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1.
Gene ; 714: 143997, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31348981

RESUMO

Based on Akt1 and Jak1 key roles in apoptosis and proliferation of many cancers, the aim of this study was to find a new gene therapy strategy by silencing of these main anti-apoptotic genes for HNSCC treatment. Cancerous HN5 and normal HUVEC cell lines were treated with Akt1 and Jak1 siRNAs alone or with each other combined with/without cisplatin. The MTS, flow cytometry, 4',6-diamidino-2-phenylindole staining, real-time PCR and ELISA methods were utilized in this study. The highest percentage of apoptosis was observed in the treatment of Jak1 siRNA/cisplatin group in cancerous HN5 cells (96.5%) where this treatment showed 12.84% apoptosis in normal HUVEC cell line. Cell viability reduced significantly to 64.57% after treatment with Akt1 siRNA in HN5 treated group. Knocking down Akt1 and Jak1 genes using siRNAs could increase levels of apoptosis and reduce proliferation rate in HNSCC indicating the powerful effects of these genes siRNAs with or without chemotherapeutic agents in HNSCC treatment. In conclusion, the combination of siRNA-mediated gene-silencing strategy can be considered as a valuable and safe approach for sensitizing cancer cells to chemotherapeutic agents thus proposed further studies regarding this issue to approve some siRNA based therapeutics for using in clinic.


Assuntos
Apoptose/genética , Proliferação de Células/genética , Neoplasias de Cabeça e Pescoço/genética , Janus Quinase 1/genética , Proteínas Proto-Oncogênicas c-akt/genética , RNA Interferente Pequeno/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Apoptose/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Cisplatino/farmacologia , Técnicas de Silenciamento de Genes/métodos , Inativação Gênica/efeitos dos fármacos , Inativação Gênica/fisiologia , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Células Endoteliais da Veia Umbilical Humana , Humanos , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico
2.
J Microbiol ; 57(6): 423-430, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31054136

RESUMO

Antibiotics have long been used for anti-infective control of bacterial infections, growth promotion in husbandry, and prophylactic protection against plant pathogens. However, their inappropriate use results in the emergence and spread of multiple drug resistance (MDR) especially among various bacterial populations, which limits further administration of conventional antibiotics. Therefore, the demand for novel anti-infective approaches against MDR diseases becomes increasing in recent years. The peptide nucleic acid (PNA)-based technology has been proposed as one of novel anti-infective and/or therapeutic strategies. By definition, PNA is an artificially synthesized nucleic acid mimic structurally similar to DNA or RNA in nature and linked one another via an unnatural pseudo-peptide backbone, rendering to its stability in diverse host conditions. It can bind DNA or RNA strands complimentarily with high affinity and sequence specificity, which induces the target-specific gene silencing by inhibiting transcription and/or translation. Based on these unique properties, PNA has been widely applied for molecular diagnosis as well as considered as a potential anti-infective agent. In this review, we discuss the general features of PNAs and their application to various bacterial pathogens as new anti-infective or antimicrobial agents.


Assuntos
Antibacterianos/farmacologia , Anti-Infecciosos/farmacologia , Ácidos Nucleicos Peptídicos/farmacologia , Anti-Infecciosos/química , Bactérias/efeitos dos fármacos , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Ácidos Nucleicos Peptídicos/química
3.
Mol Immunol ; 109: 126-133, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30928727

RESUMO

Members of the tripartite motif (TRIM) family as E3 ubiquitin ligases have been regarded as critical regulators of innate immunity and antiviral response. However, the role of TRIM7 is still elusive. Here, we provide evidence for the importance of TRIM7 in regulation of the TLR4-mediated innate response. In detail, we find that TRIM7 is highly expressed in antigen-presenting cells like macrophages. Knockdown of TRIM7 clearly inhibits the LPS-induced production of IFN-ß, TNF-α and IL-6 in macrophages. Conversely, forced expression of TRIM7 could exert an opposite effect on these pro-inflammatory cytokines. Further analysis indicates that such effect is mediated by the TLR4-associated signaling pathways including MAPKs, NF-κB and IRF3-involved pathways. Truncation of the E3 ligase domain on TRIM7 may reduce the production of pro-inflammatory cytokines, suggesting a critical role of this domain in the regulation of LPS-initiated innate response. Taken together, we report here that TRIM7 may facilitate the TLR4-mediated innate response via its E3 ligase domain in macrophages, which provides new insight into the mechanistic role of TRIM7 in innate immunity.


