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2.
Cancer Gene Ther ; 19(8): 553-7, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22653386

ABSTRACT

Chromosome 7 open reading frame 24 (C7orf24), which was identified by proteome analysis, is upregulated in various types of cancer and is associated with cellular proliferation. However, in vivo antitumor effect by knockdown of C7orf24 has not been clarified. In this study, we investigated that the antitumor effect of anti-C7orf24 small interfering RNA (siRNA) administered by needle-free jet injection (JI) on lung cancer-bearing mice. Transfection of anti-C7orf24 siRNA induced cytotoxicity in cultured human lung cancer cells through specific knockdown of C7orf24. Furthermore, JI could effectively deliver anti-C7orf24 siRNA to tumor tissues, and as a result tumor growth was significantly inhibited. Immunohistochemical analysis revealed that C7orf24 levels were significantly reduced within tumor tissues collected from anti-C7orf24 siRNA-administered mice, indicating that the knockdown of C7orf24 induced cytotoxicity in tumor tissue. In conclusion, these data show for the first time that knockdown of C7orf24 prevents tumor growth in vivo following JI-mediated the siRNA delivery.


Subject(s)
Carcinoma, Squamous Cell , Genetic Therapy , Lung Neoplasms , RNA, Small Interfering , Animals , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/therapy , Cell Line, Tumor , Chromosomes, Human, Pair 7/genetics , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Humans , Injections, Jet , Lung Neoplasms/genetics , Lung Neoplasms/therapy , Mice , Neoplasm Proteins/genetics , Open Reading Frames/genetics , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/genetics , gamma-Glutamylcyclotransferase/genetics
3.
Int J Pharm ; 383(1-2): 157-60, 2010 Jan 04.
Article in English | MEDLINE | ID: mdl-19732811

ABSTRACT

Topical application of siRNA to the skin should be an effective treatment for serious skin disorders, such as atopic dermatitis. However, it is difficult to introduce hydrophilic macromolecules, including siRNA, into the skin by conventional methods. For efficient delivery of siRNA, we examined an iontophoretic technique, since it is suitable for the delivery of charged molecules. Naked siRNA effectively accumulated in the epidermis (and not in the dermis) after iontophoretic delivery. In contrast, siRNA did not penetrate tape-stripped skin by passive diffusion. In a rat model of atopic dermatitis, skin was sensitized with ovalbumin to stimulate IL-10 mRNA expression as observed in skin lesions. Iontophoretic delivery of anti-IL-10 siRNA significantly reduced (73%) the level of IL-10 mRNA. In conclusion, we successfully delivered naked siRNA into the epidermis and concomitantly suppressed the expression of an endogenous immuno-regulatory cytokine.


Subject(s)
Dermatitis, Atopic/metabolism , Disease Models, Animal , Epidermis/metabolism , Gene Transfer Techniques , Iontophoresis/methods , RNA, Small Interfering/administration & dosage , Administration, Cutaneous , Animals , Dermatitis, Atopic/genetics , Dermatitis, Atopic/therapy , Epidermis/drug effects , Male , Ovalbumin/administration & dosage , RNA, Small Interfering/pharmacology , Rats , Rats, Inbred BN , Skin Absorption/drug effects , Skin Absorption/genetics
4.
Clin Exp Immunol ; 154(3): 384-90, 2008 Dec.
Article in English | MEDLINE | ID: mdl-19037921

ABSTRACT

In this study, we examined the role of nitric oxide (NO) in controlling vascular integrity mediated by vascular endothelial (VE)-cadherin in chronic inflammation. Periapical granulomas were analysed for the expression of inducible NO synthase (iNOS) and VE-cadherin, and more iNOS expression than VE-cadherin was shown. Human umbilical vein endothelial cells (HUVECs) were stimulated with proinflammatory cytokines and lipopolysaccharide extracted from Porphyromonas gingivalis and it induced iNOS expression, whereas it reduced VE-cadherin expression, compared with negative controls. On the other hand, pre-incubation with 1400W, an iNOS-specific inhibitor, markedly reduced iNOS expression in stimulated HUVECs and restored VE-cadherin expression to its control level, suggesting that vascular integrity was modulated in conjunction with the reduction of NO. Immunocytochemistry confirmed the functional role of NO in cultured HUVEC monolayers with or without 1400W. These data are consistent with a hypothesis suggesting that NO could attenuate VE-cadherin-mediated vascular integrity in human chronic inflammation.


