RESUMO
Background: Coronavirus disease 2019 (COVID-19) is a highly contagious respiratory disease that has posed a serious threat to people's daily lives and caused an unprecedented challenge to public health and people's health worldwide. Lung squamous cell carcinoma (LUSC) is a common type of lung malignancy with a highly aggressive nature and poor prognosis. Patients with LUSC could be at risk for COVID-19, We conducted this study to examine the potential for naringenin to develop into an ideal medicine and investigate the underlying action mechanisms of naringenin in COVID-19 and LUSC due to the anti-viral, anti-tumor, and anti-inflammatory activities of naringenin. Methods: LUSC related genes were obtained from TCGA, PharmGKB, TTD,GeneCards and NCBI, and then the transcriptome data for COVID-19 was downloaded from GEO, DisGeNET, CTD, DrugBank, PubChem, TTD, NCBI Gene, OMIM. The drug targets of Naringenin were revealed through CTD, BATMAN, TCMIP, SymMap, Chemical Association Networks, SwissTargetPrediction, PharmMapper, ECTM, and DGIdb. The genes related to susceptibility to COVID-19 in LUSC patients were obtained through differential analysis. The interaction of COVID-19/LUSC related genes was evaluated and demonstrated using STRING to develop a a COX risk regression model to screen and evaluate the association of genes with clinical characteristics. To investigate the related functional and pathway analysis of the common targets of COVID-19/LUSC and Naringenin, KEGG and GO enrichment analysis were employed to perform the functional analysis of the target genes. Finally, The Hub Gene was screened and visualized using Cytoscape, and molecular docking between the drug and the target was performed using Autodock. Results: We discovered numerous COVID-19/LUSC target genes and examined their prognostic value in LUSC patients utilizing a variety of bioinformatics and network pharmacology methods. Furthermore, a risk score model with strong predictive performance was developed based on these target genes to assess the prognosis of LUSC patients with COVID-19. We intersected the therapeutic target genes of naringenin with the LUSC, COVID-19-related targets, and identified 354 common targets, which could be used as potential target genes for naringenin to treat COVID-19/LUSC. The treatment of COVID-19/LUSC with naringenin may involve oxidative stress, anti-inflammatory, antiviral, antiviral, apoptosis, immunological, and multiple pathways containing PI3K-Akt, HIF-1, and VEGF, according to the results of the GO and KEGG enrichment analysis of these 354 common targets. By constructing a PPI network, we ascertained AKT1, TP53, SRC, MAPK1, MAPK3, and HSP90AA1 as possible hub targets of naringenin for the treatment of COVID-19/LUSC. Last but not least, molecular docking investigations showed that naringenin has strong binding activity in COVID-19/LUSC. Conclusion: We revealed for the first time the pharmacological targets and potential molecular processes of naringenin for the treatment of COVID-19/LUSC. However, these results need to be confirmed by additional research and validation in real LUSC patients with COVID-19.
Assuntos
COVID-19 , Carcinoma Pulmonar de Células não Pequenas , Carcinoma de Células Escamosas , Neoplasias Pulmonares , Humanos , Simulação de Acoplamento Molecular , Farmacologia em Rede , Fosfatidilinositol 3-Quinases , COVID-19/epidemiologia , COVID-19/genética , AntiviraisRESUMO
Objective: Mild cognitive impairment (MCI) is a preclinical and transitional stage between healthy ageing and dementia. The purpose of our study was to investigate the recent pooled global prevalence of MCI. Methods: This meta-analysis was in line with the recommendations of Cochrane's Handbook and Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020. We conducted a comprehensive search using the PubMed, Embase, Web of Science, CNKI, WFD, VIP, and CBM from their inception to March 1, 2023. Quality assessment was guided by the Agency for Healthcare Research and Quality (AHRQ) methodology checklist. The pooled global prevalence of MCI was synthesized using meta-analysis via random effect model. Subgroup analyses were performed to examine considered factors potentially associated with MCI prevalence. Results: We identified 233 studies involving 676,974 individuals aged above 50 years. All the studies rated as moderated-to-high quality. The overall prevalence of MCI was 19.7% [95% confidence interval (95% CI): 18.3-21.1%]. Subgroup analyses revealed that the global prevalence of MCI increased over time, with a significant rise [32.1% (95% CI: 22.6-41.6%)] after 2019. Additionally, MCI prevalence in hospitals [34.0% (95% CI: 22.2-45.7%)] was higher than in nursing homes [22.6% (95% CI: 15.5-29.8%)] and communities [17.9% (95% CI: 16.6-19.2%)], particularly after the epidemic of coronavirus disease 2019 (COVID-19). Conclusion: The global prevalence of MCI was 19.7% and mainly correlated with beginning year of survey and sample source. The MCI prevalence increased largely in hospitals after 2019 may be related to the outbreak of COVID-19. Further attention to MCI is necessary in the future to inform allocation of health resources for at-risk populations.
