Antitumor Effect Of Recombinant Adenovious Carrying Apoptin On Human Breast Cancer MCF-7 Cell And Proteomics Related Studies

Background:The incidence of breast cancer as a common malignant tumor in the world is increasing year by year[1].Surgery is the most common treatment for breast cancer,followed by postoperative adjuvant chemotherapy.However,the surgical trauma is large,and there are many side effects of chemotherapy,and some cancer cells also have significant drug resistance[2,3].Therefore,it is particularly important to develop new anti-tumor drugs with low toxicity and few side effects.The occurrence and development of tumors are not only reflected in the uncontrolled and abnormal differentiation of cells,but also closely related to the imbalance of tumor cell apoptosis.With the continuous development of molecular biology,scientists have found that apoptosis is not the only fate that determines cell death.In recent years,autophagy has been shown to regulate cell death together with apoptosis.In different cases,autophagy may inhibit or promote apoptosis.Apoptin is a novel anti-tumor biological protein that specifically induces apoptosis in a variety of tumor cells.Although it induces certain tumor cell apoptosis to be affirmed,its specific mechanism remains unclear,and another form of death associated with apoptosis,autophagy,has also been associated with apoptin.This experiment aimed to discover the anti-tumor effect of apoptin on tumor cell MCF-7 and its corresponding molecular mechanism.Objective:The recombinant adenovirus Ad-VT,Ad-VP3 and Ad-MOCK were constructed using human recombinant adenovirus type 5,which was constructed by our group.The recombinant viruses Ad-VT and Ad-VP3 expressing apoptin protein were used to study the killing way in MCF-7 cells.Whether it is autophagy or apoptosis,whether it is both,whether there is a connection between them and possible signal pathways.These all provide a theoretical basis for further research and development of apoptin.Research methods:MTS cell activity assay was performed to analyze the killing inhibition effect of three recombinant adenoviruses Ad-VT,Ad-VP3 and Ad-MOCK on tumor cell MCF-7 and human mammary epithelial cell MCF-10A;Using the Annexin V-FITC/PI flow cytometry of autophagy inhibitor 3-MA,qPCR and Western blot to detect quantitative assays of autophagy-related proteins LC3,P62 and Beclin-1,and identify various staining experiments for autophagy(MDC),IFA,p EGFP-LC3)and transmission electron microscopy experiments,analysis of autophagy on MCF-7 cells by Ad-VT,Ad-VP3 and Ad-MOCK;Apoptosis effects of Ad-VT,Ad-VP3 and Ad-MOCK on MCF-7 cells were analyzed by cell cycle assay,Annexin V-FITC/PI assay,Hoechst staining,JC-1 staining and transmission electron microscopy.The effects of Ad-VT,Ad-VP3 and Ad-MOCK on the migration and invasion of MCF-7 cells were analyzed by wound healing experiments and BioCoat chamber invasion assays;After proteomic experiments with recombinant adenovirus carrying Adoptin Ad-VT and Ad-VP3 acting on MCF-7 cells,the function of the differential proteins produced and the possible signaling pathways involved;MCF-7 cells containing firefly luciferase were assayed for luciferase activity and stability,and MCF-7-luc,which stably expressed luciferase,was screened in vitro.Alignment detection of biological characteristics such as growth characteristics,invasive ability and cell cycle of MCF-7-luc cells and MCF-7 cells was performed.The effects of Ad-VT,Ad-VP3 and Ad-MOCK on MCF-7-luc cells and MCF-7 cells were verified by cell inhibition rate MTS assay,cell membrane Annexin V-FITC/PI flow assay and nuclear Hoechst staining assay.After establishing a subcutaneous tumor-bearing model in mice,the tumor volume and survival time were measured by in vivo imaging experiments and vernier calipers.The effects of Ad-VT,Ad-VP3 and Ad-MOCK on tumor inhibition and survival of mice were studied.Research result:1.In vitro MTS assay showed that Ad-VT and Ad-VP3 carrying apoptin had a killing effect on human breast cancer MCF-7 cells,and had no significant killing effect on human mammary epithelial cells MCF-10A.Ad-MOCK has no killing effect on MCF-7 cells and MCF-10A.2.Flow cytometry,Western Blotting,qPCR,MDC staining,IFA and transmission electron microscopy showed that Ad-VT and Ad-VP3 carrying apoptin promoted the enhancement of autophagy in MCF-7 cells in the early stage(6h and 12h)of cell action.3.Through cell cycle detection,AnnexinV-FITC/PI flow cytometry,Annexin V-FITC/PI staining,Hoechst staining.JC-1 staining,transmission electron microscopy,it was found that Ad-VT and Ad-VP3 carrying apoptin promoted human mammary gland Cancer MCF-7 cells undergo apoptosis.4.Through wound healing experiments and BioCoat chamber invasion experiments,both Ad-VT and Ad-VP3 carrying apoptin were able to inhibit the migration and invasion of MCF-7 cells.5.Proteomics experiments with GO and KEGG analysis of differential proteins suggest that Ad-VT and Ad-VP3 carrying apoptin can promote autophagy and apoptosis of human breast cancer cell MCF-7.The FOXO1 and FOXO3a proteins are involved in the mechanism by which apoptin causes cell death,particularly the AMPK signaling pathway.6.Clone 15 strains with the highest activity,the best stability and a positive correlation with luminescence intensity(R2=0.9903)were selected for in vivo experiments.The biological characteristics of MCF-7-luc cells and MCF-7 cells were identified,which proved that the construction process had no significant effect on cell growth characteristics,invasion ability and cell cycle.MTS assay in vitro,AnnexinV-FITC/PI flow cytometry and Hoechst staining experiments confirmed that the effect of recombinant adenovirus on MCF-7-luc cells and MCF-7 cells was not significantly different.MCF-7-luc cells could be used for tumor-bearing experiments in mice.7.The results of in vivo imaging in nude mice showed that the average bioluminescence intensity of tumors was lower than that of the control group in the Ad-VP3 and Ad-VT treatment groups with apoptin,the tumor volume became smaller,and the survival time of tumor-bearing mice was prolonged.Conclusion:Recombinant adenovirus carrying apoptin can significantly inhibit the growth of human breast cancer cell MCF-7,but has no inhibitory effect on human breast normal epithelial cell MCF-10A.The recombinant adenovirus carrying apoptin mainly causes the death of MCF-7 cells in apoptotic manner,and has a significant effect on autophagy.In the early stage of action,autophagy plays a protective role on cancer cells.When the level of death gradually dominates,the apoptotic effect has exceeded the threshold of protective effect of autophagy,autophagy began to decrease,and then autophagy increased again and apoptosis together to promote MCF-7 cell death..FOXO1 protein and FOXO3a protein are involved in autophagy and apoptosis of MCF-7 cells induced by apoptin,especially the AMPK signaling pathway associated with FOXO protein.Therefore,this study suggests that AMPK may associate autophagy and apoptosis as apoptin A regulatory pathway that causes tumor cell death.