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BACKGROUND: Melanoma is the most lethal skin cancer characterized by its high metastatic potential. In the past decade, targeted and immunotherapy have brought revolutionary survival benefits to patients with advanced and metastatic melanoma, but these treatment responses are also heterogeneous and/or do not achieve durable responses. Therefore, novel therapeutic strategies for improving outcomes remain an unmet clinical need. The aim of this study was to evaluate the therapeutic potential and underlying molecular mechanisms of RC48, a novel HER2-target antibody drug conjugate, either alone or in combination with dabrafenib, a V600-mutant BRAF inhibitor, for the treatment of advanced BRAF-mutant cutaneous melanoma. METHODS: We evaluated the therapeutic efficacy of RC48, alone or in combination with dabrafenib, in BRAF-mutant cutaneous melanoma cell lines and cell-derived xenograft (CDX) models. We also conducted signaling pathways analysis and global mRNA sequencing to explore mechanisms underlying the synergistic effect of the combination therapy. RESULTS: Our results revealed the expression of membrane-localized HER2 in melanoma cells. RC48 effectively targeted and inhibited the growth of HER2-positive human melanoma cell lines and corresponding CDX models. When used RC48 and dabrafenib synergically induced tumor regression together in human BRAF-mutant melanoma cell lines and CDX models. Mechanically, our results demonstrated that the combination therapy induced apoptosis and cell cycle arrest while suppressing cell motility in vitro. Furthermore, global RNA sequencing analysis demonstrated that the combination treatment led to the downregulation of several key signaling pathways, including the PI3K-AKT pathway, MAPK pathway, AMPK pathway, and FOXO pathway. CONCLUSION: These findings establish a preclinical foundation for the combined use of an anti-HER2 drug conjugate and a BRAF inhibitor in the treatment of BRAF-mutant cutaneous melanoma.
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Antineoplásicos , Imidazóis , Imunoconjugados , Melanoma , Oximas , Neoplasias Cutâneas , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Neoplasias Cutâneas/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Fosfatidilinositol 3-Quinases , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Imunoconjugados/genética , Imunoconjugados/uso terapêutico , MutaçãoRESUMO
Due to continuous plantation of poplar, its growth and biomass accumulation may be negatively affected by the accumulation of allelochemicals such as para-hydroxybenzoic acid (pHBA) in soil. As photosynthesis is the most fundamental process in plants, it can be negatively impacted by pHBA stress. Therefore, it is crucial to improve photosynthetic capacity under pHBA stress to facilitate poplar plant growth. The mitogen-activated protein kinase (MAPK) cascade pathway is widely involved in environmental stress responses in plants. However, the regulation mechanisms of photosynthesis-related pathways by MAPK pathway genes under pHBA stress are still unclear. In this study, through transcriptome analysis and weighted gene co-expression network analysis, we observed that PeMPK7 overexpression in poplar can regulate the expression of photosynthesis-related genes and transcription factor genes, namely, WRKY1, WRKY33, and ERF3, during the early stage of pHBA stress. In addition, PeMPK7 can improve photosynthesis in poplar under long-term pHBA stress. Moreover, yeast two-hybrid and pull-down assays confirmed the interaction between PeMPK7 and PeMKK7/10. Based on these results, a schematic diagram of the pathways involved in the regulation of photosynthesis by PeMPK7 was constructed. This study provided novel insights into the molecular mechanisms of regulation of pHBA stress via MAPK cascade pathway.
