| Objective: To explore the effects of anti-cancer bioactive peptide(ABP)combined with oxaliplatin(OXA)on TOP2 A protein and its downstream Wnt/β-catenin in human gastric cancer cell lines MKN-45 and MKN-74 The influence of key factors in the signal pathway and the expression of TOP2 A in nude mice bearing gastric cancer.To preliminarily explore the possible mechanism of anti-cancer bioactive peptides combined with traditional chemotherapeutics for anti-tumor therapy.Methods: The GEPIA platform website(http://gepia.cancer-pku.cn/)was used to analyze the expression of TOP2 A in tumors and its expression in gastric cancer tissues of different pathological stages.The normal human gastric mucosal epithelial cells GES-1 and gastric cancer cell lines SGC-7901,BGC-823,MKN-45,MKN-74 were cultured in vitro,and the expression of TOP2 A protein was detected by Western-Blot method.Select MKN-45 and MKN-74 cells to be cultured in vitro and treated with control group(Control),biologically active peptide group(ABP),oxaliplatin group(OXA),and biologically active peptide combined with oxaliplatin group(AO)respectively After 24 h,observe the cell morphology of each group and use Western-Blot method to detect the expression of TOP2 A protein in each group.Forty SPF nude mice(male)aged 6 to 8 weeks old with a weight of(14±2)g were adaptively reared for 5 days,and randomly divided into control group(Control),biologically active peptide group(ABP),and oxali.Platinum group(OXA),bioactive peptide combined with oxaliplatin group(AO),10 rats in each group.MKN-45 cell suspension of 0.2m L and density of 1×106/m L was inoculated at 0.3 cm of the back of the armpit of the left forelimb of nude mice,and the tumor-like tumors could be palpated at the inoculation site in about 10 days.Modeling is successful.Drug intervention was given when the tumor body grew to about 1cm in diameter.Control group(normal saline 0.5m L/mouse),ABP group(inducible peptide 30ug/m L,0.5m L/mouse),OXA group(oxaliplatin 20ug/m L),A-O group(inducing peptide 0.25 m L/head + oxaliplatin 0.25 m L/head).The method of administration is intraperitoneal injection,once a day,for a total of 2 weeks.Two weeks later,the mice were sacrificed by neck removal,and the tumors were taken,and the tumor volume of each group was measured and the expression of TOP2 A in the tumors was detected by immunohistochemistry.The gastric cancer cell lines MKN-45 and MKN-74 were cultured in vitro and treated with Control,ABP,OXA,and AO for 24 hours,and Western-Blot method was used to detect the levels of the related factors LEF1,TCF4 and MMP7 in the Wnt/β-catenin pathway in each group.Express the situation.Results:(1)According to GEPIA data analysis,the expression of TOP2 A in gastric cancer tissues was higher than that in normal tissues(P<0.05).(2)Compared with normal human gastric mucosal epithelial cells GES-1,the expression of TOP2 A protein in the four gastric cancer cell lines SGC-7901,BGC-823,MKN-45,and MKN-74 increased(P<0.05),and MKN-The expression of TOP2 A protein in the two cell lines 45 and MKN-74 increased significantly.(3)Compared with the Control group,the MKN-45 and MKN-74 cells treated with ABP,OXA and A-O for 24 hours showed vacuoles,poorer adherence,and rounded cell morphology.(4)Comparing the tumor volume of nude mice bearing gastric cancer in each group,it was found that the tumor volume of ABP,OXA and A-O group was smaller than that of Control group(P<0.05).(5)Western-Blot detection showed that compared with the Control group,the expression of TOP2 A protein in MKN-45 and MKN-74 cells treated with AO decreased(P<0.05),while TOP2 A treated with ABP or OXA There was no significant difference in protein expression changes(P>0.05).(6)The immunohistochemical results of tumors in nude mice bearing gastric cancer showed that there was no significant difference in the expression of TOP2 A between the ABP group and the OXA group compared with the Control group(P>0.05),and the expression of TOP2 A decreased in the AO group compared with the Control group(P<0.05)(7)Western-Blot method showed that in the MKN-45 cell line,compared with the Control group,there was no significant difference in the expression of TCF4 protein after the cells were treated with ABP and OXA for 24h(P>0.05),The expression of LEF1 and MMP7 protein decreased(P<0.05).In the MKN74 cell line,compared with the Control group,the expression of TCF4 and MMP7 protein decreased after24 h of ABP treatment of cells(P<0.05),but there was no significant difference in the expression of LEF1 protein(P>0.05).Cells were treated with OXA After 24 hours,the expression of TCF4,LEF1 and MMP7 proteins all decreased(P<0.05).Compared with the Control group,the expressions of related factors TCF4,LEF1 and MMP7 in the Wnt/β-catenin pathway in MKN-45 and MKN-74 cells treated with A-O for 24 hours were decreased(P<0.05).Conclusion:(1)TOP2A is highly expressed in gastric cancer tissues and cells,suggesting that it is related to the occurrence and development of gastric cancer.(2)Inhibiting the expression of TOP2 A protein is one of the mechanisms of anti-cancer bioactive peptide combined with oxaliplatin in inhibiting the growth of gastric cancer cells and nude mice bearing gastric cancer.(3)Anti-cancer bioactive peptides combined with oxaliplatin may indirectly regulate the expression of Wnt/β-catenin signaling pathway related factors TCF4,LEF1 and MMP7 by inhibiting the expression of TOP2 A in gastric cancer cells,thereby inhibiting tumor development. |
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