RESUMO
The evolutionarily conserved extracellular signal-regulated kinase 2 (ERK2) is involved in regulating cellular signaling in both normal and pathological conditions. ERK2 expression is critical for human development, while hyperactivation is a major factor in tumor progression. Up to now, there have been no approved inhibitors that target ERK2, and as such, here we report on screening of a naturally occurring plant-based anticancerous compound-activity-target (NPACT) database for prospective ERK2 inhibitors. More than 1,500 phytochemicals were screened using in-silico molecular docking and molecular dynamics (MD) approaches. NPACT compounds with a docking score lower than a co-crystallized LHZ inhibitor (calc.-10.5 kcal/mol) were subjected to MD simulations. Binding energies (ΔGbinding) of inhibitor-ERK2 complexes over the MD course were estimated using an MM-GBSA approach. Based on MM-GBSA//100 ns MD simulations, the steroid zhankuic acid C (NPACT01034) demonstrated greater binding affinity against ERK2 protein than LHZ, with ΔGbinding values of -50.0 and -47.7 kcal/mol, respectively. Structural and energetical analyses throughout the MD course demonstrated stabilization of zhankuic acid C complexed with ERK2 protein. The anticipated ADMET properties of zhankuic acid C indicated minimal toxicity. Moreover, in-silico evaluation of fourteen ERK2 inhibitors in clinical trials demonstrated the higher binding affinity of zhankuic acid C towards ERK2 protein.
RESUMO
A large diversity of epigenetic factors, such as microRNAs and histones modifications, are known to be capable of regulating gene expression without altering DNA sequence itself. In particular, miR-1 is considered the first essential microRNA in cardiac development. In this study, miR-1 potential role in early cardiac chamber differentiation was analyzed through specific signaling pathways. For this, we performed in chick embryos functional experiments by means of miR-1 microinjections into the posterior cardiac precursors-of both primitive endocardial tubes-committed to sinoatrial region fates. Subsequently, embryos were subjected to whole mount in situ hybridization, immunohistochemistry and RT-qPCR analysis. As a relevant novelty, our results revealed that miR-1 increased Amhc1, Tbx5 and Gata4, while this microRNA diminished Mef2c and Cripto expressions during early differentiation of the cardiac sinoatrial region. Furthermore, we observed in this developmental context that miR-1 upregulated CrabpII and Rarß and downregulated CrabpI, which are three crucial factors in the retinoic acid signaling pathway. Interestingly, we also noticed that miR-1 directly interacted with Hdac4 and Calm1/Calmodulin, as well as with Erk2/Mapk1, which are three key factors actively involved in Mef2c regulation. Our study shows, for the first time, a key role of miR-1 as an epigenetic regulator in the early differentiation of the cardiac sinoatrial region through orchestrating opposite actions between retinoic acid and Mef2c, fundamental to properly assign cardiac cells to their respective heart chambers. A better understanding of those molecular mechanisms modulated by miR-1 will definitely help in fields applied to therapy and cardiac regeneration and repair.
Assuntos
Diferenciação Celular , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs , Animais , MicroRNAs/genética , MicroRNAs/metabolismo , Diferenciação Celular/genética , Embrião de Galinha , Fatores de Transcrição MEF2/metabolismo , Fatores de Transcrição MEF2/genética , Nó Sinoatrial/metabolismo , Nó Sinoatrial/citologia , Transdução de Sinais , Coração/embriologia , Coração/fisiologiaRESUMO
Micropeptides hidden in long non-coding RNAs (lncRNAs) have been uncovered to program various cell-biological changes associated with malignant transformation-glioblastoma (GBM) cascade. Here, we identified and characterized a novel hidden micropeptide implicated in GBM. We screened potential candidate lncRNAs by establishing a workflow involving ribosome-bound lncRNAs, publicly available MS/MS data, and prognosis-related lncRNAs. Micropeptide expression was detected by western blot (WB), immunofluorescence (IF), and immunohistochemistry (IHC). Cell proliferation rate was assessed by calcein/PI staining and EdU assay. Proteins interacted with the micropeptide were analyzed by proteomics after co-immunoprecipitation (Co-IP). We discovered that lncRNA AF127577.4 indeed encoded an endogenous micropeptide, named AF127577.4-ORF. AF127577.4-ORF was associated with GBM clinical grade. In vitro, AF127577.4-ORF could suppress GBM cell proliferation. Moreover, AF127577.4-ORF reduced m6A methylation level of GBM cells. Mechanistically, AF127577.4-ORF diminished ERK2 interaction with m6A reader methyltransferase like 3 (METTL3) and downregulated phosphorylated ERK (p-ERK) level. The ERK inhibitor reduced p-ERK level and downregulated METTL3 protein expression. AF127577.4-ORF weakened the stability of METTL3 protein by ERK. Also, AF127577.4-ORF suppressed GBM cell proliferation via METTL3. Our study identifies a novel micropeptide AF127577.4-ORF hidden in a lncRNA, with a potent anti-proliferating function in GBM by diminishing METTL3 protein stability by reducing the ERK2/METTL3 interaction. This micropeptide may be beneficial for development of therapeutic strategies against GBM.