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Regulação da Expressão Gênica de Plantas , Parabenos , Fotossíntese , Populus , Populus/genética , Populus/efeitos dos fármacos , Populus/fisiologia , Fotossíntese/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Estresse Fisiológico , Hidroxibenzoatos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Poluentes do Solo/toxicidadeRESUMO
This study aims to investigate the effect of silencing the CITED1 gene to regulate the PI3K/AKT pathway on the biological function of papillary thyroid carcinoma (PTC) cells and its mechanism of action. Human PTC cells SW1736 were divided into 4 groups: control group, siCITED1 group, LY294002 group and siCITED1+LY294002 group. CITED1 was silenced by transfection with siCITED1 plasmid. The PI3K/AKT pathway was inhibited by LY294002 (5 µmmol/L). Each group was determined for cell proliferation, apoptosis and invasion capabilities, as well as PI3K/AKT transcription and protein expression levels. CITED1 mRNA and protein levels in the siCITED1 group and the siCITED1+LY294002 group were significantly lower than those in the control group (P < 0.05), and the two levels were not significantly different between the LY294002 group and the control group (P > 0.05). Compared with the control group, the siCITED1 group showed remarkably lower proliferation and invasion capabilities, and remarkably higher apoptosis rate (P < 0.05). There was no significant difference in proliferation, apoptosis and invasion capabilities between the LY294002 group and the siCITED1+LY294002 group (P > 0.05), both of which had significantly lower proliferation and invasion capabilities but significantly higher apoptosis rate than the siCITED1 group (P < 0.05). PI3K and AKT protein levels in the siCITED1 group were significantly lower than those in the control group (P < 0.05). The PI3K and AKT protein levels in the LY294002 group and the siCITED1+LY294002 group were not significantly different (P > 0.05), and were significantly lower than those in the siCITED1 group (P < 0.05). In conclusion: CITED1 silence may inhibit the progression of PTC cells by inhibiting the PI3K/AKT pathway.
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Proteínas Proto-Oncogênicas c-akt , Neoplasias da Glândula Tireoide , Humanos , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/genética , Câncer Papilífero da Tireoide/genética , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Inativação GênicaRESUMO
Poplar is widely planted as an economic and ecological tree species. However, accumulation of the phenolic acid allelochemical para-hydroxybenzoic acid (pHBA) in soil is a severe threat to the growth and productivity of poplar. pHBA stress leads to excessive production of reactive oxygen species (ROS). However, it is unclear which redox-sensitive proteins are involved in the pHBA-induced cellular homeostasis regulatory mechanism. We here identified reversible redox-modified proteins and modified cysteine (Cys) sites in exogenous pHBA- and hydrogen peroxide (H2O2)-treated poplar seedling leaves by using the iodoacetyl tandem mass tag-labeled redox proteomics method. In total, 4786 redox modification sites were identified in 3176 proteins, with 104 and 91 proteins being differentially modified at 118 and 101 Cys sites in response to pHBA and H2O2 stresses, respectively. The differentially modified proteins (DMPs) were predicted to be mainly localized in the chloroplast and cytoplasm, with most proteins being enzymes with catalytic activities. The KEGG enrichment analysis of these DMPs revealed that proteins related to the MAPK signaling pathway, soluble sugar metabolism, amino acid metabolism, photosynthesis, and phagosome pathways were extensively regulated by redox modifications. Moreover, combined with our previous quantitative proteomics data, 8 proteins were upregulated and oxidized under both pHBA and H2O2 stresses. Reversible oxidation of Cys sites in these proteins might be actively responsible for the regulation of tolerance to pHBA-induced oxidative stress. Based on the aforementioned results, a redox regulatory model activated by pHBA- and H2O2-induced oxidative stress was proposed. This study conducts the first redox proteomics analysis of poplar in response to pHBA stress and provides a new insight into the mechanistic framework of reversible oxidative post-translational modifications to gain a better understanding of pHBA-induced chemosensory effects on poplar.
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Peróxido de Hidrogênio , Proteômica , Peróxido de Hidrogênio/metabolismo , Proteômica/métodos , Parabenos , Cisteína/metabolismo , Estresse Oxidativo , Processamento de Proteína Pós-Traducional , OxirreduçãoRESUMO
Mitogen-activated protein kinase (MAPK) plays a crucial role in plant stress response. Poplar is one of the most important afforestation and timber species and inevitably encounters allelopathy effects during continuous cropping. para-hydroxybenzoic acid (pHBA) is a primary soil allelochemical, which can restrict the growth and biomass of poplar. However, the involvement of MAPKs in the underlying physiological and molecular regulatory mechanisms in response to pHBA stress remains unclear. In this study, PeMPK17, a gene encoding a group D MAPK, was cloned from Populus × euramericana. PeMPK17 protein was localized in both nucleus and plasma membrane. Quantitative real-time polymerase chain reaction analysis demonstrated that PeMPK17 expression in poplar increased when treated with pHBA, PEG, and H2O2. Exogenous pHBA and H2O2 induced PeMPK17 expression mediated by reactive oxygen species (ROS). The transgenic poplar plants overexpressing PeMPK17 demonstrated attenuated phenotypic injury, higher relative water content in leaves, and lower ion leakage under pHBA stress. In transgenic poplar, the activity and expression of antioxidant enzymes including superoxide dismutase, peroxidase, and catalase increased, while the content of H2O2, O2·-, and malondialdehyde decreased. These results suggested that PeMPK17 protects cell membranes from oxidative damage by removing excess ROS. In addition, overexpression of PeMPK17 promoted osmoprotectant accumulation including soluble sugar and free proline, which may aid in the regulation of ROS balance under pHBA treatment. Furthermore, the interaction between PeMPK17 and PeMKK7 was confirmed. Collectively, these data identify the molecular mechanisms and signal pathways associated with PeMPK17 that regulate pHBA response in poplar.
