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ABSTRACT The phytochemical study of Galium tunetanum Lam., Rubiaceae, leaves led to the isolation of 13 compounds from the chloroform-methanol and the methanol extracts, including six iridoid glycosides, one non-glycoside iridoid, two p-coumaroyl iridoid glycosides, two phenolic acids, and two flavonoid glycosides. The structural determination of the isolated compounds was performed by mono- and bidimensional NMR spectroscopic data, as well as ESI-MS experiments. All compounds were isolated from this species for the first time. The anti-angiogenic effects of the isolated iridoids were also reported on new blood vessels formation using the chick embryo chorioallantoic membrane as in vivo model. Results showed that among the isolated iridoids tested at the dose of 2 µg/egg, asperuloside (1), geniposidic acid (2), and iridoid V1 (3) reduced microvessel formation of the chorioallantoic membrane on morphological observations using a stereomicroscope. The anti-angiogenic effects of the active compounds, expressed as percentages of inhibition versus control, were 67% (1), 59% (2), and 54% (3), respectively. In addition, the active compounds were able to inhibit angiogenesis in the chorioallantoic membrane assay, in a dose-dependent manner (0.5-2 µg/egg) as compared to the standard retinoic acid.
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Objective To investigate the inhibitory effects of Tum5 on the angiogenesis of human umbilical vein endothelial cells (HUVECs) and alkali-induced corneal neovascularization.Methods HUVECs in logarithmic growth phase were divided into 4 groups,cells with untreated as normal control group,cells with the infection of rAdGFP virus as rAd-GFP group,cells with the infection of rAd-Tum5 virus as rAd-Tum5 group,and cells with the infection of rAd-Tum5 virus followed by VEGF treatment as rAd-Tum5 + VEGF group.Then cell proliferation,migration,and tube formation of HUVECs were examined by CCK-8,Transwell and Matrigel assays,respectively.Sixty-four healthy male SD rats were randomly divided into 4 groups (n =16) by using random number table,and they were normal control group,alkali-burn group,alkali-burn + rAd-GFP group,and alkali-burn + rAd-Tum5 group.The alkali-burn rat model was then established except normal control group,and the normal control group received no treatment,whereas the alkali-burn,alkali-burn + rAd-GFP,and alkali-burn + rAd-Tum5 groups received subconjunctival injection of equal volumes of sterilized saline,rAd-GFP virus,and rAd-Tum5 virus,respectively following the alkaline burn.The relative area of corneal neovascularization and the number of infiltrating inflammatory cells were recorded on day 1,7,and 14 after injection.Results The CCK-8 assay showed that the proliferative rate of rAd-Tum5 group was lower than that of the normal control group and rAd-GFP group (both P ＜0.01),while rAd-Tum5 + VEGF group exhibited a significantly greater cell proliferative capability than rAd-Tum5 group (P =0.004).There were no statistical differences between rAd-Tum5 + VEGF group,normal control group and rAdGFP group (all P ＞ 0.05).Transwell assay showed the significantly lower number of migrating cells in the rAd-Tum5 group than those in the normal control group and rAdGFP group (both P ＜ 0.01).The number of migrating cells in rAd-Tum5 + VEGF group was higher than those in rAd-Tum5 group (P =0.000);however,the migration capacity had not been restored to normal level,and rAd-Tum5 + VEGF group had significant difference with normal control group and rAd-Tum5 group (both P ＜0.05).Matrigel assay showed that the number of meshes in rAd-Tum5 group was lower than that in the normal control group and rAd-GFP group (both P ＜0.01);while the number of meshes in rAd-Tum5 + VEGF group was significantly increased compared with rAd-Tum5 group(P =0.001).The density and number of corneal neovascularization increased gradually from day 1 to day 14 after alkali burn,while the relative neovascularization area in the alkali-burn + rAd-Tum5 group was significantly reduced as compared to those in the alkali-burn group and alkali-burn + rAd-GFP group on day 7 and day 14,suggesting that Tum5 could reduce the growth rate and density of corneal neovascularization,so as to inhibit corneal neovascularization induced by alkali burn.On day 7 and 14 after alkali burn,in the normal control group,the corneas were intact,no infiltrating inflammatory cells and cells were arranged in an orderly manner.On day 7 after alkali burn,there were disordered epithelial structure,corneal edema and infiltrating inflammatory cells in alkali-burn group and alkali-burn + rAd-GFP group.The number of infiltrating inflammatory cells in alkaliburn + rAd-Tum5 group was significantly lower than that in alkali-burn group and alkali-burn + rAd-GFP group (both P ＜0.01).On day 14 after alkali burn,the number of infiltrating inflammatory cells in alkali-bum,alkali-burn + rAd-GFP and alkali-burn + rAd-Tum5 group were significantly lower than those on day 7,while the corneal epitheliums were intact and dropsy was alleviated,while the number of inflammatory cells was significantly lower than that in alkali burn group and alkali-burn + rAd-GFP group,with significant difference (both P ＜ 0.01).Conelusion Tum5 can inhibit the angiogenic capability of HUVECs by VEGF pathway,as well as suppress the alkali-burn-induced corneal neovascularization and inflammatory cell infiltration in rats.
