RESUMO
BACKGROUND: Platelet adhesion and aggregation play a crucial role in arterial thrombosis and ischemic stroke. Here, we identify platelet ERO1α (endoplasmic reticulum oxidoreductase 1α) as a novel regulator of Ca2+ signaling and a potential pharmacological target for treating thrombotic diseases. METHODS: Intravital microscopy, animal disease models, and a wide range of cell biological studies were utilized to demonstrate the pathophysiological role of ERO1α in arteriolar and arterial thrombosis and to prove the importance of platelet ERO1α in platelet activation and aggregation. Mass spectrometry, electron microscopy, and biochemical studies were used to investigate the molecular mechanism. We used novel blocking antibodies and small-molecule inhibitors to study whether ERO1α can be targeted to attenuate thrombotic conditions. RESULTS: Megakaryocyte-specific or global deletion of Ero1α in mice similarly reduced platelet thrombus formation in arteriolar and arterial thrombosis without affecting tail bleeding times and blood loss following vascular injury. We observed that platelet ERO1α localized exclusively in the dense tubular system and promoted Ca2+ mobilization, platelet activation, and aggregation. Platelet ERO1α directly interacted with STIM1 (stromal interaction molecule 1) and SERCA2 (sarco/endoplasmic reticulum Ca2+-ATPase 2) and regulated their functions. Such interactions were impaired in mutant STIM1-Cys49/56Ser and mutant SERCA2-Cys875/887Ser. We found that ERO1α modified an allosteric Cys49-Cys56 disulfide bond in STIM1 and a Cys875-Cys887 disulfide bond in SERCA2, contributing to Ca2+ store content and increasing cytosolic Ca2+ levels during platelet activation. Inhibition of Ero1α with small-molecule inhibitors but not blocking antibodies attenuated arteriolar and arterial thrombosis and reduced infarct volume following focal brain ischemia in mice. CONCLUSIONS: Our results suggest that ERO1α acts as a thiol oxidase for Ca2+ signaling molecules, STIM1 and SERCA2, and enhances cytosolic Ca2+ levels, promoting platelet activation and aggregation. Our study provides evidence that ERO1α may be a potential target to reduce thrombotic events.
Assuntos
AVC Isquêmico , Trombose , Animais , Camundongos , Plaquetas/metabolismo , Sinalização do Cálcio , Dissulfetos , AVC Isquêmico/metabolismo , Ativação PlaquetáriaRESUMO
Ferroptosis is an iron-dependent regulated necrosis mediated by lipid peroxidation. Cancer cells survive under metabolic stress conditions by altering lipid metabolism, which may alter their sensitivity to ferroptosis. However, the association between lipid metabolism and ferroptosis is not completely understood. In this study, we found that the expression of elongation of very long-chain fatty acid protein 5 (ELOVL5) and fatty acid desaturase 1 (FADS1) is up-regulated in mesenchymal-type gastric cancer cells (GCs), leading to ferroptosis sensitization. In contrast, these enzymes are silenced by DNA methylation in intestinal-type GCs, rendering cells resistant to ferroptosis. Lipid profiling and isotope tracing analyses revealed that intestinal-type GCs are unable to generate arachidonic acid (AA) and adrenic acid (AdA) from linoleic acid. AA supplementation of intestinal-type GCs restores their sensitivity to ferroptosis. Based on these data, the polyunsaturated fatty acid (PUFA) biosynthesis pathway plays an essential role in ferroptosis; thus, this pathway potentially represents a marker for predicting the efficacy of ferroptosis-mediated cancer therapy.
Assuntos
Ácidos Graxos Insaturados/biossíntese , Ferroptose/fisiologia , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Ácido Araquidônico/genética , Ácido Araquidônico/metabolismo , Ácido Araquidônico/farmacologia , Carbolinas/farmacologia , Linhagem Celular Tumoral , Metilação de DNA , Dessaturase de Ácido Graxo Delta-5 , Elementos Facilitadores Genéticos , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Elongases de Ácidos Graxos/genética , Elongases de Ácidos Graxos/metabolismo , Ácidos Graxos Insaturados/genética , Ácidos Graxos Insaturados/metabolismo , Ferroptose/efeitos dos fármacos , Ferroptose/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Metabolismo dos Lipídeos/genética , Regiões Promotoras Genéticas , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/patologiaRESUMO
Endothelial progenitor cells (EPCs) promote neovascularization and tissue repair by migrating to vascular injury sites; therefore, factors that enhance EPC homing to damaged tissues are of interest. Here, we provide evidence of the prominent role of the Netrin-4 (NTN4)-Unc-5 Netrin receptor B (UNC5B) axis in EPC-specific promotion of ischemic neovascularization. Our results showed that NTN4 promoted the proliferation, chemotactic migration, and paracrine effects of small EPCs (SEPCs) and significantly increased the incorporation of large EPCs (LEPCs) into tubule networks. Additionally, NTN4 prominently augmented neovascularization in mice with hindlimb ischemia by increasing the homing of exogenously transplanted EPCs to the ischemic limb and incorporating EPCs into vessels. Moreover, silencing of UNC5B, an NTN4 receptor, abrogated the NTN4-induced cellular activities of SEPCs in vitro and blood-flow recovery and neovascularization in vivo in ischemic muscle by reducing EPC homing and incorporation. These findings suggest NTN4 as an EPC-based therapy for treating angiogenesis-dependent diseases.
