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1.
Korean J Parasitol ; 60(1): 7-14, 2022 Feb.
Article in English | MEDLINE | ID: mdl-35247949

ABSTRACT

Acanthamoeba keratitis (AK) is a rare infectious disease and accurate diagnosis has remained arduous as clinical manifestations of AK were similar to keratitis of viral, bacterial, or fungal origins. In this study, we described the production of a polyclonal peptide antibody against the adenylyl cyclase-associated protein (ACAP) of A. castellanii, and evaluated its differential diagnostic potential. Enzyme-linked immunosorbent assay revealed high titers of A. castellanii-specific IgG and IgA antibodies being present in low dilutions of immunized rabbit serum. Western blot analysis revealed that the ACAP antibody specifically interacted with A. castellanii, while not interacting with human corneal epithelial (HCE) cells and other causes of keratitis such as Fusarium solani, Pseudomonas aeruginosa, and Staphylococcus aureus. Immunocytochemistry (ICC) results confirmed the specific detection of trophozoites and cysts of A. castellanii co-cultured with HCE cells. The ACAP antibody also specifically interacted with the trophozoites and cysts of 5 other Acanthamoeba species. These results indicate that the ACAP antibody of A. castellanii can specifically detect multiple AK-causing members belonging to the genus Acanthamoeba and may be useful for differentially diagnosing Acanthamoeba infections.


Subject(s)
Acanthamoeba Keratitis , Acanthamoeba castellanii , Adenylyl Cyclases , Animals , Peptides , Rabbits , Trophozoites
2.
ACS Infect Dis ; 8(2): 271-279, 2022 02 11.
Article in English | MEDLINE | ID: mdl-34994538

ABSTRACT

Acanthamoeba is a ubiquitous and free-living protozoan pathogen responsible for causing Acanthamoeba keratitis (AK), a severe corneal infection inflicting immense pain that can result in permanent blindness. A drug-based treatment of AK has remained arduous because Acanthamoeba trophozoites undergo encystment to become highly drug-resistant cysts upon exposure to harsh environmental conditions such as amoebicidal agents (e.g., polyhexanide, chloroquine, and chlorohexidine). As such, drugs that block the Acanthamoeba encystation process could result in a successful AK treatment. Histone deacetylase inhibitors (HDACi) have recently emerged as novel therapeutic options for treating various protozoan and parasitic diseases. Here, we investigated whether novel HDACi suppress the proliferation and encystation of Acanthamoeba. Synthetic class II HDACi FFK29 (IIa selective) and MPK576 (IIb selective) dose-dependently decreased the viability of Acanthamoeba trophozoites. While these HDACi demonstrated a negligible effect on the viability of mature cysts, Acanthamoeba encystation was significantly inhibited by these HDACi. Apoptosis was slightly increased in trophozoites after a treatment with these HDACi, whereas cysts were unaffected by the HDACi exposure. The viability of human corneal cells was not affected by HDACi concentrations up to 10 µmol/L. In conclusion, these synthetic HDACi demonstrated potent amoebicidal effects and inhibited the growth and encystation of Acanthamoeba, thus highlighting their enormous potential for further development.


Subject(s)
Acanthamoeba Keratitis , Acanthamoeba castellanii , Amebicides , Acanthamoeba Keratitis/drug therapy , Acanthamoeba Keratitis/parasitology , Amebicides/pharmacology , Animals , Histone Deacetylase Inhibitors/pharmacology , Humans , Trophozoites
3.
PLoS One ; 17(1): e0262223, 2022.
Article in English | MEDLINE | ID: mdl-34986189

ABSTRACT

Contact lens usage has contributed to increased incidence rates of Acanthamoeba keratitis (AK), a serious corneal infection that can lead to blindness. Since symptoms associated with AK closely resemble those incurred by bacterial or fungal keratitis, developing a diagnostic method enabling rapid detection with a high degree of Acanthamoeba-specificity would be beneficial. Here, we produced a polyclonal antibody targeting the carboxylesterase (CE) superfamily protein secreted by the pathogenic Acanthamoeba and evaluated its diagnostic potential. Western blot analysis revealed that the CE antibody specifically interacts with the cell lysates and conditioned media of pathogenic Acanthamoeba, which were not observed from the cell lysates and conditioned media of human corneal epithelial (HCE) cells, Fusarium solani, Staphylococcus aureus, and Pseudomonas aeruginosa. High titers of A. castellanii-specific antibody production were confirmed sera of immunized mice via ELISA, and these antibodies were capable of detecting A. castellanii from the cell lysates and their conditioned media. The specificity of the CE antibody was further confirmed on A. castellanii trophozoites and cysts co-cultured with HCE cells, F. solani, S. aureus, and P. aeruginosa using immunocytochemistry. Additionally, the CE antibody produced in this study successfully interacted with 7 different Acanthamoeba species. Our findings demonstrate that the polyclonal CE antibody specifically detects multiple species belong to the genus Acanthamoeba, thus highlighting its potential as AK diagnostic tool.


