ABSTRACT
Dental pulp stem cells (DPSC) have shown osteogenic and bone regenerative potential. Improving the in situ bone regeneration potential of DPSC is crucial for their application as seed cells during bone defect reconstruction in clinics. This study aimed to develop DPSC-derived organoid-like microspheroids as effective seeds for bone tissue engineering applications. DPSC osteogenic microspheroids (70 µm diameter) were cultured in a polydimethylsiloxane-mold-based agarose-gel microwell-culture-system with or without cannabidiol (CBD)-treatment. Results of in vitro studies showed higher osteogenic differentiation potential of microspheroids compared with 2D-cultured-DPSC. CBD treatment further improved the osteogenic differentiation potential of microspheroids. The effect of CBD treatment in the osteogenic differentiation of microspheroids was more pronounced compared with that of CBD-treated 2D-cultured-DPSC. Microspheroids showed a higher degree of bone regeneration in nude mice calvarial bone defect compared to 2D-cultured-DPSC. CBD-treated microspheroids showed the most robust in situ bone regenerative potential compared with microspheroids or CBD-treated 2D-cultured-DPSC. According to mRNA sequencing, bioinformatic analysis, and confirmation study, the higher osteogenic potential of CBD-treated microspheroids was mainly attributed to WNT6 upregulation. Taken together, DPSC microspheroids have robust osteogenic potential and can effectively translate the effect of in vitro osteoinductive stimulation during in situ bone regeneration, indicating their application potential during bone defect reconstruction in clinics.
Subject(s)
Cannabidiol , Cell Differentiation , Dental Pulp , Osteogenesis , Stem Cells , Up-Regulation , Osteogenesis/drug effects , Animals , Cannabidiol/pharmacology , Stem Cells/drug effects , Stem Cells/metabolism , Stem Cells/cytology , Mice , Up-Regulation/drug effects , Dental Pulp/cytology , Dental Pulp/drug effects , Cell Differentiation/drug effects , Organoids/drug effects , Organoids/metabolism , Humans , Mice, Nude , Cells, Cultured , Bone Regeneration/drug effectsABSTRACT
Soil-transmitted helminths (STHs) are major pathogens infecting over a billion people. There are few classes of anthelmintics and there is an urgent need for new drugs. Many STHs use an unusual form of anaerobic metabolism to survive the hypoxic conditions of the host gut. This requires rhodoquinone (RQ), a quinone electron carrier. RQ is not made or used by vertebrate hosts making it an excellent therapeutic target. Here we screen 480 structural families of natural products to find compounds that kill Caenorhabditis elegans specifically when they require RQ-dependent metabolism. We identify several classes of compounds including a family of species-selective inhibitors of mitochondrial respiratory complex I. These identified complex I inhibitors have a benzimidazole core and we determine key structural requirements for activity by screening 1,280 related compounds. Finally, we show several of these compounds kill adult STHs. We suggest these species-selective complex I inhibitors are potential anthelmintics.
Subject(s)
Anthelmintics , Caenorhabditis elegans , Electron Transport Complex I , Ubiquinone/analogs & derivatives , Animals , Anthelmintics/pharmacology , Anthelmintics/chemistry , Electron Transport Complex I/antagonists & inhibitors , Electron Transport Complex I/metabolism , Caenorhabditis elegans/metabolism , Benzimidazoles/pharmacology , Benzimidazoles/chemistry , Species Specificity , Quinones/chemistry , Quinones/pharmacology , Quinones/metabolism , Biological Products/pharmacology , Biological Products/chemistryABSTRACT
Sjogren's syndrome (SS) is an autoimmune disease characterized by dysfunction of exocrine glands, such as salivary glands. However, the molecular mechanism of salivary secretion dysfunction in SS is still unclear. Given the significance of glutathione peroxidase 4 (GPX4) in cellular redox homeostasis, we hypothesized that dysregulation of GPX4 may play a pivotal role in the pathogenesis of salivary secretion dysfunction observed in SS. The salivary gland of SS patients and the SS mouse model exhibited reduced expression of the ferroptosis inhibitor GPX4 and the important protein aquaporin 5 (AQP5), which is involved in salivary secretion. GPX4 overexpression upregulated and GPX4 knockdown downregulated AQP5 expression in salivary gland epithelial cells (SGECs) and salivary secretion. Bioinformatics analysis of GSE databases from SS patients' salivary glands revealed STAT4 as a key intermediary regulator between GPX4 and AQP5. A higher level of nuclear pSTAT4 was observed in the salivary gland of the SS mouse model. GPX4 overexpression inhibited and GPX4 knockdown promoted STAT4 phosphorylation and nuclear translocation in SGECs. CHIP assay confirmed the binding of pSTAT4 within the promoter of AQP5 inhibiting AQP5 transcription. GPX4 downregulation accumulates intracellular lipid ROS in SGECs. Lipid ROS inhibitor ferrostatin-1 treatment during in vitro and in vivo studies confirmed that lipid ROS activates STAT4 phosphorylation and nuclear translocation in SGECs. In summary, the downregulated GPX4 in SGECs contributes to salivary secretion dysfunction in SS via the lipid ROS/pSTAT4/AQP5 axis. This study unraveled novel targets to revitalize the salivary secretion function in SS patients.