Assuntos
Células Progenitoras Endoteliais/metabolismo , Isquemia/metabolismo , Músculo Esquelético/metabolismo , Neovascularização Patológica/metabolismo , Receptores de Netrina/metabolismo , Netrinas/metabolismo , Animais , Células Progenitoras Endoteliais/patologia , Células Progenitoras Endoteliais/transplante , Inativação Gênica , Xenoenxertos , Membro Posterior/irrigação sanguínea , Humanos , Isquemia/genética , Isquemia/patologia , Isquemia/terapia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/patologia , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Neovascularização Patológica/terapia , Receptores de Netrina/genética , Netrinas/genéticaRESUMO
BACKGROUND: Spasmolytic polypeptide-expressing metaplasia (SPEM) is considered a precursor lesion of intestinal metaplasia and intestinal-type gastric cancer (GC), but little is known about microRNA alterations during metaplasia and GC developments. Here, we investigate miR-30a expression in gastric lesions and identify its novel target gene which is associated with the intestinal-type GC. METHODS: We conducted in situ hybridization and qRT-PCR to determine miR-30a expression in gastric tissues. miR-30a functions were determined through induction or inhibition of miR-30a in GC cell lines. A gene microarray was utilized to confirm miR-30a target genes in GC, and siRNA-mediated target gene suppression and immunostaining were performed. The Cancer Genome Atlas data were utilized to validate gene expressions. RESULTS: We found down-regulation of miR-30a during chief cell transdifferentiation into SPEM. MiR-30a level was also reduced in the early stage of GC, and its level was maintained in advanced GC. We identified a novel target gene of miR-30a and ITGA2, and our results showed that either ectopic expression of miR-30a or ITGA2 knockdown suppressed GC cell proliferation, migration, and tumorigenesis. Levels of ITGA2 inversely correlated with levels of miR-30a in human intestinal-type GC. CONCLUSION: We found down-regulation of miR-30a in preneoplastic lesions and its tumor-suppressive functions by targeting ITGA2 in GC. The level of ITGA2, which functions as an oncogene, was up-regulated in human GC. The results of this study suggest that coordination of the miR-30a-ITGA2 axis may serve as an important mechanism in the development of gastric precancerous lesions and intestinal-type GC.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinogênese/patologia , Regulação Neoplásica da Expressão Gênica , Integrina alfa2/metabolismo , Neoplasias Intestinais/patologia , MicroRNAs/genética , Neoplasias Gástricas/patologia , Animais , Apoptose , Biomarcadores Tumorais/genética , Carcinogênese/genética , Carcinogênese/metabolismo , Movimento Celular , Proliferação de Células , Feminino , Humanos , Integrina alfa2/genética , Neoplasias Intestinais/genética , Neoplasias Intestinais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Prognóstico , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Precise cell cycle regulation is critical to prevent aberrant cell proliferation and cancer progression. Cks1 was reported to be an essential accessory factor for SCFSkp2, the ubiquitin ligase that targets p27Kip1 for proteasomal degradation; these actions drive mammalian cell transition from G1 to S phase. In this study, we investigated the role played by Cks1 in the growth and progression of human hepatocellular carcinoma (HCC) cells. Silencing Cks1 expression abrogated osteopontin (OPN) expression in a p27Kip1-dependent manner in Huh7 HCC cells. OPN increased the proliferation, migration and invasion of Huh7 cells. Pharmacological inhibitor studies demonstrated that ERK1/2 signaling is responsible mainly for Cks1-mediated OPN expression. Cks1 appears to regulate ERK1/2 signaling through the expression of dual-specificity phosphatase 16 (DUSP16) because both Cks1 knockdown, which leads to DUSP16 upregulation, and DUSP16 overexpression decreased ERK1/2 phosphorylation and the resulting OPN expression. The same is true for the Cks1-mediated increases in p27Kip1, suggesting that Cks1 regulates OPN expression through activating ERK1/2 signaling either by suppressing DUSP16 expression or by a p27Kip1-dependent mechanism. Cks1 and OPN expression levels were significantly higher, but DUSP16 expression levels were significantly lower in HCC tissues than in normal liver tissues. Both Cks1 and OPN expression were negatively correlated with DUSP16 expression, whereas Cks1 expression was positively correlated with OPN expression. Moreover, combined panels for the expression levels of Cks1, DUSP16 and OPN showed significant prognostic power for the risk assessment of HCC patient overall survival. In conclusion, our data propose a novel function for Cks1 as a tumor promoter through the expression of the strongly oncogenic protein OPN in HCC.