Subject(s)
Acanthamoeba Keratitis/diagnosis , Acanthamoeba/immunology , Antibodies, Protozoan/analysis , Carboxylesterase/immunology , Culture Media, Conditioned/metabolism , Epithelium, Corneal/cytology , Acanthamoeba/classification , Acanthamoeba/growth & development , Acanthamoeba/isolation & purification , Animals , Antibodies, Protozoan/blood , Antibody Specificity , Carboxylesterase/administration & dosage , Carboxylesterase/genetics , Cell Line , Cells, Cultured , Contact Lenses/parasitology , Early Diagnosis , Epithelial Cells/cytology , Epithelial Cells/metabolism , Epithelial Cells/parasitology , Epithelium, Corneal/metabolism , Epithelium, Corneal/parasitology , Humans , Immunization , Male , Mice , Protozoan Proteins/administration & dosage , Protozoan Proteins/genetics , Protozoan Proteins/immunology
4.
Exp Parasitol ; 232: 108188, 2022 Jan.
Article in English | MEDLINE | ID: mdl-34838530

ABSTRACT

Acanthamoeba spp. feeds on bacteria, fungi, and algae to obtain nutrients from the environment. However, several pathogens can survive and multiply in Acanthamoeba. Mechanisms necessary for the survival and proliferation of microorganisms in Acanthamoeba remain unclear. The object of this study was to identify effective factors for the survival of microorganisms in Acanthamoeba. Differentially expressed genes (DEGs) in A. castellanii infected by Legionella pneumophila or Escherichia coli were identified based on mRNA sequencing. A total of 2342 and 1878 DEGs were identified in Acanthamoeba with L. pneumophila and E. coli, respectively. Among these DEGs, 502 were up-regulated and 116 were down-regulated in Acanthamoeba infected by L. pneumophila compared to those in Acanthamoeba feed on E. coli. Gene ontology analysis showed that the genes encoded small GTPase-mediated signal transduction proteins in the biological process domain, intracellular proteins in the cellular component domain, and ATP binding proteins in the molecular function domain were up-regulated while integral components of membrane proteins in the cellular component domain were down-regulated in Acanthamoeba infected by Legionella compared to those in Acanthamoeba feed on E. coli. During endosymbiosis with Legionella, Acanthamoeba showed various changes in the expression of genes supposed to be involved in phagosomal maturation. Acanthamoeba infected by Legionella also showed high expression levels of aminotransferase, methyltransferase, and cysteine proteinase but low expression levels of RNA pseudouridine synthase superfamily protein and 2OG-Fe(II) oxygenase superfamily. These results provide directions for further research to understand the survival strategy of L. pneumophila in A. castellanii.


Subject(s)
Acanthamoeba/genetics , Acanthamoeba/microbiology , Escherichia coli/physiology , Gene Expression , Legionella pneumophila/physiology , Down-Regulation , Phagocytosis/physiology , RNA, Protozoan/chemistry , Real-Time Polymerase Chain Reaction , Sequence Analysis, RNA , Symbiosis/genetics , Up-Regulation
5.
Antioxidants (Basel) ; 10(10)2021 Sep 22.
Article in English | MEDLINE | ID: mdl-34679638

ABSTRACT

Histone deacetylase inhibitors (HDACi) are emerging as anti-hepatocellular carcinoma (HCC) agents. However, the molecular mechanisms underlying HDACi-induced sensitization to oxidative stress and cell death of HCC remain elusive. We hypothesized that HDACi reduces the anti-oxidative stress capacity of HCC, rendering it more susceptible to oxidative stress and cell death. Change in the transcriptome of HCC was analyzed by RNA-seq and validated using real-time quantitative polymerase chain reaction (qPCR) and Western blot. Cell death of HCC was analyzed by fluorescence-activated cell sorting (FACS). Protein localization and binding on the target gene promoters were investigated by immunofluorescence (IF) and chromatin immunoprecipitation (ChIP), respectively. Glutathione peroxidase 8 (GPX8) was highly down-regulated in HCC upon oxidative stress and HDACi co-treatment. Oxidative stress and HDACi enhanced the expression and transcriptional activities of ER-stress-related genes. N-acetyl-cysteine (NAC) supplementation reversed the oxidative stress and HDACi-induced apoptosis in HCC. HDACi significantly enhanced the effect of ER stressors on HCC cell death. GPX8 overexpression reversed the activation of ER stress signaling and apoptosis induced by oxidative stress and HDACi. In conclusion, HDACi suppresses the expression of GPX8, which sensitizes HCC to ER stress and apoptosis by oxidative stress.

6.
Vaccines (Basel) ; 9(8)2021 Aug 18.
Article in English | MEDLINE | ID: mdl-34452044

ABSTRACT

The ongoing severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic had brought disastrous consequences throughout the entire world. While several manufactured vaccines have been approved for emergency use, continuous efforts to generate novel vaccines are needed. In this study, we developed SARS-CoV-2 virus-like particles (VLPs) containing the full length of spike (S) glycoprotein (S full), S1, or S2 together with the influenza matrix protein 1 (M1) as a core protein. Successfully constructed VLPs expressing the S full, S1, and S2 via Sf9 cell transfections were confirmed and characterized by Western blot and transmission electron microscopy (TEM). VLP immunization in mice induced higher levels of spike protein-specific IgG and its subclasses compared to naïve control, with IgG2a being the most predominant subclass. S full and S1 immune sera elicited virus-neutralizing activities, but these were not strong enough to fully inhibit receptor-ligand binding of the SARS-CoV-2. Neutralizing activities were not observed from the S2 VLP immune sera. Overall, our findings revealed that S full or S1 containing VLPs can be developed into effective vaccines.