Assuntos
Proteínas de Transporte/metabolismo , Imunidade , Macrófagos/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Células Apresentadoras de Antígenos/efeitos dos fármacos , Células Apresentadoras de Antígenos/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/genética , Citocinas/metabolismo , Regulação para Baixo/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Imunidade/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Interferon Tipo I/metabolismo , Lipopolissacarídeos , Macrófagos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Oligodesoxirribonucleotídeos/farmacologia , Domínios Proteicos , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
4.
Lipids Health Dis ; 18(1): 89, 2019 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-30954075

RESUMO

BACKGROUND: Elevation of exogenous free fatty acid (FFA) level leads to insulin resistance (IR) in liver, IR is manifested by elevated hepatic glucose production. We aim to study whether inhibition of endogenous fatty acid synthesis could decrease hepatic glucose production. METHODS: Low-passage HepG2 cells derived from human liver tissue were cultured in medium supplemented with FFA to induce IR, the influences of sterol regulatory element binding protein-1c (SREBP-1c) silencing on glucose production of HepG2 cells were investigated, and genes responsible for fatty acid and glucose metabolism were detected by real-time PCR. RESULTS: Compared with HepG2 cells cultured in normal growth medium, glucose production of HepG2 cells treated by FFA was significantly increased {[(0.28 ± 0.01) vs (0.83 ± 0.02)] umol.ug- 1 protein, n = 6 wells, P < 0.01}; the mRNA expression of phosphoenolpyruvate carboxylase kinase (PEPCK) and glucose-6-phosphatase (G6PC) in HepG2 cells increased by more than 5-fold and 3-fold, respectively; the mRNA expression of fatty acid synthase (FAS) and stearoyl-CoA desaturase-1 (SCD1) increased by approximately 4-fold and 1.1-fold, respectively; the mRNA expression of carnitine palmitoyltransferase-1 (CPT-1) changed slightly. Compared with the scrambled siRNA control, glucose production of HepG2 cells treated by FFA significantly increased after SREBP-1c silencing {[(0.018 ± 0.001) vs (0.028 ± 0.002)] umol.ug- 1 protein, n = 6 wells, P < 0.01}; the mRNA expression of PEPCK and G6PC increased by approximately 1.5-fold and 5-fold, respectively, but the mRNA expression of FAS, SCD1 and CPT-1 changed slightly. CONCLUSIONS: SREBP-1c silencing further augmented glucose production of HepG2 cells treated by FFA significantly, genes responsible for fatty acid synthesis and gluconeogenesis played an important role in this process. SREBP-1c functions not only as a lipid regulator but also plays an important role in regulation of glucose metabolism.


Assuntos
Meios de Cultura/farmacologia , Ácidos Graxos não Esterificados/farmacologia , Glucose/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Carnitina O-Palmitoiltransferase/genética , Ácido Graxo Sintases/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Resistência à Insulina/genética , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/antagonistas & inibidores , Triglicerídeos/metabolismo
5.
Artif Cells Nanomed Biotechnol ; 47(1): 685-695, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30829071

RESUMO

Plastin 3 (PLS3) overexpression may serve as a marker for predicting chemotherapeutic outcomes in drug-resistant cancer cells, but the mechanism is unclear. Herein, we show that the down-regulation of PLS3 by PLS3 gene silencing augments the sensitivity of MDA-MB-231 triple-negative breast cancer cells to paclitaxel. Interestingly, a low concentration of paclitaxel was able to induce strong apoptosis in the PLS3-silenced cells. Further study revealed that p38 MAPK signalling was responsible for the increased sensitivity to paclitaxel in these cells, as the p38 MAPK inhibitor SB203580 impaired the changes mediated by PLS3 down-regulation in response to paclitaxel. Therefore, our study identifies PLS3 as a potential target for enhancing the p38 MAPK-mediated apoptosis induced by paclitaxel. Unlike paclitaxel, Abraxane was unable to induce strong apoptosis in the PLS3-silenced cells. As PLS3 was found to be involved in the process of endocytosis in breast cancer cells, the reliance of cellular Abraxane uptake on this process may render it not as efficient as paclitaxel in PLS3-depleted tumour cells. The finding that PLS3 could be a critical regulator of paclitaxel sensitivity may have important implications for breast cancer chemotherapy.


Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Glicoproteínas de Membrana/biossíntese , Proteínas dos Microfilamentos/biossíntese , Proteínas de Neoplasias/metabolismo , Paclitaxel/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Regulação para Baixo , Feminino , Inativação Gênica/efeitos dos fármacos , Humanos , Imidazóis/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Piridinas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores
6.
Int J Mol Med ; 43(5): 2044-2054, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30896860

RESUMO

Kelch­like ECH­associated protein 1 (Keap1)/nuclear factor erythroid 2­related factor 2 (Nrf2) signaling has a protective effect on normal cells. A number of previous studies demonstrated that Keap1/Nrf2 signaling is associated with drug resistance in numerous tumors. The aim of the present study was to investigate the roles of Keap1 in renal cell carcinoma (RCC) and its effect on sensitivity to chemotherapy. Reverse transcription­quantitative polymerase chain reaction was used to detect the mRNA expression of Keap1 in 45 cases of RCC tumors and adjacent normal tissues. A total of five randomly selected patients with RCC, five RCC cell lines and normal renal tubular cells were examined to detect the protein and mRNA expressions of Keap1. The 5­year survival rate was analyzed by Kaplan­Meier analysis. The cell viability was assessed by a Cell Counting kit­8 assay. The cell apoptosis and reactive oxygen species (ROS) were determined by flow cytometry. The expressions of associated proteins were determined by western blot analysis. It was identified that in RCC tissues and RCC cell lines, the expression of Keap1 was downregulated, which was considered to be associated with poor prognosis. In total, 1 µM Axitinib significantly decreased cell viability, promoted ROS release and induced cell apoptosis in ACHN cells. Silencing Keap1 was able to reverse the inhibitory effect of Axitinib and enhance the protein expressions of Nrf2, NAD(P)H dehydrogenase [quinone] 1 and heme oxygenase 1. However, silencing Nrf2 increased the cell sensitivity to Axitinib. Under Axitinib condition, overexpressing Nrf2 was able to increase cell viability; however, overexpressing Keap1 resulted in an opposite effect. Keap1 serves as a tumor suppressor; its low expression was associated with poor prognosis and a decreased sensitivity of RCC cells to Axitinib. A possible mechanism underlying Axitinib resistance may involve Nrf2 overexpression.