Subject(s)
Aquaporin 5 , Epithelial Cells , Phospholipid Hydroperoxide Glutathione Peroxidase , Reactive Oxygen Species , STAT4 Transcription Factor , Salivary Glands , Sjogren's Syndrome , Sjogren's Syndrome/metabolism , Sjogren's Syndrome/genetics , Sjogren's Syndrome/pathology , Animals , Humans , Mice , Salivary Glands/metabolism , Salivary Glands/pathology , Aquaporin 5/metabolism , Aquaporin 5/genetics , Epithelial Cells/metabolism , Epithelial Cells/pathology , Phospholipid Hydroperoxide Glutathione Peroxidase/metabolism , Phospholipid Hydroperoxide Glutathione Peroxidase/genetics , Reactive Oxygen Species/metabolism , STAT4 Transcription Factor/metabolism , STAT4 Transcription Factor/genetics , Disease Models, Animal , Female , Down-Regulation , Male , Signal Transduction , Gene Expression Regulation , Ferroptosis/genetics , Saliva/metabolism , Middle AgedABSTRACT
BACKGROUND: Sjögren's syndrome (SS) is a chronic autoimmune disease that predominantly affects exocrine glands. Previous studies have demonstrated that upregulated interferon-gamma (IFN-γ) in SS triggers ferroptosis in salivary gland epithelial cells (SGECs), resulting in impaired salivary gland secretion. However, the immune cells responsible for secreting IFN-γ remain unclear. Therefore, this study conducted bioinformatics analysis and molecular validation to identify the origin of IFN-γ in SS salivary gland. METHODS: The 'limma' package in R software was utilized to identify differentially expressed genes (DEGs) in the human SS dataset. Subsequently, the identified DEGs were compared with the ferroptosis database and screened through Cytoscape to determine candidate genes. The cellular localization and expression patterns of candidate genes were further confirmed in the salivary gland single-cell RNA sequence (scRNA-seq) data set from healthy control and SS mice. Furthermore, in vitro and in vivo studies were performed to analyze the effect of CD4 T-secreted IFN-γ on SGECs' ferroptosis and functions. RESULTS: Upregulated TLR4, IFNG, and IL33 were screened as candidates ferroptosis ferroptosis-inducing genes in SS salivary glands. The association of IFNG and IL33 with CD4 T cells was established through immune infiltration analysis. The expression of IFN-γ on CD4 T cells was robustly higher compared with that of IL33 as evidenced by scRNA-seq and immunofluorescence co-localization. Subsequent experiments conducted on candidate genes consistently demonstrated the potent ability of IFN-γ to induce SGECs' ferroptosis and inhibit AQP5 expression. CONCLUSIONS: Our findings indicate that CD4 T cell-secreted IFN-γ in SS induces SGECs' ferroptosis and inhibits AQP5 expression.
