13. Cancer Preventive Effect of GCP

Suppressive Effect of GCP on Diethylnitrosamine (DEN)-initiated and Phenobarbital (PB)-promoted Hepatocarcinogenesis in Male ICR mice

Methods:

Fifty-six 7 weeks old mice, were divided into two groups. During the first week, the animals were injected intraperitoneally (i.p.) with DEN (50mg/kg body weight in 0.9% NaCl). During the second week through the 28^th week, the mice were given PB in their drinking water (0.09%). The mice also received a 1% solution of GCP in their drinking water throughout the whole experiment.

Results A) - Survival curve

DEN+PB treated mice gradually died starting on the 6th week of DEN injections. However, GCP treatment significantly prolonged the survival time of the mice as compared to those in the control group. The survival rates of DEN + PB was 35%, and DEN + PB+ GCP group was 68%.

Results B) The incidence of primary hepatocarcinoma and pulmonary tumor metastasis

The average number of hepatocarcinoma in DEN +PB group was 12.9, and 5.4 in the DEN +PB+ GCP group. GCP tended to reduce the development of hepato-carcinogenesis. GCP also significantly inhibited metastasis to the lungs. Finally, the average number of pulmonary tumors in DEN + PB group was 14.2, and 0.9 in the DEN + PB +GCP group (P<0.005).

The table below shows the incidence between the two groups. All of DEN+ PB group’s mice survived until the end of the experimental period, but were confirmed to have both hepatocarcinoma and pulmonary tumors. However, these incidences were decreased significantly by GCP treatment.

C) Inhibition of expression of glutathion S transferase-π (GST-π) by GCP

The identification of the placemental isozyme of GST-π as a highly expressed cytoplasmic protein is used as a marker of hepatocarcinoma. We measured protein expression levels of GST-π by western blot analysis. As shown in the figure, GCP decreased GST-π expression markedly.

D) Induction of expression of apoptosis-associated molecules by GCP

The p21 protein, which control cell progression through the cell cycle, belongs to a class of tumor suppressors. Caspase-7 has been identified as a main contributor to the execution of apoptosis and regulates the activation of DNA fragmentation enzyme. PARP is a target of the caspase protease activity associated with apoptosis. During apoptosis, PARP is cleaved from 116kDa intact then breaks into 85kDa and 25kDa fragments. Apoptosis-associated proteins such as p21, caspase-7 and PARP were increased by GCP treatment. These results suggest that induction of apoptosis is one of the mechanisms by which GCP works to suppress hepatocarcinogenesis.