RESUMO
BACKGROUND: Rhododendron pudingense, firstly discovered in Puding county of Guizhou province in 2020, have adapted to living in rocky fissure habitat, which has important ornamental and economic values. However, the genetic diversity and population structure of this species have been rarely described, which seriously affects the collection and protection of wild germplasm resources. RESULTS: In the present study, 13 pairs of primers for polymorphic microsatellite were used to investigate the genetic diversity of 65 R. pudingense accessions from six different geographic populations. A total of 254 alleles (Na) were obtained with an average of 19.5 alleles per locus. The average values of polymorphic information content (PIC), observed heterozygosity (Ho), and expected heterozygosity (He) were 0.8826, 0.4501, and 0.8993, respectively, These results indicate that the microsatellite primers adopted demonstrate good polymorphism, and the R. pudingense exhibits a high level of genetic diversity at the species level. The average genetic differentiation coefficient (Fst) was 0.1325, suggested that moderate divergence occurred in R. pudingense populations. The average values of genetic differentiation coefficient and gene flow among populations were 0.1165 and 3.1281, respectively. The analysis of molecular variance (AMOVA) indicated that most of the population differences (88%) were attributed to within-population variation. The PCoA results are consistent with the findings of the UPGMA clustering analysis, supporting the conclusion that the six populations of R. pudingense can be clearly grouped into two separate clusters. Based on Mantel analysis, we speculate that the PD population may have migrated from WM-1 and WM-2. Therefore, it is advised to protect the natural habitat of R. pudingense in situ as much as possible, in order to maximize the preservation of its genetic diversity. CONCLUSIONS: This is the first comprehensive analysis of genetic diversity and population structure of R. pudingense in Guizhou province. The research results revealed the high genetic diversity and moderate population diferentiation in this horticulture plant. This study provide a theoretical basis for the conservation of wild resources of the R. pudingense and lay the foundation for the breeding or cultivation of this new species.
Assuntos
Variação Genética , Rhododendron , Rhododendron/genética , Melhoramento Vegetal , Polimorfismo Genético , Repetições de Microssatélites/genéticaRESUMO
BACKGROUND: Peptide transporter 1 (PepT1) transports bacterial oligopeptide products and induces inflammation of the bowel. Nutritional peptides compete for the binding of intestinal bacterial products to PepT1. We investigated the mechanism of short-peptide-based enteral nutrition (SPEN) on the damage to the gut caused by the bacterial oligopeptide product muramyl dipeptide (MDP), which is transported by PepT1. The gut-lung axis is a shared mucosal immune system, and immune responses and disorders can affect the gut-respiratory relationship. METHODS AND RESULTS: Sprague-Dawley rats were gavaged with solutions containing MDP, MDP + SPEN, MDP + intact-protein-based enteral nutrition (IPEN), glucose as a control, or glucose with GSK669 (a NOD2 antagonist). Inflammation, mitochondrial damage, autophagy, and apoptosis were explored to determine the role of the PepT1-nucleotide-binding oligomerization domain-containing protein 2 (NOD2)-beclin-1 signaling pathway in the small intestinal mucosa. MDP and proinflammatory factors of lung tissue were explored to determine that MDP can migrate to lung tissue and cause inflammation. Induction of proinflammatory cell accumulation and intestinal damage in MDP gavage rats was associated with increased NOD2 and Beclin-1 mRNA expression. IL-6 and TNF-α expression and apoptosis were increased, and mitochondrial damage was severe, as indicated by increased mtDNA in the MDP group compared with controls. MDP levels and expression of proinflammatory factors in lung tissue increased in the MDP group compared with the control group. SPEN, but not IPEN, eliminated these impacts. CONCLUSIONS: Gavage of MDP to rats resulted in damage to the gut-lung axis. SPEN reverses the adverse effects of MDP. The PepT1-NOD2-beclin-1 pathway plays a role in small intestinal inflammation, mitochondrial damage, autophagy, and apoptosis.