Subject(s)
Antigens, CD/metabolism , Cadherins/metabolism , Endothelium, Vascular/metabolism , Nitric Oxide/physiology , Periapical Granuloma/metabolism , Adult , Aged , Antigens, CD/genetics , Cadherins/genetics , Cells, Cultured , Chronic Disease , Cytokines/immunology , Endothelium, Vascular/immunology , Endothelium, Vascular/pathology , Gene Expression Regulation/immunology , Humans , Lipopolysaccharides/immunology , Middle Aged , Nitric Oxide Synthase Type II/genetics , Nitric Oxide Synthase Type II/metabolism , Periapical Granuloma/immunology , Periapical Granuloma/pathology , RNA, Messenger/genetics , Umbilical Veins/cytology , Umbilical Veins/metabolism , Young Adult
5.
Trends Biotechnol ; 26(12): 668-73, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18976825

ABSTRACT

Biodiesel fuel (BDF), which refers to fatty acid alkyl esters, has attracted considerable attention as an environmentally friendly alternative fuel for diesel engines. Alkali catalysis is widely applied for the commercial production of BDF. However, enzymatic transesterification offers considerable advantages, including reducing process operations in biodiesel fuel production and an easy separation of the glycerol byproduct. The high cost of the lipase enzyme is the main obstacle for a commercially feasible enzymatic production of biodiesel fuels. To reduce enzyme associated process costs, the immobilization of fungal mycelium within biomass support particles (BSPs) as well as expression of the lipase enzyme on the surface of yeast cells has been developed to generate whole-cell biocatalysts for industrial applications.


Subject(s)
Bacterial Physiological Phenomena , Bioelectric Energy Sources/trends , Fatty Acids/biosynthesis , Fungi/physiology , Gasoline/microbiology , Genetic Enhancement/methods , Catalysis
6.
Int Endod J ; 41(5): 401-7, 2008 May.
Article in English | MEDLINE | ID: mdl-18298575

ABSTRACT

AIM: To determine whether endothelial cells (ECs) in periapical granulomas can express vascular endothelial (VE)-cadherin, CXCL8 and CXCL10 by examining with two-colour confocal laser scanning microscope. METHODOLOGY: Periapical lesions were surgically removed from patients with chronic periapical periodontitis (n = 20), and the paraffin-embedded sections were prepared after being fixed with cold acetone. The 7-mum-thick sections were stained with haematoxylin-eosin and then examined pathologically using a light microscope. The lesions diagnosed as periapical granulomas (17 specimens) were analysed further using immunofluorescence and antibodies specific for human VE-cadherin, CXCL8, and CXCL10. The slides were carefully examined using a confocal laser scanning microscope. The numbers of positive ECs were counted, and the comparison between VE-cadherin-positive ECs and CXCL8 or CXCL10 was assessed statistically using one-way ANOVA followed by a Student-Newman-Keuls test. RESULTS: The expression of CXCL8 and CXCL10 by ECs was detected in 60.4 +/- 13.4 and 67.2 +/- 13.9%, respectively. However, the percentage of VE-cadherin-expressing ECs was 40.4 +/- 10.5%, which was significantly lower (P < 0.01) than CXCL8 and CXCL10-expressing ECs. Two-colour immunofluorescence staining revealed that ECs co-expressed VE-cadherin and CXCL8 (37.4 +/- 14.1%) or CXCL10 (39.1 +/- 13.8%). CONCLUSIONS: VE-cadherin expression in ECs was lower than CXCL8 and CXCL10, suggesting that inflamed ECs in periapical granulomas could increase vascular permeability and that leukocyte chemotaxis mediated by ECs might occur. These findings may suggest the possibility that ECs could play a pivotal role in cell recruitment in periapical granulomas.


Subject(s)
Antigens, CD/biosynthesis , Cadherins/biosynthesis , Chemokine CXCL10/biosynthesis , Interleukin-8/biosynthesis , Periapical Granuloma/metabolism , Periapical Granuloma/pathology , Adult , Aged , Chemotaxis, Leukocyte , Endothelial Cells/metabolism , Female , Fluorescent Antibody Technique , Humans , Male , Microscopy, Confocal/methods , Middle Aged
7.
Br J Cancer ; 98(2): 345-55, 2008 Jan 29.
Article in English | MEDLINE | ID: mdl-18195712

ABSTRACT

Glioblastoma is characterised by invasive growth and a high degree of radioresistance. Survivin, a regulator of chromosome segregation, is highly expressed and known to induce radioresistance in human gliomas. In this study, we examined the effect of survivin suppression on radiosensitivity in malignant glioma cells, while focusing on centrosome aberration and chromosome instability (CIN). We suppressed survivin by small interfering RNA transfection, and examined the radiosensitivity using a clonogenic assay and a trypan blue exclusion assay in U251MG (p53 mutant) and D54MG (p53 wild type) cells. To assess the CIN status, we determined the number of centrosomes using an immunofluorescence analysis, and the centromeric copy number by fluorescence in situ hybridisation. As a result, the radiosensitisation differed regarding the p53 status as U251MG cells quickly developed extreme centrosome amplification (=CIN) and enhanced the radiosensitivity, while centrosome amplification and radiosensitivity increased more gradually in D54MG cells. TUNEL assay showed that survivin inhibition did not lead to apoptosis after irradiation. This cell death was accompanied by an increased degree of aneuploidy, suggesting mitotic cell death. Therefore, survivin inhibition may be an attractive therapeutic target to overcome the radioresistance while, in addition, proper attention to CIN (centrosome number) is considered important for improving radiosensitivity in human glioma.


