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1.
ACS Appl Mater Interfaces ; 12(12): 13587-13597, 2020 Mar 25.
Article in English | MEDLINE | ID: mdl-32107914

ABSTRACT

Biomaterials for tissue engineering include natural and synthetic polymers, but their clinical application is still limited due to various disadvantages associated with the use of these polymers. This uncertainty of the polymeric approach in tissue engineering launches an opportunity to address a key question: can we eliminate the disadvantages of both natural and synthetic polymers by combining them to form a synergistic relationship? To answer this question, we fabricated scaffolds from elastin, collagen, fibrin, and electrospun polycaprolactone (PCL) with different ratios. The material characterization of these scaffolds investigated degradation, water contact angle, angiogenesis by an ex ovo chorion allantoic membrane (CAM) assay, and mechanical and structural properties. Biological activity and specific differentiation pathways (MSC, adipogenic, osteogenic, myogenic, and chondrogenic) were studied by using human adipose-derived stem cells. Results indicated that all composite polymers degraded at a different rate, thus affecting their mechanical integrity. Cell-based assays demonstrated continual proliferative and viable properties of the cells on all seeded scaffolds with the particular initiation of a differentiation pathway among which the PCL/collagen/fibrin composite was the most angiogenic material with maximum vasculature. We were able to tailor the physical and biological properties of PCL-based composites to form a synergistic relationship for various tissue regeneration applications.


Subject(s)
Cell Differentiation/drug effects , Mesenchymal Stem Cells/drug effects , Neovascularization, Physiologic/drug effects , Polymers/pharmacology , Tissue Scaffolds/chemistry , Allantois/drug effects , Allantois/growth & development , Animals , Biocompatible Materials/chemical synthesis , Biocompatible Materials/chemistry , Biocompatible Materials/pharmacology , Cell Proliferation/drug effects , Cell Survival/drug effects , Chick Embryo , Chorion/drug effects , Chorion/growth & development , Collagen/chemistry , Elastin/chemistry , Fibrin/chemistry , Humans , Mesenchymal Stem Cells/cytology , Polyesters/chemistry , Polyesters/pharmacology , Polymers/chemical synthesis , Polymers/chemistry , Tissue Engineering/methods
2.
J Vet Diagn Invest ; 31(4): 581-584, 2019 Jul.
Article in English | MEDLINE | ID: mdl-31122163

ABSTRACT

Sida carpinifolia is a small subshrub that is distributed throughout Brazil and is responsible for lysosomal storage disease and occasional reproductive problems in cattle, goats, equids, sheep, and deer. We describe herein the clinical, epidemiologic, and pathologic features of hydrallantois in 3 cows naturally poisoned by S. carpinifolia in Rio Grande do Sul State, Brazil. Clinically, all cows had marked abdominal distension and mild ataxia. After natural death or euthanasia, autopsies revealed that the abdominal distension in all 3 cases was caused by severe enlargement of the uterus, which contained 100-120 L of translucent fluid within the allantois, in addition to adventitial placentation. Microscopic evaluation of the placenta revealed marked diffuse edema, sometimes with a myxomatous appearance. Neurons in the cerebellum and obex were swollen, with mild-to-moderate cytoplasmic granular vacuolation. Histochemical examination with lectins ConA, WGA, and sWGA revealed mild-to-marked staining in the cytoplasm of neurons of the cerebellum and medulla at the level of the obex, indicating the occurrence of α-mannosidosis.


Subject(s)
Allantois/drug effects , Cattle Diseases/chemically induced , Malvaceae/toxicity , Plant Poisoning/veterinary , Allantois/pathology , Animals , Brain Diseases/pathology , Brain Diseases/veterinary , Brazil/epidemiology , Cattle , Cattle Diseases/epidemiology , Female
3.
Dev Dyn ; 237(10): 2918-25, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18816835

ABSTRACT

Embryonic mouse allantoic tissue (E8.5) was cultured in hanging drops to generate a three-dimensional vascular micro-tissue. The resulting tissue spheroids had an inner network of small diameter vessels expressing platelet endothelial cell adhesion molecule-1 (PECAM-1) and an outer layer of cells expressing SMalphaA, SM22-alpha, and SM-MHC. In a subsequent phase of culture, the fusion-promoting activity of vascular endothelial growth factor (VEGF) was used to transform the inner network of small diameter endothelial tubes into a contiguous layer of cells expressing PECAM-1, CD34, and VE-cadherin that circumscribed a central lumen-like cavity. The blood vessel-like character of the VEGF-treated spheroids was further demonstrated by their physiologically relevant vasodilatory and contractile responses, including contraction induced by KCl and relaxation stimulated by high-density lipoproteins and acetylcholine-induced nitric oxide production.


