Studies On HepG-2 Cell Apoptosis Induced By Saponins Of Asparagus Through Death Receptor Pathway

Asparagus officinalis Linn is a perennial herb, as a member of Liliaceae Asparagus category, and its scientific name is asparagus. It is a kind of economic crop with a high value both as food and medicine, and its main active ingredients are saponin polysaccharides, flavonoids and amino acid. Each active ingredient has its own corresponding pharmacological effect. Early study showed that asparagus saponins had an inhibition effect on HepG-2 cells in vitro, and could induce apoptosis of HepG-2 cells through the mitochondrial pathway.In this paper, we studied the death receptor pathway of apoptosis induced by asparagus saponins, using apoptosis protein DR4, DR5/TRAIL as the starting point based on previous works. Flow cytometry results showed that, when HepG-2 tumor cells were treated for 48h with50mg/L, 100mg/L, and 200mg/L's asparagus saponins, respectively, the expression of DR4, DR5/TRAIL increased with the increase of asparagus saponin concentration, with a dose dependent manner. These results indicated that apoptosis of tumor cells induced by asparagus saponins was probably through the death receptor pathway:firstly upregulated the expression of DR4, DR5/TRAIL protein, and then activated the upstream start of Caspase-Caspase-8, which then could activate the downstream effect Caspase-Caspase-3, finally induced the apoptosis. However, the death receptor pathway mediated by DR4, DR5/TRAIL could be suppressed because of the inactivation of Caspase-8.The expression of DR4, DR5/TRAIL was not to say there was apoptosis mediated by DR4, DR5/TRAIL. ELISA assay showed that, when HepG-2 cells were treated for 48h with50mg/L, 100mg/L, and200mg/L's asparagus saponins, respectively, the activity of Caspase-8 and Caspase-3 with the increasing concentration of asparagus saponin. And the activity of Caspase-8 in HepG-2 cells treated for in the 48h treat group, while the activity of Caspase-3 was lower than that in the 48h group. All these results indicated that apoptosis of tumor cells induced by asparagus saponins was probably through the death receptor pathway:firstly upregulatied the expression of DR4, DR5/TRAIL protein, and then activated the upstream start of Caspase-Caspase-8, which then could activate the downstream effect Caspase-Caspase-3, finally induced the apoptosis.In order to prove the HepG-2 cell apoptosis induced by asparagus saponins was the combine action of the death receptor pathway and mitochondrial pathway, the link protein Bid was determined by Flow cytometry. And results showed that, when HepG-2 cells were treated for 48h with50mg/L, 100mg/L, and 200mg/L's asparagus saponins, respectively, the expression of Bid protein increased significantly with a dose dependent manner. Bid, as a member of Bcl-2 family, could be activated by Caspase-8. And Bid in cytoplasm got activated, which could downregulate the expression of Bcl-2 protein, weaken the inhibition of apoptosis, and upregulate Bax(a kind of pro-apoptosis protein), enhance its induction of apoptosis, thereby induce the release of Cyt-c of mitochondria into the cytosol to and finally enter the mitochondrial apoptosis pathway. The the two pathways converged in Caspase-3, and finally led to apoptosis.In summary, asparagus saponins could induce the expression of DR4, DR5/TRAIL protein of the death receptor pathway and joined the mitochondrial apoptosis pathway via Bid to induce the apoptosis of HepG-2 cells.