RESUMO
We developed a framework based on the software Unstructured Reaction-Diffusion Master Equation (URDME) to address tumor cells' proliferation and migration in a heterogeneous space, herein a 2D percolation cluster. A mitogenic paracrine signaling pathway is utilized phenomenologically to reveal how cells cooperate with one another. We modeled the emerging Allee effect using low seeding density culture (LSDC) assays to fit the model parameters. A Finite time scaling (FTS) function has been formulated to quantitatively analyze invasiveness of a virtual Growth-Migration (GM) system in mimicking the cancer cell growth. Through such simulation, we analyzed the GM dynamics of virtual model in mimicking the growth of BT-474 cancer cell populations in vitro in a 2D percolation cluster and calculated the successful penetration rate (SPR). By analyzing the temporal trajectories of the SPR, we could determine the critical exponents of the critical SPR scaling relation. The SPR transition point ([Formula: see text]), which is a fundamentally different from a conventional percolation transition point, is found to be negatively correlated with the invasiveness of this cancer cell. The [Formula: see text] of the three variations of the virtual GM system distinctly designated by varying paracrine-regulated Allee (PAllee) model phenotypes is 0.3408, 0.3675, and 0.4454, respectively. FTS algorithm thereon may serve as an approach to quantify invasiveness of tumor cells. Through a phenomenological paracrine model, inter-cell cooperation and mutual mitogenic boosting are enabled to elicit the Allee effect in the GM systems. The rationale behind such computationally tunable virtual mechanism can be applied to other circumstances concerning emerging processes.
Assuntos
Conceitos Matemáticos , Modelos Biológicos , Algoritmos , Simulação por Computador , DifusãoRESUMO
Environmental contaminants perfluorooctanoate (PFOA) and perfluorooctanesulfonate (PFOS) are present in human serum at the highest concentration among all per- and polyfluoroalkyl substances (PFAS). Serum concentrations as high as 500 ng and 3000 ng PFOA/ml have been detected in individuals living near contamination sites and those occupationally exposed, respectively. Animal and human studies indicated that PFOA and PFOS at these serum concentrations perturb the immune system. The aim of this study was to examine the effects of in vitro exposure of human peripheral blood mononuclear cells (PBMC) to 1, 10, or 100 µM PFOA or PFOS in a medium with serum (RPMI-1640 + 5% human AB serum) on the measurement of proliferation, T cell activation, generation of memory T cells, and cytokine production/secretion. In addition, these immune system parameters were assessed for PBMC in a serum-free medium (OpSFM), which was stimulated with phytohemagglutinin (PHA) (2.5 µg/ml) or influenza vaccine antigen (0.625 µg/ml Flu Ag). PFOS decreased proliferation stimulated by PHA or Flu Ag. With Flu Ag stimulation, PFOA and PFOS inhibited the generation of memory T cells in a concentration-dependent manner. In OpSFM, PFOA and PFOS produced no marked change in proliferation and no inhibition of T cell activation. Cytokines measured in the media with Luminex methodology indicated decreased PBMC secretion of IFN-γ by PFOA and PFOS in medium with serum, but no alteration in OpSFM. The results indicated that changes in immune parameters due to PFOA or PFOS following Flu Ag stimulation are medium (±serum) dependent.
Assuntos
Ácidos Alcanossulfônicos , Fluorocarbonos , Ácidos Alcanossulfônicos/toxicidade , Animais , Caprilatos/toxicidade , Fluorocarbonos/toxicidade , Humanos , Leucócitos Mononucleares , Mitógenos/farmacologiaRESUMO
OBJECTIVE: The aim: To investigate the clinical and pathogenetic peculiarities of formation and course of CCP and the relationship between clinical, hemodynamic and neurohumoral factors of comorbidity development in COPD combined with arterial hypertension (AH). PATIENTS AND METHODS: Materials and methods: The object of the study were 484 patients with COPD. Among them, 350 patients with CCP as a result of cardiac insufficiency/severe congestive heart failure (COPD III-IV) out of aggravation, combined with AH II stage (non-symptomatic organ damage) and 1 - 3 grades, including 55 patients (43 men, 12 women) with compensated CCP (average age 43.7 ± 3.4 years), and 295 patients (212 men and 83 women) with decompensated CCP and chronic heart failure (CHF), average age 63.2 ± 8.9 years. RESULTS: Results: It was found that the development and progression of the left and right CHF in patients with CCP combined with AH occurs due to the disorders of the central hemodynamic, progression of pulmonary hypertension, bronchial obstruction syndrome, neurohumoral and systemic immunoinflammatory activation, disorders of endothelial regulation of vascular tension, overproduction of epithelial and mitogenic growth factors, inducers of apoptosis, and is accompanied by increasing levels of natriuretic peptides. CONCLUSION: Conclusions: The main pathogenetic formation mechanisms of the heart failure on the background of CCP combined with AH are: neurohumoral and systematic immune-inflammatory activation with the development of endothelial dysfunction and (neo)angiogenesis, induction of pathological apoptosis, increase in the intrathoracic pressure, and deposition of blood in the extrathoracic tissues, which result in pulmonary and systemic hypertension, metabolic and hemodynamic remodelling and heart dysfunction.