7.
J Cancer ; 12(17): 5086-5098, 2021.
Article in English | MEDLINE | ID: mdl-34335925

ABSTRACT

Forkhead box protein M1 (FOXM1) is a pivotal regulator of G2/M cell cycle progression in many types of cancer. Previously, our study demonstrated that histone deacetylase inhibition (HDACi) sensitizes hepatocellular carcinoma cells (HCC) to oxidative stress through FOXM1 suppression. However, the mechanism underlying its suppression by HDACi still requires elucidation. We hypothesized that HDACi induce genes responsible for destabilizing and inactivating FOXM1. The transcriptome in the HepG2 was revealed by massive analysis of cDNA end (MACE). Expression of mRNA and proteins were analyzed by quantitative real-time PCR (qPCR) and western blot, respectively. Cell cycle was analyzed by fluorescence-activated cell sorting (FACS). Oxidative stress and HDACi suppressed CDK4/6 levels while enhancing CDK inhibitor 2B and 2D (CDKN2B and CDKN2D) expressions in HCC. Palbociclib, a specific inhibitor of CDK4/6, induced G2/M cell cycle arrest in HCC by down-regulating phosphorylation level of FOXM1, and its downstream target genes such as aurora kinase A (AURKA) and polo-like kinase 1 (PLK1). HDACi treatment increased the ubiquitination level of FOXM1 by suppressing ubiquitin-specific peptidase 21 (USP21), which deubiquitinates FOXM1. Inhibiting FOXM1 degradation with MG132 treatment affected neither palbociclib-induced G2/M cell cycle arrest nor expression of its target genes. Double knockdown of CDKN2B and CDKN2D reduced the oxidative stress and HDACi-induced G/2M cell cycle arrest. In conclusion, oxidative stress and HDACi synergistically cause G2/M cell cycle arrest via CDKN2 induction, which sequentially inhibits CDK4/6, FOXM1, and its downstream target genes AURKA, PLK1, and CCNB1 phosphorylation in HCC.

8.
PLoS One ; 16(4): e0250342, 2021.
Article in English | MEDLINE | ID: mdl-33891646

ABSTRACT

Accurate and rapid diagnosis of Acanthamoeba keratitis (AK) is difficult. Although the diagnostic procedure for AK has improved, further development and effective diagnostic tool utilization for AK need to continue. Chorismate mutase is a key regulatory enzyme involved in the shikimate pathway, a metabolic pathway absent in mammals but central for amino acid biosynthesis in bacteria, fungi, algae, and plants. In this study, we describe the identification and production of a polyclonal peptide antibody targeting chorismate mutase secreted by A. castellanii, which could be used for AK diagnosis. Western blot was performed using the protein lysates and conditioned media of the human corneal epithelial (HCE) cells, non-pathogenic Acanthamoeba, pathogenic Acanthamoeba, clinical isolate of Acanthamoeba spp., and other causes of keratitis such as Fusarium solani, Pseudomonas aeruginosa, and Staphylococcus aureus. Polyclonal antibodies raised against A. castellanii chorismate mutase specifically interacted with lysates of Acanthamoeba origin and their culture media, while such interactions were not observed from other samples. Acanthamoeba-specificity of chorismate mutase was also confirmed using immunocytochemistry after co-culturing Acanthamoeba with HCE cells. Specific binding of the chorismate mutase antibody to Acanthamoeba was observed, which were absent in the case of HCE cells. These results indicate that the chorismate mutase antibody of Acanthamoeba may serve as a method for rapid and differential Acanthamoeba identification.


Subject(s)
Acanthamoeba Keratitis , Acanthamoeba , Antibodies/immunology , Chorismate Mutase/immunology , Peptides/immunology , Acanthamoeba/immunology , Acanthamoeba/isolation & purification , Acanthamoeba Keratitis/diagnosis , Acanthamoeba Keratitis/parasitology , Cell Line , Epithelial Cells , Humans
9.
Heliyon ; 6(10): e05238, 2020 Oct.
Article in English | MEDLINE | ID: mdl-33088972

ABSTRACT

Legionella grows intracellularly in free-living amoeba as well as in mammalian macrophages. Until now, the overall gene expression pattern of intracellular Legionella in Acanthamoeba was not fully explained. Intracellular bacteria are capable of not only altering the gene expression of its host, but it can also regulate the expression of its own genes for survival. In this study, differentially expressed Legionella genes within Acanthamoeba during the 24 h intracellular growth period were investigated for comparative analysis. RNA sequencing analysis revealed 3,003 genes from the intracellular Legionella. Among them, 115 genes were upregulated and 1,676 genes were downregulated more than 2 fold compared to the free Legionella. Gene ontology (GO) analysis revealed the suppression of multiple genes within the intracellular Legionella, which were categorized under 'ATP binding' and 'DNA binding' in the molecular function domain. Gene expression of alkylhydroperoxidase, an enzyme involved in virulence and anti-oxidative stress response, was strongly enhanced 24 h post-intracellular growth. Amino acid ABC transporter substrate-binding protein that utilizes energy generation was also highly expressed. Genes associated with alkylhydroperoxidase, glucose pathway, and Dot/Icm type IV secretion system were shown to be differentially expressed. These results contribute to a better understanding of the survival strategies of intracellular Legionella within Acanthamoeba.

