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1.
J Mater Chem B ; 2024 Jul 02.
Article in English | MEDLINE | ID: mdl-38953113

ABSTRACT

In this paper, we explore the development of a multi-functional surface designed to tackle the challenges posed by Staphylococcus aureus (S. aureus), a common opportunistic pathogen. Infections caused by S. aureus during surgical procedures highlight the need for effective strategies to inhibit its adhesion, growth, and colonization, particularly on the surfaces of invasive medical devices. Until now, most existing research has focused on nanopillar structures (positive topographies). Uniform nanopillar arrays have been shown to control bacterial behavior based on the spacing between nanopillars. However, nanopillar structures are susceptible to external friction, impact, and force, making it challenging to maintain their antibacterial properties. Therefore, in this study, we investigate the antibacterial behavior of nanohole structures, which offer relatively superior mechanical robustness compared to nanopillars. Moreover, for applications in medical devices such as laparoscopes, there is a pressing need for surfaces that are not only transparent and flexible (or curved) but are also equipped with antibacterial properties. Our study introduces a scalable multi-functional surface that synergistically combines antibacterial and anti-fog properties. This is achieved by fabricating thin films with variously sized holes (ranging from 0.3 µm to 4 µm) using polyurethane acrylate (PUA). We assessed the activity of S. aureus on these surfaces and found that a 1 µm-diameter-hole pattern significantly reduced the presence of live S. aureus, without any detection of dead S. aureus. This bacteriostatic effect is attributed to the restricted proliferation due to the confined area provided by the hole pattern. However, the persistence of some live S. aureus on the surface necessitates further measures to minimize bacterial adhesion and enhance antibacterial effectiveness. To address this challenge, we coated the zwitterionic polymer 2-methacryloyloxyethyl phosphorylcholine (MPC) onto the nanohole pattern surface to reduce S. aureus adhesion. Moreover, in long-term experiments on surfaces, the MPC-coated effectively inhibited the colonization of S. aureus (18 h; 82%, 7 days; 83%, and 14 days; 68% antibacterial rate). By integrating PUA, MPC, and nanohole architectures into a single, flexible platform, we achieved a multi-functional surface catering to transparency, anti-fogging, and anti-biofouling requirements. This innovative approach marks a significant advancement in surface engineering, offering a versatile solution applicable in various fields, particularly in preventing S. aureus contamination in invasive medical devices like laparoscopes. The resultant surface, characterized by its transparency, flexibility, and antibacterial functionality, stands out as a promising candidate for mitigating S. aureus-related risks in medical applications.

2.
Cells ; 13(3)2024 Jan 24.
Article in English | MEDLINE | ID: mdl-38334608

ABSTRACT

Effectively targeting cancer stemness is essential for successful cancer therapy. Recent studies have revealed that SOX2, a pluripotent stem cell factor, significantly contributes to cancer stem cell (CSC)-like characteristics closely associated with cancer malignancy. However, its contradictory impact on patient survival in specific cancer types, including lung adenocarcinoma (LUAD), underscores the need for more comprehensive research to clarify its functional effect on cancer stemness. In this study, we demonstrate that SOX2 is not universally required for the regulation of CSC-like properties in LUAD. We generated SOX2 knockouts in A549, H358, and HCC827 LUAD cells using the CRISPR/Cas9 system. Our results reveal unchanged CSC characteristics, including sustained proliferation, tumor sphere formation, invasion, migration, and therapy resistance, compared to normal cells. Conversely, SOX2 knockdown using conditional shRNA targeting SOX2, significantly reduced CSC traits. However, these loss-of-function effects were not rescued by SOX2 resistant to shRNA, underscoring the potential for SOX2 protein level-independent results in prior siRNA- or shRNA-based research. Ultimately, our findings demonstrate that SOX2 is not absolutely essential in LUAD cancer cells. This emphasizes the necessity of considering cancer subtype-dependent and context-dependent factors when targeting SOX2 overexpression as a potential therapeutic vulnerability in diverse cancers.


