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1.
Anticancer Res ; 40(1): 161-168, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31892564

RESUMO

BACKGROUND: Arming of an oncolytic adenovirus (OAd) by inserting expression cassettes of therapeutic transgenes into the OAd genome is a promising approach to enhance the therapeutic effects of an OAd. Ideally, this approach would simultaneously promote the replication of an OAd in tumor cells and transgene product-mediated antitumor effects by expressing therapeutic transgenes. We previously demonstrated that knockdown of cullin 4A (CUL4A), which is an E3 ubiquitin ligase, significantly promoted adenovirus replication by increasing the c-JUN protein level. In addition, previous studies reported that CUL4A was highly expressed in various types of tumor, and was involved in tumor growth and metastasis. MATERIALS AND METHODS: In this study, we developed a novel OAd expressing a short-hairpin RNA (shRNA) against CUL4A (OAd-shCUL4A). RESULTS: OAd-shCUL4 mediated higher levels of cytotoxic effects on various types of human tumor cell than a conventional OAd. Higher levels of OAd genome copy numbers were found in the tumor cells for OAd-shCUL4A, compared with a conventional OAd. CONCLUSION: OAd-shCUL4A showed efficient antitumor effects by both enhancing OAd replication and inhibiting tumor cell growth.


Assuntos
Adenoviridae/genética , Proteínas Culina/genética , Vetores Genéticos/genética , Vírus Oncolíticos/genética , RNA Interferente Pequeno/genética , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Terapia Viral Oncolítica , Interferência de RNA , Transdução Genética
2.
Gene ; 728: 144297, 2020 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-31870788

RESUMO

DEAD-box (DDX) genes encode a group of RNA helicases that are highly conserved and ubiquitously expressed from prokaryotes to eukaryotes, and appear to participate in almost every aspect of RNA metabolism. Studies have been extensively done in yeast and human, in insect, beyond the flies, however, the information of these genes is limited. Here, we therefore identified and characterized 32 DDX genes from Locusta migratoria (L. migratoria), a crop pest. Overview of the gene structure and domain composition showed that the gene size varies significantly from one to fifteen exons, and the encoded proteins contain the conserved helicase core with various extensions at their amino and carboxyl termini. Phylogenetic trees informed that these locust DDX family members have orthologs in all insect species examined and can be classified into 30 subfamilies, all of them found counterparts in human, and most in yeast as well. Quantitative real-time PCR revealed that these genes are expressed in all stages and tissues examined, overall with higher expression level at second and third-instar nymphs and in the reproductive organs. RNA interference (RNAi) analyses showed that seven genes cause lethal phenotype when silenced, of which five lead to defective midgut and gastric caecum, indicating that these genes are essential for the survival and maintenance of normal digestive organs of locust. These data provide a foundation for further functional analysis of these DDX genes in locust.


Assuntos
RNA Helicases DEAD-box/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/genética , Locusta migratoria/genética , Interferência de RNA , Animais , RNA Helicases DEAD-box/antagonistas & inibidores , Evolução Molecular , Proteínas de Insetos/antagonistas & inibidores , Locusta migratoria/crescimento & desenvolvimento , Família Multigênica , Fenótipo , Filogenia
3.
Gut ; 69(1): 158-167, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-30833451

RESUMO

OBJECTIVE: Hepatitis D virus (HDV) is a circular RNA virus coinfecting hepatocytes with hepatitis B virus. Chronic hepatitis D results in severe liver disease and an increased risk of liver cancer. Efficient therapeutic approaches against HDV are absent. DESIGN: Here, we combined an RNAi loss-of-function and small molecule screen to uncover host-dependency factors for HDV infection. RESULTS: Functional screening unravelled the hypoxia-inducible factor (HIF)-signalling and insulin-resistance pathways, RNA polymerase II, glycosaminoglycan biosynthesis and the pyrimidine metabolism as virus-hepatocyte dependency networks. Validation studies in primary human hepatocytes identified the carbamoyl-phosphatesynthetase 2, aspartate transcarbamylase and dihydroorotase (CAD) enzyme and estrogen receptor alpha (encoded by ESR1) as key host factors for HDV life cycle. Mechanistic studies revealed that the two host factors are required for viral replication. Inhibition studies using N-(phosphonoacetyl)-L-aspartic acid and fulvestrant, specific CAD and ESR1 inhibitors, respectively, uncovered their impact as antiviral targets. CONCLUSION: The discovery of HDV host-dependency factors elucidates the pathogenesis of viral disease biology and opens therapeutic strategies for HDV cure.


