Withaferin A suppresses the expression of vascular endothelial growth factor in Ehrlich ascites tumor cells via Sp1 transcription factor Prasanna Kumar S., Shilpa P. and Bharathi P. Salimath

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Withaferin A suppresses the expression of vascular endothelial growth factor in Ehrlich ascites tumor cells via Sp1 transcription factor Prasanna Kumar S., Shilpa P. and Bharathi P. Salimath Powered By Docstoc
					Current Trends in Biotechnology and Pharmacy
Vol. 3 (2) 138-148, Apirl 2009. ISSN 0973-8916

  Withaferin A suppresses the expression of vascular endothelial
 growth factor in Ehrlich ascites tumor cells via Sp1 transcription
                               factor
                    Prasanna Kumar S., Shilpa P. and Bharathi P. Salimath*
                              Department of Studies in Biotechnology,
                   University of Mysore, Manasagangotri, Mysore-570006, India.
                               For Correspondent : Salimathuom@rediffmail.com




Abstract                                                 order to exert its antiangiogenic activity. These
       In the ayurvedic system of medicine, the          results clearly indicate the antiangiogenic potential
medicinal plant, Withania somnifera Dunal                of withaferin A in modulating antitumor activity.
(Solanaceae) finds application for numerous              Keywords: Ehrlich ascites tumor; Withaferin
ailments including cancer. This herbal plant yields      A; Angiogenesis; Sp1, VEGF.
a host of steroidal lactones called withanolides,
some of which have shown growth inhibition of            Introduction
human tumor cell lines. Withaferin A amongst                   Several natural compounds are recognized
these withanolides reportedly is very active in          as cancer chemo preventive agents. Withanalides
impairing antitumor activity. However; the               are especially well known to suppress tumor cell
underlying molecular mechanisms of this activity         growth via cell-cycle arrest and by the induction
remains still unclear. In the present study, we have     of apoptosis in several tumor cell lines (1-3).
shown that withaferin A inhibited vascular               Moreover, withaferin A inhibits endothelial cell
endothelial cell growth factor (VEGF) -induced           proliferation and angiogenesis in vitro (4).
tube formation by human umbilical vein endothelial       Angiogenesis is essential for the growth,
cells (HUVECs) and angiogenesis in chick                 progression and metastasis of solid tumors (5).
chorioallantoic membrane (CAM) assay. In                 Withaferin A, a member of the withanalides family
Ehrlich ascites tumor (EAT) model, the animals           that is present at high levels in roots and leaves
treated with withaferin A suppressed in vivo, the        of Withania somnifera plant has been found to
peritoneal angiogenesis and microvessel density.         possess antioxidant and antitumor activity (6-9).
When compared to the untreated animals, the              However, the mechanism by which withaferin A
withaferin A treated tumor bearing mice showed           suppresses angiogenesis has not been fully
                                                         elucidated.
a decrease in the volume of ascites and tumor
cell number. Quantitation of VEGF levels in ascites            Vascular endothelial growth factor (VEGF)
from withaferin A untreated or treated tumor             is a major angiogenic factor that facilitates tumor
bearing mice indicated decreased secretion of            growth and metastasis. Hypoxia is known to
VEGF in ascites from treated mice, as measured           induce the expression of VEGF gene (10, 11).
by ELISA. Studies at molecular level revealed            VEGF promoter analysis has revealed several
that withaferin A inhibits binding of Sp1                potential transcription factor-binding sites, such
transcription factor to VEGF-gene promoter, in           as hypoxia-inducible factor-1(HIF-1), activator
                                            Sp1 transcription factor
Current Trends in Biotechnology and Pharmacy                                                           139
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protein (AP)-1, AP-2, early growth response-             Materials and methods
1(Egr-1) and Sp1 (12).                                   Materials
         The GC box-binding protein, Sp1 is a                     Ehrlich ascites tumor (EAT) cells were
ubiquitous transcription factor that belongs to the      routinely maintained in Swiss albino mice in the
Sp family of transcription factors, consisting of        animal house, University of Mysore, Mysore,
Sp1, Sp2, Sp3, and Sp4 (13). Sp1 has been                India. Endothelial growth medium (EGM-2) was
implicated in the transcription of large number of       procured from Cambrex Biosciences,
genes, including housekeeping genes, tissue-             Walkersville, USA. Dulbecco’s modified Eagle’s
specific genes and genes involved in growth              medium (DMEM), fetal bovine serum (FBS),
regulation (13-15). Sp1 activities are regulated         penicillin-streptomycin and trypsin-EDTA were
by a variety of stimuli. Most of these regulations       purchased from Invitrogen, USA.T4
occur through either post-translational                  polynucleotide kinase kit was obtained from
modification or alteration of Sp1 protein                Amersham biosciences. The Sp1 oligonucleotides
abundance.                                               (5’-d (ATTCGA TCG GGG CGG GGCGAG C)-
                                                         3’) for gel shift assays were obtained from
     The principal known post-translational              Promega. Radioacive ã-[32P] ATP was obtained
modifications are phosphorylation and                    from Bhabha Atomic Research Centre (BARC),
glycosylation through the O-linkage of the               Mumbai, India. RNA isolation kit was procured
monosaccharide, N-acetylglucosamine (O-                  from Qiagen, USA. Secondary antibodies
GlcNAc) (16).                                            conjugated to alkaline phosphatase and proteinase
      Expression level of the VEGF mRNA is               inhibitors were obtained from Bangalore Genei,
tightly regulated by both transcriptional and post-      Bangalore, India. The rest of the chemicals were
transcriptional mechanisms. Recent studies have          of analytical grade of purity and were procured
demonstrated that intracellular signaling pathways       locally.
and genetic elements are involved in controlling
its expression. VEGF promoter activity is                Methods
preceded by the activation of transcription factor       Isolation of withaferin A from Withania
Sp1 (17). Therefore it is clear that a constitutive      somnifera roots
Sp1 activation is essential for the differential over
expression of VEGF, which in turn plays an                        Withania somnifera roots were collected
important role in angiogenesis and the progression       locally from Mysore, India. The plant specimens
of cancer. It has also been shown that Sp1 in            were identified and authenticated at the herbarium
particular, plays an important role in tumor             of the Department of Botany, University of
angiogenesis and contributes to the aggressive           Mysore, Mysore, India. The roots were washed,
nature of human pancreatic adenocarcinoma (18).          shade dried and powdered. One hundred grams
                                                         of the root powder was extracted in 500ml of
         In this study, we tested the hypothesis         methanol overnight. Withaferin A was isolated
that the antiangiogenic effect of withaferin A on        from the methanol extract of Withania somnifera
EAT cells involves a reduction in secretion of           roots as previously described (4). The compound
ascites fluid and expression of VEGF, which is           Withaferin A (10mg) was dissolved in 100ìl of
regulated by Sp1 transcription factor. Moreover,         DMSO and diluted 100 times with sterile distilled
we investigated the molecular mechanism by               water to make final concentration 1ìg/ìl and used
which withaferin A inhibits angiogenesis in vivo.        for subsequent experiments.

