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

Current Trends in Biotechnology and Pharmacy
Vol. 3 (2) 138-148, April 2009. ISSN 0973-8916
protein (AP)-1, AP-2, early growth response-
1(Egr-1) and Sp1 (12).
The GC box-binding protein, Sp1 is a
ubiquitous transcription factor that belongs to the
Sp family of transcription factors, consisting of
Sp1, Sp2, Sp3, and Sp4 (13). Sp1 has been
implicated in the transcription of large number of
genes, including housekeeping genes, tissuespecific
genes and genes involved in growth
regulation (13-15). Sp1 activities are regulated
by a variety of stimuli. Most of these regulations
occur through either post-translational
modification or alteration of Sp1 protein
abundance.
The principal known post-translational
modifications are phosphorylation and
glycosylation through the O-linkage of the
monosaccharide, N-acetylglucosamine (O-
GlcNAc) (16).
Expression level of the VEGF mRNA is
tightly regulated by both transcriptional and posttranscriptional
mechanisms. Recent studies have
demonstrated that intracellular signaling pathways
and genetic elements are involved in controlling
its expression. VEGF promoter activity is
preceded by the activation of transcription factor
Sp1 (17). Therefore it is clear that a constitutive
Sp1 activation is essential for the differential over
expression of VEGF, which in turn plays an
important role in angiogenesis and the progression
of cancer. It has also been shown that Sp1 in
particular, plays an important role in tumor
angiogenesis and contributes to the aggressive
nature of human pancreatic adenocarcinoma (18).
In this study, we tested the hypothesis
that the antiangiogenic effect of withaferin A on
EAT cells involves a reduction in secretion of
ascites fluid and expression of VEGF, which is
regulated by Sp1 transcription factor. Moreover,
we investigated the molecular mechanism by
which withaferin A inhibits angiogenesis in vivo.
139
Materials and methods
Materials
Ehrlich ascites tumor (EAT) cells were
routinely maintained in Swiss albino mice in the
animal house, University of Mysore, Mysore,
India. Endothelial growth medium (EGM-2) was
procured from Cambrex Biosciences,
Walkersville, USA. Dulbecco’s modified Eagle’s
medium (DMEM), fetal bovine serum (FBS),
penicillin-streptomycin and trypsin-EDTA were
purchased from Invitrogen, USA.T4
polynucleotide kinase kit was obtained from
Amersham biosciences. The Sp1 oligonucleotides
(5’-d (ATTCGA TCG GGG CGG GGCGAG C)-
3’) for gel shift assays were obtained from
Promega. Radioacive ã-[ 32 P] ATP was obtained
from Bhabha Atomic Research Centre (BARC),
Mumbai, India. RNA isolation kit was procured
from Qiagen, USA. Secondary antibodies
conjugated to alkaline phosphatase and proteinase
inhibitors were obtained from Bangalore Genei,
Bangalore, India. The rest of the chemicals were
of analytical grade of purity and were procured
locally.
Methods
Isolation of withaferin A from Withania
somnifera roots
Withania somnifera roots were collected
locally from Mysore, India. The plant specimens
were identified and authenticated at the herbarium
of the Department of Botany, University of
Mysore, Mysore, India. The roots were washed,
shade dried and powdered. One hundred grams
of the root powder was extracted in 500ml of
methanol overnight. Withaferin A was isolated
from the methanol extract of Withania somnifera
roots as previously described (4). The compound
Withaferin A (10mg) was dissolved in 100ìl of
DMSO and diluted 100 times with sterile distilled
water to make final concentration 1ìg/ìl and used
for subsequent experiments.
Prasanna et al