Proliferation Inhibition Of Human Malignant Glioblastoma U87MG And U251MG By Pulsatilla Saponin D

The monomer compounds extracted and identified from the plants are so focused fortheir low cytotoxicity to normal cells and the high apoptosis-promoting to cancer cells.The Pulsatilla saponin D widly exists in plants and especially gathered in the Chinesepulsatilla. In the present study, the Pulsatilla saponin D was investigated to detect theimpact on the U87MG and U251MG and the mechanisms of the apoptosis induced by thiscompound were both in vitro and in vivo.Part Ⅰ Effection and mechanisms of Pulsatilla saponin D on glioblastoma U87MG andU251MG cells in vitroObjective: To investigate the proliferation inbibition and apoptosis induction of thePulsatilla saponin D (that is, the blind test agent Anemone taipaiensis W6monomer) onthe human malignant glioma cell lines U87MG and U251MG in vitro, which mightprovide further basis for the malignant glioma drug research and utilization.Method:1.MTT assay: U87MG、U251MG、EVC304and normal astrocytes was used to detected the effect of the Pulsatilla saponin D. Cells were incubated with Pulsatillasaponin D at centain concentration gradient (0.219nmol/ml,0.438nmol/ml,0.876nmol/ml,1.752nmol/ml,3.504nmol/ml,7.008nmol/ml,14.016nmol/ml and28.032nmol/ml) for24h and72h. Then the IC25、IC50、IC75of the U87MG and U251MG wascalculated through their MTT colorimetric values respectively.2.Flow cytometry assay: After being treated with the Pulsatilla saponin D for12h and16h at different concentration of IC25、IC50、IC75, the U87MG and U251MG were collectedand detected for apoptosis through the Flow cytometry and the DNA contents of the theU87MG and U251MG was also measured.3.Apoptosis morphological observation: After being treated with the Pulsatillasaponin D for6h、12h and24h, the the U87MG and U251MG cells were stained by theHoshest33342and observed under the Fluorescence fiber mirror. Then the cells werecollected and centrifuged in1800r, and dipped into the glutaraldehyde for at least4hours,embedded and observed under the electron transmission microscopy. TUNEL/PI was alsoused to dected the cell apoptosis.4.Western blot analysis：For each channel,30ug of the protein for the U87MG andU251MG cells treated by the Pulsatilla saponin D were load onto the SDS–polyacrylamide gel. The expression of the Caspase-3/8、Bcl-2/Bax and Fas/Fas-L weredetected to declare the apoptotic pathway; And the LC-3Ⅱ/LC-3Ⅰwas rised as the theconcentration of the Pulsatilla saponin D increased.5.DNA fragmentation analysis：The treated U87MG and U251MG were lysed andcentrifuged at12000r for15min at4℃. Then the samples were added to the agarose geland electrophoresed at100V for4h.Result:1.MTT assay: The proliferation inhibition of the U87MG and U251MG treated by thePulsatilla saponin D were87.423%and94.034%(n=6, p<0.05) respectively at14.016nmol/ml with no obvious effect on the proliferation of EVC304and normalastrocyte. The IC50of U87MG and U251MG were7.018nmol/ml and10.132nmol/mlrespectively. 2.Through the flow cytometry analysis, the rate of the early and late apoptosis of theU87MG and U251MG cells treared by the Pulsatilla saponin D were increased, from the1.5%to57%and0.2%to25.8%respectively; Interestingly the Sub-G1peak of the cellswere rose after being treated with the Pulsatilla saponin D.3.The nuclear sharp of the U87MG and U251MG cells, were changed shape afterbeing treated with Pulsatilla saponin D, and stained by Hoechst33342in a time-and-dosedependent manner. For the TUNEL/PI stained assay, the apoptosic cells were stainedorange, and other cells were red.4.Western-blot assay: The total protein was extracted from the U87MG and U251MGcells. The expression of the Caspase-3、8、Bax、Fas/Fas-L and IC-3Ⅱ/LC-3Ⅰ wereshowed to be increased, while the level of Bax expression reduced, and the level of Bcl-2changed unobviously.5.DNA ladder：In the channels of treated U87MG and U251MG cells, the distinctiveladder pattern of DNA cleavage appeared, which was no found in the control.Part Ⅱ The effect of the Pulsatilla saponin D on giloma cells in vivo.Methods:1. Mouse modle bearing Giloma: Male Kunming mouse were picked up from theanimal center of the The Fourth Military Medical University cultured in SPF grade andweighted16.0g to18.0g. The mice were injected the200ul G422cells. After two weeks,the mice were sepreated into three groups: the blank group、the placebo group and theexperimental group. The0.9%NaCl、the culture solution included1‰DMSO and thePulsatilla saponin D were injected into the abdominal cavity of mice at the dose of0.0056mg per gram respectively. The weight of the tumor was measured after7days. Also,the same way of the experiment was repeated on the node mice.2. The tumors from the mouse mode were examined by the HE stained-TUNEL/PIassay and the electron transmission microscopy.Result:1. The weight and size of tumors for the experimental group were lighter than the theblank group and the placebo group; 2. Through HE-TUNEL/PI assay and the electron transmission microscopy, we foundthat nucleus of the apoptosic cells were deep dyeing and cytoplasmic enrichment, theapoptosic cells stained by the TUNEL/PI were yellow, which were indentified with theresults in vitro.Conclusion:1.Pulsatilla saponin D can inhibit the proliferation of human malignant glioma celllines U87MG and U251MG at time-dose dependent manner.2.Pulsatilla saponin D can induce the apoptosis of U87MG and U251MG cellsthrough the Fas/Fas-L and Mitochondrial pathway; The cell cycle was arrested at theG0/G1phase.3.Pulsatilla saponin D can induce the autophagy for U87MG and U251MG at the lowdose and short time.