                                                 Prasanna et al
Current Trends in Biotechnology and Pharmacy                                                           140
Vol. 3 (2) 138-148, Apirl 2009. ISSN 0973-8916

Human Umbilical Vein Endothelial Cells                   Tube formation assay
(HUVECs) culture                                                  Tube formation of HUVECs was
         HUVECs were purchased from                      conducted for the assay of in vitro angiogenesis.
Cambrex Biosciences, Walkersville, USA. The              The assay was performed as described in earlier
cells were cultured in 25 cm3 tissue culture flask       report (20). Briefly, a 96-well plate was coated
(NUNC, Genetix Biotech Asia, Bangalore, India)           with 50µl of Matrigel (Becton Dickinson Labware,
and grown using EGM-2 medium and endothelial             Bedford, MA), which was allowed to solidify at
cell basal medium according to the                       370C for 1 hour. HUVECs (5x 103 cells per well)
manufacturer’s protocol. Incubation was carried          were seeded on the Matrigel and cultured in EGM
out in a humidified atmosphere of 5% CO2 in air          medium containing withaferin A (3.5-14µg) for 8
at 370C. When cells reached confluency, they             hours. After incubation at 370C and 5% CO2, the
were passaged after trypsinization. HUVECs of            enclosed networks of complete tubes from five
passages 2-5 were used for the experiments.              randomly chosen fields were counted and
Animals and in vivo tumor generation                     photographed under an Olympus inverted
          Six to eight weeks old mice were               microscope (CKX40; Olympus, New York, NY)
acclimated for one week while caged in groups            connected to a digital camera at 40X
of five. Mice were housed and fed a diet of animal       magnification.
chow and water ad libitum throughout the                 Chick chorioallantoic membrane (CAM)
experiment. All experiments were conducted               assay
according to the guidelines of the Committee for
the Purpose of Control and Supervision of                         CAM assay was carried out according
Experiments on Animals (CPCSEA),                         to the detailed procedure as described by Gururaj,
Government of India. EAT cells (5×106cells/              A.E. et al. (21, 22). In brief, fertilized chicken
mouse) injected intraperitoneally grow in mice           eggs were incubated at 370C in a humidified
peritoneum forming an ascites tumor with massive         incubator. On the 11th day of development, a
abdominal swelling. The animals show a dramatic          rectangular window was made in the egg shell
increase in body weight over the growth period           and glass cover slips (6-mm diameter) saturated
and the animals succumb to the tumor burden              with 25ng/ml vascular endothelial growth factor
15-16 days after implantation. The number of cells       (VEGF) and VEGF + withaferin A (7ìg) was
increased over the 14 days of growth with                placed on the CAM and the window was closed
formation of 7-8 ml of ascites fluid with extensive      using sterile wrap. The windows were opened
neovascularization in the inner lining of peritoneal     after 48h of incubation and were inspected for
wall. EAT cells from fully grown tumor bearing           changes in the microvessel density in the area
mice were harvested from the peritoneal cavity           below the cover slip and photographed using a
of mice (19). The ascites fluid was collected in         Nikon digital camera.
isotonic saline solution containing 3.8% sodium
                                                         In vivo withaferin A treatment inhibits EAT
citrate. The cells were pelleted by centrifugation
                                                         growth
(3000 rpm for 10 min at 40C). Contaminating red
blood corpuscles if any were lysed with 0.8%                      To determine whether withaferin A
ammonium chloride. Cells were resuspended in             inhibits tumor growth and angiogenesis in EAT
0.9% saline. These cells or their aliquots were          cells in vivo, withaferin A (7mg/kg/day/mouse)
used either for transplantation or for further           and vehicle control (0.1% of DMSO) was injected
experiments.                                             into the EAT bearing mice every alternate day