Assuntos
Axitinibe/farmacologia , Carcinoma de Células Renais/patologia , Regulação para Baixo , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Fator 2 Relacionado a NF-E2/metabolismo , Regulação para Cima , Apoptose/efeitos dos fármacos , Axitinibe/administração & dosagem , Carcinoma de Células Renais/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Inativação Gênica/efeitos dos fármacos , Humanos , Prognóstico , Espécies Reativas de Oxigênio/metabolismo , Regulação para Cima/efeitos dos fármacos
7.
Cell Commun Signal ; 17(1): 22, 2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30836961

RESUMO

BACKGROUND: Autocrine motility factor (AMF) is a critical factor regulating aggressiveness of endometrial cancer (EC). Multiple pieces of evidence indicate that it is through G protein coupled estrogen receptor (GPER) signaling pathway that some growth factors promoted the migration and proliferation of tumor cells. The aim of this study is to explore the role of GPER-1 in AMF mediated regulatory mechanisms of EC recurrence and progression. METHODS: Real-Time Cell Analysis (RTCA) assays were performed to assess whether AMF depends on Autocrine motility factor recepter (AMFR) signaling in EC cells. A genome-wide expression microarray and Yeast Two-Hybrid assay were used to detect AMF and GPER-1 interaction in the context of AMFR depletion, and co-immunoprecipitation and immunofluorescence experiments were performed to confirm the physical interaction. Isobaric Tags for Relative and Absolute Quantification (iTRAQ) analysis was used for the identification of the target pathway activated by AMF-GPER-1 interaction. Cohorts of mice harboring xenografts derived from modified SPEC2 cell lines were treated with or without exogenous AMF to validate the results of previous experiments. Immunohistochemistry was performed to assess AMF and GPER-1 expression in endometrial cancer specimens and normal endometrium. RESULTS: Our data showed that GPER-1 binds to AMF and the formed complex translocates from the plasma membrane to the cytoplasm. Mechanistic investigations demonstrated that interaction between AMF and GPER-1 triggers phosphoinositide-3-kinase signaling and promotes EC cell growth. More importantly, through animal experiments and human tissue experiments, we found that AMF contributes to GPER-1-mediated EC progression, which is consistent with the above observations. CONCLUSIONS: Our work not only delineated the regulatory mechanisms of endometrial cancer progression by AMF-GPER-1-AKT signaling cascade but also laid the foundation of targeting this pathway for treating endometrial cancer.


Assuntos
Progressão da Doença , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Glucose-6-Fosfato Isomerase/farmacologia , Receptores Estrogênicos/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Animais , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Inativação Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Modelos Biológicos , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica/efeitos dos fármacos , Receptores do Fator Autócrino de Motilidade/metabolismo , Transdução de Sinais/efeitos dos fármacos
8.
Cell Commun Signal ; 17(1): 17, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30808351

RESUMO

BACKGROUND: Chondrosarcoma is a malignant cartilaginous neoplasm of the bone which resistant to radiation therapy and chemotherapy. Cyclin-dependent kinase 4 (CKD4) is highly expressed in human cancer, and palbociclib, the inhibitor of CDK4 has been used clinically under FDA approval for application in cancer therapeutic remedies. However, the level of CDK4 and the treatment possibility in chondrosarcoma require further exploration. Thus, we aim to investigate the level of CDK4 and accompanying therapeutic effects of palbociclib in chondrosarcoma. METHODS: We used immunohistochemistric analysis to evaluate human CDK4 productions in chondrosarcoma tissues. The inhibitory expression of CDK4 by siRNA or palbociclib on cell proliferation, invasion, migration, apoptosis and cycle arrest of chondrosarcoma were determined by MTT, wound healing, transwell and flow cytometry. CDK4/Rb signaling pathway were determined by western blot and Immunofluorescence assay. The inhibition effect of palbociclib on tumor growth within the bone were determined by bioluminescence imaging in vivo. RESULTS: CDK4 was found to express significantly in human chondrosarcoma samples. The enhanced levels of CDK4 were interlinked with malignant metastasis and undesirable prognosis of chondrosarcoma patients. CDK4 was also highly expressed in human chondrosarcoma cell lines and its inhibition by specific siRNA and palbociclib lead to a decrease in cell proliferation, accompanied by the phosphorylation of Rb. Furthermore, palbociclib also induced cell cycle arrest in G1 phase and decreased cell migration and invasion via CDK4/Rb signaling pathway. Administration of palbociclib in vivo could reduce tumor burden in chondrosarcoma. CONCLUSIONS: In summary, these data highlight CDK4 inhibitors, such as palbociclib, as potential promising therapeutics in the treatment of human chondrosarcoma.


