The Mechanism Of Anti-colon Cancer Effect Of RBTI Combined With Oxaliplatin Was Studied Based On MAPK Signaling Pathway

Colorectal cancer is one of the most common gastrointestinal tumors,the incidence of which increases year by year and poses a great threat to human health.It is one of the most common gastrointestinal tumors.At present,patients with colon cancer are treated mainly by surgery and supplemented by chemotherapy.Oxaliplatin,as the first-line treatment drug for colorectal cancer,its role in the treatment of colon cancer is crucial.However,the majority of patients with colorectal cancer suffer from increased mortality due to chemotherapy resistance,Therefore,it is very necessary to find new drug treatment methods with high safety,minimize adverse side effects and improve the survival rate of patients with colorectal cancer.The trypsin inhibitor(BTI)in buckwheat has potential application value in pest control,anti-tumor and so on.Our research group obtained trypsin inhibitor gene sequence from buckwheat cotyledon and constructed prokaryotic expression vector to obtain high purity recombinant buckwheat trypsin inhibition(rBTI).In vitro studies have shown that rBTI has a strong inhibitory effect on tumor cell proliferation,but has little effect on normal cells.This paper designs through further explicit the effect of combined rBTI and oxaliplatin on colon cancer cell HCT116 and DLD-1 through the effect of single action of rBTI and oxaliplatin on colon cancer survival rate.The main research results include the following aspects:1、rBTI combined with oxaliplatin can inhibit the proliferation of colon cancer HCT116 and DLD-1 cells,and affect cell morphology and clonogenesis ability.First,the results of MTT experiment showed that rBTI and oxaliplatin acting alone on HCT116 and DLD-1 could reduce the proliferation of cancer cells by dose-dependent method,and the concentration of oxaliplatin with survival rate greater than 80% was selected as the concentration of combined medication.Then,through the combination of oxaliplatin and rBTI at different concentration gradients,MTT results showed that the proliferation of colon cancer cells was significantly inhibited by the combination of drugs in a concentration-dependent manner.At the same time,the cell morphological observation experiment showed that as the concentration of the combination increases,the cell number decreases significantly and the cell morphology becomes smaller and round.Subsequent cell cloning experiments showed that in the combination group,it is significantly inhibited cell clonogenesis compared with the single group.2.Studies on the docking of rBTI and oxaliplatin with P38,JNK and ERK molecules respectively.The affinity of rBTI and oxaliplatin with P38,JNK and ERK was studied by molecular docking.The results showed that rBTI formed three,three and five hydrogen bonds with P38,JNK and ERK,respectively.Oxaliplatin formed four hydrogen bonds with P38,and the amino acid residues within 4A were GLU-81,TYR-132,ARG-136,GLU-163 and GLU-163.Oxaliplatin formed three hydrogen bonds with JNK,and the amino acid residues within 4A were LYS-121,TYR-168,TYR-171,TRP-362,ASP-364 and GLU-367.Oxaliplatin formed three hydrogen bonds with ERK,and the amino acid residues within4 A were GLU-186,TYR-187,ARG-194 and TYR-233.To provide theoretical basis for further detection of the effect of rBTI combined with oxaliplatin on MAPK signaling pathway.3.rBTI combined with oxaliplatin induces apoptosis of HCT116 and DLD-1 cells by mediating MAPK signaling pathway.Firstly,the proportion of cell apoptosis increased gradually with the increase of concentration.The results of wb and q PCR showed that,with the increase of rBTI concentration were increased,and mRNA levels were consistent.The expression of Bcl-2 at the protein level decreased gradually,as well as at the mRNA level.The results of reactive oxygen species showed that the reactive oxygen species increased with the increase of rBTI concentration in the combination group.Finally,Wb showed that with the increase of rBTI,intracellular Cytrome c protein expression was up,Cleaved caspase 9,ERK,JNK,P38,Cleaved PARP protein expression was down.These results suggest that rBTI combined with oxaliplatin induces apoptosis of HCT116 and DLD-1 cells by mediating MAPK signaling pathway.In conclusion,the combination of rBT and oxaliplatin on colon cancer cells HCT116 and DLD-1 can inhibit the proliferation of tumor cells to the greatest extent,providing a new idea and experimental basis for future clinical cancer drug combination therapy.