Subject(s)
Neovascularization, Physiologic/drug effects , Vascular Endothelial Growth Factor A/pharmacology , Actins/metabolism , Allantois/blood supply , Allantois/drug effects , Allantois/embryology , Allantois/metabolism , Animals , Endothelial Cells/metabolism , Histocompatibility Antigens/metabolism , Mice , Mice, Inbred ICR
4.
EMBO J ; 27(7): 993-1004, 2008 Apr 09.
Article in English | MEDLINE | ID: mdl-18337748

ABSTRACT

VE-cadherin is an endothelial-specific transmembrane protein concentrated at cell-to-cell adherens junctions. Besides promoting cell adhesion and controlling vascular permeability, VE-cadherin transfers intracellular signals that contribute to vascular stabilization. However, the molecular mechanism by which VE-cadherin regulates vascular homoeostasis is still poorly understood. Here, we report that VE-cadherin expression and junctional clustering are required for optimal transforming growth factor-beta (TGF-beta) signalling in endothelial cells (ECs). TGF-beta antiproliferative and antimigratory responses are increased in the presence of VE-cadherin. ECs lacking VE-cadherin are less responsive to TGF-beta/ALK1- and TGF-beta/ALK5-induced Smad phosphorylation and target gene transcription. VE-cadherin coimmunoprecipitates with all the components of the TGF-beta receptor complex, TbetaRII, ALK1, ALK5 and endoglin. Clustered VE-cadherin recruits TbetaRII and may promote TGF-beta signalling by enhancing TbetaRII/TbetaRI assembly into an active receptor complex. Taken together, our data indicate that VE-cadherin is a positive and EC-specific regulator of TGF-beta signalling. This suggests that reduction or inactivation of VE-cadherin may contribute to progression of diseases where TGF-beta signalling is impaired.


Subject(s)
Antigens, CD/metabolism , Cadherins/metabolism , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Signal Transduction/drug effects , Transforming Growth Factor beta/pharmacology , Activin Receptors, Type II/metabolism , Allantois/cytology , Allantois/drug effects , Allantois/metabolism , Animals , Cadherins/deficiency , Cell Movement/drug effects , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Cell Proliferation/drug effects , Dimerization , Embryo, Mammalian/cytology , Embryo, Mammalian/drug effects , Embryo, Mammalian/metabolism , Endothelial Cells/cytology , Humans , Kinetics , Mice , Models, Biological , Phosphorylation/drug effects , Protein Binding/drug effects , Protein Serine-Threonine Kinases/metabolism , Receptor, Transforming Growth Factor-beta Type I , Receptor, Transforming Growth Factor-beta Type II , Receptors, Transforming Growth Factor beta/metabolism , Smad2 Protein/metabolism , Smad3 Protein/metabolism , Transcription, Genetic/drug effects
5.
Reprod Domest Anim ; 43(4): 481-3, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18282213

ABSTRACT

The aim of the present study was to evaluate the clinical applications and limitations of daily low-dose oxytocin injections for the induction of parturition in pregnant at term mares, the attention was focussed on the efficacy of the treatment and on its possible negative effects on mare and foal. Three-hundred and fifty pregnant full term Standardbred mares were used: 176 were allowed to foal spontaneously, 174 were treated daily with 3.5 IU of oxytocin i.m. when mammary secretion showed a calcium concentration >or= 200 ppm. For each mare, gestational length, outcome and duration of foaling, placenta expulsion time were recorded. Physical and behavioural characteristics of each foal were also recorded. Administration of oxytocin resulted in the delivery of a normal foal within 120 min in 68.9% of treated mares: 51.3% responded to the first oxytocin administration, 14.2% to the second and 3.4% to the third. No significant difference between treated and control mares was observed in the gestational length (340 +/- 8 days vs 337 +/- 7 days), duration of foaling (10 +/- 5.6 min vs 11 +/- 4.9 min), incidence of dystocia (1.4% vs 1.7%) and failure of rupture of the allantochorion (0% vs 0.6%). No significant difference was observed in the incidence of placental retention between treated and control groups (8.1% vs 6.3%). Physical and behavioural characteristics were normal in foals of both groups. In conclusion, daily injections of low doses of oxytocin in at term mares showed only moderate efficacy for inducing parturition. However, the easy applicability and the complete safety for both mare and foal, of this method of foaling induction makes it a useful tool to simplify the management of mares in commercial stud farms.


