ABSTRACT
Accurate control of innate immune responses is required to eliminate invading pathogens and simultaneously avoid autoinflammation and autoimmune diseases. Here, we demonstrate that arginine monomethylation precisely regulates the mitochondrial antiviral-signaling protein (MAVS)-mediated antiviral response. Protein arginine methyltransferase 7 (PRMT7) forms aggregates to catalyze MAVS monomethylation at arginine residue 52 (R52), attenuating its binding to TRIM31 and RIG-I, which leads to the suppression of MAVS aggregation and subsequent activation. Upon virus infection, aggregated PRMT7 is disabled in a timely manner due to automethylation at arginine residue 32 (R32), and SMURF1 is recruited to PRMT7 by MAVS to induce proteasomal degradation of PRMT7, resulting in the relief of PRMT7 suppression of MAVS activation. Therefore, we not only reveal that arginine monomethylation by PRMT7 negatively regulates MAVS-mediated antiviral signaling in vitro and in vivo but also uncover a mechanism by which PRMT7 is tightly controlled to ensure the timely activation of antiviral defense.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Arginine/metabolism , Host-Pathogen Interactions/physiology , Immunity, Innate/physiology , Protein-Arginine N-Methyltransferases/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/immunology , Animals , DEAD Box Protein 58/metabolism , Fibroblasts/virology , HEK293 Cells , Herpes Simplex/immunology , Herpes Simplex/metabolism , Herpes Simplex/virology , Humans , Methylation , Mice , Mice, Knockout , Polyunsaturated Alkamides , Protein-Arginine N-Methyltransferases/antagonists & inhibitors , Protein-Arginine N-Methyltransferases/genetics , Protein-Arginine N-Methyltransferases/immunology , Receptors, Immunologic/metabolism , Respirovirus Infections/immunology , Respirovirus Infections/metabolism , Respirovirus Infections/virology , Tripartite Motif Proteins/genetics , Tripartite Motif Proteins/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolismABSTRACT
To effectively protect the host from viral infection while avoiding excessive immunopathology, the innate immune response must be tightly controlled. However, the precise regulation of antiviral innate immunity and the underlying mechanisms remain unclear. Here, we find that sirtuin3 (SIRT3) interacts with mitochondrial antiviral signaling protein (MAVS) to catalyze MAVS deacetylation at lysine residue 7 (K7), which promotes MAVS aggregation, as well as TANK-binding kinase I and IRF3 phosphorylation, resulting in increased MAVS activation and enhanced type I interferon signaling. Consistent with these findings, loss of Sirt3 in mice and zebrafish renders them more susceptible to viral infection compared to their wild-type (WT) siblings. However, Sirt3 and Sirt5 double-deficient mice exhibit the same viral susceptibility as their WT littermates, suggesting that loss of Sirt5 in Sirt3-deficient mice may counteract the increased viral susceptibility displayed in Sirt3-deficient mice. Thus, we not only demonstrate that SIRT3 positively regulates antiviral immunity in vitro and in vivo, likely via MAVS, but also uncover a previously unrecognized mechanism by which SIRT3 acts as an accelerator and SIRT5 as a brake to orchestrate antiviral innate immunity.
Subject(s)
Sirtuin 3 , Sirtuins , Virus Diseases , Animals , Mice , Adaptor Proteins, Signal Transducing/genetics , Immunity, Innate , Lysine , Sirtuin 3/genetics , Sirtuins/genetics , Zebrafish , Zebrafish ProteinsABSTRACT
SIRT7 is a member of the sirtuin family proteins with nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylase activity, which can inhibit the activity of hypoxia-inducible factors independently of its enzymatic activity. However, the role of SIRT7 in affecting hypoxia signaling in vivo is still elusive. Here, we find that sirt7-null zebrafish are more resistant to hypoxic conditions, along with an increase of hypoxia-responsive gene expression and erythrocyte numbers, compared with their wildtype siblings. Overexpression of sirt7 suppresses the expression of hypoxia-responsive genes. Further assays indicate that sirt7 interacts with zebrafish hif-1αa, hif-1αb, hif-2αa, and hif-2αb to inhibit their transcriptional activity and mediate their protein degradation. In addition, sirt7 not only binds to the hypoxia responsive element of hypoxia-inducible gene promoters but also causes a reduction of H3K18Ac on these promoters. Sirt7 may regulate hypoxia-responsive gene expression through its enzymatic and nonenzymatic activities. This study provides novel insights into sirt7 function and sheds new light on the regulation of hypoxia signaling by sirt7.
