ABSTRACT
'Drug abuse' has been recognized as one of the most pressing epidemics in contemporary society. Traditional research has primarily focused on understanding how drugs induce neurotoxicity or degeneration within the central nervous system (CNS) and influence systems related to reward, motivation, and cravings. However, recent investigations have increasingly shifted their attention toward the detrimental consequences of drug abuse on the blood-brain barrier (BBB). The BBB is a structural component situated in brain vessels, responsible for separating brain tissue from external substances to maintain brain homeostasis. The BBB's function is governed by cellular interactions involving various elements of the 'neurovascular unit (NVU),' such as neurons, endothelial cells, astrocytes, pericytes, and microglia. Disruption of the NVU is closely linked to serious neurodegeneration. This review provides a comprehensive overview of the harmful effects of psychostimulant drugs on the BBB, highlighting the mechanisms through which drugs can damage the NVU. Additionally, the review proposes novel therapeutic targets aimed at protecting the BBB. By understanding the intricate relationships between drug abuse, BBB integrity, and NVU function, researchers and clinicians may uncover new strategies to mitigate the damaging impact of drug abuse on brain health.
Subject(s)
Endothelial Cells , Substance-Related Disorders , Humans , Brain , Blood-Brain Barrier , Central Nervous System , Substance-Related Disorders/etiologyABSTRACT
Protein kinase C-δ (PKCδ) is a diacylglycerol-dependent, calcium-independent novel PKC isoform that is engaged in various cell signaling pathways, such as cell proliferation, apoptosis, inflammation, and oxidative stress. In this study, we searched for proteins that bind PKCδ using a yeast two-hybrid assay and identified murine arrest-defective 1 (mARD1) as a binding partner. The interaction between PKCδ and mARD1 was confirmed by glutathione S-transferase pull-down and co-immunoprecipitation assays. Furthermore, recombinant PKCδ phosphorylated full-length mARD1 protein. The NetPhos online prediction tool suggested PKCδ phosphorylates Ser80 , Ser108 , and Ser114 residues of mARD1 with the highest probability. Based on these results, we synthesized peptides containing these sites and examined their phosphorylations using recombinant PKCδ. Autoradiography confirmed these sites were efficiently phosphorylated. Consequent mass spectrometry and peptide sequencing in combination with MALDI-TOF MS/MS confirmed that Ser80 and Ser108 were major phosphorylation sites. The alanine mutations of Ser80 and Ser108 abolished the phosphorylation of mARD1 by PKCδ in 293T cells supporting these observations. In addition, kinase assays using various PKC isotypes showed that Ser80 of ARD1 was phosphorylated by PKCßI and PKCζ isotypes with the highest selectivity, while Ser108 and/or Ser114 were phosphorylated by PKCγ with activities comparable to that of the PKCδ isoform. Overall, these results suggest the possibility that PKCδ transduces signals by regulating phosphorylation of ARD1.
Subject(s)
N-Terminal Acetyltransferase A/metabolism , N-Terminal Acetyltransferase E/metabolism , Phosphorylation/physiology , Protein Kinase C-delta/metabolism , Amino Acid Sequence , Animals , Cell Line , Cell Line, Tumor , HEK293 Cells , HeLa Cells , Humans , Mice , Oxidative Stress/physiology , Peptides/metabolism , Protein Isoforms/metabolism , Serine/metabolism , Signal Transduction/physiologyABSTRACT
Disruption of colonic homeostasis caused by aberrant M1/M2 macrophage polarization and dysbiosis contributes to inflammatory bowel disease (IBD) pathogenesis. However, the molecular factors mediating colonic homeostasis are not well characterized. Here, we found that Ninjurin1 (Ninj1) limits colon inflammation by regulating macrophage polarization and microbiota composition under homeostatic conditions and during colitis development. Ninj1 deletion in mice induced hypersusceptibility to colitis, with increased prevalence of colitogenic Prevotellaceae strains and decreased immunoregulatory Lachnospiraceae strains. Upon co-housing (CoH) with WT mice, Ninj1-/- mice showed increased Lachnospiraceae and decreased Prevotellaceae abundance, with subsequent improvement of colitis. Under homeostatic conditions, M1 macrophage frequency was higher in the Ninj1-/- mouse colons than wild-type (WT) mouse colons, which may contribute to increased basal colonic inflammation and microbial imbalance. Following colitis induction, Ninj1 expression was increased in macrophages; meanwhile Ninj1-/- mice showed severe colitis development and impaired recovery, associated with decreased M2 macrophages and escalated microbial imbalance. In vitro, Ninj1 knockdown in mouse and human macrophages activated M1 polarization and restricted M2 polarization. Finally, the transfer of WT macrophages ameliorated severe colitis in Ninj1-/- mice. These findings suggest that Ninj1 mediates colonic homeostasis by modulating M1/M2 macrophage balance and preventing extensive dysbiosis, with implications for IBD prevention and therapy.