Assuntos
Insuficiência Cardíaca , Hipertensão Pulmonar , Hipertensão , Doença Pulmonar Obstrutiva Crônica , Adulto , Idoso , Feminino , Insuficiência Cardíaca/complicações , Hemodinâmica , Humanos , Hipertensão/complicações , Hipertensão Pulmonar/complicações , Masculino , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/complicaçõesRESUMO
The heterodimeric cytokine interleukin-23 (IL-23 or IL23A/IL12B) is produced by dendritic cells and macrophages and promotes the proinflammatory and regenerative activities of T helper 17 (Th17) and innate lymphoid cells. A recent study has reported that IL-23 is also secreted by lung adenoma cells and generates an inflammatory and immune-suppressed stroma. Here, we observed that proinflammatory tumor necrosis factor (TNF)/NF-κB and mitogen-activated protein kinase (MAPK) signaling strongly induce IL23A expression in intestinal epithelial cells. Moreover, we identified a strong crosstalk between the NF-κB and MAPK/ERK kinase (MEK) pathways, involving the formation of a transcriptional enhancer complex consisting of proto-oncogene c-Jun (c-Jun), RELA proto-oncogene NF-κB subunit (RelA), RUNX family transcription factor 1 (RUNX1), and RUNX3. Collectively, these proteins induced IL23A secretion, confirmed by immunoprecipitation of endogenous IL23A from activated human colorectal cancer (CRC) cell culture supernatants. Interestingly, IL23A was likely secreted in a noncanonical form, as it was not detected by an ELISA specific for heterodimeric IL-23 likely because IL12B expression is absent in CRC cells. Given recent evidence that IL23A promotes tumor formation, we evaluated the efficacy of MAPK/NF-κB inhibitors in attenuating IL23A expression and found that the MEK inhibitor trametinib and BAY 11-7082 (an IKKα/IκB inhibitor) effectively inhibited IL23A in a subset of human CRC lines with mutant KRAS or BRAFV600E mutations. Together, these results indicate that proinflammatory and mitogenic signals dynamically regulate IL23A in epithelial cells. They further reveal its secretion in a noncanonical form independent of IL12B and that small-molecule inhibitors can attenuate IL23A secretion.
Assuntos
Neoplasias Colorretais/metabolismo , Células Epiteliais/metabolismo , Subunidade p40 da Interleucina-12/metabolismo , Subunidade p19 da Interleucina-23/metabolismo , Mucosa Intestinal/metabolismo , Sistema de Sinalização das MAP Quinases , Substituição de Aminoácidos , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Células Epiteliais/patologia , Células HCT116 , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Subunidade p40 da Interleucina-12/genética , Subunidade p19 da Interleucina-23/genética , Mucosa Intestinal/patologia , Mutação de Sentido Incorreto , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismoRESUMO
Appropriately manipulating macrophage M1/M2 phenotypic transition is a promising therapeutic strategy for tissue repair after myocardial infarction (MI). Here we showed that gene ablation of hypoxia-induced mitogenic factor (HIMF) in mice (Himf-/- and HIMFflox/flox;Lyz2-Cre) attenuated M1 macrophage-dominated inflammatory response and promoted M2 macrophage accumulation in infarcted hearts. This in turn reduced myocardial infarct size and improved cardiac function after MI. Correspondingly, expression of HIMF in macrophages induced expression of pro-inflammatory cytokines; the culturing medium of HIMF-overexpressing macrophages impaired the cardiac fibroblast viability and function. Furthermore, macrophage HIMF was found to up-regulate C/EBP-homologous protein (CHOP) expression, which exaggerated the release of pro-inflammatory cytokines via activating signal transducer of activator of transcription 1 (STAT1) and 3 (STAT3) signaling. Together these data suggested that HIMF promotes M1-type and prohibits M2-type macrophage polarization by activating the CHOP-STAT1/STAT3 signaling pathway to negatively regulate myocardial repair. HIMF might thus constitute a novel target to treat MI.