ABSTRACT
Citalopram (CTP) and mirtazapine (MTP) are two typical psychoactive drugs used for the depression treatment. As emerging pollutants, CTP and MTP have raised concern because of their harmful effect on aquatic organisms. Therefore, the ecotoxicological risk of these two pollutants to aquatic organisms should be given more attention. In this study, the effects of CTP and MTP on the feeding rate, heartbeat, nutritional enzymes, and their related gene expression of D. magna were investigated under single and binary mixture pollutant exposure. Subsequently, the recovery of exposed D. magna was studied to assess the toxic persistence of those pollutants. After 24-h exposure, the ingestion rate decreased by 34.2% and 21.5%, in the group of 1.45 mg/L CTP (C-H) and binary mixture with high concentration (Mix-H), respectively. After 24-h recovery, the feeding rate of D. magna was stimulated by a compensatory response. Over the exposure period, the heartbeat rate of D. magna increased significantly in the groups of CTP, MTP, and their binary mixture with low concentration (Mix-L), and then, their heartbeat rate was recovered during the recovery period. The activity of α-amylase (AMS) and trypsin were significantly changed in most of the exposed daphnia, both during the exposure and recovery period. CTP/MTP exposure stimulated the expression of the AMS gene. MTP and Mix-H exposure inhibited the expression of the trypsin gene and the other groups stimulated its expression. After 24-h recovery, the stimulating or inhibitory effects were alleviated. There were different responses between gene expression and enzyme activity. In conclusion, our results highlighted the toxic effects at high concentrations of single and mixed pollution of CTP and MTP on the feeding rate, heartbeat, AMS and trypsin enzyme activity, and expression of related genes of D. magna to assess the environment risk of them.
Subject(s)
Daphnia , Water Pollutants, Chemical , Animals , Antidepressive Agents/pharmacology , Aquatic Organisms/metabolism , Citalopram , Cytidine Triphosphate/metabolism , Cytidine Triphosphate/pharmacology , Mirtazapine/pharmacology , Trypsin/metabolism , Trypsin/pharmacology , Water Pollutants, Chemical/metabolism , Zooplankton/metabolism , alpha-Amylases/metabolismABSTRACT
The naturally occurring nucleotide 3'-deoxy-3',4'-didehydro-cytidine-5'-triphosphate (ddhCTP) was recently found to exert potent and broad-spectrum antiviral activity. However, nucleoside 5'-triphosphates in general are not cell-permeable, which precludes the direct use of ddhCTP as a therapeutic. To harness the therapeutic potential of this endogenous antiviral nucleotide, we synthesized phosphoramidate prodrug HLB-0532247 (1) and found it to result in dramatically elevated levels of ddhCTP in cells. We compared 1 and 3'-deoxy-3',4'-didehydro-cytidine (ddhC) and found that 1 more effectively reduces titers of Zika and West Nile viruses in cell culture with minimal nonspecific toxicity to host cells. We conclude that 1 is a promising antiviral agent based on a novel strategy of facilitating elevated levels of the endogenous ddhCTP antiviral nucleotide.
Subject(s)
Antiviral Agents/pharmacology , Cytidine Triphosphate/pharmacology , West Nile virus/drug effects , Zika Virus/drug effects , Animals , Antiviral Agents/chemistry , Cell Line , Chlorocebus aethiops , Cytidine Triphosphate/chemistry , Dose-Response Relationship, Drug , Humans , Microbial Sensitivity Tests , Molecular Structure , Structure-Activity RelationshipABSTRACT
5-aza-cytidine (5-aza-C) has been shown to be a potent human immunodeficiency virus type 1 (HIV-1) mutagen that induces G-to-C hypermutagenesis by incorporation of the reduced form (i.e., 5-aza-dC, 5-aza-dCTP). Evidence to date suggests that this lethal mutagenesis is the primary antiretroviral mechanism for 5-aza-C. To investigate the breadth of application of 5-aza-C as an antiretroviral mutagen, we have conducted a comparative, parallel analysis of the antiviral mechanism of 5-aza-C between HIV-1 and gammaretroviruses - i.e., murine leukemia virus (MuLV) and feline leukemia virus (FeLV). Intriguingly, in contrast to the hallmark G-to-C hypermutagenesis observed with HIV-1, MuLV and FeLV did not reveal the presence of a significant increase in mutational burden, particularly that of G-to-C transversion mutations. The effect of 5-aza-dCTP on DNA synthesis revealed that while HIV-1 RT was not inhibited by 5-aza-dCTP even at 100 µM, 5-aza-dCTP was incorporated and significantly inhibited MuLV RT, generating pause sites and reducing the fully extended product. 5-aza-dCTP was found to be incorporated into DNA by MuLV RT or HIV-1 RT, but only acted as a non-obligate chain terminator for MuLV RT. This biochemical data provides an independent line of experimental evidence in support of the conclusion that HIV-1 and MuLV have distinct primary mechanisms of antiretroviral action with 5-aza-C. Taken together, our data provides striking evidence that an antiretroviral mutagen can have strong potency via distinct mechanisms of action among closely related viruses, unlinking antiviral activity from antiviral mechanism of action.