Assuntos
Aspartato Carbamoiltransferase/genética , Ácido Aspártico/análogos & derivados , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/genética , Di-Hidro-Orotase/genética , Receptor alfa de Estrogênio/metabolismo , Fulvestranto/farmacologia , Hepatite D Crônica/tratamento farmacológico , Ácido Fosfonoacéticos/análogos & derivados , Pirimidinas/biossíntese , Antivirais/farmacologia , Aspartato Carbamoiltransferase/antagonistas & inibidores , Aspartato Carbamoiltransferase/metabolismo , Ácido Aspártico/farmacologia , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/antagonistas & inibidores , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/metabolismo , Linhagem Celular , Di-Hidro-Orotase/antagonistas & inibidores , Di-Hidro-Orotase/metabolismo , Antagonistas do Receptor de Estrogênio/farmacologia , Receptor alfa de Estrogênio/antagonistas & inibidores , Inativação Gênica , Hepatite D Crônica/genética , Hepatite D Crônica/metabolismo , Vírus Delta da Hepatite/fisiologia , Hepatócitos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Resistência à Insulina , Estágios do Ciclo de Vida , Mutação com Perda de Função , Ácido Fosfonoacéticos/farmacologia , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Viral/metabolismo , Transdução de Sinais , Replicação Viral
4.
Insect Sci ; 27(1): 113-121, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29790281

RESUMO

The tawny crazy ant (Nylanderia fulva) is a new invasive pest in the United States. At present, its management mainly relies on the use of synthetic insecticides, which are generally ineffective at producing lasting control of the pest, necessitating alternative environmentally friendly measures. In this study, we evaluated the feasibility of gene silencing to control this ant species. Six housekeeping genes encoding actin (NfActin), coatomer subunit ß (NfCOPß), arginine kinase (NfArgK), and V-type proton ATPase subunits A (NfvATPaseA), B (NfvATPaseB) and E (NfvATPaseE) were cloned. Phylogenetic analysis revealed high sequence similarity to homologs from other ant species, particularly the Florida carpenter ant (Camponotus floridanus). To silence these genes, vector L4440 was used to generate six specific RNAi constructs for bacterial expression. Heat-inactivated, dsRNA-expressing Escherichia coli were incorporated into artificial diet. Worker ants exhibited reduced endogenous gene expression after feeding on such diet for 9 d. However, only ingestion of dsRNAs of NfCOPß (a gene involved in protein trafficking) and NfArgK (a cellular energy reserve regulatory gene in invertebrates) caused modest but significantly higher ant mortality than the control. These results suggest that bacterially expressed dsRNA can be orally delivered to ant cells as a mean to target its vulnerabilities. Improved efficacy is necessary for the RNAi-based approach to be useful in tawny crazy ant management.


Assuntos
Formigas , Genes de Insetos , Controle de Insetos/métodos , Interferência de RNA , Animais , Formigas/genética
5.
Arch Insect Biochem Physiol ; 103(1): e21636, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31612557

RESUMO

As a member of the low-density lipoprotein receptor (LDLR) superfamily, vitellogenin (Vg) receptor (VgR) is responsible for the uptake of Vg into developing oocytes and is a potential target for pest control. Here, a full-length VgR complementary DNA (named as CsVgR) was isolated and characterized in the rice stem borer, Chilo suppressalis. The composite CsVgR gene contained an open reading frame of 5,484 bp encoding a protein of 1,827 amino acid residues. Structural analysis revealed that CsVgR contained two ligand-binding domains (LBDs) with four Class A (LDLRA ) repeats in LBD1 and seven in LBD2, which was structurally different from most non-Lepidopteran insect VgRs having five repeats in LBD1 and eight in LBD2. The developmental expression analysis showed that CsVgR messenger RNA expression was first detectable in 3-day-old pupae, sharply increased in newly emerged female adults, and reached a peak in 2-day-old female adults. Consistent with most other insects VgRs, CsVgR was exclusively expressed in the ovary. Notably, injection of dsCsVgR into late pupae resulted in fewer follicles in the ovarioles as well as reduced fecundity, suggesting a critical role of CsVgR in female reproduction. These results may contribute to the development of RNA interference-mediated disruption of reproduction as a control strategy of C. suppressalis.


Assuntos
Proteínas do Ovo/genética , Mariposas/genética , Receptores de Superfície Celular/genética , Animais , Proteínas do Ovo/química , Proteínas do Ovo/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/metabolismo , Mariposas/crescimento & desenvolvimento , Mariposas/metabolismo , Ovário/crescimento & desenvolvimento , Ovário/metabolismo , Filogenia , Interferência de RNA , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Análise de Sequência de Proteína
6.
Arch Insect Biochem Physiol ; 103(1): e21642, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31667890

RESUMO

The Colorado potato beetle (Leptinotarsa decemlineata [Say]) is an insect pest that can significantly harm potato plants worldwide. Control of this insect relies heavily on chemical insecticides such as chlorantraniliprole. Nevertheless, the complete molecular signature associated with response to this compound is lacking in L. decemlineata. In this study, amplification and quantification by qRT-PCR (quantitative reverse transcription-polymerase chain reaction) of targets relevant to chlorantraniliprole were undertaken in insects exposed to this chemical. This approach showed modulation of numerous cytochrome P450s, such as CYP350D1 and CYP4Q3, as well as upregulation of microRNAs (miRNAs), including miR-1-3p and miR-305-5p, in chlorantraniliprole-exposed insects. Functional assessment of transcript targets predicted to be regulated by these miRNAs was performed and revealed their likely impact on transcriptional regulation. RNAi-based targeting of CYP350D1 notably provided preliminary evidence of its underlying implication for chlorantraniliprole response in L. decemlineata. Overall, this study strengthens the current knowledge of the molecular changes linked to chlorantraniliprole response in L. decemlineata and provides novel targets with potential relevance to chlorantraniliprole susceptibility in this insect pest of global relevance.


