The Study On Anti-Tumor Effects And Mechamisms Of Sasanquasaponin B

Object: Sasanquasaponin B is a triterpene sapaonins extracted from the roots of Camellia oleifera Abel. Studies have indicated that sasanquasaponin B had the anti-tumor effects on tumor cells, but its mechamism has been not clear. This study was investigated the anti-tumor effects and mechamisms of sasanquasaponin B in vitro and vivo.Methods: In vitro, the effect of sasanquasaponin B on cellular proliferation was measured by MTT assay; cells were exposed to concentrations of sasanquasaponin B for 24 h, stained with Giemsa and Hoechst33342 according to the mamufacturer’s instruction and detected by microscopy; To futher investigate whether the growth inhibition was due to apoptosis, the rate of apoptosis in SMMC-7721 cells and MCF-7 cells treated with sasanquasaponin B was evaluated using the Annexin V-FITC/PI assay; The effect sasanquasaponin B on cell cycle was measured by flow cytometric analysis with PI staining; Monodansylcadaverine(MDC) staining, MDC fluorescence, transmissionelectron microscopy(TEM) analysis and ROS assay were used to explored whether sasanquasaponin B induced autophagy in SMMC-7721 cells and MCF-7 cells; In addition, wesern blot analysis was performed to investigate Bcl-2, caspase3, p53 and LC3 protein express in SMMC-7721 cells and MCF-7 cells treated with sasanquasaponin B. In vivo, the anti-tumor activity of sasanquasaponin B was evaluated. in H22 tumor bearing mice.Results: Sasanquasaponin B inhibited proliferation of SMMC-7721 cells and MCF-7 cells in a time and does-dependent mammer; Compared to untreated cells, cells treated with sasanquasaponin B for 24 h and stained with giemsa showed morphological changes in whole cytoplasm and membrane; Apoptotic morphological charecteristics were founded in SMMC-7721 cells and MCF-7 cells treated with sasanquasaponin B, such as chromatin condensation and muclera fragmentation; Sasanquasaponin B induced apoptosis in SMMC-7721 cells and MCF-7 cells in a does-dependent manner; The percentage of the G2/M phase on SMMC-7721 cells was increased in a dose-dependent manner but sasanquasaponin B had no effect on cell cycle of MCF-7 cells. Meanwhile, sasanquasaponin B induced autophagy with a dose-dependent increase of the vacuole number in SMMC-7721 cells and MCF-7 cells. In addition, sasanquasaponin B could also lead to the reactive oxygen species(ROS) hypergeneration in tumour cells. Flow cytometric analysis indicated that autophagy inhibitor chloroquine inhibited apoptosis rate induced by sasanquasaponin B; Western blotting indicated that sasanquasaponin B down-regulated the expression of Bcl-2, p53 protein and up-regulated caspase3 protein. 24 h after treatment, sasanquasaponin B could promote a does-dependent express of LC3-Ⅱ /LC3- Ⅰ ratio in SMMC-7721 cells and MCF- 7 cells. The co-treatment with sasanquasaponin B and chloroquine evaluated the LC3-Ⅱ/LC3-Ⅰratio and Bcl-2, and decreased caspase 3 expression. In vivo, sasanquasaponin B effectively inhibited weight of H22 tumor tissue, with 0.5mg/kg and 1.0mg/kg resulting in 37.26% and 46.25%, respectively.Conclusion: Sasanquasaponin B induced SMMC-7721 and MCF- 7 cells apoptosis and autophagy with a dose-dependent manner. The mechanisms of sasanquasaponin B involved p53 and ROS pathway. Sasanquasaponin B had an anti-tumor effect on cancer in vitro and vivo.