Subject(s)
Animals, Newborn/anatomy & histology , Animals, Newborn/physiology , Horses/physiology , Labor, Induced/veterinary , Oxytocics/pharmacology , Oxytocin/pharmacology , Allantois/drug effects , Allantois/physiology , Animals , Calcium/blood , Cervix Uteri/drug effects , Cervix Uteri/physiology , Female , Injections, Intramuscular/veterinary , Labor, Induced/methods , Oxytocics/adverse effects , Oxytocin/adverse effects , Placenta/drug effects , Placenta/physiology , Pregnancy , Pregnancy Outcome/veterinary , Random Allocation , Time Factors
7.
J Morphol ; 269(1): 72-83, 2008 Jan.
Article in English | MEDLINE | ID: mdl-17902154

ABSTRACT

Experiments were performed on the chorio-allantoic membrane (CAM) of the chick to evaluate the effects of bombesin (BN) on vascular neoformation. In morphometrical assays, 10(-13)-10(-4) M BN promoted dose-dependent vascular development. Newly formed vessels converged toward the BN release site in a spoked wheel arrangement, suggesting a diffusion gradient mechanism. Structural and ultrastructural analysis of CAM specimens collected near the BN release site showed that both vasculogenetic and angiogenetic processes cooperated in vascular neoformation that involved committed cells from the mesenchyme (angioblasts) as well as endothelial cells. No pattern of vascular development was detected away from the BN release site. Findings from the present study emphasize the role of BN in vascular net development of respiratory organs.


Subject(s)
Allantois/drug effects , Bombesin/pharmacology , Neovascularization, Physiologic/drug effects , Allantois/cytology , Allantois/physiology , Allantois/ultrastructure , Animals , Chick Embryo , Microscopy, Electron, Transmission , Neurotransmitter Agents/pharmacology
8.
Int J Toxicol ; 27(6): 449-53, 2008 Nov.
Article in English | MEDLINE | ID: mdl-19482824

ABSTRACT

As an alternative to the standard Draize eye irritation test, the potential irritancy of compounds was evaluated by observing adverse changes that occur in chorioallantoic membrane CAM) of the hen egg (HECAM) after exposure to a test chemical placed directly on the CAM. The occurrence of hemorrhage, coagulation, and lysis in response to a test compound is the basis for employing this technique to evaluate its potential for in vivo damage to mucous membrane, in particular the eye. Irritancy is scored according to the severity and speed at which damage occurs. In the present study, five different classes of pesticides were screened for irritation potential. There was good correlation between the HECAM assay and the in vivo Draize eye irritation test. The proposed HECAM assay, which reduces the requirement for laboratory animals, could be a painless alternative to the Draize test.


Subject(s)
Allantois/drug effects , Animal Testing Alternatives , Chorion/drug effects , Eye/drug effects , Irritants/toxicity , Pesticides/toxicity , Animals , Biological Assay , Chick Embryo
9.
Mol Cancer Ther ; 6(10): 2675-85, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17938261

ABSTRACT

In the course of a blind screening program for inhibitors of angiogenesis, IB05204 (4,8-dichloro-12-phenylpyrido[5',6':4'',5'';3',2':4,5]dithieno[3'',2''-d':3,2-d]-1,2,3-ditriazine) was selected for its ability to inhibit endothelial tubule-like network formation on Matrigel. IB05204 inhibits the in vivo angiogenesis in the chorioallantoic membrane (CAM) and the mouse Matrigel plug assays. Antiangiogenic activity seems to be highly dependent on the chloro substituents because their removal results in a complete loss of the in vitro inhibitory activity of endothelial differentiation and in vivo antiangiogenic activity in CAM assay. Although IB05204 inhibits the growth of endothelial and tumor cells in culture, its antiangiogenic activity seems to be mainly dependent on the prevention of endothelial capillary-like tube formation and inhibition of endothelial migration because these effects are recorded at lower concentrations. IB05204 treatment inhibits matrix metalloproteinase-2 (MMP-2) production in endothelial and tumor cells, down-regulates endothelial cyclooxygenase-2 expression, and represses phosphorylation of endothelial Akt in response to serum stimulation, suggesting that IB05204 interferes with molecular mechanisms of cell migration and survival. IB05204 induces apoptosis in endothelial cells through cytochrome c release and caspase activation. Data here shown altogether indicate that IB05204 is a compound that interferes with several key steps of angiogenesis, making it a promising drug for further evaluation in the treatment of angiogenesis-related pathologies.


