstrated that increased apoptosis was accompanied by increased levels of Bax protein and decreased levels of Bcl-2 protein. The results of this study suggest that wogonoside may represent a potential therapeutic agent against HCC.
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BACKGROUND: Cell therapy is a promising strategy for tissue regeneration. Key to this strategy is mobilization and recruitment of exogenous or autologous stem/progenitor cells by cytokines. However, there is no effective cytokine delivery system available for clinic application, in particular for myocardial regeneration. The aim of this study was to develop a novel cytokine delivery system that is stable in solution at physiological pH. METHODS: Four groups of self-assembled chitosan oligosaccharide/heparin (CSO/H) nanoparticles were prepared with various volume ratios of chitosan oligosaccharide to heparin (5:2, 5:4, 4:15, 1:5) and characterized by laser diffraction, particle size analysis, and transmission electron microscopy. The encapsulation efficiency and loading content of two cytokines, ie, stromal cell-derived factor (SDF)-1α and vascular endothelial growth factor (VEGF) were quantified using an enzyme-linked immunosorbent assay. The biological activity of the loaded SDF-1α and VEGF was evaluated using the transwell migration assay and MTT assay. The dispersion profiles for the cytokine-loaded nanoparticles were quantified using fluorescence molecular tomography. RESULTS: CSO/H nanoparticles were prepared successfully in solution with physiological pH. The particle sizes in the four treatment groups were in the range of 96.2-210.5 nm and the zeta potential ranged from -29.4 mV to 24.2 mV. The loading efficiency in the CSO/H nanoparticle groups with the first three ratios was more than 90%. SDF-1α loaded into CSO/H nanoparticles retained its migration activity and VEGF loaded into CSO/H nanoparticles continued to show proliferation activity. The in vivo dispersion test showed that the CSO/H nanoparticles enabled to VEGF to accumulate locally for a longer period of time. CONCLUSION: CSO/H nanoparticles have a high cytokine loading capacity and allow cytokines to maintain their bioactivity for longer, are stable in an environment with physiological pH, and may be a promising cytokine delivery system for tissue regeneration.
Assuntos
Quitosana/química , Citocinas/administração & dosagem , Heparina/química , Nanopartículas/química , Animais , Proliferação de Células/efeitos dos fármacos , Quimiocina CXCL12/administração & dosagem , Quimiocina CXCL12/química , Quimiocina CXCL12/farmacologia , Citocinas/química , Sistemas de Liberação de Medicamentos/métodos , Ensaio de Imunoadsorção Enzimática , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos ICR , Microscopia Eletrônica de Transmissão , Nanopartículas/administração & dosagem , Soluções , Fator A de Crescimento do Endotélio Vascular/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/química , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Activation of K-ras and inactivation of p16 are the most frequently identified genetic alterations in human pancreatic epithelial adenocarcinoma (PDAC). Mouse models engineered with mutant K-ras and deleted p16 recapitulate key pathological features of PDAC. However, a human cell culture transformation model that recapitulates the human pancreatic molecular carcinogenesis is lacking. In this study, we investigated the role of p16 in hTERT-immortalized human pancreatic epithelial nestin-expressing (HPNE) cells expressing mutant K-ras (K-rasG12V). We found that expression of p16 was induced by oncogenic K-ras in these HPNE cells and that silencing of this induced p16 expression resulted in tumorigenic transformation and development of metastatic PDAC in an orthotopic xenograft mouse model. Our results revealed that PI3K/Akt, ERK1/2 pathways and TGFα signaling were activated by K-ras and involved in the malignant transformation of human pancreatic cells. Also, p38/MAPK pathway was involved in p16 up-regulation. Thus, our findings establish an experimental cell-based model for dissecting signaling pathways in the development of human PDAC. This model provides an important tool for studying the molecular basis of PDAC development and gaining insight into signaling mechanisms and potential new therapeutic targets for altered oncogenic signaling pathways in PDAC.
