Function And Mechanisms Of Triterpenoid Taipaienoside-A On Human Glioma U87cells

Objective Recently we isolated several novel triterpenoid saponins from the root ofAnemone taipaiensis (the species distributed locally in Qinling Mountains of China), andfound taipaienoside A possess remarkably proliferation-inhibiting activity in glioblastomacells in preliminary experiment. Our research attempted to declare how this novel saponininduced glioblastoma cells proliferation suppresses，apoptosis, as well as the molecularmechanisms involving in these biological activities via U87and VEC304cell lines assaysin vitro and provide basic evidences for new drug development.Methods1. MTT assay was used to detect the viability of U87and VEC304cell lines treated byvarious concentrations (0、0.5、1、2、4、8、16ug/ml) of taipaienoside A diluents. 2. Flow cytometric analysis was adopted to investigate distribution changes of cell cyclephases, which has been treated by taipaienoside A in various concentrations(1.13μg/ml,1.75μg/ml,2.73μg/ml) for0,8,16, or24h.3. Hoechst33342staining assay was applied to U87cells treated by variousconcentrations of diluents for6,12,18,24h. Cytomorphology change was observedfrom fluorescence microscope.4. Transmission electron microscope（TEM）was also applied to reveal ultrastructure ofU87cells treated by taipaienoside-A.5. The proportions of apoptotic cells were evaluated by Flow cytometric Annexin V/IPstaining analysis.6. To probe the apoptotic mechanisms induced by taipaienoside-A, we examined theexpression level of Fas, Fas-L, Pro-caspase8, Pro-caspase3, Bcl-2, and Bax in U87cells via Western Blot analysis.Results1. The viability of U87cells declined significantly after treated with taipaienoside-A atextremely low concentrations (1,2,4and8μg/ml) in dose dependent manner, whilehuman normal cells VEC304was not decreased significantly in compare.2. Data from flow cytometric analysis showed the numbers of cells distributed in S phasesignificantly increased accompanied with decreased proportion of cells in G1phaseafter U87cells were treated with taipaienoside-A.3. Typical apoptosis morphology changes, including nuclear fragmentation, chromatincompaction, cell shrinkage and membrane integrity loss or deformation, were observedafter U87cells treated by various concentration (0，1.13μg/ml,1.75μg/ml,2.73μg/ml)of taipaienoside-A diluents for6hours in Hoechst33342staining assays. And thisphenomenon tended to more obviously in time-and doses-dependent manners.4. Electron microscope assays showed typical and extensively condensation of chromatinadjacent to the nuclear envelope after U87cells were treated by taipaienoside-A for12h. Apoptotic body was observed more obviously after24h.5. Annexin V/PI staining assay showed that the proportions of apoptosis cells, at early stage, increased time-dependently after U87cells were treated respectively withtaipaienoside-A (1.75μg/ml) for6h,12h and24h.6. Western-blot assay detected that the expression level of Fas and Fas-L were bothsignificantly up-regulated, meanwhile the level of both pro-caspase-8and-3reducedsignificantly in time-dependent manner after treated U87cells with taipaienoside-A(1.75ug/ml IC50) for6,12,24h. Cleaved caspase8and cleaved caspase3were bothobserved, and the former peaked at6h early then the other. Bcl-2revealed nostatistically changes. The expression of Bax was undetectable in our study.Conclusion1. Taipaienoside-A induced U87cell death on extremely low concentration, while theviability of normal cell lines VEC304was not decreased. This phenomenon revealedthat taipaienoside-A possesses specific tumor suppressing activities.2. Taipaienoside-A can block U87cell cycle in S phase, revealed that taipaienosideA-induced U87cells inhibition was at least invovled with cell cycle-inhibitingactivity.While the mechanisms involved was unknown, implying that this compoundpossesses extremely research value.3. By the proof showed in this study, we confirmed that taipaienoside-A inducesapoptotic cell death on treated U87cells. And the apoptosis-inducing activity followeddoes-and time-dependent manners.4. The assays we applied to detect the mechanism of taipaienoside A-induced apoptosisrevealed that the expression level of some apoptosis-related protein on U87cellstreated by taipaienoside-A matches Fas-inducing apoptosis pathway. Therefore, wededuced that taipaienoside A-induced U87cells apoptosis was at least involved withFas/fasL associated death receptor pathway.5. All this data confirmed that taipaienoside-A involves potent apoptosis-inducing andproliferation-inhibiting activity. So we propose taipaienoside-A possesses considerablepharmaceutical research value.