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1.
J Biotechnol ; 157(1): 100-6, 2012 Jan.
Article in English | MEDLINE | ID: mdl-21893113

ABSTRACT

Aqueous extracts of Centella asiatica L. Urban were encapsulated by an edible biopolymer, gelatin, which has no effect on their cosmetic activities. The nanoparticles were w/o-type spherical liposomes that had an average diameter of 115.0nm. The encapsulation efficiency was estimated to be approximately 67%, which was relatively high for these aqueous extracts. The nanoparticles showed lower cytotoxicity (10%) in human skin fibroblast cells than the unencapsulated crude extract (15%) at 1.0mg/ml, this was possibly because a smaller amount of the extract was present in the nanoparticles. The nanoparticles efficiently reduced the expression of matrix metalloproteinase (MMP)-1 in UV-irradiated cells from 136.1% to 77.6% (UV-irradiated control) and inhibited hyaluronidase expression (>60%) at a concentration of 0.5mg/ml, which was higher than the levels produced by the unencapsulated crude extracts. The nanoparticles had a very high flux through mouse skin and also remained at relatively large concentrations in the derma when compared to the unencapsulated crude extracts. These results clearly indicate that the skin-protective activities of C. asiatica were significantly improved through the nano-encapsulation process. These findings also imply that a crude extract can be used and have the same efficacy as purified compounds, which should reduce the purification process and production costs.


Subject(s)
Centella/chemistry , Nanocapsules/chemistry , Plant Extracts/pharmacology , Protective Agents/chemistry , Analysis of Variance , Animals , Biopolymers/chemistry , Cell Line , Cell Survival/drug effects , Chickens , Cosmetics , Female , Fibroblasts , Gelatin/chemistry , Humans , Hyaluronoglucosaminidase/metabolism , Matrix Metalloproteinase 1/metabolism , Mice , Mice, Hairless , Particle Size , Plant Extracts/chemistry , Plant Extracts/pharmacokinetics , Protective Agents/pharmacokinetics , Protective Agents/pharmacology , Skin/drug effects , Skin/metabolism
2.
Cytotechnology ; 62(5): 461-72, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20936343

ABSTRACT

This work was to investigate the effect of flavonoids from Angelica gigas Nakai on the proliferation and differentiation of PC12 cells. Several solvents including hexane, chloroform, ethyl acetate, butanol and water consecutively partitioned. We determined the ethanol crude extract of Angelica gigas Nakai. The hexane fraction was shown to contain the highest number of flavonoids as follows; 21.48 mg/g and the composition of the flavonoids was as follows: 12.24 mg/g of quercetin, 4.39 mg/g of myricetin and 2.58 mg/g of catechin. In addition, this hexane fraction greatly increased both cell growth and outgrowth of the neurite, and whose effects were three times higher than those of the other fractions. The length of the neurites was measured as ca. 110 µm in adding 50 µg/mL of the hexane fraction, which was about the same as the case of adding 50 ng/mL of NGF as a positive control. This result indicates that the differentiation of PC12 cells by the addition of the hexane fraction was comparable to the case of adding NGF. The hexane fraction was also determined to prevent apoptosis of PC12 cells by suppressing DNA fragmentation. It is interesting that the mixture of three major flavonoids, quercetin, myricetin and catechin showed stronger activity on, both PC12 cell growth and neuritis outgrowth, than when adding each flavonoid alone. We believe this was due to the synergistic effects of the three flavonoids. The activities of these flavonoids from Angelica gigas Nakai are reported for the first time in this study.

3.
J Biol Chem ; 280(25): 23584-92, 2005 Jun 24.
Article in English | MEDLINE | ID: mdl-15849182

ABSTRACT

The binding of plasminogen activators and plasminogen to the cell surface results in the rapid generation of the serine protease plasmin. Plasmin is further degraded by an autoproteolytic reaction, resulting in the release of an angiostatin, A61 (Lys78-Lys468). Previously, we demonstrated that the annexin A2-S100A10 heterotetramer (AIIt) stimulates the release of A61 from plasmin by promoting the autoproteolytic cleavage of the Lys468-Gly469 bond and reduction of the plasmin Cys462-Cys541 disulfide (Kwon, M., Caplan, J. F., Filipenko, N. R., Choi, K. S., Fitzpatrick, S. L., Zhang, L., and Waisman, D. M. (2002) J. Biol. Chem. 277, 10903-10911). Mechanistically, it was unclear if AIIt promoted a conformational change in plasmin, resulting in contortion of the plasmin disulfide, or directly reduced the plasmin disulfide. In the present study, we show that AIIt thiols are oxidized during the reduction of plasmin disulfides, establishing that AIIt directly participates in the reduction reaction. Incubation of HT1080 cells with plasminogen resulted in the rapid loss of thiol-specific labeling of AIIt by 3-(N-maleimidopropionyl)biocytin. The plasminogen-dependent oxidation of AIIt could be attenuated by thioredoxin. Thioredoxin reductase catalyzed the transfer of electrons from NADPH to the oxidized thioredoxin, thus completing the flow of electrons from NADPH to AIIt. Therefore, we identify AIIt as a substrate of the thioredoxin system and propose a new model for the role of AIIt in the redox-dependent processing of plasminogen and generation of an angiostatin at the cell surface.