Subject(s)
Antiviral Agents/pharmacology , Azacitidine/analogs & derivatives , Cytidine Triphosphate/analogs & derivatives , HIV Infections/drug therapy , Leukemia, Experimental/drug therapy , Mutation/drug effects , Retroviridae Infections/drug therapy , Tumor Virus Infections/drug therapy , Animals , Azacitidine/pharmacology , Cats , Cytidine Triphosphate/pharmacology , HIV/drug effects , HIV Infections/virology , Humans , Leukemia Virus, Feline/drug effects , Leukemia Virus, Murine/drug effects , Leukemia, Experimental/virology , Mice , Mutagenesis , Mutagens , Retroviridae Infections/virology , Tumor Virus Infections/virology , Virus ReplicationABSTRACT
Viperin (RSAD2) is an antiviral radical S-adenosylmethionine (SAM) enzyme highly expressed in different cell types upon viral infection. Recently, it has been reported that the radical-SAM activity of viperin transforms cytidine triphosphate (CTP) to its analogue 3'-deoxy-3',4'-didehydro-CTP (ddhCTP). Based on biochemical studies and cell biological experiments, it was concluded that ddhCTP and its nucleoside form ddhC do not affect the cellular concentration of nucleotide triphosphates and that ddhCTP acts as replication chain terminator. However, our re-evaluation of the reported data and new results indicate that ddhCTP is not an effective viral chain terminator but depletes cellular nucleotide pools and interferes with mitochondrial activity to inhibit viral replication. Our analysis is consistent with a unifying view of the antiviral and radical-SAM activities of viperin.
Subject(s)
Mitochondria/metabolism , Nucleotides/metabolism , Proteins/metabolism , Virus Replication , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Cell Respiration , Cytidine Triphosphate/metabolism , Cytidine Triphosphate/pharmacology , Humans , Inhibitory Concentration 50 , Mitochondria/drug effects , Nucleotides/pharmacology , Oxidoreductases Acting on CH-CH Group Donors , Uridine Triphosphate/metabolism , Virus Replication/drug effectsABSTRACT
While enzyme activity is often regulated by a combination of substrate/effector availability and quaternary structure, many cytosolic enzymes may be further regulated through oligomerization into filaments. Cytidine-5'-triphosphate (CTP) synthase (CTPS) forms such filaments-a process that is promoted by the product CTP. The CTP analog and active chemotherapeutic metabolite gemcitabine-5'-triphosphate (dF-dCTP) is a potent inhibitor of CTPS; however, its effect on the enzyme's ability to form filaments is unknown. Alongside electron microscopy studies, dynamic light scattering showed that dF-dCTP induces Escherichia coli CTPS (EcCTPS) to form filaments in solution with lengths ≥30 nm in the presence of CTP or dF-dCTP. The substrate UTP blocks formation of filaments and effects their disassembly. EcCTPS variants were constructed to investigate the role of CTP-binding determinants in CTP- and dF-dCTP-dependent filament formation. Substitution of Glu 149 (i.e., E149D), which interacts with the ribose of CTP, caused reduced affinity for both CTP and dF-dCTP, and obviated filament formation. Phe 227 appears to interact with CTP through an edge-on interaction with the cytosine ring, yet the F227A and F227L variants bound CTP and dF-dCTP. F227A EcCTPS did not form filaments, while F227L EcCTPS formed shorter filaments in the presence of CTP or dF-dCTP. Hence, Phe 227 plays a role in filament formation, although replacement by a bulky hydrophobic amino acid is sufficient for limited filament formation. That dF-dCTP can induce filament formation highlights the fact that nucleotide analogs employed as chemotherapeutic agents may affect the filamentous states of enzymes and potentially alter their regulation in vivo.
Subject(s)
Antineoplastic Agents/pharmacology , Carbon-Nitrogen Ligases/chemistry , Carbon-Nitrogen Ligases/genetics , Cytidine Triphosphate/analogs & derivatives , Escherichia coli/enzymology , Binding Sites , Carbon-Nitrogen Ligases/metabolism , Cytidine Triphosphate/pharmacology , Dynamic Light Scattering , Escherichia coli/chemistry , Escherichia coli/genetics , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Microscopy, Electron, Transmission , Models, Molecular , Mutation , Protein Conformation , Protein Multimerization , Uridine Triphosphate/metabolismABSTRACT
BACKGROUND: Rhabdomyosarcoma (RMS), which can be classified as embryonal RMS (ERMS) and alveolar RMS (ARMS), represents the most frequent soft tissue sarcoma in the pediatric population; the latter shows greater aggressiveness and metastatic potential with respect to the former. Epigenetic alterations in cancer include DNA methylation changes and histone modifications that influence overall gene expression patterns. Different tumor subtypes are characterized by distinct methylation signatures that could facilitate early disease detection and greater prognostic accuracy. METHODS: A genome-wide approach was used to examine methylation patterns associated with different prognoses, and DNA methylome analysis was carried out using the Agilent Human DNA Methylation platform. The results were validated using bisulfite sequencing and 5-aza-2'deoxycytidine treatment in RMS cell lines. Some in vitro functional studies were also performed to explore the involvement of a target gene in RMS tumor cells. RESULTS: In accordance with the Intergroup Rhabdomyosarcoma Study (IRS) grouping, study results showed that distinct methylation patterns distinguish RMS subgroups and that a cluster of protocadherin genes are hypermethylated in metastatic RMS. Among these, PCDHA4, whose expression was decreased by DNA methylation, emerged as a down-regulated gene in the metastatic samples. As PCDHA4-silenced cells have a significantly higher cell proliferation rate paralleled by higher cell invasiveness, PCDHA4 seems to behave as a tumor suppressor in metastatic RMS. CONCLUSION: Study results demonstrated that DNA methylation patterns distinguish between metastatic and non-metastatic RMS and suggest that epigenetic regulation of specific genes could represent a novel therapeutic target that could enhance the efficiency of RMS treatments.