Subject(s)
Angiogenesis Inhibitors/therapeutic use , Colonic Neoplasms/blood supply , Fibrosarcoma/blood supply , Neovascularization, Pathologic/prevention & control , Triazines/therapeutic use , Allantois/blood supply , Allantois/cytology , Allantois/drug effects , Angiogenesis Inhibitors/chemistry , Animals , Aorta/cytology , Aorta/drug effects , Apoptosis/drug effects , Blotting, Western , Caspases/metabolism , Cattle , Cell Differentiation/drug effects , Cell Movement/drug effects , Chick Embryo , Chorion/blood supply , Chorion/cytology , Chorion/drug effects , Collagen/metabolism , Colonic Neoplasms/drug therapy , Colonic Neoplasms/pathology , Drug Combinations , Endothelium, Vascular/drug effects , Fibrosarcoma/drug therapy , Fibrosarcoma/pathology , Flow Cytometry , Humans , Laminin/metabolism , Matrix Metalloproteinase 2/metabolism , Mice , Neovascularization, Pathologic/pathology , Proteoglycans/metabolism , Triazines/chemistry
10.
Pharmazie ; 62(7): 534-8, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17718196

ABSTRACT

Protracted administration of topotecan (TPT), a topoisomerase I inhibitor, exhibited high anticancer efficacy both in animal models and human cancers. This phenomenon is related to the TPT-induced inhibition of angiogenesis in tumor, but the potential mechanism remains largely unknown. In the present study, we reported that TPT (1-10 microM) could inhibit angiogenesis in a dose-dependent manner in Chick embryo chorioallantoic membrane (CAM) assay. TPT showed strong inhibitory activity against proliferation on human EA.hy926 endothelial cells with an IC50 value of 0.13 microM (MTT assay), lower than that of most sensitive cancer cell lines (IC50 range, 0.17 microM to 5.1 microM). TPT could induce EA.hy926 cells undergoing apoptosis, and the percentage of apoptotic cells induced by TPT (0.05 microM-5.0 microM) were 17.9%-52.3%. The similar results were observed with AO/EB staining. Flow cytometry assay also revealed that various concentrations of TPT induced cell cycle disturbance in EA.hy926 cells. Western blotting results showed that TPT caused an obvious increase of p53 expression and a decline of ERK expression in EA.hy926 cells. In addition, the VEGF expression of PC-3 cells is inhibited by TPT in hypoxia. Altogether, inhibiting proliferation of endothelial cells and down-regulating VEGF expression in cancer cells may involve in the antiangiogenesis mechanism of TPT.


Subject(s)
Antineoplastic Agents/pharmacology , Endothelial Cells/physiology , Topotecan/pharmacology , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Vascular Endothelial Growth Factor A/biosynthesis , Acridine Orange , Allantois/drug effects , Animals , Blotting, Western , Cell Line , Cell Line, Tumor , Cell Proliferation/drug effects , Chick Embryo , Endothelial Cells/drug effects , Ethidium , Flow Cytometry , Fluorescent Dyes , Humans , Tumor Protein, Translationally-Controlled 1
11.
In Vivo ; 21(2): 297-304, 2007.
Article in English | MEDLINE | ID: mdl-17436580

ABSTRACT

BACKGROUND: TZT-1027 (Soblidotin), a microtubule-depolymerizing agent, has antivascular activity which disrupts newly formed tumor vasculature. In this study, it was investigated whether TZT-1027 has also antiangiogenic activity preventing neovascularization. MATERIALS AND METHODS: Antiangiogenic activities were evaluated in vivo in a chick embryo chorioallantoic membrane (CAM) assay and in vitro in a tube formation assay on human umbilical vein endothelial cells (HUVEC). RT-PCR and skimmed milk zymography analyses were performed to clarify the involvement of angiogenesis-related proteolytic enzymes and transcription factors. RESULTS: TZT-1027 at doses of 0.01 and 0.06 microg/egg showed potent antiangiogenic activities in the CAM assay (80% and 100% inhibition, respectively), with no lethal toxicity to the chick embryo. TZT-1027 at doses of 0.01-10 ng/mL prevented tube formation, while 1-100 ng/mL disrupted the preformed vascular tube. However, mRNA and protein expression were unchanged. CONCLUSION: TZT-1027 showed antiangiogenic activity at lower doses than it exhibited its antivascular activity. We believe it would exert its antiangiogenic activity, even if kept in a tumor at reduced concentrations to keep its antivascular activity to a minimum.