Subject(s)
Adenocarcinoma of Lung , Lung Neoplasms , Neoplastic Stem Cells , SOXB1 Transcription Factors , Humans , Adenocarcinoma of Lung/pathology , Lung Neoplasms/pathology , Neoplastic Stem Cells/pathology , RNA, Small Interfering/metabolism , SOXB1 Transcription Factors/genetics , SOXB1 Transcription Factors/metabolism
3.
Int J Mol Sci ; 24(15)2023 Jul 26.
Article in English | MEDLINE | ID: mdl-37569345

ABSTRACT

In previous work, we showed that cancer cells do not depend on glycolysis for ATP production, but they do on fatty acid oxidation. However, we found some cancer cells induced cell death after glucose deprivation along with a decrease of ATP production. We investigated the different response of glucose deprivation with two types of cancer cells including glucose insensitive cancer cells (GIC) which do not change ATP levels, and glucose sensitive cancer cells (GSC) which decrease ATP production in 24 h. Glucose deprivation-induced cell death in GSC by more than twofold after 12 h and by up to tenfold after 24 h accompanied by decreased ATP production to compare to the control (cultured in glucose). Glucose deprivation decreased the levels of metabolic intermediates of the pentose phosphate pathway (PPP) and the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) in both GSC and GIC. However, glucose deprivation increased reactive oxygen species (ROS) only in GSC, suggesting that GIC have a higher tolerance for decreased NADPH than GSC. The twofold higher ratio of reduced/oxidized glutathione (GSH/GSSG) in GIS than in GSC correlates closely with the twofold lower ROS levels under glucose starvation conditions. Treatment with N-acetylcysteine (NAC) as a precursor to the biologic antioxidant glutathione restored ATP production by 70% and reversed cell death caused by glucose deprivation in GSC. The present findings suggest that glucose deprivation-induced cancer cell death is not caused by decreased ATP levels, but rather triggered by a failure of ROS regulation by the antioxidant system. Conclusion is clear that glucose deprivation-induced cell death is independent from ATP depletion-induced cell death.


Subject(s)
Adenosine Triphosphate , Glucose , Neoplasms , Reactive Oxygen Species , Glucose/deficiency , Adenosine Triphosphate/metabolism , Pentose Phosphate Pathway , Reactive Oxygen Species/metabolism , NADP/metabolism , Glutathione/metabolism , Acetylcysteine/metabolism , Acetylcysteine/pharmacology , PC-3 Cells , Humans , Neoplasms/metabolism , Neoplasms/pathology , Cell Death
4.
Biomedicines ; 10(8)2022 Aug 06.
Article in English | MEDLINE | ID: mdl-36009455

ABSTRACT

Breast cancer has a high risk of recurrence and distant metastasis after remission. Controlling distant metastasis is important for reducing breast cancer mortality, but accomplishing this goal remains elusive. In this study, we investigated the molecular pathways underlying metastasis using cells that mimic the breast cancer distant metastasis process. HCC1143 breast cancer cells were cultured under two-dimensional (2D)-adherent, tumor sphere (TS), and reattached (ReA) culture conditions to mimic primary tumors, circulating tumor cells, and metastasized tumors, respectively. ReA cells demonstrated increased TS formation and enhanced invasion capacity compared to the original 2D-cultured parental cells. In addition, ReA cells had a higher frequency of ESA+CD44+CD24- population, which represents a stem-cell-like cell population. RNA sequencing identified the cholesterol synthesis pathway as one of the most significantly increased pathways in TS and ReA cells compared to parental cells, which was verified by measuring intracellular cholesterol levels. Furthermore, the pharmacological inhibition of the cholesterol synthesis pathway decreased the ability of cancer cells to form TSs and invade. Our results suggest that the cholesterol synthesis pathway plays an important role in the distant metastasis of breast cancer cells by augmenting TS formation and invasion capacity.