Assuntos
Quinases relacionadas a CDC2 e CDC28/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Osteopontina/biossíntese , Osteopontina/genética , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Neoplasias Hepáticas/diagnóstico , Osteopontina/metabolismoRESUMO
Adipogenesis involved in hypertrophy and hyperplasia of adipocytes is responsible for expanding the mass of adipose tissues in obese individuals. Peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα) are two principal transcription factors induced by delicate signaling pathways, including signal transducer and activator of transcription 5 (STAT5), in adipogenesis. Here, we demonstrated a novel role of ginkgetin, a biflavone from Ginkgo biloba leaves, as a STAT5 inhibitor that blocks the differentiation of preadipocytes into adipocytes. During the differentiation of 3T3-L1 cells, ginkgetin treatment during the first 2 days markedly inhibited the formation of lipid-bearing adipocytes. PPARγ and C/EBPα expression was decreased in 3T3-L1 cells during adipogenesis following ginkgetin treatment, whereas no change was observed in C/EBPß or C/EBPδ expression. Inhibition of PPARγ and C/EBPα expression by ginkgetin occurred through the prevention of STAT5 activation during the initiation phase of adipogenesis. In addition, ginkgetin-mediated the inhibition of adipogenesis was recapitulated in the differentiation of primary preadipocytes. Lastly, we confirmed the inhibitory effects of ginkgetin on the hypertrophy of white adipose tissues from high-fat diet-fed mice. These results indicate that ginkgetin is a potential anti-adipogenesis and anti-obesity drug.
Assuntos
Adipogenia/efeitos dos fármacos , Fármacos Antiobesidade/farmacologia , Fármacos Antiobesidade/uso terapêutico , Biflavonoides/farmacologia , Biflavonoides/uso terapêutico , Células 3T3-L1 , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Dieta Hiperlipídica , Ginkgo biloba , Masculino , Camundongos , Camundongos Endogâmicos C57BL , PPAR gama/genética , PPAR gama/metabolismo , Folhas de Planta , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Silica nanoparticles (SiNPs) are widely used for biosensing and diagnostics, and for the targeted delivery of therapeutic agents. Safety concerns about the biomedical and clinical applications of SiNPs have been raised, necessitating analysis of the effects of their intrinsic properties, such as sizes, shapes, and surface physicochemical characteristics, on human health to minimize risk in biomedical applications. In particular, SiNP size-associated toxicological effects, and the underlying molecular mechanisms in the vascular endothelium remain unclear. This study aimed to elucidate the detailed mechanisms underlying the cellular response to exposure to trace amounts of SiNPs and to determine applicable size criteria for biomedical application. METHODS: To clarify whether these SiNP-mediated cytotoxicity due to induction of apoptosis or necrosis, human ECs were treated with SiNPs of four different non-overlapping sizes under low serum-containing condition, stained with annexin V and propidium iodide (PI), and subjected to flow cytometric analysis (FACS). Two types of cell death mechanisms were assessed in terms of production of reactive oxygen species (ROS), endoplasmic reticulum (ER) stress induction, and autophagy activity. RESULTS: Spherical SiNPs had a diameter of 21.8 nm; this was further increased to 31.4, 42.9, and 56.7 nm. Hence, we investigated these effects in human endothelial cells (ECs) treated with these nanoparticles under overlap- or agglomerate-free conditions. The 20-nm SiNPs, but not SiNPs of other sizes, significantly induced apoptosis and necrosis. Surprisingly, the two types of cell death occurred independently and through different mechanisms. Apoptotic cell death resulted from ROS-mediated ER stress. Furthermore, autophagy-mediated necrotic cell death was induced through the PI3K/AKT/eNOS signaling axis. Together, the present results indicate that SiNPs within a diameter of < 20-nm pose greater risks to cells in terms of cytotoxic effects. CONCLUSION: These data provide novel insights into the size-dependence of the cytotoxic effects of silica nanoparticles and the underlying molecular mechanisms. The findings are expected to inform the applicable size range of SiNPs to ensure their safety in biomedical and clinical applications.