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Objective: To study the chemical constituents from Ampelopsis cantoniensis and their anti-angiogenic activities. Methods: The compounds were isolated and purified by various chromatographic techniques and their structures were elucidated by spectral analysis. The anti-angiogenic activities of the compounds isolated were evaluated using a zebrafish model. Results: Fifteen compounds were obtained from the ethyl acetate fraction in the 90% ethanol extract of A. cantoniensis and their structures were identified as cantonienol (1), nootkatone (2), aromadendrane-4β, 10β-diol (3), abscisic acid (4), 12-oxo-hardwickiic acid (5), betulinic acid (6), platanic acid (7), vanillic acid (8), resveratrol (9), nectandrin B (10), nectandrin A (11), 3, 5, 7-trihydroxychromone (12), 5, 7, 3', 4', 5'-pentahydroxyflavanone (13), taxifolin (14), and myricitrin (15). Conclusion: Compound 1 is a new sesquiterpene named cantonienol. Compounds 2-7 and 10-12 are isolated from the plants of Ampelopsis Michaux for the first time, and the other compounds are firstly reported in this plant. Compounds 9, 11, and 12 exhibit the weak anti-angiogenic activity when evaluated using a zebrafish model.
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Memora nodosa (Silva Manso) Miers é uma espécie da flora do Cerrado cujas folhas e caules são utilizados popularmente no tratamento de feridas e úlceras externas, enquanto as raízes são empregadas para dores abdominais e no tratamento da sarna. O objetivo desse trabalho foi avaliar a toxicidade aguda dos extratos etanólicos das folhas e raízes nas doses de 2000 e 5000 mg kg-1 em ratos e camundongos e a atividade cicatrizante das soluções aquosas contendo 2% desses extratos em feridas cutâneas em ratos. A contração das bordas das feridas foi avaliada por análises histológicas e morfométricas após 4, 7 e 14 dias de tratamento e por reação imunohistoquímica após 7 dias de tratamento. Os extratos etanólicos das folhas e raízes não apresentaram toxicidade na dose de 2000 mg kg-1 para ratos e camundongos e na dose de 5000 mg kg-1 para ratos. Nos camundongos, a dose de 5000 mg kg-1 dos extratos das folhas e raízes provocou alterações histológicas no fígado. Não foram observadas diferenças significativas na contração das feridas entre os grupos tratados com os extratos das folhas e das raízes e o controle após 4 e 7 dias de tratamento. Após 14 dias de tratamento, 50% dos animais tratados com o extrato das raízes apresentaram reepitelização total das feridas e reconstrução parcial dos anexos. A alantoína, isolada do extrato etanólico da raiz, pode ser considerada como um dos metabólitos secundários responsáveis pela aceleração da reepitelização.