5.
Exp Mol Med ; 53(11): 1759-1768, 2021 11.
Article in English | MEDLINE | ID: mdl-34819616

ABSTRACT

Sox2 is a core transcription factor in embryonic stem cells (ESCs), and O-GlcNAcylation is a type of post-translational modification of nuclear-cytoplasmic proteins. Although both factors play important roles in the maintenance and differentiation of ESCs and the serine 248 (S248) and threonine 258 (T258) residues of Sox2 are modified by O-GlcNAcylation, the function of Sox2 O-GlcNAcylation is unclear. Here, we show that O-GlcNAcylation of Sox2 at T258 regulates mouse ESC self-renewal and early cell fate. ESCs in which wild-type Sox2 was replaced with the Sox2 T258A mutant exhibited reduced self-renewal, whereas ESCs with the Sox2 S248A point mutation did not. ESCs with the Sox2 T258A mutation heterologously introduced using the CRISPR/Cas9 system, designated E14-Sox2TA/WT, also exhibited reduced self-renewal. RNA sequencing analysis under self-renewal conditions showed that upregulated expression of early differentiation genes, rather than a downregulated expression of self-renewal genes, was responsible for the reduced self-renewal of E14-Sox2TA/WT cells. There was a significant decrease in ectodermal tissue and a marked increase in cartilage tissue in E14-Sox2TA/WT-derived teratomas compared with normal E14 ESC-derived teratomas. RNA sequencing of teratomas revealed that genes related to brain development had generally downregulated expression in the E14-Sox2TA/WT-derived teratomas. Our findings using the Sox2 T258A mutant suggest that Sox2 T258 O-GlcNAc has a positive effect on ESC self-renewal and plays an important role in the proper development of ectodermal lineage cells. Overall, our study directly links O-GlcNAcylation and early cell fate decisions.


Subject(s)
Cell Self Renewal , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , SOXB1 Transcription Factors/metabolism , Threonine/metabolism , Alleles , Animals , Cell Differentiation/genetics , Cell Lineage , Cell Self Renewal/genetics , Cells, Cultured , Fluorescent Antibody Technique , Gene Editing , Gene Expression Regulation , Glycosylation , Mice , Mutation , Protein Processing, Post-Translational , SOXB1 Transcription Factors/genetics , Teratoma/etiology , Teratoma/metabolism , Teratoma/pathology
6.
Cancers (Basel) ; 13(3)2021 Jan 20.
Article in English | MEDLINE | ID: mdl-33498427

ABSTRACT

(1) Background: Recent advances in precision oncology research rely on indicating specific genetic alterations associated with treatment sensitivity. Developing ex vivo systems to identify cancer patients who will respond to a specific drug remains important. (2) Methods: cells from 12 patients with glioblastoma were isolated, cultured, and subjected to high-content screening. Multi-parameter analyses assessed the c-Met level, cell viability, apoptosis, cell motility, and migration. A drug repurposing screen and large-scale drug sensitivity screening data across 59 cancer cell lines and patient-derived cells were obtained from 125 glioblastoma samples. (3) Results: High-content analysis of patient-derived cells provided robust and accurate drug responses to c-Met-targeted agents. Only the cells of one glioblastoma patient (PDC6) showed elevated c-Met level and high susceptibility to the c-Met inhibitors. Multi-parameter image analysis also reflected a decreased c-Met expression and reduced cell growth and motility by a c-Met-targeting antibody. In addition, a drug repurposing screen identified Abemaciclib as a distinct CDK4/6 inhibitor with a potent c-Met-inhibitory function. Consistent with this, we present large-scale drug sensitivity screening data showing that the Abemaciclib response correlates with the response to c-Met inhibitors. (4) Conclusions: Our study provides a new insight into high-content screening platforms supporting drug sensitivity prediction and novel therapeutics screening.

8.
Cancers (Basel) ; 12(9)2020 Sep 11.
Article in English | MEDLINE | ID: mdl-32932964

ABSTRACT

Octamer-binding transcription factor 4 (Oct4) plays an important role in maintaining pluripotency in embryonic stem cells and is closely related to the malignancies of various cancers. Although posttranslational modifications of Oct4 have been widely studied, most of these have not yet been fully characterized, especially in cancer. In this study, we investigated the role of phosphorylation of serine 236 of OCT4 [OCT4 (S236)] in human germ cell tumors (GCTs). OCT4 was phosphorylated at S236 in a cell cycle-dependent manner in a patient sample and GCT cell lines. The substitution of endogenous OCT4 by a mimic of phosphorylated OCT4 with a serine-to-aspartate mutation at S236 (S236D) resulted in tumor cell differentiation, growth retardation, and inhibition of tumor sphere formation. GCT cells expressing OCT4 S236D instead of endogenous OCT4 were similar to cells with OCT4 depletion at the mRNA transcript level as well as in the phenotype. OCT4 S236D also induced tumor cell differentiation and growth retardation in mouse xenograft experiments. Inhibition of protein phosphatase 1 by chemicals or short hairpin RNAs increased phosphorylation at OCT4 (S236) and resulted in the differentiation of GCTs. These results reveal the role of OCT4 (S236) phosphorylation in GCTs and suggest a new strategy for suppressing OCT4 in cancer.

