A3
Tetrahydrobiopterin in regulation of tumour angiogenesis mediated by PI3K/Akt, eNOS and Ras pathway
Liye Chen, Jihui Wang, Eric O'Neill, Jiliang Li, Russell Leek, David Kerr, Adrian Harris, Shijie Cai
University of Oxford, UK
Background
Emerged evidence suggests endothelial nitric oxide synthase (eNOS) derived-NO
is particularly important in tumour angiogenesis and hence a novel target for
cancer treatment. eNOS activation requires tetrahydrobiopterin (BH4) as a
cofactor for NO production. However, the role of BH4 in eNOS regulation,
potentially involving phosphatidylinositol 3-kinase (PI-3K) signalling
activation, remains to be established. The effects of BH4 in tumour
angiogenesis are not known.
Method
To investigate this pathway, we augmented BH4 levels in vascular endothelial
cells by supplementing cultures with sepiaterin, a BH4 precursor for the pterin
salvage pathway synthesis. We also made a genetically modified murine
fibroblast cell line over-expressing GTP cyclohydrolase I (GTPCH, the
rate-limiting enzyme for the de novo BH4 synthesis) under doxycycline (Dox)
control and analysed the effects in a mouse xenograft.
Results
In cell cultures, sepiapterin increased Akt/eNOS phosphorylation in a dose
dependent manner in COS-7 cells (no endogenous eNOS) transfected with human
eNOS cDNA. This augmentation was abrogated by wortmannin or Ly294002, PI3K
inhibitors. eNOS/Akt phosphorylation by sepiapterin in both HUVEC and bovine
aortic endothelial cells (BAEC) was also significantly enhanced, in association
with increases in NO production, cell proliferation and migration, and
capillarity-like tube formation. Furthermore, sepiapterin greatly increased
GTP-bound wild-type Ras protein. But this effect was diminished by L-NAME, an
eNOS inhibitor. In mouse xenografts, GTPCH over-expression increased the
expression of Ki67 and CD34 in tumour tissue. Conversely, switch off of GTPCH
expression by Dox in drinking water or inhibition of its enzymatic activity by
intraperitoneal injection of DAHP (GTPCH inhibitor) significantly decreased CD34
positive endothelial cells in mouse xenografts. This study demonstrates a
critical role for BH4 in tumour angiogenesis, which is at least partially
mediated by activating the pathway of PI3K/Akt/eNOS/wild-type Ras protein in
vascular endothelial cells.
Conclusion
Our findings suggest that BH4 synthesis may be a rational target for inhibiting
tumour angiogenesis.