Assuntos
Apoptose/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Nanopartículas/toxicidade , Necrose/patologia , Transdução de Sinais/efeitos dos fármacos , Dióxido de Silício , Autofagia/efeitos dos fármacos , Células Cultivadas , Meios de Cultura , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Nanopartículas/química , Necrose/metabolismo , Tamanho da Partícula , Fosfatidilinositol 3-Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Dióxido de Silício/química , Dióxido de Silício/toxicidadeRESUMO
Certain animal and plant pathogenic bacteria have developed virulence factors including effector proteins that enable them to overcome host immunity. A plant pathogen, Pseudomonas syringae pv. tomato (Pto) secretes a large repertoire of effectors via a type III secretory apparatus, thereby suppressing plant immunity. Here, we show that Pto causes sepsis in mice. Surprisingly, the effector HopQ1 disrupted animal phagocytosis by inhibiting actin rearrangement via direct interaction with the LIM domain of the animal target protein LIM kinase, a key regulator of actin polymerization. The results provide novel insight into animal host-plant pathogen interactions. In addition, the current study firstly demonstrates that certain plant pathogenic bacteria such as Pto evade phagocytosis by animal cells due to cross-kingdom suppression of host immunity.
Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas de Bactérias/fisiologia , Fagocitose , Pseudomonas syringae/patogenicidade , Fatores de Virulência/fisiologia , Animais , Bacteriemia/microbiologia , Proteínas de Bactérias/imunologia , Interações Hospedeiro-Patógeno , Quinases Lim/metabolismo , Solanum lycopersicum/imunologia , Solanum lycopersicum/microbiologia , Camundongos , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Imunidade Vegetal , Pseudomonas syringae/imunologia , Fatores de Virulência/imunologiaRESUMO
Normal extracellular secretion of nephroblastoma overexpressed (NOV, also known as CCN3) is important for the adhesion, migration, and differentiation of cells. In previous studies, we have shown that the intracellular accumulation of CCN3 inhibits the growth of prominent neurons. Increased intracellular CCN3 can be induced through various processes, such as transcription, detoxification, and posttranslational modification. In general, posttranslational modifications are very important for protein secretion. However, it is unclear whether posttranslational modification is necessary for CCN3 secretion. In this study, we have conducted mutational analysis of CCN3 to demonstrate that its thrombospondin type-1 (TSP1) domain is important for CCN3 secretion and intracellular function. Point mutation analysis confirmed that CCN3 secretion was inhibited by cysteine (C)241 mutation, and overexpression of CCN3-C241A inhibited neuronal axonal growth in vivo. Furthermore, we demonstrated that palmitoylation is important for the extracellular secretion of CCN3 and that zinc finger DHHC-type containing 22 (ZDHHC22), a palmityoltransferase, can interact with CCN3. Taken together, our results suggest that palmitoylation by ZDHHC22 at C241 in the CCN3 TSP1 domain may be required for the secretion of CCN3. Aberrant palmitoylation induces intracellular accumulation of CCN3, inhibiting neuronal axon growth.
Assuntos
Carnitina O-Palmitoiltransferase/química , Carnitina O-Palmitoiltransferase/metabolismo , Lipoilação/fisiologia , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteína Sobre-Expressa em Nefroblastoma/química , Proteína Sobre-Expressa em Nefroblastoma/metabolismo , Neurônios/metabolismo , Animais , Sítios de Ligação , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos ICR , Neurônios/química , Neurônios/citologia , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Despite the relatively low transfection efficiency and low specific foreign protein productivity (qp) of Chinese hamster ovary (CHO) cell-based transient gene expression (TGE) systems, TGE-based recombinant protein production technology predominantly employs CHO cells for pre-clinical research and development purposes. To improve TGE in CHO cells, Epstein-Barr virus nuclear antigen-1 (EBNA-1)/polyoma virus large T antigen (PyLT)-co-amplified recombinant CHO (rCHO) cells stably expressing EBNA-1 and PyLT were established using dihydrofolate reductase/methotrexate-mediated gene amplification. The level of transiently expressed Fc-fusion protein was significantly higher in the EBNA-1/PyLT-co-amplified pools compared to control cultures. Increased Fc-fusion protein production by EBNA-1/PyLT-co-amplification resulted from a higher qp attributable to EBNA-1 but not PyLT expression. The qp for TGE-based production with EBNA-1/PyLT-co-amplified rCHO cells (EP-amp-20) was approximately 22.9-fold that of the control culture with CHO-DG44 cells. Rather than improved transfection efficiency, this cell line demonstrated increased levels of mRNA expression and replicated DNA, contributing to an increased qp. Furthermore, there was no significant difference in N-glycan profiles in Fc-fusion proteins produced in the TGE system. Taken together, these results showed that the use of rCHO cells with co-amplified expression of the viral elements EBNA-1 and PyLT improves TGE-based therapeutic protein production dramatically. Therefore, EBNA-1/PyLT-co-amplified rCHO cells will likely be useful as host cells in CHO cell-based TGE systems.