Subject(s)
Cell Adhesion Molecules, Neuronal/deficiency , Colitis/metabolism , Colitis/pathology , Gastrointestinal Microbiome/physiology , Macrophages/metabolism , Macrophages/pathology , Nerve Growth Factors/deficiency , Animals , Cell Adhesion Molecules, Neuronal/metabolism , Cell Differentiation/physiology , Cell Line, Tumor , Colitis/microbiology , Colon/metabolism , Colon/microbiology , Colon/pathology , Disease Models, Animal , Homeostasis/physiology , Humans , Inflammation/metabolism , Inflammation/microbiology , Inflammation/pathology , Inflammatory Bowel Diseases/metabolism , Inflammatory Bowel Diseases/microbiology , Inflammatory Bowel Diseases/pathology , Macrophage Activation/physiology , Male , Mice , Nerve Growth Factors/metabolism , THP-1 Cells/metabolismABSTRACT
The dysregulation of fibroblast growth factor (FGF) signaling has been implicated in tumorigenesis, tumor progression, angiogenesis, and chemoresistance. The small-molecule AZD4547 is a potent inhibitor of FGF receptors. This study was performed to investigate the antitumor effects and determine the mechanistic details of AZD4547 in ovarian cancer cells. AZD4547 markedly inhibited the proliferation and increased the apoptosis of ovarian cancer cells. AZD4547 also suppressed the migration and invasion of ovarian cancer cells under nontoxic conditions. Furthermore, it attenuated the formation of spheroids and the self-renewal capacities of ovarian cancer stem cells and exerted an antiangiogenic effect. It also suppressed in vivo tumor growth in mice. Collectively, this study demonstrated the antitumor effect of AZD4547 in ovarian cancer cells and suggests that it is a promising agent for ovarian cancer therapy.
Subject(s)
Benzamides/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Ovarian Neoplasms/drug therapy , Piperazines/pharmacology , Pyrazoles/pharmacology , Receptors, Fibroblast Growth Factor/antagonists & inhibitors , Animals , Apoptosis , Cell Movement , Cell Proliferation , Female , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Prognosis , Survival Rate , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
N-α-acetyltransferase 10 (NAA10) is an acetyltransferase that acetylates both N-terminal amino acid and internal lysine residues of proteins. NAA10 is a crucial player to regulate cell proliferation, migration, differentiation, apoptosis, and autophagy. Recently, mounting evidence presented the overexpression of NAA10 in various types of cancer, including liver, bone, lung, breast, colon, and prostate cancers, and demonstrated a correlation of overexpressed NAA10 with vascular invasion and metastasis, thereby affecting overall survival rates of cancer patients and recurrence of diseases. This evidence all points NAA10 toward a promising biomarker for cancer prognosis. Here we summarize the current knowledge regarding the biological functions of NAA10 in cancer progression and provide the potential usage of NAA10 as a prognostic marker for cancer progression.