Subject(s)
Ferroptosis , Sjogren's Syndrome , Humans , Animals , Mice , Interferon-gamma/metabolism , CD4-Positive T-Lymphocytes , Interleukin-33/metabolism , Salivary Glands , Epithelial Cells/metabolismABSTRACT
Myc belongs to a family of proto-oncogenes that encode transcription factors. The overexpression of c-Myc causes many types of cancers. Recently, we established a system for screening c-Myc inhibitors and identified antimycin A by screening the RIKEN NPDepo chemical library. The specific mechanism of promoting tumor cell metastasis by high c-Myc expression remains to be explained. In this study, we screened approximately 5,600 microbial extracts using this system and identified a broth prepared from Streptomyces sp. RK19-A0402 strongly inhibits c-Myc transcriptional activity. After purification of the hit broth, we identified compounds closely related to the aglycone of cytovaricin and had a structure similar to that of oligomycin A. Similar to oligomycin A, the hit compounds inhibited mitochondrial complex V. The mitochondria dysfunction caused by the compounds induced the production of reactive oxygen species (ROS), and the ROS activated GSK3α/ß that phosphorylated c-Myc for ubiquitination. This study provides a successful screening strategy for identifying natural products as potential c-Myc inhibitors as potential anticancer agents.
Subject(s)
Proto-Oncogene Proteins c-myc , Ubiquitin , Humans , Ubiquitin/metabolism , Reactive Oxygen Species , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , OligomycinsABSTRACT
ß-transducin repeat-containing protein (ß-TrCP) is an F-box protein subunit of the E3 Skp1-Cullin-F box (SCF) type ubiquitin-ligase complex, and provides the substrate specificity for the ligase. To find potent ligands of ß-TrCP useful for the proteolysis targeting chimera (PROTAC) system using ß-TrCP in the future, we developed a high-throughput screening system for small molecule ß-TrCP ligands. We screened the chemical library utilizing the system and obtained several hit compounds. The effects of the hit compounds on in vitro ubiquitination activity of SCFß-TrCP1 and on downstream signaling pathways were examined. Hit compounds NPD5943, NPL62020-01, and NPL42040-01 inhibited the TNFα-induced degradation of IκBα and its phosphorylated form. Hence, they inhibited the activation of the transcription activity of NF-κB, indicating the effective inhibition of ß-TrCP by the hit compounds in cells. Next, we performed an in silico analysis of the hit compounds to determine the important moieties of the hit compounds. Carboxyl groups of NPL62020-01 and NPL42040-01 and hydroxyl groups of NPD5943 created hydrogen bonds with ß-TrCP similar to those created by intrinsic target phosphopeptides of ß-TrCP. Our findings enhance our knowledge of useful small molecule ligands of ß-TrCP and the importance of residues that can be ligands of ß-TrCP.
Subject(s)
SKP Cullin F-Box Protein Ligases , beta-Transducin Repeat-Containing Proteins , Humans , beta-Transducin Repeat-Containing Proteins/metabolism , SKP Cullin F-Box Protein Ligases/metabolism , High-Throughput Screening Assays , Ligands , Cullin ProteinsABSTRACT
Sjogren's syndrome is an autoimmune disease in middle and old-aged women with a dry mucosal surface, which is caused by the dysfunction of secretory glands, such as the oral cavity, eyeballs, and pharynx. Pathologically, Sjogren's syndrome are characterized by lymphocyte infiltration into the exocrine glands and epithelial cell destruction caused by autoantibodies Ro/SSA and La/SSB. At present, the exact pathogenesis of Sjogren's syndrome is unclear. Evidence suggests epithelial cell death and the subsequent dysfunction of salivary glands as the main causes of xerostomia. This review summarizes the modes of salivary gland epithelial cell death and their role in Sjogren's syndrome progression. The molecular mechanisms involved in salivary gland epithelial cell death during Sjogren's syndrome as potential leads to treating the disease are also discussed.
