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

Saponins associated with Panax notoginseng (P. notoginseng) demonstrate significant therapeutic efficacy across multiple diseases. However, certain high-yield saponins face limited clinical applications due to their reduced pharmacological efficacy. This study synthesized and evaluated 36 saponin-1,2,3-triazole derivatives of ginsenosides Rg1/Rb1 and notoginsenoside R1 for anti-adipogenesis activity in vitro. The research revealed that the ginsenosides Rg1-1,2,3-triazole derivative a17 demonstrates superior adipogenesis inhibitory effects. Structure-activity relationships (SARs) analysis indicates that incorporating an amidyl-substituted 1,2,3-triazole into the saponin side chain via Click reaction enhances anti-adipogenesis activity. Additionally, several other derivatives exhibit general adipogenesis inhibition. Compound a17 demonstrated enhanced potency compared to the parent ginsenoside Rg1. Mechanistic investigations revealed that a17 exhibits dose-dependent inhibition of adipogenesis in vitro, accompanied by decreased expression of preadipocytes. Peroxisome proliferator-activated receptor γ (PPARγ), fatty acid synthase (FAS), and fatty acid binding protein 4 (FABP4) adipogenesis regulators. These findings establish the ginsenoside Rg1-1,2,3-triazole derivative a17 as a promising adipocyte differentiation inhibitor and potential therapeutic agent for obesity and associated metabolic disorders. This research provides a foundation for developing effective therapeutic approaches for various metabolic syndromes.
Adipogenesis/drug effects* ; Triazoles/chemical synthesis* ; Ginsenosides/chemical synthesis* ; Saponins/chemical synthesis* ; Animals ; Mice ; Structure-Activity Relationship ; PPAR gamma/genetics* ; 3T3-L1 Cells ; Adipocytes/metabolism* ; Panax notoginseng/chemistry* ; Drug Design ; Molecular Structure ; Humans ; Cell Differentiation/drug effects* ; Fatty Acid-Binding Proteins/genetics*

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

Objective: This research was performed to evaluate the effect of tebuconazole (TBZ) on reproductive organs of male rats and to assess the protective role of combined essential trace elements in alleviating the detrimental effect of TBZ on male reproductive function. Methods: For this purpose, 48 rats were exposed to 100 mg/kg TBZ, TBZ supplemented with zinc (Zn), selenium (Se), copper (Cu), and iron (Fe), TBZ + (Se + Zn); TBZ + Cu; or TBZ + Fe. The experiment was conducted for 30 consecutive days. Results: TBZ caused a significant perturbation in mineral levels and reduction in reproductive organs weights, plasma testosterone level, and testicular antioxidant enzyme activities. The TBZ-treated group also showed a significant increase in sperm abnormalities (count, motility, and viability percent), plasma follicle-stimulating hormone and luteinizing hormone concentrations, lipid peroxidation, protein oxidation, and severe DNA degradation in comparison with the controls. Histopathologically, TBZ caused testis impairments. Conversely, treatment with trace elements, in combination or alone, improved the reproductive organ weights, sperm characteristics, TBZ-induced toxicity, and histopathological modifications in testis. Conclusion TBZ exerts significant harmful effects on male reproductive system. The concurrent administration of trace elements reduces testis dysfunction, fertility, and toxicity induced by TBZ.
Animal Feed/analysis* ; Animals ; Antioxidants/metabolism* ; Diet ; Dietary Supplements/analysis* ; Fungicides, Industrial/adverse effects* ; Male ; Minerals/metabolism* ; Mutagenicity Tests ; Rats ; Rats, Wistar ; Spermatozoa/physiology* ; Testis/physiology* ; Trace Elements/metabolism* ; Triazoles/adverse effects*

OBJECTIVE: To evaluate the synergy of the Burkholderia signaling molecule cis-2-dodecenoic acid (BDSF) and fluconazole (FLU) or itraconazole (ITRA) against two azole-resistant C. albicans clinical isolates in vitro and in vivo. METHODS: Minimum inhibitory concentrations (MICs) of antibiotics against two azole-resistant C. albicans were measured by the checkerboard technique, E-test, and time-kill assay. In vivo antifungal synergy testing was performed on mice. Analysis of the relative gene expression levels of the strains was conducted by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). RESULTS: BDSF showed highly synergistic effects in combination with FLU or ITRA with a fractional inhibitory concentration index of ⪕ 0.08. BDSF was not cytotoxic to normal human foreskin fibroblast cells at concentrations of up to 300 μg/mL. The qRT-PCR results showed that the combination of BDSF and FLU/ITRA significantly inhibits the expression of the efflux pump genes CDR1 and MDR1 via suppression of the transcription factors TAC1 and MRR1, respectively, when compared with FLU or ITRA alone. No dramatic difference in the mRNA expression levels of ERG1, ERG11, and UPC2 was found, which indicates that the drug combinations do not significantly interfere with UPC2-mediated ergosterol levels. In vivo experiments revealed that combination therapy can be an effective therapeutic approach to treat candidiasis. CONCLUSION The synergistic effects of BDSF and azoles may be useful as an alternative approach to control azole-resistant Candida infections.
Antifungal Agents ; pharmacology ; Burkholderia cenocepacia ; chemistry ; Candida albicans ; drug effects ; physiology ; Candidiasis ; drug therapy ; Drug Resistance, Fungal ; Fatty Acids, Monounsaturated ; adverse effects ; Fluconazole ; pharmacology ; Humans ; Microbial Sensitivity Tests ; Triazoles ; metabolism