Subject(s)
Biomarkers, Tumor/metabolism , N-Terminal Acetyltransferase A/metabolism , N-Terminal Acetyltransferase E/metabolism , Neoplasms/pathology , Disease Progression , Humans , Neoplasms/metabolismABSTRACT
A-kinase anchor protein 12 (AKAP12) is a scaffolding protein that associates with intracellular molecules to regulate multiple signal transductions. Although the roles of AKAP12 in the central nervous system are still relatively understudied, it was previously shown that AKAP12 regulates blood-retinal barrier formation. In this study, we asked whether AKAP12 also supports the function and integrity of the blood-brain barrier (BBB). In a mouse model of focal ischemia, the expression level of AKAP12 in cerebral endothelial cells was upregulated during the acute phase of stroke. Also, in cultured cerebral endothelial cells, oxygen-glucose deprivation induced the upregulation of AKAP12. When AKAP12 expression was suppressed by an siRNA approach in cultured endothelial cells, endothelial permeability was increased along with the dysregulation of ZO-1/Claudin 5 expression. In addition, the loss of AKAP12 expression caused an upregulation/activation of the Rho kinase pathway, and treatment of Rho kinase inhibitor Y-27632 mitigated the increase of endothelial permeability in AKAP12-deficient endothelial cell cultures. These in vitro findings were confirmed by our in vivo experiments using Akap12 knockout mice. Compared to wild-type mice, Akap12 knockout mice showed a larger extent of BBB damage after stroke. However, the inhibition of rho kinase by Y-27632 tightened the BBB in Akap12 knockout mice. These data may suggest that endogenous AKAP12 works to alleviate the damage and dysfunction of the BBB caused by ischemic stress. Therefore, the AKAP12-rho-kinase signaling pathway represents a novel therapeutic target for stroke.
Subject(s)
A Kinase Anchor Proteins/metabolism , Blood-Brain Barrier/metabolism , Blood-Brain Barrier/pathology , Cell Cycle Proteins/metabolism , Ischemic Stroke/metabolism , Ischemic Stroke/pathology , Animals , Cell Membrane Permeability , Endothelium, Vascular/pathology , Mice, Inbred C57BL , Mice, Knockout , rho-Associated Kinases/metabolismABSTRACT
Arrest defective 1 (ARD1), also known as N(alpha)-acetyltransferase 10 (NAA10) was originally identified as an N-terminal acetyltransferase (NAT) that catalyzes the acetylation of N-termini of newly synthesized peptides. After that, mammalian ARD1/NAA10 expanded its' role to lysine acetyltransferase (KAT) that post-translationally acetylates internal lysine residues of proteins. ARD1/NAA10 is the only enzyme with both NAT and KAT activities. However, recent studies on the role of human ARD1/NAA10 (hARD1/NAA10) in lysine acetylation are contradictory, as crystal structure and in vitro acetylation assay results revealed the lack of KAT activity. Thus, the role of hARD1/NAA10 in lysine acetylation is still debating. Here, we found a clue that possibly explains these complicated and controversial results on KAT activity of hARD1/NAA10. Recombinant hARD1/NAA10 exhibited KAT activity, which disappeared soon in vitro. Size-exclusion analysis revealed that most recombinant hARD1/NAA10 formed oligomers over time, resulting in the loss of KAT activity. While oligomeric recombinant hARD1/NAA10 lost its ability for lysine acetylation, its monomeric form clearly exhibited lysine acetylation activity in vitro. We also characterized the KAT activity of hARD1/NAA10 that was influenced by several experimental conditions, including concentration of reactants and reaction time. Taken together, our study proves that recombinant hARD1/NAA10 exhibits KAT activity in vitro but only under accurate conditions, including reactant concentrations and reaction duration.
Subject(s)
Lysine Acetyltransferases/metabolism , N-Terminal Acetyltransferase A/metabolism , N-Terminal Acetyltransferase E/metabolism , Acetylation , Dialysis , Escherichia coli , Humans , Lysine/metabolism , N-Terminal Acetyltransferase A/genetics , N-Terminal Acetyltransferase A/isolation & purification , N-Terminal Acetyltransferase E/genetics , N-Terminal Acetyltransferase E/isolation & purification , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolismABSTRACT
Oligodendrocyte precursor cells (OPCs) give rise to oligodendrocytes in cerebral white matter. However, the underlying mechanisms that regulate this process remain to be fully defined, especially in adult brains. Recently, it has been suggested that signaling via A-kinase anchor protein 12 (AKAP12), a scaffolding protein that associates with intracellular molecules such as protein kinase A, may be involved in Schwann cell homeostasis and peripheral myelination. Here, we asked whether AKAP12 also regulates the mechanisms of myelination in the CNS. AKAP12 knockout mice were compared against wild-type (WT) mice in a series of neurochemical and behavioral assays. Compared with WTs, 2-months old AKAP12 knockout mice exhibited loss of myelin in white matter of the corpus callosum, along with perturbations in working memory as measured by a standard Y-maze test. Unexpectedly, very few OPCs expressed AKAP12 in the corpus callosum region. Instead, pericytes appeared to be one of the major AKAP12-expressing cells. In a cell culture model system, conditioned culture media from normal pericytes promoted in-vitro OPC maturation. However, conditioned media from AKAP12-deficient pericytes did not support the OPC function. These findings suggest that AKAP12 signaling in pericytes may be required for OPC-to-oligodendrocyte renewal to maintain the white matter homeostasis in adult brain. Stem Cells 2018;36:751-760.