Assuntos
Antígenos Transformantes de Poliomavirus/genética , Antígenos Nucleares do Vírus Epstein-Barr/genética , Amplificação de Genes , Técnicas de Amplificação de Ácido Nucleico/métodos , Animais , Células CHO , Cricetinae , Cricetulus , Proteínas Recombinantes/genética , TransfecçãoRESUMO
Phospho-cofilin (p-cofilin), which has a phosphate group on Ser-3, is involved in actin polymerization. Its dephosphorylated form promotes filopodia formation and cell migration by enhancing actin depolymerization. Protein phosphatase slingshot homologs (SSHs), known as dual-specificity phosphatases, catalyze hydrolytic removal of the Ser-3 phosphate group from phospho-cofilin. Aberrant SSH activity results in cancer metastasis, implicating SSHs as potential therapeutic targets for cancer metastasis. In this study, we screened 658 natural products purified from traditional oriental medicinal plants to identify three potent SSH inhibitors with submicromolar or single-digit micromolar Ki values: gossypol, hypericin, and sennoside A. The three compounds were purified from cottonseed, Saint John's wort, and rhubarb, respectively. Sennoside A markedly increased cofilin phosphorylation in pancreatic cancer cells, leading to impaired actin dynamics in pancreatic cancer cells with or without EGF stimulation and reduced motility and invasiveness in vitro and in vivo. Collaboratively, these results demonstrate that sennoside A is a novel inhibitor of SSHs and suggest that it may be valuable in the development of pharmaceutical drugs for treating cancer metastasis.
Assuntos
Antineoplásicos/farmacologia , Movimento Celular/efeitos dos fármacos , Invasividade Neoplásica/prevenção & controle , Neoplasias Pancreáticas/tratamento farmacológico , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/metabolismo , Extrato de Senna/farmacologia , Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Linhagem Celular Tumoral , Células Endoteliais da Veia Umbilical Humana , Humanos , Invasividade Neoplásica/patologia , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Pâncreas/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Fosforilação/efeitos dos fármacos , SenosídeosRESUMO
Ginsenoside Rg5 is a compound newly synthesized during the steaming process of ginseng; however, its biological activity has not been elucidated with regard to endothelial function. We found that Rg5 stimulated in vitro angiogenesis of human endothelial cells, consistent with increased neovascularization and blood perfusion in a mouse hind limb ischemia model. Rg5 also evoked vasorelaxation in aortic rings isolated from wild type and high cholesterol-fed ApoE(-/-) mice but not from endothelial nitric-oxide synthase (eNOS) knock-out mice. Angiogenic activity of Rg5 was highly associated with a specific increase in insulin-like growth factor-1 receptor (IGF-1R) phosphorylation and subsequent activation of multiple angiogenic signals, including ERK, FAK, Akt/eNOS/NO, and Gi-mediated phospholipase C/Ca(2+)/eNOS dimerization pathways. The vasodilative activity of Rg5 was mediated by the eNOS/NO/cGMP axis. IGF-1R knockdown suppressed Rg5-induced angiogenesis and vasorelaxation by inhibiting key angiogenic signaling and NO/cGMP pathways. In silico docking analysis showed that Rg5 bound with high affinity to IGF-1R at the same binding site of IGF. Rg5 blocked binding of IGF-1 to its receptor with an IC50 of â¼90 nmol/liter. However, Rg5 did not induce vascular inflammation and permeability. These data suggest that Rg5 plays a novel role as an IGF-1R agonist, promoting therapeutic angiogenesis and improving hypertension without adverse effects in the vasculature.