                                            Sp1 transcription factor
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after 6th day of tumor transplantation and growth        Preparation of nuclear extracts
of the tumor was monitored by taking the body                      Nuclear extracts were prepared
weight of the animals. Animals were sacrificed           according to the method previously described (25).
on the 14th day and the EAT cells along with             Briefly, cells (5X106) treated either with or without
ascites fluid were harvested into the beaker and         withaferin A in complete HBSS for different time
centrifuged at 3000 rpm for 10 min at 40C. The           intervals were washed with cold phosphate
pelleted cells were counted by Trypan blue dye           buffered saline and suspended in 0.5 ml of lysis
exclusion method using a haemocytometer. A               buffer (20mM HEPES, pH 7.9, 350 mM NaCl,
measure of the supernatant gave the volume of            20% Glycerol, 1% NP-40, 1 mM MgCl2, 0.5 mM
ascites fluid.                                           EGTA, 0.5 mM DTT, 1 mM Pefablock, 1µg/ml
                                                         Aprotinin, 1µg/ ml Leupeptin). The cells were
Peritoneal angiogenesis and micro vessel                 allowed to swell on ice for 10 min; the tubes were
density                                                  then vigorously mixed on a vortex mixer for 1
         After harvesting the EAT cells from             min and centrifuged at 10,000 rpm for 10 min at
control and withaferin A-treated animals, the            40C. The supernatant was immediately stored at
peritoneum was cut open and the inner lining of          -200C.
the peritoneal cavity was examined for extent of         Electrophoretic Mobility Shift Assay (EMSA)
neovasculature and photographed. Formaldehyde                     Nuclear proteins were extracted from
fixed and paraffin embedded tissues of peritoneum        EAT cells treated either with or without withaferin
from EAT bearing mice either treated or untreated        A for 60,120 and 180 min respectively. The EMSA
with withaferin A were taken and 5ìm sections            was performed as described in earlier report (26,
were prepared using automatic microtome (SLEE            27). The double stranded Sp1 consensus
Cryostat) and stained with hematoxylin and eosin.        oligonucleotide probes [5’-d (ATT CGA TCG
The images were photographed using Leitz-                GGG CGG GGC GAG C)-3’] were end-labeled
DIAPLAN microscope with CCD camera and                   with ã-[32P] ATP. Nuclear proteins (40ìg) were
the blood vessels were counted.                          incubated with 40fmoles of ã-[32P]-labeled Sp1
                                                         consensus oligonucleotides for 30min in binding
Quantitation of VEGF
                                                         buffer containing 100mM HEPES (pH 7.9),10mM
         EAT bearing mice were treated with or           MgCl 2, 125 mM KCl, 0.5mM EDTA, 4%
without withaferin A (7mg/kg/day) for 5 doses            glycerol,0.5% NP-40,1ìg of poly [dI-dC] and
on 6th, 8th, 10th and 12th day of tumor                  1mg/ml BSA. The samples were electrophoresed
transplantation. The animals were sacrificed and         in 4% non denaturing polyacrylamide gel in 0.5%
ascites fluid was collected after 24h of each dose.      TBE at room temperature for 2 hr at 200V. The
VEGF-ELISA was carried out using the ascites             gel was dried, transferred to imaging plate (IP)
fluid (21, 23, 24). In brief, 100µl of ascites from      and the image was scanned by image analyzer
tumor bearing mice either with or without                Fujifilm (FLA-5000).
withaferin A treatment, was coated using coating
                                                         Results
buffer (50 mM carbonate buffer pH 9.6) at 40C
overnight. Subsequently, wells were incubated            Withaferin A inhibits tube formation of
with anti-VEGF 165 antibodies, followed by               HUVECs induced by VEGF
incubation with secondary antibodies tagged to                    In order to verify if withaferin A interferes
alkaline phosphatase and detection using p-nitro-        directly with the formation of blood vessels by
phenyl phosphate (pNPP) as a substrate.                  HUVECs, we performed tube formation assay