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Bone morphogenetic protein 9 (BMP-9) is a member of the transforming growth factor (TGF)-beta/BMP superfamily, and we have demonstrated that it is one of the most potent BMPs to induce osteoblast differentiation of mesenchymal stem cells (MSCs). Here, we sought to investigate if canonical Wnt/beta-catenin signalling plays an important role in BMP-9-induced osteogenic differentiation of MSCs. Wnt3A and BMP-9 enhanced each other's ability to induce alkaline phosphatase (ALP) in MSCs and mouse embryonic fibroblasts (MEFs). Wnt antagonist FrzB was shown to inhibit BMP-9-induced ALP activity more effectively than Dkk1, whereas a secreted form of LPR-5 or low-density lipoprotein receptor-related protein (LRP)-6 exerted no inhibitory effect on BMP-9-induced ALP activity. beta-Catenin knockdown in MSCs and MEFs diminished BMP-9-induced ALP activity, and led to a decrease in BMP-9-induced osteocalcin reporter activity and BMP-9-induced expression of late osteogenic markers. Furthermore, beta-catenin knockdown or FrzB overexpression inhibited BMP-9-induced mineralization in vitro and ectopic bone formation in vivo, resulting in immature osteogenesis and the formation of chondrogenic matrix. Chromatin immunoprecipitation (ChIP) analysis indicated that BMP-9 induced recruitment of both Runx2 and beta-catenin to the osteocalcin promoter. Thus, we have demonstrated that canonical Wnt signalling, possibly through interactions between beta-catenin and Runx2, plays an important role in BMP-9-induced osteogenic differentiation of MSCs.
Assuntos
Osso e Ossos/citologia , Diferenciação Celular/fisiologia , Fator 2 de Diferenciação de Crescimento/fisiologia , Células-Tronco Mesenquimais/citologia , Transdução de Sinais , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animais , Linhagem Celular , Meios de Cultivo Condicionados , Humanos , CamundongosRESUMO
Marrow mesenchymal stem cells are pluripotent progenitors that can differentiate into bone, cartilage, muscle, and fat cells. Wnt signaling has been implicated in regulating osteogenic differentiation of mesenchymal stem cells. Here, we analyzed the gene expression profile of mesenchymal stem cells that were stimulated with Wnt3A. Among the 220 genes whose expression was significantly changed by 2.5-fold, we found that three members of the CCN family, CCN1/Cyr61, CCN2/connective tissue growth factor (CTGF), and CCN5/WISP2, were among the most significantly up-regulated genes. We further investigated the role of CCN1/Cyr61 in Wnt3A-regulated osteogenic differentiation. We confirmed that CCN1/Cyr61 was up-regulated at the early stage of Wnt3A stimulation. Chromatin immunoprecipitation analysis indicates that CCN1/Cyr61 is a direct target of canonical Wnt/beta-catenin signaling. RNA interference-mediated knockdown of CCN1/Cyr61 expression diminished Wnt3A-induced osteogenic differentiation. Furthermore, exogenously expressed CCN1/Cyr61 was shown to effectively promote mesenchymal stem cell migration. These findings suggest that tightly regulated CCN1/Cyr61 expression may play an important role in Wnt3A-induced osteoblast differentiation of mesenchymal stem cells.