Subject(s)
Oxygen , Sirtuins , Zebrafish Proteins , Zebrafish , Animals , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Proteolysis , Sirtuins/genetics , Sirtuins/metabolism , Zebrafish/genetics , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism , Anaerobiosis , Oxygen/metabolismABSTRACT
Prolyl hydroxylase domain (PHD)-containing enzyme 3 (PHD3) belongs to the Caenorhabditis elegans gene egl-9 family of prolyl hydroxylases. PHD3 catalyzes proline hydroxylation of hypoxia-inducible factor α (HIF-α) and promotes HIF-α proteasomal degradation through coordination with the pVHL complex under normoxic conditions. However, the relationship between PHD3 and the hypoxic response is not well understood. In this study, we used quantitative real-time PCR assay and O-dianisidine staining to characterize the hypoxic response in zebrafish deficient in phd3. We found that the hypoxia-responsive genes are upregulated and the number of erythrocytes was increased in phd3-null zebrafish compared with their wild-type siblings. On the other hand, we show overexpression of phd3 suppresses HIF-transcriptional activation. In addition, we demonstrate phd3 promotes polyubiquitination of zebrafish hif-1/2α proteins, leading to their proteasomal degradation. Finally, we found that compared with wild-type zebrafish, phd3-null zebrafish are more resistant to hypoxia treatment. Therefore, we conclude phd3 has a role in hypoxia tolerance. These results highlight the importance of modulation of the hypoxia signaling pathway by phd3 in hypoxia adaptation.
Subject(s)
Hypoxia-Inducible Factor-Proline Dioxygenases , Oxygen , Procollagen-Proline Dioxygenase , Zebrafish Proteins , Zebrafish , Animals , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Hypoxia-Inducible Factor-Proline Dioxygenases/genetics , Hypoxia-Inducible Factor-Proline Dioxygenases/metabolism , Procollagen-Proline Dioxygenase/genetics , Procollagen-Proline Dioxygenase/metabolism , Proline/metabolism , Zebrafish/genetics , Zebrafish/metabolism , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism , Gene Deletion , Oxygen/metabolismABSTRACT
The deubiquitinating enzyme OTUB1 possesses canonical deubiquitinase (DUB) activity and noncanonical, catalytic-independent activity, which has been identified as an essential regulator of diverse physiological processes. Posttranslational modifications of OTUB1 affect both its DUB activity and its noncanonical activity of binding to the E2 ubiquitin-conjugation enzyme UBC13, but further investigation is needed to characterize the full inventory of modifications to OTUB1. Here, we demonstrate that SET7, a lysine monomethylase, directly interacts with OTUB1 to catalyze OTUB1 methylation at lysine 122. This modification does not affect DUB activity of OTUB1 but impairs its noncanonical activity, binding to UBC13. Moreover, we found using cell viability analysis and intracellular reactive oxygen species assay that SET7-mediated methylation of OTUB1 relieves its suppressive role on ferroptosis. Notably, the methylation-mimic mutant of OTUB1 not only loses the ability to bind to UBC13 but also relieves its suppressive role on Tert-Butyl hydroperoxide-induced cell death and Cystine starvation/Erastin-induced cellular reactive oxygen species. Collectively, our data identify a novel modification of OTUB1 that is critical for inhibiting its noncanonical activity.