Assuntos
Acetilmuramil-Alanil-Isoglutamina , Proteína Beclina-1 , Nutrição Enteral , Lesão Pulmonar , Proteína Adaptadora de Sinalização NOD2 , Transportador 1 de Peptídeos , Ratos Sprague-Dawley , Transdução de Sinais , Animais , Transportador 1 de Peptídeos/metabolismo , Transportador 1 de Peptídeos/genética , Ratos , Proteína Beclina-1/metabolismo , Proteína Beclina-1/genética , Proteína Adaptadora de Sinalização NOD2/metabolismo , Proteína Adaptadora de Sinalização NOD2/genética , Transdução de Sinais/efeitos dos fármacos , Lesão Pulmonar/metabolismo , Masculino , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Nutrição Enteral/métodos , Apoptose/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Autofagia/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Pulmão/efeitos dos fármacos , Inflamação/metabolismoRESUMO
BACKGROUND: Cancer stem cells (CSCs) play a vital role in the occurrence, maintenance, and recurrence of solid tumors. Although, miR-145-5p can inhibit CSCs survival, poor understanding of the underlying mechanisms hamperes further therapeutic optimization for patients. Lentivirus with remarkable transduction efficiency is the most commonly used RNA carrier in research, but has shown limited tumor-targeting capability. METHODS: We have applied liposome to decorate lentivirus surface thereby yielding liposome-lentivirus hybrid-based carriers, termed miR-145-5p-lentivirus nanoliposome (MRL145), and systematically analyzed their potential therapeutic effects on liver CSCs (LCSCs). RESULTS: MRL145 exhibited high delivery efficiency and potent anti-tumor efficacy under in vitro and in vivo. Mechanistically, the overexpressed miR-145-5p can significantly suppress the self-renewal, migration, and invasion abilities of LCSCs by targeting Collagen Type IV Alpha 3 Chain (COL4A3). Importantly, COL4A3 can promote phosphorylating GSK-3ß at ser 9 (p-GSK-3ß S9) to inactivate GSK3ß, and facilitate translocation of ß-catenin into the nucleus to activate the Wnt/ß-catenin pathway, thereby promoting self-renewal, migration, and invasion of LCSCs. Interestingly, COL4A3 could attenuate the cellular autophagy through modulating GSK3ß/Gli3/VMP1 axis to promote self-renewal, migration, and invasion of LCSCs. CONCLUSIONS: These findings provide new insights in mode of action of miR-145-5p in LCSCs therapy and indicates that liposome-virus hybrid carriers hold great promise in miRNA delivery.
Assuntos
Lentivirus , Lipossomos , MicroRNAs , Células-Tronco Neoplásicas , MicroRNAs/genética , MicroRNAs/metabolismo , Lipossomos/química , Humanos , Animais , Camundongos , Lentivirus/genética , Linhagem Celular Tumoral , Células-Tronco Neoplásicas/metabolismo , Camundongos Nus , Neoplasias Hepáticas/terapia , Camundongos Endogâmicos BALB C , Movimento Celular , Glicogênio Sintase Quinase 3 beta/metabolismo , beta Catenina/metabolismo , Via de Sinalização WntRESUMO
Objective: This study aimed to validate FANCI as a potential marker for both prognosis and therapy in liver hepatocellular carcinoma. Method: FANCI expression data were acquired from GEPIA, HPA, TCGA, and GEO databases. The impact of clinicopathological features was analyzed by UALCAN. The prognosis of Liver Hepatocellular Carcinoma (LIHC) patients with highly expressed FANCI was constructed utilizing Kaplan-Meier Plotter. GEO2R was employed to identify differentially expressed genes (DEGs). Metascape was used to analyze functional pathways correlations. Protein-Protein interaction (PPI) networks were generated by Cytoscape. Furthermore, molecular complex detection (MCODE) was utilized to recognize Hub genes, which were selected to establish a prognostic model. Lastly, the relationship between FANCI and immune cell infiltration in LIHC was examined. Results: Compared to adjacent tissues, FANCI expression levels were significantly higher in LIHC tissues and were positively correlated to the cancer grade, stage, and prior hepatitis B virus (HBV) infection. High expression of FANCI was found to be associated with poor prognosis in LIHC (HR=1.89, p<0.001). DEGs that were positively correlated with FANCI were involved in various processes, including the cell cycle, VEGF pathway, immune system processes, and biogenesis of ribonucleoproteins. MCM10, TPX2, PRC1, and KIF11 were identified as key genes closely related to FANCI and poor prognosis. A reliable five-variable prognostic model was constructed with strong predictive capability. Lastly, a positive correlation was observed between FANCI expression and tumor-infiltration levels of CD8+ T cells, B cells, regulatory T (Tregs), CD4+ T helper 2 (Th2), and macrophage M2 cells. Conclusion: FANCI may hold promise as a potential biomarker for predicting prognostic outcomes, and a valuable therapeutic target for LIHC patients, with a focus on anti-proliferation, anti-chemoresistance, and combination with immunotherapy.