Assuntos
Besouros/efeitos dos fármacos , Besouros/metabolismo , Inseticidas/farmacologia , ortoaminobenzoatos/farmacologia , Animais , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação da Expressão Gênica , MicroRNAs/genética , MicroRNAs/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa
7.
Arch Insect Biochem Physiol ; 103(1): e21640, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31667893

RESUMO

Long noncoding RNAs (lncRNAs) that have immune responses to various stimuli have been identified in some insects. One type of pathogen-associated molecular pattern, double-stranded RNA (dsRNA), can trigger the RNA interference (RNAi) pathway and immune response. Interestingly, there has been no research into characterizing the relationship between lncRNA and dsRNA-induced RNAi pathways. In this study, dsRNA-induced lncRNAs were investigated in two species of lepidopteran insects, Helicoverpa armigera and Plutella xylostella, and one species of coleopteran insects, Tribolium castaneum. Between untreated group and dsRNA-induced group; 3,463 H. armigera, 6,245 P. xylostella, and 3,067 T. castaneum differentially expressed lncRNAs were identified while 156 H. armigera, 247 P. xylostella, 415 T. castaneum lncRNAs and their putative target genes showed consistent changes in gene expression. In T. castaneum, most target genes of the differentially expressed lncRNAs are enriched in the cyclic adenosine monophosphate signaling pathway, ABC transporters, and Janus kinase-signal transducers and activators of the transcription signaling pathway. Conversely, in H. armigera and P. xylostella, the differentially expressed lncRNAs were mainly enriched in the metabolic, digestive, and synthetic signaling pathways. This result indicates that dsRNA-induced lncRNA is species-dependent. We also found that both Dicer-2 and the lncRNA that targets Dicer-2 were significantly upregulated after dsRNA treatment in P. xylostella, indicating that some lncRNAs may be involved in the regulation of the core RNAi pathway in insects. Our results are the first to identify a relationship between lncRNAs and dsRNA in various insect species with different RNAi efficiencies. These results provide a reference for future study of the dsRNA-induced RNAi pathway and different RNAi efficiencies among insect species.


Assuntos
Mariposas/genética , RNA de Cadeia Dupla/farmacologia , RNA Longo não Codificante/metabolismo , Tribolium/genética , Animais , Expressão Gênica , Mariposas/metabolismo , Interferência de RNA , Transdução de Sinais , Tribolium/metabolismo
8.
Cell Physiol Biochem ; 53(S1): 52-62, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31854954

RESUMO

Kv1.3 is a voltage gated potassium channel located in the plasma membrane, as well as at intracellular levels, such as mitochondria (mitoKv1.3), nucleus and Golgi apparatus. The plasma membrane channel has been shown to be important for cell proliferation, while the mitochondrial counterpart has been related to modulation of cell death. Moreover, altered expression of Kv1.3 was observed in various tumors and Kv1.3 seems to be involved in development and progression of various cancerous forms. Recent experimental evidences have proved that pharmacological inhibition of the mitoKv1.3 succeeded in reducing up to 90% of tumor volume in vivo in orthotopic mouse model. Furthermore, mitoKv1.3 modulation could impact on cell proliferation as well as on regulation of intracellular signaling pathways. Indeed, the treatment with sub-lethal doses of mitoKv1.3 inhibitors can downregulate Wnt-ß catenin signaling by reducing mitochondrial ATP production and triggering ER-stress. In this review, we describe the role of the mitoKv1.3 in cell death, cancer and intracellular signaling. We will discuss how pharmacological modulation of mitochondrial potassium fluxes impact on mitochondrial membrane potential, reactive oxygen species production and ATP synthesis. All these changes in mitochondrial fitness are related to cell proliferation as well as to cell death and finally on cancer development and progression, so Kv1.3 (and mitoKv1.3) could be now considered a new oncological target.


Assuntos
Canal de Potássio Kv1.3/metabolismo , Mitocôndrias/metabolismo , Neoplasias/metabolismo , Animais , Apoptose , Proliferação de Células , Estresse do Retículo Endoplasmático , Canal de Potássio Kv1.3/antagonistas & inibidores , Canal de Potássio Kv1.3/genética , Neoplasias/patologia , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais
9.
J Photochem Photobiol B ; 201: 111653, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31710929

