Hederagenin from the leaves of ivy (Hedera helix L.) induces apoptosis in human LoVo colon cells through the mitochondrial pathway

Background: Colorectal Cancer has become one of the leading cause of Cancer morbidity and mortality throughout world. Hederagenin, a derivative of oleanolic acid isolated from the leaves of ivy (Hedera helix L.), has been shown to have potential anti-tumor activity. The study was conducted to evaluate whether hederagenin could induce Apoptosis of human colon Cancer LoVo cells and explore the possible mechanism.

Methods: MTT assay was used for evaluating cell viability while Annexin V-FITC/PI assay and Hoechst 33342 nuclear stainining were used for the determination of Apoptosis and mitochondrial membrane potential. DCFH-DA fluorescence staining and flow cytometry were used to measure ROS generation. Real-Time PCR and western blot analysis were performed for apoptosis-related protein expressions.

Results: MTT assay showed that hederagenin could significantly inhibit the viability of LoVo cells in a concentration-dependent and time-dependent manner by IC50 of 1.39 μM at 24 h and 1.17 μM at 48 h. The Apoptosis ratio was significantly increased to 32.46% and 81.78% by the induction of hederagenin (1 and 2 μM) in Annexin V-FITC/PI assay. Hederagenin could also induce the nuclear changes characteristic of Apoptosis by Hoechst 33342 nuclear stainining under fluorescence microscopy. DCFH-DA fluorescence staining and flow cytometry showed that hederagenin could increase significantly ROS generation in LoVo cells. Real-Time PCR showed that hederagenin induced the up-regulation of Bax and down-regulation of Bcl-2, Bcl-xL and Survivin. Western blotting analysis showed that hederagenin decreased the expressions of apoptosis-associated proteins Bcl-2, procaspase-9, procaspase-3, and polyADP- ribosepolymerase (PARP) were increased, while the expressions of Bax, Caspase-3, caspase-9 were increased. However, there was no significant change on Caspase-8.

Conclusions: These results indicated that the disruption of mitochondrial membrane potential might contribute to the Apoptosis of hederagenin in LoVo cells. Our findings suggested that hederagenin might be a promising therapeutic candidate for human colon Cancer.