Subject(s)
Centrosome/drug effects , Chromosomal Instability/drug effects , Glioma/pathology , Microtubule-Associated Proteins/antagonists & inhibitors , Neoplasm Proteins/antagonists & inhibitors , RNA, Small Interfering/pharmacology , Radiation Tolerance/drug effects , Apoptosis/drug effects , Cell Cycle/drug effects , Cell Cycle/radiation effects , Cell Death/drug effects , Cell Death/radiation effects , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/radiation effects , Down-Regulation/drug effects , Glioma/metabolism , Humans , Inhibitor of Apoptosis Proteins , Microtubule-Associated Proteins/metabolism , Neoplasm Proteins/metabolism , Radiation-Sensitizing Agents/pharmacology , Survivin , Transfection
8.
Gene Ther ; 14(8): 682-9, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17268535

ABSTRACT

This study describes a multifunctional envelope-type nano device (MEND) that mimics an envelope-type virus based on a novel packaging strategy. MEND particles contain a DNA core packaged into a lipid envelope modified with an octaarginine peptide. The peptide mediates internalization via macropinocytosis, which avoids lysosomal degradation. MEND-mediated transfection of a luciferase expression plasmid achieved comparable efficiency to adenovirus-mediated transfection, with lower associated cytotoxicity. Furthermore, topical application of MEND particles containing constitutively active bone morphogenetic protein (BMP) type IA receptor (caBmpr1a) gene had a significant impact on hair growth in vivo. These data demonstrate that MEND is a promising non-viral gene delivery system that may provide superior results to existing non-viral gene delivery technologies.


Subject(s)
Genetic Therapy/methods , Oligopeptides/genetics , Transfection/methods , Adenoviridae/genetics , Animals , Cell Line , Gene Expression , Genetic Engineering , Humans , Luciferases/analysis , Luciferases/genetics , Mice , Mice, Mutant Strains , Microscopy, Confocal , Nanoparticles , Skin/metabolism , Skin/virology , beta-Galactosidase/analysis , beta-Galactosidase/genetics
9.
Int Endod J ; 39(3): 179-84, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16507070

ABSTRACT

AIM: To clarify the mechanisms of inflammatory cell migration in human periapical granulomas by examining vascular endothelial (VE) cadherin and inducible nitric oxide synthase (iNOS)-producing cells. METHODOLOGY: Periapical tissues were obtained from patients during endodontic surgery and were divided into two portions. After fixing the tissues with acetone or 4% paraformaldehyde in phosphate-buffered saline, 5-microm-thick paraffin or cryostat sections were prepared, respectively. The paraffin sections of the inflamed tissues were evaluated histologically with haematoxylin-eosin stains. Cryostat sections of the tissue, diagnosed as periapical granulomas, were then examined by either immunohistochemistry using anti-human VE-cadherin or iNOS antibodies (Abs) for the characterization of infiltrating cells. In addition, co-localization of VE-cadherin and iNOS production was also analysed by two-colour immunofluorescence image analysis. RESULTS: Endothelial cells were strongly stained with iNOS Abs. Macrophages, lymphocytes, polymorphonuclear leucocytes and fibroblasts also exhibited iNOS production. These iNOS-positive cells accumulated around the blood vessels. On the other hand, VE-cadherin production was exhibited in only endothelial cells. Two-colour immunofluorescence image analysis using VE-cadherin and iNOS Abs demonstrated that iNOS-producing endothelial cells also showed VE-cadherin production. CONCLUSIONS: Vascular endothelial-cadherin produced by endothelial cells could be regulated by iNOS-producing cells in periapical granulomas and might play a pivotal role in vascular permeability.