10.
PLoS One ; 15(9): e0239867, 2020.
Article in English | MEDLINE | ID: mdl-32997695

ABSTRACT

Acanthamoeba keratitis (AK) is a rare disease but its prevalence throughout the globe continues to grow, primarily due to increased contact lens usage. Since early-stage symptoms associated with AK closely resemble those from other corneal infections, accurate diagnosis is difficult and this often results in delayed treatment and exacerbation of the disease, which can lead to permanent visual impairment. Accordingly, developing a rapid Acanthamoeba-specific diagnostic method is highly desired. In the present study, a rapid and differential method for AK diagnosis was developed using the secretory proteins derived from the pathogenic Acanthamoeba. Among the vast quantities of proteins secreted by the pathogenic Acanthamoeba, an open reading frame of the inosine-uridine preferring nucleoside hydrolase (IPNH) gene was obtained. After expressing and purifying the IPNH protein using the pGEX 4T-3 vector system, mice were immunized with the purified proteins for polyclonal antibody generation. Western blot was performed using protein lysates of the human corneal cell, non-pathogenic amoeba, pathogenic amoeba, and clinical amoeba isolate along with lysates from other causes of keratitis such as Staphylococcus aureus, Pseudomonas aeruginosa, and Fusarium solani to confirm Acanthamoeba-specificity. Western blot using the polyclonal IPNH antibody revealed that IPNH was Acanthamoeba-specific since these proteins were only observed in lysates of Acanthamoeba origin or its culture media. Our findings indicate that the IPNH antibody of Acanthamoeba may serve as a potential agent for rapid and differential AK diagnosis.


Subject(s)
Acanthamoeba Keratitis/diagnosis , Acanthamoeba castellanii/enzymology , Antibodies/metabolism , N-Glycosyl Hydrolases/immunology , Acanthamoeba Keratitis/parasitology , Acanthamoeba castellanii/isolation & purification , Acanthamoeba castellanii/pathogenicity , Amino Acid Sequence , Animals , Antigen-Antibody Reactions , Male , Mice , Mice, Inbred BALB C , N-Glycosyl Hydrolases/chemistry , N-Glycosyl Hydrolases/genetics , N-Glycosyl Hydrolases/metabolism , Open Reading Frames/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , Sequence Alignment
11.
Antimicrob Agents Chemother ; 64(12)2020 11 17.
Article in English | MEDLINE | ID: mdl-32928736

ABSTRACT

Treatment of Acanthamoeba keratitis (AK) is difficult because Acanthamoeba cysts are resistant to drugs, and as such, successful treatment requires an effective approach that inhibits cyst formation. Histone deacetylase inhibitors (HDACis) are involved in cell proliferation, differentiation, and apoptotic cell death. In this study, the effects of HDACis such as MPK472 and KSK64 on Acanthamoeba castellanii trophozoites and cysts were observed. MPK472 and KSK64 showed at least 60% amoebicidal activity against Acanthamoeba trophozoites at a concentration of 10 µM upon 8 h of treatment. Neither of the two HDACis affected mature cysts, but significant amoebicidal activities (36.4 and 33.9%) were observed against encysting Acanthamoeba following treatment with 5 and 10 µM HDACis for 24 h. Light microscopy and transmission electron microscopy results confirmed that the encystation of Acanthamoeba was inhibited by the two HDACis. In addition to this, low cytopathic effects on human corneal epithelial (HCE) cells were observed following treatment with MPK472 and KSK64 for 24 h. Our results indicate that the HDACis MPK472 and KSK64 could be used as new candidates for the development of an optimal therapeutic option for AK.


Subject(s)
Acanthamoeba Keratitis , Acanthamoeba castellanii , Amebicides , Acanthamoeba Keratitis/drug therapy , Amebicides/pharmacology , Animals , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylase Inhibitors/therapeutic use , Humans , Trophozoites
12.
Cells ; 9(5)2020 05 25.
Article in English | MEDLINE | ID: mdl-32466130

ABSTRACT

Respiratory syncytial virus (RSV) infection affects the lives of neonates throughout the globe, causing a high rate of mortality upon hospital admission. Yet, therapeutic options to deal with this pulmonary pathogen are currently limited. Helminth therapy has been well received for its immunomodulatory role in hosts, which are crucial for mitigating a multitude of diseases. Therefore, in this study, we used the helminth Trichinella spiralis and assessed its capabilities for modulating RSV infection as well as the inflammatory response induced by it in mice. Our results revealed that RSV-specific antibody responses were enhanced by pre-existing T. spiralis infection, which also limited pulmonary viral replication. Diminished lung inflammation, indicated by reduced pro-inflammatory cytokines and inflammatory cell influx was confirmed, as well as through histopathological assessment. We observed that inflammation-associated nuclear factor kappa-light-chain enhancement of activated B cells (NF-κB) and its phosphorylated forms were down-regulated, whereas antioxidant-associated nuclear factor erythroid 2-related factor 2 (Nrf2) protein expression was upregulated in mice co-infected with T. spiralis and RSV. Upregulated Nrf2 expression contributed to increased antioxidant enzyme expression, particularly NQO1 which relieved the host of oxidative stress-induced pulmonary inflammation caused by RSV infection. These findings indicate that T. spiralis can mitigate RSV-induced inflammation by upregulating the expression of antioxidant enzymes.