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BACKGROUND: Chinese universities are increasingly recruiting foreign students, and problem-based learning (PBL) is an effective approach to integrating those students. This study focuses on the role of intercultural sensitivity and group ethnic composition on the quality of group interaction in medical problem-based learning in China. METHODS: This paper reports an investigation of the differences in three types of group interaction (exploratory questions, cumulative reasoning, and handling conflict) among 139 s-year medical undergraduates from two backgrounds (Chinese and foreign) in a PBL setting. The roles of intercultural sensitivity, group ethnic composition, and students' personal characteristics including age, gender and ethnicity on students' perceptions of the three types of interaction were quantitatively analyzed. A 35-item questionnaire and demographic survey were administered to second year medical undergraduates. RESULTS: The results indicated that group ethnic composition was a significant negative predictor while intercultural sensitivity was a strong positive predictor of group interactions involving exploratory questions and cumulative reasoning. In addition, group heterogeneity in terms of age and ethnicity were significant predictors of group interaction. CONCLUSIONS: The findings of this study provide insights for strategically designing effective multiethnic group learning environments that encourage interaction and collaboration.
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Dinâmica de Grupo , Aprendizagem Baseada em Problemas , Humanos , Povo Asiático , China , Etnicidade , UniversidadesRESUMO
PURPOSE: The purpose of this study was to clarify the effect of ZC3H13 on the growth of papillary thyroid carcinoma (PTC). METHODS: Firstly, we used qRT-PCR and Western blot to compare the difference in the expression of ZC3H13 between normal thyroid epithelial cells and PTC cell lines. Then, ZC3H13 overexpression/knockout thyroid cancer cells were constructed by lentivirus transfection, and the effects of overexpression of ZC3H13 on the proliferation, migration and invasion of PTC cells were detected by CCK8 and transwell experiments. Lastly, MeRIP-qPCR, RIP and o Actinomycin D were used to verify that ZC3H13 regulated the expression of downstream target gene IQGAP1 through m6A modification. RESULTS: ZC3H13 expression was decreased in PTC cell lines BCPAP, KTC-1, k1, HTH83, and TPC-1. Proliferation, invasion, and migration of PTC cells were inhibited by overexpressed ZC3H13 but increased by knockdown of ZC3H13. IQGAP1 expression was suppressed by ZC3H13 overexpression but enhanced by ZC3H13 knockdown. In ZC3H13-overexpressed PTC cells, the m6A level of IQGAP1 mRNA was increased, and the IQGAP1 mRNA expression was decreased with the increasing time of Actinomycin D treatment. YTHDF2 enriched more IQGAP1 mRNA than IgG and knockdown of YTHDF2 reversed the effect of ZC3H13 overexpression on IQGAP1 mRNA stability. The xenograft tumor experiment in nude mice confirmed that the overexpression of ZC3H13 inhibited tumor growth, while overexpression of IQGAP1 could reverse the inhibitory effect of ZC3H13 overexpression on tumor growth. CONCLUSION: ZC3H13 mediates IQGAP1 mRNA degradation by promoting m6A modification of IQGAP1 mRNA, this provides a prospective therapeutic target for PTC.