Memora nodosa (Silva Manso) Miers is a Brazilian Cerrado species whose leaves and stems are commonly used to treat external wounds and ulcers and the roots are used for abdominal pain and to treat scabies. The purpose of this study was to evaluate the acute toxicity of ethanol extracts from the leaves and roots of M. nodosa at 2000 and 5000 mg kg-1 doses in rats and mice and to evaluate the healing activity of aqueous solutions containing 2% of these extracts on skin wounds in rats. The contraction of the wounds was evaluated by histological and morphometric analysis after 4, 7 and 14 days of treatment and by immunohistochemistry analysis after 7 days of treatment. The ethanol extracts of leaves and roots presented no toxicity at a 2000 mg kg-1 dose for rats and mice, and at a 5000 mg kg-1 dose for rats. Histological changes in the liver of mice were verified with a 5000 mg kg-1 dose of the leaf and root extracts. Macroscopic and histological differences were not observed in the contraction of wounds between the groups treated with leaf and root extracts and the control group after 4 and 7 days of treatment. After 14 days of treatment, 50% of the animals treated with the root extract presented total re-epithelialization of wounds and partial reconstruction of the annexes. Allantoin, isolated from the root ethanol extract, can be considered one of the secondary metabolites responsible for accelerating re-epithelialization.
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Animals , Female , Rats , Plant Roots/adverse effects , Plant Leaves/adverse effects , /analysis , Wound Healing , Plant Extracts/analysis , Bignoniaceae/classificationABSTRACT
PURPOSE: This study was designed to evaluate the angiogenic activities of gastric cancer tissue and adjacent normal gastric tissue and to analyze the correlations between the clinicopatholoic factors of gastric cancer and tumor angiogenic activity. METHODS: Sets of both tumor tissue and adjacent normal gastric tissue were sampled from 49 patients with gastric cancer at the time of gastrectomy. Each specimen was evaluated for the expression of VEGF mRNA by reverse transcriptase polymerase chain reaction (RT-PCR). For the microvessel count of each tissue sample, immunohistochemical staining was done using antiCD31 antibody. As a control group, 10 paraffin blocks of normal gastric tissue from patients with benign disease were selected and stained with the same antibody in order to count the microvessels. RESULTS: The microvessel count of the tumor tissue was higher than that of normal tissue, with a mean+/-SD of 74.10+/-30.33 and 24.69+/-10.11, respectively (p<0.001). The microvessel count of the control group was 23.40+/-6.77 and was not significantly different from that of normal tissue samples taken from patients with gastic cancer. VEGF/beta actin ratios measured from the results of RT-PCR were 0.70+/-0.32 in tumor tissue and 0.51+/-0.26 in normal tissue (p<0.001). In each tissue sample, there was a significant correlation between the microvessel count and VEGF/beta actin ratio (p<0.01 in the tumor tissue, p<0.05 in normal tissue). Micro-vessel count of tumor tissue was related with sex and types of Lauren's classification (p<0.05, p<0.01, respectively). The VEGF/beta actin ratio of tumor tissue was related to sex and degree of vascular invasion of tumor cells (p<0.05). Other clinicopathologic factors, such as age, histologic type, TNM stage, tumor depth, lymph node metastasis, and perineural invasion, were not associated with the degree of microvessel count and the level of VEGF mRNA expression. CONCLUSION: Gastric cancer shows marked angiogenic activity and the angiogenesis is related to some clinicopathologic factors. These results suggest that the clinical application of antiangiogenic agents may have a role in the treatment of gastric cancer.