RESUMO

Autophagy is an important process for maintaining intracellular homeostasis. Our previous study demonstrated that autophagy was down-regulated in ultraviolet B (UVB)-irradiated keratinocytes. Raffinose is a natural oligosaccharide that serves as a novel activator of autophagy and as a balancing agent to regulate the diversity of environmental stress. However, whether raffinose balances ultraviolet stress through the autophagy activation pathway has yet to be established. In this study, we found that raffinose treatment inhibited the LDH release and trypan blue staining in UVB-challenged human keratinocytes cell line HaCaT but did not affect the cleavage of apoptotic markers Caspase-3 and PARP, as well as translocation into nucleus of other cell death markers Endonuclease G and AIF. Moreover, we confirmed that raffinose treatment enhanced autophagy flux in an MTOR-independent manner in HaCaT cells. Importantly, decrease of LC3-II turnover in UVB-irradiated keratinocytes could be rescued by raffinose treatment, indicating that raffinose treatment increased autophagy in UVB-irradiated HaCaT cells. Furthermore, the effect on cell death by raffinose was inhibited when autophagy was suppressed with either a small interfering RNA targeting ATG5 (siATG5) or autophagic inhibitor wortmannin. In conclusion, we demonstrated that raffinose increases MTOR-independent autophagy and reduces cell death in UVB-irradiated keratinocytes. Our study indicated that the natural agent raffinose presents the potential value in opposing photodamage.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Rafinose/farmacologia , Raios Ultravioleta , Apoptose/efeitos da radiação , Autofagia/efeitos da radiação , Proteína 5 Relacionada à Autofagia/antagonistas & inibidores , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Caspase 3/metabolismo , Linhagem Celular , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo
10.
Cell Physiol Biochem ; 53(5): 747-759, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31622062

RESUMO

BACKGROUND/AIMS: Angiotensin II (Ang II) induces podocyte injury resulting in apoptosis in vitro and in vivo. However, the relationship between autophagy and apoptosis in Ang II-induced podocyte injury is unknown and the role of Ang II-induced autophagy in podocyte survival or death remains unclear. We investigated the sequential relationship between autophagy and apoptosis in Ang II-induced podocytes as well as the role of phosphatidylinositide 3-kinase (PI3-kinase). METHODS: Mouse podocytes were incubated in media containing various concentrations of Ang II and at different incubation times. The changes of podocyte autophagy and apoptosis were observed by electron microscopy, confocal imaging, western blotting, and FACS assay according to the presence of Ang II. RESULTS: Ang II enhanced the podocyte expression of the autophagic proteins, LC3A/B-II and beclin-1, and also increased the number of autophagosomes compared with control cells at early phase of 12 hours in a dose-dependent manner. This effect was inhibited by pretreatment with 3-methyladenine (3-MA), a PI3-kinase class III inhibitor. Thereafter, the Ang II-induced enhancement in autophagy decreased, whereas, podocyte apoptosis appeared later at 24 hours in concentration- and time-dependent manners in FACS and TUNEL assays. 3-MA and LY294002, a pan PI3-kinase inhibitor, further increased Ang II-induced podocyte apoptosis. Suppression of autophagy by Atg5 siRNA could induce podocyte apoptosis and further augment high-dose Ang II-induced podocyte apoptosis. CONCLUSION: These findings suggest that Ang II promotes autophagy in podocytes before apoptosis as an early adaptive cytoprotective mechanism for podocyte survival after Ang II treatment, and the transitional imbalance between autophagy and apoptosis causes podocyte injury.


Assuntos
Angiotensina II/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Autofagossomos/metabolismo , Proteína 5 Relacionada à Autofagia/antagonistas & inibidores , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Podócitos/citologia , Podócitos/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Regulação para Cima/efeitos dos fármacos
11.
Cell Physiol Biochem ; 53(4): 713-730, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31599538

RESUMO

BACKGROUND/AIMS: Renal injury related to hypertension is characterized by glomerular and tubulointerstitial damage. The overactivation of the renin-angiotensin system mainly by angiotensin II (AII) seems to be a main contributor to progressive renal fibrosis. Epithelial to mesenchymal transition (EMT) is a mechanism that promotes renal fibrosis. Owing to heat shock protein 70 (Hsp70) cytoprotective properties, the chaperone exhibits an important potential as a therapeutic target. We investigate the role of Hsp70 on Angiotensin II induced epithelial mesenchymal transition within the Losartan effect in proximal tubule cells (PTCs) from a genetic model of hypertension in rats (SHR). METHODS: Primary cell culture of PTCs from SHR and Wistar Kyoto (WKY) rats were stimulated with AII, treated with Losartan (L), (L+AII) or untreated (Cc). The functional Hsp70 role in Losartan effect, after silencing its expression by cell transfection, was determined by Immunofluorescence; Western blotting; Gelatin Zymography assays; Scratch wound assays; flow cytometry; and Live Cell Time-lapse microscopy. RESULTS: (L) and (L+AII) treatments induced highly organized actin filaments and increased cortical actin in SHR PTCs. However, SHR PTCs (Cc) and (AII) treated cells showed disorganized actin. After Hsp72 knockdown in SHR PTCs, (L) was unable to stabilize the actin cytoskeleton. We demonstrated that (L) and (L+AII) increased E-cadherin levels and decreased vinculin, α-SMA, vimentin, pERK, p38 and Smad2-3 activation compared to (AII) and (Cc) SHR PTCs. Moreover, (L) inhibited MMP-2 and MMP-9 secretion, reduced migration and cellular displacement, stabilizing intercellular junctions. Notably, (L) treatment in shHsp72 knockdown SHR PTCs showed results similar to SHR PTCs (Cc). CONCLUSION: Our results demonstrate that Losartan through Hsp70 inhibits the EMT induced by AII in proximal tubule cells derived from SHR.


