5. Anti-tumor activity of GCP
1) in vitro cell proliferation test (MTT assay)
Method: The cells used in this assay were B16/BL-6 mouse melanoma; Colon-26 mouse colon cancer, LE-1 mouse brain vascular endothelia cell, SST-2 rat mammary carcinoma, T24 human bladder cancer and Du145 human prostate cancer. GCP dissolved in 10% ethanol was adjusted into the concentration of 1 mg/ml GCP (final ethanol was less than 0.1%). The commercially available genistein was taken as positive control by the same concentration in GCP. The tumor cell suspensions were adjusted into 1 ~ 2 x 106/well and incubated at 37oC for 24 h. These samples were added into the cells at the concentration from 100 �g/ml to 0.1 �g/ml serially. The cells were cultured for additional 48 h. Cell proliferation was measured by MTT assay.
Result: The Fig. 3 showed that GCP inhibited various tumor cells proliferation at the ranges of 10 ~ 50 �g/ml concentrations and it is suggested that there might be other cytotoxicitive substances in GCP.
Fig. 3 Effect of GCP on tumor cell proliferation in different
tumor cell lines2) in vivo tumor-bearing mice
Method: Female C57BL6/Jcl mice (8 weeks old), 20 g of body weight approx., were used in the experiments. Syngeneic B16/BL-6 mouse melanoma cells, a transplantable tumor cell line, were used. B16/BL-6 cells (1x106) were inoculated subcutaneously at day 0. In experiment 1, GCP was administered one week later after the tumor inoculation by taken diet (3.0 g diet containing 1 mg genistein per day) for 14 days. In experiment 2, GCP was given by oral administration at the same day of the tumor inoculation (0.3 ml GCP containing 1 mg genistein per day) for 21 days. The tumors were removed at the day 21. The tumor weights were measured.
Result: GCP administration significantly inhibited the tumor growth in both experiments (Fig. 4)
Fig. 4 Inhibition of tumor growth in tumor-bearing mice by GCP
3) Inhibitory effects on angiogenesis by GCP
1. in vivo angiogenesis assay
Method: BALB/c mouse and Colon-26 mouse colon carcinoma cell line were used in the experiment. Stick the filters on the both side of millipore ring (14 mm in out-diameter, 10 mm in inner-diameter) by MF cement and dry it over. Adjusted the cultured Colon-26 cell suspension in PBS (1 x 107 /ml). Filled the millipore chamber by injection of 0.2 ml of the cell suspension through the injection hole and filled in the hole with nylon bar and kept in cold PBS until use. For the negative control, the chamber was filled with PBS. The chamber was inoculated into the back of the mouse subcutaneously and close wound with clips. The mice were oralkt administrated 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.
Result: The results showed that tumor cells in the chamber induced new blood vessels (angiogenesis) as compared with PBS filled chamber, but there were not so many new vessels found in the GCP-treated mouse. It is suggested GCP treatment inhibited the tumor-induced angiogenesis in vivo (Fig. 5).
Fig. 5 In vivo inhibitory effects on angiogenesis by GCP
Control
Non-treated
GCP-treated
Vessels Area 3854
7200 2494 Ratio (%) 12.80
26.89 10.59 2. ex ovo angiogenesis assay by chick choriollantoic membrane (CAM)
Method: This assay is based on the development of a vascular system in the vitelline membrane, which is, upon opening of the egg's shell, directly accessible for observation. When placed onto the vitelline membrane, antiangiogenic substances can inhibit angiogenesis. The GCP dissolved in 10% ethanol were adjusted into the 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 taken as positive control. One % ethanol in PBS was taken as control. Distributions of blood vessels were photographed and analyzed by NIH Image.
Result: GCP significantly inhibited the formation of novel vessels whereas commercial genistein control did not exert such significant angiogenesis inhibitions (Fig. 6). This result suggested that GCP has strong anti-angiogenesis activity.
Fig. 6 Inhibitory effect on angiogenesis by GCP ex ovo
Group No. Area SD Mean Area (%) Inhibition (%) Control 3 87167 1394 38.31 ----- Genistein 3 34300 1536 14.82 61.31 GCP 3 11862 1279 5.21 86.39
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