7. Anti-tumor Activity of GCP

To test the cytotoxic effects of GCP in cancer cells, we performed following in vitro and in vivo experiments as well as performed clinical trials in hospitals.

A) In vitro cell proliferation test (MTT assay)

The cells used in this assay were: B16/BL-6 mouse melanoma; Colon-26 mouse colon cancer, LE-1 mouse brain vascular endothelial cells; SST-2 rat mammary carcinoma; T24 human bladder cancer; and Du145 human prostate cancer. GCP was firstly dissolved in DMSO, the concentration of GCP was adjusted by culture medium according to their genistein content in GCP (GCP contains about 60 mg of genistein/gram GCP). Final concentration of DMSO was less than 0.1%. The commercially available genistein was used as a control in the same concentration as GCP. The tumor cell suspension was adjusted to 1-2 x10⁴ and incubated at 37ºC for 24 hours. These samples were then added to the cells at a concentration of 100 µg/ml to 0.1 µg/ml one after the other. The cells were cultured for additional 48 hours. Cell proliferation was measured by MTT assay.

Results GCP inhibited various tumor cell proliferations in the range of 10-50 µg/ml which suggests that there might be other cytotoxic substances in GCP.

B) In vivo trial: Tumor-bearing mice

Method: 8 weeks old female C57BL6/Jcl mice weighing approximately 20g, were used in the experiments. Syngeneic B16/BL-6 mouse melanoma cells, transplantable tumor cell lines, were used. B16/BL-6 cells (1x10⁶) were inoculated subcutaneously at day 0. In the first experiment, the mice consumed 3g GCP (containing 1 mg genistein) in their diet for 14 days, starting one week after tumor inoculation. In the second experiment, 0.3 ml GCP (containing 1 mg genistein) was given orally on the same day of tumor inoculation for 21 days. The tumors were removed, weighed, and measured on day 21

Results: GCP administration significantly inhibited tumor growth in both experiments suggesting that GCP has preventive effect as well as therapeutic effect on cancer treatment.

C) Clinical Treatment for Cancer Patients：Case Studies

Currently, GCP clinical trials have been performed on nearly 200 cancer patients and in about 30 civil hospitals. The patients were selected according to medical doctor’s opinion and patient’s desire to partake in the trial. Patients partaking were not required to stop their conventional therapies such as surgery, chemotherapy, radiation, and other immunotherapy. GCP was administrated in the dose of 2-4 gram per day (0.25 gram per capsule). The following are some of the case studied which showed good response to GCP:

Chemotherapy(-), TAE (Transcatheter arterial embolization),.and other immunotherapy- AHCC. Josendaiho-Ton

Effectiveness of GCP：CR, PS=0

No tumor can be found

Case 2: N.M. ♀ 52 years old （S/HP）

Ovarian Cancer, recurrence, Stage Ⅲ

Carcinomatous pleuritis, carcinomatous peritonitis.

Chemotherapy（2000.11), Operation（1997.5.26),

and with other immotherapy：AHCC, Josendaiho-Ton

GCP：August 29, 2000 to present, 2g per day

Tumor marker：CA-125（＜35U/ml）

Effectiveness of GCP: PR, PS=0

Tumor marker decreased suddenly and the chemotherapy became more effective.

T2N0M0, Operation (5/98).

No operation after recurrence.

Tumor marker decreased suddenly and the chemotherapy became more effective. General condition has greatly improved.

Tumor marker decreased suddenly and the chemotherapy became more effective. General condition has greatly improved.

Summary: The case study results described above indicate that GCP exerts its anti-cancer effects in various kinds of cancers. In all the abovementioned cases,

the decrease in tumor marker and improvement in performance status occurred after GCP treatment began, suggesting that combining GCP with other therapies such as low-dose chemotherapy might increase the effects of GCP as well as chemotherapy. These results are identical to other reports showing that combining genistein with chemotherapy more readily induces apoptosis of cancer cells. GCP is the expected adjunctive cancer treatment of the future.