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  3. Evaluation of the effects of some peptides binding to Trk B receptor on Sk-ov-3 and Ov-car-3 cell lines

Evaluation of the effects of some peptides binding to Trk B receptor on Sk-ov-3 and Ov-car-3 cell lines

Number of pages: 106 File Format: word File Code: 32078
Year: 2013 University Degree: Master's degree Category: Medical Sciences
Tags/Keywords: Binding peptides - breast cancer - cancer - Carcinogen - leukemia - Peptide - Prostate cancers - TrkB receptor - tumor
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  • Summary of Evaluation of the effects of some peptides binding to Trk B receptor on Sk-ov-3 and Ov-car-3 cell lines

    Master's Thesis

    Medical Biotechnology Group

    Persian summary
    Cancer has been made as the second cause of death after heart diseases, one of the most important proteins of the oncogene is TREKB, whose specific ligand is BDNF (Brain derived neutrophilic factor). Overexpression of this receptor has been observed in prostate, multiple myeloma, ovarian, and thyroid cancers. BDNF, by binding to the TRK B receptor, causes dimerization in this receptor and creates signals in the path of angiogenesis and increases the number of cells. The aim of this study is to design peptides against the TRK B receptor (Tropomyosin Receptor B Kinase) as an inhibitor, in This study was first made in the Insilico part of the peptide library using the sequence tolerance and backrub method, and the optimization of the peptide energy was done using the Monte Carlo method, and the peptides with maximum stability based on energy were selected by the R software, and then the three-dimensional structure of the peptides was determined using the molecular dynamics method using the Hyperchem 7 software, and the docking of these peptides was completed by the HADDOCK software, and their connection method was completed by the software. Lighplot was analyzed and the three-dimensional structure of these peptides with the receptor was displayed by PYMOL software, and finally the sequence of 2 of the best peptides with the lowest energy were synthesized by the Danish company Tag copeghene. Traxin at a concentration of 200 nanomolar was studied as a positive control on the RPMI822, Sk-ov-3, Ov-car-3U266 cell line, and then flow cytometry was performed for the cells with the FITC-Annexin V kit at concentrations of 450 and 350 nanomolar for peptide one and two and at a concentration of 200 nanomolar for cyclotaxin, then the proteins after The treatment was performed using lysis buffer, lysed and their proteins were extracted, and the protein concentration was determined by Bradford spectrometry device, and then western blotting was performed to check the protein expression level after the treatment.
    The findings showed that the designed peptides have a high binding affinity to the TrkB receptor and inhibited cell growth in the mentioned cell line, and the results of this study showed that inhibition of the Trk B receptor It can lead to stopping the growth of cancer cells.

     

     
    Abstract

     

    Cancer is the second cause of death after heart diseases in the world. One of the important oncogene proteins is tropomyosin related kinase (TRK) whose ligand is brain derived neutrophilic factor. (BDNF). Over-expression of Trk B was observed in thyroid, ovarian cancer, prostate cancer and multiple myeloma.
    BDNF binding to TrkB lead to Trk B dimerization and activation of angiogenesis and increasing proliferation signalings.
    The objective of this study is to design new peptides against Trk B receptor as an inhibitor. Firstly, in silico section, a peptide library was generated by using sequence tolerance and backrub protocols. Then, peptides were energy minimized by means of Monte Carlo methods. Finally, the highest stable peptides were chosen based on the lowest energy score in an R package. Three dimensional structures of the peptides were predicted by hyperchem 7 software. Binding pattern of designed peptides with receptor was investigated using HADDOCK web server. Finally, two peptide sequences were synthesized by Cophgen Company in Denmark.
    In the experimental section, cell linens (skov-3, ov-car-3, U266, PRMI-8226) were treated four concentrations of peptides: 50, 200, 350, 500 nanomolar (nM). The cytotoxic effects of the peptides were evaluated using MTT assay.