Assuntos
Carcinoma Hepatocelular , Anemia de Fanconi , Hepatite B , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Prognóstico , Proteínas de Grupos de Complementação da Anemia de FanconiRESUMO
Non-small cell lung cancer (NSCLC) is a common clinical malignant tumor with limited therapeutic drugs. Leading by cytotoxicity against NSCLC cell lines (A549 and PC9), bioactivity-guided isolation of components from Peganum harmala seeds led to the isolation of pegaharoline A (PA). PA was elucidated as a structurally novel aniline derivative, originating from tryptamine with a pyrrole ring cleaved and the degradation of carbon. Biological studies showed that PA significantly inhibited NSCLC cell proliferation, suppressed DNA synthesis, arrested the cell cycle, suppressed colony formation and HUVEC angiogenesis, and blocked cell invasion and migration. Molecular docking and surface plasmon resonance (SPR) demonstrated PA could bind with CD133, correspondingly decreased CD133 expression to activate autophagy via inhibiting the PI3K/AKT/mTOR pathway, and increased ROS levels, Bax, and cleaved caspase-3 to promote apoptosis. PA could also decrease p-cyclinD1 and p-Erk1/2 and block the EMT pathway to inhibit NSCLC cell growth, invasion, and migration. According to these results, PA could inhibit NSCLC cell growth by blocking PI3K/AKT/mTOR and EMT pathways. This study provides evidence that PA has a promising future as a candidate for developing drugs for treating NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Peganum , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Simulação de Acoplamento Molecular , Neoplasias Pulmonares/tratamento farmacológico , Apoptose , Autofagia , Compostos de Anilina/farmacologiaRESUMO
Objective To screen antigen targets for immunotherapy by analyzing over-expressed genes, and to identify significant pathways and molecular mechanisms in esophageal cancer by using bioinformatic methods such as enrichment analysis, protein-protein interaction (PPI) network, and survival analysis based on the Gene Expression Omnibus (GEO) database.Methods By screening with highly expressed genes, we mainly analyzed proteins MUC13 and EPCAM with transmembrane domain and antigen epitope from TMHMM and IEDB websites. Significant genes and pathways associated with the pathogenesis of esophageal cancer were identified using enrichment analysis, PPI network, and survival analysis. Several software and platforms including Prism 8, R language, Cytoscape, DAVID, STRING, and GEPIA platform were used in the search and/or figure creation.Results Genes MUC13 and EPCAM were over-expressed with several antigen epitopes in esophageal squamous cell carcinoma (ESCC) tissue. Enrichment analysis revealed that the process of keratinization was focused and a series of genes were related with the development of esophageal cancer. Four genes including ALDH3A1, C2, SLC6A1,and ZBTB7C were screened with significant P value of survival curve.Conclusions Genes MUC13 and EPCAM may be promising antigen targets or biomarkers for esophageal cancer. Keratinization may greatly impact the pathogenesis of esophageal cancer. Genes ALDH3A1, C2, SLC6A1,and ZBTB7C may play important roles in the development of esophageal cancer.
Assuntos
Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Humanos , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/metabolismo , Molécula de Adesão da Célula Epitelial/genética , Molécula de Adesão da Célula Epitelial/metabolismo , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização IntracelularRESUMO
BACKGROUND: The early clinical diagnosis of spinal infections in elderly patients with recessive or atypical symptoms is difficult. Klebsiella aerogenes is a common opportunistic bacterium that can infect the respiratory tract, urinary tract, and even the central nervous system. However, whether it can infect the lumbar spine has not been previously described. CASE PRESENTATION: In this paper, we report the case of a 69-year-old female patient with osteoporosis who was initially diagnosed with hemolytic anemia. Later, she was diagnosed with K. aerogenes infection of the lumbar spine based on imaging combined with blood culture and metagenome next-generation sequencing (mNGS) detection. After precise medication, the lumbar degeneration was improved. CONCLUSIONS: Bacterial infection should therefore be considered in cases of lumbar degenerative disease in middle-aged and elderly patients.
Assuntos
Enterobacter aerogenes , Infecções por Klebsiella , Idoso , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Infecções por Klebsiella/diagnóstico , Vértebras Lombares , Metagenoma , Pessoa de Meia-IdadeRESUMO
Hepatocellular carcinoma (HCC) is a major subtype of primary liver cancer with a high mortality rate. Pyroptosis and autophagy are crucial processes in the pathophysiology of HCC. Searching for efficient drugs targeting pyroptosis and autophagy with lower toxicity is useful for HCC treatment. Mallotucin D (MLD), a clerodane diterpenoid from Croton crassifolius, has not been previously reported for its anticancer effects in HCC. This study aims to evaluate the inhibitory effects of MLD in HCC and explore the underlying mechanism. We found that the cell proliferation, DNA synthesis, and colony formation of HepG2 cells and the angiogenesis of HUVECs were all greatly inhibited by MLD. MLD caused mitochondrial damage and decreased the TOM20 expression and mitochondrial membrane potential, inducing ROS overproduction. Moreover, MLD promoted the cytochrome C from mitochondria into cytoplasm, leading to cleavage of caspase-9 and caspase-3 inducing GSDMD-related pyroptosis. In addition, we revealed that MLD activated mitophagy by inhibiting the PI3K/AKT/mTOR pathway. Using the ROS-scavenging reagent NAC, the activation effects of MLD on pyroptosis- and autophagy-related pathways were all inhibited. In the HepG2 xenograft model, MLD effectively inhibited tumor growth without detectable toxicities in normal tissue. In conclusion, MLD could be developed as a candidate drug for HCC treatment by inducing mitophagy and pyroptosis via promoting mitochondrial-related ROS production.