Assuntos
Adenocarcinoma/patologia , Carcinogênese/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Regulação para Baixo , Genes ras/genética , Neoplasias Pancreáticas/patologia , Adenocarcinoma/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica , Senescência Celular , Inibidor p16 de Quinase Dependente de Ciclina/deficiência , Feminino , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Camundongos , Mutação , Metástase Neoplásica , Neoplasias Pancreáticas/genéticaRESUMO
PURPOSE: The most common genetic lesions in pancreatic ductal adenocarcinoma (PDAC) have been identified. However, significant gaps still exist in our understanding of how such genetic alterations act in concert to induce PDAC development. In this study, we investigated the mechanism of tumorigenic transformation in the immortalized human pancreatic ductal epithelial (HPDE) cell line by sequentially introducing PDAC signature alterations into this cell line. EXPERIMENTAL DESIGN: The phenotype for stable expression of mutant K-ras, Her2, p16/p14shRNA, and Smad4shRNA in HPDE cells was examined by assays for cell proliferation, migration, invasion, soft agar, and orthotopic tumorigenesis. The mechanisms of tumorigenic transformation were further explored by gene expression profiling and pathway analyses. RESULTS: The transformed cells exhibited enhanced proliferation, migration, and invasion, displayed anchorage-independent growth in soft agar, and grew orthotopic tumors with some histopathologic features of PDAC. We found that Smad4 played key roles in the tumorigenic transformation of HPDE cells. We further found that MDM2 and Bmi-1 were overexpressed in the tumorigenic HPDE cells and that Bmi-1 overexpression was regulated by Smad4. Ingenuity Pathway Analysis software analysis of microarray data revealed that dysregulation of integrin-linked kinase signaling and the cell cycle were the most significant changes involved in tumorigenic transformation. Altogether, this cell culture model closely recapitulated human pancreatic carcinogenesis from gene lesions, activation of specific signaling pathways, and some histopathologic features. CONCLUSION: The combination of activated K-ras and Her2 with inactivated p16/p14 and Smad4 was sufficient and essential to transform HPDE cells, thus revealing the potential tumorigenic mechanism.
Assuntos
Carcinoma Ductal Pancreático/etiologia , Transformação Celular Neoplásica , Ductos Pancreáticos/patologia , Neoplasias Pancreáticas/etiologia , Animais , Linhagem Celular Transformada , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/genética , Modelos Animais de Doenças , Expressão Gênica , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes ras , Humanos , Camundongos , Mutação , Ductos Pancreáticos/metabolismo , Interferência de RNA , Receptor ErbB-2/genética , Transdução de Sinais , Proteína Smad4/genéticaRESUMO
In most eukaryotes the subunit 2 of cytochrome c oxidase (COX2) is encoded in intact mitochondrial genes. Some green algae, however, exhibit split cox2 genes (cox2a and cox2b) encoding two polypeptides (COX2A and COX2B) that form a heterodimeric COX2 subunit. Here, we analyzed the distribution of intact and split cox2 gene sequences in 39 phylogenetically diverse green algae in phylum Chlorophyta obtained from databases (28 sequences from 22 taxa) and from new cox2 data generated in this work (23 sequences from 18 taxa). Our results support previous observations based on a smaller number of taxa, indicating that algae in classes Prasinophyceae, Ulvophyceae, and Trebouxiophyceae contain orthodox, intact mitochondrial cox2 genes. In contrast, all of the algae in Chlorophyceae that we examined exhibited split cox2 genes, and could be separated into two groups: one that has a mitochondrion-localized cox2a gene and a nucleus-localized cox2b gene ("Scenedesmus-like"), and another that has both cox2a and cox2b genes in the nucleus ("Chlamydomonas-like"). The location of the split cox2a and cox2b genes was inferred using five different criteria: differences in amino acid sequences, codon usage (mitochondrial vs. nuclear), codon preference (third position frequencies), presence of nucleotide sequences encoding mitochondrial targeting sequences and presence of spliceosomal introns. Distinct green algae could be grouped according to the form of cox2 gene they contain: intact or fragmented, mitochondrion- or nucleus-localized, and intron-containing or intron-less. We present a model describing the events that led to mitochondrial cox2 gene fragmentation and the independent and sequential migration of cox2a and cox2b genes to the nucleus in chlorophycean green algae. We also suggest that the distribution of the different forms of the cox2 gene provides important insights into the phylogenetic relationships among major groups of Chlorophyceae.