Subject(s)
Cadherins/analysis , Endothelial Cells/metabolism , Nitric Oxide Synthase Type II/analysis , Periapical Granuloma/metabolism , Adult , Aged , Animals , Antigens, CD , Female , Goats , Humans , Male , Middle Aged , Periapical Granuloma/pathology , Rabbits
10.
J Immunol Methods ; 288(1-2): 123-34, 2004 May.
Article in English | MEDLINE | ID: mdl-15183091

ABSTRACT

This paper presents an improved method of isolating, culturing and cryopreserving human monocytes in large quantity with high purity using standard laboratory centrifuges. Monocytes were isolated from 300 to 360 ml of heparinized human blood using a Double Density technique employing Ficoll Isopaque and 46% iso-osmotic Percoll. Yields of monocytes ranged from 75 to 205 million (from 300 to 360 ml of blood) with an average purity of 90.6%. The ability of fresh or frozen monocytes to adhere to endothelial cells in the presence of oxidized L-alpha-1-palmitoyl-2-arachidonosyl-sn-glycero-3-phosphocholine (oxPAPC) or lipopolysaccharide (LPS) did not differ and no significant difference in response to the chemotactic stimulant N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) was observed. We define a useful method for the culture and differentiation of fresh or frozen monocytes isolated by this method, into macrophages as judged by morphology, expression of the macrophage marker SRA-1 and induction of inflammatory genes TNF-alpha, IL-6 and COX-2. Also, fresh or frozen Double Density isolated cells can be successfully differentiated into dendritic cells in the presence of GM-CSF and IL-4 as judged by the expression of the hallmark surface proteins CD1a and DC-sign and the absence of CD14. This method also yields a pure population of lymphocytes.


Subject(s)
Cell Differentiation/physiology , Chemotaxis/physiology , Clinical Laboratory Techniques/methods , Cryopreservation/methods , Monocytes/physiology , Cell Adhesion/physiology , Cell Culture Techniques/methods , Dendritic Cells/physiology , Humans , Macrophages/physiology
11.
Appl Microbiol Biotechnol ; 65(3): 301-5, 2004 Aug.
Article in English | MEDLINE | ID: mdl-15052375

ABSTRACT

In the present study, we used gene manipulation to construct a recombinant Aspergillus oryzae strain overexpressing lipase and investigated its application to the optical resolution of chiral compounds. A. oryzae niaD300, which was derived from the wild-type strain RIB40, was used as the host strain. The tglA gene, which encodes a triacylglycerol lipase, was cloned from the A. oryzae niaD300 chromosomal genome, then reintroduced, with and without a secretion-signal sequence, into the genome and expressed under the control of the improved glaA promoter of plasmid pNGA142. The resulting recombinant strain overexpressing A. oryzae lipase was immobilized within biomass-support particles and used as a whole-cell biocatalyst. The immobilized lipase-overexpressing strain with secretion-signal sequence showed high activity and was used to selectively synthesize (R)-1-phenylethyl acetate from (RS)-1-phenylethanol and vinyl acetate. After 48 h reaction at 30 degrees C with molecular sieve 4A, the yield and enantiomeric excess (%ee) of (R)-1-phenylethyl acetate reached approximately 90 and 95%ee, respectively. The whole-cell biocatalyst for optical resolution of chiral compounds produced in this study maintained its activity over 25 batch-reaction cycles.


Subject(s)
Aspergillus oryzae/enzymology , Lipase/metabolism , Phenylethyl Alcohol/metabolism , Recombinant Proteins/metabolism , Aspergillus oryzae/genetics , Cells, Immobilized/enzymology , Cells, Immobilized/metabolism , Esterification , Gene Expression Regulation, Fungal , Lipase/genetics , Stereoisomerism , Vinyl Compounds/metabolism
12.
Biochemistry (Mosc) ; 69(1): 50-7, 2004 Jan.
Article in English | MEDLINE | ID: mdl-14972018

ABSTRACT

Tocopheryl succinate (TS), a succinyl ester of alpha-tocopherol (alpha-T), has been reported to have various biological activities. In this communication, we review the current findings about TS including our recent studies of its effects on nitric oxide (NO) and superoxide (O2-) generations implicated in cancer and atherosclerosis. First, we investigated the effect of TS on NO production in vascular smooth muscle cells (VSMC) under atherosclerosis-like conditions using lipopolysaccharide (LPS) and interferon-gamma (IFN). TS enhanced LPS/IFN-dependent NO production, but alpha-T itself did not. The enhancement by TS of NO production was inhibited by alpha-T but not by antioxidants such as ascorbic acid and 2[3]-t-butyl-4-hydroxyanisole (BHA). TS enhanced the amount of protein kinase Calpha (PKCalpha) in VSMC, and PKC inhibitors inhibited TS-enhanced NO production, suggesting that the enhancing effect of TS on NO production is caused by up-regulation of PKC. Second, we found that TS induced apoptosis in VSMC associated with increase in O2- generation via NADPH-dependent oxidase. We further observed that a mouse breast cancer cell line C127I was more susceptible for TS-induced apoptosis than a mouse breast normal cell line NmuMG, and that superoxide dismutase, alpha-T, and BHA inhibited TS-caused morphological cell damage in C127I. From these results, O2- itself and/or other reactive oxygen species are assumed to associate with TS-induced cell toxicity, and antioxidative defense systems are supposed to be lowered in cancer cells. Finally, we found that intravenous injection of TS vesicles completely inhibited the growth of melanoma cells B16-F1 inoculated on the back of hairless mice and enhanced their survival time.


Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Nitric Oxide/metabolism , Superoxides/metabolism , Vitamin E/analogs & derivatives , Vitamin E/pharmacology , Animals , Antineoplastic Agents/chemistry , Humans , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/enzymology , Myocytes, Smooth Muscle/metabolism , Nitric Oxide/biosynthesis , Tocopherols , Vitamin E/chemistry
13.
Br J Cancer ; 89(9): 1802-11, 2003 Nov 03.
Article in English | MEDLINE | ID: mdl-14583787

ABSTRACT

It is well established that cells synchronised at the G1-S phase are highly radiosensitive. In this study, p16-null human glioma cell lines were induced into G1 cell cycle arrest by adenovirus-mediated p16 gene transfer, and examined for radiation-induced cell killing. Clonogenic analysis and trypan blue extraction test showed that the p16 gene transfer enhanced radiation-induced cell killing in p16-null glioma cell lines. TUNEL assays and pulse-field gel electrophoresis confirmed that the radiation-induced cell killing of p16-transfected cells could be caused by a nonapoptotic mechanism. Gimsa staining demonstrated that irradiation alone or Ax-mock infection plus irradiation results in a slight increase in the frequency of cells with abnormal nucleus, compared to unirradiated uninfected or Ax-mock infected cells. However, Ax-hp16 or Ax-hp21 infection alone modestly increased the frequency of cells with abnormal nucleus (especially bi- and multinucleation), and 4-Gy irradiation of Ax-hp16 or Ax-hp21 infected cells substantially enhanced this frequency. These results suggest that there exists some unknown interaction between radiation and p16 in cytoplasm/membranes, which decreases cytokinesis and promotes abnormal nucleation. Thus, p16 expression prevented radiation-induced apoptosis by promoting abnormal nucleation, thereby leading to another mode of cell death.


Subject(s)
Cell Nucleus/radiation effects , Gene Transfer Techniques , Genes, p16/radiation effects , Glioma/genetics , Glioma/pathology , Adenoviridae/genetics , Apoptosis/radiation effects , Cell Line, Tumor , Cell Nucleus/pathology , Electrophoresis, Gel, Pulsed-Field , Genetic Vectors , Humans , In Situ Nick-End Labeling , Radiation, Ionizing , Transfection
14.
Arterioscler Thromb Vasc Biol ; 21(11): 1770-6, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11701464

ABSTRACT

Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (ox-PAPC), a component of minimally modified low density lipoprotein, induces monocyte adhesion to endothelial cells. It is not known whether the upstroke slopes of pulsatile flow, defined as shear stress slew rates (tau(r)/tauT)), can regulate monocyte binding to ox-PAPC-treated bovine aortic endothelial cells (BAECs). At 60 cycles per minute, ox-PAPC-treated BAECs were exposed to 3 conditions representing known vascular conditions: (1) high shear stress slew rates (tau(r)/tau(T)=293 dyne. cm(-2). s(-1)), with time-averaged shear stress=50 dyne/cm(2); (2) low shear stress slew rate (tau(r)/tau(t)=71 dyne. cm(-2). s(-1)), with identical time-averaged shear stress; and (3) reversing oscillating flow (0+/-2.6 mm Hg). Reverse transcription-polymerase chain reaction and quantification were performed for monocyte chemoattractant protein-1 (MCP-1) mRNA expression. High tau(r)/tau(t) reduced monocyte binding to ox-PAPC-treated BAECs by 64+/-3.2% compared with static conditions, and low tau(r)/tau(t) reduced monocyte binding by 31+/-3.4%, whereas oscillating flow increased monocyte binding by 22+/-1.7% (P<0.005). High partial tau(r)/tau(t) downregulated MCP-1 expression by 33+/-8%, and low partial tau(r)/tau(t) downregulated MCP-1 expression by 15+/-4%, but oscillating flow upregulated MCP-1 by 13+/-5%. These results suggest that shear stress slew rates regulate monocyte binding by modulating the expression of a potent monocyte chemoattractant.