Assuntos
Morte Celular Autofágica , Carcinoma Hepatocelular , Croton , Diterpenos Clerodânicos , Neoplasias Hepáticas , Humanos , Morte Celular Autofágica/efeitos dos fármacos , Carcinoma Hepatocelular/metabolismo , Proliferação de Células/efeitos dos fármacos , Croton/química , Diterpenos Clerodânicos/farmacologia , Células Hep G2/efeitos dos fármacos , Células Hep G2/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Piroptose/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Acquired drug resistance decreases the efficacy of gefitinib after approximately 1 year of treatment in non-small-cell lung cancer (NSCLC). Autophagy is a process that could lead to cell death when it is prolonged. Thus, we investigated a drug combination therapy of gefitinib with rapamycin-a cell autophagy activator-in gefitinib-resistant NSCLC cell line H1975 to improve the therapeutic efficacy of gefitinib in advanced NSCLC cells through acute cell autophagy induction. Cell viability and tumor formation assays indicated that rapamycin is strongly synergistic with gefitinib inhibition, both in vitro and in vivo. Mechanistic studies demonstrated that EGFR expression and cell autophagy decreased under gefitinib treatment and were restored after the drug combination therapy, indicating a potential cell autophagy-EGFR positive feedback regulation. To further optimize the delivery efficiency of the combinational agents, we constructed an anti-EGFR aptamer-functionalized nanoparticle (NP-Apt) carrier system. The microscopic observation and cell proliferation assays suggested that NP-Apt achieved remarkably targeted delivery and cytotoxicity in the cancer cells. Taken together, our results suggest that combining rapamycin and gefitinib can be an efficacious therapy to overcome gefitinib resistance in NSCLC, and targeted delivery of the drugs using the aptamer-nanoparticle carrier system further enhances the therapeutic efficacy of gefitinib.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Nanopartículas , Autofagia , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Combinação de Medicamentos , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/metabolismo , Gefitinibe/farmacologia , Gefitinibe/uso terapêutico , Humanos , Neoplasias Pulmonares/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/uso terapêutico , Sirolimo/farmacologia , Sirolimo/uso terapêuticoRESUMO
Choriocarcinoma is characterized by malignant proliferation and transformation of trophoblasts and is currently treated with systemic chemotherapeutic agents. The lack of specific targets for chemotherapeutic agents results in indiscriminate drug distribution. In our study, we aimed to delineate the mechanism by which G protein-coupled receptor 1 (GPR1) regulates the development of choriocarcinoma and thus investigated GPR1 as a prospective chemotherapeutic target. In this study, GPR1 expression levels were examined in several trophoblast cell lines. We found significantly higher GPR1 expression in choriocarcinoma cells (JEG3 and BeWo) than in normal trophoblast cells (HTR-8/SVneo). Additionally, we studied the role of GPR1 in choriocarcinoma in vitro and in vivo. GPR1 knockdown suppressed proliferation, invasion, and Akt and ERK phosphorylation in vitro and slowed tumor growth in vivo. Interestingly, GPR1 overexpression promoted increased proliferation, invasion, and Akt and ERK phosphorylation in vitro. Furthermore, we identified a specific GPR1-binding seven-amino acid peptide, LRH7-G3, that might also suppress choriocarcinoma in vitro and in vivo through phage display. Our study is the first to report that GPR1 may play a role in regulating choriocarcinoma progression through the Akt and ERK pathways. GPR1 could be a promising potential pharmaceutical target for choriocarcinoma.
Assuntos
Coriocarcinoma/metabolismo , Coriocarcinoma/patologia , Receptores Acoplados a Proteínas G/fisiologia , Neoplasias Uterinas/metabolismo , Neoplasias Uterinas/patologia , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Invasividade Neoplásica/patologia , GravidezRESUMO
The Nogo-66 receptor (NgR1), a receptor for Nogo-A, contributes to the inhibition of axonal regeneration in the adult central nervous system after traumatic injuries. Thus, NgR1 has been considered a critical target in axon regeneration therapy. Here, we identified a specific NgR1 antagonist peptide (HIYTALV, named NAP2) which promotes neurite regeneration in vitro from a phage display heptapeptide library. NAP2 was co-localized with NgR1 on the surface of PC12 cells and cerebellar granule cells (CGCs) by immunofluorescence assay. Horseradish peroxidase (HRP)-streptavidin-biotin assay further showed that NAP2 binds to NgR1 and the dissociation constant (Kd) was 0.45 µM Functional analyses indicated that NAP2 could reduce the inhibitory effects of Nogo-66 on neurite outgrowth in differentiated PC12 cells and CGCs by blocking the Nogo-66-induced activation of Rho-associated coiled coil-containing protein kinase (ROCK), collapsin response mediator protein 2 (CRMP2) and myosin light chain (MLC). Taken together, the small molecule NgR1 antagonist peptide NAP2 (MW: 815.98Da) has a potential ability in crossing blood brain barrier and will be a promising therapeutic agent for the treatment of spinal cord injury and neurodegenerative diseases.
