The effect of kojic acid derivatives with new synthesized antithyrosinase activity on cytotoxicity and cell death in the A375 malign melanoma cell line

In this thesis study, kojic acid derivative compounds which are tyrosinase inhibitors were investigated. The mechanisms that lead to death and molecular death pathways in the cells caused by which compound with the strongest cytotoxic properties against A375 human malignant melanoma cells have been examined. As a result of the SRB test conducted for cytotoxicity, it was observed that Compound-15, whose IC50 value was measured as 9.81 μM, effectively killed living cells. The enzymatic inhibition of Compound-15 was determined spectrophotometrically using the cytosol of B16F10 mice cells and kojic acid as a control. According to spectrophotometry results, it was demonstrated that Compound-15 could compete with kojic acid. Furthermore, the IC50 values of kojic acid and Compound-15 were obtained as 59.20 μM and 76.79 μM respectively. The apoptosis test was carried out and as stated in test results, Compound-15 took the cells to the last stage of apoptosis. Then, to determine which apoptotic genes in A375 cells affected by Compound-15, RNA was purified from cells, and cDNA was synthesized. Apoptotic p53 and Bax, anti-apoptotic Bcl-2 as well as Jnk, Mdm2 and Mdr1 gene levels were examined. Bcl-2 levels decreased 0.5 times and Bax levels increased 2.4 times compared to control. Thus, it has been observed that Compound-15 leads cells to apoptosis in 24 hours. The ratio of Mdm2 in which p53 showed early activity, increased 3 times compared to p53, supports the idea that increased p53 level in cells may have been taken under control. The results obtained in the first 24 hours for the Mdr-1 gene were high. No significant change was observed in the Jnk gene compared to the control. The death pathway triggered by Compound-15 in melanoma cells was determined by flow cytometry and according to the results, it can be said that the late apoptosis rate of 58.8% leads A375 cells to more effective programmed death. As a result of the western blot and β-Actin, p53 and Mdr1 protein analysis performed on A375 cells, it was concluded that the findings were consistent with the gene expression results.