Subject(s)
Deubiquitinating Enzymes , Ferroptosis , Histone-Lysine N-Methyltransferase , Ubiquitin-Conjugating Enzymes , Deubiquitinating Enzymes/metabolism , Lysine/metabolism , Protein Binding , Reactive Oxygen Species/metabolism , Ubiquitination , Humans , Histone-Lysine N-Methyltransferase/metabolismABSTRACT
Hypoxia-inducible factor (HIF)1α, a main transcriptional regulator of the cellular response to hypoxia, also plays important roles in oxygen homeostasis of aerobic organisms, which is regulated by multiple mechanisms. However, the full cellular response to hypoxia has not been elucidated. In this study, we found that expression of SMYD3, a methyltransferase, augments hypoxia signaling independent of its enzymatic activity. We demonstrated SMYD3 binds to and stabilizes HIF1α via co-immunoprecipitation and Western blot assays, leading to the enhancement of HIF1α transcriptional activity under hypoxia conditions. In addition, the stabilization of HIF1α by SMYD3 is independent of HIF1α hydroxylation by prolyl hydroxylases and the intactness of the von Hippel-Lindau ubiquitin ligase complex. Furthermore, we showed SMYD3 induces reactive oxygen species accumulation and promotes hypoxia-induced cell apoptosis. Consistent with these results, we found smyd3-null zebrafish exhibit higher hypoxia tolerance compared to their wildtype siblings. Together, these findings define a novel role of SMYD3 in affecting hypoxia signaling and demonstrate that SMYD3-mediated HIF1α stabilization augments hypoxia signaling, leading to the impairment of hypoxia tolerance.
Subject(s)
Histone-Lysine N-Methyltransferase , Hypoxia , Methyltransferases , Zebrafish Proteins , Animals , Hypoxia/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Methyltransferases/metabolism , Signal Transduction , Von Hippel-Lindau Tumor Suppressor Protein/genetics , Von Hippel-Lindau Tumor Suppressor Protein/metabolism , Zebrafish/metabolism , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
Egg-laying defective nine 1 (EGLN1) functions as an oxygen sensor to catalyze prolyl hydroxylation of the transcription factor hypoxia-inducible factor-1 α under normoxia conditions, leading to its proteasomal degradation. Thus, EGLN1 plays a central role in the hypoxia-inducible factor-mediated hypoxia signaling pathway; however, the posttranslational modifications that control EGLN1 function remain largely unknown. Here, we identified that a lysine monomethylase, SET7, catalyzes EGLN1 methylation on lysine 297, resulting in the repression of EGLN1 activity in catalyzing prolyl hydroxylation of hypoxia-inducible factor-1 α. Notably, we demonstrate that the methylation mimic mutant of EGLN1 loses the capability to suppress the hypoxia signaling pathway, leading to the enhancement of cell proliferation and the oxygen consumption rate. Collectively, our data identify a novel modification of EGLN1 that is critical for inhibiting its enzymatic activity and which may benefit cellular adaptation to conditions of hypoxia.
Subject(s)
Histone-Lysine N-Methyltransferase , Hypoxia-Inducible Factor 1, alpha Subunit , Hypoxia-Inducible Factor-Proline Dioxygenases , Lysine , Animals , Catalysis , Humans , Hydroxylation , Hypoxia/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Hypoxia-Inducible Factor-Proline Dioxygenases/genetics , Hypoxia-Inducible Factor-Proline Dioxygenases/metabolism , Lysine/metabolism , Methylation , Oxygen/metabolism , Protein Processing, Post-TranslationalABSTRACT
Enzyme-responsive drug delivery systems have drawn much attention in the field of cancer theranostics due to their high sensitivity and substrate specificity under mild conditions. In this study, an amphiphilic polymer T1 is reported, which contains a tetraphenylethene unit and a poly(ethylene glycol) chain linked by an esterase-responsive phenolic ester bond. In aqueous solution, T1 formed stable micelles via self-assembly, which showed an aggregation-induced emission enhancement of 32-fold at 532 nm and a critical micelle concentration of 0.53 µM as well as esterase-responsive activity. The hydrophobic drug doxorubicin (DOX) was efficiently encapsulated into the micelles with a drug loading of 21%. In the presence of the esterase, the selective decomposition of drug-loaded T1 micelles was observed, and DOX was subsequently released with a half-life of 5 h. In vitro antitumor studies showed that T1@DOX micelles exhibited good therapeutic effects on HeLa cells, while normal cells remained mostly intact. In vivo anticancer experiments revealed that T1@DOX micelles indeed suppressed tumor growth and had reduced side effects compared to DOX·HCl. The present work showed the potential clinical application of esterase-responsive drug delivery in cancer therapy.
