Investigation of molecular and therapeutic effects of Macrovipera lebetinus venom on human colorectal carcinoma cells

Cancer is an environmental or inherited disease of the genes that control the proliferation, differentiation and death of abnormal cells. The traditional treatment methods used today to treat colorectal cancer (CRC), which occurs in the large intestine tissue, also damage healthy cells in the patient's body, lowering the patient's immunity. For patients with a low immune system, CRC treatment is inadequate and not promising due to various side effects. Scientists have aimed to develop alternative or complementary treatment methods that are naturally produced against colon tumors that are resistant to treatments due to the presence of mutated KRAS, BRAF, FAS and TRAIL genes and that do not harm healthy cells and show cytotoxic properties against tumor cells. The presence of metal ions and peptide-containing substances such as phospholipase A2 (PLA2), metalloproteinase (SVMP), serineproteinase (SVSP) and L- amino oxidase (LAAO) in complex snake venoms for new treatment methods in line with the researches draws attention to the strong potential of their therapeutic effect. In particular, studies conducted against various types of cancer show that snake venoms belonging to the Elapidae and Viperidae families are promising for cancer treatment by showing effects such as activating ROS release, initiating apoptosis mechanism, inhibiting integrin activity and inhibiting metastasis and colony formation on tumor cells. In this thesis, the molecular and therapeutic effects of Macrovipera lebetinus snake venom of Viperidae family on colorectal carcinoma cells were investigated. Large viper snakes collected from Soğmatar village of Şanlıurfa province were bitten into a sterile collection container covered with parafilm and milking was performed by squeezing the venom glands. After milking, the snake venom was powdered by lyophilization to preserve the potency and stability of the venom. The rich peptide and protein content of the powdered raw Macrovipera lebetinus snake venom was characterized by SDS-PAGE method, RP-HPLC chromatography method, pH analysis, bicinchoninic acid (BCA) total protein quantification, UV-Vis and FT-IR spectroscopic methods. The cytotoxicity analysis of crude big viper snake venom was determined by Alamar Blue method on human colon adenocarcinoma DLD-1 and human healthy colon CCD-18Co cell lines under in vitro conditions. After calculating the half-kill dose (IC50) of crude Macrovipera lebetinus snake venom in DLD-1 cell line, apoptotic effect, metastasis and colony formation capacity were investigated under in vitro conditions. In order to evaluate the effects of crude Macrovipera lebetinus venom on DLD-1 cell line at molecular level, protein expression and gene expression studies were performed by Western blot and qRT-PCR methods, respectively. In line with the findings obtained in the thesis study, as a result of the characterization studies of the poison, peptide-containing protein samples were determined by SDS-PAGE method and 15 distinct protein fractions consistent with the literature were determined. In order to investigate the cytotoxic effects in more detail, 29 different fractions were determined by RP-HPLC chromatography method. The total protein amount of our acidic venom was calculated at a concentration of 2538.75 μg/mL and showed fluorescence at maximum absorption at 280 nm. In order to elucidate the biomolecular mechanism of our raw snake venom, FT-IR analysis revealed the presence of 11 different peak vibrations consistent with the literature and the presence of peptide/protein structures, amines, nucleosides, carbohydrates and inorganic elements. The presence of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) residues surrounding the venom gland lumen was also proven. At the end of the measurement, it was determined that there was a dose-dependent decrease in the viability of snake venom-treated human adenocarcinoma cell line DLD-1 and human healthy colon CCD-18Co cells. IC50 values were calculated as 6.12 μg/mL and 15 μg/mL, respectively. Compared to DLD-1 colon cancer cell line, healthy human colon cells have very low cytotoxic effect. It inhibited the migration rate of DLD-1 cells by 88.28% and colony formation by 71.51% (p<0.0001) compared to the control group. As a result of apoptosis analysis, the early apoptosis rate was 13.07% and the late apoptosis rate was 0.96%, leading DLD-1 cells to apoptosis. Poison treatment increased the expression of 74 genes and decreased the expression of 4 genes in the human apoptosis panel gene study in DLD-1 cell line. It decreased the expression of 72 genes and increased the expression of 1 gene in the human colon cancer panel gene study in DLD-1 cell line compared to the control group. In protein expression studies, it decreased BcL-2 (0.56-fold) protein expression and increased P53 (1.08-fold) and BAX (1.25-fold) protein expressions in the venom colon adenocarcinoma cell line. According to the data in the thesis study, if the cause of CRC formation in the person is the result of environmental factors or hereditary factors, our snake venom, which increases or decreases the effective gene and protein mechanisms, is thought to be a treatment-responsive agent by reducing resistance. Keywords: Macrovipera lebetinus, Colon cancer, Cytotoxicity, Apoptosis, Gene and protein expression, Snake venom