    The annexin V-FITC and P1 staining and FACS analysis were used for investigating the apoptosis in treated cells. The cell signaling pathways were studied via evaluation of expression of AKT, EIF4E, MAPK using immune-blotting. In this work, Cyclotraxin (200nM) used as a positive control in all of experiments. It reproduces and destroys healthy tissues. Cancer cells are separated from normal cell division and growth. Cancer is known as the second cause of death in the world after heart attack, and in fact, cancer is considered a deadly factor due to its high death rate, and according to global statistics in 2011, about 1,596,670 people were infected and about 571,950 people died due to this disease (1). Statistics have shown that 1 out of every 4 Americans die from concussion, and in Iran, concussion is the third cause of death (2). The most common cancers include lung, stomach, colorectal, liver and breast cancer (3, 4). Cancer has two aspects: environmental (about 90%) and genetic (about 10%), and the environmental causes include smoking, diets and obesity, radiation, infection, stress, lack of physical activity, and environmental pollution (5). Tobacco has several carcinogenic agents, including nitrosamine and aromatic hydrocarbons, which are directly related to lung and larynx cancer, and diet and lack of physical activity are the cause of 30% of cancers (6, 7). Ionizing and non-ionizing radiation are related to cancer. DNA damage by irradiation leads to gene activation, silencing and deletion in the chromosome. Heredity does not play an important role in many cancers, but some mutations in the BRCA1 gene lead to breast and ovarian cancer, which leads to an imbalance in cell production and cell death, which are two important aspects of the cell cycle (8, 9). Genetic factors are classified into 2 groups, oncogenes that lead to cell growth and another group that plays an important role in inhibiting cell growth and division are known as tumor suppressors (10, 11). The most important oncogenes are RAS, WNT, MYC, ERK, and TRK (12), and tumor suppressors include p21, p27, p53, p16, p15, p19, and p18, which have been found in various cancers (13, 14). Of course, it should be noted that genetic changes are necessary to cause some cancers, and these mutations occur to a different extent in different cancers. Recently, it has been shown that a number of proto-oncogenes and tumor suppressors in the cell are controlled by the ubiquitin-proteasome degradation system (15, 16), among other factors that play a role in causing cancer, we can mention epigenetic changes including hypomethylation and hypermethylation at the DNA level (17, 18), hypermethylation of the promoter region of suppressor genes leads to a decrease in the translation of these genes, which allows the cell to Let the runaway grow.  Mutated genes play a role in cell growth and death, cell-cell interactions and cell signaling pathways, cell cycle disorder is considered as a very important factor in cancer pathology (19, 20), such that mutations of genes such as p53 have been seen in 50% of cancer cases (21, 22). Loss of function of tumor suppressor genes, which causes excessive and abnormal cell growth. Tumors are classified into two groups, which include benign and malignant tumors (23). Cancer has different types: carcinoma that originates from epithelial cells, which includes the most cases of cancer (24, 25), sarcoma, which originates from connective tissues such as bone and cartilage, etc., which actually originates from mesenchymal cells, and endo-leukemia, which originates from two types of hematopoietic cells. and lymphoma cells originate and include about 30% of tumors in children, germ cell tumors are seen in the cells of the testis, ovary and blastoma (which originates from placental cells) (26, 27).

  • Contents & References of Evaluation of the effects of some peptides binding to Trk B receptor on Sk-ov-3 and Ov-car-3 cell lines

    List:

    Cancer. 6

    Cancer treatment. 8

    TrkB receptor and its function. 9

    Trk B and cancer. 11

    Structure of Trk B. 12

    Trk receptor binding sites. 13

    Neurotrophic factors and their function 13

    BDNF protein and its function. 15

    Trk B inhibitor drugs. 17

    Peptide therapy. 17

    Bioinformatics. 28

    Flow cytometry. 29

    Western Blot 30

    Materials and methods 32

    Peptide library design. 33

    Creation of flexible peptide skeletons. 34

    Classification of produced peptides based on energy. 35

    Selecting the best peptides based on energy. 36

    Creating the three-dimensional structure of the most stable peptides 36

    Connection of the designed peptides with Trk B receptor. 36

    Investigation of peptide and receptor interactions. 38

    Laboratory section. 38

    Solutions 38

    Cell culture. 38

    Cellular passage (sub-culture) 39

    Adherent cells. 39

    Suspant cells. 40

    Freeze. 40

    Decomplemented FBS preparation method for use in cell culture. 41

    To melt. 41

    Cell counting. 41

    MTT Assay. 42

    Used concentrations for peptide. 43

    Flow cytometry. 43

    Protein extraction. 44

    Materials used to harvest cells. 45

    Protein measurement by Bradford method. 47

    Steps of western blotting. 47

    SDS-PAGE gel preparation 48

    Preparation of proteins for loading in SDS-PAGE. 50

    Transfer step. 51

    Preparation of solutions. 52

    Preparation of pvdf paper 53

    Blotting step. 53

    Peptide library design. 55

    IC50% of cells 57

    3D structure prediction. 58

    ligand-receptor interaction. 59

    Amino acids participating in the interaction. 61

    Showing interactions of Trk B receptor with designed peptides. 62

    Investigating the toxic effect of synthesized peptides by cell survival test. 65

    Flow cytometry results. 71

    Western blot results. 77

    Peptide and receptor interactions. 79

    Discussion. 82

    Sources 90

    Source:

     

     

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Evaluation of the effects of some peptides binding to Trk B receptor on Sk-ov-3 and Ov-car-3 cell lines
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