Subject(s)
Micelles , Polyethylene Glycols , Humans , Polyethylene Glycols/chemistry , HeLa Cells , Esterases , Drug Carriers/chemistry , Doxorubicin/pharmacology , Doxorubicin/therapeutic use , Polymers/chemistry , Drug Delivery Systems , Hydrogen-Ion ConcentrationABSTRACT
TNFR-associated factor 6 (TRAF6) not only recruits TBK1/IKKε to MAVS upon virus infection but also catalyzes K63-linked polyubiquitination on substrate or itself, which is critical for NEMO-dependent and -independent TBK1/IKKε activation, leading to the production of type I IFNs. The regulation at the TRAF6 level could affect the activation of antiviral innate immunity. In this study, we demonstrate that zebrafish prmt2, a type I arginine methyltransferase, attenuates traf6-mediated antiviral response. Prmt2 binds to the C terminus of traf6 to catalyze arginine asymmetric dimethylation of traf6 at arginine 100, preventing its K63-linked autoubiquitination, which results in the suppression of traf6 activation. In addition, it seems that the N terminus of prmt2 competes with mavs for traf6 binding and prevents the recruitment of tbk1/ikkε to mavs. By zebrafish model, we show that loss of prmt2 promotes the survival ratio of zebrafish larvae after challenge with spring viremia of carp virus. Therefore, we reveal, to our knowledge, a novel function of prmt2 in the negative regulation of antiviral innate immunity by targeting traf6.
Subject(s)
Immunity, Innate/immunology , Protein-Arginine N-Methyltransferases/immunology , Rhabdoviridae Infections/immunology , TNF Receptor-Associated Factor 6/immunology , Animals , Rhabdoviridae/immunology , ZebrafishABSTRACT
Stimuli-responsive drug delivery systems (DDSs) based on amphiphilic polymers have attracted much attention. In this study, we reported an innovative H2O2-responsive amphiphilic polymer (TBP), bearing a H2O2-sensitive phenylboronic ester, AIE fluorophore tetraphenylethene (TPE) hydrophobic, and polyethylene glycol hydrophilic (PEG) moieties. TBP could self-assemble into micelles with an encapsulation efficiency as high as 74.9% for doxorubicin (DOX) in aqueous solution. In the presence of H2O2, TBP micelles was decomposed by oxidation, hydrolysis and rearrangement, leading to almost 80% DOX release from TBP@DOX micelles. TBP and the corresponding degradation products were biocompatible, while TBP@DOX micelles only displayed obvious toxicity toward cancer cells. Drug delivery process was clearly monitored by confocal laser scanning microscopic (CLSM) and flow cytometry (FCM) analysis. Moreover, in vivo anticancer study showed that TBP@DOX micelles were accumulated in tumor region of nude mice and effectively inhibited tumor growth. The results suggested that the reported H2O2-responsive amphiphilic polymer displayed great potential in drug delivery and tumor therapy.
Subject(s)
Antibiotics, Antineoplastic/pharmacology , Doxorubicin/pharmacology , Drug Delivery Systems , Hydrogen Peroxide/chemistry , Polymers/chemistry , Surface-Active Agents/chemistry , Animals , Antibiotics, Antineoplastic/chemistry , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Doxorubicin/chemistry , Drug Liberation , Drug Screening Assays, Antitumor , Female , HEK293 Cells , HeLa Cells , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Molecular Structure , Protein Aggregates , Structure-Activity RelationshipABSTRACT
A series of unnatural tripeptides, each consisting of two aromatic γ-amino acid residues and an Ï-amino acid residue, are designed to probe their folding into hairpin conformations. The Ï-amino acid residues, with aliphatic or aromatic spacers of different sizes, serve as the loop of the hairpins. Studies based on one-dimensional (1D) 1H NMR performed at different concentrations, solvent polarity, and temperature, along with 2D-NMR studies, demonstrated that the doubly H-bonded aromatic γ-amino acid residues play important roles in driving these tripeptides into the hairpin conformation. The loop based on 5-aminovaleric acid, which offers a four-carbon (CH2)4 spacer, enhanced the stability of the corresponding hairpin, while loops having a shorter, a longer and a more rigid spacer disfavored the formation of the hairpins. Results from computational studies are in good agreement with the experimental observations. Furthermore, the crystal structure of peptide 1b revealed the expected hairpin conformation in the solid state. This turn motif, which contains H-bonded aromatic γ-amino acid residues as the core unit and an Ï-amino acid residue serving as the loop, provides a new platform that can be used to obtain a variety of turn conformations by incorporating diverse amino acids into the loops.