Subject(s)
Cell Adhesion Molecules, Neuronal/genetics , DNA Methylation , Gene Expression Regulation, Neoplastic , Neuropeptides/genetics , Receptors, Cell Surface/genetics , Rhabdomyosarcoma/genetics , Rhabdomyosarcoma/pathology , Azacitidine/analogs & derivatives , Azacitidine/pharmacology , Biopsy , Cell Line, Tumor , Cluster Analysis , Cytidine Triphosphate/analogs & derivatives , Cytidine Triphosphate/pharmacology , Epigenesis, Genetic/drug effects , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/drug effects , Genome-Wide Association Study , Humans , Hydroxamic Acids/pharmacology , Neoplasm Metastasis , Promoter Regions, Genetic , Protocadherins , TranscriptomeABSTRACT
CTP synthase (CTPS) catalyzes the conversion of UTP to CTP and is a target for the development of antiviral, anticancer, antiprotozoal, and immunosuppressive agents. Exposure of cell lines to the antineoplastic cytidine analogue gemcitabine causes depletion of intracellular CTP levels, but the direct inhibition of CTPS by its metabolite gemcitabine-5'-triphosphate (dF-dCTP) has not been demonstrated. We show that dF-dCTP is a potent competitive inhibitor of Escherichia coli CTPS with respect to UTP [Ki =(3.0±0.1)â µm], and that its binding affinity exceeds that of CTP ≈75-fold. Site-directed mutagenesis studies indicated that Glu149 is an important binding determinant for both CTP and dF-dCTP. Comparison of the binding affinities of the 5'-triphosphates of 2'-fluoro-2'-deoxycytidine and 2'-fluoro-2'-deoxyarabinocytidine revealed that the 2'-F-arabino group contributes markedly to the strong binding of dF-dCTP. Geminal 2'-F substitution on UTP (dF-dUTP) did not result in an increase in binding affinity with CTPS. Remarkably, CTPS catalyzed the conversion of dF-dUTP into dF-dCTP, thus suggesting that dF-dCTP might be regenerated in vivo from its catabolite dF-dUTP.
Subject(s)
Carbon-Nitrogen Ligases/antagonists & inhibitors , Cytidine Triphosphate/analogs & derivatives , Enzyme Inhibitors/pharmacology , Carbon-Nitrogen Ligases/metabolism , Cytidine Diphosphate/analogs & derivatives , Cytidine Triphosphate/chemistry , Cytidine Triphosphate/pharmacology , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemistry , Escherichia coli/enzymology , Molecular Structure , Structure-Activity RelationshipABSTRACT
ALS-8112 is the parent molecule of ALS-8176, a first-in-class nucleoside analog prodrug effective in the clinic against respiratory syncytial virus (RSV) infection. The antiviral activity of ALS-8112 is mediated by its 5'-triphosphate metabolite (ALS-8112-TP, or 2'F-4'ClCH2-cytidine triphosphate) inhibiting the RNA polymerase activity of the RSV L-P protein complex through RNA chain termination. Four amino acid mutations in the RNA-dependent RNA polymerase (RdRp) domain of L (QUAD: M628L, A789V, L795I, and I796V) confer in vitro resistance to ALS-8112-TP by increasing its discrimination relative to natural CTP. In this study, we show that the QUAD mutations specifically recognize the ClCH2 group of ALS-8112-TP. Among the four mutations, A789V conferred the greatest resistance phenotype, which was consistent with its putative position in the active site of the RdRp domain. AZ-27, a non-nucleoside inhibitor of RSV, also inhibited the RdRp activity, with decreased inhibition potency in the presence of the Y1631H mutation. The QUAD mutations had no effect on the antiviral activity of AZ-27, and the Y1631H mutation did not significantly increase the discrimination of ALS-8112-TP. Combining ALS-8112 with AZ-27 in vitro resulted in significant synergistic inhibition of RSV replication. Overall, this is the first mechanistic study showing a lack of cross-resistance between mutations selected by different classes of RSV polymerase inhibitors acting in synergy, opening the door to future potential combination therapies targeting different regions of the L protein.
Subject(s)
Antiviral Agents/pharmacology , Benzazepines/pharmacology , Cytidine Triphosphate/analogs & derivatives , DNA-Directed RNA Polymerases/antagonists & inhibitors , Drug Resistance, Viral/genetics , Niacinamide/analogs & derivatives , Point Mutation , Viral Proteins/antagonists & inhibitors , Cell Line, Tumor , Cytidine Triphosphate/pharmacology , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Drug Combinations , Drug Synergism , Epithelial Cells/drug effects , Epithelial Cells/virology , Gene Expression , Humans , Niacinamide/pharmacology , RNA, Viral/antagonists & inhibitors , RNA, Viral/biosynthesis , RNA, Viral/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
The male genital tract is a potential site of viral persistence. Therefore, adequate concentrations of antiretrovirals are required to eliminate HIV replication in the genital tract. Despite higher zidovudine (ZDV) and lamivudine (3TC) concentrations in seminal plasma (SP) than in blood plasma (BP) (SP/BP drug concentration ratios of 2.3 and 6.7, respectively), we have previously reported lower relative intracellular concentrations of their active metabolites, zidovudine triphosphate (ZDV-TP) and lamivudine triphosphate (3TC-TP), in seminal mononuclear cells (SMCs) than in peripheral blood mononuclear cells (PBMCs) (SMC/PBMC drug concentration ratios of 0.36 and 1.0, respectively). Here, we use population pharmacokinetic (PK) modeling-based methods to simultaneously describe parent and intracellular metabolite PK in blood, semen, and PBMCs and SMCs. From this model, the time to steady state in each matrix was estimated, and the results indicate that the PK of 3TC-TP and ZDV-TP in PBMCs are different from the PK of the two in SMCs and different for the two triphosphates. We found that steady-state conditions in PBMCs were achieved within 2 days for ZDV-TP and 3 days for 3TC-TP. However, steady-state conditions in SMCs were achieved within 2 days for ZDV-TP and 2 weeks for 3TC-TP. Despite this, or perhaps because of it, ZDV-TP in SMCs does not achieve the surrogate 50% inhibitory concentration (IC50) (as established for PBMCs, assuming SMC IC50 = PBMC IC50) at the standard 300-mg twice-daily dosing. Mechanistic studies are needed to understand these differences and to explore intracellular metabolite behavior in SMCs for other nucleoside analogues used in HIV prevention, treatment, and cure.