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MicroRNAs , Neoplasias da Glândula Tireoide , Camundongos , Animais , Humanos , Câncer Papilífero da Tireoide/genética , Câncer Papilífero da Tireoide/patologia , MicroRNAs/genética , Camundongos Nus , Dactinomicina/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Invasividade Neoplásica , Movimento Celular , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , RNA Mensageiro , Regulação Neoplásica da Expressão Gênica , Proteínas Nucleares , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Cryopreservation of single seminiferous tubule is significant for fertility preservation, especially for male patients with cryptorchidism, Y-chromosome deletion and orchitis. However, there are few studies on cryopreservation of single seminiferous tubule. This study proposes several improved strategies for cryopreservation of single seminiferous tubule in mice. First, single seminiferous tubule was cryopreserved with modified slow freezing and vitrification methods. The results showed that the apoptosis negative rates of spermatogenic cells in single seminiferous tubule with modified slow freezing method were significantly higher than vitrification with plastic slide. Then, plastic slide and two metal vitrification carriers with high thermal conductivity, copper mesh and aluminum foil, were used to vitrify single seminiferous tubule. The metal carriers could improve the outcome of vitrification than plastic slide. The apoptosis negative rates of spermatogenic cells in the aluminum foil group was significantly higher than that in the copper mesh group. Finally, single seminiferous tubule was perfused with CPAs by micro-injection technique and vitrified. The results of cryo-microscopy experiments showed that the ice crystals formed inside the injected seminiferous tubule was reduced during the cooling process, the apoptosis negative rate of spermatocytes, spermatids and Sertoli cells were significantly higher than that of the non-injection group, indicating that the injection technique can effectively improve the effect of vitrification. This study has potential clinical value for in-vitro culture of spermatogonial stem cells and autologous testicular tissue grafting in patients with azoospermia.
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Criopreservação , Espermatogênese , Alumínio , Animais , Cobre , Criopreservação/métodos , Humanos , Masculino , Camundongos , Plásticos , Túbulos Seminíferos , VitrificaçãoRESUMO
A new potentiometric sensor based on modified carbon paste electrode (CPE) was prepared for the sensitive and selective detection of total residual chlorine (TRC) in simulated electrolytically-treated ballast water (BW). The modified CPE was prepared using ferrocene (Fc) as the sensing species and paraffin oil as the binder. It is revealed that the addition of Fc can significantly shorten the response time and improve the reproducibility, selectivity, and stability of the sensor. The open circuit potential of the Fc-CPE is in linear proportion to the logarithm of TRC within the TRC concentration range from 1 mgâdm-3 to 15 mgâdm-3. In addition, the Fc-CPE sensor exhibits good selectivity to TRC over a wide concentration range of the possible co-exiting interference ions in seawater. The Fc-CPE electrode can be used as a convenient and reliable sensor for the continuous monitoring of TRC during the electrolytic treatment of BW.
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BACKGROUND: Papillary thyroid cancer (PTC) is a type of malignant tumor with excellent prognosis, accounting for more than 80% of thyroid cancer. Recently, numerous studies illustrated the importance of N 6-methyladenosine (m6A) RNA modification to tumorigenesis, but it has never been reported in PTC. METHODS: We downloaded data from The Cancer Genome Atlas (TCGA) and analyzed RNA expression, single nucleotide polymorphisms (SNPs) and copy number variations (CNVs) of 19 m6A RNA methylation regulators in PTC. Then we used nonnegative matrix factorization (NMF) to cluster patients into two m6A subtypes and compared them in overall survival (OS) and disease-free survival (DFS). The Weighted correlation network analysis (WGCNA) and univariate Cox proportional hazard model (CoxPH) were used to select genes for the construction of a m6A-related signature. The accuracy and prognostic value of this signature were validated by using receiver operating characteristic (ROC) curves, K-M (Kaplan-Meier) survival analysis, univariant and multivariant analyses. RESULTS: CNVs and differential expression of m6A regulators were observed in PTC patients. Especially IGF2BP2 (Insulin-like growth factor 2 mRNA binding protein 2), which was most significantly overexpressed in tumor tissue. We chose 4 genes in the m6A-related module from WGCNA: IGF2BP2, STT3A, MTHFD1 and GSTM4, and used them to construct a m6A-related signature. The prognostic value of this signature was validated, and risk scores provided by the signature was the independent prognostic factor for PTC. A nomogram was also provided for clinical usage. CONCLUSIONS: We performed a comprehensive evaluation of the m6A RNA modification landscape of PTC and explored its underlying mechanisms. Our m6A-related signature was of great significance in predicting the DFS of patients with PTC. And IGF2BP2 was a gene worthy for further analysis as its strong correlation with DFS and clinical phenotypes of PTC.