Subject(s)
Allantois/drug effects , Angiogenesis Inhibitors/pharmacology , Chorion/drug effects , Endothelium, Vascular/drug effects , Oligopeptides/pharmacology , Animals , Antineoplastic Agents/pharmacology , Chick Embryo , DNA Primers , Humans , Reverse Transcriptase Polymerase Chain Reaction , Umbilical Veins , Urokinase-Type Plasminogen Activator/genetics
12.
FEBS Lett ; 581(5): 962-6, 2007 Mar 06.
Article in English | MEDLINE | ID: mdl-17316624

ABSTRACT

We investigate here the anti-angiogenic properties of the synthetic compound myo-inositol trispyrophosphate (ITPP). By increasing oxy-haemoglobin dissociation, ITPP has the potential to counteract the effects of hypoxia, a critical regulator of angiogenesis and cancer progression. ITPP inhibited angiogenesis of the chorioallantoic membrane (CAM), as analyzed with an original program dedicated to automated quantification of angiogenesis in this model. ITPP also markedly reduced tumor progression and angiogenesis in an experimental model of U87 glioma cell nodules grafted onto the CAM. These results point out the potential of ITPP for the development of a new class of anti-angiogenic and anti-cancer compounds.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Inositol Phosphates/pharmacology , Neovascularization, Pathologic/prevention & control , Neovascularization, Physiologic/drug effects , Allantois/blood supply , Allantois/drug effects , Animals , Cell Line, Tumor , Chick Embryo , Chorion/blood supply , Chorion/drug effects , Glioma/blood supply , Glioma/drug therapy , Humans , Neoplasm Transplantation , Transplantation, Heterologous
13.
Anticancer Drugs ; 18(2): 127-37, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17159599

ABSTRACT

Single-domain antibodies are attractive as tumor-targeting vehicles because of their much smaller size than intact antibody molecules. Lidamycin is a macromolecular antitumor antibiotic, which consists of a labile enediyne chromophore (AE) and a noncovalently bound apoprotein (LDP). An enediyne-energized fusion protein VH-LDP-AE composed of single-domain antibody directed against type IV collagenase and lidamycin was prepared by a novel two-step method including DNA recombination and molecular reconstitution. VH-LDP-AE demonstrated extremely potent cytotoxicity to cancer cells and marked antiangiogenic activity in vitro. In the mouse hepatoma 22 model, drugs were administered intravenously as a single dose on day 1 with maximal tolerated doses. VH-LDP-AE (0.25 mg/kg) suppressed the tumor growth by 95.9%, whereas lidamycin (0.05 mg/kg) and mitomycin (1 mg/kg) by 79.6 and 51.1%, respectively. In the HT-1080 xenograft model in nude mice, drugs were given intravenously as a single dose on day 4 after tumor implantation. VH-LDP-AE at 0.25 mg/kg suppressed tumor growth by 76% (P<0.05) compared with that of lidamycin at 0.05 mg/kg (53%) on day 18. No obvious toxic effects were observed in all groups during treatments. The results showed that energized fusion protein VH-LDP-AE was more effective than lidamycin and mitomycin. These properties, together with its much smaller size than conventional antibody-based agents, suggested that VH-LDP-AE would be a promising candidate for cancer-targeting therapy. In addition, the two-step approach could serve as a new technology platform for making a series of highly potent engineered antibody-based drugs for a variety of cancers.