Subject(s)
Anti-HIV Agents/pharmacokinetics , Cytidine Triphosphate/analogs & derivatives , Dideoxynucleotides/pharmacokinetics , Lamivudine/analogs & derivatives , Leukocytes, Mononuclear/metabolism , Models, Statistical , Semen/metabolism , Thymine Nucleotides/pharmacokinetics , Zidovudine/analogs & derivatives , Adult , Anti-HIV Agents/pharmacology , Biological Availability , Biological Transport , Blood Cells/drug effects , Blood Cells/metabolism , Blood Cells/pathology , Blood Cells/virology , Computer Simulation , Cytidine Triphosphate/pharmacokinetics , Cytidine Triphosphate/pharmacology , Dideoxynucleotides/pharmacology , HIV Infections/drug therapy , HIV Infections/pathology , HIV Infections/virology , HIV-1/drug effects , HIV-1/physiology , Humans , Lamivudine/pharmacokinetics , Lamivudine/pharmacology , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/pathology , Leukocytes, Mononuclear/virology , Male , Semen/cytology , Semen/drug effects , Semen/virology , Thymine Nucleotides/pharmacology , Time Factors , Zidovudine/pharmacokinetics , Zidovudine/pharmacologyABSTRACT
Respiratory syncytial virus (RSV) causes severe lower respiratory tract infections, yet no vaccines or effective therapeutics are available. ALS-8176 is a first-in-class nucleoside analog prodrug effective in RSV-infected adult volunteers, and currently under evaluation in hospitalized infants. Here, we report the mechanism of inhibition and selectivity of ALS-8176 and its parent ALS-8112. ALS-8176 inhibited RSV replication in non-human primates, while ALS-8112 inhibited all strains of RSV in vitro and was specific for paramyxoviruses and rhabdoviruses. The antiviral effect of ALS-8112 was mediated by the intracellular formation of its 5'-triphosphate metabolite (ALS-8112-TP) inhibiting the viral RNA polymerase. ALS-8112 selected for resistance-associated mutations within the region of the L gene of RSV encoding the RNA polymerase. In biochemical assays, ALS-8112-TP was efficiently recognized by the recombinant RSV polymerase complex, causing chain termination of RNA synthesis. ALS-8112-TP did not inhibit polymerases from host or viruses unrelated to RSV such as hepatitis C virus (HCV), whereas structurally related molecules displayed dual RSV/HCV inhibition. The combination of molecular modeling and enzymatic analysis showed that both the 2'F and the 4'ClCH2 groups contributed to the selectivity of ALS-8112-TP. The lack of antiviral effect of ALS-8112-TP against HCV polymerase was caused by Asn291 that is well-conserved within positive-strand RNA viruses. This represents the first comparative study employing recombinant RSV and HCV polymerases to define the selectivity of clinically relevant nucleotide analogs. Understanding nucleotide selectivity towards distant viral RNA polymerases could not only be used to repurpose existing drugs against new viral infections, but also to design novel molecules.
Subject(s)
Antiviral Agents/pharmacology , Cytidine Triphosphate/analogs & derivatives , Cytidine Triphosphate/pharmacology , DNA-Directed RNA Polymerases/metabolism , Respiratory Syncytial Virus Infections/drug therapy , Respiratory Syncytial Virus, Human/drug effects , Virus Replication/drug effects , Animals , Chlorocebus aethiops , Humans , RNA, Viral/genetics , Respiratory Syncytial Virus Infections/virology , Respiratory Syncytial Virus, Human/genetics , Viral Proteins/geneticsABSTRACT
Methylation of CpG islands in promoter gene regions is frequently observed in lymphomas. DNA methylation is established by DNA methyltransferases (DNMTs). DNMT1 maintains methylation patterns, while DNMT3A and DNMT3B are critical for de novo DNA methylation. Little is known about the expression of DNMTs in lymphomas. DNMT3A and 3B genes can be regulated post-transcriptionally by miR-29 family. Here, we demonstrated for the first time the overexpression of DNMT1 and DNMT3B in Burkitt lymphoma (BL) tumor samples (69% and 86%, respectively). Specifically, the treatment of two BL cell lines with the DNMT inhibitor 5-aza-dC decreased DNMT1 and DNMT3B protein levels and inhibited cell growth. Additionally, miR-29a, miR-29b and miR-29c levels were significantly decreased in the BL tumor samples. Besides, the ectopic expression of miR-29a, miR-29b and miR-29c reduced the DNMT3B expression and miR-29a and miR-29b lead to increase of p16(INK4a) mRNA expression. Altogether, our data suggest that deregulation of DNMT1, DNMT3B and miR29 may be involved in BL pathogenesis.