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Núcleo Celular/genética , Clorófitas/genética , Ciclo-Oxigenase 2/genética , Modelos Genéticos , Filogenia , Sequência de Aminoácidos , Sequência de Bases , Análise por Conglomerados , Códon/genética , Biologia Computacional , Primers do DNA/genética , DNA Mitocondrial/genética , Funções Verossimilhança , Dados de Sequência Molecular , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
Many genetic and molecular alterations, such as K-ras mutation and NF-kappaB activation, have been identified in pancreatic cancer. However, the mechanisms by which pancreatic cancer metastasizes still remain to be determined. Although we previously showed that the tropomyosin-related kinase B (TrkB) was significantly correlated with the development of liver metastasis, its function in pancreatic cancer metastasis remained unresolved. In the present study, we showed that overexpressed TrkB is an alternatively spliced transcript variant of TrkB (TrkBT1) with a unique COOH-terminal 12-amino acid sequence and is mainly localized in the cytoplasm. Our results showed that overexpression of Flag-tagged TrkBT1 but not a Flag-tagged TrkBT1 COOH-terminal deletion mutant (Flag-TrkBT1DeltaC) in nonmetastatic pancreatic cancer cells enhanced cell proliferation, promoted formation of colonies in soft agar, stimulated tumor cell invasion, and induced liver metastasis in an orthotopic xenograft mouse model of pancreatic cancer. TrkBT1 interacted with Rho GDP dissociation inhibitor (GDI) in vivo, but Flag-TrkBT1DeltaC did not. Furthermore, overexpression of Flag-TrkBT1 and knockdown of RhoGDI expression by RhoGDI short hairpin RNAs promoted RhoA activation, but Flag-TrkBT1DeltaC overexpression did not. Therefore, our results showed that TrkBT1 overexpression induces liver metastasis of pancreatic cancer and uncovered a unique signaling mechanism by which TrkBT1 sequesters GDI and activates RhoA signaling.
Assuntos
Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Neoplasias Hepáticas/secundário , Neoplasias Pancreáticas/patologia , Receptor trkB/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Caderinas/biossíntese , Caderinas/genética , Adesão Celular/fisiologia , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Ativação Enzimática , Feminino , Humanos , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/metabolismo , Subunidades Proteicas , Receptor trkB/biossíntese , Receptor trkB/genética , Transplante Heterólogo , Regulação para CimaRESUMO
Pancreatic cancer is an aggressive malignancy with morbidity rates almost equal to mortality rates because of the current lack of effective treatment options. Here, we describe a targeted approach to treating pancreatic cancer with effective therapeutic efficacy and safety in noninvasive imaging models. We developed a versatile expression vector "VISA" (VP16-GAL4-WPRE integrated systemic amplifier) and a CCKAR (cholecystokinin type A receptor) gene-based, pancreatic-cancer-specific promoter VISA (CCKAR-VISA) composite to target transgene expression in pancreatic tumors in vivo. Targeted expression of BikDD, a potent proapoptotic gene driven by CCKAR-VISA, exhibited significant antitumor effects on pancreatic cancer and prolonged survival in multiple xenograft and syngeneic orthotopic mouse models of pancreatic tumors with virtually no toxicity.
