OBJECTIVES: Metastasis is the primary cause of death in osteosarcoma, and current clinical treatments remain limited. BRD4, a key epigenetic regulator, has shown therapeutic promise in various cancers through its inhibition. However, the mechanistic role of BRD4 in osteosarcoma remains poorly understood. This study aims to elucidate the molecular mechanisms by which BRD4 regulate osteosarcoma progression and to explore novel therapeutic strategies. METHODS: Immunofluorescence was used to assess BRD4 expression levels in a tissue microarray containing 80 osteosarcoma samples from different patients. The Gene Expression Omnibus (GEO) dataset (GSE42352, containing survival data from 88 osteosarcoma patients) was downloaded to perform Kaplan-Meier survival analysis based on BRD4 gene expression levels. In vivo, an orthotopic intramedullary osteosarcoma model was established using HOS cells in C57 mice, followed by treatment with varying doses of the BRD4 inhibitor (+)-JQ1. Micro-CT, 3D reconstruction of bone tissue, and HE staining were employed to evaluate pathological changes in bone and intestinal lymph nodes. In vitro, cell viability was measured using the methyl thiazolyl tetrazolium (MTT) assay, while colony formation and Transwell assays assessed proliferative and invasive capacities. Chromatin-bound BRD4 was analyzed via co-immunoprecipitation combined with mass spectrometry (Co-IP/MS), and O-GlcNAc glycosylation sites and glycan chains of BRD4 were identified using Co-IP with Nano-LC MS/MS. Real-time PCR and Western blotting were used to analyze the relative mRNA and protein expression levels of target genes, respectively. RESULTS: BRD4 was positively expressed in 61.25% (49/80) of osteosarcoma tissues. Patients with high BRD4 expression exhibited significantly shorter survival times (P<0.05). In the orthotopic mouse model, intervention with (+)-JQ1, a potent and commonly used BETi, significantly inhibited tumor growth in vivo and reduced bone destruction (P<0.05). (+)-JQ1 treatment significantly suppressed the proliferation (P<0.001), invasion (P<0.001), and migration (P<0.05) of HOS cells. In osteosarcoma cells, BRD4 exhibited O-GlcNAc modifications at both N- and C- C-termini, particularly at Thr73, which is essential for protein stability. This modification also contributed to the activation of the EGFR tyrosine kinase inhibitor resistance pathway (KEGG Pathway: hsa01521). (+)-JQ1 treatment displaced BRD4 from enhancers and downregulated the transcription of pathway-related genes, such as EGFR and PDGFC, thereby suppressing the malignant behavior of osteosarcoma cells. CONCLUSIONS BRD4 promotes osteosarcoma progression via O-GlcNAc modification at Thr73 and plays a crucial role in tumor growth and metastasis.
Osteosarcoma/drug therapy* ; Humans ; Transcription Factors/metabolism* ; Animals ; Cell Cycle Proteins ; Mice ; Bone Neoplasms/drug therapy* ; Azepines/pharmacology* ; Cell Line, Tumor ; Cell Proliferation/drug effects* ; Triazoles/pharmacology* ; Mice, Inbred C57BL ; Nuclear Proteins/metabolism* ; Gene Expression Regulation, Neoplastic ; Male ; Bromodomain Containing Proteins