Assuntos
Proteínas da Mielina/antagonistas & inibidores , Regeneração Nervosa , Neuritos/efeitos dos fármacos , Oligopeptídeos/farmacologia , Animais , Células Cultivadas , Cerebelo/citologia , Cerebelo/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Ligantes , Proteínas da Mielina/metabolismo , Cadeias Leves de Miosina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuritos/metabolismo , Neuritos/fisiologia , Proteínas Nogo , Células PC12 , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Quinases Associadas a rho/metabolismoRESUMO
BACKGROUND: The basic helix-loop-helix transcription factor HAND1 is essential for cardiac development and structural remodeling, and mutations in HAND1 have been causally linked to various congenital heart diseases. However, whether genetically compromised HAND1 predisposes to dilated cardiomyopathy (DCM) in humans remains unknown. METHODS: The whole coding region and splicing junctions of the HAND1 gene were sequenced in 140 unrelated patients with idiopathic DCM. The available family members of the index patient carrying an identified mutation and 260 unrelated ethnically matched healthy individuals used as controls were genotyped for HAND1. The functional effect of the mutant HAND1 was characterized in contrast to its wild-type counterpart by using a dual-luciferase reporter assay system. RESULTS: A novel heterozygous HAND1 mutation, p.R105X, was identified in a family with DCM transmitted as an autosomal dominant trait, which co-segregated with DCM in the family with complete penetrance. The nonsense mutation was absent in 520 control chromosomes. Functional analyses unveiled that the mutant HAND1 had no transcriptional activity. Furthermore, the mutation abolished the synergistic activation between HAND1 and GATA4, another crucial cardiac transcription factors that has been associated with various congenital cardiovascular malformations and DCM. CONCLUSIONS: This study firstly reports the association of HAND1 loss-of-function mutation with increased susceptibility to DCM in humans, which provides novel insight into the molecular mechanisms underpinning DCM.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Cardiomiopatia Dilatada/diagnóstico , Cardiomiopatia Dilatada/genética , Predisposição Genética para Doença , Mutação/genética , Animais , Estudos de Casos e Controles , Feminino , Genótipo , Células HeLa , Humanos , Luciferases , Masculino , Camundongos , Pessoa de Meia-Idade , Células NIH 3T3 , Linhagem , FenótipoRESUMO
Alzheimer's disease (AD) is an age-related neurodegenerative disorder in which amyloid ß (Aß) peptide accumulates in the brain. The receptor for advanced glycation end product (RAGE) is a cellular binding site for Aß peptide and mediates amyloid ß-induced perturbations in cerebral vessels, neurons, and microglia in AD. Here, we identified a specific high-affinity RAGE inhibitor (APDTKTQ named RP-1) from a phage display library. RP-1 bound to RAGE and inhibited Aß peptide-induced cellular stress in human neuroblastoma SH-SYSY cells in vitro. Three amino acids in RP-1 are identical to those in the Aß peptide. RP-1 shows high homology to the 16-23 (KLVFFAED) regions in Aß peptide and high-affinity RAGE. Functional analyses indicated that RP-1 significantly reduced the level of reactive oxygen species (ROS) and ROS products and that it enhanced catalase and glutathione peroxidase (GPx) activity. Furthermore, it inactivated caspase3 and caspase9 and inhibited the upregulation of RAGE, nuclear factor-κB (NF-κB), and beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1) protein expression. In addition, RP-1 activated the PI3K/AKT signaling pathway, inhibiting the interaction between Bax and Bcl-2. Our data suggest that RP-1 is a potent RAGE blocker that effectively controls the progression of Aß peptide-mediated brain disorders and that it may have potential as a disease-modifying agent for AD.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Biblioteca de Peptídeos , Receptor para Produtos Finais de Glicação Avançada/antagonistas & inibidores , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/química , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Bacteriófagos/metabolismo , Sítios de Ligação , Encéfalo/fisiologia , Caspases/genética , Caspases/metabolismo , Catalase/genética , Linhagem Celular Tumoral , Glutationa Peroxidase/metabolismo , Humanos , NF-kappa B/genética , Neurônios/fisiologia , Oligopeptídeos/isolamento & purificação , Oligopeptídeos/metabolismo , Fragmentos de Peptídeos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptor para Produtos Finais de Glicação Avançada/genética , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Transdução de SinaisRESUMO