Assuntos
Indutores da Angiogênese/farmacologia , Ginsenosídeos/farmacologia , Membro Posterior/irrigação sanguínea , Isquemia/tratamento farmacológico , Receptor IGF Tipo 1/agonistas , Vasodilatação/efeitos dos fármacos , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Regulação da Expressão Gênica , Membro Posterior/efeitos dos fármacos , Membro Posterior/patologia , Humanos , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Isquemia/genética , Isquemia/metabolismo , Isquemia/patologia , Camundongos , Neovascularização Fisiológica , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais , Técnicas de Cultura de Tecidos , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismoRESUMO
The heterogeneity and genetic instability of ovarian cancer cells often lead to the development of drug resistance, closely related with the increased cancer-related mortality. In this study, we investigated the role of dual-specificity phosphatase 1 (DUSP1) in the development of the resistance in human ovarian cancer cells against paclitaxel. Overexpression of DUSP1 in HeyA8 human ovarian cancer cells (HeyA8-DUSP1) up-regulated the expression of the drug efflux pump, p-glycoprotein. Consequently, HeyA8-DUSP1 cells are highly resistant to paclitaxel, with the resistance comparable to that of a multi-drug resistance cell line (HeyA8-MDR). Moreover, over expression of DUSP1 significantly increased the activation of p38 MAPK, leaving the activation of ERK1/2 and JNK1/2 unaffected. Pharmacological suppression of p38 MAPK activity prevents the up-regulation of p-glycoprotein expression and the consequent resistance against paclitaxel in HeyA8-DUSP1 cells. By contrast, HeyA8-MDR cells expressed a significantly higher level of DUSP1, but treatment with small interference RNA against DUSP1 significantly suppressed the expression of p-glycoprotein and the resistance against paclitaxel in HeyA8-MDR cells. Ectopic expression of MKK3, an upstream activator of p38 MAPK, significantly up-regulated the expression of p-glycoprotein and increased the consequent resistance against paclitaxel in HeyA8 cells. Collectively, these data indicated that DUSP1 may induce the resistance against paclitaxel through the p38 MAPK-mediated overexpression of p-glycoprotein in human ovarian cancer cells.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Fosfatase 1 de Especificidade Dupla/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Paclitaxel/administração & dosagem , Antineoplásicos Fitogênicos/administração & dosagem , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/fisiologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Ovarianas/patologia , Resultado do TratamentoRESUMO
BACKGROUND & AIMS: Gallbladder carcinoma (GBC) is the most common malignancy of the biliary tract and one of the most lethal forms of human cancer. However, there is limited information about the molecular pathogenesis of GBC. Here, we examined the functional role of the tumor suppressor N-myc downstream-regulated gene 2 (NDRG2) and the underlying molecular mechanisms of disease progression in GBC. METHODS: Clinical correlations between NDRG2 expression and clinicopathological factors were determined by immunohistochemical analysis of tumor tissues from 86 GBC patients. Biological functions of NDRG2 and NDRG2-mediated signaling pathways were determined in GBC cell lines with NDRG2 knockdown or overexpression. RESULTS: Loss of NDRG2 expression was an independent predictor of decreased survival and was significantly associated with a more advanced T stage, higher cellular grade, and lymphatic invasion in patients with GBC. GBC cells with loss of NDRG2 expression showed significantly enhanced proliferation, migration, and invasiveness in vitro, and tumor growth and metastasis in vivo. Loss of NDRG2 induced the expression of matrix metalloproteinase-19 (MMP-19), which regulated the expression of Slug at the transcriptional level. In addition, MMP-19-induced Slug, increased the expression of a receptor tyrosine kinase, Axl, which maintained Slug expression through a positive feedback loop, and stabilized epithelial-mesenchymal transition of GBC cells. CONCLUSIONS: The results of our study help to explain why the loss of NDRG2 expression is closely correlated with malignancy of GBC. These results strongly suggest that NDRG2 could be a favorable prognostic indicator and promising target for therapeutic agents against GBC.
Assuntos
Transição Epitelial-Mesenquimal/genética , Neoplasias da Vesícula Biliar/genética , Neoplasias da Vesícula Biliar/patologia , Metaloproteinases da Matriz Secretadas/metabolismo , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Idoso , Animais , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/fisiologia , Feminino , Neoplasias da Vesícula Biliar/metabolismo , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Modelos Biológicos , Invasividade Neoplásica/genética , Transplante de Neoplasias , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Fatores de Transcrição da Família Snail , Proteínas Supressoras de Tumor/antagonistas & inibidores , Regulação para Cima , Receptor Tirosina Quinase AxlRESUMO
Understanding the molecular networks that regulate adipogenesis is crucial for gaining insight into obesity and identifying medicinal targets thereof is necessary for pharmacological interventions. However, the identity and molecular actions of activators that promote the early development of adipocytes are still largely unknown. Here, we demonstrate a novel role for phosphoprotein phosphatase 1CB (PPP1CB) as a potent adipogenic activator that promotes adipocyte differentiation. PPP1CB expression increased in vitro during the early phase of 3T3-L1 adipogenesis and in the murine model of high-fat diet-induced obesity. Depletion of PPP1CB dramatically suppressed the differentiation of 3T3-L1 cells into mature adipocytes, with a concomitant change in adipocyte marker genes and significantly inhibited clonal expansion. We also showed that knockdown of PPP1CB caused a significant decrease in C/EBPδ expression, which in turn resulted in attenuation of PPARγ, C/EBPα, adiponectin, and aP2. In addition, we elucidated the functional significance of PPP1CB by linking p38 activation to C/EBPδ expression in early adipogenesis. Overall, our findings demonstrate a novel function of PPP1CB in promoting adipogenesis and suggest that PPP1CB may be a promising therapeutic target for treatment of obesity and obesity-related diseases.