Subject(s)
A Kinase Anchor Proteins/metabolism , Cell Cycle Proteins/metabolism , Cell Differentiation/physiology , Neural Stem Cells/cytology , Oligodendroglia/metabolism , White Matter/metabolism , A Kinase Anchor Proteins/genetics , Aging , Animals , Cell Cycle Proteins/genetics , Cell Proliferation/physiology , Cells, Cultured , Culture Media, Conditioned , Mice, Knockout , Myelin Sheath/metabolism , Neurogenesis/physiology , Oligodendroglia/cytology , White Matter/cytologyABSTRACT
On the basis of deguelin, a series of the B,C-ring truncated surrogates with N-substituted amide linkers were investigated as HSP90 inhibitors. The structure activity relationship of the template was studied by incorporating various substitutions on the nitrogen of the amide linker and examining their HIF-1α inhibition. Among them, compound 57 showed potent HIF-1α inhibition and cytotoxicity in triple-negative breast cancer lines in a dose-dependent manner. Compound 57 downregulated expression and phosphorylation of major client proteins of HSP90 including AKT, ERK and STAT3, indicating that its antitumor activity was derived from the inhibition of HSP90 function. The molecular modeling of 57 demonstrated that 57 bound well to the C-terminal ATP-binding pocket in the open conformation of the hHSP90 homodimer with hydrogen bonding and pi-cation interactions. Overall, compound 57 is a potential antitumor agent for triple-negative breast cancer as a HSP90 C-terminal inhibitor.
Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , HSP90 Heat-Shock Proteins/antagonists & inhibitors , Rotenone/analogs & derivatives , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Female , HSP90 Heat-Shock Proteins/metabolism , Humans , Models, Molecular , Molecular Structure , Rotenone/chemical synthesis , Rotenone/chemistry , Rotenone/pharmacology , Structure-Activity RelationshipABSTRACT
Thiazolidinedione is a five-membered heterocycle that is widely used in drug discovery endeavors. In this study, we report the design, synthesis, and biological evaluation of a series of thiazolidinedione-based HDAC6 inhibitors. In particular, compound 6b exerts an excellent inhibitory activity against HDAC6 with an IC50 value of 21 nM, displaying a good HDAC6 selectivity over HDAC1. Compound 6b dose-dependently induces the acetylation level of α-tubulin via inhibition of HDAC6 in human neuroblastoma SH-SY5Y cell line. Moreover, compound 6b efficiently reverses methamphetamine-induced morphology changes of SH-SY5Y cells via regulating acetylation landscape of α-tubulin. Collectively, compound 6b represents a novel HDAC6-isoform selective inhibitor and demonstrates promising therapeutic potential for the treatment of methamphetamine addiction.