OBJECTIVES: To investigate the effect of BRD4 inhibition on migration of esophageal squamous cell carcinoma (ESCC) cells with low acetyl-CoA carboxylase 1 (ACC1) expression. METHODS: ESCC cell lines with lentivirus-mediated ACC1 knockdown or transfected with a negative control sequence (shNC) were treated with DMSO, JQ1 (a BRD4 inhibitor), co-transfection with shNC-siBRD4 or siNC with additional DMSO or C646 (an ahistone acetyltransferase inhibitor) treatment, or JQ1combined with 3-MA (an autophagy inhibitor). BRD4 mRNA expression in the cells was detected using RT-qPCR. The changes in cell proliferation, migration, autophagy, and epithelial-mesenchymal transition (EMT) were examined with CCK8 assay, Transwell migration assay, and Western blotting. RESULTS: ACC1 knockdown did not significantly affect BRD4 expression in the cells but obviously increased their sensitivity to JQ1. JQ1 treatment at 1 and 2 μmol/L significantly inhibited ESCC cell proliferation, while JQ1 at 0.2 and 2 μmol/L promoted cell migration. The cells with ACC1 knockdown and JQ1 treatment showed increased expresisons of vimentin and Slug and decreased expression of E-cadherin. BRD4 knockdown promoted migration of ESCC cells, and co-transfection with shACC1 and siBRD4 resulted in increased vimentin and Slug expressions and decreased E-cadherin expression in the cells. C646 treatment of the co-transfected cells reduced acetylation levels, decreased vimentin and Slug expressions, and increased E-cadherin expression. Treatment with JQ1 alone obviously increased LC3A/B-II levels in the cells either with or without ACC1 knockdown. In the cells with ACC1 knockdown and JQ1 treatment, additional 3-MA treatment significantly decreased the expressions of vimentin, Slug and LC3A/B-II and increased the expression of E-cadherin. CONCLUSIONS BRD4 inhibition promotes autophagy of ESCC cells via a histone acetylation-dependent mechanism, thereby enhancing EMT and ultimately increasing cell migration driven by ACC1 deficiency.
Humans ; Cell Movement ; Transcription Factors/metabolism* ; Esophageal Neoplasms/metabolism* ; Cell Line, Tumor ; Cell Cycle Proteins ; Azepines/pharmacology* ; Epithelial-Mesenchymal Transition ; Carcinoma, Squamous Cell/metabolism* ; Esophageal Squamous Cell Carcinoma ; Triazoles/pharmacology* ; Nuclear Proteins/genetics* ; Cell Proliferation ; Acetyl-CoA Carboxylase/genetics* ; Transfection ; Autophagy ; Bromodomain Containing Proteins

OBJECTIVE: To investigate the effects and mechanism of bromodomain and extra-terminal (BET) inhibitor JQ1 on the double-expressor lymphoma (DEL) cell lines. METHODS: Protein expressions of cMyc and BCL-2 in 3 lymphoma cell lines were checked by Western blot so as to identify DEL cell lines. CCK-8 assay was used to detect the effects of JQ1 on anti-proliferation in the DEL cell lines. Western blot and RT-PCR were used to measure the protein and mRNA expressions of cMyc, BCL-2 and BCL-6 in DEL cell lines which treated by JQ1. Flow cytometry was used to detect the effect of JQ1 on cell apoptosis. RESULTS: Based on the expressions of cMyc and BCL-2, the SU-DHL6 and OCILY3 cell lines were confirmed as DEL cell lines. CCK-8 assay showed that the proliferation of DEL cell lines was inhibited by JQ1, which was similar to non-DEL cell lines and mainly regulated the expression of cMyc and BCL-6 but not BCL-2. JQ1 had no effects on apoptosis in the DEL cell lines. CONCLUSION BET inhibitor JQ1 has anti-tumor effect in the DEL cell lines, thus providing evidence for the therapeutic potential of BET inhibitor JQ1.
Apoptosis ; Azepines/pharmacology* ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Proto-Oncogene Proteins c-myc/metabolism* ; Sincalide/pharmacology* ; Triazoles/pharmacology* ; Xenograft Model Antitumor Assays