Assuntos
Adipócitos/enzimologia , Adipogenia/genética , Gorduras na Dieta/administração & dosagem , Obesidade/genética , Proteína Fosfatase 1/metabolismo , Células 3T3-L1 , Adipócitos/patologia , Adiponectina/genética , Adiponectina/metabolismo , Animais , Proteína delta de Ligação ao Facilitador CCAAT/genética , Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Diferenciação Celular , Dieta Hiperlipídica , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/enzimologia , Obesidade/etiologia , Obesidade/patologia , Proteína Fosfatase 1/antagonistas & inibidores , Proteína Fosfatase 1/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Thioredoxin-interacting protein (TXNIP) has multiple functions, including tumor suppression and involvement in cell proliferation and apoptosis. However, its role in the inflammatory process remains unclear. In this report, we demonstrate that Txnipâ»/â» mice are significantly more susceptible to lipopolysaccharide (LPS)-induced endotoxic shock. In response to LPS, Txnipâ»/â» macrophages produced significantly higher levels of nitric oxide (NO) and inducible nitric oxide synthase (iNOS), and an iNOS inhibitor rescued Txnipâ»/â» mice from endotoxic shock-induced death, demonstrating that NO is a major factor in TXNIP-mediated endotoxic shock. This susceptibility phenotype of Txnipâ»/â» mice occurred despite reduced IL-1ß secretion due to increased S-nitrosylation of NLRP3 compared to wild-type controls. Taken together, these data demonstrate that TXNIP is a novel molecule that links NO synthesis and NLRP3 inflammasome activation during endotoxic shock.
Assuntos
Proteínas de Transporte/metabolismo , Inflamassomos/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico/metabolismo , Choque Séptico/metabolismo , Tiorredoxinas/metabolismo , Animais , Proteínas de Transporte/genética , Inflamassomos/genética , Lipopolissacarídeos/toxicidade , Camundongos , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR , Óxido Nítrico/genética , Óxido Nítrico Sintase Tipo II/genética , Choque Séptico/induzido quimicamente , Choque Séptico/genética , Tiorredoxinas/genéticaRESUMO
Nanoparticles are of great interest due to their wide variety of biomedical and bioengineering applications. However, they affect cellular differentiation and/or intracellular signaling when applied and exposed to target organisms or cells. The brown adipocyte is a cell type important in energy homeostasis and thus closely related to obesity. In this study, we assessed the effects of silica nanoparticles (SNPs) on brown adipocyte differentiation. The results clearly showed that brown adipocyte differentiation was significantly repressed by exposure to SNPs. The brown adipocyte-specific genes as well as mitochondrial content were also markedly reduced. Additionally, SNPs led to suppressed p38 phosphorylation during brown adipocyte differentiation. These effects depend on the size of SNPs. Taken together, these results lead us to suggest that SNP has anti-brown adipogenic effect in a size-dependent manner via regulation of p38 phosphorylation.
Assuntos
Nanopartículas/química , Dióxido de Silício/química , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Células 3T3-L1 , Adipócitos Marrons/citologia , Adipócitos Marrons/metabolismo , Adipócitos Marrons/patologia , Animais , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Canais Iônicos/genética , Canais Iônicos/metabolismo , Camundongos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Nanopartículas/toxicidade , Nanopartículas/ultraestrutura , PPAR gama/genética , PPAR gama/metabolismo , Tamanho da Partícula , Fosforilação/efeitos dos fármacos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Desacopladora 1RESUMO
Tumor angiogenesis is essential for tumor invasive growth and metastasis, and generates abnormal vascular structures unlike developmental neovessel formation. To reduce tumor vascular abnormalities such as leakage and perivascular cell coverage deficiency that limit cancer therapy effectiveness, novel therapeutic approaches focus on vessel normalization. We have previously shown that Dickkopf-1 (DKK1), a Wnt antagonist, inhibits and its homolog DKK2 enhances, angiogenesis in normal tissues. In the present study, we investigated the effects of DKK1 and DKK2 on tumor growth and angiogenesis. Treatment of B16F10 melanoma-bearing mice with adenovirus expressing DKK1 significantly reduced tumor growth but DKK2 increased growth compared with controls. Similar pattern of tumor growth was observed in endothelial-specific DKK1 and DKK2 transgenic mice. Interestingly, tumor vascular density and perfusion were significantly decreased by DKK1 but increased by DKK2. Moreover, coverage of blood vessels by pericytes was reduced by DKK1, while DKK2 increased it. We further observed that DKK1 diminished retinal vessel density and increased avascular area in an in vivo murine model of oxygen-induced retinopathy, whereas DKK2 showed opposite results. These findings demonstrate that DKK1 and DKK2 have differential roles in normalization and functionality of tumor blood vessels, in addition to angiogenesis.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Melanoma/irrigação sanguínea , Melanoma/metabolismo , Proteínas de Neoplasias/biossíntese , Neovascularização Patológica/metabolismo , Adenoviridae , Animais , Linhagem Celular Tumoral , Feminino , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Melanoma/genética , Melanoma/patologia , Camundongos , Camundongos Transgênicos , Proteínas de Neoplasias/genética , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Transdução GenéticaRESUMO
