10.
GCP’s Inhibitory Effects on Angiogenesis
Angiogenesis is the growth of new blood vessels from existing
microvasculature. Tumor growth is a complex interrelationship between tumor
hypoxia and tumor angiogenisis. Tumor hypoxia accelerates angiogenesis and tumor
growth depends on the process of angiogenesis. Therefore, inhibition of angiogenesis
is an important means of controlling tumor growth. Genistein has been widely
reported to inhibit angiogenesis. To reveal whether GCP would have an effect
on inhibition of angiogenesis, we performed the following experiments:
A) GCP Inhibits Angiogenesis in vivo: Dorsal Skinfold Chamber Assay
Method:
BALB/c mouse and Colon-26 mouse carcinoma cell lines were used in the experiment.
The filters were put on both side of the millipore ring (14 mm in out-diameter,
10 mm in inner-diameter) by MF cement and dried over it. The cultured Colon-26
cell suspension in PBS (1 x 107 /ml) was adjusted. The millipore
chamber was injected with 0.2 ml of cell suspension via the injection hole and
filled with nylon bar and kept at cold temperature PBS until used. For the negative
control, the chamber was filled with PBS. The chamber was inoculated into the
mouse subcutaneously and the wound was closed with clips. The mice were orally
administered with/without GCP (0.3 g/day). After 5 days, the chamber was taken
out and the distribution of vessels around the chamber was photographed and
analyzed by NIH Image.
Results: The results showed that tumor cells in the
chamber induced new blood vessels (angiogenesis) as compared with the PBS filled
chamber, but there were not many new vessels found in the GCP-treated mouse.
This suggests that GCP treatment inhibited the tumor-induced angiogenesis in
vivo.
B) GCP Inhibit Angiogenesis ex ovo : Chick chorioallantoic
membrane (CAM)
Method:
This assay is based on the development of a vascular system in the vitelline
membrane which is at the surface of the egg’s shell upon opening, directly
accessible for observation. When placed onto the vitelline membrane, anti-angiogenic
substances can inhibit angiogenesis. The GCP dissolved in 10% ethanol was adjusted
into a concentration of 1 mg/ml genistein by PBS (ethanol concentration was
less than 1%) and added into the eggs through the windows. Commercial genistein
(1 mg/ml ) was used as positive control, and 1 % ethanol in PBS was used as
a control. Distributions of blood vessels were photographed and analyzed by
NIH Image.
Result: GCP significantly inhibited the formation of
new vessels whereas commercial genistein control did not exert such significant
angiogenesis inhibition. This result suggested that GCP has strong anti-angiogenesis
activity, and stronger activity than genestein.