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/deficiência , Macrófagos/metabolismo , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Regeneração , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Fibroblastos/metabolismo , Fibroblastos/patologia , Deleção de Genes , Mediadores da Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/patologia , Fenótipo , Células RAW 264.7 , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismoRESUMO
Insulin resistance is associated with an increased risk of several metabolic disorders including type 2 diabetes, hypertension and cardiovascular diseases. Advances over the last decade have expanded our understanding of the molecular mechanisms underlying insulin resistance; however, many details of the mechanisms causing insulin resistance remain unknown. Recently, attention has shifted toward the role of epigenetics in insulin resistance. In this regard, acetylation of the histone tails has been widely investigated for its role in influencing both metabolic and mitogenic cascades of insulin signaling. More specifically, histone acetyltransferases and histone deacetylases, as major modulators of chromatin accessibility and gene expression, have been studied to determine a possible interconnectivity between the special effects of lysine acetylation status and tyrosine phosphorylation networks on the target proteins of downstream pathways involved in both metabolic and mitogenic cascades of insulin signaling. There is accumulating evidence for the post-translational modification effects of IGFR, InsR, IRS1/2, PI3K, Akt, GLUT4, FoxO, PGC-1α, PPAR, AMPK and MAPKs on insulin resistance and glucose homeostasis. In this paper, we review the importance of acetylation of these factors in the regulation of insulin signaling and glucose metabolism, with a primary focus on the target proteins of downstream signaling of insulin. We also provide an update on the interplay between epigenetic modification and the cellular genome in the context of insulin signaling and describe the possible effect of the environment on this epigenetic regulation.
RESUMO
The role of proNGF, the precursor of nerve growth factor (NGF), in the biology of adult neural stem cells (aNSCs) is still unclear. Here, we analyzed adult hippocampal neurogenesis in AD11 transgenic mice, in which the constitutive expression of anti-NGF antibody leads to an imbalance of proNGF over mature NGF. We found increased proliferation of progenitors but a reduced neurogenesis in the AD11 dentate gyrus (DG)-hippocampus (HP). Also in vitro, AD11 hippocampal neural stem cells (NSCs) proliferated more, but were unable to differentiate into morphologically mature neurons. By treating wild-type hippocampal progenitors with the uncleavable form of proNGF (proNGF-KR), we demonstrated that proNGF acts as mitogen on aNSCs at low concentration. The mitogenic effect of proNGF was specifically addressed to the radial glia-like (RGL) stem cells through the induction of cyclin D1 expression. These cells express high levels of p75NTR , as demonstrated by immunofluorescence analyses performed ex vivo on RGL cells isolated from freshly dissociated HP-DG or selected in vitro from NSCs by leukemia inhibitory factor. Clonogenic assay performed in the absence of mitogens showed that RGLs respond to proNGF-KR by reactivating their proliferation and thus leading to neurospheres formation. The mitogenic effect of proNGF was further exploited in the expansion of mouse-induced neural stem cells (iNSCs). Chronic exposure of iNSCs to proNGF-KR increased their proliferation. Altogether, we demonstrated that proNGF acts as mitogen on hippocampal and iNSCs. Stem Cells 2019;37:1223-1237.