Subject(s)
Sjogren's Syndrome , Xerostomia , Female , Humans , Middle Aged , Aged , Sjogren's Syndrome/metabolism , Sjogren's Syndrome/pathology , Salivary Glands/pathology , Autoantibodies , Xerostomia/complications , Epithelial Cells/metabolism , Epithelial Cells/pathologyABSTRACT
Alzheimer's disease (AD) is the most common neurodegenerative condition in civilizations worldwide. The distinctive occurrence of amyloid-beta (Aß) accumulation into insoluble fibrils is part of the disease pathophysiology with Aß42 being the most toxic and aggressive Aß species. The polyphenol, p-Coumaric acid (pCA), has been known to boost a number of therapeutic benefits. Here, pCA's potential to counteract the negative effects of Aß42 was investigated. First, pCA was confirmed to reduce Aß42 fibrillation using an in vitro activity assay. The compound was next examined on Aß42-exposed PC12 neuronal cells and was found to significantly decrease Aß42-induced cell mortality. pCA was then examined using an AD Drosophila melanogaster model. Feeding of pCA partially reversed the rough eye phenotype, significantly lengthened AD Drosophila's lifespan, and significantly enhanced the majority of the AD Drosophila's mobility in a sex-dependent manner. The findings of this study suggest that pCA may have therapeutic benefits for AD.
Subject(s)
Alzheimer Disease , Animals , Alzheimer Disease/drug therapy , Alzheimer Disease/genetics , Drosophila , Drosophila melanogaster , Amyloid beta-Peptides , Peptide FragmentsABSTRACT
c-Myc is a critical regulator of cell proliferation and growth. Elevated levels of c-Myc cause transcriptional amplification, leading to various types of cancers. Small molecules that specifically inhibit c-Myc-dependent regulation are potentially invaluable for anticancer therapy. Because c-Myc does not have enzymatic activity or targetable pockets, researchers have attempted to obtain small molecules that inhibit c-Myc cofactors, activate c-Myc repressors, or target epigenetic modifications to regulate the chromatin of c-Myc-addicted cancer without any clinical success. In this study, we screened for c-Myc inhibitors using a cell-dependent assay system in which the expression of c-Myc and its transcriptional activity can be inferred from monomeric Keima and enhanced GFP fluorescence, respectively. We identified one mitochondrial inhibitor, antimycin A, as a hit compound. The compound enhanced the c-Myc phosphorylation of threonine-58, consequently increasing the proteasome-mediated c-Myc degradation. The mechanistic analysis of antimycin A revealed that it enhanced the degradation of c-Myc protein through the activation of glycogen synthetic kinase 3 by reactive oxygen species (ROS) from damaged mitochondria. Furthermore, we found that the inhibition of cell growth by antimycin A was caused by both ROS-dependent and ROS-independent pathways. Interestingly, ROS-dependent growth inhibition occurred only in the presence of c-Myc, which may reflect the representative features of cancer cells. Consistently, the antimycin A sensitivity of cells was correlated to the endogenous c-Myc levels in various cancer cells. Overall, our study provides an effective strategy for identifying c-Myc inhibitors and proposes a novel concept for utilizing ROS inducers for cancer therapy.
Subject(s)
Antimycin A , Proteolysis , Proto-Oncogene Proteins c-myc , Antimycin A/pharmacology , Cell Line, Tumor , High-Throughput Screening Assays , Phosphorylation , Proto-Oncogene Proteins c-myc/antagonists & inhibitors , Proto-Oncogene Proteins c-myc/metabolism , Reactive Oxygen Species/metabolism , Threonine/metabolism , Proteolysis/drug effects , Transcription, Genetic/drug effects , Antineoplastic Agents/pharmacology , HCT116 Cells , HeLa Cells , Cell Survival/drug effects , HumansABSTRACT
Alzheimer's disease (AD) is the most prevalent type of dementia globally. The accumulation of amyloid-beta (Aß) extracellular senile plaques in the brain is one of the hallmark mechanisms found in AD. Aß42 is the most damaging and aggressively aggregating Aß isomer produced in the brain. Although Aß42 has been extensively researched as a crucial peptide connected to the development of the characteristic amyloid fibrils in AD, the specifics of its pathophysiology are still unknown. Therefore, the main objective was to identify novel compounds that could potentially mitigate the negative effects of Aß42. 3-[[(3S)-1,2,3,4-Tetrahydroisoquinoline-3-carbonyl]amino]propanoic acid (THICAPA) was identified as a ligand for Aß42 and for reducing fibrillary Aß42 aggregation. THICAPA also improved cell viability when administered to PC12 neuronal cells that were exposed to Aß42. Additionally, this compound diminished Aß42 toxicity in the current AD Drosophila model by rescuing the rough eye phenotype, prolonging the life span, and enhancing motor functions. Through next-generation RNA-sequencing, immune response pathways were downregulated in response to THICAPA treatment. Thus, this study suggests THICAPA as a possible disease-modifying treatment for AD.