Introduction: Tularemia, caused by the bacterium Francisella tularensis, poses health risks to humans and can spread through a variety of routes. It has also been classified as a Tier 1 Select agent by the CDC, highlighting its potential as a bioterrorism agent. Moreover, it is difficult to diagnose in a timely fashion, owing to the non-specific nature of tularemia infections. Rapid, sensitive, and accurate detection methods are required to reduce mortality rates. We aimed to develop antibodies directed against the outer membrane protein A of F. tularensis (FopA) for rapid and accurate diagnosis of tularemia. Methods: We used a baculovirus insect cell expression vector system to produce the FopA antigen and generate anti-FopA antibodies through immunization of BALB/c mice. We then employed hybridoma and phage display technologies to screen for antibodies that could recognize unique epitopes on FopA. Result: Two monoclonal antibodies, 6B12 and 3C1, identified through phage display screening specifically bound to recombinant FopA in a dose-dependent manner. The binding affinity of the anti-FopA 6B12 and 3C1 antibodies was observed to have an equilibrium dissociation constant of 1.76 × 10-10 M and 1.32 × 10-9 M, respectively. These antibodies were used to develop a sandwich ELISA system for the diagnosis of tularemia. This assay was found to be highly specific and sensitive, with detection limits ranging from 0.062 ng/mL in PBS to 0.064 ng/mL in skim milk matrices. Discussion: Our findings demonstrate the feasibility of a novel diagnostic approach for detecting F. tularensis based on targeting FopA, as opposed to existing tests that target the bacterial lipopolysaccharide.
Assuntos
Anticorpos Antibacterianos , Anticorpos Monoclonais , Proteínas da Membrana Bacteriana Externa , Francisella tularensis , Camundongos Endogâmicos BALB C , Proteínas Recombinantes , Tularemia , Tularemia/diagnóstico , Animais , Francisella tularensis/imunologia , Francisella tularensis/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/genética , Anticorpos Monoclonais/imunologia , Camundongos , Imunoensaio/métodos , Sensibilidade e Especificidade , Feminino , Técnicas de Visualização da Superfície Celular , Epitopos/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/genética , Hibridomas , Baculoviridae/genéticaRESUMO
Precise regulation of kinases and phosphatases is crucial for human metabolic homeostasis. This study aimed to investigate the roles and molecular mechanisms of protein tyrosine phosphatase type IVA1 (PTP4A1) in regulating hepatosteatosis and glucose homeostasis. Method: Ptp4a1-/- mice, adeno-associated virus encoding Ptp4a1 under liver-specific promoter, adenovirus encoding Fgf21, and primary hepatocytes were used to evaluate PTP4A1-mediated regulation in the hepatosteatosis and glucose homeostasis. Glucose tolerance test, insulin tolerance test, 2-deoxyglucose uptake assay, and hyperinsulinemic-euglycemic clamp were performed to estimate glucose homeostasis in mice. The staining, including oil red O, hematoxylin & eosin, and BODIPY, and biochemical analysis for hepatic triglycerides were performed to assess hepatic lipids. Luciferase reporter assays, immunoprecipitation, immunoblots, quantitative real-time polymerase chain reaction, and immunohistochemistry staining were conducted to explore the underlying mechanism. Results: Here, we found that deficiency of PTP4A1 aggravated glucose homeostasis and hepatosteatosis in mice fed a high-fat (HF) diet. Increased lipid accumulation in hepatocytes of Ptp4a1-/- mice reduced the level of glucose transporter 2 on the plasma membrane of hepatocytes leading to a diminution of glucose uptake. PTP4A1 prevented hepatosteatosis by activating the transcription factor cyclic adenosine monophosphate-responsive element-binding protein H (CREBH)/fibroblast growth factor 21 (FGF21) axis. Liver-specific PTP4A1 or systemic FGF21 overexpression in Ptp4a1-/- mice fed an HF diet restored the disorder of hepatosteatosis and glucose homeostasis. Finally, liver-specific PTP4A1 expression ameliorated an HF diet-induced hepatosteatosis and hyperglycemia in wild-type mice. Conclusions: Hepatic PTP4A1 is critical for regulating hepatosteatosis and glucose homeostasis by activating the CREBH/FGF21 axis. Our current study provides a novel function of PTP4A1 in metabolic disorders; hence, modulating PTP4A1 may be a potential therapeutic strategy against hepatosteatosis-related diseases.