Study On The Apoptosis Of HCT116 Cell Lines Induced By Flavonoids From Spina Gleditsiae

Abstract Objective:Apoptosis refers to the cells in certain physiological or pathological conditions,follow their own procedures,the initiative by the death of gene regulation process,inducing Apoptosis is an important way to treat cancer. Flavonoids from Spina Gleditsiae(FSG)is separated from the Chinese herb Spina Gleditsiae, preliminary results show, FSG has a variety of tumor cell proliferation significantly. In this study, HCT116 cell is selected from the drug-sensitive human cancer cells,by detecting the FSG on cultured human colon cancer cells HCT116 proliferation, morphological changes, the rate of apoptosis and Caspase-3 activity levels of apoptosis were observed from cellular and molecular biology of tumor cell apoptosis induced by FSG, providing experimental evidence for tumor therapy. Methods:Human colon cancer HCT116 cells were cultured with RPMI-1640 medium contained 10% standard fetal bovine serum in 37℃humidified atmosphere with 5%CO2 and passaged every 3 days as a model, Cells in loganthmic phase cells were used for experiments,then treat HCT116 cells with different concentrations(12.5,25,50,100,200)mg·L-1 of FSG after 24,48 and 72 hours'.The HCT116 cell proliferation rate was detected with CCK-8 kit (Cell Counting Kit-8),and drug delt HCT116 cells were treated with conventional HE staining and Hoechst 33258 staining to observe the induction of apoptosis morphological changes;flow cytometry AnnexinV/PI double staining assay was used for caculating the quantification of apoptotic cell apoptosis;and caculating experimental data uses statistical software.Detecting the apoptosis ultrastructure of FSG by transmission electron microscopy;detecting the apoptotic cells' Caspase-3 protein activity induced by FSG with Caspase-3 activity assay kit. Results:After 24,48 and 72 hours'treated in different concentrations (12.5,25,50,100,200)mg·L-1 of HCT116 cells by FSG, the inhibition rates were (0.04±0.1)%,(3.22±1.11)%,(21.71±4.71)%,(47.24±7.56)% and (78.03±10.38)%, drugs 48 hours,the strongest growth inhibition, IC50 is(104±0.96)mg·L-1. Compared with the control group,FSG can inhibit the proliferation of HCT116 cells,with the increase in drug concentration, the stronger the inhibition; FSG role in HCT116 cells after 48 hours,HE staining showed varying degrees of nuclear condensation, cell body smaller, stained stained chromatin condensation,cell attachment rate has dropped and so changed.Hoechst 33258 staining showed that the solution of different concentrations of FSG intervention on HCT116 cells, showing nuclear enrichment, dense clumps of strong fluorescence,nuclear fragmentation and apoptotic bodies and other morphological characteristics of apoptosis.AnnexinV/PI double staining by flow cytometry showed that apoptosis, FSG can induce apoptosis in HCT116 cells.The effect of FSG at the different concentration of (12.5,25,50,100,200)mg·L-1,the apoptosis rates were (8.07±2.19)%,(14.55±3.04)%,(20.83±2.73)%,(35.61±3.76)% and(43.90±7.52)%, and the apoptosis rates increased with the drug concentration increasing larger, and the negative control group,the difference was statistically significant (P<0.01). Transmission electron microscopy analysis showed that, after treated with (25,50 and 100)mg·L-1 FSG,the cells showed mitochondrial swelling, cytoplasmic condensation,nuclear condensation, nuclear chromatin aggregation in the inner nuclear membrane into a block or a new group change shape of apoptosis and so on.Compared with the control group,Caspase-3 protein activity was significantly increased (P<0.05),that may be involved in the induced HCT116 cells to apoptosis is the mechanism.Conclusion:FSG can inhibit the proliferation of human colon cancer HCT116 cells in a dosage dependent; FSG can induce apoptosis in human colon cancer cells HCT116,and enhance the activity of Caspase-3 protein; the activity of the enhanced Caspase-3 protein maybe involved in the FSG inhibition of HCT116 cell proliferation and induced apoptosis mechanism.