Activation of caspase-3 protease during the process of ursolic acid and its derivative-induced apoptosis.

The apoptosis-inducing effect of the triterpene saponins, namely, ursolic acid and its natural derivative, methyl-ursolate beta-D-glucoside on A431 human epidermoid carcinoma cells was studied. The cells treated with 5-50 microg/ml of ursolic acid resulted in a dose- and time-dependent decrease in cell number, due to an increase of apoptotic cells as evidenced by MTT assay together with morphological changes. The highest dose (50 microg/ml) of ursolic acid resulted in approximately 90% inhibition in tumor cell growth after 96 hours of treatment and 60% of apoptosis after 48 hours. To the contrary, when the same treatment was carried out with methyl-ursolate beta-D-glucoside, after 96 hours of treatment the percentage of cell growth inhibition was found to be only 30% at the dose of 50 microg/ml and the value of apoptosis did not exceed 10%. Similarly to these results, ursolic acid effectively induced proteolytic activation of caspase-3 protease in a dose-dependent manner while its derivative showed only weak activity in this enzyme assay. The addition of DEVD-CHO prior to ursolic acid and methyl-ursolate beta-D-glucoside treatment effectively prevented the loss of triterpenes-induced viability. In summary, the triterpene saponins investigated contain an apoptotic-inducing activity in A431 cells and in the case of ursolic acid it is associated with proteolytic activation of caspase-3 and/or other similar caspases. Our results also indicated that methylation of COOH-28 together with the glycosylation of C3 of ursolic acid have a strong impact on its antitumor activity.