Subject(s)
Antibodies/chemistry , Antibodies/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Allantois/chemistry , Allantois/drug effects , Amino Acid Sequence , Angiogenesis Inhibitors/chemical synthesis , Angiogenesis Inhibitors/pharmacology , Animals , Antineoplastic Agents/chemical synthesis , Blotting, Western , Cell Line, Tumor , Chick Embryo , Collagenases/chemistry , Collagenases/metabolism , Enzyme-Linked Immunosorbent Assay , Female , Genetic Vectors , Indicators and Reagents , Mice , Mice, Inbred BALB C , Mice, Nude , Microscopy, Fluorescence , Microtubules , Molecular Sequence Data , Recombinant Fusion Proteins/chemical synthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/pharmacology
14.
Cancer Res ; 66(17): 8779-87, 2006 Sep 01.
Article in English | MEDLINE | ID: mdl-16951194

ABSTRACT

Inhibitors of tumor angiogenesis and metastasis are increasingly emerging as promising agents for cancer therapy. Recently, heparanase inhibitors have offered a new avenue for such work because heparanase is thought to be critically involved in the metastatic and angiogenic potentials of tumor cells. Here, we report that oligomannurarate sulfate (JG3), a novel marine-derived oligosaccharide, acts as a heparanase inhibitor. Our results revealed that JG3 significantly inhibited tumor angiogenesis and metastasis, both in vitro and in vivo, by combating heparanase activity via binding to the KKDC and QPLK domains of the heparanase molecule. The JG3-heparanase interaction was competitively inhibited by low molecular weight heparin (4,000 Da) but not by other glycosaminoglycans. In addition, JG3 abolished heparanase-driven invasion, inhibited the release of heparan sulfate-sequestered basic fibroblast growth factor (bFGF) from the extracellular matrix, and repressed subsequent angiogenesis. Moreover, JG3 inactivated bFGF-induced bFGF receptor and extracellular signal-regulated kinase 1/2 phosphorylation and blocked bFGF-triggered angiogenic events by directly binding to bFGF. Thus, JG3 seems to inhibit both major heparanase activities by simultaneously acting as a substrate mimetic and as a competitive inhibitor of heparan sulfate. These findings suggest that JG3 should be considered as a promising candidate agent for cancer therapy.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Enzyme Inhibitors/pharmacology , Fibroblast Growth Factor 2/physiology , Glucuronidase/antagonists & inhibitors , Mannans/pharmacology , Neoplasm Metastasis/prevention & control , Neovascularization, Pathologic/prevention & control , Allantois/drug effects , Allantois/physiology , Animals , Aorta , Cattle , Cell Movement/drug effects , Chorion/drug effects , Chorion/physiology , Enzyme Inhibitors/chemical synthesis , Fibroblast Growth Factor 2/drug effects , Fibroblast Growth Factor 2/metabolism , Humans , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/physiology , Neoplasm Invasiveness/prevention & control , Rats , Surface Plasmon Resonance
15.
Food Chem Toxicol ; 44(8): 1184-90, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16793484

ABSTRACT

This study was carried out to evaluate the effect of bacteriocin produced by Lactococcus sp. HY 449 against skin-inflammatory bacteria such as Staphylococcus epidermidis ATCC 12228, Staphylococcus aureus ATCC 65389, Streptococcus pyogenes ATCC 21059, and Propionibacterium acnes ATCC 6919. The spot-on-the-lawn method was used to determine the antimicrobial activity of bacteriocin against indicator strains on the human skin. The bacteriocin produced by Lactococcus sp. HY 449 inhibited the growth of S. epidermidis ATCC 12228, S. aureus ATCC 65389, Strep. pyogenes ATCC 21059, and P. acnes ATCC 6919. The treatment of crude bacteriocin caused a rapid inactivation of P. acnes ATCC 6919. The LC50 of bacteriocin on human fibroblast was ca. 50mg/ml at which the inhibition of cell proliferation was not observed. Neither any irritations nor allergic reactions by the bacteriocin were evident in a human patch test. The bacteriocin produced by Lactococcus sp. HY 449 may be a useful antimicrobial substance to control the growth of P. acnes and to prevent skin inflammation and acne.


Subject(s)
Bacteriocins/pharmacology , Dermatitis/microbiology , Gram-Positive Bacterial Infections/microbiology , Lactococcus/metabolism , Skin Diseases, Infectious/microbiology , Adult , Allantois/drug effects , Animals , Bacteriocins/isolation & purification , Bacteriocins/toxicity , Chick Embryo , Cosmetics , Dermatitis/drug therapy , Dermatitis, Irritant/etiology , Female , Fibroblasts/drug effects , Gram-Positive Bacterial Infections/drug therapy , Humans , Microbial Sensitivity Tests , Propionibacterium acnes/drug effects , Skin Diseases, Infectious/drug therapy , Skin Tests , Staphylococcus aureus/drug effects , Staphylococcus epidermidis/drug effects , Streptococcus pyogenes/drug effects
16.
Carcinogenesis ; 27(12): 2455-63, 2006 Dec.
Article in English | MEDLINE | ID: mdl-16777983