Subject(s)
Annexin A2/metabolism , S100 Proteins/metabolism , Thioredoxins/metabolism , Biopolymers , Blotting, Western , Cell Line , Culture Media, Conditioned , Electrophoresis, Polyacrylamide Gel , Humans , Oxidation-Reduction , Plasminogen/metabolism , Substrate Specificity
4.
Int J Exp Pathol ; 85(4): 201-12, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15312125

ABSTRACT

Summary Diffuse distribution of small, faintly staining, beaded deposits of rat immunoglobulin M (IgM) around the glomerular capillary blood vessels, and a more intensely staining larger deposition in the mesangium, were observed on the kidney sections of normal rats. As glomerular-fixed nephritogenic antigens are known to be present on the epithelial aspect of the glomerular basement membrane (GBM), especially at the soles of foot processes and at the slit pores, it was assumed that the IgM antibodies were directed against these antigens. Investigation by immunofluorescent antibody double-staining techniques of rat kidney sections obtained from normal and rabbit anti-FX1A-injected rats stained for the nephritogenic antigen showed that a number of antigenic sites in the glomeruli and in the mesangium shared antibody hits by heterologous rabbit IgG and autologous rat IgM antibodies. Most sites in the glomeruli stained specifically for rat IgM or rabbit IgG, but preferentially for the latter. The intensely fluorescent mesangial deposits stained mainly for rat IgM, indicating that at these sites the antigenic material was virtually saturated, while areas at the entry to the mesangial space also stained for rabbit IgG, indicating that at these locations free nephritogenic epitopes were still available for reaction with the anti-FX1A antibody. Western blot analysis have shown that the rabbit anti-rat FX1A IgG and the rat anti-rat KF3 IgM antibodies are directed against the same renal tubular-derived antigen with a molecular weight of 70,000. These experimental findings collectively demonstrate that the heterologous IgG and autologous IgM antibodies are directed against the same nephritogenic antigen, which is found in the glomeruli, the mesangium and the proximal convoluted tubules. Thus, the IgM autoantibody has a possible physiological role but, in addition, there is evidence of active immunophagocytic events, manifested in a rapid and continuous entrapment and expulsion of macromolecules after their processing by the mesangial cells of normal and passive Heymann nephritis rats.


Subject(s)
Autoantibodies/analysis , Autoimmune Diseases/immunology , Glomerulonephritis/immunology , Immunoglobulin M/analysis , Kidney Glomerulus/blood supply , Animals , Autoantigens/analysis , Capillaries/immunology , Fluorescent Antibody Technique/methods , Glomerular Mesangium/immunology , Kidney Glomerulus/immunology , Male , Rats , Rats, Sprague-Dawley
5.
J Biol Chem ; 279(10): 8723-31, 2004 Mar 05.
Article in English | MEDLINE | ID: mdl-14672933

ABSTRACT

Annexin A2 (ANXA2) is a Ca(2+)-binding protein that is up-regulated in virally transformed cell lines and in human tumors. Here, we show that ANXA2 binds directly to both ribonucleotide homopolymers and human c-myc RNA. ANXA2 was shown to bind specifically to poly(G) with high affinity (K(d) = 60 nM) and not to poly(A), poly(C), or poly(U). The binding of ANXA2 to poly(G) required Ca(2+) (A(50%) = 10 microM). The presence of RNA in the immunoprecipitates of ANXA2 isolated from HeLa cells established that ANXA2 formed a ribonucleoprotein complex in vivo. Sucrose gradient analysis showed that ANXA2 associates with ribonucleoprotein complexes and not with polyribosomes. Reverse transcriptase-PCR identified c-myc mRNA as a component of the ribonucleoprotein complex formed by ANXA2 in vivo, and binding studies confirmed a direct interaction between ANXA2 and c-myc mRNA. Transfection of LNCaP cells with the ANXA2 gene resulted in the up-regulation of c-Myc protein. These findings identify ANXA2 as a Ca(2+)-dependent RNA-binding protein that interacts with the mRNA of the nuclear oncogene, c-myc.