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

BACKGROUND/AIMS: The treatment of chronic myeloid leukemia (CML) has achieved impressive success since the development of the Bcr-Abl tyrosine kinase inhibitor, imatinib mesylate. Nevertheless, resistance to imatinib has been observed, and a substantial number of patients need alternative treatment strategies. METHODS: We have evaluated the effects of deferasirox, an orally active iron chelator, and imatinib on K562 and KU812 human CML cell lines. Imatinib-resistant CML cell lines were created by exposing cells to gradually increasing concentrations of imatinib. RESULTS: Co-treatment of cells with deferasirox and imatinib induced a synergistic dose-dependent inhibition of proliferation of both CML cell lines. Cell cycle analysis showed an accumulation of cells in the subG1 phase. Western blot analysis of apoptotic proteins showed that co-treatment with deferasirox and imatinib induced an increased expression of apoptotic proteins. These tendencies were clearly identified in imatinib-resistant CML cell lines. The results also showed that co-treatment with deferasirox and imatinib reduced the expression of BcrAbl, phosphorylated Bcr-Abl, nuclear factor-kappaB (NF-kappaB) and beta-catenin. CONCLUSIONS: We observed synergistic effects of deferasirox and imatinib on both imatinib-resistant and imatinib-sensitive cell lines. These effects were due to induction of apoptosis and cell cycle arrest by down-regulated expression of NF-kappaB and beta-catenin levels. Based on these results, we suggest that a combination treatment of deferasirox and imatinib could be considered as an alternative treatment option for imatinib-resistant CML.
Antineoplastic Agents/*pharmacology ; Apoptosis/drug effects ; Apoptosis Regulatory Proteins/metabolism ; Benzoates/*pharmacology ; Cell Proliferation/drug effects ; Dose-Response Relationship, Drug ; Drug Resistance, Neoplasm/*drug effects ; G1 Phase Cell Cycle Checkpoints/drug effects ; Humans ; Imatinib Mesylate/*pharmacology ; Iron Chelating Agents/*pharmacology ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/*drug therapy/metabolism ; Protein Kinase Inhibitors/*pharmacology ; Signal Transduction/drug effects ; Triazoles/*pharmacology

OBJECTIVE: To explore the effect of compound malt pills (CMP) on polycystic ovarian syndrome (PCOS) rat model induced by letrozole and the underlying mechanisms.
 METHODS: To establish a PCOS rat model, 48 female SD rats aged 6 weeks were randomly divided into 6 groups (n=8): A normal group, a model control group, a positive control group, a low-dose CMP group, a middle-dose CMP group, and a high-dose CMP group. Rats were treated for 21 days after the PCOS model was successfully established. Ovarian morphology changes were observed, and the expressions of ERα and ERβ was examined by immunohistochemistry, Western blot and RT-PCR, respectively.
 RESULTS: Compared with the normal group, the number of follicular cystic dilatation in the model control group was increased and the granulosa cells were decreased. After the treatment, the number of follicular cystic dilatation was reduced compared with the model control group, but the primordial follicles, corpus luteum and granulosa cells were increased. The expressions of ERα and ERβ in the model control group were significantly decreased (P<0.01), which were increased in the intervention groups (P<0.05 or P<0.01).
 CONCLUSION CMP may play a role in the treatment of PCOS by regulating the expressions of ERα and ERβ.
Animals ; Corpus Luteum ; drug effects ; Disease Models, Animal ; Drugs, Chinese Herbal ; pharmacology ; Estrogen Receptor alpha ; metabolism ; Estrogen Receptor beta ; metabolism ; Female ; Granulosa Cells ; drug effects ; Letrozole ; Nitriles ; adverse effects ; Ovarian Follicle ; drug effects ; Polycystic Ovary Syndrome ; chemically induced ; metabolism ; Rats ; Rats, Sprague-Dawley ; Triazoles ; adverse effects

OBJECTIVETo evaluate the inhibitory effect of genistin combined with anastrozole on the growth and apoptosis of breast tumor tissue, and to study their anti-cancer mechanism by using the model of 7,12-dimethylbenz [alpha] anthracene (DMBA)-induced mammary tumors following ovariectomy in Sprague-Dawley (SD) rats.

METHODSThe DMBA induced postmenopausal SD rats were randomly divided into the control group, the genistein group, the anastrozole group, and the genistein combined with anastrozole group. The growth of tumors was observed in each group. The proliferation index and apoptosis index of tumor cells were determined. Moreover, estradiol (E2) and 17beta-HSD1 mRNA levels were determined by ELISA and RT-PCR respectively.

RESULTSThe tumor growth was inhibited in the genistein group and the anastrozole group. The inhibitory ratio was significantly higher in the genistein combined with anastrozole group (P < 0.05). Compared with the control group, levels of E2 and 17beta-HSD1 mRNA decreased more significantly in the genistein combined with anastrozole group (P < 0.05).

CONCLUSIONSGenistein could suppress the growth of mammary tumors in postmenopausal rats. It showed synergistic effect when combined with anastrozole, which resulted in reduced levels of E2 and 17beta-HSD1 mRNA. It had inhibitory effect on the growth of breast tumors.

17-Hydroxysteroid Dehydrogenases ; metabolism ; Animals ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Estradiol ; metabolism ; Female ; Genistein ; administration & dosage ; pharmacology ; Mammary Neoplasms, Experimental ; chemically induced ; pathology ; Nitriles ; administration & dosage ; pharmacology ; Ovariectomy ; Postmenopause ; Rats ; Rats, Sprague-Dawley ; Triazoles ; administration & dosage ; pharmacology