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BACKGROUND: Spontaneous bacterial peritonitis (SBP) and bacterascites (BA) represent frequent and serious complications in cirrhosis patients with ascites. However, few detailed data are available regarding the clinical and bacteriological feature of SBP or BA patients in China. METHODS: We retrospectively analyzed bacteriological and clinical characteristics of patients with SBP and BA at Beijing 302 Hospital in China from January 2012 to December 2015. RESULTS: A total of 600 patients with SBP (n = 408) or BA (n = 192) were enrolled. Patients with BA appeared to have a less severe clinical manifestation and lower mortality rate than patients with SBP. Gram-negative bacteria formed the majority of pathogens in SBP (73.9%) and BA (55.8%) cases. Higher ascitic fluid polymorphonuclear leucocytes (PMN) count and hepatocellular carcinoma were independent risk factors for BA episode progressing to SBP. The concentration of blood urea nitrogen (BUN) was independent risk factor for 30-day mortality of BA patients. For patients with SBP, the independent risk factors for 30-day mortality were age, Model for End-Stage Liver Disease (MELD) score, septic shock and hepatocellular carcinoma. Patients with third-generation cephalosporin or carbapenems resistant infection had a significantly lower survival probability. There were significant differences in clinical characteristics and outcome among the major bacteria. Multivariate analysis showed that patients infected with Klebsiella spp. had higher hazard ratio of 30-day mortality. CONCLUSION: Our study reported the bacteriological and clinical characteristics of patients with SBP and BA. Higher ascitic fluid PMN count and hepatocellular carcinoma were found to be independent risk factors for BA episode progressed to SBP. Outcome of ascitic fluid infection in patients with cirrhosis was influenced by the type of bacteria and antimicrobial susceptibility.
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Ascite/microbiologia , Infecções Bacterianas/etiologia , Peritonite/etiologia , Adulto , Idoso , Ascite/tratamento farmacológico , Ascite/etiologia , Ascite/mortalidade , Líquido Ascítico/microbiologia , Líquido Ascítico/patologia , Povo Asiático , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/mortalidade , Carcinoma Hepatocelular/complicações , Carcinoma Hepatocelular/microbiologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Feminino , Bactérias Gram-Negativas/isolamento & purificação , Humanos , Cirrose Hepática/complicações , Cirrose Hepática/microbiologia , Neoplasias Hepáticas/complicações , Neoplasias Hepáticas/microbiologia , Masculino , Pessoa de Meia-Idade , Peritonite/tratamento farmacológico , Peritonite/mortalidade , Prognóstico , Estudos Retrospectivos , Fatores de RiscoRESUMO
It is well known that the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/TNFSF10) is specifically expressed in various tumor cells, but less or no expression in most normal tissues and cells. While TRAIL engages with its native death receptors, TRAIL receptor 1 (TRAIL-R1) or 2 (TRAIL-R2), usually elicits the tumor cell death by apoptosis. In this study, we report that a novel humanized monoclonal antibody against TRAIL-R2 (named as zaptuzumab) well remain the biological activity of the parental mouse antibody AD5-10 inducing cell death in various cancer cells, but little effect on normal cells. Zaptuzumab also markedly inhibited the tumor growth in the mouse xenograft of NCI-H460 without toxicity to the liver and kidney, and the efficacy of tumor suppression was increased significantly while it combined with cis-dichlorodiamineplatinum. Especially, 131 I-labeled zaptuzumab injected into mouse tail vein specifically targeted to the xenograft of the lung cancer cells. Confocal analysis showed that zaptuzumab bound with TRAIL-R2 on cell surface could be quickly internalized and transferred into the lysosome. Furthermore, zaptuzumab possessed a high level of antibody-dependent cytotoxicity as well as complement-dependent cytotoxicity. Study on the mechanisms of cell death induced by zaptuzumab showed that it efficiently induced both caspase-dependent apoptosis and autophagic cell death. These data suggest that the humanized anti-TRAIL-R2 monoclonal antibody or the second generation of the antibody may have an important clinical usage for cancer immunotherapy. © 2017 IUBMB Life, 69(9):735-744, 2017.