                                                 Prasanna et al
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in vitro. The HUVECs were plated on the                     Withaferin A inhibits VEGF induced neovas-
Matrigel. The HUVECs in the basal medium could              cularization on chick chorioallantoic membrane
not form tubes and VEGF was used to induce                           CAM assay is one of the widely used
the tube formation. In the positive control group           validation assays for formation of new blood
stimulated with VEGF (10ng), HUVECs rapidly                 vessels. In order to further verify if withaferin A
aligned with one another and formed tube-like               is an inhibitor of new blood vessel formation,
structures resembling a capillary plexus within 8           withaferin A was applied on chorioallantoic
hours, after VEGF treatment. However,                       membrane of chick embryo to test its in vivo
treatment with withaferin A prevented VEGF -                antiangiogenic activity. In the CAM assay model
stimulated tube formation of HUVECs in a                    withaferin A induced avasculature zone formation
concentration (3.5-14µg) - dependent manner                 in the developing embryos. Notably newly formed
(Fig. 1). Meanwhile, no cytotoxicity was observed           microvessels were regressed around the area of
under this concentration range of withaferin A              withaferin A treated CAM (Fig. 2).
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.


                                                            Fig. 2: Effect of withaferin A on neovascularization
                                                                    in the chick CAM
                                                                  Withaferin A was applied on CAM of 11-day-
                                                            old 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.