Assuntos
Giro Denteado/citologia , Hipocampo/citologia , Mitógenos/farmacologia , Fator de Crescimento Neural/farmacologia , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Precursores de Proteínas/farmacologia , Animais , Anticorpos/genética , Anticorpos/imunologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Fator Inibidor de Leucemia/farmacologia , Camundongos Transgênicos , Fator de Crescimento Neural/imunologia , Fator de Crescimento Neural/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Precursores de Proteínas/imunologia , Precursores de Proteínas/metabolismoRESUMO
Antheraea mylitta arylphorin protein was extracted from the silk gland of fifth instar larvae and purified by ammonium sulphate precipitation, ion-exchange, and gel filtration chromatography. The N-terminal sequencing of ten amino acids (NH2-SVVHPPHHEV-COOH) showed similarity with Antheraea pernyi arylphorin. Based on N-terminal and C-terminal A. pernyi arylphorin sequences, primers were designed, and A. mylitta arylphorin cDNA was cloned by RT-PCR from silk gland mRNA. Sequencing of complete cDNA including 25 nucleotides at 5' UTR (obtained by 5' RACE) showed that it consisted of an ORF of 2115 nucleotides which could encode a protein of 704 amino acids (predominantly aromatic residues) having molecular weight 83 kDa. Homology modelling was done using A. pernyi arylphorin as a template. Cloned arylphorin cDNA was expressed in E. coli and recombinant His-tagged protein was purified by Ni-NTA affinity chromatography. Analysis of tissue-specific expression of arylphorin by real-time PCR showed maximum expression in the fat body followed by silk gland and integument. 5' flanking region (759 bp) of arylphorin gene was amplified by inverse PCR and the full length gene (5359 nucleotides) containing five exons and four introns was cloned from the A. mylitta genomic DNA and sequenced. Polyclonal antibody was raised against purified arylphorin and more native arylphorin protein (500 kDa) was purified from the fat body by antibody affinity chromatography. Study of mitogenic effect of native and chymotrypsin hydrolysate of arylphorin on different insect cell lines showed that arylphorin could be used as serum substitute for in vitro cultivation of insect cells.
Assuntos
Regiões 5' não Traduzidas , Corpo Adiposo/metabolismo , Regulação da Expressão Gênica , Genes de Insetos , Proteínas de Insetos , Mariposas , Animais , Proteínas de Insetos/biossíntese , Proteínas de Insetos/química , Proteínas de Insetos/genética , Mariposas/genética , Mariposas/metabolismoRESUMO
Insulin resistance (IR) is a shared pathological condition among type 2 diabetes, obesity, cardiovascular disease, and other metabolic disorders. It is growing significantly all over the world and consequently, a substantial effort is needed for developing the potential novel diagnostics and therapeutics. An insulin signaling pathway is tightly modulated by different mechanisms including the epigenetic modifications. Today, a deal of great attention has been shifted towards the regulatory role of noncoding RNAs on target proteins of the insulin signaling pathway. Noncoding RNAs are a major area of the epigenetics which control gene expression at the posttranscriptional levels and include a large class of microRNAs (miRNAs). With this in view, many studies have implicated the mediatory effects of miRNAs on the downstream metabolic and mitogenic proteins of the insulin signaling pathway. Since providing new biomarkers for the early diagnosis of IR and related metabolic traits are very significant, we intended to review the possible role of miRNAs in the regulation of the insulin signaling pathway, with a primary focus on the downstream target proteins of the metabolic and mitogenic cascades.
Assuntos
Insulina/metabolismo , MicroRNAs/metabolismo , Animais , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Resistência à Insulina/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Doxorubicin (Dox)-induced cardiotoxicity, a limiting factor in the use of Dox to treat cancer, can be mitigated by the mitogenic factor FGF2 in vitro, via a heme oxygenase 1 (HO-1)-dependent pathway. HO-1 upregulation was reported to require protein kinase CK2 activity. We show that a mutant non-mitogenic FGF2 (S117A-FGF2), which does not activate CK2, is cardioprotective against acute cardiac ischemic injury. We now investigate the potential of S117A-FGF2 to protect cardiomyocytes against acute Dox injury and decrease Dox-induced upregulation of oxidized phospholipids. The roles of CK2 and HO-1 in cardiomyocyte protection are also addressed.Rat neonatal cardiomyocyte cultures were used as an established in vitro model of acute Dox toxicity. Pretreatment with S117A-FGF2 protected against Dox-induced: oxidative stress; upregulation of fragmented and non-fragmented oxidized phosphatidylcholine species, measured by LC/MS/MS; and cardiomyocyte injury and cell death measured by LDH release and a live-dead assay. CK2 inhibitors (TBB and Ellagic acid), did not affect protection by S117A-FGF2 but prevented protection by mitogenic FGF2. Furthermore, protection by S117A-FGF2, unlike that of FGF2, was not prevented by HO-1 inhibitors and S117A-FGF2 did not upregulate HO-1. Protection by S117A-FGF2 required the activity of FGF receptor 1 and ERK.We conclude that mitogenic and non-mitogenic FGF2 protect from acute Dox toxicity by common (FGFR1) and distinct, CK2/HO-1- dependent or CK2/HO-1-independent (respectively), pathways. Non-mitogenic FGF2 merits further consideration as a preventative treatment against Dox cardiotoxicity.