Subject(s)
Inflammation/pathology , Respiratory Syncytial Virus Infections/parasitology , Respiratory Syncytial Virus Infections/virology , Trichinella spiralis/physiology , Trichinellosis/parasitology , Trichinellosis/virology , Animals , Antibody Specificity/immunology , Antioxidants/metabolism , Cell Line , Female , Humans , Lung/virology , Mice, Inbred BALB C , Pneumonia/complications , Pneumonia/immunology , Pneumonia/parasitology , Pneumonia/virology , Respiratory Syncytial Virus Infections/complications , Respiratory Syncytial Virus Infections/pathology , Trichinellosis/complications , Trichinellosis/immunology , Up-Regulation/genetics , Virus Replication/physiology
13.
Dis Model Mech ; 13(5)2020 05 27.
Article in English | MEDLINE | ID: mdl-32179549

ABSTRACT

High-salt intake and high-fructose intake are risk factors for hypertension via oxidative stress and inflammation. T helper (Th)17 lymphocytes play an important role in the development of hypertension. Here, we tested the hypothesis that activation of pathogenic Th17 lymphocytes induces hypertension after high-fructose intake in Dahl salt-sensitive (SS) but not Dahl salt-resistant (SR) rats. Eight-week-old male SS and SR rats were offered 20% fructose solution or tap water only for 4 weeks. Systolic blood pressure was measured by the tail-cuff method. T lymphocyte [Th17 and T regulatory (Treg)] profiling was determined via flow cytometry. The expression of Th17-related (IL-17A, IL-17RA, IL-23R and RORγt) and Treg-related (IL-10, CD25, FOXP3 and TGFß) factors were measured via ELISA or qRT-PCR. Th17 lymphocytes isolated from high-fructose-fed SS rats were intraperitoneally injected into recipient SS and SR rats, and recombinant IL-23 protein was subcutaneously injected into SS and SR rats to induce hypertension.High-fructose intake induced hypertension via the activation of pathogenic Th17 lymphocytes in SS but not SR rats. Injection of activated Th17 lymphocytes isolated from fructose-fed SS rats induced hypertension via increase of serum IL-17A only in recipient SS rats. In addition, injection of IL-23 induced hypertension via activation of pathogenic Th17 lymphocytes only in SS rats.Thus, activation of pathogenic Th17 lymphocytes induces hypertension after high-fructose intake in SS but not SR rats. These results indicate that immunologic tolerance plays an important role in protection against hypertension in SR rats.


Subject(s)
Hypertension/immunology , Lymphocyte Activation/immunology , Th17 Cells/immunology , Animals , Blood Pressure , Body Weight , Cytokines/blood , Cytokines/metabolism , Forkhead Transcription Factors/metabolism , Fructose , Glucose Intolerance/blood , Glucose Intolerance/complications , Glucose Intolerance/immunology , Hypertension/blood , Hypertension/complications , Immediate-Early Proteins/metabolism , Interleukin-23/metabolism , Male , Models, Biological , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Rats, Inbred Dahl , Signal Transduction , Systole , T-Lymphocytes, Regulatory/immunology
14.
Exp Parasitol ; 210: 107833, 2020 Mar.
Article in English | MEDLINE | ID: mdl-31935358

ABSTRACT

Safety precautions prior to contact lens usage is essential for preventing Acanthamoeba keratitis. Contact lens disinfecting solutions containing 3% hydrogen peroxide (H2O2) are known to exert amoebicidal effect against Acanthamoeba. Yet, these solutions need to be neutralized to prevent ocular irritation, which consequently may result in incomplete disinfection. In this study, amoebicidal effect of tert-butyl hydroperoxide (tBHP) was investigated and its efficacy was compared to those of hydrogen peroxide (H2O2). H2O2 and tBHP showed dose dependent amoebicidal effect, however high concentration of these compounds demonstrated cytotoxicity in human corneal epithelial (HCE) cells. To reduce their cytotoxicity, the concentrations of both compounds were diluted to 50 µM and subsequently combined with 10 µM vorinostat to enhance amoebicidal effect. Addition of vorinostat induced high amoebicidal effect against Acanthamoeba trophozoites, even at low concentrations of H2O2 or tBHP. Cellular damage induced by combined treatment of H2O2 or tBHP with vorinostat in Acanthamoeba were determined by assessing cell cycle arrest and apoptosis via FACS analysis. While 50 µM H2O2 combined with 10 µM vorinostat showed 36.26% cytotoxicity on HCE cells during 24 h exposure, 50 µM tBHP with 10 µM vorinostat did not show cytotoxicity on HCE cells. These findings suggest that the application of tBHP and vorinostat for Acanthamoeba keratitis treatment and contact lens disinfection system is highly plausible.