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Modelos Biológicos , Neoplasias Pancreáticas/genética , Animais , Camundongos , Camundongos Endogâmicos C57BL , Receptores da Colecistocinina/genética , TransgenesRESUMO
BACKGROUND: Constitutive activation of nuclear factor-kappaB (NF-kappaB) is a frequent molecular alteration in pancreatic cancer and a number of studies have suggested that constitutive NF-kappaB activity plays a key role in the aggressive behavior of this disease. In an attempt to identify an effective therapeutic agent for pancreatic cancer, the authors studied the role of FUT-175, a synthetic serine protease inhibitor, in the inhibition of NF-kappaB activation and the induction of apoptotic responses. METHODS: To examine the effect of FUT-175 on the inhibition of NF-kappaB and the induction of apoptosis in pancreatic cancer cell lines, Western and Northern blot analyses, electromobility shift (EMSA), luciferase reporter gene, DNA fragmentation, immunoprecipitation, in vitro kinase, small interfering RNA (siRNA), and chromatin immunoprecipitation (ChIP) assays were performed. RESULTS: In a time-dependent and dose-dependent manner, FUT-175 inhibited IkappaBalpha phosphorylation and NF-kappaB activation, thereby inhibiting the antiapoptotic activity of NF-kappaB. Simultaneously, FUT-175 up-regulated the expression of tumor necrosis factor receptor-1 (TNFR1), which in turn activated the proapoptotic caspase-8 and Bid pathways and induced apoptosis in pancreatic cancer cells. FUT-175-induced activation of Fas-associated death domain (FADD) and caspase-8 was suppressed by RNA interference-mediated inhibition of TNFR1 expression. Furthermore, expression of the transcription factor PEA3 was up-regulated by FUT-175 and was involved in FUT-175-mediated TNFR1 expression. CONCLUSIONS: These results suggested a possible mechanism by which FUT-175 may disrupt interconnected signaling pathways by both suppressing the NF-kappaB antiapoptotic activity and inducing TNFR-mediated apoptosis. Supported by this unique function as a NF-kappaB inhibitor and apoptosis inducer, this well-established synthetic serine protease inhibitor with as-of-yet poorly understood mechanisms of actions appears to be a potentially therapeutic agent for pancreatic cancer.
Assuntos
Adenocarcinoma/patologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Guanidinas/farmacologia , Neoplasias Pancreáticas/patologia , Inibidores de Serina Proteinase/farmacologia , Benzamidinas , Caspase 8/metabolismo , Linhagem Celular Tumoral/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Proteína de Domínio de Morte Associada a Fas/metabolismo , Humanos , Proteínas I-kappa B/metabolismo , NF-kappa B/efeitos adversos , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Fatores de Transcrição/metabolismoRESUMO
PURPOSE: Pancreatic adenocarcinoma is currently the fourth leading cause of cancer death in the United States, and most pancreatic cancers develop locally advanced disease or metastasis at the time of diagnosis. The mechanisms by which it invades and metastasizes are not known. EXPERIMENTAL DESIGN: To identify the genes involved in pancreatic cancer metastasis, we analyzed the gene expression profiles between highly metastatic Colo357L3.6pl and parental Colo357FG pancreatic cancer cell lines using cDNA microarrays and confirmed differential gene expression by reverse transcription-PCR, Western blotting, and immunologic analysis of 54 samples from pancreatic cancer patients. The correlation with clinical outcome was also examined. The possible signaling pathways involved with tropomyosin-related kinase B (TrkB) were analyzed. RESULTS: Our findings showed that TrkB was overexpressed in the highly metastatic Colo357L3.6pl cells, which correlated with perineural invasion (P = 0.026), positive retroperitoneal margin (P = 0.0005), and shorter latency to development of liver metastasis (Cox proportional hazard ratio, 0.3; 95% confidence interval, 0.1-0.8; P = 0.01) in patient samples. Extracellular signal-regulated kinases 1 and 2 were activated and Elk-1 and AP-1 DNA binding activity was induced in Colo357L3.6pl cells. Furthermore, interleukin 8 and vascular endothelial growth factor were more strongly expressed in Colo357L3.6pl than Colo357FG cells, and these findings were confirmed in Colo357L3.6pl and Colo357FG orthotopic tumors. CONCLUSION: These results suggest that overexpression of TrkB and activation of mitogen-activated protein kinase and AP-1, which may in turn induce the expression of vascular endothelial growth factor and interleukin 8, may mediate the cardinal clinical features of locally aggressive growth and metastasis of pancreatic cancer. Our results also imply that TrkB receptor may be a novel therapeutic target for pancreatic cancer.