Assuntos
Proliferação de Células , Glioblastoma , Metiltransferases , Proteína Quinase 1 Ativada por Mitógeno , RNA Longo não Codificante , Humanos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Linhagem Celular Tumoral , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/genética , Metiltransferases/metabolismo , Metiltransferases/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Peptídeos/metabolismoRESUMO
Parkinson's disease (PD) is a complex neurological disease associated with the degeneration of dopaminergic neurons. Oxidative stress is a key player in instigating apoptosis in dopaminergic neurons. To improve the survival of neurons many dietary phytochemicals have gathered significant attention recently. Thus, the present study implements a comprehensive network pharmacology approach to unravel the mechanisms of action of dietary phytochemicals that benefit disease management. A literature search was performed to identify ligands (i.e., comprising dietary phytochemicals and Food and Drug Administration pre-approved PD drugs) in the PubMed database. Targets associated with selected ligands were extracted from the search tool for interactions of chemicals (STITCH) database. Then, the construction of a gene-gene interaction (GGI) network, analysis of hub-gene, functional and pathway enrichment, associated transcription factors, miRNAs, ligand-target interaction network, docking were performed using various bioinformatics tools together with molecular dynamics (MD) simulations. The database search resulted in 69 ligands and 144 unique targets. GGI and subsequent topological measures indicate histone acetyltransferase p300 (EP300), mitogen-activated protein kinase 1 (MAPK1) or extracellular signal-regulated kinase (ERK)2, and CREB-binding protein (CREBBP) as hub genes. Neurodegeneration, MAPK signaling, apoptosis, and zinc binding are key pathways and gene ontology terms. hsa-miR-5692a and SCNA gene-associated transcription factors interact with all the 3 hub genes. Ligand-target interaction (LTI) network analysis suggest rasagiline and baicalein as candidate ligands targeting MAPK1. Rasagiline and baicalein form stable complexes with the Y205, K330, and V173 residues of MAPK1. Computational molecular insights suggest that baicalein and rasagiline are promising preclinical candidates for PD management.
Assuntos
Indanos , Farmacologia em Rede , Doença de Parkinson , Humanos , Ligantes , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/genética , Compostos Fitoquímicos/farmacologia , Simulação de Acoplamento MolecularRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Acute lung injury (ALI) ranks among the deadliest pulmonary diseases, significantly impacting mortality and morbidity. Presently, the primary treatment for ALI involves supportive therapy; however, its efficacy remains unsatisfactory. Strictosamide (STR), an indole alkaloid found in the Chinese herbal medicine Nauclea officinalis (Pierre ex Pit.) Merr. & Chun (Wutan), has been found to exhibit numerous pharmacological properties, particularly anti-inflammatory effects. AIM OF THE STUDY: This study aimes to systematically identify and validate the specific binding proteins targeted by STR and elucidate its anti-inflammatory mechanism in lipopolysaccharide (LPS)-induced ALI. MATERIALS AND METHODS: Biotin chemical modification, protein microarray analysis and network pharmacology were conducted to screen for potential STR-binding proteins. The binding affinity was assessed through surface plasmon resonance (SPR), cellular thermal shift assay (CETSA) and molecular docking, and the anti-inflammatory mechanism of STR in ALI treatment was assessed through in vivo and in vitro experiments. RESULTS: Biotin chemical modification, protein microarray and network pharmacology identified extracellular-signal-regulated kinase 2 (ERK2) as the most important binding proteins among 276 candidate STR-interacting proteins and nuclear factor-kappaB (NF-κB) pathway was one of the main inflammatory signal transduction pathways. Using SPR, CETSA, and molecular docking, we confirmed STR's affinity for ERK2. In vitro and in vivo experiments demonstrated that STR mitigated inflammation by targeting ERK2 to modulate the NF-κB signaling pathway in LPS-induced ALI. CONCLUSIONS: Our findings indicate that STR can inhibit the NF-κB signaling pathway to attenuate LPS-induced inflammation by targeting ERK2 and decreasing phosphorylation of ERK2, which could be a novel strategy for treating ALI.
