ABSTRACT
The serum response factor (SRF) binds to coactivators, such as myocardin-related transcription factor-A (MRTF-A), and mediates gene transcription elicited by diverse signaling pathways. SRF/MRTF-A-dependent gene transcription is activated when nuclear MRTF-A levels increase, enabling the formation of transcriptionally active SRF/MRTF-A complexes. The level of nuclear MRTF-A is regulated by nuclear G-actin, which binds to MRTF-A and promotes its nuclear export. However, pathways that regulate nuclear actin levels are poorly understood. Here, we show that MICAL-2, an atypical actin-regulatory protein, mediates SRF/MRTF-A-dependent gene transcription elicited by nerve growth factor and serum. MICAL-2 induces redox-dependent depolymerization of nuclear actin, which decreases nuclear G-actin and increases MRTF-A in the nucleus. Furthermore, we show that MICAL-2 is a target of CCG-1423, a small molecule inhibitor of SRF/MRTF-A-dependent transcription that exhibits efficacy in various preclinical disease models. These data identify redox modification of nuclear actin as a regulatory switch that mediates SRF/MRTF-A-dependent gene transcription.
Subject(s)
Cell Nucleus/metabolism , Microfilament Proteins/metabolism , Oxidoreductases/metabolism , Serum Response Factor/metabolism , Signal Transduction , Actins/metabolism , Amino Acid Sequence , Anilides/pharmacology , Animals , Benzamides/pharmacology , Cell Line , Cells, Cultured , DNA-Binding Proteins/metabolism , Gene Knockdown Techniques , Humans , Mice , Microfilament Proteins/analysis , Microfilament Proteins/genetics , Mixed Function Oxygenases/analysis , Mixed Function Oxygenases/genetics , Mixed Function Oxygenases/metabolism , Molecular Sequence Data , Nerve Growth Factor/metabolism , Neurites/metabolism , Oncogene Proteins, Fusion/metabolism , Oxidation-Reduction , Oxidoreductases/analysis , Oxidoreductases/genetics , Rats , Sequence Alignment , Trans-Activators , Transcription, Genetic , ZebrafishABSTRACT
TRPV1 is a Ca(2+)-permeable channel studied mostly as a pain receptor in sensory neurons. However, its role in other cell types is poorly understood. Here we found that TRPV1 was functionally expressed in CD4(+) T cells, where it acted as a non-store-operated Ca(2+) channel and contributed to T cell antigen receptor (TCR)-induced Ca(2+) influx, TCR signaling and T cell activation. In models of T cell-mediated colitis, TRPV1 promoted colitogenic T cell responses and intestinal inflammation. Furthermore, genetic and pharmacological inhibition of TRPV1 in human CD4(+) T cells recapitulated the phenotype of mouse Trpv1(-/-) CD4(+) T cells. Our findings suggest that inhibition of TRPV1 could represent a new therapeutic strategy for restraining proinflammatory T cell responses.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Inflammation/immunology , Lymphocyte Activation/immunology , Receptors, Antigen, T-Cell/metabolism , TRPV Cation Channels/genetics , Anilides/pharmacology , Animals , CD4-Positive T-Lymphocytes/cytology , Calcium/metabolism , Calcium Channels/immunology , Calcium Signaling/drug effects , Calcium Signaling/immunology , Capsaicin/pharmacology , Cells, Cultured , Cinnamates/pharmacology , Colitis/immunology , Humans , Interleukin-10/genetics , Intestines/immunology , Intestines/pathology , Lymphocyte Activation/drug effects , Mice , Mice, Inbred C57BL , Mice, Knockout , Sensory System Agents/pharmacology , TRPV Cation Channels/antagonists & inhibitors , TRPV Cation Channels/biosynthesisABSTRACT
This study aimed to explore the effects of peroxisome proliferator-activated receptor γ (PPARγ) inhibition on fracture healing of nonunion and the underlying mechanisms. Bone marrow mesenchymal stem cells (BMSCs) were treated with PPARγ antagonist GW9662 (5 µM, 10 µM). Alkaline phosphatase (ALP) staining and Alizarin Red S was used to assess early stage of osteogenesis and osteogenic differentiation. GW9662 (1 mg/kg/day) were administered intraperitoneally into the rats with bone fracture. Bone healing processes in the rat femur fracture model were recorded and assessed by radiographic methods on Weeks 8, 14, and 20 postoperation. Osteogenesis and angiogenesis at the fracture sites were evaluated by radiographic and histological methods on postoperative Week 20. GW9662 treatment increased ALP activity and Alp mRNA expression in rat BMSCs. Moreover, GW9662 administration increased matrix mineralization and mRNA and protein levels of Bmp2 and Runx2 in the BMSCs. In addition, GW9662 treatment improved radiographic score in the fracture rats and increased osteogenesis-related proteins, including type I collagen, osteopontin, and osteoglycin, in the bone tissues of the fracture sites. In conclusion, PPARγ inhibition promotes osteogenic differentiation of rat BMSCs, as well as improves the fracture healing of rats through Bmp2/Runx2 signaling pathway in the rat model of bone fracture.