Assuntos
Condrossarcoma/tratamento farmacológico , Condrossarcoma/enzimologia , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Terapia de Alvo Molecular , Piperazinas/uso terapêutico , Piridinas/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Condrossarcoma/genética , Condrossarcoma/patologia , Quinase 4 Dependente de Ciclina/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Metástase Neoplásica , Fosforilação/efeitos dos fármacos , Piperazinas/farmacologia , Prognóstico , Piridinas/farmacologia , Proteína do Retinoblastoma/metabolismo , Carga Tumoral
9.
Vision Res ; 156: 66-72, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30684501

RESUMO

Mutation of FOXC1 causes Axenfeld-Rieger Syndrome (ARS) with early onset or congenital glaucoma. We assessed retinal ganglion cell (RGC) number in zebrafish due to CRISPR-mediated mutation and antisense inhibition of two-forkhead box transcription factors, foxc1a and foxc1b. These genes represent duplicated homologues of human FOXC1. Using a CRISPR induced null mutation in foxc1b, in combination with antisense inhibition of foxc1a, we demonstrate reduced cell number in the retinal ganglion cell layer of developing zebrafish eyes. As early as 5 days post fertilization (dpf), fewer RGCs are found in foxc1b homozygous mutants injected with foxc1a morpholinos, and a thinner optic nerve results. Our data illustrates that foxc1 is required for the expression of atonal homolog 7 (atoh7), a gene that is necessary for RGC differentiation. As markers of differentiated RGCs (pou4f2) are downregulated in foxc1b-/- mutants injected with foxc1a morpholinos and no cell death is observed, our results are consistent with defects in the differentiation of RGCs leading to reduced cell number, as opposed to increased cell death of RGCs or off targets effects of morpholino injection. Our zebrafish model demonstrates that aberrant regulation of RGC number could act in concert with other known glaucoma risk factors to influence the development of congenital and early onset glaucoma due to FOXC1 mutation.


Assuntos
Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Mutação , Nervo Óptico/embriologia , Células Ganglionares da Retina/patologia , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Animais , Axônios/patologia , Contagem de Células , Morte Celular , Diferenciação Celular , Proteínas de Ligação a DNA/genética , Embrião não Mamífero , Inativação Gênica/efeitos dos fármacos , Glaucoma/embriologia , Glaucoma/genética , Hibridização In Situ , Morfolinos/farmacologia , Reação em Cadeia da Polimerase , Transfecção
10.
Int J Mol Sci ; 20(2)2019 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-30654447

RESUMO

Human Dental Pulp Stem Cells (hDPSCs) represent a type of adult mesenchymal stem cells that have the ability to differentiate in vitro in several lineages such as odontoblasts, osteoblasts, chondrocytes, adipocytes and neurons. In the current work, we used hDPSCs as the experimental model to study the role of recombinant prion protein 23⁻231 (recPrPC) in the neuronal differentiation process, and in the signal pathway activation of ERK 1/2 and Akt. We demonstrated that recPrPC was able to activate an intracellular signal pathway mediated by extracellular-signal-regulated kinase 1 and 2 (ERK 1/2) and protein kinase B (Akt). Moreover, in order to understand whether endogenous prion protein (PrPC) was necessary to mediate the signaling induced by recPrPC, we silenced PrPC, demonstrating that the presence of endogenous PrPC was essential for ERK 1/2 and Akt phosphorylation. Since endogenous PrPC is a well-known lipid rafts component, we evaluated the role of these structures in the signal pathway induced by recPrPC. Our results suggest that lipid rafts integrity play a key role in recPrPC activity. In fact, lipid rafts inhibitors, such as fumonisin B1 and MßCD, significantly prevented ERK 1/2 and Akt phosphorylation induced by recPrPC. In addition, we investigated the capacity of recPrPC to induce hDPSCs neuronal differentiation process after long-term stimulation through the evaluation of typical neuronal markers expression such as B3-Tubulin, neurofilament-H (NFH) and growth associated protein 43 (GAP43). Accordingly, when we silenced endogenous PrPC, we observed the inhibition of neuronal differentiation induced by recPrPC. The combined data suggest that recPrPC plays a key role in the neuronal differentiation process and in the activation of specific intracellular signal pathways in hDPSCs.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Células-Tronco Mesenquimais/citologia , Neurônios/citologia , Fragmentos de Peptídeos/farmacologia , Príons/farmacologia , Proteínas Recombinantes/farmacologia , Adolescente , Biomarcadores/metabolismo , Polpa Dentária/citologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Inativação Gênica/efeitos dos fármacos , Humanos , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Adulto Jovem
11.
Mol Immunol ; 107: 29-40, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30639476