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Stomach NeoplasmsABSTRACT
PURPOSE: We measured the expression of midkine (MK) gene and biophenotypes in brain tumor cell lines and tumor tissues. MATERIALS AND METHODS: We used the cell lines purchased from ATCC: two glioblastoma cell lines (T98G, U87MG), rat bladder tumor cell line (NBT-II), NIH/3T3, and two endothelial cell lines [(human umbilical vein endothelial cell (HUVEC) and fetal bovine heart endothelial cell (FBHE)]. RNA was taken from 4 cancer tissues of glioblastoma multiforme. Tissue cytosol was obtained from 5 cancer patients and 2 tissues of epilepsy patients. Pentosanpolysulfate(PPS) was used as a competitive inhibitor of heparin-binding growth factor (HBGF). MK and pleiotrophin (PTN) mRNA expression was tested by Northem hybridization, uPA and PAI-1 levels were measured by ELISA (Monozyme, Netherlands). Cross-feeding assay was done to measure the activity of endothelial cell growth stimulation induced by cancer cell lines. RESULTS: T98G cell line expressed both MK and PTN while U87MG expressed only PTN. In cross-feeding assay, endothelial cell growth increased in proportion to the number of administered feeding tumor cells, T98G and U87MG. This phenomenon was found in HUVEC, but not in FBHE. Conditioned media of T98G and U87MG showed similar stimulatory effect on the growth of NIH/3T3 cells. T98G cell showed higher excretion rate of uPA into conditioned media while U87MG showed higher excretion rate of PAI-1 into conditioned media. 20% of growth inhibition was induced on T98G cell with 100 pg/ml PPS, while 40% of growth inhibition was induced with 10 pg/ml PPS on U87MG cell. In four cancer tissues, thee expressed MK. In cancer tissue cytosol, uPA and PAI-1 expressions varied in individuals. No PAI-1 was found in non-tumor tissues. CONCLUSION: MK expression was found in brain tumor cell lines and tumor tissues. Modulation of biophenotypes (angiogenic activity and growth) by PPS in tumor cells with MK expression suggested a possible biotherapy in brain tumor targeting growth factor.
Subject(s)
Animals , Humans , Rats , Biological Therapy , Brain Neoplasms , Cell Line , Culture Media, Conditioned , Cytosol , Endothelial Cells , Enzyme-Linked Immunosorbent Assay , Epilepsy , Glioblastoma , Heart , Plasminogen Activator Inhibitor 1 , RNA , RNA, Messenger , Umbilical Veins , Urinary Bladder NeoplasmsABSTRACT
PURPOSE: This study was carried out to investigate the correlation among tumor angiogenetic activity, epithelial membrane antigen (EMA) reactivity and various clinicopathologic parameters. We also evaluated the validity of both as an independent prognostic factor in patients with papillary thyroid carcinoma. MATERIALS & METHODS: We studied 120 patients out of 727 patients with papillary thyroid casrcinoma who underwent thyroidectomy at our institute from January 1986 to December 1994. The age of the patients ranged from 14 to 80 years with a mean of 48.2 years. There were 24 males and 96 females (M:F=1:4). The paraffin embedded tissues of these patients were stained with the monoclonal antibodies against factor VIII related antigen, antigen CD34 to highlight microvessels and against EMA to show immunoreactivity. We measured microvessel density (MVD) in the area of highest vascular density at 200 times of magnification (0.785 mm2 per field). The positive cells for EMA were counted as percentages of the whole cell population and the degree of reaction was rated on a five-point scale. RESULTS: Mean MVDs and EMA reactivities by location of tissue per field were 64.8+/-18.9, 1.97+/-0.74, in the center of the tumor; 41.3+/-15.3, 1.55+/-0.68 in the periphery of tumor; and 22.1+/-14.4, 1.09+/-0.75 in normal thyroid tissue, respectively. In relation to TNM stage, only the MVDs of patients with stage IV disease were higher than those of other disease stages with statistical significance (p<.05). In relation to DeGroot stage, the MVDs of patients with stage IV disease was also higher than others with statistical significance (p<.005). There were no significant differences in MVD and EMA reactivity between the two groups of low risk (n=77) and high risk (n=43) by AMES scale. The MVDs and EMA reactivities of patients with local recurrence (n=23) and death (n=7) during the follow-up period had no statistical significance against those patients without recurrence and living patients. CONCLUSION: Tumor angiogenic activity and EMA reactivity in papillary thyroid carcinoma did not correlate with TNM stage, DeGroot stage, AMES score, local recurrence, and patient death. However, MVD was significantly higher in patients with distant metastasis and may be useful in predicting the distant metastasis in papillary thyroid carcinoma.