Subject(s)
Anilides , Cell Differentiation , Fracture Healing , Mesenchymal Stem Cells , Osteogenesis , PPAR gamma , Animals , Male , Rats , Anilides/pharmacology , Bone Morphogenetic Protein 2 , Cell Differentiation/drug effects , Cells, Cultured , Core Binding Factor Alpha 1 Subunit/metabolism , Core Binding Factor Alpha 1 Subunit/genetics , Fracture Healing/drug effects , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/cytology , Osteogenesis/drug effects , PPAR gamma/antagonists & inhibitors , PPAR gamma/metabolism , Rats, Sprague-DawleyABSTRACT
Platensilin, platensimycin, and platencin are potent inhibitors of ß-ketoacyl-acyl carrier protein synthase (FabF) in the bacterial and mammalian fatty acid synthesis system, presenting promising drug leads for both antibacterial and antidiabetic therapies. Herein, a bioinspired skeleton reconstruction approach is reported, which enables the unified synthesis of these three natural FabF inhibitors and their skeletally diverse analogs, all stemming from a common ent-pimarane core. The synthesis features a diastereoselective biocatalytic reduction and an intermolecular Diels-Alder reaction to prepare the common ent-pimarane core. From this intermediate, stereoselective Mn-catalyzed hydrogen atom-transfer hydrogenation and subsequent Cu-catalyzed carbenoid C-H insertion afford platensilin. Furthermore, the intramolecular Diels-Alder reaction succeeded by regioselective ring opening of the newly formed cyclopropane enables the construction of the bicyclo[3.2.1]-octane and bicyclo[2.2.2]-octane ring systems of platensimycin and platencin, respectively. This skeletal reconstruction approach of the ent-pimarane core facilitates the preparation of analogs bearing different polycyclic scaffolds. Among these analogs, the previously unexplored cyclopropyl analog 47 exhibits improved antibacterial activity (MIC80 = 0.0625 µg/mL) against S. aureus compared to platensimycin.
Subject(s)
Adamantane , Aminobenzoates , Aminophenols , Anilides , Polycyclic Compounds , Aminophenols/chemistry , Aminophenols/pharmacology , Aminophenols/chemical synthesis , Polycyclic Compounds/chemistry , Polycyclic Compounds/pharmacology , Polycyclic Compounds/chemical synthesis , Adamantane/chemistry , Adamantane/pharmacology , Adamantane/chemical synthesis , Adamantane/analogs & derivatives , Anilides/pharmacology , Anilides/chemistry , Anilides/chemical synthesis , Aminobenzoates/pharmacology , Aminobenzoates/chemistry , Aminobenzoates/chemical synthesis , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Staphylococcus aureus/drug effects , Molecular Structure , Cycloaddition Reaction , Microbial Sensitivity Tests , Stereoisomerism , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistryABSTRACT
BACKGROUND: Pediatric-type diffuse high-grade glioma (pHGG) is the most frequent malignant brain tumor in children and can be subclassified into multiple entities. Fusion genes activating the MET receptor tyrosine kinase often occur in infant-type hemispheric glioma (IHG) but also in other pHGG and are associated with devastating morbidity and mortality. METHODS: To identify new treatment options, we established and characterized two novel orthotopic mouse models harboring distinct MET fusions. These included an immunocompetent, murine allograft model and patient-derived orthotopic xenografts (PDOX) from a MET-fusion IHG patient who failed conventional therapy and targeted therapy with cabozantinib. With these models, we analyzed the efficacy and pharmacokinetic properties of three MET inhibitors, capmatinib, crizotinib and cabozantinib, alone or combined with radiotherapy. RESULTS: Capmatinib showed superior brain pharmacokinetic properties and greater in vitro and in vivo efficacy than cabozantinib or crizotinib in both models. The PDOX models recapitulated the poor efficacy of cabozantinib experienced by the patient. In contrast, capmatinib extended survival and induced long-term progression-free survival when combined with radiotherapy in two complementary mouse models. Capmatinib treatment increased radiation-induced DNA double-strand breaks and delayed their repair. CONCLUSIONS: We comprehensively investigated the combination of MET inhibition and radiotherapy as a novel treatment option for MET-driven pHGG. Our seminal preclinical data package includes pharmacokinetic characterization, recapitulation of clinical outcomes, coinciding results from multiple complementing in vivo studies, and insights into molecular mechanism underlying increased efficacy. Taken together, we demonstrate the groundbreaking efficacy of capmatinib and radiation as a highly promising concept for future clinical trials.
Subject(s)
Brain Neoplasms , Glioma , Proto-Oncogene Proteins c-met , Xenograft Model Antitumor Assays , Animals , Humans , Glioma/pathology , Glioma/drug therapy , Glioma/genetics , Glioma/therapy , Proto-Oncogene Proteins c-met/antagonists & inhibitors , Proto-Oncogene Proteins c-met/genetics , Proto-Oncogene Proteins c-met/metabolism , Mice , Brain Neoplasms/pathology , Brain Neoplasms/drug therapy , Brain Neoplasms/genetics , Brain Neoplasms/radiotherapy , Benzamides/pharmacology , Benzamides/therapeutic use , Cell Line, Tumor , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Female , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Pyridines/pharmacology , Pyridines/therapeutic use , Crizotinib/pharmacology , Crizotinib/therapeutic use , Disease Models, Animal , Child , Neoplasm Grading , Anilides/pharmacology , Imidazoles , TriazinesABSTRACT
Excessive scar formation such as hypertrophic scars and keloids, resulting from trauma or surgical procedures, present a widespread concern for causing disfigurement, discomfort, and functional limitations. Macrophages play pivotal roles in maintaining tissue homeostasis, orchestrating tissue development, repair, and immune responses, and its transition of function and phenotype plays a critical role in regulating the balance between inflammation and tissue regeneration, which is central to cutaneous scar formation. Recent evidence suggests the involvement of Sonic Hedgehog (SHH) in the induction of anti-inflammatory M2-like macrophage phenotypes within tumor microenvironments. In our study, we observed increased SHH expression in human hypertrophic scars, prompting an investigation into its influence on macrophage polarization, efferocytosis, and cutaneous scar formation. Our findings reveal that SHH can enhance oxidative phosphorylation (OXPHOS) in macrophages, augment macrophage efferocytosis, and promote M2 polarization, finally contributing to the progression of cutaneous scar formation. Notably, targeting SHH signaling with vismodegib exhibited promising potential in mitigating scar formation by reversing the effects of enhanced OXPHOS and M2 polarization in macrophages. In conclusion, this study underscores the critical roles of macrophage metabolism, particularly OXPHOS, efferocytosis and SHH signaling in cutaneous scar formation. Understanding these mechanisms provides new avenues for potential interventions and scar prevention strategies.
