| ObjectiveTo screen the anti-cancer activities of Daphniphyllum alkaloids from Daphniphyllum oldhami and calycinum in vitro, obtain the effective anti-tumor chemical composition which were investigated to explore the anti-tumor mechanism of action, and provide the scientific basis for developing new anticancer drugs from these compounds.Methods1. Anti-cancer activities in vitro was investigated by MTT assay on five human tumor cell lines A-549, MCF-7, HepG-2, HeLa and U-251, and half maximal inhibitory concentration (IC50) of 8 Daphniphyllum alkaloids was measured in the 24h,48h,72h after acted on the tumor cells.2. In order to better observe drugs effect in different time or concentrations and time-effect relationship and dose-effect relationship changes, four kinds of Daphniphyllum alkaloids (Deoxycalyciphylline B, Daphnioldhanin D, Daphnioldhanin E, daphnioldhanine H) that could inhibit the proliferation of tumor cell were reduced the span of a concentration gradient of the compounds (2-fold dilution) in the next experiment.3. To study the the anti-tumor mechanism of Daphnioldhanin E that has the best anti-tumor activity, optical microscope observation of Wright-Giemsa staining, fluorescence microscope observation of FDA-PI staining, DNA agarose gel electrophoresis, flow cytometry, protein analysis and other means were conducted.Results1. Preliminary screening of the anti-tumor activity of the Daphniphyllum alkaloids The results showed that four kinds of alkaloids (deoxycalyciphylline B, Daphnioldhanin D, Daphnioldhanin E, daphnioldhanine H) displayed a certain inhibiting action on proliferation of above five tumor cells,especially on the HepG-2 cells,IC50 were 4.94,2.24,1.17,3.44μg/ mL in the 72h separately;2. The study of time-effect relationship and dose-effect relationship With increasing drug concentrations, the inhibition rate of four kinds of the Daphniphyllum alkaloids on HepG-2 cells was increased and the inhibition had a good time-effect relationship and dose-effect relationship. In addition, IC50 of the 4 compounds were less than 10μg/mL in the 72h, had a clear dose-dependent relationship, in which the role of Daphnioldhanin E was the best and the IC50 reached 5.70,1.29,0.75μg/mL in the 24h,48h,72h separately;3. Research for the mechanism of action①Morphology:Under an optical microscope of Wright-Giemsa staining we observed the volumeof HepG -2 cell increased, the nucleus was clearly visible and surrounded by a large number of cytoplasmic vacuoles, but the cell membranes remained intact after the cells were treated with small doses of Daphnioldhanin E (10μg/mL). With the increase of drug concentration and treatment time, cell membrane integrity was destroyed, and ultimately caused cell nuclear fragmentation, and accompanied by significantly reducing the density of the cells.②DNA ladder electrophoresis:Agarose gel electrophoresis appeared a large number of small fragments of 50-200bp and no apoptotic characteristic DNA ladder after the treatment of Daphnioldhanin E on HepG-2 cells.③Flow cytometry:Flow cytometry detected that apoptotic rate of early apoptotic cells was no significant change (P> 0.05) than normal cells and apoptotic rate of later apoptotic cells or necrosis rate obviously increased(P<0.05) after small doses of Daphnioldhanin E (10μg/mL) on HepG-2 cells in the 24,48,72h, the difference have statistical significance;while apoptotic rate of early apoptotic cells increased than normal cells (P<0.05) the high dose group (30μg/mL) at above three time points, but was not significant and were respectively (6.34±0.89)%, (9.16±0.32)%, (11.67±0.40)%, apoptotic rate of later apoptotic cells or necrosis rate also visible increased(P<0.05), the difference have statistical significance④Fluorescence microscope:Fluorescence microscope displayed that control group and Daphnioldhanin E group based in green fluorescence, indicated that cell membrane of two groups of cells did not damaged and PI failed to stain, suggested the cells were not a large number of necrosis in the early of Daphnioldhanin E on HepG-2 cells;while green fluorescence of apoptotic group visible increased, suggested cell death or later apoptotic also added.⑤Study of antitumor activity of apoptosis inhibitor Z-VAD-FMK on Daphnioldhanin E:Two concentrations of Daphnioldhanin E were not the impact by Z-VAD-FMK but showed specific killing effect. Caspase-3 activity detected that Daphnioldhanin E treatment group compared with the control group, caspase-3 activity of HepG-2 cells was no obvious change, indicated that cell caspase-3 had not been activated in the course of cell death.Conclusion1. Four of eight Daphniphyllum alkaloids (deoxycalyciphylline B, Daphnioldhanin D, Daphnioldhanin E, daphnioldhanine H) on five kinds of tissue-derived tumor cell lines significantly inhibited, and showed time and dose dependent, which inhibitory activity of Daphnioldhanin E on HepG-2 cells was the best;2. Daphnioldhanin E impossibly influenced the proliferation of HepG-2 cells by Paraptosis and brought into full play of anti-tumor, might develop into a new antitumor drugs. |
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