Nogo-66, a hydrophilic loop of 66 amino acids flank two hydrophobic domains of the Nogo-A C terminus, interacts with the Nogo-66 receptor (NgR) to exert numerous functions in the central nervous system (CNS). Nogo-66 has important roles in aspects of neuronal development, including cell migration, axon guidance, fasciculation, and dendritic branching, and in aspects of CNS plasticity, including oligodendrocyte differentiation and myelination. Here, the small ubiquitin-related modifier (SUMO) was fused to the target gene, Nogo-66, and the construct was expressed in Escherichia coli (E. coli). Under the optimal fermentation conditions, the soluble expression level of the fusion protein was 33 % of the total supernatant protein. After cleaving the fusion proteins with SUMO protease and purifying them by Ni-NTA affinity chromatography, the yield and purity of recombinant Nogo-66 obtained by 10-L scale fermentation were 23 ± 1.5 mg/L and greater than 93 %, respectively. The authenticity of the recombinant Nogo-66 was confirmed by an electrospray ionization-mass spectrometry analysis. The functional analyses indicated that the recombinant Nogo-66 was capable of binding the NgR specifically. The immunofluorescence results showed that the recombinant Nogo-66 could significantly inhibit neurite outgrowth of rat pheochromocytoma (PC12) cells stimulated by nerve growth factor and cerebellar granule cells (CGCs). Furthermore, Nogo-66 inhibited neurite outgrowth by increasing the level of phosphorylated Rho-associated coiled-coil-containing protein kinase 2 (ROCK2), collapsin response mediator protein 2 (CRMP2), and myosin light chain (MLC). This study provided a feasible and convenient production method for generating sufficient recombinant Nogo-66 for experimental and clinical applications.
Assuntos
Proteínas da Mielina/metabolismo , Neuritos/efeitos dos fármacos , Proteína SUMO-1/metabolismo , Animais , Linhagem Celular Tumoral , Cromatografia de Afinidade , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular , Proteínas da Mielina/genética , Proteínas da Mielina/isolamento & purificação , Cadeias Leves de Miosina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nogo , Ligação Proteica , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteína SUMO-1/genética , Espectrometria de Massas por Ionização por Electrospray , Quinases Associadas a rho/metabolismoRESUMO
It has been reported that acidic fibroblast growth factor (aFGF) is expressed in breast cancer and via interactions with fibroblast growth factor receptors (FGFRs) to promote the stage and grade of the disease. Thus, aFGF/FGFRs have been considered essential targets in breast cancer therapy. We identified a specific aFGF-binding peptide (AGNWTPI, named AP8) from a phage display heptapeptide library with aFGF after four rounds of biopanning. The peptide AP8 contained two (TP) amino acids identical and showed high homology to the peptides of the 182-188 (GTPNPTL) site of high-affinity aFGF receptor FGFR1. Functional analyses indicated that AP8 specifically competed with the corresponding phage clone A8 for binding to aFGF. In addition, AP8 could inhibit aFGF-stimulated cell proliferation, arrested the cell cycle at the G0/G1 phase by increasing PA2G4 and suppressing Cyclin D1 and PCNA, and blocked the aFGF-induced activation of Erk1/2 and Akt kinase in both breast cancer cells and vascular endothelial cells. Therefore, these results indicate that peptide AP8, acting as an aFGF antagonist, is a promising therapeutic agent for the treatment of breast cancer.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Fator 1 de Crescimento de Fibroblastos/metabolismo , Peptídeos/química , Peptídeos/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Mama/efeitos dos fármacos , Mama/metabolismo , Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Biblioteca de Peptídeos , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
INTRODUCTION: Streptococcus suis (S. suis) disease is a zoonotic infection caused by invasive S. suis and can lead to meningitis, septic shock, arthritis, and endocarditis. Early treatment is the key to reducing mortality. However, clinical manifestations of most cases are atypical, severely limiting rapid diagnosis and treatment. CASE REPORT: Here, we report a 74-year-old female patient diagnosed with S. suis infection. The main symptoms were hearing loss, lumbago, and scattered ecchymosis of the lower extremities and trunk. Blood non-specific infection indexes were significantly increased and platelets were significantly decreased; however, no pathogens were obtained from routine blood culture. Finally, the S. suis infection was confirmed by metagenomic next-generation sequencing (mNGS) of blood and cerebrospinal fluid. After antibiotic treatment, the limb and trunk scattered ecchymosis and lumbago symptoms were significantly relieved, but the hearing did not recover. CONCLUSIONS: Human infection with S. suis is rare in central cities, and it is easy to misdiagnose, especially in cases with atypical early symptoms. mNGS technology, combined with clinical observation, is helpful to clarify the direction of diagnosis and treatment, which is conducive to patient recovery.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Metagenômica , Infecções Estreptocócicas , Streptococcus suis , Humanos , Streptococcus suis/genética , Streptococcus suis/isolamento & purificação , Feminino , Idoso , Infecções Estreptocócicas/diagnóstico , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/tratamento farmacológico , Metagenômica/métodos , Antibacterianos/uso terapêuticoRESUMO