Subject(s)
Hedgehog Proteins , Macrophages , Oxidative Phosphorylation , Phagocytosis , Hedgehog Proteins/metabolism , Macrophages/metabolism , Macrophages/drug effects , Humans , Oxidative Phosphorylation/drug effects , Animals , Phagocytosis/drug effects , Cicatrix, Hypertrophic/metabolism , Cicatrix, Hypertrophic/pathology , Mice , Signal Transduction/drug effects , Cicatrix/pathology , Cicatrix/metabolism , Mice, Inbred C57BL , Anilides/pharmacology , Pyridines/pharmacology , EfferocytosisABSTRACT
Despite the efficacy of Hedgehog pathway inhibitors in the treatment of basal cell carcinoma (BCC)1, residual disease persists in some patients and may contribute to relapse when treatment is discontinued2. Here, to study the effect of the Smoothened inhibitor vismodegib on tumour clearance, we have used a Ptch1-Trp53 mouse model of BCC3 and found that mice treated with vismodegib harbour quiescent residual tumours that regrow upon cessation of treatment. Profiling experiments revealed that residual BCCs initiate a transcriptional program that closely resembles that of stem cells of the interfollicular epidermis and isthmus, whereas untreated BCCs are more similar to the hair follicle bulge. This cell identity switch was enabled by a mostly permissive chromatin state accompanied by rapid Wnt pathway activation and reprogramming of super enhancers to drive activation of key transcription factors involved in cellular identity. Accordingly, treatment of BCC with both vismodegib and a Wnt pathway inhibitor reduced the residual tumour burden and enhanced differentiation. Our study identifies a resistance mechanism in which tumour cells evade treatment by adopting an alternative identity that does not rely on the original oncogenic driver for survival.
Subject(s)
Anilides/pharmacology , Carcinoma, Basal Cell/pathology , Cell Differentiation/drug effects , Hedgehog Proteins/antagonists & inhibitors , Pyridines/pharmacology , Signal Transduction/drug effects , Skin Neoplasms/pathology , Anilides/administration & dosage , Anilides/therapeutic use , Animals , Carcinoma, Basal Cell/drug therapy , Carcinoma, Basal Cell/metabolism , Cell Proliferation/drug effects , Epidermal Cells/drug effects , Epidermal Cells/metabolism , Epidermal Cells/pathology , Hair Follicle/drug effects , Hair Follicle/metabolism , Hair Follicle/pathology , Hedgehog Proteins/metabolism , Humans , Mice , Pyridines/administration & dosage , Pyridines/therapeutic use , Skin Neoplasms/drug therapy , Skin Neoplasms/metabolism , Smoothened Receptor/metabolism , Stem Cells/drug effects , Stem Cells/metabolism , Stem Cells/pathology , Wnt Signaling Pathway/drug effectsABSTRACT
Basal cell carcinoma (BCC) is the most frequent cancer in humans and results from constitutive activation of the Hedgehog pathway1. Several Smoothened inhibitors are used to treat Hedgehog-mediated malignancies, including BCC and medulloblastoma2. Vismodegib, a Smoothened inhibitor, leads to BCC shrinkage in the majority of patients with BCC3, but the mechanism by which it mediates BCC regression is unknown. Here we used two genetically engineered mouse models of BCC4 to investigate the mechanisms by which inhibition of Smoothened mediates tumour regression. We found that vismodegib mediates BCC regression by inhibiting a hair follicle-like fate and promoting the differentiation of tumour cells. However, a small population of tumour cells persists and is responsible for tumour relapse following treatment discontinuation, mimicking the situation found in humans5. In both mouse and human BCC, this persisting, slow-cycling tumour population expresses LGR5 and is characterized by active Wnt signalling. Combining Lgr5 lineage ablation or inhibition of Wnt signalling with vismodegib treatment leads to eradication of BCC. Our results show that vismodegib induces tumour regression by promoting tumour differentiation, and demonstrates that the synergy between Wnt and Smoothened inhibitors is a clinically relevant strategy for overcoming tumour relapse in BCC.