Subject(s)
Acanthamoeba/drug effects , Antiprotozoal Agents/pharmacology , Cell Cycle Checkpoints/drug effects , Vorinostat/pharmacology , tert-Butylhydroperoxide/pharmacology , Acanthamoeba/cytology , Acanthamoeba/genetics , Anti-Infective Agents, Local/pharmacology , Apoptosis/drug effects , Cells, Cultured , Cornea/cytology , Cornea/drug effects , Cornea/parasitology , DNA, Protozoan/drug effects , DNA, Protozoan/physiology , Drug Combinations , Epithelial Cells/cytology , Epithelial Cells/drug effects , Epithelial Cells/parasitology , Humans , Hydrogen Peroxide/pharmacology
15.
Free Radic Biol Med ; 147: 129-138, 2020 02 01.
Article in English | MEDLINE | ID: mdl-31870798

ABSTRACT

Oxidative stress resistance in cancer cells has contributed to multi-drug resistance, which poses a serious challenge to cancer therapy. To surmount this, combinatorial treatment involving anticancer drugs and histone deacetylase inhibitors (HDACi) have emerged as a chemotherapeutic option. Yet, HDACi's role in redox states of cancer cells still requires elucidation. In the present study, we hypothesized that HDACi sensitizes cancer cells to oxidative stress and results in G2/M cell cycle arrest. Cell viability and cell cycle were analyzed using Cell Counting Kit 8 (CCK8) and fluorescent activated cell sorting (FACS), respectively. The transcriptomes of cells were investigated by massive analysis of cDNA end (MACE). Expression of mRNA and proteins were analyzed by quantitative real-time PCR (qPCR) and Western blot, respectively. Intracellular oxidative stress induced by tert-Butyl hydroperoxide (tBHP) reduced cell viability and resulted in G2/M cell cycle arrest in a dose-dependent manner in hepatocellular carcinoma (HCC) cells. The effects of sorafenib on cell cycle arrest and HCC viability were enhanced through HDACi treatment. MACE revealed that genes related to progression of G2/M cell cycle including Foxm1, Aurka, Plk1, and Ccnb1 were significantly down-regulated in tBHP and HDACi-treated HepG2 cells. Inhibition of FOXM1 with thiostrepton also resulted in reduced cell viability and expression of FOXM1 target genes such as Aurka, Plk1, and Ccnb1. These results indicate that HDACi sensitizes HepG2 cells to oxidative stress and results in G2/M cell cycle arrest via down-regulation of FOXM1, which plays a key role in progression of G2/M cell cycle.


Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Apoptosis , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/genetics , Cell Cycle Checkpoints , Cell Line, Tumor , Cell Proliferation , Histone Deacetylases , Humans , Liver Neoplasms/drug therapy , Liver Neoplasms/genetics , Oxidative Stress
16.
Cornea ; 39(2): 245-249, 2020 Feb.
Article in English | MEDLINE | ID: mdl-31724982

ABSTRACT

PURPOSE: The aim of this study was to reduce the cytotoxicity and improve the amoebicidal effect of polyhexamethylene biguanide (PHMB) at low concentrations by combining it with histone deacetylase (HDAC) inhibitors. METHODS: To reduce the cytotoxic effect on human corneal epithelial (HCE) cells, the concentration of PHMB was reduced to 0.0002%. To enhance the amoebicidal effect of PHMB, HDAC inhibitors such as suberoylanilide hydroxamic acid, MS275, or MC1568 were combined with it. Acanthamoeba and HCE cells were treated with 3 combinations to evaluate the amoebicidal and cytotoxic effects. Microscopy and fluorescence-activated cell sorting analysis were performed to investigate the apoptotic cell death of Acanthamoeba by these combinatorial treatments. RESULTS: The low concentration of PHMB (0.0002%) alone demonstrated no cytopathic effects (CPEs) on HCE cells. Three combinatorial treatments using 0.0002% PHMB with 10 µM suberoylanilide hydroxamic acid, 10 µM MS275, or 10 µM MC1568 showed higher amoebicidal effects on A. castellanii trophozoites than PHMB alone. Fluorescence-activated cell sorting analysis confirmed that HDAC inhibitors increased the apoptotic cell death of Acanthamoeba. Mild CPEs were observed from HCE cells cotreated with PHMB and the HDAC inhibitors after 24 hours of exposure. CONCLUSIONS: Combinatorial treatments showed high amoebicidal effects on Acanthamoeba and low CPEs on HCE cells, which suggests their potential application for Acanthamoeba keratitis treatment.