Subject(s)
Alzheimer Disease , Tetrahydroisoquinolines , Animals , Alzheimer Disease/drug therapy , Alzheimer Disease/metabolism , Drosophila/metabolism , Propionates/pharmacology , Amyloid beta-Peptides/metabolism , Peptide Fragments , Tetrahydroisoquinolines/pharmacologyABSTRACT
BACKGROUND: Restoration of salivary gland function in Sjogren's syndrome (SS) is still a challenge. Dental pulp stem cells (DPSCs) derived exosomes had shown anti-inflammatory, anti-oxidative, immunomodulatory, and tissue function restorative abilities. However, the salivary gland function restoration potential of DPSCs-derived exosomes (DPSC-Exos) during SS has not been investigated yet. METHODS: DPSC-Exos was isolated by ultracentrifugation methods and characterized. Salivary gland epithelial cells (SGEC) were treated with interferon-gamma (IFN-γ) to mimic SS in vitro and cultured with or without DPSC-Exos. SGEC survival and aquaporin 5 (AQP5) expression were analyzed. mRNA sequencing and bioinformatics analysis were performed in IFN-γ vs. DPSC-Exos+ IFN-γ treated SGEC. Non-obese diabetic (NOD)/ltj female mice (SS model), were intravenously administered with DPSC-Exos, and salivary gland functions and SS pathogenicity were analyzed. Furthermore, the mRNA sequencing and bioinformatics predicted mechanism of the therapeutic effect of DPSC-Exos was further investigated both in vitro and in vivo using RT-qPCR, Western blot, immunohistochemistry, immunofluorescence, flowcytometry analysis. RESULTS: DPSC-Exos partially rescued IFN-γ triggered SGEC death. IFN-γ inhibited AQP5 expression in SGEC and DPSC-Exos reversed this effect. Transcriptome analysis showed GPER was the upregulated DEG in DPSC-Exos-treated SGEC with a positive correlation with salivary secretion-related DEGs. Pathway enrichment analysis revealed that DEGs were mainly attributed to estrogen 16 alpha-hydroxylase activity, extracellular exosome function, cAMP signaling, salivary secretion, and estrogen signaling. Intravenous injection of DPSC-Exos in NOD/ltj mice alleviated the SS syndrome as indicated by the increased salivary flow rate, attenuated glandular inflammation, and increased AQP5 expression. GPER was also upregulated in the salivary gland of DPSC-Exos-treated NOD/ltj mice compared with the PBS-treated NOD/ltj mice. IFN-γ+DPSC-Exos-treated SGEC showed higher expression of AQP5, p-PKA, cAMP, and intracellular Ca2+ levels compared with IFN-γ-treated SGEC. These effects were reversed by the inhibition of GPER. CONCLUSIONS: Our results showed that DPSC-Exos revitalize salivary gland epithelial cell function during SS via the GPER-mediated cAMP/PKA/CREB pathway suggesting the possible therapeutic potential of DPSC-Exos in SS-treatment.
