The Effect And Mechanism Of Bovine Lactoferrin Antimicrobial Peptide Anti-gastric Cancer

Objective To investigate the effect of lactoferrin antimicrobial peptide（Lfcin B）on the proliferation,the cell cycle and apoptosis of human gastric cancer cell line MGC803,and the growth of transplanted tumor in vivo,and provide a basis for the development of new ways and new methods to treat gastric cancer.Methods From the two aspects to implement the experiment.In vitro experiments,Lfcin B was used in gastric cancer cell line MGC803 to detect its effect on cell proliferation,apoptosis and cycle,and to detect its effect on related gene expression.Specific experimental procedure were as follows,1.The proliferation of MGC803 cells at different concentrations of Lfcin B were evaluated by CCK-8 assay for 24,48 and 72 h,respectively.2.Flow cytometry（FCM）was performed to detect the effect of Lfcin B on MGC803 cells apoptosis.3.The expression of apoptotic-specific genes,such as bcl-2,bax and caspase-3 was assessed by RT-PCR and Western blot analysis.In vivo experiments were carried out to construct animal model of nude mice（BALB/c An N-nu/nu）bearing human gastric cancer,experimental procedure as follows: 1.MGC803 cells were inoculated into nude mice subcutaneously for modeling and divided into four groups: normal saline group,Lfcin B high dose group,Lfcin B low dose group,cisplatin control group.High dose and low dose of Lfcin B were injected intraperitoneally into the abdomen of nude mice to record the growth of transplanted tumor.2.The transplanted tumor tissue was used for immunohistochemistry to observe the changes of apoptosis-related protein index.3.TUNEL experiments were performed to detect the apoptotic status in different concentration groups.4.The changes of different proteins in tissues were detected by Western blot.Results In vitro experimental,1.Different concentrations of Lfcin B on gastric cancer MGC803 cells proliferation all had inhibitory effect,but the inhibition is not obvious at24 h,then the maximum inhibition rate can reach（30.62 ± 0.72）% at 48 h.72 h inhibition rate was（42.06±0.72）%,and the difference was significant compared with the negative control group（P <0.05,P<0.01）.2.Lfcin B could promote the apoptosis of gastric cancer MGC803 cells,and the apoptotic rate was the highest at 72 h with（25.78± 1.74）% and had a time and dose-dependent（P <0.05,P <0.01）,DDP group is the main role of early cell apoptosis.3.Cell cycle results showed that the number of G0/ G1 phase cells decreased slightly,S phase cells gradually increased compared with the negative control group,The above results indicated that Lfcin B in 10^-610_-2 mg / ml concentration could affect the cycle of MGC803 cells,which may act on the S/G2 phase,then cells were blocked in the S phase,which with the role of prolonged time-dependent,but the overall trend is not particularly obvious.4.Lfcin B significantly inhibited m RNA expression of bcl-2 gene and promoted the m RNA expression of bax and caspase-3 genes in MGC803 cells,and decreased or increased with the increase of Lfcin B concentration（P <0.05,P <0.01）.5.Western blot results showed that Lfcin B could efficiently inhibit the expression of Bcl-2 protein and promote the expression of Caspase-3 and Bax protein（P <0.05,P <0.01）.In vivo experimental,1.According to the tumor weight,tumor volume changes indicated that Lfcin B could inhibit the growth of transplanted tumor,but although the high dose of Lfcin B was doubled than low dose,the anti-tumor effect was not equal growth,but similar to the same.2.Immunohistochemistry showed that Bcl-2 protein was expressed in the negative control group and was expressed in the low dose group and almost no expression in the high dose group.While Bax and Caspase-3 were almost nonexistent in the control group and higher in the high and low dose groups.3.The apoptotic index（AI）was（1.84 ±0.39）%,（8.46±1.01）%,（11.63±1.92）%,（16.54±2.81）%,respectively.4.The expression of Bax and Caspase-3 protein increased,and the expression of Bcl-2 protein decreased with the increase of dose,and the difference was significant compared with the negative group（P <0.05,P <0.01）.Conclusion 1.Lfcin B can significantly inhibit the growth of MGC803 gastric cancer cell line,induce apoptosis,and act on S/G2 phase of MGC803 cell cycle,so as to block it in S phase.2.Lfcin B significantly inhibit the growth of transplanted tumor,and can induce apoptosis of transplanted tumor cells.