Subject(s)
Drug Discovery , Histone Deacetylase 6/antagonists & inhibitors , Histone Deacetylase Inhibitors , Thiazolidinediones , Amphetamine-Related Disorders/drug therapy , Amphetamine-Related Disorders/enzymology , Cell Line, Tumor , Histone Deacetylase 6/chemistry , Histone Deacetylase 6/metabolism , Histone Deacetylase Inhibitors/chemical synthesis , Histone Deacetylase Inhibitors/chemistry , Histone Deacetylase Inhibitors/pharmacology , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/chemistry , Isoenzymes/metabolism , Thiazolidinediones/chemical synthesis , Thiazolidinediones/chemistry , Thiazolidinediones/pharmacologyABSTRACT
The use of activated sludge process for biological treatment of domestic and industrial wastewaters generates large amounts of sewage sludge, which is regarded as problematic biowaste. Conventional waste treatment methods such as landfilling and ocean pumping have been used to dispose the unwanted sludge, but this practice is no longer recommended due to serious secondary pollution and strict environmental regulations. Hydrothermal treatment represents a promising alternative that has attracted attention in recent years. In this study, batch experiments of hydrothermal treatment of domestic sewage sludge were conducted under varying conditions (temperature of 150-300⯰C, reaction time of 0.5-3.0â¯h, and sludge concentration of 5-30â¯g/L). A statistical study of the responses, including disintegration degree and concentration of dissolved compounds, was performed using a response surface methodology. Optimal conditions for hydrothermal treatment of sewage sludge were obtained through mathematical modeling.
Subject(s)
Models, Theoretical , Sewage , Temperature , Waste Disposal, FluidABSTRACT
Metabolomics is a powerful tool used to understand comprehensive changes in the metabolic response and to study the phenotype of an organism by instrumental analysis. It most commonly involves mass spectrometry followed by data mining and metabolite assignment. For the last few decades, hair has been used as a valuable analytical sample to investigate retrospective xenobiotic exposure as it provides a wider window of detection than other biological samples such as saliva, plasma, and urine. Hair contains functional metabolomes such as amino acids and lipids. Moreover, segmental analysis of hair based on its growth rate can provide information on metabolic changes over time. Therefore, it has great potential as a metabolomics sample to monitor chronic diseases, including drug addiction or abnormal conditions. In the current review, the latest applications of hair metabolomics in animal studies and clinical settings are highlighted. For this purpose, we review and discuss the characteristics of hair as a metabolomics sample, the analytical techniques employed in hair metabolomics and the consequence of hair metabolome alterations in recent studies. Through this, the value of hair as an alternative biological sample in metabolomics is highlighted.
Subject(s)
Hair/metabolism , Metabolome , Metabolomics , Animals , Chromatography, High Pressure Liquid , Humans , Mass Spectrometry , Metabolomics/methodsABSTRACT
Interleukin 4 (IL-4) can suppress delayed-type hypersensitivity reactions (DTHRs), including organ-specific autoimmune diseases in mice and humans. Despite the broadly documented antiinflammatory effect of IL-4, the underlying mode of action remains incompletely understood, as IL-4 also promotes IL-12 production by dendritic cells (DCs) and IFN-γ-producing T(H)1 cells in vivo. Studying the impact of IL-4 on the polarization of human and mouse DCs, we found that IL-4 exerts opposing effects on the production of either IL-12 or IL-23. While promoting IL-12-producing capacity of DCs, IL-4 completely abrogates IL-23. Bone marrow chimeras proved that IL-4-mediated suppression of DTHRs relies on the signal transducer and activator of transcription 6 (STAT6)-dependent abrogation of IL-23 in antigen-presenting cells. Moreover, IL-4 therapy attenuated DTHRs by STAT6- and activating transcription factor 3 (ATF3)-dependent suppression of the IL-23/T(H)17 responses despite simultaneous enhancement of IL-12/TH1 responses. As IL-4 therapy also improves psoriasis in humans and suppresses IL-23/T(H)17 responses without blocking IL-12/T(H)1, selective IL-4-mediated IL-23/T(H)17 silencing is promising as treatment against harmful inflammation, while sparing the IL-12-dependent T(H)1 responses.
Subject(s)
Antigen-Presenting Cells/immunology , Gene Silencing , Inflammation/physiopathology , Interleukin-23/genetics , Interleukin-4/physiology , Th17 Cells/immunology , HumansABSTRACT
We previously reported that PP242 (dual inhibitor of mTORC1/2) plus curcumin induced apoptotic cell death through lysosomal membrane permeabilization (LMP)-mediated autophagy. However, the relationship between ER stress and apoptotic cell death by combined PP242 and curcumin treatment remains unknown. In the present study, we found that combined PP242 and curcumin treatment induced cytosolic Ca2+ release and ER stress. Interestingly, pretreatment with the chemical chaperones (TUDCA and 4-PBA) and knockdown of CHOP and ATF4 by siRNA did not abolish combined treatment-induced apoptosis in renal carcinoma cells. These results suggest that combined treatment with mTORC1/2 inhibitor and curcumin induces ER stress which is not essential for apoptotic cell death. Furthermore, overexpression of HSP70 significantly inhibited PP242 plus curcumin-induced LMP and apoptosis, but the protective effect was abolished by K77R mutation of acetylation site of HSP70. Taken together, our results reveal that regulation of HSP70 through K77 acetylation plays role in combined PP242 and curcumin treatment-induced apoptosis.