9.
Exp Mol Med ; 51(12): 1-11, 2019 12 06.
Article in English | MEDLINE | ID: mdl-31811117

ABSTRACT

Glioblastoma (GBM) is the most lethal primary brain tumor with few treatment options. The survival of glioma-initiating cells (GICs) is one of the major factors contributing to treatment failure. GICs frequently produce and respond to their own growth factors that support cell proliferation and survival. In this study, we aimed to identify critical autocrine factors mediating GIC survival and to evaluate the anti-GBM effect of antagonizing these factors. Proteomic analysis was performed using conditioned media from two different patient-derived GBM tumor spheres under a growth factor-depleted status. Then, the antitumor effects of inhibiting an identified autocrine factor were evaluated by bioinformatic analysis and molecular validation. Proteins secreted by sphere-forming GICs promote cell proliferation/survival and detoxify reactive oxygen species (ROS). Among these proteins, we focused on midkine (MDK) as a clinically significant and pathologically relevant autocrine factor. Antagonizing MDK reduced the survival of GBM tumor spheres through the promotion of cell cycle arrest and the consequent apoptotic cell death caused by oxidative stress-induced DNA damage. We also identified PCBP4, a novel molecular predictor of resistance to anti-MDK treatment. Collectively, our results indicate that MDK inhibition is an important therapeutic option by suppressing GIC survival through the induction of ROS-mediated cell cycle arrest and apoptosis.


Subject(s)
Central Nervous System/metabolism , Glioblastoma/metabolism , Midkine/metabolism , RNA-Binding Proteins/metabolism , Apoptosis/genetics , Apoptosis/physiology , Cell Cycle/genetics , Cell Cycle/physiology , Computational Biology , DNA Damage/genetics , DNA Damage/physiology , Humans , In Vitro Techniques , Reactive Oxygen Species/metabolism , Sequence Analysis, RNA
10.
Cancers (Basel) ; 11(12)2019 Nov 22.
Article in English | MEDLINE | ID: mdl-31771104

ABSTRACT

Glioblastoma is a highly aggressive and lethal brain tumor, with limited treatment options. Abnormal activation of the neddylation pathway is observed in glioblastoma, and the NEDD8-activating enzyme (NAE) inhibitor, MLN4924, was previously shown to be effective in glioblastoma cell line models. However, its effect has not been tested in patient-derived glioblastoma stem cells. We first analyzed public data to determine whether NEDD8 pathway proteins are important in glioblastoma development and patient survival. NAE1 and UBA3 levels increased in glioblastoma patients; high NEDD8 levels were associated with poor clinical outcomes. Immunohistochemistry results also supported this result. The effects of MLN4924 were evaluated in 4 glioblastoma cell lines and 15 patient-derived glioblastoma stem cells using high content analysis. Glioblastoma cell lines and patient-derived stem cells were highly susceptible to MLN4924, while normal human astrocytes were resistant. In addition, there were various responses in 15 patient-derived glioblastoma stem cells upon MLN4924 treatment. Genomic analyses indicated that MLN4924 sensitive cells exhibited enrichment of Extracellular Signal Regulated Kinase (ERK) and Protein kinase B (AKT, also known as PKB) signaling. We verified that MLN4924 inhibits ERK and AKT phosphorylation in MLN4924 sensitive cells. Our findings suggest that patient-derived glioblastoma stem cells in the context of ERK and AKT activation are sensitive and highly regulated by neddylation inhibition.