ABSTRACT
Nitric oxide (NO) is a messenger molecule in organisms, participating in the regulation of many biological processes. The abnormal expression of NO is often observed in a variety of diseases, including cerebral ischemia, atherosclerosis, and cancer. However, a suitable tool that can directly and sensitively detect NO in vitro and in vivo is important for understanding its various biological functions. In this report, a new fluorescent probe for nitric oxide, DHP-4, was prepared, based on dihydropyridine-coumarin. DHP-4 was able to greatly enhance the fluorescence of NO, but did not affect the fluorescence emissions of other reactive oxygen species and nitrogen species, demonstrating its highly selective and sensitive response to NO. The probe generated stable optical signals in a buffer solution at pH values ranging from 3 to 10. In addition, DHP-4 could detect NO directly, showed low cellular toxicity, and was successfully applied to determine NO in Raw 264.7 cells, indicating its great potential as a tool for investigating the biological roles of NO in vivo.
Subject(s)
Coumarins/chemistry , Dihydropyridines/chemistry , Fluorescent Dyes/analysis , Fluorescent Dyes/chemistry , Nitric Oxide/analysis , Animals , Cell Survival/drug effects , Cells, Cultured , Coumarins/analysis , Coumarins/pharmacology , Dihydropyridines/analysis , Dihydropyridines/pharmacology , Dose-Response Relationship, Drug , Fluorescent Dyes/chemical synthesis , Fluorescent Dyes/pharmacology , Mice , Molecular Structure , Optical Imaging , RAW 264.7 CellsABSTRACT
Nano drug delivery is a promising domain in biomedical theranostics and has aroused more and more attention in recent years. We report here an amphiphilic polymer TPG1, bearing a H2O2-sensitive benzil and an AIE fluorophore tetraphenylethene (TPE) unit, which is able to self-assemble into spherical nanosized micelles in aqueous solution. Doxorubicin (DOX) can be encapsulated into TPG1 micelles efficiently with the loading capability of up to 59% by weight. The benzil moiety could be cleaved via the Baeyer-Villiger type reaction in the presence of H2O2, leading to the decomposition of TPG1 micelles and release of DOX. In vitro studies indicated that DOX-loaded TPG1 micelles can be internalized by cancer cells, followed by unloading encapsulated DOX under the stimulation of H2O2. The drug release process can be monitored by the AIE fluorescence from the degradation products containing a TPE moiety. MTT assays against HeLa and HepG2 cancer cells demonstrated that DOX-loaded micelles showed good anticancer efficacy. The polymer TPG1 and the corresponding decomposed products showed great biocompatibility. Our data suggest that TPG1 has the potential to be employed for the controlled drug delivery system.