Subject(s)
Dental Pulp , Exosomes , Salivary Glands , Sjogren's Syndrome , Humans , Animals , Mice , Dental Pulp/cytology , Cells, Cultured , Exosomes/metabolism , Female , Mice, Inbred NOD , Interferon-gamma/pharmacology , Salivary Glands/cytology , Epithelial Cells/metabolism , Sjogren's Syndrome/therapyABSTRACT
The elevated level of interferon-γ (IFN-γ) in Sjogren's syndrome (SS) triggers salivary gland epithelial cells (SGEC) death. However, the underlying mechanisms of IFN-γ-induced SGEC death modes are still not fully elucidated. We found that IFN-γ triggers SGEC ferroptosis via Janus kinase/signal transducer and activator of transcription 1 (JAK/STAT1)-mediated inhibition of cystine-glutamate exchanger (System Xc-). Transcriptome analysis revealed that ferroptosis-related markers are differentially expressed in SS human and mouse salivary glands with distinct upregulation of IFN-γ and downregulation of glutathione peroxidase 4 (GPX4) and aquaporin 5 (AQP5). Inducing ferroptosis or IFN-γ treatment in the Institute of cancer research (ICR) mice aggravated and inhibition of ferroptosis or IFN-γ signaling in SS model non-obese diabetic (NOD) mice alleviated ferroptosis in the salivary gland and SS symptoms. IFN-γ activated STAT1 phosphorylation and downregulated system Xc- components solute carrier family 3 member 2 (SLC3A2), glutathione, and GPX4 thereby triggering ferroptosis in SGEC. JAK or STAT1 inhibition in SGEC rescued IFN-γ-downregulated SLC3A2 and GPX4 as well as IFN-γ-induced cell death. Our results indicate the role of ferroptosis in SS-related death of SGEC and SS pathogenicity.
Subject(s)
Ferroptosis , Sjogren's Syndrome , Animals , Humans , Mice , Epithelial Cells/metabolism , Ferroptosis/genetics , Interferon-gamma/metabolism , Mice, Inbred NOD , Salivary Glands/metabolism , Sjogren's Syndrome/genetics , Sjogren's Syndrome/metabolism , STAT1 Transcription Factor/genetics , STAT1 Transcription Factor/metabolism , Janus Kinases/metabolismABSTRACT
Diffuse large B-cell lymphoma (DLBCL) is a highly heterogeneous malignancy. Epidemiologically, the incidence of DLBCL is higher in men, and the female sex is a favorable prognostic factor, which can be explained by estrogen. This study aimed to explore the potential targets of the estrogen receptor (ER) signaling pathway and provide a meaningful way to treat DLBCL patients. Datasets were obtained from the Gene Expression Omnibus (GEO) to identify differentially expressed genes (DEGs). Representative gene sets estrogen receptor pathways, and growth regulatory pathways were identified based on Gene Set Enrichment Analysis (GSEA) analysis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used for function and pathway analysis. STRING and Cytoscape were used to construct the interaction network, and the MCODE plug-in performed the module analysis. GEPIA, TCGA, and LOGpc databases were used for expression and predictive analysis. The Human Protein Atlas (HPA) database was used to analyze the protein expression levels, cBioPortal was used to explore genetic alterations, and ROC analysis and prognostic assessment were used to predict the diagnostic value of genes. Finally, BJAB cells were treated with ER inhibitor fulvestrant and specific shRNA, and the expression of hub genes was verified by RT-qPCR. We identified 81 overlapping DEGs and CDC6, CDC20, KIF20A, STIL, and TOP2A as novel biomarkers affecting the prognosis of DLBCL. In addition, the STAT and KRAS pathways are considered potential growth regulatory pathways. These results hold promise for new avenues for the treatment of DLBCL patients.
ABSTRACT
BACKGROUND: Sjogren's syndrome (SS) is an autoimmune disorder characterized by the destruction of exocrine glands, resulting in dry mouth and eyes. Currently, there is no effective treatment for SS, and the mechanisms associated with inadequate salivary secretion are poorly understood. METHODS: In this study, we used NOD mice model to monitor changes in mice's salivary secretion and water consumption. Tissue morphology of the submandibular glands was examined by H&E staining, and Immunohistochemical detected the expression of AQP5 (an essential protein in salivary secretion). Global gene expression profiling was performed on submandibular gland tissue of extracted NOD mice model using RNA-seq. Subsequently, a series of bioinformatics analyses of transcriptome sequencing was performed, including differentially expressed genes (DEGs) identification, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, PPI network construction, hub gene identification, and the validity of diagnostic indicators using the dataset GSE40611. Finally, IFN-γ was used to treat the cells, the submandibular gland tissue of NOD mice model was extracted, and RT-qPCR was applied to verify the expression of hub genes. RESULTS: We found that NOD mice model had reduced salivary secretion and increased water consumption. H&E staining suggests acinar destruction and basement membrane changes in glandular tissue. Immunohistochemistry detects a decrease in AQP5 immunostaining within acinar. In transcriptome sequencing, 42 overlapping DEGs were identified, and hub genes (REN, A2M, SNCA, KLK3, TTR, and AZGP1) were identified as initiating targets for insulin signaling. In addition, insulin signaling and cAMP signaling are potential pathways for regulating salivary secretion and constructing a regulatory relationship between target-cAMP signaling-salivary secretion. CONCLUSION: The new potential targets and signal axes for regulating salivary secretion provide a strategy for SS therapy in a clinical setting.