11.
Cell Death Dis ; 9(8): 792, 2018 07 18.
Article in English | MEDLINE | ID: mdl-30022047

ABSTRACT

Testing new ways to identify untapped opportunities for glioblastoma therapies remains highly significant. Amplification and overexpression of MDM2 gene is frequent in glioblastoma and disrupting the MDM2-p53 interaction is a promising strategy to treat the cancer. RG7112 is the first-in class inhibitor and recently discovered AMG232 is the most potent MDM2 inhibitor known to date. Here, we compared the effects of these two clinical MDM2 inhibitors in six glioblastoma cell lines and ten patient-derived glioblastoma stem cells. Targeted sequencing of the TP53, MDM2 genes and whole transcriptome analysis were conducted to verify genetic status associated with sensitivity and resistance to the drugs. Although TP53 wild-type glioblastoma cell lines are similarly sensitive to AMG232 and RG7112, we found that four TP53 wild-type out of ten patient-derived glioblastoma cells are much more sensitive to AMG232 than RG7112 (average IC50 of 76 nM vs. 720 nM). Among these, 464T stem cells containing MDM2 gene amplification were most sensitive to AMG232 with IC50 of 5.3 nM. Moreover, AMG232 exhibited higher selectivity against p53 wild-type cells over p53 mutant stem cells compared to RG7112 (average selectivity of 512-fold vs. 16.5-fold). Importantly, we also found that AMG232 is highly efficacious in three-dimensional (3D) tumor spheroids growth and effectively inhibits the stemness-related factors, Nestin and ZEB1. Our data provide new evidence that glioblastoma stem cells have high susceptibility to AMG232 suggesting the potential clinical implications of MDM2 inhibition for glioblastoma treatment. These will facilitate additional preclinical and clinical studies evaluating MDM2 inhibitors in glioblastoma and direct further efforts towards developing better MDM2-targeted therapeutics.


Subject(s)
Acetates/pharmacology , Apoptosis/drug effects , Enzyme Inhibitors/pharmacology , Piperidones/pharmacology , Proto-Oncogene Proteins c-mdm2/antagonists & inhibitors , Acetates/chemistry , Cell Line, Tumor , Enzyme Inhibitors/chemistry , Glioblastoma/metabolism , Glioblastoma/pathology , Humans , Imidazolines/chemistry , Imidazolines/pharmacology , Mutation , Nestin/metabolism , Piperidones/chemistry , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Zinc Finger E-box-Binding Homeobox 1/metabolism
12.
Oncotarget ; 6(24): 20145-59, 2015 Aug 21.
Article in English | MEDLINE | ID: mdl-26023737

ABSTRACT

Glioblastoma (GBM) is the most lethal brain cancer with profound genomic alterations. While the bona fide tumor suppressor genes such as PTEN, NF1, and TP53 have high frequency of inactivating mutations, there may be the genes with GBM-suppressive roles for which genomic mutation is not a primary cause for inactivation. To identify such genes, we employed in vivo RNAi screening approach using the patient-derived GBM xenograft models. We found that Nemo-Like Kinase (NLK) negatively regulates mesenchymal activities, a characteristic of aggressive GBM, in part via inhibition of WNT/ß-catenin signaling. Consistent with this, we found that NLK expression is especially low in a subset of GBMs that harbors high WNT/mesenchymal activities. Restoration of NLK inhibited WNT and mesenchymal activities, decreased clonogenic growth and survival, and impeded tumor growth in vivo. These data unravel a tumor suppressive role of NLK and support the feasibility of combining oncogenomics with in vivo RNAi screen.


Subject(s)
Brain Neoplasms/genetics , Glioblastoma/genetics , Mitogen-Activated Protein Kinases/genetics , Mitogen-Activated Protein Kinases/metabolism , Animals , Brain Neoplasms/enzymology , Brain Neoplasms/pathology , Cell Proliferation/genetics , Female , Glioblastoma/enzymology , Glioblastoma/pathology , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Mice , Mice, Inbred BALB C , Mice, Nude , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , RNA Interference
13.
Immunology ; 120(2): 173-81, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17054728

ABSTRACT

In studying the mechanisms underlying the susceptibility of the kidney to candidal infection, we previously reported that the reduced production of cytokines [i.e. tumour necrosis factor-alpha (TNF-alpha)] via platelet-activating factor (PAF)-induced activation of nuclear factor-kappaB (NF-kappaB) renders the organ susceptible to the fungal burden. In this study, we investigated the possibility that pathogenic Candida albicans may evade clearance and perhaps even multiply by inhibiting elements in the signalling pathway that lead to the production of TNF-alpha. The fungal burden of pathogenic C. albicans in the kidneys was 10(4)-10(5)-fold higher than that of a non-pathogenic strain. PAF-induced early activation of NF-kappaB and TNF-alpha mRNA expression were both observed in the kidneys of mice infected with non-pathogenic strains of C. albicans, but not in mice infected with pathogenic strains. Impairment of PAF-mediated early NF-kappaB activation following infection with pathogenic C. albicans was associated with the prevention of activation of the enzyme cytosolic phospholipase A(2) (cPLA(2)) as well as the upstream pathway of cPLA(2), p38 mitogen-activated protein kinase. Collectively, these findings indicate that C. albicans exerts its pathogenicity through impairing the production of anticandidal cytokines by preventing cPLA(2) activity. This novel mechanism provides insight into understanding pathogenic C. albicans and perhaps identifies a target for its treatment.


