RESUMO
Background: Diabetes-associated cognitive decline (DACD) is one of the nervous system dysfunctions induced by diabetes mellitus with cognitive impairment as the major symptom. In a previous preliminary proteomic study, we found that endoplasmic reticulum processing and PI3K-Akt signaling pathway might be impaired in DACD pathogenesis. In addition, growth factor receptor-bound protein 2 might be a crucial protein as a molecular target of the neuroprotective effects of ZiBuPiYin recipe (ZBPYR). Methods: In this study, 6-8 weeks aged db/db mice were treated with excipients or ZBPYR for 6 weeks. Body weight and RBG were recorded weekly. Oral glucose tolerance and insulin tolerance tests were used to assess insulin sensitivity. Morris water maze (MWM) tests were used to assess memory function. The expression of Grb2, Gab2, Akt, and GSK3ß in mouse hippocampus and cerebral cortex were analyzed by Western blotting. Results: ZBPYR not only significantly reduced RGB and improved glucose tolerance and insulin resistance, but also improved spatial cognition in DACD mice. The expression of Grb2 and Gab2 in hippocampus and cerebral cortex of db/db mice was upregulated after treated with ZBPYR, and then affected the PI3K/Akt signaling pathway, and inhibited GSK3ß overactivity. Conclusions: This study showed that ZBPYR could enhance the memory and learning ability of db/db mice. Such neuroprotective effect might be related to the activation of Grb2-PI3K/Akt signaling which might provide a novel therapeutic target for the clinical treatment of DACD.
Assuntos
Córtex Cerebral/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Proteína Adaptadora GRB2/metabolismo , Fármacos Neuroprotetores/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Glicemia , Córtex Cerebral/metabolismo , Resistência à Insulina/fisiologia , Masculino , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
The present state of cancer chemotherapy is unsatisfactory. New anticancer drugs that marginally improve the survival of patients continue to be developed at an unsustainably high cost. The study aimed to elucidate the effects of insulin (INS), an inexpensive drug with a convincing safety profile, on the susceptibility of colon cancer to chemotherapeutic agents: 5-fluorouracil (FU), oxaliplatin (OXA), irinotecan (IRI), cyclophosphamide (CPA) and docetaxel (DOC). To examine the effects of insulin on cell viability and apoptosis, we performed an in vitro analysis on colon cancer cell lines Caco-2 and SW480. To verify the results, we performed in vivo analysis on mice bearing MC38 colon tumors. To assess the underlying mechanism of the therapy, we examined the mRNA expression of pathways related to the signaling downstream of insulin receptors (INSR). Moreover, we performed Western blotting to confirm expression patterns derived from the genetic analysis. For the quantification of circulating tumor cells in the peripheral blood, we used the maintrac method. The results of our study show that insulin-pretreated colon cancer cells are significantly more susceptible to commonly used chemotherapeutics. The apoptosis ratio was also enhanced when INS was administered complementary to the examined drugs. The in vivo study showed that the combination of INS and FU resulted in significant inhibition of tumor growth and reduction of the number of circulating tumor cells. This combination caused a significant downregulation of the key signaling substrates downstream of INSR. The results indicate that the downregulation of PIK3CA (phosphatidylinositol 3-kinase catalytic subunit alpha), which plays a critical role in cell signaling and GRB2 (growth factor receptor-bound protein 2), a regulator of cell proliferation and differentiation may be responsible for the sensitizing effect of INS. These findings were confirmed at protein levels by Western blotting. In conclusion, these results suggest that INS might be potentially applied to clinical use to enhance the therapeutic effectiveness of chemotherapeutic drugs. The findings may become a platform for the future development of new and inexpensive strategies for the clinical chemotherapy of tumors.