Assuntos
Angiotensina II/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/metabolismo , Losartan/farmacologia , Citoesqueleto de Actina/efeitos dos fármacos , Animais , Caderinas/metabolismo , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Adesões Focais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Proteínas de Choque Térmico HSP70/genética , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Vinculina/metabolismo
12.
Cell Physiol Biochem ; 53(4): 701-712, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31592599

RESUMO

BACKGROUND/AIMS: Cholinergic signalling mediated by the activation of muscarinic and nicotinic receptors has been described in the literature as a classic and important signalling pathway in the regulation of the inflammatory response. Recent research has investigated the role of acetylcholine, the physiological agonist of these receptors, in the control of energy homeostasis at the central level. Studies have shown that mice that do not express acetylcholine in brain regions regulating energy homeostasis present with excessive weight gain and hyperphagia. However, it has not yet been well-described in the literature which cholinergic receptor subunits are involved in this response; moreover, the signalling pathways responsible for the observed effects are not fully delineated. The hypothalamus is the regulating centre of energy homeostasis, and the α7 subunit of the nicotinic acetylcholine receptor (α7nAChR) is highly expressed in this region. When active, α7nAChR recruits proteins such as JAK2/STAT3 to mediate its signalling; the same intracellular components are required by leptin, an anorexigenic hormone. The aim of the present study was to evaluate the role of the hypothalamic α7nAChR in the control of energy homeostasis. METHODS: The work was performed on Swiss male mice. Initially, using immunofluorescent staining on brain sections, the presence of α7nAChR in hypothalamic cells regulating energy homeostasis was evaluated. Animals were submitted to stereotaxis in the lateral ventricle and intracerebroventricular stimulation (ICV) was used for the administration of an agonist (PNU) or antagonist (α-bungarotoxin) of α7nAChR. Metabolic parameters were evaluated and the expression of neuropeptides was evaluated in the hypothalamus by real-time PCR and western blot. The expression of hypothalamic neuropeptides was evaluated in mice treated with siRNA or inhibitors of JAK2/STAT3 (AG490 and STATTIC) proteins. We also evaluated food intake in α7nAChR knockout animals (α7KO). Additionally, in mouse hypothalamic cell culture (the mypHoA-POMC/GFP lineage), we evaluated the expression of neuropeptides and pSTAT3 after stimulation with PNU. RESULTS: Our results indicate co-localisation of α7nAChR with α-MSH, AgRP and NPY in hypothalamic cells. Pharmacological activation of α7nAChR reduced food intake and increased hypothalamic POMC expression and decreased NPY and AgRP mRNA levels and the protein content of pAMPK. Inhibition of α7nAChR with an antagonist increased the mRNA content of NPY and AgRP. Inhibition of α7nAChR with siRNA led to the suppression of POMC expression and an increase in AgRP mRNA levels. α7KO mice showed no changes in food intake. Inhibition of proteins involved in the JAK2/STAT3 signalling pathway reversed the effects observed after PNU stimulation. POMC-GFP cells, when treated with PNU, showed increased POMC expression and nuclear translocation of pSTAT3. CONCLUSION: Thus, selective activation of α7nAChR is able to modulate important markers of the response to food intake, suggesting that α7nAChR activation can suppress the expression of orexigenic markers and favour the expression of anorexics using the intracellular JAK2/STAT3 machinery.


Assuntos
Proteína Relacionada com Agouti/metabolismo , Janus Quinase 2/metabolismo , Pró-Opiomelanocortina/metabolismo , Fator de Transcrição STAT3/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Proteína Relacionada com Agouti/genética , Animais , Benzamidas/farmacologia , Compostos Bicíclicos com Pontes/farmacologia , Bungarotoxinas/farmacologia , Linhagem Celular , Ingestão de Alimentos/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Pró-Opiomelanocortina/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/genética , Transdução de Sinais/efeitos dos fármacos , Receptor Nicotínico de Acetilcolina alfa7/agonistas , Receptor Nicotínico de Acetilcolina alfa7/antagonistas & inibidores , Receptor Nicotínico de Acetilcolina alfa7/genética
13.
Chem Biol Interact ; 314: 108848, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31610156