ABSTRACT

Rigorous and systematic pre-clinical studies are necessary and essential to establish the efficacy and safety of Oriental herbs and formulas in order to transform traditional herbal practices into evidence-based medicine. Here we evaluated the anti-cancer activities of the ethanol extract of Ka-mi-kae-kyuk-tang (KMKKT), a formula of ten Oriental herbs, with a battery of in vitro and in vivo mechanism-based biomarkers involving angiogenesis, apoptosis and metastasis. The results show that KMKKT suppressed the vascular endothelial responses by inhibiting basic fibroblast growth factor (bFGF)-induced ERK1/2 phosphorylation, cell migration as well as tube formation in the human umbilical vein endothelial cell model, and decreased the hypoxia-induced HIF1alpha and vascular epithelial growth factor (VEGF) expression in the mouse Lewis lung carcinoma (LLC) cells in vitro, and inhibited the bFGF-induced angiogenesis in chick chorioallantoic membrane model, and in the Matrigel plugs in mice. Intraperitoneal delivery of KMKKT potently inhibited the growth of the subcutaneously inoculated LLC cells in syngenic mice. In addition, KMKKT inhibited the invasion ability of the mouse colon 26-L5 cancer cells in vitro and decreased their formation of liver metastasis when intraportally inoculated in syngenic mice. Furthermore, KMKKT suppressed the growth of the human PC-3 prostate cancer xenografts in athymic nude mice and averted the cancer-related body weight loss. The in vivo cancer growth suppression was associated with a decreased microvessel density and VEGF abundance as well as an increased PARP cleavage and the TUNEL-positive apoptosis. Together, our data support broad-spectra in vivo anti-cancer activities of KMKKT targeting angiogenesis, apoptosis and metastasis without any adverse effect on the body weight. This formula merits serious consideration for further evaluation for the chemoprevention and treatment of cancers of multiple organ sites.


Subject(s)
Apoptosis/drug effects , Neoplasm Metastasis/prevention & control , Neovascularization, Pathologic/prevention & control , Plant Extracts/pharmacology , Allantois/drug effects , Animals , Cell Division/drug effects , Cell Movement/drug effects , Cell Survival/drug effects , Cells, Cultured , Chick Embryo , Chorion/drug effects , Colonic Neoplasms/pathology , Disease Models, Animal , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Humans , Korea , Liver Neoplasms/pathology , Medicine, East Asian Traditional , Mice , Mice, Inbred BALB C , Umbilical Veins
17.
Biochem Biophys Res Commun ; 345(4): 1398-404, 2006 Jul 14.
Article in English | MEDLINE | ID: mdl-16723120

ABSTRACT

The bioactivity, refolding, and multimer formation of endostatin, particularly of recombinant endostatin produced from bacteria, are proved challenging for clinical application. In order to determine the biological activity of recombinant endostatin multimer, first, we expressed endostatin in Escherichia coli and purified it with ion-exchange chromatography. The purified active protein could elicit multimer formation spontaneously, but still has comparable activity. Aim to determine the anti-angiogenic activity of multimer endostatin, by use of RP-HPLC, we then successfully separated endostatin monomer and multimer for subjecting to anti-angiogenesis assay. The results from CAM (chorioallantoic membrane) inhibition assay showed that both monomer and multimer suppressed CAM vascularization significantly. At the dosage of 0.8 microg, inhibition rates of multimeric and monomeric proteins were about 58% and 38%, respectively. Multimeric endostatin exerted a higher activity than monomeric endostatin (p < 0.05). However, when the protein dosage is less than 0.4 microg/ml, there is no significance between their inhibition rates (p > 0.05), although both of them show a high inhibition effect in contrast to control. The results from HUVEC proliferation assay also showed similar effects at dosages of 0.6 and 1.6 microg/ml, multimer exerted a higher activity on inhibition of HUVEC proliferation comparing with monomer (p < 0.05). In conclusion, our results suggest that endostatin multimer has a comparable or higher bioactivity and multimerization will not affect its bioactivity, implying that endostatin activity is insensitive to structure conformation contributed by disulfide bonds.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Endostatins/pharmacology , Allantois/blood supply , Allantois/drug effects , Animals , Cell Line , Cell Proliferation/drug effects , Chick Embryo , Chorion/blood supply , Chorion/drug effects , Chromatography, High Pressure Liquid/methods , Dimerization , Electrophoresis, Polyacrylamide Gel , Endostatins/chemistry , Endostatins/genetics , Endothelial Cells/cytology , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Escherichia coli/genetics , Humans , Mice , NIH 3T3 Cells , Neovascularization, Physiologic/drug effects , Platelet Endothelial Cell Adhesion Molecule-1/analysis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/pharmacology , Solubility
18.
Lab Invest ; 85(12): 1507-16, 2005 Dec.
Article in English | MEDLINE | ID: mdl-16200076