Subject(s)
Anilides/pharmacology , Anilides/therapeutic use , Carcinoma, Basal Cell/drug therapy , Carcinoma, Basal Cell/pathology , Neoplasm Recurrence, Local , Pyridines/pharmacology , Pyridines/therapeutic use , Receptors, G-Protein-Coupled/metabolism , Anilides/administration & dosage , Animals , Carcinoma, Basal Cell/genetics , Cell Differentiation/drug effects , Cell Lineage/drug effects , Disease Models, Animal , Female , Hair Follicle/cytology , Hair Follicle/drug effects , Hedgehog Proteins/antagonists & inhibitors , Hedgehog Proteins/metabolism , Humans , Male , Mice , Neoplasm Recurrence, Local/prevention & control , Patched-1 Receptor/deficiency , Pyridines/administration & dosage , Recurrence , Secondary Prevention , Skin Neoplasms/drug therapy , Skin Neoplasms/genetics , Skin Neoplasms/pathology , Smoothened Receptor/antagonists & inhibitors , Withholding Treatment , Wnt Proteins/antagonists & inhibitors , Wnt Proteins/metabolism , Wnt Signaling Pathway/drug effectsABSTRACT
Endothelial dysfunction is associated with vascular disease and results in disruption of endothelial barrier function and increased sensitivity to apoptosis. Currently, there are limited treatments for improving endothelial dysfunction. Activated protein C (aPC), a promising therapeutic, signals via protease-activated receptor-1 (PAR1) and mediates several cytoprotective responses, including endothelial barrier stabilization and anti-apoptotic responses. We showed that aPC-activated PAR1 signals preferentially via ß-arrestin-2 (ß-arr2) and dishevelled-2 (Dvl2) scaffolds rather than G proteins to promote Rac1 activation and barrier protection. However, the signaling pathways utilized by aPC/PAR1 to mediate anti-apoptotic activities are not known. aPC/PAR1 cytoprotective responses also require coreceptors; however, it is not clear how coreceptors impact different aPC/PAR1 signaling pathways to drive distinct cytoprotective responses. Here, we define a ß-arr2-mediated sphingosine kinase-1 (SphK1)-sphingosine-1-phosphate receptor-1 (S1PR1)-Akt signaling axis that confers aPC/PAR1-mediated protection against cell death. Using human cultured endothelial cells, we found that endogenous PAR1 and S1PR1 coexist in caveolin-1 (Cav1)-rich microdomains and that S1PR1 coassociation with Cav1 is increased by aPC activation of PAR1. Our study further shows that aPC stimulates ß-arr2-dependent SphK1 activation independent of Dvl2 and is required for transactivation of S1PR1-Akt signaling and protection against cell death. While aPC/PAR1-induced, extracellular signal-regulated kinase 1/2 (ERK1/2) activation is also dependent on ß-arr2, neither SphK1 nor S1PR1 are integrated into the ERK1/2 pathway. Finally, aPC activation of PAR1-ß-arr2-mediated protection against apoptosis is dependent on Cav1, the principal structural protein of endothelial caveolae. These studies reveal that different aPC/PAR1 cytoprotective responses are mediated by discrete, ß-arr2-driven signaling pathways in caveolae.
Subject(s)
Phosphotransferases (Alcohol Group Acceptor)/metabolism , Protein C/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Receptor, PAR-1/metabolism , Sphingosine-1-Phosphate Receptors/metabolism , beta-Arrestin 2/metabolism , Anilides/pharmacology , Apoptosis/physiology , Endothelial Cells/physiology , Enzyme Inhibitors/pharmacology , Gene Expression Regulation/drug effects , Heterocyclic Compounds, 3-Ring/pharmacology , Humans , Lactones/pharmacology , Methanol/pharmacology , Organophosphonates/pharmacology , Phosphotransferases (Alcohol Group Acceptor)/genetics , Platelet Aggregation Inhibitors/pharmacology , Protein C/genetics , Proto-Oncogene Proteins c-akt/genetics , Pyridines/pharmacology , Pyrrolidines/pharmacology , Receptor, PAR-1/genetics , Sphingosine-1-Phosphate Receptors/genetics , Sulfones/pharmacology , beta-Arrestin 2/geneticsABSTRACT
Human inflammatory breast cancer (IBC) and canine inflammatory mammary cancer (IMC) are highly aggressive neoplastic diseases that share numerous characteristics. In IBC and IMC, chemotherapy produces a limited pathological response and anti-androgen therapies have been of interest for breast cancer treatment. Therefore, the aim was to evaluate the effect of a therapy based on bicalutamide, a non-steroidal anti-androgen, with doxorubicin and docetaxel chemotherapy on cell proliferation, migration, tumor growth, and steroid-hormone secretion. An IMC-TN cell line, IPC-366, and an IBC-TN cell line, SUM149, were used. In vitro assays revealed that SUM149 exhibited greater sensitivity, reducing cell viability and migration with all tested drugs. In contrast, IPC-366 exhibited only significant in vitro reductions with docetaxel as a single agent or in different combinations. Decreased estrogen levels reduced in vitro tumor growth in both IMC and IBC. Curiously, doxorubicin resulted in low efficacy, especially in IMC. In addition, all drugs reduced the tumor volume in IBC and IMC by increasing intratumoral testosterone (T) levels, which have been related with reduced tumor progression. In conclusion, the addition of bicalutamide to doxorubicin and docetaxel combinations may represent a potential treatment for IMC and IBC.