RESUMO

Yes-associated protein (YAP) is a significant downstream protein in the Hippo signaling pathway with important functions in cell proliferation, apoptosis, invasion and migration. YAP also plays a role in the progression and development of various liver diseases. In hepatic fibrosis development and reversion, the proliferation and apoptosis of activated hepatic stellate cells (HSCs) play a critical role. However, the contribution of YAP to hepatic fibrosis progression and reversion and the underlying mechanism have not been investigated. Here we investigated the expression and function of YAP in the proliferation and apoptosis of activated HSCs. We found that YAP expression was increased in liver fibrosis tissues from CCl4-induced model mice and restored to normal level after stopping CCl4 injection and 6 weeks of spontaneously recovery. YAP expression was elevated in HSC-T6 cells treated with TGF-ß1 and recovered after MDI treatment. Silencing of YAP inhibited the activation and proliferation of HSC-T6 cells stimulated by TGF-ß1. In addition, the apoptosis of activated HSC-T6 cells silenced for YAP was slightly enhanced. Furthermore, over-expression of YAP repressed the reversion of activated HSC-T6 cells mediated by MDI reversal. We found that HSC-T6 cells activated by TGF-ß1 showed higher levels of nuclear YAP compared with MDI-treated cells, indicating that YAP was activated in HSC-T6 cells treated by TGF-ß1. We also found that loss of YAP attenuated Wnt/ß-catenin pathway activity in activated HSC-T6 cells. Treatment of VP, an inhibitor of the YAP-TEAD complex, reduced both activation and proliferation of HSC-T6 cells and increased apoptosis. Together these results indicated that reduced expression of YAP contributes to acquisition of the quiescent phenotype in HSCs. Our results suggest that YAP may be a useful target in HSCs activation and reversion.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Fosfoproteínas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Tetracloreto de Carbono , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Inativação Gênica/efeitos dos fármacos , Células Estreladas do Fígado/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos Endogâmicos C57BL , Modelos Biológicos , Fator de Crescimento Transformador beta1/metabolismo , Verteporfina/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos
12.
J Nanobiotechnology ; 17(1): 11, 2019 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-30670041

RESUMO

BACKGROUND: We developed a non-viral vector, a combination of HIV-1 Tat peptide modified with histidine and cysteine (mTat) and polyethylenimine, jetPEI (PEI), displaying the high efficiency of plasmid DNA transfection with little toxicity. Since the highest efficiency of INTERFERin (INT), a cationic amphiphilic lipid-based reagent, for small interfering RNA (siRNA) transfection among six commercial reagents was shown, we hypothesized that combining mTat/PEI with INT would improve transfection efficiency of siRNA delivery. To elucidate the efficacy of the hybrid vector for siRNA silencing, ß-actin expression was measured after siRNA ß-actin was transfected with mTat/PEI/INT or other vectors in HSC-3 human oral squamous carcinoma cells. RESULTS: mTat/PEI/INT/siRNA produced significant improvement in transfection efficiency with little cytotoxicity compared to other vectors and achieved ≈ 100% knockdown of ß-actin expression compared to non-treated cells. The electric charge of mTat/PEI/INT/siRNA was significantly higher than INT/siRNA. The particle size of mTat/PEI/INT/siRNA was significantly smaller than INT/siRNA. Filipin III and ß-cyclodextrin, an inhibitor of caveolae-mediated endocytosis, significantly inhibited mTat/PEI/INT/siRNA transfection, while chlorpromazine, an inhibitor of clathrin-mediated endocytosis, did not inhibit mTat/PEI/INT/siRNA transfection. Furthermore, the transfection efficiency of mTat/PEI/INT at 4 °C was significantly lower than 37 °C. CONCLUSIONS: These findings demonstrated the feasibility of using mTat/PEI/INT as a potentially attractive non-viral vector for siRNA delivery.


Assuntos
Técnicas de Transferência de Genes , Peptídeos/química , Polietilenoimina , RNA Interferente Pequeno/administração & dosagem , Linhagem Celular , Endocitose/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Humanos
13.
Proc Natl Acad Sci U S A ; 116(1): 233-238, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30559205

RESUMO

The composition of the gut microbiota is largely determined by environmental factors including the host diet. Dietary components are believed to influence the composition of the gut microbiota by serving as nutrients to a subset of microbes, thereby favoring their expansion. However, we now report that dietary fructose and glucose, which are prevalent in the Western diet, specifically silence a protein that is necessary for gut colonization, but not for utilization of these sugars, by the human gut commensal Bacteroides thetaiotaomicron Silencing by fructose and glucose requires the 5' leader region of the mRNA specifying the protein, designated Roc for regulator of colonization. Incorporation of the roc leader mRNA in front of a heterologous gene was sufficient for fructose and glucose to turn off expression of the corresponding protein. An engineered strain refractory to Roc silencing by these sugars outcompeted wild-type B. thetaiotaomicron in mice fed a diet rich in glucose and sucrose (a disaccharide composed of glucose and fructose), but not in mice fed a complex polysaccharide-rich diet. Our findings underscore a role for dietary sugars that escape absorption by the host intestine and reach the microbiota: regulation of gut colonization by beneficial microbes independently of supplying nutrients to the microbiota.


Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Bacteroides thetaiotaomicron/efeitos dos fármacos , Carboidratos da Dieta/farmacologia , Açúcares da Dieta/farmacologia , Microbioma Gastrointestinal/efeitos dos fármacos , Animais , Proteínas de Bactérias/metabolismo , Frutose/administração & dosagem , Frutose/farmacologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Glucose/administração & dosagem , Glucose/farmacologia , Camundongos , Polissacarídeos/administração & dosagem , Polissacarídeos/farmacologia , Simbiose/efeitos dos fármacos
14.
Gene Ther ; 26(3-4): 75-85, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30538307

RESUMO

Gene silencing for plasmid-based vectors and the underlying mechanism are critical factors for development of effective gene therapy. The objective of this study is to explore the role of epigenetic regulation for transgene expression. Two reporter genes, mouse interleukin 10 and human secreted alkaline phosphatase under the control of human cytomegalovirus immediate early promoter for expression, were delivered to mouse liver by hrodydynamics-based procedure and reporter gene expression was monitored. Reporter gene expression reached its peak level one day after gene delivery and declined progressively thereafter, reaching the minimal level in about 3 weeks. Intra-peritoneal injection of vorinostat, valproic acid or sodium butyrate, the known histone deacetylase inhibitors, resulted in a dose-dependent reactivation of reporter gene expression. Repeated administration of histone deacetylase inhibitors blocked gene silencing and maintained reporter gene expression. Mechanistic studies reveal that reactivation of reporter genes is corelated with hyperacetylation of histones H3 and H4, and elevated binding of TATA-box binding protein to the promoter region. These results suggest that epigenetic regulation plays a critical role in controlling transgene expression in vivo and demonstrate that enzymes involved in epigenetic regulation such as histone deacetylase could serve as a target to achieve controlled transgene expression for gene therapy.


Assuntos
Inativação Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Fosfatase Alcalina/genética , Animais , Epigênese Genética/genética , Feminino , Regulação da Expressão Gênica/genética , Inativação Gênica/fisiologia , Genes Reporter/genética , Vetores Genéticos , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histonas , Humanos , Interleucina-10/genética , Camundongos , Regiões Promotoras Genéticas/genética , Transgenes/genética , Ácido Valproico
15.
Biochem Biophys Res Commun ; 508(4): 1168-1174, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30554661

RESUMO

Cardiovascular disease is one of the leading causes of death in the elderly, and novel therapeutic targets against atherogenesis are urgent. The initiation of atherosclerotic changes of monocyte adhesion on the vascular endothelium and subsequent foam cell formation are noteworthy pathophysiologies when searching for strategies to prevent the progression of age-related atherosclerosis. We report the significance of the deubiquitinating enzyme cylindromatosis (CYLD) in vascular remodeling by interference with inflammatory responses regulated by NF-κB signaling. The purpose of this study was to elucidate the pathological functions of CYLD in the early phase of atherogenesis associated with aging. Treatment with inflammatory cytokines induced endogenous CYLD in aortic endothelial cells (HAECs) and THP-1 cells. siRNA-mediated CYLD silencing led to enhanced monocyte adhesion along with increased adhesion molecules in HAECs treated with TNFα. In siRNA-mediated CYLD silenced RAW 264.7 macrophages treated with oxidized LDL (oxLDL), augmented lipid accumulation was observed, along with increased expression of the class A macrophage scavenger receptor (SR-A), lectin-like oxidized LDL receptor-1 (LOX-1), CD36, fatty acid binding protein 4 (FABP4), the cholesterol ester synthase acyl-CoA cholesterol acyltransferase (ACAT1), MCP-1, and IL-1ß and decreased expression of scavenger receptor class B type I (SR-BI). Intriguingly, CYLD gene expression was significantly reduced in bone marrow-derived macrophages of aged mice compared that of young mice, as well as in senescent HAECs compared with young cells. These findings suggest that age-related attenuation of CYLD expression in endothelial cells (ECs) and macrophages triggers the initiation of age-related atherogenesis by exacerbating monocyte adhesion on the endothelium and foam cell formation. CYLD in the vasculature may be a novel therapeutic target, especially in the early preventive intervention against the initiation of age-related atherogenesis.


Assuntos
Envelhecimento/patologia , Aterosclerose/fisiopatologia , Cisteína Endopeptidases/metabolismo , Enzima Desubiquitinante CYLD/metabolismo , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Macrófagos/metabolismo , Envelhecimento/genética , Animais , Aterosclerose/genética , Aterosclerose/patologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Adesão Celular/efeitos dos fármacos , Citocinas/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotélio Vascular/efeitos dos fármacos , Células Espumosas/efeitos dos fármacos , Células Espumosas/metabolismo , Inativação Gênica/efeitos dos fármacos , Humanos , Mediadores da Inflamação/metabolismo , Lipoproteínas LDL/farmacologia , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Células RAW 264.7 , RNA Interferente Pequeno/metabolismo , Células THP-1 , Regulação para Cima/efeitos dos fármacos
16.
Iran Biomed J ; 23(1): 21-33, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30041514