Subject(s)
Doxorubicin/pharmacology , Drug Delivery Systems , Fluorescent Dyes/chemistry , Hydrogen Peroxide/pharmacology , Phenylglyoxal/analogs & derivatives , Polymers/pharmacology , Stilbenes/chemistry , Cell Survival/drug effects , Doxorubicin/chemistry , Drug Carriers/chemistry , Drug Carriers/pharmacology , HeLa Cells , Hep G2 Cells , Humans , Hydrogen Peroxide/chemistry , Micelles , Molecular Structure , Optical Imaging , Phenylglyoxal/chemistry , Phenylglyoxal/pharmacology , Polymers/chemistryABSTRACT
BACKGROUND: Gastrointestinal motility disorder has been demonstrated to be regulated by acupuncture treatment. The mechanisms underlying the effects of acupuncture stimulation of abdominal and lower limb acupoints on gastrointestinal motility have been thoroughly studied; however, the physiology underlying the effects of acupuncture on the forelimbs to mediate gastrointestinal motility requires further exploration. The aim of this study was to determine whether electroacupuncture (EA) at LI11 promotes jejunal motility, whether the parasympathetic pathway participates in this effect, and if so, which somatic afferent nerve fibres are involved. METHODS: A manometric balloon was used to observe jejunal motility. The effects and mechanisms of EA at LI11 were explored in male Sprague-Dawley rats with or without drug administration (propranolol, clenbuterol, acetylcholine, and atropine) and with or without vagotomy. Three types of male mice (ß1ß2 receptor-knockout [ß1ß2-/-] mice, M2M3 receptor-knockout [M2M3-/-] mice and wild-type [WT] mice) were also studied by using different EA intensities (1, 2, 4, 6, and 8 mA). A total of 72 rats and 56 mice were included in the study. RESULTS: EA at LI11 increased the contractile amplitude of jejunal motility in the majority of both rats and mice. However, EA at LI11 did not enhance jejunal motility in rats administered atropine, rats that underwent vagotomy, and M2M3-||/- mice (at all intensities). In WT mice, EA at LI11 significantly increased jejunal motility at all intensities except 1 mA, and a plateau was reached at intensities greater than 4 mA. CONCLUSION: Our results suggest that EA at LI11 promotes jejunal motility primarily by exciting the parasympathetic pathway, and that Aδ-fibres and C-fibres may play important roles in the process.
Subject(s)
Electroacupuncture , Gastrointestinal Diseases/therapy , Jejunum/physiopathology , Parasympathetic Nervous System/physiopathology , Acupuncture Points , Acupuncture Therapy , Animals , Gastrointestinal Diseases/physiopathology , Gastrointestinal Motility , Humans , Male , Mice, Knockout , Rats , Rats, Sprague-DawleyABSTRACT
The ability to detect and visualize cellular events and associated biological analytes is essential for the understanding of their physiological and pathological functions. Cysteine (Cys) plays a crucial role in biological systems and lysosomal homeostasis. This puts forward higher requirements on the performance of the probe. Herein, we rationally designed a coumarin-based probe for the reversible, specific, sensitive, and rapid detection of Cys based on pH regulating reactivity. The obtained probe (ECMA) introduces a morpholine moiety to target lysosomes, and α,ß-unsaturated-ketone with an electron-withdrawing CN group served as a reversible reaction site for Cys. Importantly, ECMA was successfully applied to the real-time monitoring of Cys dynamics in living cells. Furthermore, cell imaging clearly revealed that exogenous Cys could induce the up-regulation of lysosomal ROS, which provided a powerful tool for investigating the relationship between oxidative stress and lysosomal Cys.
Subject(s)
Cysteine , Fluorescent Dyes , Lysosomes , Oxidative Stress , Lysosomes/metabolism , Lysosomes/chemistry , Fluorescent Dyes/chemistry , Fluorescent Dyes/chemical synthesis , Cysteine/chemistry , Cysteine/metabolism , Oxidative Stress/drug effects , Humans , Hydrogen-Ion Concentration , HeLa Cells , Optical Imaging , Molecular Structure , Coumarins/chemistry , Reactive Oxygen Species/metabolismABSTRACT
The combination of small-interfering RNA (siRNA)-mediated gene silencing and chemotherapeutic agents for lung cancer treatment has attracted widespread attention in terms of a greater therapeutic effect, minimization of systemic toxicity, and inhibition of multiple drug resistance (MDR). In this work, three amphiphiles, CBN1-CBN3, were first designed and synthesized as a camptothecin (CPT) conjugate and gene condensation agents by the combination of CPT prodrugs and di(triazole-[12]aneN3) through the ROS-responsive phenylborate ester and different lengths of alkyl chains (with 6, 9, 12 carbon chains for CBN1-CBN3, respectively). CBN1-CBN3 were able to be self-assembled into liposomes with an average diameter in the range of 320-240 nm, showing the ability to effectively condense siRNA. Among them, CBN2, with a nine-carbon alkyl chain, displayed the best anticancer efficiency in A549 cells. In order to give nanomedicines a stealth property and PEGylation/dePEGylation transition, a GSH-responsive PEGylated TPE derivative containing a disulfide linkage (TSP) was further designed and prepared. A combination of CBN2/siRNA complexes and DOPE with TSP resulted in GSH/ROS dual-responsive lipid-polymer hybrid nanoparticles (CBN2-DP/siRNA NPs). In present GSH and H2O2, CBN2-DP/siRNA NPs were decomposed, resulting in the controlled release of CPT drug and siRNA. In vitro, CBN2-DP/siPHB1 NPs showed the best anticancer activity for suppression of about 75% of A549 cell proliferation in a serum medium. The stability of CBN2-DP/siRNA NPs was significantly prolonged in blood circulation, and they showed effective accumulation in the A549 tumor site through an enhanced permeability and retention (EPR) effect. In vivo, CBN2-DP/siPHB1 NPs demonstrated enhanced synergistic cancer therapy efficacy and tumor inhibition as high as 71.2%. This work provided a strategy for preparing lipid-polymer hybrid NPs with GSH/ROS dual-responsive properties and an intriguing method for lung cancer therapy.