Subject(s)
Acanthamoeba castellanii/drug effects , Apoptosis/drug effects , Biguanides/pharmacology , Disinfectants/pharmacology , Epithelium, Corneal/drug effects , Histone Deacetylase Inhibitors/pharmacology , Acanthamoeba castellanii/metabolism , Annexin A5/metabolism , Benzamides/pharmacology , Biguanides/administration & dosage , Cells, Cultured , Disinfectants/administration & dosage , Drug Synergism , Epithelium, Corneal/metabolism , Flow Cytometry , Fluorescein-5-isothiocyanate , Humans , Hydroxamic Acids/pharmacology , Microscopy, Fluorescence , Pyridines/pharmacology , Pyrroles/pharmacology , Vorinostat/pharmacology
17.
Vet Parasitol ; 273: 60-66, 2019 Sep.
Article in English | MEDLINE | ID: mdl-31442895

ABSTRACT

Maternal antibody transmission via placenta and breastmilk are known to confer protection in infants. In this study, we investigated the maternal immunity transmission in pups delivered by rats infected with Trichinella spiralis and assessed the resulting resistance against subsequent parasitic infection. Our results revealed that parasite-specific IgG, IgG1 and IgG2a antibodies were present in pups prior to breastmilk ingestion (pre-milk), in which IgG and IgG1 antibodies persisted until week 8 after birth while parasite-specific IgG2a antibodies only lasted until week 4. After weaning on week 3, pups delivered by T. spiralis-infected dam and subsequently challenge-infected (immune-challenge) were found to possess higher mucosal IgG antibodies than control groups, whereas mucosal IgA levels were not significantly different across all groups. T. spiralis excretory-secretory antigen was discovered to react with pup sera until week 8, correlating with the resistance against parasitic infection which is represented by lessened worm burden. Upon T. spiralis infection at weeks 3 and 8, lower levels of eosinophil responses were detected in immune-challenge pups compared to naïve-challenge pups, indicating correlates of resistances in which ADCC may be involved. Findings from the present study demonstrate that resistances against T. spiralis infection in pups can be acquired by maternally-derived IgG, IgG1 and IgG2a antibody transmission through the placenta and breastmilk from T. spiralis-infected dam, which lasts until week 8.


Subject(s)
Disease Resistance/immunology , Immunity, Maternally-Acquired/immunology , Trichinella spiralis/immunology , Trichinellosis/immunology , Animals , Antibodies, Helminth/blood , Antigens, Helminth/metabolism , Female , Helminth Proteins/metabolism , Milk/immunology , Placenta/immunology , Pregnancy , Rats
18.
Clin Exp Pharmacol Physiol ; 46(3): 226-236, 2019 03.
Article in English | MEDLINE | ID: mdl-30099761

ABSTRACT

Histone deacetylases (HDACs) are a vast family divided into four major classes: class I (1, 2, 3, and 8), class II (4, 5, 6, 7, 9 and 10), class III (sirtuin family) and class IV (HDAC11). HDAC inhibition attenuates cardiac hypertrophy through suppression of the mechanistic target of rapamycin complex1 (mTORC1) signaling. HDAC inhibitors upregulate the expression of tuberous sclerosis complex 2 (TSC2), an mTORC1 inhibitor. However, the molecular mechanism underlying HDAC inhibitor-mediated upregulation of TSC2 is unclear. We hypothesized that an HDAC inhibitor, CG200745 (CG), ameliorates cardiac hypertrophy through the inhibition of mTORC1 signaling by upregulating of the CCAAT/enhancer-binding protein-ß (C/EBP-ß)/TSC2 pathway. To establish a cardiac hypertrophy model, deoxycorticosterone acetate (DOCA, 40 mg/kg/wk) was subcutaneously injected for 4 weeks into Sprague-Dawley rats. All rats were unilaterally nephrectomized and had free access to drinking water containing 1% NaCl with or without CG of different concentrations. The expression level of TSC2 and C/EBP-ß was measured by quantitative real-time PCR (qRT-PCR) and western blot analysis. Acetylation of C/EBP-ß was analyzed by immunoprecipitation. The recruitment of C/EBP-ß and polymerase II (Pol II) on TSC2 promoter region was analyzed by chromatin immunoprecipitation (ChIP). CG treatment increased the expression of TSC2. In addition, CG treated rats showed an increased in the expression and acetylation of C/EBP-ß, owing to the increase in the recruitment of C/EBP-ß and Pol II at Tsc2 gene promoter. Thus, CG ameliorates cardiac hypertrophy through the inhibition of mTORC1 signaling via upregulation of the C/EBP-ß/TSC2 pathway in DOCA-induced hypertensive rats.


Subject(s)
CCAAT-Enhancer-Binding Protein-beta/metabolism , Heart/drug effects , Histone Deacetylase Inhibitors/pharmacology , Hydroxamic Acids/pharmacology , Myocardium/pathology , Naphthalenes/pharmacology , Tuberous Sclerosis Complex 2 Protein/metabolism , Up-Regulation/drug effects , Acetylation/drug effects , Animals , CCAAT-Enhancer-Binding Protein-beta/genetics , Cardiotonic Agents/pharmacology , Desoxycorticosterone Acetate/adverse effects , Hypertrophy/chemically induced , Hypertrophy/metabolism , Hypertrophy/pathology , Hypertrophy/prevention & control , Male , Mechanistic Target of Rapamycin Complex 1/metabolism , Myocardium/metabolism , Promoter Regions, Genetic/genetics , Rats , Rats, Sprague-Dawley , Tuberous Sclerosis Complex 2 Protein/genetics , Ventricular Remodeling/drug effects
19.
Biomed Pharmacother ; 109: 921-929, 2019 Jan.
Article in English | MEDLINE | ID: mdl-30551546

