April Journal-2009.p65 - Association of Biotechnology and Pharmacy
April Journal-2009.p65 - Association of Biotechnology and Pharmacy
April Journal-2009.p65 - Association of Biotechnology and Pharmacy
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Current Trends in <strong>Biotechnology</strong> <strong>and</strong> <strong>Pharmacy</strong><br />
Vol. 3 (2) 138-148, <strong>April</strong> 2009. ISSN 0973-8916<br />
after 6th day <strong>of</strong> tumor transplantation <strong>and</strong> growth<br />
<strong>of</strong> the tumor was monitored by taking the body<br />
weight <strong>of</strong> the animals. Animals were sacrificed<br />
on the 14th day <strong>and</strong> the EAT cells along with<br />
ascites fluid were harvested into the beaker <strong>and</strong><br />
centrifuged at 3000 rpm for 10 min at 4 0 C. The<br />
pelleted cells were counted by Trypan blue dye<br />
exclusion method using a haemocytometer. A<br />
measure <strong>of</strong> the supernatant gave the volume <strong>of</strong><br />
ascites fluid.<br />
Peritoneal angiogenesis <strong>and</strong> micro vessel<br />
density<br />
After harvesting the EAT cells from<br />
control <strong>and</strong> withaferin A-treated animals, the<br />
peritoneum was cut open <strong>and</strong> the inner lining <strong>of</strong><br />
the peritoneal cavity was examined for extent <strong>of</strong><br />
neovasculature <strong>and</strong> photographed. Formaldehyde<br />
fixed <strong>and</strong> paraffin embedded tissues <strong>of</strong> peritoneum<br />
from EAT bearing mice either treated or untreated<br />
with withaferin A were taken <strong>and</strong> 5ìm sections<br />
were prepared using automatic microtome (SLEE<br />
Cryostat) <strong>and</strong> stained with hematoxylin <strong>and</strong> eosin.<br />
The images were photographed using Leitz-<br />
DIAPLAN microscope with CCD camera <strong>and</strong><br />
the blood vessels were counted.<br />
Quantitation <strong>of</strong> VEGF<br />
EAT bearing mice were treated with or<br />
without withaferin A (7mg/kg/day) for 5 doses<br />
on 6th, 8th, 10th <strong>and</strong> 12th day <strong>of</strong> tumor<br />
transplantation. The animals were sacrificed <strong>and</strong><br />
ascites fluid was collected after 24h <strong>of</strong> each dose.<br />
VEGF-ELISA was carried out using the ascites<br />
fluid (21, 23, 24). In brief, 100µl <strong>of</strong> ascites from<br />
tumor bearing mice either with or without<br />
withaferin A treatment, was coated using coating<br />
buffer (50 mM carbonate buffer pH 9.6) at 4 0 C<br />
overnight. Subsequently, wells were incubated<br />
with anti-VEGF 165<br />
antibodies, followed by<br />
incubation with secondary antibodies tagged to<br />
alkaline phosphatase <strong>and</strong> detection using p-nitrophenyl<br />
phosphate (pNPP) as a substrate.<br />
141<br />
Preparation <strong>of</strong> nuclear extracts<br />
Nuclear extracts were prepared<br />
according to the method previously described (25).<br />
Briefly, cells (5X10 6 ) treated either with or without<br />
withaferin A in complete HBSS for different time<br />
intervals were washed with cold phosphate<br />
buffered saline <strong>and</strong> suspended in 0.5 ml <strong>of</strong> lysis<br />
buffer (20mM HEPES, pH 7.9, 350 mM NaCl,<br />
20% Glycerol, 1% NP-40, 1 mM MgCl 2<br />
, 0.5 mM<br />
EGTA, 0.5 mM DTT, 1 mM Pefablock, 1µg/ml<br />
Aprotinin, 1µg/ ml Leupeptin). The cells were<br />
allowed to swell on ice for 10 min; the tubes were<br />
then vigorously mixed on a vortex mixer for 1<br />
min <strong>and</strong> centrifuged at 10,000 rpm for 10 min at<br />
4 0 C. The supernatant was immediately stored at<br />
-20 0 C.<br />
Electrophoretic Mobility Shift Assay (EMSA)<br />
Nuclear proteins were extracted from<br />
EAT cells treated either with or without withaferin<br />
A for 60,120 <strong>and</strong> 180 min respectively. The EMSA<br />
was performed as described in earlier report (26,<br />
27). The double str<strong>and</strong>ed Sp1 consensus<br />
oligonucleotide probes [5’-d (ATT CGA TCG<br />
GGG CGG GGC GAG C)-3’] were end-labeled<br />
with ã-[ 32 P] ATP. Nuclear proteins (40ìg) were<br />
incubated with 40fmoles <strong>of</strong> ã-[ 32 P]-labeled Sp1<br />
consensus oligonucleotides for 30min in binding<br />
buffer containing 100mM HEPES (pH 7.9),10mM<br />
MgCl 2,<br />
125 mM KCl, 0.5mM EDTA, 4%<br />
glycerol,0.5% NP-40,1ìg <strong>of</strong> poly [dI-dC] <strong>and</strong><br />
1mg/ml BSA. The samples were electrophoresed<br />
in 4% non denaturing polyacrylamide gel in 0.5%<br />
TBE at room temperature for 2 hr at 200V. The<br />
gel was dried, transferred to imaging plate (IP)<br />
<strong>and</strong> the image was scanned by image analyzer<br />
Fujifilm (FLA-5000).<br />
Results<br />
Withaferin A inhibits tube formation <strong>of</strong><br />
HUVECs induced by VEGF<br />
In order to verify if withaferin A interferes<br />
directly with the formation <strong>of</strong> blood vessels by<br />
HUVECs, we performed tube formation assay<br />
Prasanna et al