ABSTRACT
Cancer cells intrinsically proliferate in an autonomous manner; however, the expansion of cancer cell areas in a tissue is known to be regulated by surrounding nontransformed cells. Whether these nontransformed cells can be targeted to control the spread of cancer cells is not understood. In this study, we established a system to evaluate the cancer-inhibitory activity of surrounding nontransformed cells and screened chemical compounds that could induce this activity. Our findings revealed that lonidamine (LND) and domperidone (DPD) inhibited expansion of oncogenic foci of KRASG12D-expressing transformed cells, whereas they did not inhibit the proliferation of monocultured KRASG12D-expressing cells. Live imaging revealed that LND and DPD suppressed the movement of nontransformed cells away from the attaching cancer cells. Moreover, we determined that LND and DPD promoted stress fiber formation, and the dominant-negative mutant of a small GTPase RhoA relieved the suppression of focus expansion, suggesting that RhoA-mediated stress fiber formation is involved in the inhibition of the movement of nontransformed cells and focus expansion. In conclusion, we suggest that elucidation of the mechanism of action of LND and DPD may lead to the development of a new type of drug that could induce the anticancer activity of surrounding nontransformed cells.
Subject(s)
Antineoplastic Agents , Domperidone , Indazoles , Neoplasms , Domperidone/pharmacology , Indazoles/pharmacology , Antineoplastic Agents/pharmacology , Neoplasms/drug therapy , Neoplasms/pathology , Animals , Mice , Epithelial Cells , Mammary Glands, Animal/cytology , Drug Screening Assays, AntitumorABSTRACT
In Arabidopsis thaliana, the Sigma factor B regulator RsbQ-like family of α/ß hydrolases contains the strigolactone (SL) receptor DWARF14 (AtD14), the karrikin receptor KARRIKIN INSENSITIVE2 (AtKAI2), and DWARF14-LIKE2 (AtDLK2), a protein of unknown function. Despite very similar protein folds, AtD14 and AtKAI2 differ in size and architecture of their ligand binding pockets, influencing their substrate specificity. We present the 1.5 Å crystal structure of AtDLK2, revealing the smallest ligand binding pocket in the protein family, bordered by two unique glycine residues. We identified a gatekeeper residue in the protein's lid domain and present a pyrrolo-quinoline-dione compound that inhibits AtDLK2's enzymatic activity.
ABSTRACT
Leptospirosis is a zoonotic disease caused by pathogenic Leptospira spp., with global implications primarily in tropical countries. However, the mechanisms of leptospiral pathogenesis are still not fully known and not all virulence factors (VFs) have been identified. Budding yeast, Saccharomyces cerevisiae is a popular eukaryotic model which has been used to identify bacterial VFs that target the conserved eukaryotic cellular processes. In this study, we screened for putative VFs of L. interrogans, one of the dominant species causing leptospirosis, by expressing candidate VFs in budding yeast and examining their impact on yeast growth in a high-throughput format. From an initial selection of 288 L. interrogans ORFs, we screened 226 candidate VFs in a yeast growth inhibition assay and identified nine putative VFs in four categories (adhesion, enzymatic, host structure interaction, and immunogenicity). Notably, LIC10280 was highly toxic even when expressed at low copies. We also observed specific subcellular localization for several putative VFs. This study shows that there are still potential L. interrogans VFs that await discovery. KEY POINTS: ⢠High-throughput cloning and expression of leptospiral proteins in yeast. ⢠Heterologous expression of nine leptospiral proteins inhibited yeast growth. ⢠An uncharacterized protein LIC10280 maybe a putative VF for further validation.