Subject(s)
Curcumin/pharmacology , HSP70 Heat-Shock Proteins/metabolism , Mechanistic Target of Rapamycin Complex 1/antagonists & inhibitors , Mechanistic Target of Rapamycin Complex 2/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Acetylation , Apoptosis/drug effects , Calcium/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Curcumin/chemistry , Drug Synergism , Endoplasmic Reticulum Stress/drug effects , Humans , Protein Kinase Inhibitors/chemistry , Signal Transduction/drug effectsABSTRACT
Thyroid hormones (THs) as a therapeutic intervention to treat obesity has been tried but the effect of THs on body weight and the mechanistic details of which are far from clear. This study was designed to determine and elucidate the mechanistic details of metabolic action of THs in high-fat diet (HFD) fed Sprague Dawley (SD) rats. Rats were made surgically hypothyroid (thyroidectomy, Thx). Body weights and food and water intake profoundly decreased in HFD fed thyroidectomized group (HN Thx). Results showed that delayed insulin response, increased total cholesterol, high-density lipoprotein, and low-density lipoprotein in HN Thx. Unexpectedly, however, Thx reduced serum and hepatic triglyceride concentrations. Further studies revealed that Thx dramatically increased circulating GLP-1 as well as increased expressions of GLP-1 in small intestine. Diminished hepatic expressions of lipogenic genes, were observed in HN Thx group. Beta-catenin and glutamine synthetase, a known target of ß-catenin, were up-regulated in the liver of HN Thx group. The expressions of gluconeogenic genes G6P and PCK were reduced in the liver of HN Thx group. The results may suggest that surgery-induced hypothyroidism increases GLP-1, the actions of which may in part be responsible for the reduction in water intake, appetite and hepatic steatosis.
Subject(s)
Eating , Fatty Liver/metabolism , Fatty Liver/surgery , Glucagon-Like Peptide 1/metabolism , Thyroid Hormones/metabolism , Thyroidectomy , Animals , Appetite Regulation , Diet, High-Fat , Dietary Fats , Fatty Liver/diagnosis , Male , Rats , Rats, Sprague-Dawley , Treatment OutcomeABSTRACT
Based on the lead compound L-80 (compound 2), a potent heat shock protein 90 (HSP90) inhibitor, a series of C-ring truncated deguelin analogs were designed, synthesized and evaluated for Hypoxia Inducible Factor-1α (HIF-1α) inhibition as a primary screening method. Their structure-activity relationship was investigated in a systematic manner by varying the A/B ring, linker and D/E ring, respectively. Among the synthesized inhibitors, compound 5 exhibited potent HIF-1α inhibition in a dose-dependent manner and significant antitumor activity in human non-small cell lung carcinoma (H1299), with better activities than L-80. It also inhibited in vitro hypoxia-mediated angiogenic processes in human retinal microvascular endothelial cells (HRMEC). The docking study of 5 showed a similar binding mode as L-80: it occupied the C-terminal ATP-binding pocket of HSP90, indicating that the anticancer and antiangiogenic activities of 5 were derived from HIF-1α destabilization by inhibiting the C-terminal ATP-binding site of hHSP90.