                                                            Withaferin A inhibits growth of EAT cells
                                                            and secretion of ascites in vivo
                                                                    Initially, proliferation of tumor cells in
                                                            mice was monitored by measuring the weight of
Fig. 1: Effect of withaferin A on VEGF induced HUVECs       the animals every day. A decrease in body weight
        tube formation                                      in withaferin A treated animals was observed
                                                            when compared to the increased body weight of
      HUVECs (5X103 cells) cultured in EGM medium
                                                            the untreated tumor bearing mice. It was also
with 3.5µg, 7µg and 14 µg withaferin A was added to
the Matrigel coated 96 wells plate. After incubation        observed that withaferin A lessened the tumor
for 8 hours at 37 0 C, capillary networks were              burden considerably in a dose dependent manner
photographed and quantified (Magnification: X40).           showing the optimum activity at 7mg/kg/dose. Cell
Concentration dependent inhibition of tube formation        number was counted after each dose of withaferin
by withaferin A was recorded. All datas are presented       A treatment. In control group, which is tumor
as mean from three different experiments with triplicates   bearing mice not treated with withaferin A, the
and means of ± S.E.M. P<0.05 vs control.                    number of EAT cells increased exponentially. In
                                               Sp1 transcription factor
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contrast, in the withaferin A treated group, the                 Withaferin A inhibits angiogenesis in vivo
number of cells were drastically decreased (Fig.                          Sprouting of new blood vessels is evident
3A). This implied that withaferin A inhibit tumor                in the inner peritoneal lining of EAT bearing mice.
cell growth in vivo. The volume of ascites was                   The peritoneal lining of EAT bearing animals and
also measured using ascites from EAT bearing                     withaferin A treated mice was inspected for the
as well as withaferin A treated EAT bearing                      effect on tumor-induced peritoneal
animals. The results indicated that withaferin A                 neovascularization. EAT bearing mice treated with
treatment reduces the secretion of ascites fluid                 withaferin A showed decreased peritoneal
(Fig. 3B). It is indicative from this data that                  angiogenesis when compared to the extent of
withaferin A is probably capable of inhibiting the               peritoneal angiogenesis in untreated EAT bearing
proliferation of tumor cell growth in vivo.                      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. 4: Withaferin A inhibits angiogenesis and
                                                                          microvessel density
                                                                 A) Inhibition of peritoneal angiogenesis.
                                                                 EAT bearing mice were treated with and without
Fig. 3: Effect of withaferin A on EAT cell number and
                                                                 withaferin A for four doses (7mg/kg/animal). The mice
        ascites volume in vivo
                                                                 were sacrificed and the peritoneal lining was observed
                                                                 for extent of neovascularization. We presented
       EAT cells (5X106 cells/animal, i.p.) were injected into   representative photograph of peritoneum.
mice. After 6 days of tumor transplantation, withaferin A
(7mg/kg/animal) was injected on days 7th, 9th, 11th and          B) Reduction in microvessel density (MVD)
13th. The animals were sacrificed on days 8th, 10th, 12th        The peritoneum of control as well as withaferin A
and 14th. EAT cells were collected along with ascites fluid.     treated EAT bearing mice was embedded in paraffin
Cells were counted in haemocytometer (A) and ascites             and 5ìm sections were taken using microtome. The
volume was measured (B). At least five mice were used in         sections were stained with hematoxyline and eosine
each group and the results obtained are an average of three      and observed for microvessel density. Arrows indicate
individual experiments and means of ± S.E.M.                     the microvessels.
                                                        Prasanna et al
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Withaferin A inhibits VEGF secretion in                  performed. The results indicated that withaferin
ascites fluid of EAT bearing mice                        A inhibits binding of Sp1 transcription factor to
          In order to verify whether withaferin A        the proximal promoter region of the VEGF gene.
induced inhibition of neovascularization and             In contrast, there was prominent band showing
microvessel density is due to decreased secretion        the binding of Sp1 to the proximal promoter region
of VEGF by EAT cells, we have quantified the             (Fig. 6) when nuclear extract from tumor bearing
secreted VEGF in ascites fluid of control and            mice was used.
different doses of withaferin A treated animals
by ELISA. It is evident in Fig. 5 that withaferin A
inhibits production of VEGF in EAT cells. In EAT
bearing mice, 1280ng of VEGF was detected in
ascites. However in case of withaferin A treated
animal ascites 220ng of VEGF was detected per
mouse.




                                                         Fig. 6: Effect of withaferin A on Sp1-DNA binding
                                                                 activity
                                                          Nuclear extracts were prepared from EAT cells
                                                         untreated and treated with withaferin A. Sp1-DNA
                                                         binding activity was assayed by EMSA using Sp1
Fig. 5: Effect of withaferin A on in vivo production     oligonucleotides.
        and expression of VEGF
EAT bearing mice were injected with withaferin A (7mg/   Discussion
kg/animal) for four doses and ascites fluid was                   Plant and dietary products contribute to
collected after sacrificing the animal every alternate   about one-third of potential anticancer drugs and
day after each dose of treatment. ELISA was carried      the preventive effects of plant-based diets on
out to quantitate VEGF in ascites fluid using anti-
                                                         tumorigenesis and other chronic diseases have
VEGF165 antibodies. Animals bearing EAT cells not
treated with withaferin A was used as control.
                                                         been well documented (28). Withania somnifera
                                                         (L.) Duna1 commonly known as Ashwagandha
Withaferin A inhibits Sp1 DNA binding                    (family Solanaceae) is extensively used in many
activity in EAT cells                                    indigenous preparations. W. somnifera is
         To verify for the involvement of                reported to have anti-inflammatory (29), anti-
transcription factor Sp1 in withaferin A induced         arthritic (30) and anti-tumor (31) activities.
inhibition of VEGF gene expression, an                   Withaferin A, a withanolide was isolated and
electrophoretic mobility shift assay was                 reported to be antiangiogenic and anti-tumor active