RESUMO

Cardiomyocyte injury induced by acute myocardial infarction contributes to myocardial dysfunction. Accumulating evidence has demonstrated that pleckstrin homology domain leucine-rich repeat protein phosphatase 2 (PHLPP2) is a cytoprotective protein that protects against various adverse injuries. However, whether PHLPP2 participates in regulating myocardial-infarction-induced cardiomyocyte injury remains unknown. In the present study, we aimed to investigate the biological role and molecular mechanism of PHLPP2 in regulating hypoxia-induced cardiomyocyte injury. Cardiomyocytes were cultured in an anaerobic chamber for 24 h to induce hypoxic injury in vitro. The expression of PHLPP2 was determined by real-time quantitative PCR and Western blot analysis. Cell viability was measured by MTT assay. Cell apoptosis was assessed by TUNEL and caspase-3 activity assays. Intracellular reactive oxygen species (ROS) levels were measured by DCFH-DA probe. PHLPP2 expression was highly upregulated in hypoxia-injured cardiomyocytes. Inhibition of PHLPP2 by small interfering RNA (siRNA)-mediated gene silencing significantly improved the viability of hypoxia-injured cardiomyocytes and attenuated hypoxia-induced apoptosis and ROS production. In contrast, PHLPP2 overexpression exacerbated hypoxia-induced apoptosis and ROS production in cardiomyocytes. Mechanism research revealed that PHLPP2 silencing increased the phosphorylation of glycogen synthase kinase (GSK)-3ß and promoted the nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2). In addition, PHLPP2 inhibition promoted Nrf2/antioxidant response element (ARE) transcriptional activity. However, Nrf2 silencing markedly reversed PHLPP2-inhibition-mediated cardioprotection, while GSK-3ß inhibition partially blocked the PHLPP2-overexpression-induced adverse effect. Taken together, these findings demonstrate that PHLPP2 inhibition alleviates hypoxia-induced cardiomyocyte injury by reinforcing Nrf2/ARE antioxidant signaling via inactivating GSK-3ß, a pathway that highlights the importance of the PHLPP2/GSK-3ß/Nrf2/ARE signaling axis in regulation of cardiomyocyte injury. Our study suggests a potential relevance for PHLPP2 in acute myocardial infarction, and this protein may serve as a promising target for cardioprotection.


Assuntos
Elementos de Resposta Antioxidante/genética , Hipóxia Celular , Fator 2 Relacionado a NF-E2/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular , Regulação para Baixo , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta/metabolismo , Camundongos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Fator 2 Relacionado a NF-E2/genética , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/genética , Fosforilação , Pirimidinas/farmacologia , Pirróis/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
14.
Cell Physiol Biochem ; 53(4): 731-745, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31613064

RESUMO

BACKGROUND/AIMS: 3-Deazaneplanocin, DZNep, has been reported to inhibit the EZH2 histone methylase and to induce cell apoptosis in chondrosarcomas (CS). The present study aims to confirm the therapeutic potential of EZH2 inhibitors and investigate the molecular mechanisms of DZNep in chondrosarcomas. METHODS: CS cell lines and primary cultures were used. Apoptosis was investigated using PARP cleavage, caspase 3/7 activity, or Apo2.7 expression. S-adenosylhomocysteine (SAH) and S-adenosylmethionine (SAM) were quantified by UHPLC-MS/MS. Differentially expressed genes in treated-chondrosarcomas and chondrocytes were researched by microarray analysis. RESULTS: DZNep induced apoptosis in chondrosarcomas both in vivo and in vitro. However, this effect was not correlated to EZH2 expression nor activity, and EZH2 knock-down by siRNA did not reduce CS viability. Additionally, the reduction of H3K27me3 induced by GSK126 or tazemetostat (EPZ-6438) did not provoke chondrosarcoma death. However, as expected, DZNep induced SAH accumulation and reduced SAM:SAH ratio. Further, microarray analysis suggests a key role of EGFR in antitumoral effect of DZNep, and pharmacological inhibition of EGFR reduced chondrosarcoma survival. CONCLUSION: EZH2 is not an adequate target for chondrosarcoma treatment. However, DZNep induces apoptosis in chondrosarcomas in vitro and in vivo, by a mechanism likely mediated though EGFR expression. Consequently, it would be worth initiating clinical trials to evaluating efficiency to S-adenosylhomocysteine hydrolase or EGFR inhibitors in patients with chondrosarcomas.


Assuntos
Adenosina/análogos & derivados , Regulação para Baixo/efeitos dos fármacos , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Adenosina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Condrossarcoma/metabolismo , Condrossarcoma/patologia , Dano ao DNA/efeitos dos fármacos , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Histonas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Nus , Mapas de Interação de Proteínas/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , S-Adenosil-Homocisteína/metabolismo
15.
Cell Physiol Biochem ; 53(4): 656-686, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31573152

RESUMO

BACKGROUND/AIMS: Despite recent advances in melanoma drug discovery, the average overall survival of patients with late stage metastatic melanoma is approximately 3 years, suggesting a need for approaches that identify new melanoma targets. We have previously reported a discovery of novel anti-melanoma compound 2155-14 (Onwuha-Ekpete et al., J Med Chem. 2014 Feb 27; 57(4):1599-608). In the report presented herein we aim to identify its target(s) and mechanism of action. METHODS: We utilized biotinylated analog of 2155-14 to pull down its targets from melanoma cells. Proteomics in combination with western blot were used to identify the targets. Mechanism of action of 2155-14 was determined using flow cytometry, RT-PCR, microscopy, western blot, and enzymatic activity assays. Where applicable, one-way analysis of variance (ANOVA) was used followed by Dunnett post hoc test. RESULTS: In the present study, we identified ATP-dependent RNA helicase DDX1 and heterogeneous nuclear ribonucleoproteins (hnRNPs) H1, H2 and A2/B1 as targets of anti-melanoma compound 215514. To the best of our knowledge, this is a first report suggesting that these proteins could be targeted for melanoma therapy. Mechanistic investigations showed that 2155-14 induces ER stress leading to potentiation of basal autophagy resulting in melanoma cell death in BRAF and NRAS mutated melanoma cells. CONCLUSION: Identification of mode of action of 2155-14 may provide insight into novel therapies against a broad range of melanoma subtypes. These studies were enabled by the novel probe derived from a mixture-based library, an important class of chemical biology tools for discovering novel targets.