Subject(s)
Camptothecin , Lung Neoplasms , Nanoparticles , RNA, Small Interfering , Reactive Oxygen Species , Animals , Humans , Mice , A549 Cells , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Biocompatible Materials/chemistry , Biocompatible Materials/pharmacology , Biocompatible Materials/chemical synthesis , Camptothecin/chemistry , Camptothecin/pharmacology , Cell Proliferation/drug effects , Cell Survival/drug effects , Drug Screening Assays, Antitumor , Glutathione/chemistry , Glutathione/metabolism , Lipids/chemistry , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Lung Neoplasms/metabolism , Materials Testing , Molecular Structure , Nanoparticles/chemistry , Particle Size , Prohibitins , Reactive Oxygen Species/metabolism , RNA, Small Interfering/chemistryABSTRACT
The level of sulfur dioxide (SO2) and viscosity in mitochondria play vital roles in various physiological and pathological processes. Abnormalities in mitochondrial SO2 and viscosity are closely associated with numerous biological diseases. It is of great significance to develop novel fluorescence probes for simultaneous detection of SO2 and viscosity within mitochondria. Herein, we have developed a water-soluble, mitochondrial-targeted and near-infrared fluorescent probe, CMBT, for the simultaneous detection of SO2 and viscosity. The probe CMBT incorporates benzothiazolium salt as a mitochondrial targeting moiety and 7-diethylaminocoumarin as a rotor for viscosity detection, respectively. Based on the prompt reaction between nucleophilic HSO3-/SO32- and the backbone of the benzothiazolium salt derivative, probe CMBT displayed high sensitivity and selectivity toward SO2 with a limit of detection as low as 0.17 µM. As viscosity increased, the twisted intramolecular charge transfer (TICT) process was restricted, resulting in fluorescence emission enhancement at 690 nm. Moreover, probe CMBT demonstrated exceptional mitochondrial targeting ability and was successfully employed to image variations of SO2 and viscosity in living cells and mice. The work highlights the great potential of the probe as a convenient tool for revealing the relationship between SO2 and viscosity in biological systems.
Subject(s)
Fluorescent Dyes , Mitochondria , Sulfur Dioxide , Sulfur Dioxide/analysis , Sulfur Dioxide/chemistry , Fluorescent Dyes/chemistry , Animals , Mitochondria/chemistry , Mitochondria/metabolism , Viscosity , Mice , Humans , Optical Imaging/methods , HeLa Cells , Limit of DetectionABSTRACT
Despite good hepatitis B virus (HBV) inhibition by nucleoside analogs (NAs), cases of hepatocellular carcinoma (HCC) still occur. This study proposed a non-invasive predictive model to assess HCC risk in patients with chronic hepatitis B (CHB) receiving NAs treatment. Data were obtained from a hospital-based retrospective cohort registered on the Platform of Medical Data Science Academy of Chongqing Medical University, from 2013 to 2019. A total of 501 patients under NAs treatment had their FIB-4 index updated semiannually by recalculation based on laboratory values. Patients were divided into three groups based on FIB-4 index values: < 1.45, 1.45-3.25, and ≥ 3.25. Subsequently, HCC incidence was reassessed every six months using Kaplan-Meier curves based on the updated FIB-4 index. The median follow-up time of CHB patients after receiving NAs treatment was 2.5 years. HCC incidences with FIB-4 index < 1.45, 1.45-3.25, and ≥ 3.25 were 1.18%, 1.32%, and 9.09%, respectively. Dynamic assessment showed that the percentage of patients with FIB-4 index < 1.45 significantly increased semiannually (P < 0.001), and of patients with FIB-4 index ≥ 3.25 significantly decreased (P < 0.001). HCC incidence was the highest among patients with FIB-4 index ≥ 3.25. The FIB-4 index effectively predicted HCC incidence, and its dynamic assessment could be used for regular surveillance to implement early intervention and reduce HCC risk.