Assuntos
Antineoplásicos/uso terapêutico , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Neoplasias Colorretais/tratamento farmacológico , Proteína Adaptadora GRB2/antagonistas & inibidores , Insulina/farmacologia , Animais , Western Blotting , Células CACO-2/efeitos dos fármacos , Células CACO-2/metabolismo , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias Colorretais/metabolismo , Ciclofosfamida/uso terapêutico , Docetaxel/uso terapêutico , Regulação para Baixo/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Fluoruracila/uso terapêutico , Proteína Adaptadora GRB2/metabolismo , Humanos , Irinotecano/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Células Neoplásicas Circulantes/efeitos dos fármacos , Células Neoplásicas Circulantes/metabolismo , Oxaliplatina/uso terapêuticoRESUMO
The focal adhesion kinase-growth factor receptor 2 (FAK-Grb2) protein-protein interaction is implicated in pathogenesis of stress-induced cardiac hypertrophy. The focal adhesion targeting (FAT) domain of FAK unfolds to form a structural intermediate that interacts with a multibinding hot spot in the SH2 domain of Grb2. Disruption of the Grb2-FAT interaction is a therapeutic strategy for prevention of pathological cardiac hypertrophy. A pharmacophore was generated on the basis of structural and electrostatic properties of FAT bound to FAK using the Forge tool (Cresset). This pharmacophore was used as a query for Blaze server (Cresset) to screen a selectively enriched chemical library of 4,32,508 small molecules. The compounds selected were further filtered by hierarchical flexible docking approach using AutoDock v4. From the favorably docked compounds, five were selected on the basis of good adsorption, distribution, metabolism, excretion, and toxicity (ADMET) properties using SwissADME, MedChem Designer v.3, and MOLINSPIRATION. Stability of the binding mode of the inhibitors was further confirmed by molecular dynamic simulation study with AMBER v15 for a simulation time of 50 ns in aqueous environment. PM2307 was identified as the best inhibitor in terms of pharmacophoric features, dock score, and in silico ADMET analysis. The calculated binding affinity of PM2307 was better than that of the FAT-Grb2 complex as well as a previously reported small molecule inhibitor. PM2307 is also a quinolyl derivative sharing a similar scaffold with ofloxacin drugs, asserting its drug-like properties. Thus, it was proposed as a lead compound for development of drugs for pathological cardiac hypertrophy.
Assuntos
Cardiomegalia/tratamento farmacológico , Proteína-Tirosina Quinases de Adesão Focal/antagonistas & inibidores , Proteína Adaptadora GRB2/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Cardiomegalia/metabolismo , Avaliação Pré-Clínica de Medicamentos , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Proteína Adaptadora GRB2/metabolismo , Humanos , Ligantes , Modelos Moleculares , Ligação Proteica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/químicaRESUMO
BACKGROUND: Overexpression or mutation of the epidermal growth factor receptor (EGFR) potently enhances the growth of many solid tumors. Tumor cells frequently display resistance to mechanistically-distinct EGFR-directed therapeutic agents, making it valuable to develop therapeutics that work by additional mechanisms. Current EGFR-targeting therapeutics include antibodies targeting the extracellular domains, and small molecules inhibiting the intracellular kinase domain. Recent studies have identified a novel prone extracellular tetrameric EGFR configuration, which we identify as a potential target for drug discovery. METHODS: Our focus is on the prone EGFR tetramer, which contains a novel protein-protein interface involving extracellular domain III. This EGFR tetramer is computationally targeted for stabilization by small molecule ligand binding. This study performed virtual screening of a Life Chemicals, Inc. small molecule library of 345,232 drug-like compounds against a molecular dynamics simulation of protein-protein interfaces distinct to the novel tetramer. One hundred nine chemically diverse candidate molecules were selected and evaluated using a cell-based high-content imaging screen that directly assessed induced internalization of the EGFR effector protein Grb2. Positive hits were further evaluated for influence on phosphorylation of EGFR and its effector ERK1/2. RESULTS: Fourteen hit compounds affected internalization of Grb2, an adaptor responsive to EGFR activation. Most hits had limited effect on cell viability, and minimally influenced EGFR and ERK1/2 phosphorylation. Docked hit compound poses generally include Arg270 or neighboring residues, which are also involved in binding the effective therapeutic cetuximab, guiding further chemical optimization. CONCLUSIONS: These data suggest that the EGFR tetrameric configuration offers a novel cancer drug target.
Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/química , Proteína Adaptadora GRB2/metabolismo , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cetuximab/farmacologia , Avaliação Pré-Clínica de Medicamentos , Receptores ErbB/metabolismo , Cloridrato de Erlotinib/farmacologia , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Fosforilação/efeitos dos fármacos , Domínios e Motivos de Interação entre Proteínas , Transdução de SinaisRESUMO
Panduratin A (PA), a cyclohexanyl chalcone from Boesenbergia rotunda (L.) Mansf. was shown to possess anti-angiogenic effects in our previous study. In the present study, the molecular targets and anti-angiogenic mechanisms of PA on human umbilical vein endothelial cells (HUVECs) were identified using an iTRAQ-based quantitative proteomics approach. A total of 263 proteins were found to be differentially regulated in response to treatment with PA. Ingenuity Pathway Analysis revealed that cellular growth and proliferation, protein synthesis, RNA post-transcriptional modification, cellular assembly and organization and cell-to-cell signaling and interaction were the most significantly deregulated molecular and cellular functions in PA-treated HUVECs. PA inhibited the expressions of ARPC2 and CTNND1 that are associated with the formation of actin cytoskeleton, focal adhesion and cellular protrusions. In addition, PA down-regulated CD63, GRB-2, ICAM-2 and STAB-1 that are implicated in adhesion, migration and tube formation of endothelial cells. The differential expressions of three targets, namely, ARPC2, CDK4, and GRB-2 were validated by western blot analyses. Furthermore, PA inhibited G1-S progression, and resulted in G0/G1 arrest in HUVECs. The blockage in cell cycle progression was accompanied with the suppression of mTOR signaling. Treatment of HUVECs with PA resulted in decreased phosphorylation of ribosomal S6 and 4EBP1 proteins, the two downstream effectors of mTOR signaling. We further showed that PA is able to inhibit mTOR signaling induced by VEGF, a potent inducer of angiogenesis. Taken together, by integrating quantitative proteomic approach, we identified protein targets in which PA mediates its anti-angiogenic effects. The present study thus provides mechanistic evidence to the previously reported multifaceted anti-angiogenic effects of PA. Our study further identified mTOR signaling as an important target of PA, and therefore highlights the potential of PA for therapeutic intervention against angiogenesis-related pathogenesis, particularly, metastatic malignancy.
Assuntos
Inibidores da Angiogênese/farmacologia , Chalconas/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Proteoma/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Proliferação de Células/efeitos dos fármacos , Quinase 4 Dependente de Ciclina/metabolismo , Proteína Adaptadora GRB2/metabolismo , Humanos , Proteômica , Rizoma/química , Zingiberaceae/químicaRESUMO
The Siberian hamster, Phodopus sungorus, undergoes a striking seasonal cycle of leptin sensitivity and body weight regulation, but the molecular mechanism and relevance to human leptin insensitivity are unknown. Here we show that nuclear translocation of phospho-STAT3 in the hypothalamus is rapidly stimulated by leptin to a greater extent in hamsters held in short-day length (SD) as compared to long-day length (LD). Intriguingly, effects of leptin on STAT3 appeared to be in part limited to nuclear translocation of phospho-STAT3 associated with the cell surface rather than phosphorylation of STAT3. The number of phospho-ERK cells within the hypothalamus was unaffected by either photoperiod or leptin. However, proximal to ERK phosphorylation, hypothalamic SH2-containing tyrosine phosphatase (SHP2) and the small growth factor receptor-binding protein (GRB2), which act as competitive negative modulators on binding of SOCS3 to leptin receptor (LRb)-associated Tyr985, were increased in SD compared to LD. Our findings suggest that activation of STAT3 by leptin may be dependent on interaction of stimulatory SHP2/GRB2 as well as inhibitory SOCS3 on the level of competitive binding to LRb-associated Tyr985. This hypothetical mechanism may represent the molecular identity of seasonally induced adjustments in leptin sensitivity and may be applied to investigating leptin sensitivity in other rodent models.