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Anticorpos Monoclonais Humanizados/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/antagonistas & inibidores , Células A549 , Animais , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Apoptose/imunologia , Humanos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Camundongos , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/imunologia , Ligante Indutor de Apoptose Relacionado a TNF/antagonistas & inibidores , Ligante Indutor de Apoptose Relacionado a TNF/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
African cactiform Hoodia gordonii (Asclepiadaceae) has been used for thousands of years by Xhomani Bushmen as an anorexant during hunting trips and has been proposed as a new agent for the management of body weight. However, its in vivo targets and molecular mechanisms remain elusive. GPR119, a G protein-coupled receptor highly expressed in pancreatic ß cells and intestinal L cells, has been demonstrated to facilitate glucose-stimulated insulin secretion (GSIS) and represents a novel and attractive target for the therapy of metabolic disorders. Here, we disclose that Gordonoside F (a steroid glycoside isolated from H. gordonii), but not the widely known P57, activates specifically GPR119. Successful synthesis of Gordonoside F facilitates further characterization of this compound. Gordonoside F promotes GSIS both in vitro and in vivo and reduces food intake in mice. These effects are mediated by GPR119 because GPR119 knockout prevents the therapeutic effects of Gordonoside F. Interestingly, the appetite-suppressing effect of Hoodia extract was also partially blocked by GPR119 knockout. Our results demonstrate for the first time, to our knowledge, that GPR119 is a direct target and one of the major mechanisms underlying the therapeutic effect of the popular "weight loss" herb H. gordonii. Given the long history of safe application of this herb in weight control, it is foreseeable that the novel scaffold of Gordonoside F provides a promising opportunity to develop new drugs in treating metabolic diseases.
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Apocynaceae/química , Doenças Metabólicas/tratamento farmacológico , Extratos Vegetais/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Redução de Peso/efeitos dos fármacos , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Ingestão de Alimentos/efeitos dos fármacos , Teste de Tolerância a Glucose , Glicosídeos/química , Glicosídeos/farmacologia , Células HEK293 , Humanos , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Masculino , Doenças Metabólicas/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estrutura Molecular , Terapia de Alvo Molecular/métodos , Fitoterapia/métodos , Extratos Vegetais/química , Plantas Medicinais/química , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genética , Esteroides/química , Esteroides/farmacologiaRESUMO
AIM: GPR119 is a G protein-coupled receptor (GPCR) that is highly expressed in pancreatic ß-cells and intestinal L-cells and facilitates glucose-stimulated insulin secretion (GSIS). GPR119 may represent a novel target for the treatment of metabolic disorders. Here, we sought to identify novel small-molecule GPR119 agonists. METHODS: A cell-based high-throughput screening assay was established using HEK293 cells stably expressing GPR119 and pCRE-luc reporter plasmid (HEK293/GPR119/pCRE-luc). A compound library composed of 1440 compounds was screened. Mouse ß-cell line MIN-6 and isolated mouse islets were used to evaluate the effects of candidate compounds on GSIS in vitro. RESULTS: Three compounds with novel structures (ZSY-04, -06, and -13) were found to activate GPR119-mediated signaling and to induce GPR119 desensitization. The EC50 values of ZSY-04, -06, and -13 in stimulating intracellular cAMP accumulation in HEK293/GPR119 cells were 2.758, 3.046, and 0.778 µmol/L, respectively. Furthermore, all three compounds displayed high selectivity for GPR119, and did not activate other 9 GPCRs tested. Moreover, all three compounds significantly increased GSIS in both MIN-6 mouse ß-cells and isolated mouse islets at concentration of 10 µmol/L. CONCLUSION: Three novel small-molecule GPR119 agonists (ZSY-04, -06, and -13) with high receptor selectivity and capacity to induce GSIS in vitro were discovered. These compounds are potential candidates to be structurally optimized into drugs for the treatment of type 2 diabetes.