ABSTRACT

Inhibition of histone deacetylase (HDAC) suppresses inflammation of pancreatic islets and apoptosis of ß-cells. However, the underlying molecular mechanism is unclear. In the present study, we demonstrate that MGCD0103 (MGCD), an HDAC inhibitor, protects the pancreas from streptozotocin (STZ)-induced oxidative stress and cell death. Sprague-Dawley rats were intraperitoneally injected with STZ (40 mg/kg) to induce type I diabetes. MGCD (10 µg/day) was infused with osmotic mini-pump for 4 weeks. Pancreatic insulin and macrophage infiltration were analyzed by immunohistochemistry. Cellular level of reactive oxygen species (ROS) was evaluated with fluorescence-activated cell sorting. Tetramethylrhodamine ethyl ester was used to analyze mitochondrial membrane potential. Activation of caspase-3 was analyzed by western blotting. Chromatin immunoprecipitation was performed to investigate the binding affinity of specificity protein 1 (SP1) on the promoters of target genes. mRNA expression was analyzed by quantitative real-time polymerase chain reaction. As a result, we found that MGCD infusion ameliorated STZ-induced hyperglycemia, islet deformation, decreased insulin level, and macrophage infiltration. STZ injection promoted the production of ROS, which induced caspase activity and ß-cell death. 4-Hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL), a mimetic of superoxide dismutase (SOD), reduced STZ-induced caspase activity and ß-cell death. MGCD treatment increased SOD expression and histone acetylation level on promoters. Infusion of MGCD promoted acetylation of SP1 and its enrichment on SOD promoters. Thus, MGCD protects pancreatic ß-cells from STZ-induced oxidative stress and cell death through the induction of antioxidant enzymes such as SODs.


Subject(s)
Benzamides/administration & dosage , Histone Deacetylase Inhibitors/administration & dosage , Insulin-Secreting Cells/drug effects , Oxidative Stress/drug effects , Pancreas/drug effects , Pyrimidines/administration & dosage , Streptozocin/toxicity , Animals , Cell Death/drug effects , Cell Death/physiology , Dose-Response Relationship, Drug , HEK293 Cells , Hep G2 Cells , Humans , Infusion Pumps , Insulin-Secreting Cells/metabolism , Male , Oxidative Stress/physiology , Pancreas/metabolism , Pancreas/pathology , Random Allocation , Rats , Rats, Sprague-Dawley
20.
Int J Mol Sci ; 19(11)2018 Nov 09.
Article in English | MEDLINE | ID: mdl-30424007

ABSTRACT

Type 2 diabetes mellitus (T2DM) is a chronic disease manifested by hyperglycemia. It is essential to effectively control hyperglycemia to prevent complications of T2DM. Here, we hypothesize that repression of transcriptional activity of forkhead box O1 (FoxO1) via histone deacetylase inhibitors (HDACi) ameliorates hyperglycemia in T2DM rats. METHODS: Male Long-Evans Tokushima Otsuka (LETO) and Otsuka Long-Evans Tokushima Fatty (OLETF) rats aged 14 weeks were administered sodium valproate (VPA, 0.71% w/v) dissolved in water for 20 weeks. Electrophoretic mobility shift assay (EMSA) and luciferase assay were performed for elucidation of transcriptional regulation through acetylation of FoxO1 by HDACi. RESULTS: VPA attenuated blood glucose levels in accordance with a decrease in the expression of gluconeogenic genes in hyperglycemic OLETF rats. It has been shown that HDAC class I-specific and HDAC class IIa-specific inhibitors, as well as pan-HDAC inhibitors decrease FoxO1 enrichment at the cis-element of target gene promoters. Mutations in FoxO1 prevent its acetylation, thereby increasing its transcriptional activity. HDAC3 and HDAC4 interact with FoxO1, and knockdown of HDAC3, HDAC4, or their combination increases FoxO1 acetylation, thereby decreasing the expression of gluconeogenic genes. CONCLUSIONS: These results indicate that HDACi attenuates the transcriptional activity of FoxO1 by impeding deacetylation, thereby ameliorating hyperglycemia in T2DM rats.


Subject(s)
Diabetes Mellitus, Experimental/drug therapy , Diabetes Mellitus, Type 2/drug therapy , Diabetes Mellitus, Type 2/genetics , Forkhead Box Protein O1/genetics , Histone Deacetylase Inhibitors/therapeutic use , Hyperglycemia/drug therapy , Hyperglycemia/genetics , Transcription, Genetic , Acetylation , Animals , Diabetes Mellitus, Experimental/genetics , Forkhead Box Protein O1/metabolism , Gluconeogenesis/drug effects , Gluconeogenesis/genetics , Glucose/toxicity , Glucose-6-Phosphate/metabolism , Hep G2 Cells , Histone Deacetylases/metabolism , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Male , Phosphoenolpyruvate Carboxykinase (GTP)/genetics , Phosphoenolpyruvate Carboxykinase (GTP)/metabolism , Rats, Inbred OLETF , Repressor Proteins , Transcription, Genetic/drug effects , Valproic Acid/administration & dosage , Valproic Acid/pharmacology , Valproic Acid/therapeutic use
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