Subject(s)
Burkitt Lymphoma/genetics , DNA (Cytosine-5-)-Methyltransferases/biosynthesis , MicroRNAs/biosynthesis , Adolescent , Azacitidine/analogs & derivatives , Azacitidine/pharmacology , Burkitt Lymphoma/pathology , Cell Line, Tumor , Child , Child, Preschool , Cyclin-Dependent Kinase Inhibitor p16/biosynthesis , Cyclin-Dependent Kinase Inhibitor p16/genetics , Cytidine Triphosphate/analogs & derivatives , Cytidine Triphosphate/pharmacology , DNA (Cytosine-5-)-Methyltransferase 1 , DNA (Cytosine-5-)-Methyltransferases/antagonists & inhibitors , DNA Methylation , Female , Gene Expression Regulation, Neoplastic , Humans , Male , Promoter Regions, Genetic , RNA, Messenger/biosynthesis , DNA Methyltransferase 3BABSTRACT
As a result of alternative splicing and differential promoter usage, RASSF5 exists in at least three isoforms (RASSF5A-RASSF5C), which may play different roles in tumorigenesis. The present study was to detect the role of RASSF5A, B and C in esophageal squamous cell carcinoma (ESCC) and clarify the critical CpG sites of RASSF5A, in order to clarify more information on the role of RASSF5 with regard to the pathogenesis of ESCC. Frequent silencing of RASSF5A but not RASSF5B and RASSF5C were found in esophageal cancer cell lines and the silencing of RASSF5A may be reversed by 5-Aza-dC or TSA treatment. The aberrant CpG island 1 methylation of RASSF5A induces silencing of its expression in TE13 cell line. Decreased mRNA and protein expression of RASSF5A was observed in ESCC tumor tissues and was associated with RASSF5A CpG island 1 methylation status. Unlike RASSF5A, expression variation of RASSF5B and RASSF5C was not found in ESCC tissues. Aberrant promoter methylation of RASSF5C was also not found in ESCC. RASSF5A methylation and protein expression were independently associated with ESCC patients' survival. These data indicated that the inactivation of RASSF5A through CpG island 1 methylation may play an important role in ESCC carcinogenesis, RASSF5A may be a functional tumor suppressor and may serve as a prognostic biomarker for ESCC.
Subject(s)
Biomarkers, Tumor/genetics , Carcinoma, Squamous Cell/genetics , Esophageal Neoplasms/genetics , Monomeric GTP-Binding Proteins/genetics , Adaptor Proteins, Signal Transducing , Adult , Aged , Alternative Splicing/genetics , Apoptosis/genetics , Apoptosis Regulatory Proteins , Azacitidine/analogs & derivatives , Azacitidine/pharmacology , Biomarkers, Tumor/metabolism , Carcinoma, Squamous Cell/mortality , Carcinoma, Squamous Cell/pathology , Cell Line, Tumor , Cell Proliferation/genetics , Cytidine Triphosphate/analogs & derivatives , Cytidine Triphosphate/pharmacology , DNA Methylation , Esophageal Neoplasms/mortality , Esophageal Neoplasms/pathology , Esophagus/metabolism , Female , Genes, Tumor Suppressor , Genetic Predisposition to Disease , Humans , Hydroxamic Acids/pharmacology , Male , Middle Aged , Monomeric GTP-Binding Proteins/metabolism , Neoplasm Invasiveness/genetics , Prognosis , Promoter Regions, Genetic/genetics , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Synthesis Inhibitors/pharmacology , RNA, Messenger, Stored/biosynthesisABSTRACT
Glucose uptake by peripheral tissues such as skeletal muscles and adipocytes is important in the maintenance of glucose homeostasis. We previously demonstrated that P2Y6 receptor (P2Y6R) agonists protect pancreatic islet cells from apoptosis and stimulate glucose-dependent insulin release. Here, we investigated the effects of P2Y6R activation on glucose uptake in insulin target tissues. An agonist of the P2Y6R, P1-(5'-uridine)-P3-(5'-N4-methoxycytidine)-triphosphate (MRS2957), significantly increased the uptake of [3H]2-deoxyglucose in mouse C2C12 myotubes and 3T3-L1 adipocytes, and this stimulation was significantly decreased by a selective P2Y6R antagonist N,Nâ³-1,4-butanediyl-bis[N'-(3-isothiocyanatophenyl)thiourea] (MRS2578). Pre-incubation with Compound C (an inhibitor of 5'-AMP-activated protein kinase, AMPK), or AMPK siRNA abolished the stimulatory effect of MRS2957 on glucose uptake. Also, MRS2957 (60 min incubation) increased recruitment of the facilitated glucose transporter-4 (GLUT4) to the cell membrane, which was blocked by MRS2578. Treatment of C2C12 myotubes with MRS2957 induced significant phosphorylation of AMPK, which increase GLUT4 expression through histone deacetylase (HDAC)5 signaling. Glucose uptake in primary mouse adipocytes from wild-type mice was stimulated upon P2Y6R activation by either MRS2957 or native agonist UDP, and the P2Y6R effect was antagonized by MRS2578. However, in adipocytes from P2Y6R-knockout mice P2Y6R agonists had no effect on glucose uptake, and there was no change in the glucose uptake by insulin. Our results indicate that the P2Y6R promotes glucose metabolism in peripheral tissues, which may be mediated through AMPK signaling.