Subject(s)
Cell Adhesion , Endothelium, Vascular/physiology , Lipoproteins, LDL/pharmacology , Monocytes/physiology , Animals , Cattle , Cells, Cultured , Chemokine CCL2/biosynthesis , Chemokine CCL2/genetics , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Kinetics , Phosphatidylcholines/pharmacology , RNA, Messenger/biosynthesis , Stress, Mechanical
15.
Anticancer Res ; 21(4A): 2537-45, 2001.
Article in English | MEDLINE | ID: mdl-11724319

ABSTRACT

BACKGROUND: Deletions and point mutations of the p16 gene are detectable in more than 50% of ovarian cancer cells. In this study, we examined the effect of p16 gene transduction on the growth of ovarian cancer cells and on the effect of anti-cancer agents. MATERIALS AND METHODS: p16-null human ovarian cancer cell lines, SKOV-3 and OVCAR-5, were used in this study. We transduced the full-length human p16 gene using recombinant adenovirus (AxCA-hp16). RESULTS: The spontaneous growth of these cells was significantly inhibited by hp16 transduction. MTT assay revealed that AxCA-hp16 infection induced chemoresistance in both cell lines. Flow cytometric analysis revealed that only hp16 -transduced SKOV-3, were arrested at the G1-phase for 3 days whereas those infected with AxCA-mock and OVCAR-5 infected with both recombinant viruses did not. Western blot analysis showed increased microtubule-associated proteins 4 (MAP4) in both cell lines. CONCLUSION: These results suggest that in SKOV-3 cells, G1-arrest induced by p16-transduction prevents paclitaxel- and vindesine-induced cell death, and in OVCAR-5 cells, the other unknown mechanisms play a role of chemoresistance.


Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Genes, p16 , Ovarian Neoplasms/drug therapy , Paclitaxel/pharmacology , Transduction, Genetic , Vindesine/pharmacology , Adenoviridae/genetics , Cell Division/drug effects , Cell Division/genetics , Combined Modality Therapy , Cyclin-Dependent Kinase Inhibitor p16/biosynthesis , Cyclin-Dependent Kinase Inhibitor p16/genetics , Cyclin-Dependent Kinase Inhibitor p16/physiology , Dose-Response Relationship, Drug , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Female , Genetic Vectors/genetics , Humans , Microtubule-Associated Proteins/biosynthesis , Ovarian Neoplasms/genetics , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Tumor Cells, Cultured
16.
J Biol Chem ; 276(48): 44444-9, 2001 Nov 30.
Article in English | MEDLINE | ID: mdl-11579088

ABSTRACT

The oxidation of apolipoprotein B-containing lipoproteins and cell membrane lipids is believed to play an integral role in the development of fatty streak lesions, an initial step in atherogenesis. We have previously shown that two antioxidant-like enzymes, paraoxonase (PON)-1 and PON3, are high density lipoprotein-associated proteins capable of preventing the oxidative modification of low density lipoprotein (LDL) (Reddy, S. T., Wadleigh, D. J., Grijalva, V., Ng, C., Hama, S., Gangopadhyay, A., Shih, D. M., Lusis, A. J., Navab, M., and Fogelman, A. M. (2001) Arterioscler. Thromb. Vasc. Biol. 21, 542-547). In the present study, we demonstrate that PON2 (i) is not associated with high density lipoprotein; (ii) has antioxidant properties; and (iii) prevents LDL lipid peroxidation, reverses the oxidation of mildly oxidized LDL (MM-LDL), and inhibits the ability of MM-LDL to induce monocyte chemotaxis. The PON2 protein was overexpressed in HeLa cells using the tetracycline-inducible ("Tet-On") system, and its antioxidant capacity was measured in a fluorometric assay. Cells that overexpressed PON2 showed significantly less intracellular oxidative stress following treatment with hydrogen peroxide or oxidized phospholipid. Moreover, cells that overexpressed PON2 were also less effective in oxidizing and modifying LDL and, in fact, were able to reverse the effects of preformed MM-LDL. Our results suggest that PON2 possesses antioxidant properties similar to those of PON1 and PON3. However, in contrast to PON1 and PON3, PON2 may exert its antioxidant functions at the cellular level, joining the host of intracellular antioxidant enzymes that protect cells from oxidative stress.


Subject(s)
Antioxidants/pharmacology , Aryldialkylphosphatase , Esterases/biosynthesis , Esterases/physiology , Lipoproteins, LDL/metabolism , Arteries/metabolism , Blotting, Northern , Blotting, Western , Cells, Cultured , Chemotaxis , Cloning, Molecular , Contrast Media/pharmacology , Doxycycline/pharmacology , Endothelium, Vascular/metabolism , Fluorescein/pharmacology , HeLa Cells , Humans , Lipoproteins, HDL/metabolism , Oxidative Stress , Oxygen/metabolism , Phospholipids/metabolism , Spectrometry, Fluorescence , Time Factors , Tissue Distribution , Transfection
17.
Biochim Biophys Acta ; 1528(1): 25-30, 2001 Sep 03.
Article in English | MEDLINE | ID: mdl-11514094

ABSTRACT

We investigated the mechanism of cell toxicity of alpha-tocopheryl hemisuccinate (TS). TS concentration- and time-dependently induced the lactate dehydrogenase release and DNA fragmentation of rat vascular smooth muscle cells (VSMC). Exogenous addition of superoxide dismutase, but not catalase, significantly inhibited the cell toxicity of TS. The NADPH-dependent oxidase activity of VSMC was stimulated by TS treatment. The cell toxicity of TS was inhibited by NADPH oxidase inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride. Consequently, TS-induced apoptosis of VSMC was suggested to be caused by exogenous O(2)(-) generated via the oxidase system activated with TS.


