April Journal-2009.p65 - Association of Biotechnology and Pharmacy

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Current Trends in Biotechnology and Pharmacy Vol. 3 (2) 138-148, April 2009. ISSN 0973-8916 in vitro. The HUVECs were plated on the Matrigel. The HUVECs in the basal medium could not form tubes and VEGF was used to induce the tube formation. In the positive control group stimulated with VEGF (10ng), HUVECs rapidly aligned with one another and formed tube-like structures resembling a capillary plexus within 8 hours, after VEGF treatment. However, treatment with withaferin A prevented VEGF - stimulated tube formation of HUVECs in a concentration (3.5-14µg) - dependent manner (Fig. 1). Meanwhile, no cytotoxicity was observed under this concentration range of withaferin A used in the assay. Withaferin A was shown to interfere with the ability of HUVECs to form the in vitro vessel-like tubes, one of the important traits of the endothelial cells. 142 Withaferin A inhibits VEGF induced neovascularization on chick chorioallantoic membrane CAM assay is one of the widely used validation assays for formation of new blood vessels. In order to further verify if withaferin A is an inhibitor of new blood vessel formation, withaferin A was applied on chorioallantoic membrane of chick embryo to test its in vivo antiangiogenic activity. In the CAM assay model withaferin A induced avasculature zone formation in the developing embryos. Notably newly formed microvessels were regressed around the area of withaferin A treated CAM (Fig. 2). Fig. 2: Effect of withaferin A on neovascularization in the chick CAM Withaferin A was applied on CAM of 11-dayold chicken embryo. After 48h of incubation, the applied area was inspected for changes in neovascularization. The arrows indicate the applied area. The data shown represents the result of an experiment, which was done using maximum six eggs in each group. All photographs were taken at same magnification. Fig. 1: Effect of withaferin A on VEGF induced HUVECs tube formation HUVECs (5X10 3 cells) cultured in EGM medium with 3.5µg, 7µg and 14 µg withaferin A was added to the Matrigel coated 96 wells plate. After incubation for 8 hours at 37 0 C, capillary networks were photographed and quantified (Magnification: X40). Concentration dependent inhibition of tube formation by withaferin A was recorded. All datas are presented as mean from three different experiments with triplicates and means of ± S.E.M. P
Current Trends in Biotechnology and Pharmacy Vol. 3 (2) 138-148, April 2009. ISSN 0973-8916 contrast, in the withaferin A treated group, the number of cells were drastically decreased (Fig. 3A). This implied that withaferin A inhibit tumor cell growth in vivo. The volume of ascites was also measured using ascites from EAT bearing as well as withaferin A treated EAT bearing animals. The results indicated that withaferin A treatment reduces the secretion of ascites fluid (Fig. 3B). It is indicative from this data that withaferin A is probably capable of inhibiting the proliferation of tumor cell growth in vivo. 143 Withaferin A inhibits angiogenesis in vivo Sprouting of new blood vessels is evident in the inner peritoneal lining of EAT bearing mice. The peritoneal lining of EAT bearing animals and withaferin A treated mice was inspected for the effect on tumor-induced peritoneal neovascularization. EAT bearing mice treated with withaferin A showed decreased peritoneal angiogenesis when compared to the extent of peritoneal angiogenesis in untreated EAT bearing mice (Fig. 4A). Histopathological staining of peritoneum sections from the EAT bearing group appeared well vascularized. In contrast withaferin A treated peritoneum sections were characterized by a pronounced decrease in micro vessel density and the caliber of detectable vascular channels. While tumor bearing peritoneum sections showed 17 ± 1.2 blood vessels, withaferin A treated peritoneum showed 6.8 ± 1.3 blood vessels (Fig. 4B). Fig. 3: Effect of withaferin A on EAT cell number and ascites volume in vivo EAT cells (5X10 6 cells/animal, i.p.) were injected into mice. After 6 days of tumor transplantation, withaferin A (7mg/kg/animal) was injected on days 7th, 9th, 11th and 13th. The animals were sacrificed on days 8th, 10th, 12th and 14th. EAT cells were collected along with ascites fluid. Cells were counted in haemocytometer (A) and ascites volume was measured (B). At least five mice were used in each group and the results obtained are an average of three individual experiments and means of ± S.E.M. Fig. 4: Withaferin A inhibits angiogenesis and microvessel density A) Inhibition of peritoneal angiogenesis. EAT bearing mice were treated with and without withaferin A for four doses (7mg/kg/animal). The mice were sacrificed and the peritoneal lining was observed for extent of neovascularization. We presented representative photograph of peritoneum. B) Reduction in microvessel density (MVD) The peritoneum of control as well as withaferin A treated EAT bearing mice was embedded in paraffin and 5ìm sections were taken using microtome. The sections were stained with hematoxyline and eosine and observed for microvessel density. Arrows indicate the microvessels. Prasanna et al
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April Journal-2009.p65 - Association of Biotechnology and Pharmacy

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