RESUMO

Background: Targeted co-delivery of siRNA and a chemotherapeutic drug is an attractive approach to cancer drug design and treatment. This study was carried out to design an anti-Mucin1 aptamer (Apt)-conjugated chitosan nanoparticle (NP) for targeted co-delivery of insulin-like growth factor receptor 1 (IGF-1R) Silencer siRNA and docetaxel (DTX) to SKBR3 cells. Methods: Characterization of nano-drugs, cellular uptake of NPs, cell viability, and gene expression studies were evaluated based on metastatic breast cancer cells. Results: The results of this study showed that NPs had spherical and smooth morphology with 110-118 nm in size and had positive zeta potential (12-14 mV). siRNA and DTX were considerably loaded into NPs. The appropriate conjugation of the Apt to the NPs was affirmed by gel electrophoresis. The Apt-conjugated NPs were observed to enhance the cellular uptake of NPs into the SKBR3 cells. Although the combination treatment significantly decreased the cell viability of SKBR3 cells, the augmentative effect was observed when Apt was conjugated to NPs. Furthermore, Apt-conjugated NPs dramatically reduced the genetic expression of IGF-1R, signal transducers and activators of transcription 3 (STAT3), matrix metalloproteinases (MMP9), and vascular growth factor (VEGF). Conclusion: The targeted NPs may augment the targeting of pathways involved in tumorigenesis and metastasis of breast cancer. Therefore, more animal model experiments are needed to further clarify the efficacy and safety of this functionalized nanodrug.


Assuntos
Aptâmeros de Nucleotídeos/química , Neoplasias da Mama/patologia , Quitosana/química , Docetaxel/farmacologia , Mucina-1/metabolismo , Nanopartículas/química , RNA Interferente Pequeno/administração & dosagem , Receptores de Somatomedina/metabolismo , Animais , Neoplasias da Mama/genética , Células CHO , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Docetaxel/administração & dosagem , Liberação Controlada de Fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Heparina/química , Humanos , Nanopartículas/ultraestrutura , Metástase Neoplásica , Tamanho da Partícula , Soro/metabolismo , Eletricidade Estática
17.
Artigo em Inglês | MEDLINE | ID: mdl-30528668

RESUMO

The crosstalk between peroxisome proliferator-activated receptor α (PPARα) and estrogenic pathways are shared from fish to humans. Salmonid fish had an additional genome duplication, and two PPARα isoforms (PPARαBa and PPARαBb) were previously identified. Since a negative regulation between estrogen signaling and PPARα was described, a post-transcriptional gene silencing for PPARαBb was designed in primary brown trout hepatocytes. The aims of the study were to: (i) decipher the effects of PPARαBb knock-down on peroxisome morphology and on mRNA expression of potential target genes, and (ii) to assess the cross-interferences caused by an estrogenic compound (17α-ethinylestradiol - EE2) and a PPARα agonist (Wy-14,643 - Wy) using the established knock-down model. A knock-down efficiency of 70% was achieved for PPARαBb and its silencing significantly reduced the volume density of peroxisomes, but did not alter mRNA levels of the studied genes. Exposure to Wy did not change peroxisome morphology or mRNA expression, but under silencing conditions Wy rescued the volume density of peroxisomes to control levels, and increased acyl-coenzyme A oxidase 1-3l (Acox1-3l) mRNA. Exposure to EE2 caused a reduction of peroxisome volume density, but under silencing conditions this effect was abolished and ApoA1 mRNA level was diminished. The morphological alterations of peroxisomes by WY and EE2 demonstrated that obtained results are PPARαBb dependent, and suggest the regulation of unknown downstream targets of PPARαBb. In summary, PPARαBb is involved in the control of peroxisome size and/or number, which opens future opportunities to explore its regulation and molecular targets.


Assuntos
Estrogênios/farmacologia , Proteínas de Peixes , Inativação Gênica/efeitos dos fármacos , Hepatócitos/metabolismo , Subunidade 1 do Complexo Mediador/biossíntese , PPAR alfa , Pirimidinas/farmacologia , Animais , Proteínas de Peixes/agonistas , Proteínas de Peixes/biossíntese , Hepatócitos/citologia , Humanos , PPAR alfa/agonistas , PPAR alfa/biossíntese , Cultura Primária de Células , Truta
18.
Int J Nanomedicine ; 13: 4333-4344, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30087564

RESUMO

Background: Effective endosomal escape is still a critical bottleneck for intracellular delivery of small interfering RNAs (siRNAs) to maximize their therapeutic efficacy. To overcome this obstacle, we have developed a photothermally triggered system by using the near-infrared (NIR) irradiation to achieve "on-demand" endosomal escape and subsequent siRNA release into cytoplasm. Materials and methods: Herein, the poly-L-lysine (PLL) was successfully conjugated with melanin to obtain melanin-poly-L-lysine (M-PLL) polymer as a siRNA vehicle. The melanin was an efficient photothermal sensitizer, and the positive pendant amino groups of PLL could condense siRNAs to form stable complexes by electrostatic interactions. Results and discussion: Inspired by its excellent photothermal conversion efficiency, the melanin was first involved in the siRNA delivery system. Confocal laser scanning microscopic observation revealed that after cellular uptake the photothermally induced endosomal escape could facilitate siRNAs to overcome endosomal barrier and be delivered into cytoplasm, which resulted in significant silence in the luciferase expression over the NIR- and melanin-free controls. Moreover, the anti-survivin siRNA-loaded M-PLL nanoparticles displayed great inhibitory effect on 4T1 tumor growth in vitro and in vivo. Conclusion: These findings suggest that the M-PLL-mediated siRNA delivery is a promising candidate for therapeutic siRNA delivery and shows improved effect for cancer therapy via enhanced endosomal escape.


