| Abstract [eng] |
Master‘s thesis „Combined effects of semaglutide and berberine on oxidative stress in an SK-MEL-28 cells In Vitro model“. Supervisor assist. dr. Aušra Repečkienė. The thesis was completed by Antanas Klapatauskis. Vilnius University, Faculty of medicine, Institute of Biomedical Sciences, Centre of Pharmacy. The aim: to investigate and compare the effects of semaglutide and berberine on melanoma cell metabolic activity, oxidative stress induction and migration activity in an in vitro cell culture model, including their combinations with various substances on cell migration in in vitro cell culture models. Objectives: To evulate and investigate the cytotoxic effect of semaglutide and berberine combination on SK-MEL-28 melanoma cells. To determine reactive oxygen species level changes induced by semaglutide and berberine combination in melanoma cells. To investigate and compare the effects of different semaglutide and berberine combinations on SK-MEL-28 cell migratory activity in vitro. To assess the potential relationship between oxidative stress changes and melanoma cell viability and migratory activity. Methods: The object of the study was the human melanoma cell line SK-MEL-28 and were cultured in T-25 flasks with the required supplements. Cell growth took place in an incubator at 37°C. All procedures were carried out under sterile conditions using sterilized solutions and equipment. The procedures were performed in a laminar flow cabinet. Cell viability and concentration were determined according to standard procedures. A cell counting chamber with Neubauer grid and trypan blue stain was used. This method is based on the permeability of the dye into non-viable cells. The cells were analyzed using a microplate reader and dedicated software. Next, the cells were cryopreserved. After cryopreservation, the cells were prepared for the exposure experiment. At this stage, they were divided into control and experimental groups. After group allocation, the cell growth rate was determined using the CCK-8 kit. The procedure was carried out according to the manufacturer’s instructions. Once the cell growth rate had been determined, cell migration was assessed in vitro. After the migration assay, intracellular ROS levels were determined in SK-MEL-28 cells. All experiments were performed at least three times to ensure reliability, and statistical analysis of the data was then carried out. The results of the conducted study showed that the combination of 0,6 µM semagliutide+100µM berberine statistically significantly inhibited melanoma cell proliferation, while lower concentrations of this combination didn‘t show significant cytotoxic effects on SK-MEL-28 cells. Following ROS level determination, all semaglutide and berberine combination concentractions markedly promoted ROS formation, which potetntially induced oxidative stress in melanoma cells. According to migration results, all semaglutide and berberine combinations most strongly inhibited migration compared to individual substance concentrations of semaglutide (0,6 µM) and berberine (25µM,50 µM,100µM), which suggests a possible synergistic effect of these active substances on melanoma cell migration inhibition. Conclusions: It was established that melanoma cell prolifertion was statistically significantly inhibited by the highest concentration of semaglutide with berberine (0,6 µM semagliutide+ 100µM berberine). All tested semaglutide and berberine combinations promoted ROS formation after 24 hours, while after 48 hours ROS level decreased, regardless of combination. It was determined that semaglutide and berberine combinations inhibited SK-MEL-28 melanoma cells migration, and the strength of the effect depended on concentration. It was demonstrated that oxidative stress changes are associated with decreased melanoma cell viability and migration inhibition. Therefore, it can be concluded that the effect of semaglutide combinations may be linked to oxidative stress mechanisms. |