Assuntos
Lesão Pulmonar Aguda , NF-kappa B , Alcaloides de Vinca , Humanos , NF-kappa B/metabolismo , Lipopolissacarídeos/toxicidade , Biotina/metabolismo , Biotina/farmacologia , Biotina/uso terapêutico , Simulação de Acoplamento Molecular , Transdução de Sinais , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/metabolismo , Anti-Inflamatórios/efeitos adversos , Inflamação/tratamento farmacológico , Pulmão/metabolismoRESUMO
Severe acute pancreatitis (SAP) is an inflammatory disease of the pancreas with a high mortality rate. Macrophages play a crucial role in the pathogenesis of pancreatitis. Tectoridin (Tec) is a highly active isoflavone with anti-inflammatory pharmacological activity. However, the role of Tec in the SAP process is not known. The purpose of this study was to investigate the therapeutic effect and potential mechanism of Tec on SAP. To establish SAP mice by intraperitoneal injection of caerulein and Lipopolysaccharide (LPS), the role of Tec in the course of SAP was investigated based on histopathology, biochemical indicators of amylase and lipase and inflammatory factors. The relationship between Tec and macrophage polarization was verified by immunofluorescence, real-time quantitative PCR and Western blot analysis. We then further predicted the possible targets and signal pathways of action of Tec by network pharmacology and molecular docking, and validated them by in vivo and in vitro. In this study, we demonstrated that Tec significantly reduced pancreatic injury in SAP mice, and decreased serum levels of amylase and lipase. The immunofluorescence and Western blot analysis showed that Tec promoted macrophage M2 polarization. Network pharmacology and molecular docking predicted that Tec may target ERK2 for the treatment of SAP, and in vivo and in vitro experiments proved that Tec inhibited the ERK MAPK signal pathway. In summary, Tec can target ERK2, promote macrophage M2 polarization and attenuate pancreatic injury, Tec may be a potential drug for the treatment of SAP.
Assuntos
Isoflavonas , Pancreatite , Camundongos , Animais , Pancreatite/induzido quimicamente , Pancreatite/tratamento farmacológico , Pancreatite/metabolismo , Ceruletídeo/efeitos adversos , Doença Aguda , Simulação de Acoplamento Molecular , Isoflavonas/farmacologia , Isoflavonas/uso terapêutico , Macrófagos/metabolismo , Amilases , LipaseRESUMO
[This corrects the article DOI: 10.3389/fonc.2023.1144951.].
RESUMO
AIM: In the present study, we sought to explore potential differences in the expression and promoter methylation of mitogen-activated protein kinase 1 (MAPK1) between tumor and marginal cells of breast cancer lesions. METHODS: A total of 50 randomly selected patients with breast cancer (BCa) undergoing needle biopsy were enrolled. Clinical specimens containing both tumor and marginal cells were collected and preserved. After DNA extraction using specific primers, MAPK1 mRNA and promoter methylation were measured with spectrophotometry at 260/280 nm absorption wavelengths. To deliver a comparative analysis, data from The Cancer Genome Atlas (TCGA) program regarding breast cancer (BRCA), were downloaded from Xena Functional Genomics Explorer and separately analyzed. The suitability of MAPK1 expression and promoter methylation as biomarkers for BCa was analyzed with receiver operating characteristic (ROC) curves. RESULTS: We found a positive correlation between tumor stage and MAPK1 expression (P-value: 0.029) in BCa. Likewise, MAPK1 expression was significantly associated with lymph node metastasis (P-value: 0.018). There was a significant difference in the expression of MAPK1 mRNA between tumor and marginal cells of BCa and BRCA (P-value < 0.001). However, we did not find any statistically significant difference in MAPK1 promoter methylation between tumor and marginal cells of both BCa and BRCA. With an area under the curve (AUC) of 0.71, the diagnostic accuracy of MAPK1 expression in BCa and BRCA was validated. However, MAPK1 promoter methylation was not found to be a suitable biomarker. CONCLUSION: Our findings suggest that while MAPK1 expression, might be a promising biomarker for evaluating oncogenic activity in patients suspected of BCa. We were not able to detect a prognostic/diagnostic role for MAPK1 promoter methylation.
Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/patologia , Metilação de DNA , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Biomarcadores , RNA Mensageiro/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismoRESUMO
Sphingosylphosphorylcholine (SPC) is one of the bioactive phospholipids that has many cellular functions such as cell migration, adhesion, proliferation, angiogenesis, and Ca²⁺ signaling. Recent studies have reported that SPC induces invasion of breast cancer cells via matrix metalloproteinase-3 (MMP-3) secretion leading to WNT activation. Thrombospondin-1 (TSP-1) is a matricellular and calcium-binding protein that binds to a wide variety of integrin and non-integrin cell surface receptors. It regulates cell proliferation, migration, and apoptosis in inflammation, angiogenesis and neoplasia. TSP-1 promotes aggressive phenotype via epithelial mesenchymal transition (EMT). The relationship between SPC and TSP-1 is unclear. We found SPC induced EMT leading to mesenchymal morphology, decrease of E-cadherin expression and increases of N-cadherin and vimentin. SPC induced secretion of thrombospondin-1 (TSP-1) during SPC-induced EMT of various breast cancer cells. Gene silencing of TSP-1 suppressed SPC-induced EMT as well as migration and invasion of MCF10A cells. An extracellular signal-regulated kinase inhibitor, PD98059, significantly suppressed the secretion of TSP-1, expressions of N-cadherin and vimentin, and decrease of E-cadherin in MCF10A cells. ERK2 siRNA suppressed TSP-1 secretion and EMT. From online PROGgene V2, relapse free survival is low in patients having high TSP-1 expressed breast cancer. Taken together, we found that SPC induced EMT and TSP-1 secretion via ERK2 signaling pathway. These results suggests that SPC-induced TSP-1 might be a new target for suppression of metastasis of breast cancer cells.
Assuntos
Humanos , Apoptose , Neoplasias da Mama , Caderinas , Movimento Celular , Proliferação de Células , Transição Epitelial-Mesenquimal , Inativação Gênica , Inflamação , Metástase Neoplásica , Fenótipo , Fosfolipídeos , Fosfotransferases , Receptores de Superfície Celular , Recidiva , RNA Interferente Pequeno , Trombospondina 1 , VimentinaRESUMO
Sphingosylphosphorylcholine (SPC) is one of the bioactive phospholipids that has many cellular functions such as cell migration, adhesion, proliferation, angiogenesis, and Ca²⁺ signaling. Recent studies have reported that SPC induces invasion of breast cancer cells via matrix metalloproteinase-3 (MMP-3) secretion leading to WNT activation. Thrombospondin-1 (TSP-1) is a matricellular and calcium-binding protein that binds to a wide variety of integrin and non-integrin cell surface receptors. It regulates cell proliferation, migration, and apoptosis in inflammation, angiogenesis and neoplasia. TSP-1 promotes aggressive phenotype via epithelial mesenchymal transition (EMT). The relationship between SPC and TSP-1 is unclear. We found SPC induced EMT leading to mesenchymal morphology, decrease of E-cadherin expression and increases of N-cadherin and vimentin. SPC induced secretion of thrombospondin-1 (TSP-1) during SPC-induced EMT of various breast cancer cells. Gene silencing of TSP-1 suppressed SPC-induced EMT as well as migration and invasion of MCF10A cells. An extracellular signal-regulated kinase inhibitor, PD98059, significantly suppressed the secretion of TSP-1, expressions of N-cadherin and vimentin, and decrease of E-cadherin in MCF10A cells. ERK2 siRNA suppressed TSP-1 secretion and EMT. From online PROGgene V2, relapse free survival is low in patients having high TSP-1 expressed breast cancer. Taken together, we found that SPC induced EMT and TSP-1 secretion via ERK2 signaling pathway. These results suggests that SPC-induced TSP-1 might be a new target for suppression of metastasis of breast cancer cells.