Subject(s)
Candida albicans/pathogenicity , Candidiasis/enzymology , Kidney Diseases/enzymology , Phospholipases A/metabolism , p38 Mitogen-Activated Protein Kinases/physiology , Animals , Enzyme Inhibitors/pharmacology , Female , Gene Expression Regulation , Kidney Diseases/metabolism , Kidney Diseases/microbiology , Mice , Mice, Inbred BALB C , NF-kappa B/metabolism , Phospholipases A2 , Phosphorylation , Platelet Activating Factor/antagonists & inhibitors , Platelet Activating Factor/physiology , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/genetics , Virulence , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors
14.
Curr Drug Deliv ; 2(4): 401-6, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16305443

ABSTRACT

The development of novel 'new generation' vaccine systems that is based on proteins, peptides or DNA is of great current interest. However, due to the lower efficiencies of these new generation vaccines, they are seldomly used alone. Rather, their formulations often contain adjuvants, either to enhance the immune responses or to reduce dosing. The present chapter will provide a brief overview of the recent advances in peptide-based cancer vaccine adjuvants, focusing mainly on Liposome-Protamine-DNA (LPD) nanoparticle-mediated antigen delivery.


Subject(s)
Antigens/administration & dosage , DNA/immunology , Drug Delivery Systems , Lipids/immunology , Protamines/immunology , Vaccines, DNA/administration & dosage , Vaccines, DNA/immunology , Adjuvants, Immunologic , Animals , Cancer Vaccines/administration & dosage , Cancer Vaccines/immunology , DNA/administration & dosage , DNA/chemistry , Dendritic Cells/drug effects , Dendritic Cells/immunology , Humans , Lipids/administration & dosage , Lipids/chemistry , Protamines/administration & dosage , Protamines/chemistry , Vaccines, Subunit/administration & dosage , Vaccines, Subunit/immunology
15.
Clin Diagn Lab Immunol ; 12(11): 1285-91, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16275942

ABSTRACT

Porphyromonas gingivalis is one of the suspected periodontopathic bacteria. The lipopolysaccharide (LPS) of P. gingivalis is a key factor in the development of periodontitis. Inflammatory cytokines play important roles in the gingival tissue destruction that is a characteristic of periodontitis. Macrophages are prominent at chronic inflammatory sites and are considered to contribute to the pathogenesis of periodontitis. Xylitol stands out and is widely believed to possess anticaries properties. However, to date, little is known about the effect of xylitol on periodontitis. The aim of the present study was to determine tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta) expression when RAW 264.7 cells were stimulated with P. gingivalis LPS (hereafter, LPS refers to P. gingivalis LPS unless stated otherwise) and the effect of xylitol on the LPS-induced TNF-alpha and IL-1beta expression. The kinetics of TNF-alpha and IL-1beta levels in culture supernatant after LPS treatment showed peak values at 1 h (TNF-alpha) and 2 to 4 h (IL-1beta), respectively. NF-kappaB, a transcription factor, was also activated by LPS treatment. These cytokine expressions and NF-kappaB activation were suppressed by pretreatment with pyrrolidine dithiocarbamate (an inhibitor of NF-kappaB). Pretreatment with xylitol inhibited LPS-induced TNF-alpha and IL-1beta gene expression and protein synthesis. LPS-induced mobilization of NF-kappaB was also inhibited by pretreatment with xylitol in a dose-dependent manner. Xylitol also showed inhibitory effect on the growth of P. gingivalis. Taken together, these findings suggest that xylitol may have good clinical effect not only for caries but also for periodontitis by its inhibitory effect on the LPS-induced inflammatory cytokine expression.