Assuntos
Regulação da Expressão Gênica , Proteínas Imediatamente Precoces/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismo , Proteínas Wnt/metabolismo , Adenoviridae/genética , Fosfatase Alcalina/análise , Western Blotting , Diferenciação Celular , Linhagem Celular , Movimento Celular , Imunoprecipitação da Cromatina , Proteína Rica em Cisteína 61 , Perfilação da Expressão Gênica , Células HCT116 , Humanos , Proteínas Imediatamente Precoces/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Reação em Cadeia da Polimerase , Análise Serial de Proteínas , Interferência de RNA , Transdução de SinaisRESUMO
The cold-inducible protein RBM3 mediates hypothermic neuroprotection against nitric oxide (NO)-induced cell death. Meanwhile, it is well-known that cyclooxygenase-2 (COX-2) is upregulated by RBM3 in several types of cells; however, it is still unclear whether COX-2 contributes to the neuroprotective effects of mild hypothermia/RBM3 against NO-induced cell death. Using human SH-SY5Y neuroblastoma cells, it was revealed that NO remarkably downregulates the expression of COX-2 at both mRNA and protein levels. When COX-2 was silenced using siRNA technique, cells became more sensitive to NO-induced cell death. Conversely, the overexpression of COX-2 significantly prevented NO-induced cell death in SH-SY5Y cells, indicating a pro-survival role of COX-2. Upon mild hypothermia pre-treatment, COX-2 was notably induced at both mRNA and protein levels; however, COX-2 silencing abrogated hypothermia-related neuroprotection against NO-induced cell death. Furthermore, it was revealed that either silencing or overexpression of RBM3 had no effects on the expression of COX-2 in SH-SY5Y cells. These findings suggest that mild hypothermia could protect neuroblastoma cells against NO-induced cell death by inducing COX-2 in a RBM3-independent manner.
Assuntos
Temperatura Baixa , Ciclo-Oxigenase 2/metabolismo , Neurônios/metabolismo , Óxido Nítrico/toxicidade , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Osteosarcoma (OS) is the most common primary malignancy of bone. Here, we investigated a possible role of defective osteoblast differentiation in OS tumorigenesis. We found that basal levels of the early osteogenic marker alkaline phosphatase (ALP) activity were low in OS lines. Osteogenic regulators Runx2 and OSX, and the late marker osteopontin (OPN) expressed at low levels in most OS lines, indicating that most OS cells fail to undergo terminal differentiation. Furthermore, OS cells were refractory to osteogenic BMP-induced increases in ALP activity. Osteogenic BMPs were shown to upregulate early target genes, but not late osteogenic markers OPN and osteocalcin (OC). Furthermore, osteogenic BMPs failed to induce bone formation from human OS cells, rather effectively promoted OS tumor growth in an orthotopic OS model. Exogenous expression of early target genes enhanced BMP-stimulated OS tumor growth, whereas osteogenic BMP-promoted OS tumor growth was inhibited by exogenous Runx2 expression. These results suggest that alterations in osteoprogenitors may disrupt osteogenic differentiation pathway. Thus, identifying potential differentiation defects in OS tumors would allow us to reconstruct the tumorigenic events in osteoprogenitors and to develop rational differentiation therapies for clinical OS management.
Assuntos
Proteínas Morfogenéticas Ósseas/fisiologia , Diferenciação Celular , Divisão Celular/fisiologia , Osteogênese/fisiologia , Osteossarcoma/patologia , Fosfatase Alcalina/metabolismo , Animais , Linhagem Celular , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Humanos , Camundongos , Camundongos Endogâmicos C3H , Osteocalcina/genética , Osteopontina/genética , Osteossarcoma/enzimologia , Osteossarcoma/genéticaRESUMO
Bone formation during skeletal development involves a complex coordination among multiple cell types and tissues. Bone is of crucial importance for the human body, providing skeletal support, and serving as a home for the formation of hematopoietic cells and as a reservoir for calcium and phosphate. Bone is also continuously remodeled in vertebrates throughout life. Osteoblasts and osteoclasts are specialized cells responsible for bone formation and resorption, respectively. Early development of the vertebrate skeleton depends on genes that control the distribution and proliferation of cells from cranial neural crest, sclerotomes, and lateral plate mesoderm into mesenchymal condensations, where cells differentiate to osteoblasts. Significant progress has been made over the past decade in our understanding of the molecular framework that controls osteogenic differentiation. A large number of morphogens, signaling molecules, and transcriptional regulators have been implicated in regulating bone development. A partial list of these factors includes the Wnt/beta-catenin, TGF-beta/BMP, FGF, Notch and Hedgehog signaling pathways, and Runx2, Osterix, ATF4, TAZ, and NFATc1 transcriptional factors. A better understanding of molecular mechanisms behind osteogenic differentiation would not only help us to identify pathogenic causes of bone and skeletal diseases but also lead to the development of targeted therapies for these diseases.