Subject(s)
Apoptosis , Muscle, Smooth, Vascular/drug effects , Superoxides/metabolism , Vitamin E/analogs & derivatives , Vitamin E/toxicity , Animals , Ascorbic Acid , Catalase , Cells, Cultured , Cytochrome c Group/chemistry , DNA Fragmentation , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , L-Lactate Dehydrogenase/analysis , Muscle, Smooth, Vascular/enzymology , NADPH Oxidases/antagonists & inhibitors , NADPH Oxidases/metabolism , Oxygen/analysis , Rats , Superoxide Dismutase , Superoxides/analysis , Tocopherols , Vitamin E/antagonists & inhibitors , Vitamin E/chemistry
18.
J Lipid Res ; 42(8): 1308-17, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11483633

ABSTRACT

We have developed a novel and rapid cell-free assay of the ability of HDL to prevent the formation of or inactivate oxidized phospholipids. HDL was tested for its ability to inhibit the oxidation of LDL, or inhibit the oxidation of l-alpha-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC) by hydroperoxyoctadecadienoic acid (HPODE), or inactivate oxidized PAPC (Ox-PAPC). In each case the fluorescent signal generated in the presence of the test substances and the test HDL was determined. As little as 2.5 microg of normal human HDL cholesterol significantly inhibited the fluorescent signal generated by Ox-PAPC; results did not differ regardless of whether the HDL was prepared by gel electrophoresis, fast protein liquid chromatography, or dextran sulfate precipitation. HDL from each of 27 patients with coronary atherosclerosis failed to inhibit the fluorescent signal generated by a control LDL, whereas HDL from each of 31 matched normal subjects with the same levels of HDL cholesterol significantly inhibited the signal. Results from an established cell-based assay (Navab, M., S. Hama, J. Cooke, G. M. Anantharamaiah, M. Chaddha, L. Jin, G. Subbanagounder, K. F. Faull, S. T. Reddy, N. E. Miller, and A. M. Fogelman. 2000. J. Lipid Res. 41: 1481-1494) were identical. HDL from the patients also failed to inhibit the fluorescent signal generated from PAPC plus HPODE (10 of 10 patients) whereas HDL from matched controls (8 of 8 patients) significantly inhibited the fluorescent signal. We conclude that this new assay has the potential to allow widespread testing of the hypothesis that HDL that is dysfunctional in preventing the formation or inactivating oxidized phospholipids may play an important role in the development of atherosclerosis.


Subject(s)
Lipid Peroxides/antagonists & inhibitors , Lipoproteins, HDL/blood , Lipoproteins, HDL/pharmacology , Animals , Antioxidants/pharmacology , Aorta , Cell-Free System , Chemotaxis, Leukocyte/drug effects , Clusterin , Coculture Techniques , Coronary Artery Disease/blood , Endothelium, Vascular , Female , Fluoresceins , Fluorescent Dyes , Glycoproteins/pharmacology , Humans , Linoleic Acids/pharmacology , Lipid Peroxidation/drug effects , Lipoproteins, LDL/blood , Male , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Molecular Chaperones/pharmacology , Monocytes/physiology , Muscle, Smooth, Vascular , Oxidation-Reduction , Phospholipid Ethers/chemistry , Phospholipid Ethers/metabolism , Spectrometry, Fluorescence
19.
J Lipid Res ; 42(7): 1096-104, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11441137