Subject(s)
Adipocytes/metabolism , Cytidine Triphosphate/pharmacology , Dinucleoside Phosphates/pharmacology , Glucose/metabolism , Muscle Fibers, Skeletal/metabolism , Receptors, Purinergic P2/metabolism , 3T3-L1 Cells , AMP-Activated Protein Kinases/metabolism , Animals , Cell Line , Gene Expression Regulation/drug effects , Glucose Transporter Type 4/metabolism , Isothiocyanates/pharmacology , Mice , Receptors, Purinergic P2/genetics , Signal Transduction/drug effects , Thiourea/analogs & derivatives , Thiourea/pharmacologyABSTRACT
Cytidine triphosphate (CTP) synthetase (CTPS) (EC number 6.3.4.2) is a key enzyme involved in de novo synthesis of CTP. It catalyzes the rate-limiting step of the process due to the product inhibition effects on the enzyme. In this study, a novel CTPS from Corynebacterium glutamicum ATCC 13032 (CgCTPS) was cloned, expressed and characterized. A series of mutagenesis in its N-terminal ammonia ligase (ALase) domain was performed in order to reduce CTP product inhibition. All single mutation variants (D160E, E162A, E168K) lowered product inhibition by lowering the enzyme's binding affinity for CTP. The homology model of CgCTPS showed that D160E mutant caused steric hindrance for the pyrimidine ring of CTP stacking, E162A disrupted the hydrogen bond between CTP ribose and side chain and D168K caused minor localized structure perturbations of CTP binding pocket. The triple mutant of CTPS (D160E-E162A-E168K) with halved Km, doubled Vmax and the 23.5-fold increased IC50 for CTP shows a potential for use in industrial-scale CTP production by its better performance in enzyme kinetics and product inhibition.
Subject(s)
Carbon-Nitrogen Ligases/antagonists & inhibitors , Carbon-Nitrogen Ligases/metabolism , Cytidine Triphosphate/metabolism , Cytidine Triphosphate/pharmacology , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Mutagenesis, Site-Directed , Amino Acid Sequence , Binding Sites , Biocatalysis , Carbon-Nitrogen Ligases/chemistry , Carbon-Nitrogen Ligases/genetics , Cloning, Molecular , Corynebacterium glutamicum/enzymology , Corynebacterium glutamicum/genetics , Escherichia coli/genetics , Hydrogen-Ion Concentration , Inhibitory Concentration 50 , Kinetics , Metals/pharmacology , Models, Molecular , Molecular Sequence Data , Mutation , Protein Structure, Tertiary , TemperatureABSTRACT
The epigenetic suppression of Wnt antagonists (sFRPs, DKKs, and WIF-1) causes the activation of both ß-catenin and target genes, which play an important role in cell proliferation, metastasis, and angiogenesis. This study is aimed to investigate, on transcriptional and protein levels, the synergic effects of unaccompanied and/or combined use of 5-aza-2'-deoxycytidine (DAC, 5-aza-dC), trichostatin A (TSA), and gemcitabine+cisplatin chemotherapeutic agents on the apoptotic pathway of human bladder cancer cell line T24. The anti-tumor effects of gemcitabine (0-500 nM), cisplatin (0-10 µM), DAC (10 µM), and TSA (300 nM) alone and/or together on T24 cells were determined by WST-1. ELISA method was used to analyze the effects of unaccompanied and combined use of gemcitabine+cisplatin, DAC, and TSA on cell proliferation and determine the cytotoxic and apoptotic dosages at the level of H3 histone acetylation. Methylation-specific PCR was used to evaluate methylation profiles of Wnt antagonist gene (WIF-1). In the case of unaccompanied and/or combined use of specified drugs, the variations in the expression levels of CTNNB1, GSK3ß, c-MYC, CCND1, CASP-3, CASP-8, CASP-9, BCL2L1, and WIF-1 genes were determined by quantitative real-time PCR. Our results indicate that through inhibition of DNA methylation, expression of ß-catenin and Wnt antagonist re-activation and expressions of canonical Wnt/ß-catenin pathway target genes, c-myc and cyclin D1 (CCND1), have decreased. In addition, DAC, TSA, and gemcitabine+cisplatin combination caused an increase in GSK3ß mRNA levels, which in turn significantly decreased CCND1 mRNA levels. Moreover, BCL2L1, an anti-apoptotic gene, was downregulated significantly. Meanwhile, both CASP-3 mRNA and active caspase-3 protein levels increased with respect to control (p<0.01). The results revealed that use of quadruplicate gemcitabine+cisplatin+DAC+TSA combination led to a reduced inhibition of canonical Wnt/ß-catenin pathway and reduced cell proliferation. Our findings may offer a new approach to consider in the treatment of bladder cancer.
Subject(s)
Apoptosis/drug effects , Azacitidine/analogs & derivatives , Cytidine Triphosphate/analogs & derivatives , Epigenomics , Gene Expression Regulation, Neoplastic/drug effects , Hydroxamic Acids/pharmacology , Wnt Proteins/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Azacitidine/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Cyclin D/genetics , Cytidine Triphosphate/pharmacology , Dose-Response Relationship, Drug , Genes, myc/genetics , Humans , Urinary Bladder Neoplasms/drug therapy , beta Catenin/geneticsABSTRACT
Cancer/testis (CT) antigens, which are expressed in various cancer cells but not in normal cells except germline cells of the testis, have been used as targets for cancer vaccine therapy. 5-Aza-2'-deoxycytidine (DAC), a potent inhibitor of genomic and promoter-specific DNA methylation, inhibits DNA methyltransferase activity and is reported to induce the expression of certain CT antigens by the demethylation of promoter CpG islands of the treated cells. Here, using DAC-treated cancer cells, we searched for novel attractive target molecules that would be useful for cancer immunotherapy and found a meiosis-specific protein, meiosis specific with OB domains (MEIOB), to be a novel CT antigen. Indeed, the MEIOB gene is expressed only in the testis and not in other normal tissues. The mRNA expression of MEIOB was greatly enhanced in several lung cancer cell lines after the treatment with DAC. Furthermore, we identified a variety of helper epitopes of the MEIOB antigen, which were recognized by MEIOB antigen-specific T cells in a HLA-restriction manner. Finally, we demonstrated that IFN-γ production of MEIOB peptide-specific helper T cells in response to HLA-matched cancer cells was greatly augmented by treatment with DAC and IFN-γ. Taken together, these findings show DAC to be a promising tool for finding novel CT antigens and for developing a future novel combination cancer vaccine chemotherapy.