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Descoberta de Drogas , Hipoglicemiantes/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Ilhotas Pancreáticas/efeitos dos fármacos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas , Animais , Células CHO , Sinalização do Cálcio/efeitos dos fármacos , Cricetulus , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Glucose/metabolismo , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Hipoglicemiantes/química , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Camundongos , Estrutura Molecular , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Reprodutibilidade dos Testes , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Relação Estrutura-Atividade , Fatores de Tempo , Técnicas de Cultura de Tecidos , TransfecçãoRESUMO
AIM: TGR5 is a G protein-coupled receptor that is expressed in intestinal L-cells and stimulates glucagon-like peptide 1 (GLP-1) secretion. TGR5 may represent a novel target for the treatment of metabolic disorder. Here, we sought to design and synthesize a series of TGR5 agonists derived from the natural product betulinic acid. METHODS: A series of betulinic acid derivatives were designed and synthesized. A cAMP assay was established using a HEK293 cell line expressing human TGR5. Luciferase reporter assay was established using HEK293 cells transfected with plasmids encoding human FXR and luciferase reporter. A human intestinal L-cell line NCI-H716 was used to evaluate the effects of the betulinic acid derivatives on GLP-1 secretion in vitro. RESULTS: Biological data revealed that the 3-α-OH triterpenoids consistently show increased potency for TGR5 compared to their 3-ß-OH epimers. 3-OH esterification increased the lipophilicity and TGR5 activity of 3-α betulinic derivatives and enhanced the activity differences between 3-α and 3-ß derivatives. The 3-α-acyloxy betulinic acids also exhibited a significant dose-dependent GLP-1 secretion effect. CONCLUSION: This study demonstrates that highly lipophilic 3-epi-betulinic acid derivatives can be potent and selective TGR5 agonists with improved cellular efficacy, and our research here provides a new strategy for the design and development of potent TGR5 agonists.
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Receptores Acoplados a Proteínas G/agonistas , Triterpenos/farmacologia , Administração Oral , Animais , AMP Cíclico/metabolismo , Genes Reporter , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Células HEK293 , Células Hep G2 , Humanos , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Estrutura Molecular , Triterpenos Pentacíclicos , Ratos , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Acoplados a Proteínas G/genética , Relação Estrutura-Atividade , Transfecção , Triterpenos/administração & dosagem , Triterpenos/síntese química , Triterpenos/farmacocinética , Ácido BetulínicoRESUMO
In order to investigate the change rules and response characteristics of growth status on each component of poplar seedling followed by continuous cropping generations and growth period, we clear the biomass distribution pattern of poplar seedling, adapt continuous cropping, and provide theoretical foundation and technical reference on cultivation management of poplar seedling, the first generation, second generation, and third generation continuous cropping poplar seedlings were taken as study objects, and the whole poplar seedling was harvested to measure and analyze the change of each component biomass on different growth period poplar leaves, newly emerging branches, trunks and root system, and so forth. The results showed that the whole biomass of poplar seedling decreased significantly with the leaf area and its ratio increased, and the growth was inhibited obviously. The biomass aboveground was more than that underground. The ratios of leaf biomass and newly emerging branches biomass of first continuous cropping poplar seedling were relatively high. With the continuous cropping generations and growth cycle increasing, poplar seedling had a growth strategy to improve the ratio of root-shoot and root-leaf to adapt the limited soil nutrient of continuous cropping.
Assuntos
Biomassa , Folhas de Planta/crescimento & desenvolvimento , Raízes de Plantas/crescimento & desenvolvimento , Populus/crescimento & desenvolvimento , Plântula/crescimento & desenvolvimentoRESUMO
Oocyte vitrification has become a widely adopted method in clinical practice. However, the solidification behavior and its impact on oocytes during the ultrarapid cooling process remain poorly understood. In this study, we established a system and methodology to observe crystallization behavior in oocytes during quench cooling and warming. Subsequently, the threshold concentration of cryoprotective agents (CPAs) required for oocyte vitrification was determined through a visualization method. The results demonstrated that the ice front could not be observed in the image sequence when using 16.5% DMSO +16.5% EG during high-speed quench cooling (2821.58°C/min). Finally, oocytes were encapsulated with an antifreezing hydrogel (7.5% EG +7.5% DMSO +0.5% alginate) and subjected to high-speed quench cooling. No ice crystals appeared in the antifreezing hydrogel-encapsulated oocytes at a low concentration of osmotic CPA (2.4 M). This research opens up new possibilities for oocyte vitrification with a reduced concentration of CPA.