Subject(s)
Anilides , Cell Proliferation , Docetaxel , Inflammatory Breast Neoplasms , Mammary Neoplasms, Animal , Nitriles , Tosyl Compounds , Tosyl Compounds/pharmacology , Humans , Animals , Female , Nitriles/pharmacology , Nitriles/therapeutic use , Cell Line, Tumor , Anilides/pharmacology , Dogs , Inflammatory Breast Neoplasms/drug therapy , Inflammatory Breast Neoplasms/pathology , Inflammatory Breast Neoplasms/metabolism , Cell Proliferation/drug effects , Docetaxel/pharmacology , Mammary Neoplasms, Animal/drug therapy , Mammary Neoplasms, Animal/pathology , Mammary Neoplasms, Animal/metabolism , Doxorubicin/pharmacology , Mice , Cell Survival/drug effects , Cell Movement/drug effects , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Xenograft Model Antitumor Assays , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , TestosteroneABSTRACT
Chikungunya virus (CHIKV) epidemics around the world have created public health concern with the unavailability of effective drugs and vaccines. This emphasizes the need for molecular understanding of host-virus interactions for developing effective targeted antivirals. Microarray analysis was carried out using CHIKV strain (Prototype and Indian) infected Vero cells and two host isozymes, MAPK activated protein kinase 2 (MK2) and MAPK activated protein kinase 3 (MK3) were selected for further analysis. The substrate spectrum of both enzymes is indistinguishable and covers proteins involved in cytokines production, endocytosis, reorganization of the cytoskeleton, cell migration, cell cycle control, chromatin remodeling and transcriptional regulation. Gene silencing and drug treatment were performed in vitro and in vivo to unravel the role of MK2/MK3 in CHIKV infection. Gene silencing of MK2 and MK3 abrogated around 58% CHIKV progeny release from the host cell and a MK2 activation inhibitor (CMPD1) treatment demonstrated 68% inhibition of viral infection suggesting a major role of MAPKAPKs during late CHIKV infection in vitro. Further, it was observed that the inhibition in viral infection is primarily due to the abrogation of lamellipodium formation through modulation of factors involved in the actin cytoskeleton remodeling pathway. Moreover, CHIKV-infected C57BL/6 mice demonstrated reduction in the viral copy number, lessened disease score and better survivability after CMPD1 treatment. In addition, reduction in expression of key pro-inflammatory mediators such as CXCL13, RAGE, FGF, MMP9 and increase in HGF (a CHIKV infection recovery marker) was observed indicating the effectiveness of the drug against CHIKV. Taken together it can be proposed that MK2 and MK3 are crucial host factors for CHIKV infection and can be considered as important target for developing effective anti-CHIKV strategies.
Subject(s)
Actins/metabolism , Anilides/pharmacology , Antiviral Agents/pharmacology , Chikungunya Fever/prevention & control , Chikungunya virus/drug effects , Cytoskeleton/drug effects , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Protein Serine-Threonine Kinases/antagonists & inhibitors , Tetrahydronaphthalenes/pharmacology , Actins/drug effects , Animals , Chikungunya Fever/virology , Chlorocebus aethiops , Male , Mice , Mice, Inbred C57BL , Vero Cells , Virus ReleaseABSTRACT
Acute liver injury is a common disease without effective therapy in humans. We sought to evaluate a combination therapy of insulin-like growth factor 1 (IGF-I) and BTP-2 in a mouse liver injury model induced by lipopolysaccharide (LPS). We chose this model because LPS is known to increase the expression of the transcription factors related to systemic inflammation (i.e., NFκB, CREB, AP1, IRF 3, and NFAT), which depends on calcium signaling. Notably, these transcription factors all have pleiotropic effects and account for the other observed changes in tissue damage parameters. Additionally, LPS is also known to increase the genes associated with a tissue injury (e.g., NGAL, SOD, caspase 3, and type 1 collagen) and systemic expression of pro-inflammatory cytokines. Finally, LPS compromises vascular integrity. Accordingly, IGF-I was selected because its serum levels were shown to decrease during systemic inflammation. BTP-2 was chosen because it was known to decrease cytosolic calcium, which is increased by LPS. This current study showed that IGF-I, BTP-2, or a combination therapy significantly altered and normalized all of the aforementioned LPS-induced gene changes. Additionally, our therapies reduced the vascular leakage caused by LPS, as evidenced by the Evans blue dye technique. Furthermore, histopathologic studies showed that IGF-I decreased the proportion of hepatocytes with ballooning degeneration. Finally, IGF-I also increased the expression of the hepatic growth factor (HGF) and the receptor for the epidermal growth factor (EGFR), markers of liver regeneration. Collectively, our data suggest that a combination of IGF-I and BTP-2 is a promising therapy for acute liver injury.