Subject(s)
Interleukin-1/antagonists & inhibitors , Interleukin-1/biosynthesis , Lipopolysaccharides/pharmacology , Porphyromonas gingivalis/immunology , Sweetening Agents/pharmacology , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/biosynthesis , Xylitol/pharmacology , Animals , Cariostatic Agents/pharmacology , Cell Line , Humans , Interleukin-1/genetics , Lipopolysaccharides/antagonists & inhibitors , Mice , NF-kappa B/metabolism , Porphyromonas gingivalis/drug effects , Porphyromonas gingivalis/growth & development , Tumor Necrosis Factor-alpha/genetics
16.
Mol Pharm ; 2(1): 22-8, 2005.
Article in English | MEDLINE | ID: mdl-15804174

ABSTRACT

A novel and improved vaccine delivery system and/or adjuvant is actively sought to enhance the potency of vaccines. Previously, we reported that strong antitumor immunity could be generated when a peptide antigen was incorporated into LPD (cationic liposome-polycation-pDNA) nanoparticles. In this study, we found that both the cationic liposome and DNA are required for the full immunostimulation activity of LPD. The unique ability of LPD to readily move into local lymphoid tissues and to activate antigen-presenting cells might be responsible for its strong immunostimulatory activity. Moreover, cationic liposome stimulates the expression of CD80/CD86 on dendritic cells (DCs), but not the release of TNF-alpha from DCs, suggesting the existence of a NF-kappaB-independent immunostimulation pathway for cationic lipids such as DOTAP.


Subject(s)
Immunization , Liposomes/administration & dosage , Nanostructures , Protamines/administration & dosage , Vaccines, DNA/administration & dosage , Animals , Antigen-Presenting Cells/immunology , Antigens, CD/metabolism , B7-1 Antigen/immunology , B7-2 Antigen , Cell Line , Cholesterol/administration & dosage , Cytotoxicity Tests, Immunologic , Dendritic Cells/cytology , Dendritic Cells/immunology , Drug Combinations , Fatty Acids, Monounsaturated/administration & dosage , Female , Interferon-gamma/metabolism , Luciferases/metabolism , Lymph Nodes/immunology , Lymphoid Tissue/immunology , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred C57BL , Oxazoles/administration & dosage , Plasmids , Pyrimidinones/administration & dosage , Quaternary Ammonium Compounds/administration & dosage , Spleen/cytology , Spleen/immunology , Tumor Necrosis Factor-alpha/metabolism
17.
Pharm Res ; 21(6): 1018-25, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15212168

ABSTRACT

PURPOSE: Previously, our laboratory has reported that liposome-protamine-DNA (LPD) nanoparticle is an effective delivery system for tumor-associated antigens. Mannan, which potentially targets antigen-presenting cells, was coated on LPD to further enhance its antitumor activity. METHODS: Cholesterol-conjugated mannan was coated on LPD. The abilities of mannan-coated LPD to target antigen-presenting cells, to activate dendritic cells, and to induce antitumor immunity were investigated and compared to those of LPD alone. RESULTS: Both in vitro and in vivo uptake of LPD showed that mannan-coated LPD particles were preferably taken up by dendritic cells and macrophages. In addition, the expression of co-stimulatory molecules CD80/CD86 on DC2.4 cells after co-incubation with mannan-coated LPD was significantly higher than that after co-incubation with LPD. A model major histocompatibility complex class I-restricted peptide antigen from HPV 16 E7 protein was incorporated into LPD to immunize mice against the growth of TC-1 tumor cells expressing E7 protein. Coating with mannan significantly enhanced both preventive and therapeutic activities of LPD/E7. Finally, the release of IFN-gamma from isolated splenocytes was significantly enhanced when mice were immunized with mannan-coated LPD/E7 than with LPD/E7 alone. CONCLUSION: Targeting of the LPD/E7 to local draining lymph nodes by mannan is partially responsible for the enhanced anti-tumor activity.


Subject(s)
DNA/metabolism , Liposomes/metabolism , Mannans/metabolism , Oncogene Proteins, Viral/chemistry , Oncogene Proteins, Viral/immunology , Protamines/metabolism , Animals , Antigens, CD/immunology , Antigens, CD/metabolism , B7-1 Antigen/immunology , B7-1 Antigen/metabolism , B7-2 Antigen , Cells, Cultured , Cholesterol/chemical synthesis , Cholesterol/immunology , Cholesterol/metabolism , DNA/chemistry , Dendritic Cells/cytology , Dendritic Cells/drug effects , Dendritic Cells/immunology , Disease Models, Animal , Gene Expression/drug effects , Gene Transfer Techniques , Humans , Liposomes/chemical synthesis , Liposomes/immunology , Mannans/chemistry , Mannans/immunology , Membrane Glycoproteins/immunology , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred Strains , Nanotechnology , Papillomavirus E7 Proteins , Particle Size , Protamines/chemical synthesis , Protamines/immunology , Protein Binding
18.
J Biol Chem ; 277(47): 44715-21, 2002 Nov 22.
Article in English | MEDLINE | ID: mdl-12235132