Assuntos
Cardiotônicos/farmacologia , Caseína Quinase II/metabolismo , Citoproteção/efeitos dos fármacos , Doxorrubicina/toxicidade , Fator 2 de Crescimento de Fibroblastos/farmacologia , Heme Oxigenase-1/metabolismo , Miócitos Cardíacos/patologia , Animais , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Modelos Biológicos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Oxirredução , Fosfolipídeos/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: Spirocerca lupi is a nematode that parasitizes vertebrates in particular canids, by forming nodules in the thoracic cavity specifically in the oesophagus. In 25% of Spirocerca infections of the domestic dog, nodules progress from inflammatory to pre-neoplastic to sarcomatous neoplasia. With the mechanism of neoplastic transformation being incompletely understood, this study investigates if S. lupi parasite proteinaceous secretory/excretory products (ESPs) play a role in the neoplastic transformation. METHODS: To facilitate collection of ESPs, we maintained naturally harvested adult parasites in the laboratory under artificial conditions. Media in which the parasites were grown was subsequently evaluated for the presence of proteinaceous compounds using a mass spectroscopy library as well as for their ability to be mitogenic in primary murine fibroblastic cells. RESULTS: Chromatrography of the ethyl acetate extracted incubation media showed the presence of 9 protein compounds, of which three were identified as non-specific proteins isolated from Nematostella vectensis, Caenorhabditis brenneri and Sus scrofa, with the rest being unknown. Acetone, methanol, hexane and ethylacetate extracted culture media were unable to induce a mitogenic change in primary murine fibroblasts in comparison to the controls. CONCLUSION: While no mitogenic effect was evident, further studies are required to understand the role of worm excretory/secretory products on clastogenesis under chronic exposure. In addition, while not of primary importance for this study, the observed duration of parasite survival indicates that ex vivo studies on S. lupi are possible. For the latter we believe that the worm culture method can be further optimized if longer survival times are required.
Assuntos
Fibroblastos/parasitologia , Proteínas de Helminto/fisiologia , Mitógenos/fisiologia , Thelazioidea/patogenicidade , Animais , Transformação Celular Neoplásica , Células Cultivadas , Cães/parasitologia , Feminino , Proteínas de Helminto/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Thelazioidea/crescimento & desenvolvimento , Thelazioidea/isolamento & purificaçãoRESUMO
Gaining the full activity of the insulin receptor (IR) requires the proteolytic cleavage of its proform by intra-Golgi furin-like activity. In mammalian cells, IR is expressed as two isoforms (IRB and IRA) that are responsible for insulin action. However, only IRA transmits the growth-promoting and mitogenic effects of insulin-like growth factor 2. Here we demonstrate that the two IR isoforms are similarly cleaved by furin, but when this furin-dependent maturation is inefficient, IR proforms move to the cell surface where the proprotein convertase PACE4 selectively supports IRB maturation. Therefore, in situations of impaired furin activity, the proteolytic maturation of IRB is greater than that of IRA, and accordingly, the amount of phosphorylated IRB is also greater than that of IRA. We highlight the ability of a particular proprotein convertase inhibitor to effectively reduce the maturation of IRA and its associated mitogenic signaling without altering the signals emanating from IRB. In conclusion, the selective PACE4-dependent maturation of IRB occurs when furin activity is reduced; accordingly, the pharmacological inhibition of furin reduces IRA maturation and its mitogenic potential without altering the insulin effects.
Assuntos
Fator de Crescimento Insulin-Like II/metabolismo , Pró-Proteína Convertases/metabolismo , Receptor de Insulina/metabolismo , Serina Endopeptidases/metabolismo , Células 3T3-L1 , Animais , Proliferação de Células , Furina/genética , Furina/metabolismo , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Camundongos , Pró-Proteína Convertases/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptor de Insulina/genética , Serina Endopeptidases/genéticaRESUMO
Basal cell carcinoma (BCC) is the most common cancer worldwide, and its current treatment options are insufficient and toxic. Surprisingly, unlike several other malignancies, chemopreventive efforts against BCC are almost lacking. Silibinin, a natural agent from milk thistle seeds, has shown strong efficacy against several cancers including ultraviolet radiation-induced skin (squamous) cancer; however, its potential activity against BCC is not yet examined. Herein, for the first time, we report the efficacy of silibinin and its oxidation product 2,3-dehydrosilibinin (DHS) against BCC both in vitro and in vivo using ASZ (p53 mutated) and BSZ (p53 deleted) cell lines derived from murine BCC tumors. Both silibinin and DHS significantly inhibited cell growth and clonogenicity while inducing apoptosis in a dose- and time-dependent manner, with DHS showing higher activity at lower concentrations. Both agents also inhibited the mitogenic signaling by reducing EGFR, ERK1/2, Akt, and STAT3 phosphorylation and suppressed the activation of transcription factors NF-κB and AP-1. More importantly, in an ectopic allograft model, oral administration of silibinin and DHS (200 mg/kg body weight) strongly inhibited the ASZ tumor growth by 44% and 71% (P < 0.05), respectively, and decreased the expression of proliferation biomarkers (PCNA and cyclin D1) as well as NF-κB p50 and c-Fos in the tumor tissues. Taken together, these results provide the first evidence for the efficacy and usefulness of silibinin and its derivative DHS against BCC, and suggest the need for additional studies with these agents in pre-clinical and clinical BCC chemoprevention and therapy models.