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Desenvolvimento Ósseo , Osso e Ossos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco/citologia , Adipócitos/metabolismo , Animais , Diferenciação Celular , Linhagem da Célula , Humanos , Modelos Biológicos , Osteoblastos , Osteogênese , Transdução de Sinais , Transcrição GênicaRESUMO
American ginseng (Panax quinquefolius L., Araliaceae) possesses anti-cancer potential and is one of the most commonly used herbal medicines in the United States. Ginsenoside Rg3, one of the saponins in American ginseng, has been shown to inhibit tumor growth. In this study, we sought to characterize the downstream genes targeted by American ginseng extracts in HCT-116 human colorectal cancer cells. We first demonstrated that the content of Rg3 in American ginseng steamed at 120 degrees C for 2 h (referred to as S2h) was significantly increased when compared with that of the unsteamed ginseng. Both S2h and Rg3 exhibited antiproliferative effects on HCT-116 cells. Using the Affymetrix high density genechips containing more than 40,000 genes and ESTs, the gene expression profiling of HCT-116 cells were assayed. Microarray data indicated that the expression levels of 76 genes were changed significantly after treatment with S2h or Rg3, whereby it was found that 52 of the 76 genes were up-regulated while the remaining 24 were down-regulated. Ingenuity pathways analysis of top functions affected by both S2h and Rg3 were carried out. The most effected pathway is the Ephrin receptor pathway. To validate the microarray data, quantitative real-time PCR of six candidate target genes was conducted, whereby it was found that three genes were up-regulated (AKAPA8L, PMPCB and PDE5A) and three were down-regulated (PITPNA, DUS2L and RIC8A). Although further studies are needed to elucidate the mechanisms of action, our findings should expand the understanding of the molecular framework of American ginseng as an anti-cancer agent.
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Antineoplásicos Fitogênicos/farmacologia , Neoplasias Colorretais/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Ginsenosídeos/farmacologia , Panax , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Análise por Conglomerados , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Relação Dose-Resposta a Droga , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes/efeitos dos fármacos , Células HCT116 , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Extratos Vegetais/farmacologia , Raízes de Plantas , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estados UnidosRESUMO
Osteosarcoma is the most common nonhematologic malignancy of bone in children and adults. The peak incidence occurs in the second decade of life, with a smaller peak after age 50. Osteosarcoma typically arises around the growth plate of long bones. Most osteosarcoma tumors are of high grade and tend to develop pulmonary metastases. Despite clinical improvements, patients with metastatic or recurrent diseases have a poor prognosis. Here, we reviewed the current understanding of human osteosarcoma, with an emphasis on potential links between defective osteogenic differentiation and bone tumorigenesis. Existing data indicate osteosarcoma tumors display a broad range of genetic and molecular alterations, including the gains, losses, or arrangements of chromosomal regions, inactivation of tumor suppressor genes, and the deregulation of major signaling pathways. However, except for p53 and/or RB mutations, most alterations are not constantly detected in the majority of osteosarcoma tumors. With a rapid expansion of our knowledge about stem cell biology, emerging evidence suggests osteosarcoma should be regarded as a differentiation disease caused by genetic and epigenetic changes that interrupt osteoblast differentiation from mesenchymal stem cells. Understanding the molecular pathogenesis of human osteosarcoma could ultimately lead to the development of diagnostic and prognostic markers, as well as targeted therapeutics for osteosarcoma patients.
