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