                                            Sp1 transcription factor
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principle from Withania somnifera. A recent              expression. Withaferin A is already reported as a
study demonstrated that the anti-angiogenic effect       potent inhibitor of NF-êB and AP-1 DNA binding
of withaferin A was due to the inhibition of             activity (4, 36, 37).
endothelial cell proliferation (4). However, the
detailed molecular mechanisms involved in the                      Recent studies indicated that Sp1
anti-angiogenic effect of withaferin A were not          transcription factor plays an important role in
clearly understood. In this study we investigated        VEGF expression and tumor angiogenesis. A
the anti-angiogenic effects of withaferin A both         region between nucleotide-109 and -61 of the
in vitro and in vivo model. Withaferin A                 VEGF promoter and its intact Sp1-binding sites
suppressed human endothelial cell- tube formation        were required for the inhibition of VEGF promoter
which is one of the hallmarks of angiogenesis            activity. In this study, we found that withaferin A
indicating that withaferin A inhibits endothelial cell   treatment reduced Sp1 DNA binding activity to
proliferation. This may be due to the induction of       the proximal promoter region of VEGF gene in a
HUVECs apoptosis by withaferin A (4). Further,           time dependent manner. It was shown recently
in Ehrlich ascites tumor bearing mice and also by        that celecoxib inhibits VEGF expression and
using several ex-vivo and cell based validation          reduces angiogenesis and metastasis of human
assays, we observed that withaferin A besides            pancreatic cancer via suppression of Sp1 (38).
inhibiting the growth of the tumor suppressed            Sp1 suppression was closely correlated with
peritoneal angiogenesis and microvessel density          reduced VEGF level. Withaferin A has been
by down regulating VEGF gene expression and              reported as potent inhibitor of PKC and TNF
VEGF secretion into the ascites of tumor bearing         dependent IêB kinase â, which subsequently
mice. It also inhibited neoangiogenesis induced          blocks NF-êB nuclear translocation (3, 37). PKC
by VEGF in CAM assay indicating that withaferin          isoforms are also involved in the activation of Sp1,
A targets both tumor and endothelial cell to exert       NF-êB and AP-1 in B16F1 murine melanoma
its anti proliferative and antiangiogenic activities.    cells (39, 40).
                                                                  In summary, our experiments have
         Increased VEGF expression is closely            shown that inhibition of VEGF secretion and tumor
associated with an increase in microvessel density       microvessel formation is one of the potential
(32). VEGF being a permeability factor plays             mechanisms by which withaferin A suppresses
fundamental role in the fluid accumulation and           the growth of Ehrlich ascites tumor. Additionally,
tumor growth in ascites tumor. By secreting              the suppression of VEGF secretion appears to
VEGF, ascites tumor enhances the permeability            be as a consequence of altered Sp1
of preexisting microvessel lining of peritoneal          transactivation and inhibition of VEGF gene
cavity to stimulate ascites formation thereby            expression. The data clearly indicates a novel
tumor progression. Inhibition of fluid accumulation,     mechanism for the antiangiogenic activity of
tumor growth and microvessel density by                  withaferin A and also substantiate the important
neutralization of VEGF has been demonstrated             role of Sp1 in tumor biology and the biological
underlying the importance of VEGF in malignant           basis for the development of new Sp1-targeting
ascites formation (33-35). Our results indicated         agents for cancer treatment.
that there was decrease in the VEGF secretion
in withaferin A treated animals. Inhibition of VEGF      Acknowledgements
secretion could be due to inhibition of activity of             The authors thank Department of Science
transcription factors NF-êB, AP-1 or Sp1 which           and Technology (FIST), Government of India,
are involved in the regulation of VEGF gene              India, University Grants Commission,

                                                 Prasanna et al
Current Trends in Biotechnology and Pharmacy                                                            146
Vol. 3 (2) 138-148, Apirl 2009. ISSN 0973-8916

Government of India, India, and Department of                   brain frontal cortex and striatum. Indian
Atomic Energy (BNRS), for financial support to                  Journal of Experimental Biology, 35: 236-
perform this work. We thank Dr. H.N. Yejurvedi,                 239.
In-charge, animal facility, Department of Zoology,       7.     Bhattacharya, A., Ghosal, S. and
University of Mysore for providing animals for                  Bhattacharya, S.K. (2001). Anti-oxidant
the experiment.                                                 effect      of     Withania    somnifera
                                                                glycowithanolides in chronic footshock
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