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Neoplasias Ósseas/genética , Neoplasias Ósseas/fisiopatologia , Diferenciação Celular/fisiologia , Células-Tronco Mesenquimais/citologia , Osteossarcoma/genética , Osteossarcoma/fisiopatologia , Inibidor p16 de Quinase Dependente de Ciclina/genética , Humanos , Neurofibromatose 2/genética , Proto-Oncogenes/genética , RecQ Helicases/genética , Proteína do Retinoblastoma/genética , Transdução de Sinais/genética , Proteína Supressora de Tumor p14ARF/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
RNA interference (RNAi)-mediated gene silencing has become a valuable tool for functional studies, reverse genomics, and drug discoveries. One major challenge of using RNAi is to identify the most effective short interfering RNAs (siRNAs) sites of a given gene. Although several published bioinformatic prediction models have proven useful, the process to select and validate optimal siRNA sites for a given gene remains empirical and laborious. Here, we developed a fluorescence-based selection system using a retroviral vector backbone, namely pSOS, which was based on the premise that candidate siRNAs would knockdown the chimeric transcript between GFP and target gene. The expression of siRNA was driven by the opposing convergent H1 and U6 promoters. This configuration simplifies the cloning of duplex siRNA oligonucleotide cassettes. We demonstrated that GFP signal reduction was closely correlated with siRNA knockdown efficiency of human beta-catenin, as well as with the inhibition of beta-catenin/Tcf4 signaling activity. The pSOS should not only facilitate the selection and validation of candidate siRNA sites, but also provide efficient delivery tools of siRNAs via viral vectors in mammalian cells. Thus, the pSOS system represents an efficient and user-friendly strategy to select and validate siRNA target sites.
Assuntos
Interferência de RNA , RNA Interferente Pequeno/genética , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , DNA/genética , Expressão Gênica , Genes Reporter , Técnicas Genéticas , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Humanos , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Transfecção , beta Catenina/genéticaRESUMO
PURPOSE: Panax notoginseng is a commonly used Chinese herb. Although a few studies have found that notoginseng shows anti-tumor effects, the effect of this herb on colorectal cancer cells has not been investigated. 5-Fluorouracil (5-FU) is a chemotherapeutic agent for the treatment of colorectal cancer that interferes with the growth of cancer cells. However, this compound has serious side effects at high doses. In this study, using HCT-116 human colorectal cancer cell line, we investigated the possible synergistic anti-cancer effects between notoginseng flower extract (NGF) and 5-FU on colon cancer cells. METHODS: The anti-proliferation activity of these modes of treatment was evaluated by MTS cell proliferation assay. Apoptotic effects were analyzed by using Hoechst 33258 staining and Annexin-V/PI staining assays. The anti-proliferation effects of four major single compounds from NGF, ginsenosides Rb1, Rb3, Rc and Rg3 were also analyzed. RESULTS: Both 5-FU and NGF inhibited proliferation of HCT-116 cells. With increasing doses of 5-FU, the anti-proliferation effect was slowly increased. The combined usage of 5-FU 5 microM and NGF 0.25 mg/ml, significantly increased the anti-proliferation effect (59.4 +/- 3.3%) compared with using the two medicines separately (5-FU 5 microM, 31.1 +/- 0.4%; NGF 0.25 mg/ml, 25.3 +/- 3.6%). Apoptotic analysis showed that at this concentration, 5-FU did not exert an apoptotic effect, while apoptotic cells induced by NGF were observed, suggesting that the anti-proliferation target(s) of NGF may be different from that of 5-FU, which is known to inhibit thymidilate synthase. CONCLUSIONS: This study demonstrates that NGF can enhance the anti-proliferation effect of 5-FU on HCT-116 human colorectal cancer cells and may decrease the dosage of 5-FU needed for colorectal cancer treatment.
Assuntos
Proliferação de Células/efeitos dos fármacos , Fluoruracila/farmacologia , Ginsenosídeos/farmacologia , Panax notoginseng/química , Animais , Antimetabólitos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Colorretais/patologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Medicamentos de Ervas Chinesas/química , Flores/química , Ginsenosídeos/química , Ginsenosídeos/isolamento & purificação , Células HCT116 , Humanos , Estrutura Molecular , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Raízes de Plantas/química , RatosRESUMO
Efficacious bone regeneration could revolutionize the clinical management of many bone and musculoskeletal disorders. Bone morphogenetic proteins (BMPs) can regulate the differentiation of mesenchymal stem cells into cartilage, bone, tendon/ligament, and fat lineages. Early data documented the osteogenic potential of rhBMP2 and rhBMP7/OP-1. However, prior to this work that summarized several of our recent studies, no comprehensive analysis had been undertaken to characterize relative osteogenic activity of all BMPs. Using recombinant adenoviruses expressing 14 BMPs, we have demonstrated that, besides BMP2 and BMP7, BMP6 and BMP9 exhibit the highest osteogenic activity both in vitro and in vivo. We further demonstrated that several BMPs may exert synergistic effect on osteogenic differentiation, and that osteogenic BMPs produce a distinct set of molecular fingerprints during osteogenic differentiation. The reported work should expand our current understanding of BMP functions during osteogenic differentiation. It is conceivable that osteogenic BMPs (i.e., BMP2, 4, 6, 7, and 9) may be used to formulate synergistic pairs among themselves and/or with other less osteogenic BMPs for efficacious bone regeneration in clinical settings.