Subject(s)
Anilides , Chemical and Drug Induced Liver Injury, Chronic , Chemical and Drug Induced Liver Injury , Insulin-Like Growth Factor I , Thiadiazoles , Anilides/metabolism , Anilides/pharmacology , Animals , Chemical and Drug Induced Liver Injury/drug therapy , Chemical and Drug Induced Liver Injury, Chronic/metabolism , Disease Models, Animal , Inflammation/metabolism , Insulin-Like Growth Factor I/metabolism , Insulin-Like Growth Factor I/pharmacology , Lipopolysaccharides/pharmacology , Liver/metabolism , Mice , Thiadiazoles/metabolism , Thiadiazoles/pharmacologyABSTRACT
A significant fraction of patients with advanced prostate cancer treated with androgen deprivation therapy experience relapse with relentless progression to lethal metastatic castration-resistant prostate cancer (mCRPC). Immune checkpoint blockade using antibodies against cytotoxic-T-lymphocyte-associated protein 4 (CTLA4) or programmed cell death 1/programmed cell death 1 ligand 1 (PD1/PD-L1) generates durable therapeutic responses in a significant subset of patients across a variety of cancer types. However, mCRPC showed overwhelming de novo resistance to immune checkpoint blockade, motivating a search for targeted therapies that overcome this resistance. Myeloid-derived suppressor cells (MDSCs) are known to play important roles in tumour immune evasion. The abundance of circulating MDSCs correlates with prostate-specific antigen levels and metastasis in patients with prostate cancer. Mouse models of prostate cancer show that MDSCs (CD11b+Gr1+) promote tumour initiation and progression. These observations prompted us to hypothesize that robust immunotherapy responses in mCRPC may be elicited by the combined actions of immune checkpoint blockade agents together with targeted agents that neutralize MDSCs yet preserve T-cell function. Here we develop a novel chimaeric mouse model of mCRPC to efficiently test combination therapies in an autochthonous setting. Combination of anti-CTLA4 and anti-PD1 engendered only modest efficacy. Targeted therapy against mCRPC-infiltrating MDSCs, using multikinase inhibitors such as cabozantinib and BEZ235, also showed minimal anti-tumour activities. Strikingly, primary and metastatic CRPC showed robust synergistic responses when immune checkpoint blockade was combined with MDSC-targeted therapy. Mechanistically, combination therapy efficacy stemmed from the upregulation of interleukin-1 receptor antagonist and suppression of MDSC-promoting cytokines secreted by prostate cancer cells. These observations illuminate a clinical path hypothesis for combining immune checkpoint blockade with MDSC-targeted therapies in the treatment of mCRPC.
Subject(s)
Immunotherapy/methods , Prostatic Neoplasms, Castration-Resistant/immunology , Prostatic Neoplasms, Castration-Resistant/therapy , Anilides/pharmacology , Anilides/therapeutic use , Animals , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , Chimera , Cytokines/immunology , Cytokines/metabolism , Disease Models, Animal , Drug Synergism , Female , Humans , Imidazoles/pharmacology , Imidazoles/therapeutic use , Lymphocytes, Tumor-Infiltrating/drug effects , Lymphocytes, Tumor-Infiltrating/immunology , Male , Mice , Molecular Targeted Therapy , Myeloid-Derived Suppressor Cells/cytology , Myeloid-Derived Suppressor Cells/drug effects , Myeloid-Derived Suppressor Cells/immunology , Phosphoinositide-3 Kinase Inhibitors , Prostatic Neoplasms, Castration-Resistant/pathology , Pyridines/pharmacology , Pyridines/therapeutic use , Quinolines/pharmacology , Quinolines/therapeutic use , Signal Transduction/drug effects , Tumor Microenvironment/drug effects , Tumor Microenvironment/immunologyABSTRACT
Medulloblastoma is the most common malignant brain tumor in children. Here we describe a medulloblastoma model using Induced pluripotent stem (iPS) cell-derived human neuroepithelial stem (NES) cells generated from a Gorlin syndrome patient carrying a germline mutation in the sonic hedgehog (SHH) receptor PTCH1. We found that Gorlin NES cells formed tumors in mouse cerebellum mimicking human medulloblastoma. Retransplantation of tumor-isolated NES (tNES) cells resulted in accelerated tumor formation, cells with reduced growth factor dependency, enhanced neurosphere formation in vitro, and increased sensitivity to Vismodegib. Using our model, we identified LGALS1 to be a GLI target gene that is up-regulated in both Gorlin tNES cells and SHH-subgroup of medulloblastoma patients. Taken together, we demonstrate that NES cells derived from Gorlin patients can be used as a resource to model medulloblastoma initiation and progression and to identify putative targets.
Subject(s)
Hedgehog Proteins/metabolism , Medulloblastoma/genetics , Neural Stem Cells/physiology , Anilides/pharmacology , Animals , Basal Cell Nevus Syndrome/genetics , Basal Cell Nevus Syndrome/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/physiology , Galectin 1/genetics , Galectin 1/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Hedgehog Proteins/genetics , Humans , Mice , Neoplasms, Experimental , Patched-1 Receptor/genetics , Pyridines/pharmacologyABSTRACT
Scirrhous gastric cancer frequently develops into peritoneal carcinomatosis with malignant ascites, leading to an extremely poor prognosis. We had demonstrated that paracrine hepatocyte growth factor (HGF)-induced MET activation promotes peritoneal carcinomatosis with ascites formation. The vascular endothelial growth factor (VEGF) receptor (VEGFR)/VEGF axis facilitates tumor progression and formation of malignant ascites. This study investigated the role of MET and VEGFR2 in the development of peritoneal carcinomatosis with malignant ascites. Cabozantinib is a dual inhibitor of MET and VEGFR2. We examined the effects of cabozantinib on MET- and VEGFR2-mediated progression of peritoneal carcinomatosis in human scirrhous gastric cancer in vitro and in vivo. Cabozantinib inhibited HGF-stimulated proliferation of scirrhous cancer cell lines NUGC4 and GCIY, with a high potential to generate peritoneal carcinomatosis with ascites fluid, as well as the constitutive proliferation of MKN45 cells with MET amplification. Cabozantinib also inhibited the phosphorylation of both MET and VEGFR2 in scirrhous cancer cells and HGF- or VEGF-stimulated HUVECs. It effectively reduced ascitic fluid and prolonged the survival of NUGC4-inoculated nude mice. In clinical specimens, malignant ascites fluid from patients with peritoneal carcinomatosis contained high levels of HGF and VEGF. Our results strongly suggest that MET- and VEGFR2-mediated signaling pathways play pivotal roles in the pathogenesis of peritoneal carcinomatosis in scirrhous gastric cancer. Thus, the dual blockade of MET and VEGFR2 signaling may be a potential therapeutic maneuver for peritoneal carcinomatosis in scirrhous gastric cancer.