Assuntos
Adenocarcinoma/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Pancreáticas/metabolismo , Proteínas Quinases/metabolismo , Receptor trkB/metabolismo , Idoso , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Ativação Enzimática , Feminino , Perfilação da Expressão Gênica , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Metástase Linfática , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Invasividade Neoplásica/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptor trkB/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas Elk-1 do Domínio etsRESUMO
RelA, the p65 subunit of NF-kappaB transcription factors, plays a key role in regulation of antiapoptotic and proapoptotic responses. However, the downstream target genes regulated by RelA-NF-kappaB in the initiation of proapoptotic signaling were not identified. We previously showed that RelA-NF-kappaB functioned as a proapoptotic factor by activating the p53-signaling pathway in response to doxycycline-induced superoxide. In the present study, we demonstrate that the ability of doxycycline/superoxide to induce expression of polo-like kinase 3 (Plk3) depends on NF-kappaB activity. We identified a kappaB binding site in the promoter of Plk3, and this kappaB site is directly involved in its induction by the RelA-NF-kappaB complex. Plk3 formed a complex with p53 and was involved in the phosphorylation of p53 on Ser-20 in response to superoxide. Inhibition of Plk3 expression by Plk3 small interfering RNA suppressed the doxycycline/superoxide-mediated apoptosis. Overexpression of wild-type Plk3 in HCT116 p53+/+ cells induced rapid apoptosis, whereas overexpression of wild-type Plk3 in HCT116 p53-/- cells and the kinase-defective mutant Plk3(K91R) in p53+/+ cells induced delayed onset of apoptosis. Furthermore, mutagenesis of Plk3 showed that the N-terminal domain (amino acids 1-26) is essential for the induction of delay onset of apoptosis. These data show that Plk3 is a RelA-NF-kappaB-regulated gene that induces apoptosis in both p53-dependent and -independent signaling pathways, suggesting a possible mechanism for RelA-NF-kappaB-regulated proapoptotic responses.
Assuntos
Apoptose , Regulação da Expressão Gênica , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Sítios de Ligação , Northern Blotting , Western Blotting , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Inibidor de Quinase Dependente de Ciclina p21 , Fragmentação do DNA , Doxiciclina/farmacologia , Genes p53 , Humanos , Proteínas I-kappa B/metabolismo , Imuno-Histoquímica , Luciferases/metabolismo , Microscopia de Fluorescência , Modelos Biológicos , Inibidor de NF-kappaB alfa , NF-kappa B/fisiologia , Fosforilação , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina/química , Transdução de Sinais , Fatores de Tempo , Fator de Transcrição RelA , Transfecção , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de TumorRESUMO
Nuclear factor kappaB (NF-kappaB) and activator protein 1 (AP-1) transcription factors regulate many important biological and pathological processes. Activation of NF-kappaB is regulated by the inducible phosphorylation of NF-kappaB inhibitor IkappaB by IkappaB kinase. In contrast, Fos, a key component of AP-1, is primarily transcriptionally regulated by serum responsive factors (SRFs) and ternary complex factors (TCFs). Despite these different regulatory mechanisms, there is an intriguing possibility that NF-kappaB and AP-1 may modulate each other, thus expanding the scope of these two rapidly inducible transcription factors. To determine whether NF-kappaB activity is involved in the regulation of fos expression in response to various stimuli, we analyzed activity of AP-1 and expression of fos, fosB, fra-1, fra-2, jun, junB, and junD, as well as AP-1 downstream target gene VEGF, using MDAPanc-28 and MDAPanc-28/IkappaBalphaM pancreatic tumor cells and wild-type, IKK1-/-, and IKK2-/- murine embryonic fibroblast cells. Our results show that elk-1, a member of TCFs, is one of the NF-kappaB downstream target genes. Inhibition of NF-kappaB activity greatly decreased expression of elk-1. Consequently, the reduced level of activated Elk-1 protein by extracellular signal-regulated kinase impeded constitutive, serum-, and superoxide-inducible c-fos expression. Thus, our study revealed a distinct and essential role of NF-kappaB in participating in the regulation of elk-1, c-fos, and VEGF expression.