Assuntos
Humanos , Apoptose , Neoplasias da Mama , Caderinas , Movimento Celular , Proliferação de Células , Transição Epitelial-Mesenquimal , Inativação Gênica , Inflamação , Metástase Neoplásica , Fenótipo , Fosfolipídeos , Fosfotransferases , Receptores de Superfície Celular , Recidiva , RNA Interferente Pequeno , Trombospondina 1 , VimentinaRESUMO
PURPOSE: BRAF mutation and expression of extracellular signal regulated kinase (ERK) are linked with colorectal carcinogenesis through the serrated pathway. BRAF and ERK1/2 play important roles in the activation of mitogen-activated protein (MAP) kinase signaling pathways. The present study investigated the clinicopathologic outcomes of BRAF mutation and ERK1/2 expression in patients with colorectal cancer (CRC) and the possibility of using them as prognostic indicators. METHODS: Dual-priming oligonucleotide-based multiplex polymerase chain reaction for BRAF(V600E) mutation and immunohistochemical analysis of ERK1/2 were performed using 65 formalin-fixed, paraffin-embedded samples from patients with CRC. We analyzed the dependences of the clinicopathologic features on BRAF mutation and ERK1/2 expression. RESULTS: Out of 65 samples from CRC patients, BRAF mutation was detected in 3 (4.6%). The 3 patients with BRAF mutation presented with T3 CRC with lymph node metastasis (stage III) showing moderately or poorly differentiated histology. ERK1 and ERK2 were positively detected in 73.8% and 15.4% of the patients with CRC, respectively. ERK1 expression was significantly correlated with lymph node metastasis (P = 0.049). ERK2 expression was significantly correlated with tumor emboli (P < 0.05), tumor invasion (P = 0.035), lymph node metastasis (P = 0.017), and stage (P = 0.02). CONCLUSION: BRAF mutation and ERK1/2 expression may be associated with advanced or more aggressive CRC. These molecular markers might play prognostic roles in CRC developed through the serrated pathway.
Assuntos
Humanos , Adenocarcinoma , Carcinogênese , Neoplasias Colorretais , Linfonodos , Reação em Cadeia da Polimerase Multiplex , Metástase Neoplásica , FosfotransferasesRESUMO
Objective By investigating the effects of AT1 and ERK2 signaling pathway in CHF,to study the effect of Guanxin-Shutong capsule on AT1,ERK2 expression in rats with chronic heart failure (CHF).Methods The CHF rat models were setup by coronary artery ligation,exhausting swimming and reducing feeding.Divided CHF rat methods into a model group,a lisinopril group,Qi-benefiting with Chinese medicine group,blood activating with Chinese medicine group,Qi benefiting and blood activating with Chinese medicine group(QBBA group) and Guanxin-Shutong capsule group.Rats without left coronary artery ligation were set for sham operated group.Real-time quantitative PCR technique and immunohistochemical method were adopted to detect AT1,ERK2 changes of CHF rats.Results AT1,ERK2 expression increased obviously in the model group compared with the sham operated group,and differences were statistically significant (P<0.01).After treatment,AT1,ERK2 expression reduced significantly in QBBA group and Guanxin-Shutong capsule group than the lisinopril group.(P>0.05).And therapeutic effect of Guanxin-Shutong capsule group was much better than other Chinese medicine treated group (P<0.01).Conclusion Guanxin-Shutong capsule would achieve the goal of treating chronic heart failure By inhibiting the expression of AT1 and ERK2 in organizations,and inhibiting or reversing ventricular remodeling process.
RESUMO
Objective To study the effect of anti-epileptic,nootropic drugs on the expression of NCAM and ERK2 in the hippocampus changes on the epileptic rats with cognitive dysfunction.Methods A total of 120Wistar rats were used.20 controls and 100 in which epilepticus with cognitive dysfunction were randomly assigned to 5 groups (n =20/group) that received daily treatments for 30 days with either (1) saline (epilepsy),(2) carbamazine (traditional anti-epileptic),(3) oxcarbazine (new anti-epileptic),(4) aniracetam (brain protective),or (5) donepezil (nootopic).Spatial learning and memory were assessed with a Morris Water Maze (MWM).Hippocampus tissue was assessed for NCAM1 and ERK-2 mRNAs by RT-PCR and proteins by immunochemistry.Results The mean escape latency of the place navigation test:EP group ((67.14 ± 7.37)s)was all higher than NS group (35.78 ± 4.84 s)and there was statistical significance (P < 0.01),carbamazepine group ((81.23 ± 9.46)s) > EP group((67.14 ±7.37)s) > donepezi group((53.75 ±6.74) s) (P<0.01).Immunohistochemical and RT-PCR result:carbamazepine < oxcarbazepine < epilepsy < aniracetam < donepezi group.Compared with control group,donepezil group > control group (P < 0.01),aniracetam group > control group (P < 0.05).Conclusion ERK-2 expression is decreased and NCAM 1 expression is increased in the hippocampus in the epileptic rats.Thus,both are involved in cognitive dysfunction.Carbamazepine aggravates cognitive dysfunction,whereas donepezil improves cognitive dysfunction associated with epilepsy.