Subject(s)
Anilides , Peritoneal Neoplasms , Proto-Oncogene Proteins c-met , Pyridines , Stomach Neoplasms , Vascular Endothelial Growth Factor Receptor-2 , Anilides/pharmacology , Animals , Ascites/drug therapy , Cell Line, Tumor , Humans , Mice , Mice, Nude , Peritoneal Neoplasms/drug therapy , Peritoneal Neoplasms/metabolism , Peritoneal Neoplasms/pathology , Proto-Oncogene Proteins c-met/antagonists & inhibitors , Pyridines/pharmacology , Signal Transduction/drug effects , Stomach Neoplasms/drug therapy , Stomach Neoplasms/metabolism , Stomach Neoplasms/pathology , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitorsABSTRACT
R-bicalutamide is a first-line therapy used to treat prostate cancer (PCa) inhibiting the androgen receptor (AR) which plays an important role in the development and the progression of PCa. However, after a protracted drug administration, many patients develop a form of androgen insensitivity since R-bicalutamide starts to exhibit some agonistic properties lead by the W741L AR mutation in the ligand-binding pocket even if the mechanism of the antagonist-agonist switch is still not clear. To study the drug-resistant mechanism, we explored the structural effects of the antagonist R-bicalutamide on the homodimer stability considering both the AR wild-type and W741L employing molecular dynamic (MD) simulations. The results obtained indicate that the binding of R-bicalutamide in the two AR monomers induces a great instability in the homodimer, which may determine the monomer's dissociation preventing AR migration into the nucleus and avoiding the transcriptional activity. If the W741L mutation occurs, the homodimer tends to have a behaviour close to the agonistic system where the two monomers are tightly bound, which may explain the effect of the W741L in drug insensitivity from a structural point of view.
Subject(s)
Prostatic Neoplasms , Receptors, Androgen , Androgen Antagonists , Anilides/pharmacology , Cell Line, Tumor , Humans , Male , Molecular Dynamics Simulation , Nitriles/pharmacology , Nitriles/therapeutic use , Prostatic Neoplasms/metabolism , Receptors, Androgen/metabolism , Tosyl Compounds/pharmacologyABSTRACT
Spreading depolarization is a slowly propagating wave of massive cellular depolarization associated with acute brain injury and migraine aura. Genetic studies link depolarizing molecular defects in Ca2+ flux, Na+ current in interneurons, and glial Na+-K+ ATPase with spreading depolarization susceptibility, emphasizing the important roles of synaptic activity and extracellular ionic homeostasis in determining spreading depolarization threshold. In contrast, although gene mutations in voltage-gated potassium ion channels that shape intrinsic membrane excitability are frequently associated with epilepsy susceptibility, it is not known whether epileptogenic mutations that regulate membrane repolarization also modify spreading depolarization threshold and propagation. Here we report that the Kcnq2/Kv7.2 potassium channel subunit, frequently mutated in developmental epilepsy, is a spreading depolarization modulatory gene with significant control over the seizure-spreading depolarization transition threshold, bi-hemispheric cortical expression, and diurnal temporal susceptibility. Chronic DC-band cortical EEG recording from behaving conditional Kcnq2 deletion mice (Emx1cre/+::Kcnq2flox/flox) revealed spontaneous cortical seizures and spreading depolarization. In contrast to the related potassium channel deficient model, Kv1.1-KO mice, spontaneous cortical spreading depolarizations in Kcnq2 cKO mice are tightly coupled to the terminal phase of seizures, arise bilaterally, and are observed predominantly during the dark phase. Administration of the non-selective Kv7.2 inhibitor XE991 to Kv1.1-KO mice partly reproduced the Kcnq2 cKO-like spreading depolarization phenotype (tight seizure coupling and bilateral symmetry) in these mice, indicating that Kv7.2 currents can directly and actively modulate spreading depolarization properties. In vitro brain slice studies confirmed that Kcnq2/Kv7.2 depletion or pharmacological inhibition intrinsically lowers the cortical spreading depolarization threshold, whereas pharmacological Kv7.2 activators elevate the threshold to multiple depolarizing and hypometabolic spreading depolarization triggers. Together these results identify Kcnq2/Kv7.2 as a distinctive spreading depolarization regulatory gene, and point to spreading depolarization as a potentially significant pathophysiological component of KCNQ2-linked epileptic encephalopathy syndromes. Our results also implicate KCNQ2/Kv7.2 channel activation as a potential adjunctive therapeutic target to inhibit spreading depolarization incidence.