Assuntos
Criopreservação , Crioprotetores , Hidrogéis , Gelo , Oócitos , Vitrificação , Oócitos/metabolismo , Oócitos/citologia , Animais , Hidrogéis/química , Camundongos , Crioprotetores/farmacologia , Crioprotetores/química , Criopreservação/métodos , Feminino , CristalizaçãoRESUMO
BACKGROUND: Papillary thyroid carcinoma (PTC) accounts for about 85% of thyroid cancer cases. Transmembrane protein 252 (TMEM252) is a gene encoding a transmembrane protein that has only been reported to be associated with triple-negative breast cancer. Herein, we first elucidated the physiological roles and possible regulatory proteins of TMEM252 in PTC pathogenesis. METHODS: Quantitative real-time polymerase chain reaction, western blot, and immunohistochemical analyses were utilized to ascertain the relative TMEM252 expression in PTC and surrounding normal tissues. Functional investigations involved CCK-8 viability assay, EdU incorporation assay for proliferation, transwell assays for migration and invasion, and an in vivo tumor development assessment to evaluate the TMEM252-mediated regulation of tumor formation. RESULTS: Our results first revealed diminished TMEM252 transcript and protein expressions in PTC tissues and cell lines. TMEM252 overexpression suppressed cell proliferation through reducing p53, p21, and p16 expression. Conversely, TMEM252 depletion has opposite effects in PTC cells both in vivo. Additionally, the upregulation of TMEM252 demonstrated cell migration and invasion suppression by impeding the epithelial-mesenchymal transition (EMT) process via inhibition of the Notch pathway. Furthermore, overexpression of TMEM252 suppressed tumor growth in vivo. CONCLUSION: Our study elucidates that TMEM252 suppresses PTC progression by modulating the Notch pathway. These findings underscore TMEM252 is a potential therapeutic target in managing PTC.
RESUMO
Colorectal cancer (CRC) is the third most common cancer associated with a poor prognosis. Effective targeted therapy alone or in combination for treating advanced CRC remains to be a major clinical challenge. Here, we propose the therapeutic efficacy and molecular mechanism underlying RC48, a FDA-approved anti-HER2 antibody conjugate via a cleavable linker to the microtubule inhibitor monomethyl auristatin E (MMAE), either alone or in combination with gemcitabine (GEM) in various models of HER2-positive advanced CRC. Our findings demonstrated that HER2 was widely expressed and located on the plasma membrane of CRC patient specimens, PDX xenograft tumors and cell lines. It confirmed that RC48 alone significantly targeted and eradicated HER2 positive CRC tumor in these models. Moreover, we screened a panel of FDA-approved first-line chemotherapy drugs in vitro. We found that GEM exhibited stronger antiproliferative activity compared to the other first-line anti-cancer agents. Furthermore, combination therapy of RC48 and GEM significantly showed synergetic antitumor activity in vitro and in vivo. To gain further mechanistic insights into the combination therapy, we performed RNA-seq analysis. The results revealed that combination treatment of RC48 and GEM regulated multiple signaling pathways, such as PI3K-AKT, MAPK, p53, Foxo, apoptosis, cell cycle and cell senescence, etc., to exert its antitumor activity in CRC cells. Collectively, these preclinical findings demonstrated that RC48 alone or combinational therapy exerted promising antitumor activity, and meriting the preclinical framework for combinational therapy of anti-HER2 drug conjugate drug and chemotherapy drugs for HER2-positive patients with advanced CRC.
Assuntos
Neoplasias Colorretais , Imunoconjugados , Humanos , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Fosfatidilinositol 3-Quinases , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Anticorpos , GencitabinaRESUMO
Endometrial carcinoma (EC) is a prevalent gynecological tumor in women, and its treatment and prevention are significant global health concerns. The mutations in DNA polymerase ε (POLE) are recognized as key features of EC and may confer survival benefits in endometrial cancer patients undergoing anti-PD-1/PD-L1 therapy. However, the anti-tumor mechanism of POLE mutations remains largely elusive. This study demonstrates that the hot POLE P286R mutation impedes endometrial tumorigenesis by inducing DNA breakage and activating the cGAS-STING signaling pathway. The POLE mutations were found to inhibit the proliferation and stemness of primary human EC cells. Mechanistically, the POLE mutants enhance DNA damage and suppress its repair through the interaction with DNA repair proteins, leading to genomic instability and the upregulation of cytoplasmic DNA. Additionally, the POLE P286R mutant also increases cGAS level, promotes TBK1 phosphorylation, and stimulates inflammatory gene expression and anti-tumor immune response. Furthermore, the POLE P286R mutation inhibits tumor growth and facilitates the infiltration of cytotoxic T cells in human endometrial cancers. These findings uncover a novel mechanism of POLE mutations in antagonizing tumorigenesis and provide a promising direction for effective cancer therapy.