Assuntos
Apoptose , Autofagia , RNA Helicases DEAD-box/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , RNA Helicases DEAD-box/antagonistas & inibidores , RNA Helicases DEAD-box/genética , Avaliação Pré-Clínica de Medicamentos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Ribonucleoproteínas Nucleares Heterogêneas/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Melanoma/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos
16.
Cell Physiol Biochem ; 53(4): 606-622, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31550088

RESUMO

BACKGROUND/AIMS: Adenosine release and connexin (Cx) hemichannel activity are enhanced in the respiratory epithelium during pathophysiological events such as inflammation. We analysed the interplay between Cx channels and adenosine signalling in human respiratory airway epithelium using the Calu-3 cell line as a model. METHODS: The Cx hemichannel activity in Calu-3 cells was evaluated by dye uptake assays. The expressed Cx isoforms and adenosine receptor subtypes were identified by PCR and western blot analysis. Pharmacological and molecular biological experiments were performed to analyse the involvement of the different adenosine receptor subtypes, the induced signalling pathways and the contribution of specific Cx isoforms to the hemichannel activity. RESULTS: The adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) increased the dye uptake rate in Calu-3 cells. The pannexon and Cx hemichannel inhibitor carbenoxolone (CBX) did not supress the dye uptake at pannexin-specific concentrations (100 µM). High CBX concentrations or the inhibitor La3+, both effective on Cx hemichannels, were needed to inhibit the dye uptake. The NECA-related increase of dye uptake depended on enhanced cAMP synthesis and subsequent activation of the protein kinase A (PKA) as shown by quantification of cAMP levels and pharmacological inhibition of the adenylyl cyclase and the PKA. Further pharmacological inhibition as well as knockdown experiments with specific siRNA showed that the A2B adenosine receptor was the subtype mainly responsible for the increased dye uptake. The NECA-related increase of the dye uptake rate correlated with a decrease of Cx43 mRNA and an increase of Cx26 mRNA content in the cells as well as Cx26 protein synthesis and was inhibited by Cx26 knockdown using Cx26 siRNA. Of note, a siRNA-induced knockdown of Cx43 increased the content of Cx26 mRNA and correspondingly the dye uptake rate. CONCLUSION: The Calu-3 cell model shows that stimulation of the A2B adenosine receptor subtype activates synthesis of cAMP. cAMP activates PKA and induces thereby an increase in Cx26 and a decrease in Cx43 mRNA levels. As a result, the synthesis of Cx26 is reinforced, leading to an enhanced Cx hemichannel activity. The report identifies a mechanism that integrates adenosine release and Cx hemichannel activity and shows how adenosine signalling and Cx channels may act together to promote persistent inflammation, which is observed in several chronic diseases of the respiratory airway.


Assuntos
Conexina 26/metabolismo , Receptor A2B de Adenosina/metabolismo , Agonistas do Receptor A2 de Adenosina/farmacologia , Carbenoxolona/farmacologia , Linhagem Celular , Conexina 26/antagonistas & inibidores , Conexina 26/genética , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Espectroscopia Dielétrica , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptor A2B de Adenosina/química , Receptor A2B de Adenosina/genética , Transdução de Sinais/efeitos dos fármacos
17.
J Agric Food Chem ; 67(42): 11607-11615, 2019 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-31560536

RESUMO

ζ-carotene desaturase (ZDS) is a key enzyme in carotenoid biosynthesis and plays an important role in plant photosynthesis. We characterized an albino leaf-color mutant obtained from ethyl methanesulfonate treatment: albino and seedling lethality 1 (ale1). The material contains a chloroplast thylakoid defect where photosynthetic pigments declined and reactive oxygen species accumulated resulting in ale1 death within 3 weeks. Positional cloning and sequencing revealed that there was a single base substitution in ALE1, which encoded a ZDS involved in carotenoid biosynthesis. RNAi and complementation tests confirmed the identity of ALE1. Subcellular localization showed that the ALE1 protein is localized in the chloroplast. Expression analysis indicated that the genes involved in chlorophyll and carotenoid biosynthesis were downregulated. We conclude that ALE1 plays an important role in chloroplast and plant growth in rice.