ABSTRACT: Introduction: Intestinal flora and the translocation of its products, such as muramyl dipeptide (MDP), are common causes of sepsis. MDP is a common activator of the intracellular pattern recognition receptor NOD2, and MDP translocation can cause inflammatory damage to the small intestine and systemic inflammatory responses in rats. Therefore, this study investigated the effects of MDP on the intestinal mucosa and distant organs during sepsis and the role of the NOD2/AMPK/LC3 pathway in MDP-induced mitochondrial dysfunction in the intestinal epithelium. Methods: Fifty male Sprague Dawley rats were randomly divided into five treatment groups: lipopolysaccharide (LPS) only, 1.5 and 15 mg/kg MDP+LPS, and 1.5 and 15 mg/kg MDP+short-peptide enteral nutrition (SPEN)+LPS. The total caloric intake was the same per group. The rats were euthanized 24 h after establishing the model, and peripheral blood and small intestinal mucosal and lung tissues were collected. Results: Compared to the LPS group, both MDP+LPS groups had aggravated inflammatory damage to the intestinal mucosal and lung tissues, increased IL-6 and MDP production, increased NOD2 expression, decreased AMPK and LC3 expression, increased mitochondrial reactive oxygen species production, and decreased mitochondrial membrane potential. Compared to the MDP+LPS groups, the MDP+SPEN+LPS groups had decreased IL-6 and MDP production, increased AMPK and LC3 protein expression, and protected mitochondrial and organ functions. Conclusions: MDP translocation reduced mitochondrial autophagy by regulating the NOD2/AMPK/LC3 pathway, causing mitochondrial dysfunction. SPEN protected against MDP-induced impairment of intestinal epithelial mitochondrial function during sepsis.
Assuntos
Acetilmuramil-Alanil-Isoglutamina , Mucosa Intestinal , Mitocôndrias , Proteína Adaptadora de Sinalização NOD2 , Ratos Sprague-Dawley , Animais , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Masculino , Ratos , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Proteína Adaptadora de Sinalização NOD2/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Citocinas/metabolismo , Lipopolissacarídeos/toxicidade , Sepse/metabolismo , Interleucina-6/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Inflamação/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND: Podoplanin (PDPN) is a highly conserved, mucin-type protein specific to the lymphatic system. Overexpression of PDPN is associated with the progression of various solid tumors, and plays an important roles in the tumor microenvironment by regulating the immune system. However, the role of PDPN-mediated signal activation in the progression of melanoma is still unknown. METHODS: PDPN expression was first analyzed in 112 human melanoma tissue microarrays and melanoma cell lines. Functional experiments including proliferation, clone formation, migration, and metastasis were utilized to identify the suppressive effects of PDPN. The Ph.D.TM-12 Phage Display Peptide Library was used to obtain a PDPN antagonist peptide, named CY12-RP2. The immunofluorescence, SPR assay, and flow cytometry were used to identify the binding specificity of CY12-RP2 with PDPN in melanoma cells. Functional and mechanistic assays in vivo and in vitro were performed for discriminating the antitumor and immune activation effects of CY12-RP2. RESULTS: PDPN was overexpressed in melanoma tissue and cells, and inhibited melanoma cells proliferation, migration, and metastasis by blocking the EMT and Wnt/ß-catenin pathway. PDPN antagonistic peptide, CY12-RP2, could specifically bind with PDPN, suppressing melanoma various functions inducing apoptosis in both melanoma cells and 3D spheroids. CY12-RP2 also enhanced the anti-tumor capacity of PBMC, and inhibited melanoma cells growth both in xenografts and allogeneic mice model. Moreover, CY12-RP2 could inhibit melanoma lung metastasis, and abrogated the immunosuppressive effects of PDPN by increasing the proportion of CD3 + CD4 + T cells, CD3 + CD8 + T cells, CD49b + Granzyme B + NK cells, and CD11b + CD86 + M1-like macrophages and the levels of IL-1ß, TNF-α, and IFN-γ. CONCLUSIONS: This study has demonstrated the important role of PDPN in the progression of melanoma and formation of immunosuppressive environment, and provided a potential approach of treating melanoma using the novel CY12-RP2 peptide. In melanoma, PDPN is overexpressed in the cancer cells, and promotes melanoma cells growth and metastasis through activating the Wnt/ß-catenin pathway. Treatment with the PDPN antagonistic peptide CY12-RP2 could not only inhibit the melanoma growth and metastasis both in vitro and in vivo through Wnt/ß-catenin pathway blockade, but also abrogate the immunosuppressive effects of PDPN through modulating immune cells.