ABSTRACT

Lebestatin, a new member of the lysine-threonine-serine (KTS)-disintegrin family, was purified to homogeneity from Tunisian snake (Macrovipera lebetina) venom. It is a single-chain polypeptide composed of 41 amino acids. The amino-acid sequence of lebestatin shows that it displays a pattern of cysteines similar to other short disintegrins, but contains the sequence KTS rather than RGD in its integrin-binding loop. Lebestatin presents a high homology with obtustatin and viperistatin. Lebestatin interacts specifically with the alpha1beta1 integrin. It was thus able to inhibit both adhesion and migration of PC12 and alpha1beta1 integrin-expressing CHO cells (CHO-alpha1) to type I and IV collagens. This disintegrin also affected adhesion and migration of endothelial cells and exhibited an anti-angiogenic effect in vivo when using the 8-day-old embryo chick chorioallantoic membrane model.


Subject(s)
Cell Adhesion/drug effects , Cell Movement/drug effects , Disintegrins/pharmacology , Neovascularization, Physiologic/drug effects , Viper Venoms/chemistry , Viperidae , Allantois/blood supply , Allantois/drug effects , Amino Acid Sequence , Animals , CHO Cells/drug effects , CHO Cells/metabolism , Cell Line, Tumor , Chick Embryo , Cricetinae , Cricetulus , Disintegrins/isolation & purification , Dose-Response Relationship, Drug , Humans , Integrin alpha1beta1/antagonists & inhibitors , Molecular Sequence Data , Molecular Structure , PC12 Cells/drug effects , PC12 Cells/metabolism , Rats
19.
Phytother Res ; 19(4): 323-6, 2005 Apr.
Article in English | MEDLINE | ID: mdl-16041727

ABSTRACT

The antiinflammatory effects of ethanol and aqueous extracts from Drosera rotundifolia and from Drosera madagascariensis were compared in vivo in the HET-CAM assay. Both extracts from D. rotundifolia and the ethanol extract from D. madagascariensis showed remarkable efficacy at doses of 500 microg/pellet. The inhibition of the inflammation by the extracts was stronger than that by 50 microg hydrocortisone/pellet. In contrast, there was only a very weak effect observed at a dose of 500 microg/pellet of the water extract from D. madagascariensis. The chemical analyses of the extracts showed that the effect cannot be attributed to naphthoquinones, but might be due to flavonoids. Ellagic acid obviously plays an important role in the antiangiogenic effect of the Drosera extracts.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Drosera , Inflammation/prevention & control , Phytotherapy , Plant Extracts/pharmacology , Allantois/drug effects , Animals , Anti-Inflammatory Agents, Non-Steroidal/administration & dosage , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Biological Assay , Chick Embryo , Dose-Response Relationship, Drug , Humans , Plant Components, Aerial , Plant Extracts/administration & dosage , Plant Extracts/therapeutic use
20.
Cell Biol Int ; 29(7): 583-5, 2005 Jul.
Article in English | MEDLINE | ID: mdl-15979904

ABSTRACT

The exact function of osteocalcin (OC), a protein synthesized by osteoblasts during the matrix mineralization phase, is still unknown. In this study we investigated the capacity of OC to promote vasoproliferation in chick embryo chorioallantoic membrane (CAM), a well established in vivo assay for angiogenesis and anti-angiogenesis. The results showed that OC stimulates angiogenesis and that the response is similar to that obtained with FGF-2, a well-known angiogenic cytokine. It has previously been demonstrated that OC is involved in bone repair, so the angiogenic activity reported here might also play a crucial role in bone formation.


Subject(s)
Allantois/drug effects , Chorion/drug effects , Neovascularization, Physiologic/drug effects , Osteocalcin/pharmacology , Allantois/blood supply , Animals , Chick Embryo , Chorion/blood supply , Fibroblast Growth Factor 2/pharmacology , Gelatin Sponge, Absorbable
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