Assuntos
Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Carcinoma Basocelular/metabolismo , Transdução de Sinais/efeitos dos fármacos , Silimarina/farmacologia , Neoplasias Cutâneas/metabolismo , Fatores de Transcrição/metabolismo , Aloenxertos , Animais , Antineoplásicos/química , Antioxidantes/química , Apoptose/efeitos dos fármacos , Carcinoma Basocelular/tratamento farmacológico , Carcinoma Basocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Humanos , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , Silibina , Silimarina/química , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/patologia , Fator de Transcrição AP-1/metabolismo , Ensaio Tumoral de Célula-TroncoRESUMO
Platelet-derived growth factor-D (PDGF-D) was recently identified, and acts as potent mitogen for mesenchymal cells. PDGF-D also induces cellular transformation and promotes tumor growth. However, the functional role of PDGF-D in adipose-derived stem cells (ASCs) has not been identified. Therefore, we primarily investigated the autocrine and paracrine roles of PDGF-D in this study. Furthermore, we identified the signaling pathways and the molecular mechanisms involved in PDGF-D-induced stimulation of ASCs. It is of interest that PDGF-B is not expressed, but PDGF-D and PDGF receptor-ß are expressed in ASCs. PDGF-D showed the strongest mitogenic effect on ASCs, and PDGF-D regulates the proliferation and migration of ASCs through the PI3K/Akt pathways. PDGF-D also increases the proliferation and migration of ASCs through generation of mitochondrial reactive oxygen species (mtROS) and mitochondrial fission. mtROS generation and fission were mediated by p66Shc phosphorylation, and BCL2-related protein A1 and Serpine peptidase inhibitor, clade E, member 1 mediated the proliferation and migration of ASCs. In addition, PDGF-D upregulated the mRNA expression of diverse growth factors such as vascular endothelial growth factor A, fibroblast growth factor 1 (FGF1), FGF5, leukemia inhibitory factor, inhibin, beta A, interleukin 11, and heparin-binding EGF-like growth factor. Therefore, the preconditioning of PDGF-D enhanced the hair-regenerative potential of ASCs. PDGF-D-induced growth factor expression was attenuated by a pharmacological inhibitor of mitogen-activated protein kinase pathway. In summary, PDGF-D is highly expressed by ASCs, where it acts as a potent mitogenic factor. PDGF-D also upregulates growth factor expression in ASCs. Therefore, PDGF-D can be considered a novel ASC stimulator, and used as a preconditioning agent before ASC transplantation.
Assuntos
Tecido Adiposo/metabolismo , Regulação da Expressão Gênica/fisiologia , Linfocinas/biossíntese , Fator de Crescimento Derivado de Plaquetas/biossíntese , Células-Tronco/metabolismo , Tecido Adiposo/citologia , Células Cultivadas , Citocinas/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Linfocinas/farmacologia , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/efeitos dos fármacos , Dinâmica Mitocondrial/fisiologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/biossíntese , Espécies Reativas de Oxigênio/metabolismo , Células-Tronco/citologiaRESUMO
The toxicological profile of insulins is exclusively due to exaggerated pharmacology resulting in hypoglycemic findings. Insulin analogues displaying modifications and aimed at improving pharmacokinetics do not induce different toxicity. The main target is the brain displaying neuronal necrosis. Wallerian degeneration of nerves occurs rarely after severe hypoglycemia. These findings are of potential human relevance; nevertheless, these changes are induced in normoglycemic animals whereas diabetic patients suffer from hyperglycemia. Therefore, it is usually not difficult to achieve a therapeutic window for subsequent use in patients. Based upon this and in the absence of classical toxicity, there has been no scientific need for diabetic animal models. A greater challenge is the mitogenicity already inherent with regular insulin. Thus, the focus for preclinical safety evaluation of analogues is to demonstrate that modifications in regular insulin do not result in enhanced mitogenicity. The approaches used to assess the mitogenic potential of insulin analogues have changed over time driven by scientific progression and changes within the regulatory environment. Therefore, in vitro and in vivo evaluation of cell proliferation has become common practice, and to date there has been no evidence that the mitogenic potential of insulin analogues may be increased compared to regular insulin.