Subject(s)
Cancer Vaccines/immunology , DNA-Binding Proteins/immunology , Immunotherapy, Adoptive/methods , Lung Neoplasms/immunology , Lung Neoplasms/therapy , Peptide Fragments/immunology , T-Lymphocytes, Helper-Inducer/immunology , Amino Acid Sequence , Antigens, Neoplasm/immunology , Azacitidine/analogs & derivatives , Azacitidine/pharmacology , Cancer Vaccines/therapeutic use , Cell Line, Tumor , Cytidine Triphosphate/analogs & derivatives , Cytidine Triphosphate/pharmacology , DNA Methylation , DNA-Binding Proteins/genetics , Epitope Mapping , Gene Expression Regulation, Neoplastic , HLA Antigens/metabolism , Humans , Interferon-gamma/metabolism , Lymphocyte Activation/drug effects , Male , Meiosis/immunology , Peptide Fragments/genetics , T-Cell Antigen Receptor Specificity/immunology , T-Lymphocytes, Helper-Inducer/drug effects , Testis/immunologyABSTRACT
microRNAs (miRNAs) have been reported to play a crucial role in regulating a variety of genes pivotal for tumor metastasis. miR-126 is well known as one of the angiogenesis regulatory miRNAs. Recent studies have reported controversial roles of miR-126 in tumor progression. In this study, we sought to investigate the potential roles of miR-126 in colorectal cancer (CRC). By real-time PCR, miR-126 was shown to be downregulated in primary CRC tissues and cell lines. Restoration of miR-126 in CRC cells inhibited cell growth, migration and invasion. Using both in silico prediction and immunoblotting, we found that vascular endothelial growth factor (VEGF) was a target of miR-126. The interaction of miR-126 on the 3'UTR of VEGF mRNA was validated by luciferase reporter assay. Mechanistically, we found that the silencing of miR-126 was induced by promoter methyl-ation of its host gene, EGFL7. Treatment with 5-aza-CdR restored miR-126 expression and thereby led to a decline in VEGF expression. Functionally, due to suppression of VEGF, enhanced miR-126 expression inhibited tumor neovasculature triggered by CRC cells. In conclusion, our findings suggest that DNA methylation-induced silencing of miR-126 contributes, at least in part, to tumor invasion and angiogenesis in CRC, through upregulation of VEGF expression. miR-126 may be a potential target for the therapeutic strategy against CRC.
Subject(s)
Colorectal Neoplasms/pathology , MicroRNAs/genetics , Neoplasm Invasiveness/genetics , Neovascularization, Pathologic/genetics , Vascular Endothelial Growth Factor A/metabolism , 3' Untranslated Regions/genetics , Azacitidine/analogs & derivatives , Azacitidine/pharmacology , Calcium-Binding Proteins , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation , Colorectal Neoplasms/genetics , Cytidine Triphosphate/analogs & derivatives , Cytidine Triphosphate/pharmacology , DNA Methylation/genetics , EGF Family of Proteins , Endothelial Growth Factors/genetics , Epigenesis, Genetic/genetics , Gene Expression Regulation, Neoplastic , HCT116 Cells , HEK293 Cells , HT29 Cells , Humans , MicroRNAs/biosynthesis , MicroRNAs/metabolism , Neoplasm Invasiveness/pathology , Promoter Regions, Genetic , RNA, Messenger/metabolism , Vascular Endothelial Growth Factor A/geneticsABSTRACT
Placental trophoblast invasion involves a cellular transition from epithelial to mesenchymal phenotype. Cytotrophoblasts undergo epithelial to mesenchymal transition (EMT) when differentiating into extravillous trophoblasts and gaining the capacity of invasion. In this research, we investigated the role of DNA methylation in trophoblasts during this EMT. First, using BeWo and HTR8/SVneo cell lines as models of cytotrophoblasts and extravillous trophoblasts, respectively, we analyzed the gene expression and DNA methylation status of the known epithelial marker genes, E-Cadherin and Cytokeratin7. We found that, in HTR8/SVneo cells, both genes were silenced and their promoters hypermethylated, as compared with the high-level gene expression and promoter hypomethylation observed in BeWo cells. This result suggests that dynamic DNA methylation of epithelial marker genes plays a critical role in the trophoblast EMT process. To verify these results, we treated HTR8/SVneo cells with 5-aza-dC, a known inhibitor of DNA methyltransferase, for three days. Five-Aza-dC treatment significantly increased the expression of epithelial marker genes and slightly decreased the expression of mesenchymal genes, as detected by qRT-PCR, immunocytochemistry and Western blot. Furthermore, 5-aza-dC treated HTR8/SVneo cells changed their morphology from mesenchymal into epithelial phenotype, indicating that 5-aza-dC induced mesenchymal to epithelial transition. Lastly, we examined the effect of 5-aza-dC on trophoblast migration and invasion capacity. We applied 5-aza-dC to HTR8/SVneo cells in trans-well cell migration and invasion assays and found that 5-aza-dC treatment decreased trophoblast migration and invasion capacity. In conclusion, DNA methylation of epithelial marker genes represents a molecular mechanism for the process of trophoblast EMT.