Subject(s)
Brain/physiology , Cortical Spreading Depression/physiology , KCNQ2 Potassium Channel/metabolism , Nerve Tissue Proteins/metabolism , Anilides/pharmacology , Animals , Brain/drug effects , Bridged Bicyclo Compounds/pharmacology , Carbamates/pharmacology , Cortical Spreading Depression/drug effects , KCNQ2 Potassium Channel/agonists , KCNQ2 Potassium Channel/deficiency , Mice , Mice, Inbred C57BL , Mice, Transgenic , Nerve Tissue Proteins/agonists , Nerve Tissue Proteins/deficiency , Organ Culture Techniques , Phenylenediamines/pharmacologyABSTRACT
Electric field (EF) directed cell migration (electrotaxis) is known to occur in glioblastoma multiforme (GBM) and neural stem cells, with key signalling pathways frequently dysregulated in GBM. One such pathway is EGFR/PI3K/Akt, which is down-regulated by peroxisome proliferator activated receptor gamma (PPARγ) agonists. We investigated the effect of electric fields on primary differentiated and glioma stem cell (GSCs) migration, finding opposing preferences for anodal and cathodal migration, respectively. We next sought to determine whether chemically disrupting Akt through PTEN upregulation with the PPARγ agonist, pioglitazone, would modulate electrotaxis of these cells. We found that directed cell migration was significantly inhibited with the addition of pioglitazone in both differentiated GBM and GSCs subtypes. Western blot analysis did not demonstrate any change in PPARγ expression with and without exposure to EF. In summary we demonstrate opposing EF responses in primary GBM differentiated cells and GSCs can be inhibited chemically by pioglitazone, implicating GBM EF modulation as a potential target in preventing tumour recurrence.
Subject(s)
Cell Movement/genetics , Neoplastic Stem Cells/metabolism , Neuroglia/metabolism , PPAR gamma/genetics , Taxis Response , Anilides/pharmacology , Cell Differentiation/drug effects , Cell Line, Tumor , Electricity , Electrodes , Electromagnetic Fields , ErbB Receptors/genetics , ErbB Receptors/metabolism , Gene Expression Regulation, Neoplastic , Humans , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/pathology , Neuroglia/drug effects , Neuroglia/pathology , PPAR gamma/agonists , PPAR gamma/metabolism , PTEN Phosphohydrolase/genetics , PTEN Phosphohydrolase/metabolism , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Pioglitazone/pharmacology , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Signal TransductionABSTRACT
Developmental signals of the Hedgehog (Hh) and Wnt families are transduced across the membrane by Frizzledclass G-protein-coupled receptors (GPCRs) composed of both a heptahelical transmembrane domain (TMD) and an extracellular cysteine-rich domain (CRD). How the large extracellular domains of GPCRs regulate signalling by the TMD is unknown. We present crystal structures of the Hh signal transducer and oncoprotein Smoothened, a GPCR that contains two distinct ligand-binding sites: one in its TMD and one in the CRD. The CRD is stacked a top the TMD, separated by an intervening wedge-like linker domain. Structure-guided mutations show that the interface between the CRD, linker domain and TMD stabilizes the inactive state of Smoothened. Unexpectedly, we find a cholesterol molecule bound to Smoothened in the CRD binding site. Mutations predicted to prevent cholesterol binding impair the ability of Smoothened to transmit native Hh signals. Binding of a clinically used antagonist, vismodegib, to the TMD induces a conformational change that is propagated to the CRD, resulting in loss of cholesterol from the CRD-linker domain-TMD interface. Our results clarify the structural mechanism by which the activity of a GPCR is controlled by ligand-regulated interactions between its extracellular and transmembrane domains.
Subject(s)
Extracellular Space/metabolism , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/metabolism , Anilides/chemistry , Anilides/metabolism , Anilides/pharmacology , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , Binding Sites/genetics , Cholesterol/metabolism , Cholesterol/pharmacology , Crystallography, X-Ray , Cysteine/chemistry , Cysteine/genetics , Cysteine/metabolism , Hedgehog Proteins/metabolism , Humans , Ligands , Models, Molecular , Protein Binding/genetics , Protein Stability/drug effects , Protein Structure, Tertiary/drug effects , Protein Structure, Tertiary/genetics , Pyridines/chemistry , Pyridines/metabolism , Pyridines/pharmacology , Receptors, G-Protein-Coupled/antagonists & inhibitors , Receptors, G-Protein-Coupled/genetics , Signal Transduction/drug effects , Smoothened ReceptorABSTRACT
Background: Kartogenin is a heterocyclic compound able to promote the proliferation, migration, and differentiation of various cell types and induce cartilage-like tissue regeneration. However, the role of kartogenin in hair follicles (HFs), remains unknown. We therefore investigated the effects of kartogenin on the regulation of hair growth and hair growth cycle transition. Methods: The effects of kartogenin on the proliferation, cell cycle status, and migration of primary human outer root sheath cells (ORSCs) were evaluated by MTS assay, flow cytometry, Transwell® and scratch assays, respectively. We exposed ORSCs to kartogenin (1 µM) and determined changes in mRNA and protein levels of transforming growth factor (TGF)-ß2/Smad signaling molecules by reverse transcription polymerase chain reaction, western blotting, and immunofluorescence. We also examined the effects of kartogenin (10 µM) on HFs in mice by histology following cutaneous injection. Results: Kartogenin enhanced ORSC proliferation and migration function in a dose-dependent manner, and downregulated the expression of TGF-ß2/Smad signaling molecules in vitro. Injection of kartogenin delayed catagen phase and increased regenerated hair length in mice in vivo. Conclusions: Kartogenin modulates HF growth and regulates the hair cycle and the TGF-ß2/Smad signaling pathway, providing a potential new approach for the treatment of hair loss.