Assuntos
Antineoplásicos/farmacologia , Endossomos/metabolismo , Técnicas de Transferência de Genes , Luz , RNA Interferente Pequeno/administração & dosagem , Temperatura Ambiente , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Feminino , Expressão Gênica , Inativação Gênica/efeitos dos fármacos , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Luciferases/metabolismo , Melaninas/química , Camundongos Endogâmicos BALB C , Nanopartículas/administração & dosagem , Nanopartículas/ultraestrutura , Metástase Neoplásica , Polilisina/química , RNA Interferente Pequeno/genética , Proteínas Repressoras/metabolismo , Survivina
19.
Int J Nanomedicine ; 13: 3713-3728, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29983564

RESUMO

Background: Graphene oxide (GO) has attracted intensive interest in biological and medical fields in recent years due to its unique physical, chemical, and biological properties. In our previous work, we proved that GO could deliver small interfering RNA (siRNA) into cells and downregulate the expression of the desired gene. Methods: This study investigated the potential of a modified GO nanocarrier for co-delivery of siRNA and doxorubicin (DOX) for enhanced cancer therapy. Fourier transform infrared spectroscopy, laser particle size analyzer, UV-visible spectroscopy, gel electrophoresis retardation, and in vitro release assay were studied. Results: The results of real-time polymerase chain reaction revealed that the expression of vascular endothelial growth factor (VEGF) mRNA was decreased 46.84%±3.72% (mean ± SD). Enzyme-linked immunosorbent assay indicated that the expression of VEGF protein was down-regulated to 52.86%±1.10% (mean ± SD) in vitro. In vivo tumor growth assay GO-poly-l-lysine hydrobromide/folic acid (GPF)/DOX/siRNA exhibited gene silencing and tumor inhibition (66.95%±2.35%, mean ± SD) compared with naked siRNA (1.62%±1.47%, mean ± SD) and DOX (33.63%±5.85%, mean ± SD). GPF/DOX/siRNA exhibited no testable cytotoxicity. Conclusion: The results indicated that co-delivery of siRNA and DOX by GPF could be a promising application in tumor clinical therapy.


Assuntos
Doxorrubicina/uso terapêutico , Sistemas de Liberação de Medicamentos , Grafite/química , Nanopartículas/química , Neoplasias/tratamento farmacológico , RNA Interferente Pequeno/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Doxorrubicina/administração & dosagem , Doxorrubicina/química , Doxorrubicina/farmacologia , Liberação Controlada de Fármacos , Ácido Fólico/química , Inativação Gênica/efeitos dos fármacos , Células HeLa , Humanos , Camundongos Endogâmicos ICR , Nanopartículas/ultraestrutura , Neoplasias/patologia , Polilisina/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Ribonuclease Pancreático/antagonistas & inibidores , Espectrofotometria Ultravioleta , Espectroscopia de Infravermelho com Transformada de Fourier , Eletricidade Estática , Carga Tumoral/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/genética
20.
Med Sci Monit ; 24: 3772-3781, 2018 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-29867072

RESUMO

BACKGROUND Salivary pleomorphic adenoma is one of the most common salivary gland tumors. It has a relatively high tendency to recur and a high risk of malignant transformation. The present study aimed to study the effect of XT-I gene silencing on the implanting growth of salivary pleomorphic adenoma. MATERIAL AND METHODS Primary cultures of SPA cells and fibroblasts from the same patient were assessed. The adenovirus vector Ad-shRNA-XT-I was constructed and transfected into SPA cells. The expression of XT-I gene and XT-I protein was detected by real-time PCR and Western blot. The contents of proteoglycans were detected. The SPA cells transfected with Ad-shRNA-XT-I (group SPA-XT-I) and Ad-shRNA-HK (group SPA-HK), as well as without transfection (group SPA), were implanted into ADM scaffold with fibroblasts and then transferred into 18 BALB/C-nu nude mice for 3 months. RESULTS Primary cultures showed SPA cells were positive for human CK and S-100 protein and the fibroblasts were positive for human vimentin. The expressions of XT-I gene and protein were decreased by 51% and 51.31%, respectively. The content of proteoglycans was reduced by 48.45%. The results of the implanting growth in vitro and in vivo of nude mice indicated that no tumors grew in the SPA-XT-I group, whereas SPA grew in groups SPA-HK and SPA positive for human a-SMA, S-100 protein, and calponin. CONCLUSIONS XT-I gene silencing effectively inhibited the implanting growth of SPA.


Assuntos
Adenoma Pleomorfo/genética , Pentosiltransferases/genética , Pentosiltransferases/fisiologia , Adenoma Pleomorfo/metabolismo , Adulto , Animais , Fibroblastos/metabolismo , Inativação Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica/genética , Recidiva Local de Neoplasia/genética , Cultura Primária de Células , Interferência de RNA , RNA Interferente Pequeno/genética , Neoplasias das Glândulas Salivares/genética , Neoplasias das Glândulas Salivares/metabolismo
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