Cytotoxic effects of cuphiin D₁ on the growth of human cervical carcinoma and normal cells

Cuphiin D₁ (CD₁), macrocyclic hydrolyzable tannin isolated from Cuphea hyssopifolia, has been shown to exert an antitumor effect both in vitro and in vivo. Furthermore, CD₁ significantly inhibited the growth of the human cervical carcinoma, i.e. HeLa, cells and showed less cytotoxicity to normal primary-cultured cervical fibroblasts. In this study, we explored the cytotoxic mechanism of CD₁ on HeLa cells. The cytotoxic effects of CD₁ showed dose-dependency at 3.15∼100 μg/ml on HeLa for 12, 24, 48 and 72 hours and with an IC₅₀ value at 14.2 μg/ml for 48 hours. However, the IC₅₀ value of CD₁ in primary-cultured normal cervical fibroblasts was 74.5 μg/ml. Therefore, the selectivity shown by CD₁ is ascribed to differences in growth speeds between normal and tumor cells. HeLa cells treated with 50 μg/ml CD₁ for 24 hours exhibited chromatin condensation, indicating the occurrence of apoptosis. Flow cytometric analysis demonstrated the presence of apoptotic cells with low DNA content among HeLa cells. CD₁ also caused DNA fragmentation and inhibited Bcl-2, pro-caspase 3, and inactived PARP expression in HeLa cells. These results suggest that the inhibition of Bcl-2 expression in HeLa cells might account for the mechanism of CD₁-induced apoptosis.