ABSTRACT

We have recently shown that a class A amphipathic peptide 5F with increased amphipathicity protected mice from diet-induced atherosclerosis (Garber et al. J. Lipid Res. 2001. 42: 545-552). We have now examined the effects of increasing the hydrophobicity of a series of homologous class A amphipathic peptides, including 5F, on physical and functional properties related to atherosclerosis inhibition by systematically replacing existing nonpolar amino acids with phenylalanine. The peptides, based on the sequence Ac-D-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F-NH(2) (Ac-18A-NH(2) or 2F) were: 3F(3)(Ac-F(3)18A-NH(2)), 3F(14)(Ac-F(14)18A-NH(2)), 4F(Ac-F(3,14)18A-NH(2)), 5F(Ac-F(11,14,17) 18A-NH(2)), 6F(Ac-F(10,11,14,17)18A-NH(2)), and 7F(Ac-F(3,10,11,14,17) 18A-NH(2)). Measurements of aqueous solubility, HPLC retention time, exclusion pressure for penetration into an egg phosphatidylcholine (EPC) monolayer, and rates of EPC solubilization revealed an abrupt increase in the hydrophobicity between peptides 4F and 5F; this was accompanied by increased ability to associate with phospholipids. The peptides 6F and 7F were less effective, indicating a limit to increased hydrophobicity for promoting lipid interaction in these peptides. Despite this marked increase in lipid affinity, these peptides were less effective than apoA-I in activating the plasma enzyme, lecithin:cholesterol acyltransferase, with 5F activating LCAT the best (80% of apoA-I). Peptides 4F, 5F, and 6F were equally potent in inhibiting LDL-induced monocyte chemotactic activity. These studies suggest that an appropriate balance between peptide-peptide and peptide-lipid interactions is required for optimal biological activity of amphipathic peptides. These studies provide a rationale for the design of small apoA-I-mimetics with increased potency for atherosclerosis inhibition.


Subject(s)
Apolipoprotein A-I/pharmacology , Peptides/chemistry , Peptides/pharmacology , Phosphatidylcholine-Sterol O-Acyltransferase/drug effects , Phospholipids/chemistry , Animals , Apolipoprotein A-I/chemistry , Apolipoprotein A-I/metabolism , Binding Sites/physiology , Cells, Cultured , Chemotaxis/drug effects , Cholesterol/metabolism , Cholesterol, LDL/pharmacology , Circular Dichroism , Enzyme Activation/physiology , Humans , Intercellular Signaling Peptides and Proteins , Mice , Monocytes/physiology , Peptides/analysis , Peptides/metabolism , Phosphatidylcholine-Sterol O-Acyltransferase/metabolism , Phosphatidylcholines/chemistry , Phosphatidylcholines/metabolism , Phospholipids/metabolism , Solubility
20.
Circulation ; 103(18): 2283-8, 2001 May 08.
Article in English | MEDLINE | ID: mdl-11342478

ABSTRACT

BACKGROUND: Viruses have been identified as one of a variety of potential agents that are implicated in atherogenesis. METHODS AND RESULTS: C57BL/6J mice were killed before or 2, 3, 5, 7, or 9 days after intranasal infection with 10(5) plaque-forming units (pfu) of Influenza A strain WSN/33. Peak infectivity in lungs was reached by 72 hours, and it returned to baseline by 9 days. No viremia was observed at any time. The activities of paraoxonase and platelet-activating factor acetylhydrolase in HDL decreased after infection and reached their lowest levels 7 days after inoculation. The ability of HDL from infected mice to inhibit LDL oxidation and LDL-induced monocyte chemotactic activity in human artery wall cell cocultures decreased with time after inoculation. Moreover, as the infection progressed, LDL more readily induced monocyte chemotaxis. Peak interleukin-6 and serum amyloid A plasma levels were observed at 2 and 7 days after inoculation. HDL apoA-I levels did not change. ApoJ and ceruloplasmin levels in HDL peaked 3 days after infection. Ceruloplasmin remained elevated throughout the time course, whereas apoJ levels decreased toward baseline after the third day. CONCLUSIONS: We conclude that alterations in the relative levels of paraoxonase, platelet-activating factor acetylhydrolase, ceruloplasmin, and apoJ in HDL occur during acute influenza infection, causing HDL to lose its anti-inflammatory properties.


Subject(s)
Inflammation/blood , Inflammation/virology , Influenza, Human/blood , Lipoproteins, HDL/metabolism , 1-Alkyl-2-acetylglycerophosphocholine Esterase , Acute Disease , Acute-Phase Reaction/metabolism , Acute-Phase Reaction/virology , Animals , Apolipoproteins/blood , Arteries/cytology , Arteries/drug effects , Arteries/metabolism , Aryldialkylphosphatase , Cells, Cultured , Ceruloplasmin/analysis , Ceruloplasmin/metabolism , Chemotaxis/drug effects , Clusterin , Disease Models, Animal , Esterases/analysis , Esterases/metabolism , Female , Glycoproteins/analysis , Glycoproteins/metabolism , Humans , Influenza A virus/growth & development , Influenza A virus/isolation & purification , Influenza, Human/virology , Interleukin-6/blood , Lipoproteins, HDL/chemistry , Lipoproteins, HDL/pharmacology , Lipoproteins, LDL/blood , Lipoproteins, LDL/pharmacology , Macrophages/drug effects , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Molecular Chaperones/analysis , Molecular Chaperones/metabolism , Monocytes/drug effects , Monocytes/metabolism , Phospholipases A/analysis , Phospholipases A/metabolism , Serum Amyloid A Protein
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