| Objective:Gastric cancer(GC)is the fifth most common malignant tumor in the world and the third leading cause of cancer death in both sexes.Although the diagnosis and treatment of GC has progressed in recent decades,the prognosis of patients with GC is still very poor,especially In China,where the five-year survival rate is only about 30%.In our previous studies,we synthesized and identified 24 3’,4’,5’-trimethoxyflavonoid benzimidazole derivatives,of which compound 15(FB-15)is a potential anti-tumor active compound,In this essay,we studied the effects and mechanisms of 3’,4’,5’-trimethoxyflavone benzimidazole derivative 15(FB-15)on the apoptosis of human gastric cancer HGC-27 cells,opens up new ideas and provides theoretical basis for gastric cancer Drug treatment.Methods: CCK-8 was used to detect the survival rate of human gastric cancer HGC-27 cells by FB-15;The clone formation assay was used to detect the proliferation of human gastric cancer HGC-27 cells by FB-15;flow cytometry was used to detect the effect of FB-15 on cell cycle and apoptosis of human gastric cancer HGC-27 cells;flow cytometry was used to detect the effect of FB-15 on mitochondrial potential of human gastric cancer HGC-27 cells;Caspase-3,Caspase-8 and Caspase-9 kits were used to detect the effect of FB-15 on Caspase activity in human gastric cancer HGC-27 cells;Western blot was used to detect the expression of Bad,Bax,Bcl-XL and Bcl-2 proteins;The acute toxicity test of FB-15 by intraperitoneal injection on BABL/c-nu nude mice.Results: CCK-8 results showed that FB-15 had a very significant killing effect on human gastric cancer HGC-27 cells at FB-15 concentrations of 8,16,32,64 and 128μmol/L,and it was better than the positive control drug;The concentration of FB-15 increased,and the killing effect on human gastric cancer HGC-27 cells was stronger and dose-dependent.Under the established FB-15 concentration culture conditions,the longer the incubation time is,the killing effect of FB-15 on human gastric cancer HGC-27 cells is more significant is in a time-dependent manner.Cell cloning experiments showed that FB-15 treatment could significan-tly inhibit the proliferation of human gastric cancer HGC-27 cells in vitro in a dose-dependent manner.The cell cycle results of flow cytometry showed that the proportion of cells in G0/G1 phase was significantly increased and had a concentration effect relationship(from 74.52±0.38% to 85.86±2.05%),while the proportion of cells in S phase and G2/M phase significantly reduced and had a concentra-tion effect relationship(S phase decreased from 6.25 ± 1.92% to 2.32 ± 0.47%;G2 / M phase decreased from 13.55 ± 2.86% to 6.87 ± 1.70%),and there was a statistically significant difference(P < 0.01).It indicated that FB-15 may induce cell apoptosis in human gastric cancer HGC-27 cells by inhibiting cell entry into S phase and blocking cell mitosis in G0/G1 phase,and it has a dose-dependent effect.Flow cytometry showed that the apoptosis rate and total apoptotic rate were significantly increased after FB-15 treatment compared with the control group(the late apoptosis rate increased from 4.72±1.21% to 18.25±16.47%;The total apoptotic rate increased from 16.41±4.05% to 31.88±13.17%,and the difference was statistically significant(P<0.05),indicating that FB-15 induced apoptosis of human gastric cancer HGC-27 cells in a dose-dependent manner.The results of flow cytometry detection of mitochondrial potential show-ed that human gastric cancer HGC-27 cells JC-1 monomer(green fluores-cence)after treatment with different concentrations of FB-15(10,20,40 μmol/L)for 24 h compared with the control group(%)increased from 0.720± 0.364% to 2.710 ± 0.416%,and the difference was statistically significant(P< 0.01),with a dose-dependent effect.It indicated that FB-15 can cause mitoc-hondrial membrane potential loss in human gastric cancer HGC-27 cells,thereby inducing mitochondrial dysfunction.Caspase-3,Caspase-8 and Caspase-9 kits were used to detect Caspase activity.The results showed that after treatment with different concentrations of FB-15(10,20,40μmol/L)for 24 h Caspase-3 activity was 40.263±23.250 rose to 186.591±8.140,Caspase-8 activity increased from 30.544±13.464 to 219.077±13.698,Caspase-9 activity increased from 25.744±8.685 to 154.195±11.171,the difference was statistically significant(P<0.01).It indicated that FB-15 could increase the activities of Caspase-3,Caspase-8 and Caspase-9 in human gastric cancer HGC-27 cells in a dose-dependent manner.Western blot analysis of Bcl-2 family protein showed that the expression of Bad and Bax was up-regulated and Bcl-XL and Bcl-2 expression were down-regulated in a dose-dependent manner after FB-15 treatment of human gastric cancer HGC-27 cells for 24 hours compared with β-actin internal reference,Bax/Bcl-2 value increased from 0.184±0.030 to 1.865±0.522,confirming that FB-15 destroyed the balance of Bcl-2 family and induces apoptosis of human gastric cancer HGC-27 cells,and is detected by flow cytometry,the results of apoptotic detection were consistent.The acute toxicity test of FB-15 by intraperitoneal injection on BABL/c-nu nude mice showed that there was no obvious abnormality in spontaneous activity of saline and FB-15 mice within 0-4 hours after intraperitoneal administration.After continuous administration for 14 days after administration,no animal death was observed in the saline group and the FB-15 group;compared with the control group,the mice in the FB-15 administration group had no statistical difference in body weight on days 4-14,and the animal changes were small.The normal weight gain range of the rats;gross anatomical examination showed no obvious abnormalities on the surface and cut surface of each organ.It was confirmed that under the conditions of this experiment,BABL/c-nu nude mice were injected intraperitoneally with FB-15,and no obvious toxic reaction dose was 150 mg/kg(7.5 mg/mL).Conclusion:1、FB-15 can inhibit the proliferation of human gastric cancer HGC-27 cells;2、the mechanism of apoptosis is to activate the endogenous pathway through the disorder of Bcl-2 family. |
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