Bromodomain-containing 4 (BRD4) has been considered as an important requirement for disease maintenance and an attractive therapeutic target for cancer therapy. This protein can be targeted by JQ1, a selective small-molecule inhibitor. However, few studies have investigated whether BRD4 influenced acute promyelocytic leukemia (APL), and whether BRD4 had interaction with promyelocytic leukemia-retinoic acid receptor α (PML/RARα) fusion protein to some extent. Results from cell viability assay, cell cycle analysis, and Annexin-V/PI analysis indicated that JQ1 inhibited the growth of NB4 cells, an APL-derived cell line, and induced NB4 cell cycle arrest at G1 and apoptosis. Then, we used co-immunoprecipitation (co-IP) assay and immunoblot to demonstrate the endogenous interaction of BRD4 and PML/RARα in NB4 cells. Moreover, downregulation of PML/RARα at the mRNA and protein levels was observed upon JQ1 treatment. Furthermore, results from the RT-qPCR, ChIP-qPCR, and re-ChIP-qPCR assays showed that BRD4 and PML/RARα co-existed on the same regulatory regions of their target genes. Hence, we showed a new discovery of the interaction of BRD4 and PML/RARα, as well as the decline of PML/RARα expression, under JQ1 treatment.
Apoptosis ; drug effects ; Azepines ; pharmacology ; Cell Differentiation ; Down-Regulation ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Leukemia, Promyelocytic, Acute ; drug therapy ; genetics ; Nuclear Proteins ; genetics ; Promyelocytic Leukemia Protein ; genetics ; RNA, Messenger ; genetics ; Retinoic Acid Receptor alpha ; genetics ; Transcription Factors ; genetics ; Triazoles ; pharmacology ; Tumor Cells, Cultured

To investigate the effect of bromodomain and extra-terminal (BET) protein inhibitor JQ1 on expression of autoimmune-related genes in CD4+T cells from patients with systemic lupus erythematosus (SLE).
 Methods: Peripheral CD4+T cells were isolated by positive selection with CD4 microbeads. The percentage of CD4+T cells were detected by flow cytometry. CD4+T cells were treated by JQ1 at 100 nm/L for 6, 24, 48 h. The expression of T cell-related genes was measured by quantitative real-time PCR (qPCR). The secretion levels of cytokines in culture supernatant were measured by ELISA at 48 h.
 Results: The percentage of CD4+T cells isolated by CD4 microbeads is 97.2%. Compared with the control group, the mRNA expression levels of IFNG, IL-17F, IL-21, CXCR5 and FOXP3 were down-regulated at 6, 24 and 48 h (P<0.05), and IL-17A mRNA level was decreased at 6 and 24 h (P<0.01); while IL-4 mRNA level was up-regulated at 24, 48 h (P<0.01), and TGF-β1 mRNA level was up-regulated at 6 and 48 h (P<0.05) in SLE CD4+T cells treated with JQ1. The secretion levels of IFN-γ and IL-21 in JQ1-treated group were decreased significantly (P<0.05), while the secretion levels of IL-4 and TGF-β were up-regulated compared with control group (P<0.05).
 Conclusion: JQ1 can reverse the immune dysregulation and improve the immunity homeostasis in CD4+T cells from patients with SLE.
Azepines ; pharmacology ; CD4 Lymphocyte Count ; CD4-Positive T-Lymphocytes ; cytology ; drug effects ; metabolism ; Cytokines ; analysis ; metabolism ; Flow Cytometry ; Humans ; Interferon-gamma ; metabolism ; Lupus Erythematosus, Systemic ; immunology ; metabolism ; Proteins ; antagonists & inhibitors ; RNA, Messenger ; metabolism ; Time Factors ; Transforming Growth Factor beta1 ; Triazoles ; pharmacology

OBJECTIVETo investigate the molecular mechanism of action of BET bromodomain inhibitor JQ1 in treating airway remodeling in asthmatic mice.

METHODSA total of 24 mice were randomly divided into control group, ovalbumin (OVA)-induced asthma group (OVA group), and JQ1 intervention group (JQ1+OVA group), with 8 mice in each group. OVA sensitization/challenge was performed to establish a mouse model of asthma. At 1 hour before challenge, the mice in the JQ1+OVA group were given intraperitoneal injection of JQ1 solution (50 μg/g). Bronchoalveolar lavage fluid (BALF) and lung tissue samples were collected at 24 hours after the last challenge, and the total number of cells and percentage of eosinophils in BALF were calculated. Pathological staining was performed to observe histopathological changes in lung tissue. RT-PCR and Western blot were used to measure the mRNA and protein expression of E-cadherin and vimentin during epithelial-mesenchymal transition (EMT).

