ABSTRACT
Objective: To screen the differentially expressed proteins of saponins in Pulsatillae Radix inhibiting the proliferation and induce apoptosis on NCI-H460 tumor cells based on proteome technology using nano LC-LTQ-Orbitrap-MS/MS, and preliminarily speculate the potential mechanism. Method: NCI-H460, SK-OV-3 and SGC-7901 tumor cells were cultured in vitro. Methylthiazoletetrazolium (MTT) assay was used to detect the inhibitory rate of saponins in Pulsatillae Radix on three tumor cell lines. Effect of saponins in Pulsatillae Radix on apoptosis was analyzed by Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining flow cytometry and 4',6-diamidino-2-phenylindole (DAPI) staining. Apoptosis was analyzed using flow cytometry and DAPI stain. Nano LC-LTQ-Orbitrap-MS/MS was used to investigate the changes in the protein profiles on NCI-H460 cells treated with saponins in Pulsatillae Radix. Proteins exhibiting differential expression were analyzed by DAVID Bioinformatics Resources 6.8 and Kyoto encyclopedia of genes and genomes (KEGG) database. The differentially expressed proteins were verified by Western blot. Result: Saponins in Pulsatillae Radix could inhibit the proliferation of NCI-H460, SK-OV-3 and SGC-7901 tumor cells and induce apoptosis of NCI-H460 tumor cells. Effect of Saponins in Pulsatillae Radix on the proliferation and apoptosis of NCI-H460 tumor cells was mainly related to the regulation of biological function of ribosome, glycolysis/gluconeogenesis and other biological processes. It was possible to induce apoptosis of NCI-H460 tumor cells by interfering mitogen-activated protein kinase (MAPK) signaling pathway and regulating the Caspase pathway. Conclusion: Saponins in Pulsatillae Radix can inhibit the proliferation and induce the apoptosis of NCI-H460 tumor cells, the mechanism may be related to the intervention of MAPK signaling pathway and the regulation of Caspase pathway. These findings are helpful to elucidate the molecular mechanism of the anti-tumor effect of saponins in Pulsatillae Radix.
ABSTRACT
Objective:To study the mechanism of anti-proliferative effect of lupeol on highly metastatic human hepatocellular car-cinoma HCCLM3 cells. Methods:CCK-8 assay was performed to evaluate the effects of lupeol at different concentration on cell viability in 12-48 h. Caspase inhibitors were used to identify the subtypes of caspases activated during lupeol-induced cell death. The effects of lupeol on the mRNA expression of caspase family and Bcl-2 related genes were detected by real-time PCR. The effects of lupeol on HC-CLM3 cell phase distribution were investigated by flow cytometry. Results:Compared with the control group, lupeol could inhibit HC-CLM3 cell proliferation in a concentration-dependent manner with IC50 of 93 μmol·L-1 in 24h. The number of HCCLM3 cells in the period of G2/M was increased by 1-fold when the lupeol concentration was within 60-100 μmol·L-1 . Lupeol could activate the path-way of caspase, and the mRNA expression of caspase-3 was elevated by 50%-150% when compared with that in the control group. Mo-reover, the mRNA expression of p53 and Bax were increased above 1-fold by lupeol at 100 μmol·L-1 , and the Bcl-2 and PARP ex-pression were significantly suppressed by lupeol at 60-100 μmol·L-1(P<0. 05 or P<0. 01). Conclusion:The results indicate that lupeol has anti-proliferative effect on the liver cancer cells, which is beneficial to the prevention and treatment of liver cancer.
ABSTRACT
Objective: To investigate the neuroprotective effects of paeoniflorin (PF) against cell death induced by hydrogen peroxide (H2O2) in human neuroblastoma cells and its mechanisms. Methods: H2O2 was used to induce SH-SY5Y cell damages, and the cell survival rate was detected by CCK-8 assay; the cell morphologic changes were observed by inverted optical microscope; the apoptosis was tested using Hoechst 33258 staining; flow cytometer (FCM) and propidium iodide staining were used to analyze the apoptosis and cell cycle alteration; reactive oxygen species (ROS) production was determined by 2', 7'-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence; lactic dehydrogenase (LDH) release was detected by reaction of diaphorase and INT; 8-OHdG production was determined by enzyme-linked immunosorbent assay (ELISA); caspase-3 activity was determined by caspase-3 catalyzing the specific substrate. Results: Compared with control group, after the treatment with H2O2 (200 μmol/L) for 24 h, the viability and proliferation index of CH-SY5Y cells were significantly decreased (P < 0.01, 0.05), the apoptosis rate and content of 8-OHdG were increased (P < 0.01), the LDH resease and ROS production were increased (P < 0.01); the activity of caspase 3 was increased (P < 0.01). Compared with H2O2 injury group, PF (20-40 μmol/L) significantly ameliorated the changes in SH-SY5Y cells induced by H2O2 in concentration-related manner (P < 0.05). PF (10 μmol/L) did not significantly change the above indexes except the cell viability, ROS, and caspase-3 activity induced by H2O2 (P < 0.05). Conclusion: PF has the significant protective effect against the H2O2-induced cell injury, which may be related to eliminatinging ROS, alleviating DNA oxidative damage, regulation of cell cycle, and inhibition of apoptosis of caspase pathway activation.