Assuntos
Proteína Adaptadora GRB2/metabolismo , Hipotálamo/metabolismo , Janus Quinase 2/metabolismo , Leptina/metabolismo , Phodopus/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Animais , Núcleo Celular , Cricetinae , Feminino , Regulação da Expressão Gênica , Hipotálamo/citologia , Masculino , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Fotoperíodo , Isoformas de Proteínas/metabolismo , Transporte Proteico , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , RNA Mensageiro/metabolismo , Receptores para Leptina/metabolismo , Fator de Transcrição STAT3/genética , Proteínas Supressoras da Sinalização de Citocina/metabolismoRESUMO
BACKGROUND: Diet plays a role on the development of the immune system, and polyunsaturated fatty acids can modulate the expression of a variety of genes. Human milk contains conjugated linoleic acid (CLA), a fatty acid that seems to contribute to immune development. Indeed, recent studies carried out in our group in suckling animals have shown that the immune function is enhanced after feeding them with an 80:20 isomer mix composed of c9,t11 and t10,c12 CLA. However, little work has been done on the effects of CLA on gene expression, and even less regarding immune system development in early life. RESULTS: The expression profile of mesenteric lymph nodes from animals supplemented with CLA during gestation and suckling through dam's milk (Group A) or by oral gavage (Group B), supplemented just during suckling (Group C) and control animals (Group D) was determined with the aid of the specific GeneChip(®) Rat Genome 230 2.0 (Affymettrix). Bioinformatics analyses were performed using the GeneSpring GX software package v10.0.2 and lead to the identification of 89 genes differentially expressed in all three dietary approaches. Generation of a biological association network evidenced several genes, such as connective tissue growth factor (Ctgf), tissue inhibitor of metalloproteinase 1 (Timp1), galanin (Gal), synaptotagmin 1 (Syt1), growth factor receptor bound protein 2 (Grb2), actin gamma 2 (Actg2) and smooth muscle alpha actin (Acta2), as highly interconnected nodes of the resulting network. Gene underexpression was confirmed by Real-Time RT-PCR. CONCLUSIONS: Ctgf, Timp1, Gal and Syt1, among others, are genes modulated by CLA supplementation that may have a role on mucosal immune responses in early life.
Assuntos
Perfilação da Expressão Gênica , Ácidos Linoleicos Conjugados/farmacologia , Linfonodos/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Animais Recém-Nascidos , Animais Lactentes , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Suplementos Nutricionais , Feminino , Proteína Adaptadora GRB2/genética , Proteína Adaptadora GRB2/metabolismo , Galanina/genética , Galanina/metabolismo , Redes Reguladoras de Genes , Linfonodos/crescimento & desenvolvimento , Linfonodos/imunologia , Mesentério , Gravidez , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Sinaptotagmina I/genética , Sinaptotagmina I/metabolismo , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismoRESUMO
Muc4 serves as an intramembrane ligand for the receptor tyrosine kinase ErbB2. The time to complex formation and the stoichiometry of the complex were determined to be <15 min and 1:1 by analyses of Muc4 and ErbB2 coexpressed in insect cells and A375 tumor cells. In polarized CACO-2 cells, Muc4 expression causes relocalization of ErbB2, but not its heterodimerization partner ErbB3, to the apical cell surface, effectively segregating the two receptors. The apically located ErbB2 is phosphorylated on tyrosines 1139 and 1248. The phosphorylated ErbB2 in CACO-2 cells recruits the cytoplasmic adaptor protein Grb2, consistent with previous studies showing phosphotyrosine 1139 to be a Grb2 binding site. To address the issue of downstream signaling from apical ErbB2, we analyzed the three MAPK pathways of mammalian cells, Erk, p38, and JNK. Consistent with the more differentiated phenotype of the CACO-2 cells, p38 phosphorylation was robustly increased by Muc4 expression, with a consequent activation of Akt. In contrast, Erk and JNK phosphorylation was not changed. The ability of Muc4 to segregate ErbB2 and other ErbB receptors and to alter downstream signaling cascades in polarized epithelial cells suggests that it has a role in regulating ErbB2 in differentiated epithelia.