RESULTSCompared with the control group, the OVA group had marked infiltration of inflammatory cells in the airway, thickening of the airway wall, increased secretion of mucus, and increases in the total number of cells and percentage of eosinophils in BALF (P<0.01). Compared with the OVA group, the JQ1+OVA group had significantly alleviated airway inflammatory response and significant reductions in the total number of cells and percentage of eosinophils in BALF (P<0.01). Compared with the control group, the OVA group had significant reductions in the mRNA and protein expression of E-cadherin and significant increases in the mRNA and protein expression of vimentin (P<0.01); compared with the OVA group, the JQ1+OVA group had significant increases in the mRNA and protein expression of E-cadherin and significant reductions in the mRNA and protein expression of vimentin (P<0.01); there were no significant differences in these indices between the JQ1+OVA group and the control group (P>0.05).

CONCLUSIONSMice with OVA-induced asthma have airway remodeling during EMT. BET bromodomain inhibitor JQ1 can reduce airway inflammation, inhibit EMT, and alleviate airway remodeling, which provides a new direction for the treatment of asthma.

Airway Remodeling ; drug effects ; Animals ; Asthma ; drug therapy ; Azepines ; pharmacology ; Cadherins ; analysis ; genetics ; Epithelial-Mesenchymal Transition ; Female ; Mice ; Nuclear Proteins ; antagonists & inhibitors ; Ovalbumin ; immunology ; RNA, Messenger ; analysis ; Transcription Factors ; antagonists & inhibitors ; Triazoles ; pharmacology ; Vimentin ; analysis ; genetics

The objective of the present study was to establish a method based on principal component analysis (PCA) for the study of transdermal delivery of multiple components in Chinese medicine, and to choose the best penetration enhancers for the active fraction of Xiangfusiwu decoction (BW) with this method. Improved Franz diffusion cells with isolated rat abdomen skins were carried out to experiment on the transdermal delivery of six active components, including ferulic acid, paeoniflorin, albiflorin, protopine, tetrahydropalmatine and tetrahydrocolumbamine. The concentrations of these components were determined by LC-MS/MS, then the total factor scores of the concentrations at different times were calculated using PCA and were employed instead of the concentrations to compute the cumulative amounts and steady fluxes, the latter of which were considered as the indexes for optimizing penetration enhancers. The results showed that compared to the control group, the steady fluxes of the other groups increased significantly and furthermore, 4% azone with 1% propylene glycol manifested the best effect. The six components could penetrate through skin well under the action of penetration enhancers. The method established in this study has been proved to be suitable for the study of transdermal delivery of multiple components, and it provided a scientific basis for preparation research of Xiangfusiwu decoction and moreover, it could be a reference for Chinese medicine research.
Administration, Cutaneous ; Alkenes ; pharmacology ; Animals ; Azepines ; pharmacology ; Benzophenanthridines ; isolation & purification ; pharmacokinetics ; Berberine Alkaloids ; isolation & purification ; pharmacokinetics ; Bridged-Ring Compounds ; isolation & purification ; pharmacokinetics ; Coumaric Acids ; isolation & purification ; pharmacokinetics ; Drug Combinations ; Drug Synergism ; Drugs, Chinese Herbal ; administration & dosage ; chemistry ; isolation & purification ; pharmacokinetics ; Glucosides ; isolation & purification ; pharmacokinetics ; In Vitro Techniques ; Male ; Monoterpenes ; isolation & purification ; pharmacokinetics ; Permeability ; Plants, Medicinal ; chemistry ; Principal Component Analysis ; Rats ; Rats, Sprague-Dawley ; Skin Absorption ; drug effects