Subject(s)
Leptospira interrogans , Leptospira , Leptospirosis , Fungal Proteins/metabolism , Humans , Leptospira interrogans/genetics , Leptospira interrogans/metabolism , Leptospirosis/metabolism , Leptospirosis/microbiology , Leptospirosis/pathology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Virulence Factors/genetics , Virulence Factors/metabolismABSTRACT
The identification, structure-activity relationships (SARs), and biological effects of new antimalarials consisting of a 2,3,4,9-tetrahydro-1H-ß-carboline core, a coumarin ring, and an oxyalkanoyl linker are described. A cell-based phenotypic approach was employed in this search for novel antimalarial drugs with unique modes of action. Our screening campaign of the RIKEN compound library succeeded in the identification of the known tetrahydro-ß-carboline derivative (4e) as a hit compound showing significant in vitro activity. SAR studies on this chemical series led to the discovery of compound 4h having a (R)-methyl group on the oxyacetyl linker with potent inhibition of parasite growth (IC50 = 2.0 nM). Compound 4h was also found to exhibit significant in vivo antimalarial effects in mouse models. Furthermore, molecular modeling studies on 4e, 4h, and its diastereomer (4j) suggested that the (R)-methyl group of 4h forces the preferential adoption of a specific conformer which is considered to be an active conformer.
Subject(s)
Antimalarials , Animals , Antimalarials/pharmacology , Carbolines/chemistry , Carbolines/pharmacology , Coumarins/pharmacology , Mice , Structure-Activity RelationshipABSTRACT
Xerostomia is a common symptom in menopausal women, suggesting the role of sex steroids in disease development. Shreds of literature had reported the potential use of herbal extracts to relieve xerostomia. However, a cocktail of multiple components in herbal extract makes it difficult to understand the exact mechanism of action. Aquaporin5 (AQP5), the specific aquaporin expressed in salivary glands, plays an important role in salivary secretion as a downstream of estrogen signaling. In this study, we aimed to unravel a single active herbal component as a therapeutic for xerostomia and investigate its mechanism of action. The effects of apigenin (flavonoid), dauricine (alkaloids), protopine (alkaloids), and lentinan (polysaccharides) on AQP5 transcription were screened in vitro. Only apigenin robustly induced AQP5 transcription and expression, and this effect was even robust compared to the effect of estradiol (E2, a positive control). Overexpression of estrogen receptor α (ERα) in the human salivary gland cell line (HSG) upregulated the AQP5 transcription and expression and the knockdown ERα reversed this effect, suggesting the role of ERα signaling on AQP5 activation in HSG cells. Docking results showed apigenin-specific binding sites in ERα. We further analyzed the therapeutic effect of apigenin on ovariectomized mice as a xerostomia model. The saliva secretion in the xerostomia group was reduced to one-third of the sham group, whereas the apigenin or E2 treatment for 12 weeks reversed this effect. Meanwhile, the water consumption in the xerostomia group was augmented obviously compared to the sham group, whereas the water consumption in the apigenin and E2 group was declined to the level of the sham group. Immunohistochemistry of submandibular glands revealed the downregulation of AQP5 expression in xerostomia mice compared to control. Apigenin, or E2 treatment, upregulated AQP5 expression in xerostomia mice. In conclusion, apigenin, a single active component of herbal extract, upregulated AQP5 expression in HSG cells via activation of ERα signaling and restored saliva flow rates in OVX mice. These results revealed apigenin as a single active component of herbal extract with the potential to treat xerostomia.
ABSTRACT
Glutathione peroxidase 4 (GPX4) is an intracellular enzyme that oxidizes glutathione while reducing lipid peroxides and is a promising target for cancer therapy. To date, several GPX4 inhibitors have been reported to exhibit cytotoxicity against cancer cells. However, some cancer cells are less sensitive to the known GPX4 inhibitors. This study aimed to explore compounds showing synergistic effects with GPX4 inhibitors. We screened a chemical library and identified a compound named NPD4928, whose cytotoxicity was enhanced in the presence of a GPX4 inhibitor. Furthermore, we identified ferroptosis suppressor protein 1 as its target protein. The results indicate that NPD4928 enhanced the sensitivity of various cancer cells to GPX4 inhibitors, suggesting that the combination might have therapeutic potential via the induction of ferroptosis.