Assuntos
Proteínas Morfogenéticas Ósseas/farmacologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteoblastos/citologia , Animais , Biomarcadores , Proteína Morfogenética Óssea 2 , Proteína Morfogenética Óssea 3 , Proteína Morfogenética Óssea 6 , Proteína Morfogenética Óssea 7 , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Perfilação da Expressão Gênica , Fator 2 de Diferenciação de Crescimento , Fatores de Diferenciação de Crescimento , Sequências Hélice-Alça-Hélice , Humanos , Injeções Intramusculares , Rim/citologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Nus , Ossificação Heterotópica/induzido quimicamente , Ossificação Heterotópica/genética , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologiaRESUMO
This study was designed to determine the changes in saponin content in American ginseng berries after treatment by heating and to assess the anticancer effects of the extracts. After steaming treatment (100-120 degrees C for 1 h, and 120 degrees C for 0.5-4 h), the content of seven ginsenosides, Rg1, Re, Rb1, Rc, Rb2, Rb3, and Rd, decreased; the content of five ginsenosides, Rh1, Rg2, 20R-Rg2, Rg3, and Rh2, increased. Rg3, a previously identified anticancer ginsenoside, increased significantly. Two hours of steaming at 120 degrees C increased the content of ginsenoside Rg3 to a greater degree than other tested ginsenosides. When human colorectal cancer cells were treated with 0.5 mg/mL steamed berry extract (120 degrees C 2 h), the antiproliferation effects were 97.8% for HCT-116 and 99.6% for SW-480 cells. At the same treatment concentration, the effects of unsteamed berry extract were 34.1% for HCT-116 and 4.9% for SW-480 cells. After staining with Hoechst 33258, apoptotic cells increased significantly by treatment with steamed berry extract compared with unheated extracts. Induction of apoptosis activity was confirmed by flow cytometry after staining with annexin V/PI. The steaming of American ginseng berries augments ginsenoside Rg3 content and increases the antiproliferative effects on two human colorectal cancer cell lines.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Frutas/química , Ginsenosídeos/análise , Ginsenosídeos/farmacologia , Temperatura Alta , Panax/química , Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , HumanosRESUMO
Pluripotent mesenchymal stem cells (MSCs) are bone marrow stromal progenitor cells that can differentiate into osteogenic, chondrogenic, adipogenic, and myogenic lineages. We previously demonstrated that bone morphogenetic protein (BMP) 9 is one of the most potent and yet least characterized BMPs that are able to induce osteogenic differentiation of MSCs both in vitro and in vivo. Here, we conducted gene expression-profiling analysis and identified that Hey1 of the hairy/Enhancer of split-related repressor protein basic helix-loop-helix family was among the most significantly up-regulated early targets in BMP9-stimulated MSCs. We demonstrated that Hey1 expression was up-regulated at the immediate early stage of BMP9-induced osteogenic differentiation. Chromatin immunoprecipitation analysis indicated that Hey1 may be a direct target of the BMP9-induced Smad signaling pathway. Silencing Hey1 expression diminished BMP9-induced osteogenic differentiation both in vitro and in vivo and led to chondrogenic differentiation. Likewise, constitutive Hey1 expression augmented BMP9-mediated bone matrix mineralization. Hey1 and Runx2 were shown to act synergistically in BMP9-induced osteogenic differentiation, and Runx2 expression significantly decreased in the absence of Hey1, suggesting that Runx2 may function downstream of Hey1. Accordingly, the defective osteogenic differentiation caused by Hey1 knockdown was rescued by exogenous Runx2 expression. Thus, our findings suggest that Hey1, through its interplay with Runx2, may play an important role in regulating BMP9-induced osteoblast lineage differentiation of MSCs.