ABSTRACT

The nuclear factor-kappaB (NF-kappaB) is an important transcription factor necessary for initiating and sustaining inflammatory and immune reactions. The inducers of NF-kappaB are well characterized, but the molecular mechanisms underlying multiple in vivo NF-kappaB activation processes are poorly understood. The injection of lipopolysaccharide resulted in a biphasic activation of NF-kappaB during the 18-h observation period in various organs of mice. The early and late phases of NF-kappaB activation occurred at 0.5-2 h and 8-12 h, respectively. Platelet-activating factor, which is released in response to lipopolysaccharide injection, was responsible for the activation of the early phase of NF-kappaB. The early NF-kappaB activity led to the induction of proinflammatory cytokines, tumor necrosis factor (TNF), and interleukin (IL)-1beta, which are known to be efficient inducers of NF-kappaB. Using the TNF knockout and IL-1 receptor knockout mice, we found that TNF and IL-1beta had a role in the second phase activation of NF-kappaB. These cytokines did promote the synthesis of platelet-activating factor, which in turn induced the secondary activation of NF-kappaB. These observations describe a novel autoregulatory molecular mechanism for the biphasic activation of NF-kappaB.


Subject(s)
Lipopolysaccharides/pharmacology , NF-kappa B/metabolism , Platelet Activating Factor/metabolism , Animals , Azepines/pharmacology , Cell Line , Female , Interleukin-1/genetics , Interleukin-1/metabolism , Macrophages, Peritoneal/drug effects , Macrophages, Peritoneal/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Platelet Activating Factor/antagonists & inhibitors , Pyridinium Compounds/pharmacology , Receptors, Interleukin-1/genetics , Receptors, Interleukin-1/metabolism , Signal Transduction/physiology , Time Factors , Triazoles/pharmacology , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism
19.
Cancer Res ; 62(6): 1809-14, 2002 Mar 15.
Article in English | MEDLINE | ID: mdl-11912159

ABSTRACT

This study investigated the mechanisms of platelet-activating factor (PAF)-induced angiogenesis in a mouse model of Matrigel implantation. PAF induced a dose- and time-dependent angiogenic response. Inhibitors of nuclear factor (NF) kappaB expression or action, including antisense oligonucleotides to the p65 subunit of NFkappaB (p65 antisense) and antioxidants such as alpha-tocopherol and N-acetyl-L-cysteine, significantly reduced PAF-induced angiogenesis. In human umbilical vein endothelial cells, PAF-induced mRNA expression and protein synthesis of various NFkappaB-dependent angiogenic factors, such as tumor necrosis factor-alpha, interleukin-1alpha, basic fibroblast growth factor, and vascular endothelial growth factor (VEGF). The PAF-induced expression of the above mentioned factors was inhibited by p65 antisense or antioxidants. A significant inhibition of the angiogenic effect of PAF was achieved by anti-VEGF antibodies or soluble VEGF receptors such as KDR and flt-1 but not by antibodies against tumor necrosis factor-alpha, interleukin-1alpha, or basic fibroblast growth factor. These data indicate that PAF enhances angiogenesis through inducing NFkappaB activation, which in turn promotes the production of angiogenic factors such as VEGF.


Subject(s)
NF-kappa B/physiology , Neovascularization, Physiologic/physiology , Platelet Activating Factor/physiology , Animals , Antioxidants/pharmacology , Endothelial Growth Factors/biosynthesis , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Endothelium, Vascular/physiology , Female , Growth Substances/biosynthesis , Humans , Lymphokines/biosynthesis , Mice , Mice, Inbred BALB C , NF-kappa B/antagonists & inhibitors , NF-kappa B/genetics , Neovascularization, Physiologic/drug effects , Oligonucleotides, Antisense/pharmacology , Platelet Activating Factor/pharmacology , Recombinant Proteins/pharmacology , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors
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