Two steroidal saponins from Camassia cusickii induce L1210 cell death through the apoptotic mechanism

Two steroidal saponins, tigogenin hexasaccharide-1 (TGHS-1, (25R)-5α-spirostan-3β-yl 4-O-[2-O-[3-O(α-L-rhamnopyranosyl)-β-D-glucopyranosyl]-3-O-[4-O- (α-L-rhamnopyranosyl)-β-D-glucopyranosyl]-β-D- glucopyranosyl]- β-D-galactopyranoside) and tigogenin hexasaccharide-2 (TGHS-2, (25R)-5α-spirostan-3β-yl 4-O-[2-O-[3-O- (β-D-glucopyranosyl)-β-D-glucopyranosyl]-3-O-[4-O-(α-L- rhamnopyranosyl)-β-D-glucopyranosyl]-β-D-glucopyranosyl]- β-D-galactopyranoside), were isolated from the fresh bulbs of Camassia cusickii. In murine leukemic L1210 cells, both compounds showed cytotoxicity with an EC₅₀ value of 0.06 μM. The morphological observation revealed that TGHS-1 and TGHS-2 induced shrinkage in cell soma and chromatin condensation, suggesting apoptotic cell death. The cell death was confirmed to be apoptosis by Annexin V binding to phosphatidylserine in the cell membrane and excluding propidium iodide. A typical apoptotic DNA ladder and the cleavage of caspase-3 were observed after treatment with TGHS-1 and TGHS-2. In the presence of both the compounds, cells with sub-G₁ DNA content were detected by flow cytometric analysis, indicating that TGHS-1 and TGHS-2 (each EC₅₀ value of 0.1 μM) are the most powerful apoptotic saponins known. These results suggest that TGHS-1 and TGHS-2 induce apoptotic cell death through caspase-3 activation.