Subject(s)
Antiviral Agents , Carcinoma, Hepatocellular , Hepatitis B, Chronic , Liver Cirrhosis , Liver Neoplasms , Humans , Hepatitis B, Chronic/complications , Hepatitis B, Chronic/drug therapy , Male , Female , Retrospective Studies , Antiviral Agents/therapeutic use , Middle Aged , Adult , Risk Factors , Nucleosides/therapeutic use , Incidence , Risk AssessmentABSTRACT
Oxygen is essential for aerobic organisms, but little is known about its role in antiviral immunity. Here, we report that during responses to viral infection, hypoxic conditions repress antiviral-responsive genes independently of HIF signaling. EGLN1 is identified as a key mediator of the oxygen enhancement of antiviral innate immune responses. Under sufficient oxygen conditions, EGLN1 retains its prolyl hydroxylase activity to catalyze the hydroxylation of IRF3 at proline 10. This modification enhances IRF3 phosphorylation, dimerization and nuclear translocation, leading to subsequent IRF3 activation. Furthermore, mice and zebrafish with Egln1 deletion, treatment with the EGLN inhibitor FG4592, or mice carrying an Irf3 P10A mutation are more susceptible to viral infections. These findings not only reveal a direct link between oxygen and antiviral responses, but also provide insight into the mechanisms by which oxygen regulates innate immunity.
Subject(s)
Hypoxia-Inducible Factor-Proline Dioxygenases , Immunity, Innate , Interferon Regulatory Factor-3 , Oxygen , Proline , Zebrafish , Animals , Hypoxia-Inducible Factor-Proline Dioxygenases/metabolism , Hypoxia-Inducible Factor-Proline Dioxygenases/genetics , Interferon Regulatory Factor-3/metabolism , Hydroxylation , Humans , Proline/metabolism , Mice , Oxygen/metabolism , HEK293 Cells , Phosphorylation , Mice, Knockout , Signal Transduction , Mice, Inbred C57BLABSTRACT
A novel dual-responsive nanoparticle (NP) system was aimed to be developed for the co-delivery of camptothecin (CPT) and plasmid encoding TNF-related apoptosis-inducing ligand (pTRAIL) DNA in cancer therapy. The combination of the prodrug CPT and the nucleic acid condensing di-(triazole-[12]aneN3) unit with 4-nitrobenzyl ester through alkyl chains resulted in three nitroreductase (NTR) responsive amphiphiles, CNN1-CNN3 (with 5, 8, and 11 carbon chains, respectively). Among them, CNN2 was the most effective in inhibiting the proliferation of HeLa cells in the presence of fusogenic lipid DOPE. The NPs composed of CNN2, pDNA, and DOPE were further co-assembled with ROS-responsive thioketal-linked amphiphilic polymer (TTP) to afford the core-shell NPs (CNN2-DT/pDNA) with an average size of 118 nm, which exhibited high drug-loading capacity, excellent serum tolerance, and good biocompatibility. In the presence of ROS, NTR, and NADH, the core-shell NPs were decomposed, leading to the efficient release of 80% CPT and abundant pDNA. The self-assembly and delivery process of CNN2-DT NPs and DNA were clearly observed through the AIE fluorescent imaging. In vitro and in vivo results demonstrated that the CNN2-DT/pTRAIL NPs synergistically promoted 68% apoptosis of tumor cells and inhibited tumor growth with negligible toxic side effects. This study showed that the combination of prodrug and nucleic acid through dual-responsive core-shell NPs provide a spatially and temporally-controlled strategy for cancer therapy.