Assuntos
Melanoma , Animais , Camundongos , Humanos , Melanoma/patologia , beta Catenina/metabolismo , Leucócitos Mononucleares/metabolismo , Via de Sinalização Wnt , Proliferação de Células , Linhagem Celular Tumoral , Peptídeos/farmacologia , Movimento Celular , Transição Epitelial-Mesenquimal , Microambiente Tumoral , Proteínas de Membrana/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Glicoproteínas de Membrana/metabolismoRESUMO
BACKGROUND AND AIMS: Isogarcinol, a natural compound extracted from the fruits of Garcinia oblongifolia, has potential chemopreventive activity. This study aimed to elucidate the anti-tumor effects and mechanism of action of isogarcinol on nasopharyngeal carcinoma (NPC). METHODS: Isogarcinol was isolated from Garcinia oblongifolia by using chromatographic separation. The anti-tumor effects of isogarcinol in NPC cells were tested by MTT assay, flow cytometry, wound healing assay, western blotting, transwell assay, colony formation assay, immunofluorescence, and transmission electron microscopy (TEM). The anti-tumor efficacy in vivo was evaluated in NPC cells xenograft models. RESULTS: Functional studies revealed that isogarcinol inhibited the proliferation, colony formation, migration and invasion abilities of NPC cells in vitro. Isogarcinol caused mitochondrial damage to overproduce reactive oxygen species through reducing the mitochondrial membrane potential and ΔΨm. Isogarcinol also substantially inhibited NPC cells growth in a xenograft tumor model without any obvious toxicity when compared with paclitaxel (PTX). Mechanistic studies have illustrated that isogarcinol increased the Bax/Bcl-2 ratio, cleaved caspase-3, and cytoplasmic cytochrome C levels to induce mitochondrial apoptosis. The ROS overproduction by isogarcinol could suppress EMT pathway via decreasing the levels of p-Akt and Snail. Furthermore, isogarcinol promoted the conversion of LC3-â to LC3-â ¡, but increased p62 level to block autophagic flux, resulting in the accumulation of damaged mitochondria to promote autophagic cell death in NPC cells. CONCLUSION: This study provides a new theoretical foundation for the anti-tumor application of Garcinia oblongifolia and confirms that isogarcinol could be developed as a candidate drug for NPC treatment with low toxicity.
Assuntos
Antineoplásicos Fitogênicos , Garcinia , Camundongos Nus , Mitocôndrias , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas , Humanos , Garcinia/química , Animais , Mitocôndrias/efeitos dos fármacos , Linhagem Celular Tumoral , Carcinoma Nasofaríngeo/tratamento farmacológico , Neoplasias Nasofaríngeas/tratamento farmacológico , Antineoplásicos Fitogênicos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Morte Celular Autofágica/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Ensaios Antitumorais Modelo de Xenoenxerto , Camundongos , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Frutas/químicaRESUMO
Curcin can inhibit the proliferation of tumor cells and promote tumor cell apoptosis, but the cytotoxicity of curcin is not selective for tumors or normal cells. In order to enhance the targeting of the anti-tumor ability of curcin, a transferrin receptor (TfR) binding peptide, TfRBP9, was fused with curcin. The curcin-TfRBP9 gene was cloned into pQE-30 and the recombinant vector pQE-30-curcin-TfRBP9 was established. Then the recombinant vector pQE-30-curcin-TfRBP9 was transferred into Escherichia coli M15. After being induced by 0.5mM IPTG for 6h at 37°C, the expressed quantity of the recombinant protein was about 30% of the total protein. Recombinant curcin-TfRBP9 was expressed in the form of an inclusion body. After dissolution, purification and renaturation, the purity of the recombinant curcin-TfRBP9 reached 95%. Immunofluorescence analysis showed that the TfRBP9 significantly enhanced the ability of the curcin binding to HepG2, and was enriched in the cytoplasm. The curcin-TfRBP9 fusion protein had significant proliferation inhibition effects on the HepG2 cells that over-expressed transferrin receptors, had lower inhibitory effects on the SKBR-3 cells that expressed low transferrin receptors, and had the lowest inhibitory effects on the LO-2 cells that were normal human liver cells. Compared with curcin, the curcin-TfRBP9 induced higher apoptosis rates in the HepG2 cells.