Subject(s)
Antineoplastic Agents/pharmacology , HSP90 Heat-Shock Proteins/antagonists & inhibitors , Neovascularization, Pathologic/drug therapy , Rotenone/analogs & derivatives , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , HSP90 Heat-Shock Proteins/metabolism , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/antagonists & inhibitors , Models, Molecular , Molecular Structure , Neovascularization, Pathologic/pathology , Rotenone/chemical synthesis , Rotenone/chemistry , Rotenone/pharmacology , Structure-Activity RelationshipABSTRACT
The clinical benefit of current anticancer regimens for lung cancer therapy is still limited due to moderate efficacy, drug resistance, and recurrence. Therefore, the development of effective anticancer drugs for first-line therapy and for optimal second-line treatment is necessary. Because the 90-kDa molecular chaperone heat shock protein (Hsp90) contributes to the maturation of numerous mutated or overexpressed oncogenic proteins, targeting Hsp90 may offer an effective anticancer therapy. Here, we investigated antitumor activities and toxicity of a novel deguelin-derived C-terminal Hsp90 inhibitor, designated L80. L80 displayed significant inhibitory effects on the viability, colony formation, angiogenesis-stimulating activity, migration, and invasion of a panel of non-small cell lung cancer cell lines and their sublines with acquired resistance to paclitaxel with minimal toxicity to normal lung epithelial cells, hippocampal cells, vascular endothelial cells, and ocular cells. Biochemical analyses and molecular docking simulation revealed that L80 disrupted Hsp90 function by binding to the C-terminal ATP-binding pocket of Hsp90, leading to the disruption of the interaction between hypoxia-inducible factor (HIF)-1α and Hsp90, downregulation of HIF-1α and its target genes, including vascular endothelial growth factor (VEGF) and insulin-like growth factor 2 (IGF2), and decreased the expression of various Hsp90 client proteins. Consistent with these in vitro findings, L80 exhibited significant antitumor and antiangiogenic activities in H1299 xenograft tumors. These results suggest that L80 represents a novel C-terminal Hsp90 inhibitor with effective anticancer activities with minimal toxicities.
Subject(s)
Antineoplastic Agents/administration & dosage , Antineoplastic Agents/chemical synthesis , Benzopyrans/administration & dosage , Carcinoma, Non-Small-Cell Lung/drug therapy , HSP90 Heat-Shock Proteins/chemistry , Lung Neoplasms/drug therapy , Quinolines/administration & dosage , Rotenone/analogs & derivatives , Animals , Antineoplastic Agents/pharmacology , Benzopyrans/chemical synthesis , Benzopyrans/pharmacology , Carcinoma, Non-Small-Cell Lung/metabolism , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Gene Expression Regulation, Neoplastic/drug effects , HSP90 Heat-Shock Proteins/antagonists & inhibitors , Human Umbilical Vein Endothelial Cells , Humans , Lung Neoplasms/metabolism , Mice , Mice, SCID , Quinolines/chemical synthesis , Quinolines/pharmacology , Rotenone/administration & dosage , Rotenone/chemical synthesis , Rotenone/pharmacology , Signal Transduction/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Ninjurin1 is a homotypic adhesion molecule that contributes to leukocyte trafficking in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. However, in vivo gene deficiency animal studies have not yet been done. Here, we constructed Ninjurin1 knock-out (KO) mice and investigated the role of Ninjurin1 on leukocyte trafficking under inflammation conditions such as EAE and endotoxin-induced uveitis. Ninjurin1 KO mice attenuated EAE susceptibility by reducing leukocyte recruitment into the injury regions of the spinal cord and showed less adhesion of leukocytes on inflamed retinal vessels in endotoxin-induced uveitis mice. Moreover, the administration of a custom-made antibody (Ab26-37) targeting the Ninjurin1 binding domain ameliorated the EAE symptoms, showing the contribution of its adhesion activity to leukocyte trafficking. In addition, we addressed the transendothelial migration (TEM) activity of bone marrow-derived macrophages and Raw264.7 cells according to the expression level of Ninjurin1. TEM activity was decreased in Ninjurin1 KO bone marrow-derived macrophages and siNinj1 Raw264.7 cells. Consistent with this, GFP-tagged mNinj1-overexpressing Raw264.7 cells increased their TEM activity. Taken together, we have clarified the contribution of Ninjurin1 to leukocyte trafficking in vivo and delineated its direct functions to TEM, emphasizing Ninjurin1 as a beneficial therapeutic target against inflammatory diseases such as multiple sclerosis.