Subject(s)
Annexin A2/analysis , RNA-Binding Proteins/analysis , Annexin A2/metabolism , Binding Sites , Cell Line , Humans , Protein Binding , Proto-Oncogene Proteins c-myc/biosynthesis , Proto-Oncogene Proteins c-myc/genetics , RNA/genetics , RNA/metabolism , RNA-Binding Proteins/metabolism , Up-Regulation
6.
FASEB J ; 17(2): 235-46, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12554702

ABSTRACT

The defining characteristic of a tumor cell is its ability to escape the constraints imposed by neighboring cells, invade the surrounding tissue, and metastasize to distant sites. This invasive property of tumor cells is dependent on activation of proteases at the cell surface. Most cancer cells secrete the urokinase-type plasminogen activator, which converts cell-bound plasminogen to plasmin. Here we address the issue of whether the plasminogen binding protein, p11, plays a significant role in this process. Transfection of human HT1080 fibrosarcoma cells with the human p11 gene in the antisense orientation resulted in a loss of p11 protein from the cell surface and concomitant decreases in cellular plasmin production, ECM degradation, and cellular invasiveness. The transfected cells demonstrated reduced development of lung metastatic foci in SCID mice. In contrast, HT1080 cells transfected with the p11 gene in the sense orientation displayed increased cell surface p11 protein and concomitant increases in cellular plasmin production, as well as enhanced ECM degradation and enhanced cellular invasiveness. The p11 overexpressing cells showed enhanced development of lung metastatic foci. These data establish that changes in the extracellular expression of the plasminogen receptor protein, p11, dramatically affect tumor cell-mediated pericellular proteolysis.


Subject(s)
Annexin A2 , Calcium-Binding Proteins/physiology , Fibrinolysin/biosynthesis , Fibrosarcoma/metabolism , S100 Proteins , Animals , Calcium-Binding Proteins/genetics , Cell Line , Cell Movement/drug effects , Extracellular Space/metabolism , Fibrosarcoma/pathology , Genetic Vectors/genetics , Humans , Microscopy, Fluorescence , Neoplasm Invasiveness , Plasminogen Activators/metabolism , Retroviridae/genetics , Transfection , Tumor Cells, Cultured/drug effects
7.
Cancer Res ; 63(2): 348-53, 2003 Jan 15.
Article in English | MEDLINE | ID: mdl-12543787

ABSTRACT

The human reovirus is an oncolytic virus that specifically targets cancer cells with an activated Ras pathway. Because it is replication competent and highly specific for cancer cells, this virus has the potential to be an effective antimetastatic cancer agent through remote site delivery. In this study, we exploited the ability of reovirus to replicate in murine cells to test the efficacy of this virus in eliminating distal and/or metastatic tumors in immune-competent mice. We found that i.v. therapy with reovirus not only inhibited metastatic tumor growth but also led to a significant improvement in animal survival. Combining i.v. reovirus treatment with immune suppression (cyclosporine A or anti-CD4/anti-CD8 antibodies) resulted in further reduction in tumor size and a considerable prolongation in survival, compared with viral therapy alone. Combined therapy was also effective in overcoming a preexisting immunity to reovirus (a common occurrence in humans and thus a potential impediment to oncolytic effectiveness) to induce metastatic tumor regression. This is the first study to use systemic delivery of an oncolytic agent in conjunction with immune-suppressive drugs to effectively prolong animal survival. Altogether, our results suggest that i.v. reovirus therapy may present a feasible, novel alternative in the treatment of metastatic cancer in humans.


Subject(s)
Neoplasms, Experimental/therapy , Neoplasms, Experimental/virology , Retroviridae/physiology , Animals , Carcinoma, Lewis Lung/therapy , Carcinoma, Lewis Lung/virology , Combined Modality Therapy , Cyclosporine/pharmacology , Cytopathogenic Effect, Viral , Disease Models, Animal , Female , Immunosuppressive Agents/pharmacology , Lung Neoplasms/secondary , Lung Neoplasms/therapy , Lung Neoplasms/virology , Mammary Neoplasms, Experimental/therapy , Mammary Neoplasms, Experimental/virology , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Retroviridae/immunology
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