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Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/fisiologia , Fator 2 de Diferenciação de Crescimento/metabolismo , Fatores de Diferenciação de Crescimento/metabolismo , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Calcificação Fisiológica/fisiologia , Proteínas de Ciclo Celular/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Perfilação da Expressão Gênica/métodos , Fator 2 de Diferenciação de Crescimento/genética , Fatores de Diferenciação de Crescimento/genética , Humanos , Células-Tronco Mesenquimais/citologia , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Osteoblastos/citologia , Osteogênese/fisiologia , Proteínas Smad/genética , Proteínas Smad/metabolismo , Regulação para Cima/fisiologiaRESUMO
Pluripotent mesenchymal stem cells (MSCs) are bone marrow stromal progenitor cells that can differentiate into osteogenic, chondrogenic, adipogenic, and myogenic lineages. Several signaling pathways have been shown to regulate the lineage commitment and terminal differentiation of MSCs. Here, we conducted a comprehensive analysis of the 14 types of bone morphogenetic protein (BMPs) for their abilities to regulate multilineage specific differentiation of MSCs. We found that most BMPs exhibited distinct abilities to regulate the expression of Runx2, Sox9, MyoD, and PPARgamma2. Further analysis indicated that BMP-2, BMP-4, BMP-6, BMP-7, and BMP-9 effectively induced both adipogenic and osteogenic differentiation in vitro and in vivo. BMP-induced commitment to osteogenic or adipogenic lineage was shown to be mutually exclusive. Overexpression of Runx2 enhanced BMP-induced osteogenic differentiation, whereas knockdown of Runx2 expression diminished BMP-induced bone formation with a decrease in adipocyte accumulation in vivo. Interestingly, overexpression of PPARgamma2 not only promoted adipogenic differentiation, but also enhanced osteogenic differentiation upon BMP-2, BMP-6, and BMP-9 stimulation. Conversely, MSCs with PPARgamma2 knockdown or mouse embryonic fibroblasts derived from PPARgamma2(-/-) mice exhibited a marked decrease in adipogenic differentiation, coupled with reduced osteogenic differentiation and diminished mineralization upon BMP-9 stimulation, suggesting that PPARgamma2 may play a role in BMP-induced osteogenic and adipogenic differentiation. Thus, it is important to understand the molecular mechanism behind BMP-regulated lineage divergence during MSC differentiation, as this knowledge could help us to understand the pathogenesis of skeletal diseases and may lead to the development of strategies for regenerative medicine.
Assuntos
Adipogenia/fisiologia , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular/fisiologia , Células-Tronco Mesenquimais/fisiologia , Osteogênese/fisiologia , Células-Tronco Pluripotentes/fisiologia , Isoformas de Proteínas/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/genética , Linhagem da Célula , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Masculino , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Knockout , Camundongos Nus , PPAR gama/genética , PPAR gama/metabolismo , Isoformas de Proteínas/genética , Transplante de Células-TroncoRESUMO
Wnt signaling plays an important role in regulating cell proliferation and differentiation. De-regulation of these signaling pathways has been implicated in many human diseases, ranging from cancers to skeletal disorders. Wnt proteins are a large family of secreted factors that bind to the Frizzled receptors and LRP5/6 co-receptors and initiate complex signaling cascades. Over the past two decades, our understanding of Wnt signaling has been significantly improved due to the identification of many key regulators and mediators of these pathways. Given that Wnt signaling is tightly regulated at multiple cellular levels, these pathways themselves offer ample nodal points for targeted therapeutics. Here, we focus on our current understanding of these pathways, the associations of Wnt signaling with human disorders, and the opportunities to target key components of Wnt signaling for rational drug discovery.
Assuntos
Doença , Tratamento Farmacológico/métodos , Regulação da Expressão Gênica , Modelos Biológicos , Transdução de Sinais/fisiologia , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Diferenciação Celular/fisiologia , Proliferação de Células , Humanos , Transdução de Sinais/genética , beta Catenina/metabolismoRESUMO
Recombinant adenoviruses provide a versatile system for gene expression studies and therapeutic applications. We have developed an approach that simplifies the generation and production of such viruses called the AdEasy system. A recombinant adenoviral plasmid is generated with a minimum of enzymatic manipulations, employing homologous recombination in bacteria rather than in eukaryotic cells. After transfection of such plasmids into a mammalian packaging cell line, viral production is conveniently followed with the aid of GFP encoded by a gene incorporated into the viral backbone. This system has expedited the process of generating and testing recombinant adenoviruses for a variety of purposes. In this protocol, we describe the practical aspects of using the AdEasy system for generating recombinant adenoviruses. The full protocol usually takes 4-5 weeks to complete.