Assuntos
Hipoglicemiantes/toxicidade , Insulinas/toxicidade , Testes de Toxicidade , Animais , HumanosRESUMO
Fibroblast growth factor 20 (FGF20) has a wide range of biological activities; its expression is most pronounced in neural tissues where it has functions in development and neuroprotection. Given these activities, interest in the clinical applications of FGF20 is rising, which will lead to increasing demand for active recombinant human FGF20 (rhFGF20). To improve the production of rhFGF20, an artificial gene encoding fgf20 was cloned into pET3a and expressed in E. coli BL21(DE3)pLysS. By optimizing induction conditions, we successfully induced large amounts of insoluble rhFGF20. Following solubilization and refolding of the rhFGF20 from inclusion bodies, it was purified by HiTrap heparin affinity chromatography to a purity of over 96% with a yield of 218 mg rhFGF20/100 g wet cells. The purified rhFGF20 could stimulate proliferation of both NIH 3T3 cells and PC-12 cells, measured by the MTT assay. In a model of Aß25-35-induced apoptosis on PC-12 cells, rhFGF20 had a clear protective effect. RT-PCR and Western blot analysis of apoptosis-related genes and proteins revealed that the FGF20-derived protective mechanism was likely due to the relief of endoplasmic reticulum stress (ER stress). In conclusion, the approach described here may be a better means to produce active rhFGF20 in good quantity, thereby allowing for its future pharmacological and clinical use.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fatores de Crescimento de Fibroblastos/biossíntese , Fármacos Neuroprotetores/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Peptídeos beta-Amiloides , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Precipitação Química , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica , Humanos , Corpos de Inclusão/química , Camundongos , Células NIH 3T3 , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacologia , Células PC12 , Fragmentos de Peptídeos , Plasmídeos/química , Plasmídeos/metabolismo , Redobramento de Proteína , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , SolubilidadeRESUMO
In the developing cerebellum, the proliferation and differentiation of glial and neuronal cell types depend on the modulation of the sonic hedgehog (Shh) signaling pathway. The vertebrate G-protein-coupled receptor 37-like 1 (GPR37L1) gene encodes a putative G-protein-coupled receptor that is expressed in newborn and adult cerebellar Bergmann glia astrocytes. This study shows that the ablation of the murine Gpr37l1 gene results in premature down-regulation of proliferation of granule neuron precursors and precocious maturation of Bergmann glia and Purkinje neurons. These alterations are accompanied by improved adult motor learning and coordination. Gpr37l1(-/-) mice also exhibit specific modifications of the Shh signaling cascade. Specific assays show that in Bergmann glia cells Gpr37l1 is associated with primary cilium membranes and it specifically interacts and colocalizes with the Shh primary receptor, patched 1. These findings indicate that the patched 1-associated Gpr37l1 receptor participates in the regulation of postnatal cerebellum development by modulating the Shh pathway.