Assuntos
Cloroplastos/enzimologia , Oryza/crescimento & desenvolvimento , Oxirredutases/genética , Proteínas de Plantas/genética , Clorofila/metabolismo , Cloroplastos/genética , Regulação da Expressão Gênica de Plantas , Oryza/enzimologia , Oryza/genética , Oxirredutases/metabolismo , Fotossíntese , Proteínas de Plantas/metabolismo , Interferência de RNA , Plântula/enzimologia , Plântula/genética , Plântula/crescimento & desenvolvimento
18.
Curr Top Med Chem ; 19(23): 2081-2097, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31486755

RESUMO

Kirsten rat sarcoma 2 viral oncogene homolog (KRAS) is the most commonly mutated oncogene in human cancer. The developments of many cancers depend on sustained expression and signaling of KRAS, which makes KRAS a high-priority therapeutic target. Scientists have not successfully developed drugs that target KRAS, although efforts have been made last three decades. In this review, we highlight the emerging experimental strategies of impairing KRAS membrane localization and the direct targeting of KRAS. We also conclude the combinatorial therapies and RNA interference technology for the treatment of KRAS mutant cancers. Moreover, the virtual screening approach to discover novel KRAS inhibitors and synthetic lethality interactors of KRAS are discussed in detail.


Assuntos
Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Proteína Oncogênica p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Antineoplásicos/química , Humanos , Mutação , Neoplasias/genética , Proteína Oncogênica p21(ras)/genética , Proteína Oncogênica p21(ras)/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Interferência de RNA
19.
Arch Virol ; 164(11): 2747-2759, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31502079

RESUMO

RNA silencing is a major antiviral mechanism in plants, which is counteracted by virus-encoded proteins with silencing suppression activity. ORFs encoding putative silencing suppressor proteins that share no structural or sequence homology have been identified in the genomes of four criniviruses. In this study, we investigated the RNA silencing suppression activity of several proteins encoded by the RNA1 (RdRp, p22) and RNA2 (CP, CPm and p26) of cucurbit chlorotic yellows virus (CCYV) using co-agroinfiltration assays on Nicotiana benthamiana plants. Our results indicate that p22 is a suppressor of local RNA silencing that does not interfere with cell-to-cell movement of the RNA silencing signal or with systemic silencing. Furthermore, comparisons of the suppression activity of CCYV p22 with that of two other well-known crinivirus suppressors (CYSDV p25 and ToCV p22) revealed that CCYV p22 is a weaker suppressor of local RNA silencing than the other two proteins. Finally, a comparative sequence analysis of the p22 genes of seven Greek CCYV isolates was performed, revealing a high level of conservation. Taken together, our research advances our knowledge about plant-virus interactions of criniviruses, an emergent group of pathogens that threatens global agriculture.


Assuntos
Crinivirus/genética , Interferência de RNA/fisiologia , RNA Viral/genética , Tabaco/virologia , Proteínas do Core Viral/genética , Genoma Viral/genética , Interações Hospedeiro-Patógeno/genética , Doenças das Plantas/virologia
20.
Pestic Biochem Physiol ; 159: 118-126, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31400773

RESUMO

In the plant-insect arms race, plants synthesize toxic compounds to defend against herbivorous insects, whereas insects employ cytochrome P450 monooxygenases (P450s) to detoxify these phytotoxins. As ubiquitous environmental contaminants, heavy metals can be easily absorbed by plants and further accumulated in herbivorous insects through the food chains, resulting in tangible consequences for plant-insect interactions. However, whether heavy metals can influence P450 activities and thereby cause further effects on larval tolerance to phytotoxins remains unknown. In this study, we shown that prior exposure to copper (Cu) enhanced larval tolerance to xanthotoxin in Spodoptera litura, a major polyphagous pest of agriculture. P450 activities were induced in larvae exposed to Cu or xanthotoxin, and a midgut specific expressed P450 gene, CYP6B50 was cross-induced after exposure to these two toxic xenobiotics. Knocking down CYP6B50 by RNA interference (RNAi) rendered the larvae more sensitive to xanthotoxin. As defense against oxidative stress following metal exposure has been demonstrated to affect insecticide resistance, the reactive oxygen species (ROS) generation and antioxidant enzyme activities were assessed. Cu exposure caused the accumulation of hydrogen peroxide (H2O2) and enhanced the activities of superoxide dismutase (SOD) and peroxidase (POD) in larval midgut. In addition, two antioxidant response elements (AREs) were identified from the CYP6B50 promoter, indicating that Cu-induced CYP6B50 expression may be related to the ROS burst. Application of ROS scavenger N-acetylcysteine (NAC) effectively suppressed CYP6B50 expression, inhibited P450 activities and impaired larval tolerance to xanthotoxin that had been induced by Cu. These results indicate that the increase in CYP6B50 expression regulated by Cu-induced H2O2 generation contributed to the enhancement of larval tolerance to xanthotoxin in S. litura. Ingestion of heavy metals from their host plants can inadvertently boost the counter-defense system of herbivorous insects to protect themselves against plant defensive toxins.


Assuntos
Cobre/farmacologia , Peróxido de Hidrogênio/metabolismo , Metoxaleno/farmacologia , Spodoptera/efeitos dos fármacos , Spodoptera/metabolismo , Animais , Elementos de Resposta Antioxidante/genética , Elementos de Resposta Antioxidante/fisiologia , Sistema Enzimático do Citocromo P-450/metabolismo , Peroxidase/genética , Peroxidase/metabolismo , Interferência de RNA , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo
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