Subject(s)
Cadherins/genetics , DNA Methylation , Epithelial-Mesenchymal Transition/genetics , Gene Expression Regulation , Keratin-7/genetics , Pregnancy Trimester, First , Trophoblasts/pathology , Azacitidine/analogs & derivatives , Azacitidine/pharmacology , Cell Line , Cytidine Triphosphate/analogs & derivatives , Cytidine Triphosphate/pharmacology , DNA-Cytosine Methylases/antagonists & inhibitors , Female , Humans , Pregnancy , Trophoblasts/metabolismABSTRACT
Nucleoside analogs are a significant class of anti-cancer agent. As prodrugs, they terminate the DNA synthesis upon transforming to their active triphosphate metabolites. We have encapsulated a biologically activate nucleotide analog (i.e. gemcitabine triphosphate (GTP)), instead of the nucleoside (i.e. gemcitabine) derivative, into a novel Lipid/Calcium/Phosphate nanoparticle (LCP) platform. The therapeutic efficacy of LCP-formulated GTP was evaluated in a panel of human non-small-cell lung cancer (NSCLC) and human pancreatic cancer models after systemic administrations. GTP-loaded LCPs induced cell death and arrested the cell cycle in the S phase. In vivo efficacy studies showed that intravenously injected GTP-loaded LCPs triggered effective apoptosis of tumor cells, significant reduction of tumor cell proliferation and cell cycle progression, leading to dramatic inhibition of tumor growth, with little in vivo toxicity. Broadly speaking, the current study offers preclinical proof-of-principle that many active nucleotide or phosphorylated nucleoside analogs could be encapsulated in the LCP nanoplatform and delivered systemically for a wide variety of therapeutic applications.
Subject(s)
Antineoplastic Agents/administration & dosage , Antineoplastic Agents/therapeutic use , Carcinoma, Non-Small-Cell Lung/drug therapy , Cytidine Triphosphate/analogs & derivatives , Lung Neoplasms/drug therapy , Nanoparticles/chemistry , Pancreatic Neoplasms/drug therapy , Animals , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Calcium/chemistry , Carcinoma, Non-Small-Cell Lung/enzymology , Carcinoma, Non-Small-Cell Lung/pathology , Caspases/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Cytidine Triphosphate/administration & dosage , Cytidine Triphosphate/pharmacokinetics , Cytidine Triphosphate/pharmacology , Cytidine Triphosphate/therapeutic use , Enzyme Activation/drug effects , Female , Humans , Lipids/chemistry , Lung Neoplasms/enzymology , Lung Neoplasms/pathology , Mice , Mice, Nude , Nanoparticles/ultrastructure , Pancreatic Neoplasms/enzymology , Pancreatic Neoplasms/pathology , Particle Size , Phosphates/chemistry , Poly(ADP-ribose) Polymerases/metabolism , Polyethylene Glycols/chemistry , S Phase/drug effects , Static Electricity , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: ATP is an extracellular signaling molecule with many ascribed functions in sensory systems, including the olfactory epithelium. The mechanism(s) by which ATP is released in the olfactory epithelium has not been investigated. Quantitative luciferin-luciferase assays were used to monitor ATP release, and confocal imaging of the fluorescent ATP marker quinacrine was used to monitor ATP release via exocytosis in Swiss Webster mouse neonatal olfactory epithelial slices. RESULTS: Under control conditions, constitutive release of ATP occurs via exocytosis, hemichannels and ABC transporters and is inhibited by vesicular fusion inhibitor Clostridium difficile toxin A and hemichannel and ABC transporter inhibitor probenecid. Constitutive ATP release is negatively regulated by the ATP breakdown product ADP through activation of P2Y receptors, likely via the cAMP/PKA pathway. In vivo studies indicate that constitutive ATP may play a role in neuronal homeostasis as inhibition of exocytosis inhibited normal proliferation in the OE. ATP-evoked ATP release is also present in mouse neonatal OE, triggered by several ionotropic P2X purinergic receptor agonists (ATP, αßMeATP and Bz-ATP) and a G protein-coupled P2Y receptor agonist (UTP). Calcium imaging of P2X2-transfected HEK293 "biosensor" cells confirmed the presence of evoked ATP release. Following purinergic receptor stimulation, ATP is released via calcium-dependent exocytosis, activated P2X1,7 receptors, activated P2X7 receptors that form a complex with pannexin channels, or ABC transporters. The ATP-evoked ATP release is inhibited by the purinergic receptor inhibitor PPADS, Clostridium difficile toxin A and two inhibitors of pannexin channels: probenecid and carbenoxolone. CONCLUSIONS: The constitutive release of ATP might be involved in normal cell turn-over or modulation of odorant sensitivity in physiological conditions. Given the growth-promoting effects of ATP, ATP-evoked ATP release following injury could lead to progenitor cell proliferation, differentiation and regeneration. Thus, understanding mechanisms of ATP release is of paramount importance to improve our knowledge about tissue homeostasis and post-injury neuroregeneration. It will lead to development of treatments to restore loss of smell and, when transposed to the central nervous system, improve recovery following central nervous system injury.