Structure Comparison Of Bacillus Thuringiensis Protein Cry51Aa1 With PS4 And Screening Of Its Anticancer Spectrum

Objective: â‘  To compare the evolutionary relationship and consistency&difference of structure between Cry5Aa1 from Bacillus thuringiensis and anticancer parasporin proteins, and further speculate cytotoxicity of Cry51Aa1 against human cancer cells; â‘¡ Testing anticancer activity and screening the spectrum of Cry51Aa1, to select 1-2 kinds of sensitive cell types and discuss the mechanism of Cry51Aa1 in killing cancer cells, which could lead Cry51Aa1 towards possible medical application.Methods: â‘  Bioinformatic data bases were used to analyze physical and chemical propertities and get its amino acid sequence, at the same time, the sequences of 6 groups of 19 anticancer parasporin proteins were exported. The maximum likelihood clustering method was used to construct family phylogenetic tree among Cry51Aa1 and PS proteins, which would help to identify possible homologous protein of Cry51Aa1 in PSs; Comparison between the primary and secondary structure parameters, and SWISS-MODEL homology modeling for three domain spatial structures comparison were done between Cry51Aa1 and the protein which had the highest similarity with it. â‘¡Cry51Aa1 was purified between applied into target cancer cells( including 12 kinds of human cancer cells, 2 human normal cells,1 kind of mice cancer cell and 3 kinds of mice normal cells),MTT assay was used to test cytotoxicity of Cry51Aa1 against different cells. After getting sensitive cells, time and concentration factors on affection of cell survival/inhibition were tested; â‘¢ Simple Cell migration experiment was performed to test the inhibition in cell mobility; flow cytometry(FCM)and Western blot(WB)were used in final steps to explore whether apoptosis exist in the process of Cry51Aa1 on killing target cancer cells.Results: â‘  From the amino acid sequence clustering results established by evolutionary tree diagram, Cry51Aa1 was considered having the highest amino acid sequence similarity with PS4, which reached 36%, and boostrap model test result was more than 70, which showed good credibility. Structural similarity was further reflected in the protein models, especially in alpha carbon atoms( CA),with root mean square(RMS) = 0.92. â‘¡ Cry51Aa1 could cause cell bulling and shrinking in target cells, which complied with Cry51Aa1 concentration, while the percentage of cell bulling declined with time and turned into shrinking;â‘¢ Differences existed between different cell types, with cancer cells presenting higher toxicity than normal cells; Difference also performed in different cancer cell systems, such as digestive system and genital system. Cell mobility was significantly inhibited in Cry51Aa1 group than the control group.The anticancer spectrum found in this research including gastric cancer cells SGC-7901, gallbladder cancer cells GBC-SD and RBE of the digestive system; others were cervical cancer cell line Hela, HEC and endometrial cancer cells and breast cancer cell line HBL-100 of the reproductive system. â‘£ Apoptosis was observed when high concentration of Cry51Aa1 was applied, at the same time, Caspase family was considered playing important role in causing cell apoptosis, and could inhibit cell moving compared with control group.Conclusions: â‘  Cry51Aa1 could be listed into the same family as anticancer parasporal proteins, it has the highest genetic relationship with parasporin-4 and they have some similarity in structure; â‘¡ Cry51Aa1 showed cytotoxicity to some kinds of Humam Cancer cells, other than normal cells. Differences exist in toxicity of Cry51Aa1 against different tumor system and cell types from the same system; Anticancer spectrum of Cry51Aa1 found including 3 cell lines of the digestive system and 4 cell lines from the reproductive system. But further researches are expected to confirm the specific cytotoxicity of Cry51Aa1;â‘¢Cry51Aa1 could cause changes in cell shape and activity in target cell and show a dose response relationship, with positive relationship between inhibition rate and protein concentration; â‘£Cry51Aa1 could kill target cells possibly by Caspase-induced apoptosis, and may also inhibit cell migration.