Subject(s)
Bone Marrow Cells/metabolism , Cell Adhesion Molecules, Neuronal/metabolism , Cell Movement , Encephalomyelitis, Autoimmune, Experimental/metabolism , Macrophages/metabolism , Nerve Growth Factors/metabolism , Animals , Antibodies, Neutralizing/pharmacology , Bone Marrow Cells/pathology , Cell Adhesion Molecules, Neuronal/antagonists & inhibitors , Cell Adhesion Molecules, Neuronal/genetics , Cell Line , Disease Susceptibility , Encephalomyelitis, Autoimmune, Experimental/genetics , Encephalomyelitis, Autoimmune, Experimental/pathology , Encephalomyelitis, Autoimmune, Experimental/therapy , Macrophages/pathology , Mice , Mice, Knockout , Multiple Sclerosis/genetics , Multiple Sclerosis/metabolism , Multiple Sclerosis/pathology , Multiple Sclerosis/therapy , Nerve Growth Factors/antagonists & inhibitors , Nerve Growth Factors/geneticsABSTRACT
Ninjurin1 is involved in the pathogenesis of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, by mediating leukocyte extravasation, a process that depends on homotypic binding. However, the precise regulatory mechanisms of Ninjurin1 during inflammation are largely undefined. We therefore examined the pro-migratory function of Ninjurin1 and its regulatory mechanisms in macrophages. Interestingly, Ninjurin1-deficient bone marrow-derived macrophages exhibited reduced membrane protrusion formation and dynamics, resulting in the impairment of cell motility. Furthermore, exogenous Ninjurin1 was distributed at the membrane of filopodial structures in Raw264.7 macrophage cells. In Raw264.7 cells, RNA interference of Ninjurin1 reduced the number of filopodial projections, whereas overexpression of Ninjurin1 facilitated their formation and thus promoted cell motility. Ninjurin1-induced filopodial protrusion formation required the activation of Rac1. In Raw264.7 cells penetrating an MBEC4 endothelial cell monolayer, Ninjurin1 was localized to the membrane of protrusions and promoted their formation, suggesting that Ninjurin1-induced protrusive activity contributed to transendothelial migration. Taking these data together, we conclude that Ninjurin1 enhances macrophage motility and consequent extravasation of immune cells through the regulation of protrusive membrane dynamics. We expect these findings to provide insight into the understanding of immune responses mediated by Ninjurin1.
Subject(s)
Cell Adhesion Molecules, Neuronal/physiology , Cell Movement/physiology , Macrophages/physiology , Nerve Growth Factors/physiology , Animals , Cell Adhesion/physiology , Cell Adhesion Molecules, Neuronal/deficiency , Cell Adhesion Molecules, Neuronal/genetics , Cell Line , Cell Membrane/physiology , Cells, Cultured , Endothelial Cells/physiology , Gene Knockdown Techniques , Inflammation/etiology , Inflammation/physiopathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Biological , Nerve Growth Factors/deficiency , Nerve Growth Factors/genetics , Neuropeptides/metabolism , Pseudopodia/physiology , RNA Interference , rac1 GTP-Binding Protein/metabolismABSTRACT
It is now relatively well accepted that the cerebrovascular system does not merely provide inert pipes for blood delivery to the brain. Cerebral endothelial cells may compose an embedded bunker of trophic factors that contribute to brain homeostasis and function. Recent findings suggest that soluble factors from cerebral endothelial cells nourish neighboring cells, such as neurons and astrocytes. Although data are strongest in supporting mechanisms of endothelial-neuron and/or endothelial-astrocyte trophic coupling, it is likely that similar interactions also exist between cerebral endothelial cells and oligodendrocyte lineage cells. In this mini-review, we summarize current advances in the field of endothelial-oligodendrocyte trophic coupling. These endothelial-oligodendrocyte interactions may comprise the oligovascular niche to maintain their cellular functions and sustain ongoing angiogenesis/oligodendrogenesis. Importantly, it should be noted that the cell-cell interactions are not static-the trophic coupling is disturbed under acute phase after brain injury, but would be recovered in the chronic phase to promote brain remodeling and repair. Oligodendrocyte lineage cells play critical roles in white matter function, and under pathological conditions, oligodendrocyte dysfunction lead to white matter damage. Therefore, a deeper understanding of the mechanisms of endothelial-oligodendrocyte trophic coupling may lead to new therapeutic approaches for white matter-related diseases, such as stroke or vascular dementia.