Assuntos
Cerebelo/crescimento & desenvolvimento , Neuroglia/fisiologia , Desempenho Psicomotor/fisiologia , Células de Purkinje/fisiologia , Receptores Acoplados a Proteínas G/genética , Animais , Western Blotting , Proliferação de Células , Cerebelo/citologia , Primers do DNA/genética , Imunofluorescência , Deleção de Genes , Vetores Genéticos/genética , Proteínas Hedgehog/metabolismo , Imunoprecipitação , Hibridização In Situ , Camundongos , Camundongos Knockout , Mitógenos/metabolismo , Receptores Patched , Receptor Patched-1 , Receptores de Superfície Celular/metabolismoRESUMO
The cellular response to mitogens is tightly regulated via transcriptional and post-transcriptional mechanisms to rapidly induce genes that promote proliferation and efficiently attenuate their expression to prevent malignant growth. RNase L is an endoribonuclease that mediates diverse antiproliferative activities, and tristetraprolin (TTP) is a mitogen-induced RNA-binding protein that directs the decay of proliferation-stimulatory mRNAs. In light of their roles as endogenous proliferative constraints, we examined the mechanisms and functional interactions of RNase L and TTP to attenuate a mitogenic response. Mitogen stimulation of RNase L-deficient cells significantly increased TTP transcription and the induction of other mitogen-induced mRNAs. This regulation corresponded with elevated expression of serum-response factor (SRF), a master regulator of mitogen-induced transcription. RNase L destabilized the SRF transcript and formed a complex with SRF mRNA in cells providing a mechanism by which RNase L down-regulates SRF-induced genes. TTP and RNase L proteins interacted in cells suggesting that RNase L is directed to cleave TTP-bound RNAs as a mechanism of substrate specificity. Consistent with their concerted function in RNA turnover, the absence of either RNase L or TTP stabilized SRF mRNA, and a subset of established TTP targets was also regulated by RNase L. RNase L deficiency enhanced mitogen-induced proliferation demonstrating its functional role in limiting the mitogenic response. Our findings support a model of feedback regulation in which RNase L and TTP target SRF mRNA and SRF-induced transcripts. Accordingly, meta-analysis revealed an enrichment of RNase L and TTP targets among SRF-regulated genes suggesting that the RNase L/TTP axis represents a viable target to inhibit SRF-driven proliferation in neoplastic diseases.
Assuntos
Proliferação de Células/efeitos dos fármacos , Endorribonucleases/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Mitógenos/farmacologia , Estabilidade de RNA/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Animais , Proliferação de Células/fisiologia , Regulação da Expressão Gênica/fisiologia , Células HEK293 , Células HeLa , Humanos , Camundongos , Modelos Biológicos , Estabilidade de RNA/fisiologia , Transcrição Gênica/fisiologia , Tristetraprolina/metabolismoRESUMO
Radiotherapy of is well established and frequently utilized in prostate cancer (PCa) patients. However, recurrence following therapy and distant metastases are commonly encountered problems. Previous studies underline that, in addition to its therapeutic effects, ionizing radiation (IR) increases the vascularity and invasiveness of surviving radioresistant cancer cells. This invasive phenotype of radioresistant cells is an upshot of IR-induced pro-survival and mitogenic signaling in cancer as well as endothelial cells. Here, we demonstrate that a plant flavonoid, silibinin can radiosensitize endothelial cells by inhibiting expression of pro-angiogenic factors. Combining silibinin with IR not only strongly down-regulated endothelial cell proliferation, clonogenicity and tube formation ability rather it strongly (p<0.001) reduced migratory and invasive properties of PCa cells which were otherwise marginally affected by IR treatment alone. Most of the pro-angiogenic (VEGF, iNOS), migratory (MMP-2) and EMT promoting proteins (uPA, vimentin, N-cadherin) were up-regulated by IR in PCa cells. Interestingly, all of these invasive and EMT promoting actions of IR were markedly decreased by silibinin. Further, we found that potentiated effect was an end result of attenuation of IR-activated mitogenic and pro-survival signaling, including Akt, Erk1/2 and STAT-3, by silibinin.
Assuntos
Indutores da Angiogênese/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Neoplasias da Próstata/patologia , Radiação Ionizante , Silimarina/farmacologia , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos da radiação , Movimento Celular , Proliferação de Células , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos da radiação , Humanos , Masculino , Recidiva Local de Neoplasia , Fenótipo , Silibina , Fator A de Crescimento do Endotélio Vascular/metabolismo , CicatrizaçãoRESUMO
Biological activities of alkali extracted (Barium hydroxide: BE-480 kDa, Potassium hydroxide: KE1-1080 and KE2-40 kDa), purified arabinoxylans (AX) from the finger millet bran varying in their molecular weight, phenolic acid content, arabinose to xylose ratios were evaluated for their immune-stimulatory activities using murine lymphocytes and peritoneal exudate macrophages. All three purified AX displayed significant (p < 0.001) mitogenic activity and activation of macrophages including phagocytosis. Among these BE has shown higher enhancing lymphocyte proliferation (>2 fold) and macrophage phagocytosis than KE1 and KE2. The above results clearly documented that the immunostimulatory activity of arabinoxylans is directly proportional to the amount of ferulic acid content (0.11 mg/100 g), whereas molecular weight as well as arabinose/xylose ratio, did not have any bearing. Purified AX from the finger millet bran can be explored as a potent natural immunomodulator.