GCP Research

Proceedings from the 8^thSymposium of AHCC and GCP Research Association

A) GCP: Genistein Combined Polysaccharide with anti-angiogenesis and anti-tumor activities in vitro and in vivo

Lan YUAN, Takehito MIURA, Buxiang SUN, Mayumi YOSHITA, Hajime FUJII, Kenichi KOSUNA. Laboratory of Biochemistry, Amino Up Chemical. Co. Ltd., Sapporo 004-0839, Japan.

In this study, Genistein Combined Polysaccharide (GCP) was studied for its anti-angiogenensis activity in vitro, in vivo, and ex ovo. GCP was found to inhibit 50% of mouse brain vascular endothelial cell LE-1 on their proliferation in vitro. In three-dimensional collagen gels for angiogenesis in vitro assay, GCP inhibited the new blood vessel formation. The rate of growth of micriovessels from the perimeter of rat aortic rings embedded in fibrin gel was measured. GCP inhibited 96 % of microvessels growth as compared with the PBS treated group. Both of the inhibitions were much higher than that for purified genistein. A chamber angiogenesis assay in vivo was performed using mouse colon cancer cells, Colon-26, inoculated into the back of the mouse subcutaneously. The results showed that tumor cells in the chamber induced new blood vessels (angiogenesis), yet they were significantly inhibited in the GCP-treated mice. GCP also inhibited chick embryo chorioallantoic membrane (CAM) angiogenesis ex ovo in a dose-dependent manner and was more effective than commercial isoflavone. In the anti-tumor activity studies, GCP inhibited several kinds of tumor cells by induction of apoptosis. GCP significantly inhibited the tumor growth in both melanoma and sarcoma tumor bearing animal models. These results strongly suggest that GCP is an effective antiangiogenic agent that can be used in cancer treatment.

Key Words: isoflavone, genistein, polysaccharide, tumor, angiogenesis

B) Studies on GCP’s ability to inhibit angiogenesis

Paul F. Davis, Bei Xu. Bioactivity Investigation Group, Wellington School of Medicine, P.O. Box 7343, Wellington South, New Zealand

Cancerous tumors need an adequate blood supply in order to grow. Thus, inhibiting the growth of new blood vessels will slow the growth of cancers. Isoflavones such as genistein and GCP contain sugars, polysaccharides, and amino acids. Thus, it is predicted that GCP will inhibit the development of new blood vessels (angiogenesis) and be useful in the treatment of cancers. When mixed with water, some of the GCP dissolves. Testing of this solution at different concentrations showed that it was anti-angiogenic. This suggested that there was a constituent other than genistein that was responsible for this inhibition. When GCP is dissolved in dimethylsulfoxide (DMSO) there was greater than 90% inhibition of angiogenesis at 1.5mg/ml. This was the equivalent of 10 g/ml of genistein. In order to investigate this further, GCP was extracted with ethanol. About 36.5% of the GCP and more than 95% of the genistein dissolved in this solution. However when this was tested in the angiogenic assay, it could not be evaluated since the fibrin gel matrix dissolved. The ethanol-insoluble fraction was extracted with water. This solution contained 48% GCP (mostly sugars and amino acids) but only 1.9% of the genistein was as strongly anti-angiogenic with 50% inhibition of 25 g/ml.

These results and other observations confirmed that GCP is potently anti-angiogenic with both genistein and the water-soluble constituents contributing to its anti-angiogenic properties.

C) Various effects of the co-administration of AHCC and GCP in vitro and in vivo

Buxiang Sun, Ph.D., Amino Up Chemical Co. LTD.; Xubao Shi, Ph.D., Research Scientist, Medical Center of UC Davis; Ralph deVere White, MD., Director of Cancer Center, Medical Center of UC Davis; Robert Hackman, Ph.D., Research Professor, Nutrition Department of UC Davis

Studies have demonstrated that GCP has anti-angiogenic properties which suggests its role as an anti-tumor substance. AHCC, another substance, has show to increase immunoreactivity. Here, we investigated their synergistic anti-tumor effects in vitro and in vivo. Six human cancer cell lines and two mouse carcinoma cell lines were used in the studies. Of eight cell lines, four stem from prostate (LNCaP, PC3, DU145 and TSU-pr1), one from bladder (T24), one from bone (Saos-2), one from lung (3LL), and one from colon (Colon 26). MTT assay was used to measure the inhibitory effects on cell growth. It was found that both GCP and GCP+AHCC could inhibit the growth of all eight cell lines tested in dose-dependent manners, but AHCC did not show obvious inhibitory effect on these cell lines except Colon 26. We examined the expression levels of VEGF using ELISA, and found that GCP can down-regulate the VEGF expression depending on the cell lines. We observed that GCP and AHCC induced apoptosis in several cell lines. PC3 cells were treated with these reagents, and Western blotting analysis of PARP protein and TUNEL assay were used to examine the apoptosis of tumor cells. GCP+AHCC induced obvious apoptotic death when compared to GCP and AHCC alone. Furthermore, a similar effect was observed in T24 and 3LL cells. In addition, GCP+AHCC was also found to up-regulate p21 and down-regulate VEGF in PC-3 cells. GCP+AHCC shows a synergistic effect on inhibition of tumor growth in animal models. Nude mice bearing PC3 were separately treated with GCP+AHCC, GCP and AHCC. Although the inhibitory effects on the tumor growth were observed in all three groups, the inhibition in GCP+AHCC group was much more obvious than in other two groups. The synergistic effect on tumor growth inhibition was also observed in 3LL cells and in Sarcoma 180 cells. When the treatment was stopped in AHCC group and GCP+AHCC group, the PC3 tumors in both groups showed the same growth rate as in the control group untreated. In summary, GCP and AHCC show anti-tumor effects by inducing apoptosis and/or anti-angiogenesis in these cell lines tested. The anti-tumor effects can be increased by using a combination administration of GCP and AHCC.

D) A Mixture of Basidiomycetes Polysaccharide and Genistein (GCP) Inhibits Proliferation and Induces Apoptosis in Human Prostate Cancer Cells in vitro and in vivo

R. Buttyan, Aaron E. Katz, Y. Cao, T. Dorai, C. Olsson. Department of Urology and Pathology, Columbia University, College of Physicians and Surgeons, New York, New York 10032.

In our studies at Columbia University in New York, we have performed several experiments in the laboratory using GCP. It is clear that GCP can have a dramatic effect on Prostate Cancer Cells. GCP can cause Prostatic Cancer Cells to stop growing and dividing. This finding has been duplicated in both cell culture and animal models. While the exact mechanism of actions is unknown, we have found that GCP can block an important regulator of cell cycle function, known as p27. In addition, when cancer cells are exposed to GCP, they undergo a mechanism of cell death known as apoptosis. The death of these cells can occur early, often within a few hours after exposure to GCP. In animals implanted with Prostate Tumors, GCP caused significant regression of these tumors.

GCP may have a number of potential roles in fighting cancer, as well as in cancer prevention. This is an exciting new compound from Japan and may play a significant role in the management of cancer here in the United States.

Proceedings

1. Yuan L. Inhibition of tumor proliferation and angiogenesis by Genistein Combined Polysaccharide in vitro and in vivo. The First International China Angiogenesis Program, Molecular Strategies and clinical development. November 1999, Beijing, China.

2. Miura T, and Yuan L. et al. Anti-tumor and anti-angiogenesis effects of soybean isofalvone aglycones and extracts from cultured Basidiomycetes mycelium. Nippon Nogeikagaku Kaishi. 74: 68, 2000.