Farnesoid X receptor (FXR) belongs to the nuclear receptor superfamily. It is highly related to the formation of metabolic syndrome and the glucose homeostasis, and therefore represents an important drug target against metabolic diseases and diabetes. In recent years, great progress has been made in the agonists, antagonists, and crystal structures of FXR. The diverse FXR ligands and their structure-activity relationship are reviewed in this article. The advances in the crystal structures of FXR in complex with different ligands are also introduced.
Animals ; Anticholesteremic Agents ; chemical synthesis ; chemistry ; pharmacology ; Azepines ; chemical synthesis ; chemistry ; pharmacology ; Benzene Derivatives ; chemical synthesis ; chemistry ; pharmacology ; Chenodeoxycholic Acid ; analogs & derivatives ; chemical synthesis ; chemistry ; pharmacology ; Crystallization ; Humans ; Indoles ; chemical synthesis ; chemistry ; pharmacology ; Isoxazoles ; chemical synthesis ; chemistry ; pharmacology ; Ligands ; Molecular Structure ; Multienzyme Complexes ; chemical synthesis ; chemistry ; pharmacology ; Pregnenediones ; chemical synthesis ; chemistry ; pharmacology ; Receptors, Cytoplasmic and Nuclear ; agonists ; antagonists & inhibitors ; metabolism ; Structure-Activity Relationship

OBJECTIVETo study the effect of different penetration enhancers on the in vitro transdermal permeation of 1-tethrahydropalmatine (L-THP) through rat skin.

METHODBoth natural and chemical synthesis penetration enhancers were applied singly or jointly to investigate the skin permeation rates of l-THP. The skin permeation profiles were evaluated by Valian-Chien permeation cells with isolated rat skin. HPLC-UV method was established to determine the concentration of l-THP in samples.

RESULTAs chemical synthesis penetration enhancer was used alone, 8% azone was observed to be the optimal penetration enhancer with the maximal penetration rate of 21.153 microg x cm(-20 x h(-1). Although 2% menthol crystal or 5% eucalyptus oil was effective as a natural penetration enhancer when used alone, the average penetration rate reached only half of that of 8% azone. The penetration potency of either menthol oil or menthol crystal combined with 8% azone was more effective than that of azone alone (P < 0.05).

CONCLUSIONEither menthol oil or menthol crystal combined with 8% azone is effective on transdermal penetration of l-THP in vitro. There is significant synergistic effect when natural penetration enhancers combined with chemical synthesis penetration enhancers.

Administration, Cutaneous ; Animals ; Azepines ; pharmacology ; Berberine Alkaloids ; analysis ; pharmacokinetics ; Drug Synergism ; Eucalyptus ; chemistry ; Male ; Menthol ; pharmacology ; Oils, Volatile ; pharmacology ; Permeability ; drug effects ; Rats ; Rats, Sprague-Dawley ; Skin ; drug effects ; metabolism ; Skin Absorption ; drug effects

OBJECTIVETo investigate the different permeation enhancers on the transdermal permeation of Xiao'er Niuhuang tuire cataplasms (XNTC).

METHODUsing improved franz-type diffusion cell with excised rat skin in vitro as the transdermal barrier, the content of permeated geniposide was determined by HPLC to study the kinetic parameters such as cumulative permeation quantity and permeation rate.

RESULTThe result showed that the process of penetrating of geniposide in XNTC through skin could be in accordance with zero-rade releasing equation and XNTC was stable during the course of experiment.

CONCLUSION5% Propylene glycol (PG)-azone (2:3) has the best permeation-enhancing effect, and the results provided a primary basis for the future research on Xiao'er Niuhuang tuire cataplasms.

Animals ; Azepines ; pharmacology ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; chemistry ; In Vitro Techniques ; Iridoids ; chemistry ; Pharmaceutical Vehicles ; pharmacology ; Propylene Glycol ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Skin ; drug effects ; metabolism ; Skin Absorption ; drug effects