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 Human Umbilical Vein Endothelial Cells (HUVECs) culture HUVECs were purchased from Cambrex Biosciences, Walkersville, USA. The cells were cultured in 25 cm 3 tissue culture flask (NUNC, Genetix Biotech Asia, Bangalore, India) and grown using EGM-2 medium and endothelial cell basal medium according to the manufacturer’s protocol. Incubation was carried out in a humidified atmosphere of 5% CO 2 in air at 37 0 C. When cells reached confluency, they were passaged after trypsinization. HUVECs of passages 2-5 were used for the experiments. Animals and in vivo tumor generation Six to eight weeks old mice were acclimated for one week while caged in groups of five. Mice were housed and fed a diet of animal chow and water ad libitum throughout the experiment. All experiments were conducted according to the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India. EAT cells (5×10 6 cells/ mouse) injected intraperitoneally grow in mice peritoneum forming an ascites tumor with massive abdominal swelling. The animals show a dramatic increase in body weight over the growth period and the animals succumb to the tumor burden 15-16 days after implantation. The number of cells increased over the 14 days of growth with formation of 7-8 ml of ascites fluid with extensive neovascularization in the inner lining of peritoneal wall. EAT cells from fully grown tumor bearing mice were harvested from the peritoneal cavity of mice (19). The ascites fluid was collected in isotonic saline solution containing 3.8% sodium citrate. The cells were pelleted by centrifugation (3000 rpm for 10 min at 4 0 C). Contaminating red blood corpuscles if any were lysed with 0.8% ammonium chloride. Cells were resuspended in 0.9% saline. These cells or their aliquots were used either for transplantation or for further experiments. 140 Tube formation assay Tube formation of HUVECs was conducted for the assay of in vitro angiogenesis. The assay was performed as described in earlier report (20). Briefly, a 96-well plate was coated with 50µl of Matrigel (Becton Dickinson Labware, Bedford, MA), which was allowed to solidify at 37 0 C for 1 hour. HUVECs (5x 10 3 cells per well) were seeded on the Matrigel and cultured in EGM medium containing withaferin A (3.5-14µg) for 8 hours. After incubation at 37 0 C and 5% CO2, the enclosed networks of complete tubes from five randomly chosen fields were counted and photographed under an Olympus inverted microscope (CKX40; Olympus, New York, NY) connected to a digital camera at 40X magnification. Chick chorioallantoic membrane (CAM) assay CAM assay was carried out according to the detailed procedure as described by Gururaj, A.E. et al. (21, 22). In brief, fertilized chicken eggs were incubated at 37 0 C in a humidified incubator. On the 11th day of development, a rectangular window was made in the egg shell and glass cover slips (6-mm diameter) saturated with 25ng/ml vascular endothelial growth factor (VEGF) and VEGF + withaferin A (7ìg) was placed on the CAM and the window was closed using sterile wrap. The windows were opened after 48h of incubation and were inspected for changes in the microvessel density in the area below the cover slip and photographed using a Nikon digital camera. In vivo withaferin A treatment inhibits EAT growth To determine whether withaferin A inhibits tumor growth and angiogenesis in EAT cells in vivo, withaferin A (7mg/kg/day/mouse) and vehicle control (0.1% of DMSO) was injected into the EAT bearing mice every alternate day Sp1 transcription factor
Current Trends in Biotechnology and Pharmacy Vol. 3 (2) 138-148, April 2009. ISSN 0973-8916 after 6th day of tumor transplantation and growth of the tumor was monitored by taking the body weight of the animals. Animals were sacrificed on the 14th day and the EAT cells along with ascites fluid were harvested into the beaker and centrifuged at 3000 rpm for 10 min at 4 0 C. The pelleted cells were counted by Trypan blue dye exclusion method using a haemocytometer. A measure of the supernatant gave the volume of ascites fluid. Peritoneal angiogenesis and micro vessel density After harvesting the EAT cells from control and withaferin A-treated animals, the peritoneum was cut open and the inner lining of the peritoneal cavity was examined for extent of neovasculature and photographed. Formaldehyde fixed and paraffin embedded tissues of peritoneum from EAT bearing mice either treated or untreated with withaferin A were taken and 5ìm sections were prepared using automatic microtome (SLEE Cryostat) and stained with hematoxylin and eosin. The images were photographed using Leitz- DIAPLAN microscope with CCD camera and the blood vessels were counted. Quantitation of VEGF EAT bearing mice were treated with or without withaferin A (7mg/kg/day) for 5 doses on 6th, 8th, 10th and 12th day of tumor transplantation. The animals were sacrificed and ascites fluid was collected after 24h of each dose. VEGF-ELISA was carried out using the ascites fluid (21, 23, 24). In brief, 100µl of ascites from tumor bearing mice either with or without withaferin A treatment, was coated using coating buffer (50 mM carbonate buffer pH 9.6) at 4 0 C overnight. Subsequently, wells were incubated with anti-VEGF 165 antibodies, followed by incubation with secondary antibodies tagged to alkaline phosphatase and detection using p-nitrophenyl phosphate (pNPP) as a substrate. 141 Preparation of nuclear extracts Nuclear extracts were prepared according to the method previously described (25). Briefly, cells (5X10 6 ) treated either with or without withaferin A in complete HBSS for different time intervals were washed with cold phosphate buffered saline and suspended in 0.5 ml of lysis buffer (20mM HEPES, pH 7.9, 350 mM NaCl, 20% Glycerol, 1% NP-40, 1 mM MgCl 2 , 0.5 mM EGTA, 0.5 mM DTT, 1 mM Pefablock, 1µg/ml Aprotinin, 1µg/ ml Leupeptin). The cells were allowed to swell on ice for 10 min; the tubes were then vigorously mixed on a vortex mixer for 1 min and centrifuged at 10,000 rpm for 10 min at 4 0 C. The supernatant was immediately stored at -20 0 C. Electrophoretic Mobility Shift Assay (EMSA) Nuclear proteins were extracted from EAT cells treated either with or without withaferin A for 60,120 and 180 min respectively. The EMSA was performed as described in earlier report (26, 27). The double stranded Sp1 consensus oligonucleotide probes [5’-d (ATT CGA TCG GGG CGG GGC GAG C)-3’] were end-labeled with ã-[ 32 P] ATP. Nuclear proteins (40ìg) were incubated with 40fmoles of ã-[ 32 P]-labeled Sp1 consensus oligonucleotides for 30min in binding buffer containing 100mM HEPES (pH 7.9),10mM MgCl 2, 125 mM KCl, 0.5mM EDTA, 4% glycerol,0.5% NP-40,1ìg of poly [dI-dC] and 1mg/ml BSA. The samples were electrophoresed in 4% non denaturing polyacrylamide gel in 0.5% TBE at room temperature for 2 hr at 200V. The gel was dried, transferred to imaging plate (IP) and the image was scanned by image analyzer Fujifilm (FLA-5000). Results Withaferin A inhibits tube formation of HUVECs induced by VEGF In order to verify if withaferin A interferes directly with the formation of blood vessels by HUVECs, we performed tube formation assay Prasanna et al
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