Assuntos
Regulação da Expressão Gênica , NF-kappa B/metabolismo , Transdução de Sinais/fisiologia , Fator de Transcrição AP-1/metabolismo , Animais , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Doxiciclina/metabolismo , Humanos , Quinase I-kappa B , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Células-Tronco/citologia , Células-Tronco/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas Elk-1 do Domínio etsRESUMO
BACKGROUND: Currently, pancreatic adenocarcinoma is the fourth leading cause of cancer-related death in the United States. Despite the advances in pancreatic carcinoma research, patients with this devastating disease have a very poor prognosis. To identify the gene expression profile of pancreatic carcinoma, an important step in the process of developing new diagnostic and therapeutic strategies, the authors investigated the alteration of gene expression in this disease. METHODS: The authors analyzed a public serial analysis of gene expression (SAGE) database and examined in greater detail the expression of synuclein-gamma mRNA in several pancreatic carcinoma cell lines and tumor tissue samples by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and Northern blot analysis. The expression of synuclein-gamma protein was investigated further by immunohistochemical and Western blot analyses using tumor cell lines, tumor tissue, and serum samples. RESULTS: Synuclein-gamma mRNA was overexpressed in 11 of 12 pancreatic carcinoma cell lines, including AsPc-1, MDAPanc28, Capan-1, Capan-2, PANC-1, HS766T, MDAPanc3, MDAPanc48, Colo357FG, MiaPaCa2, CFPac1, and BxPc3. The expression of synuclein-gamma protein was detectable in 8 of 12 pancreatic carcinoma cell lines (67%) and in 22 of 32 pancreatic tumor tissue samples (69%) by Western blot analysis. On immunohistochemical staining, synuclein-gamma protein was present in 61% of the tumor tissue samples examined from patients with Stage I and II pancreatic carcinoma. The overexpression of synuclein-gamma is correlated with perineural and lymph node invasion. Synuclein-gamma protein also was detectable by Western blot in serum samples from 21 of 56 patients (38%) with pancreatic carcinoma. CONCLUSIONS: Synuclein-gamma, which initially was described as a breast carcinoma-specific gene involved in invasion, metastasis, and chemotherapy resistance, was frequently overexpressed in pancreatic carcinoma. Overexpression of synuclein-gamma may play a role in pancreatic carcinoma invasion. Further studies will be necessary to determine the role of synuclein-gamma in pancreatic carcinoma.
Assuntos
Adenocarcinoma/metabolismo , Biomarcadores Tumorais/análise , Proteínas do Tecido Nervoso/biossíntese , Neoplasias Pancreáticas/metabolismo , Adenocarcinoma/sangue , Adenocarcinoma/patologia , Northern Blotting , Western Blotting , Linhagem Celular Tumoral , Expressão Gênica , Humanos , Imuno-Histoquímica , Metástase Linfática , Proteínas do Tecido Nervoso/sangue , Neoplasias Pancreáticas/sangue , Neoplasias Pancreáticas/patologia , Prognóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SinucleínasRESUMO
Both pro- and antiapoptotic activities of NF-kappaB transcription factor have been observed; however, less is known about the mechanism by which NF-kappaB induces apoptosis. To elucidate how NF-kappaB regulates proapoptotic signaling, we performed functional analyses using wild-type, ikk1(-/-), ikk2(-/-), rela(-/-) murine fibroblasts, MDAPanc-28/Puro, MDAPanc-28/IkappaBalphaM, and HCT116/p53(+/+) and HCT116/p53(-/-) cells with investigational anticancer agent doxycycline as a superoxide inducer for generating apoptotic stimulus. In this report, we show that doxycycline increased superoxide generation and subsequently activated NF-kappaB, which in turn up-regulated p53 expression and increased the stability and DNA binding activity of p53. Consequently, NF-kappaB-dependent p53 activity induced the expression of p53-regulated genes PUMA and p21(waf1) as well as apoptosis. Importantly, lack of RelA, IKK, and p53 as well as expression of a dominant negative IkappaBalpha (IkappaBalphaM) inhibited NF-kappaB-dependent p53 activation and apoptosis. The doxycycline-induced NF-kappaB activation was not inhibited in HCT116/p53(-/-) cells. Our results demonstrate that NF-kappaB plays an essential role in activation of wild-type p53 tumor suppressor to initiate proapoptotic signaling in response to overgeneration of superoxide. Thus, these findings reveal a mechanism of NF-kappaB-regulated proapoptotic signaling.