Investigation of the angiogenesis effect of P90RSK inhibitors on invitro and in vivo assays

Angiogenesis is a process that refers to the formation of new blood vessels from existing ones and is necessary for tissue development and repair under physiological conditions. However, uncontrolled progression of this process can play a role in the development of many diseases, particularly cancer, as well as cardiovascular and ocular diseases. Currently, the number of anti-angiogenic agents used in the treatment of angiogenesis-related diseases is limited. Research into the development of new molecules and treatment strategies is still ongoing today. 90 kDa ribosomal S6 kinases (p90RSK, RSK) are proteins belonging to the serine/threonine kinase family that are involved in the regulation of fundamental cellular processes such as cell growth, cell motility, cell cycle, and cell survival. Abnormal activation of p90RSK plays a critical role in the development of various cancers, such as kidney and lung cancer, and in cardiovascular diseases. Therefore, targeting p90RSK stands out as an attractive approach, particularly for the treatment of angiogenesis-related diseases and cancer. BI-D1870 is the best-known p90RSK inhibitor and targets RSK1-4 isoforms. Although the effects of this molecule in different diseases have been investigated, there are only a limited number of studies demonstrating its effects on angiogenesis. Although LJH685, a new-generation derivative of BI-D1870, has been reported to exhibit anticancer activity, there are no studies in the literature on its effect on angiogenesis. Similarly, although the protective effects of BIX 02565, an RSK2 inhibitor, in cardiovascular diseases have been reported, its relationship with angiogenesis has not been studied in detail. This thesis aims to comprehensively evaluate the effects of BI-D1870, LJH685, and BIX 02565 on angiogenesis for the first time using in vitro (cytotoxicity, wound healing, flow cytometry of tissue formation), ex vivo (aortic ring), and in vivo (CAM) methods. The cytotoxic effects of p90RSK inhibitors were first examined in the EA.hy926 endothelial cell line using SRB and MTT methods. The findings showed that all three inhibitors produced a dose- and time-dependent cytotoxic effect, which became particularly pronounced at 48 hours. Cell migration, wound healing, and tube formation assays were performed using the doses determined by cytotoxicity assays as a reference. In wound healing experiments, it was observed that the doses of BI-D1870 (10 µM), BIX 02565 (25 µM), and LJH685 (50 µM) significantly inhibited cell migration, and BI-D1870, in particular, prevented the closure of the wound area, causing the area to expand. Cell cycle analysis was performed using flow cytometry. BI-D1870 was found to arrest cells in the G2/M phase, while BIX 02565 arrested cells in the S phase. LJH685 did not show a significant phase-specific effect on the cell cycle. These results suggest that the inhibitors inhibit endothelial cell proliferation through different molecular pathways. The anti-angiogenic effects obtained in vitro experiments were confirmed by ex vivo rat aortic ring and in vivo CAM (Chick Embryo Chorioallantoic Membrane) models. In aortic ring experiments, all three inhibitors completely suppressed vascular sprouting and did not allow new vessel formation throughout the experiment. In the CAM model, BI-D1870 and LJH685 reduced vascular density in a dose-dependent manner, while BIX 02565 reduced it in a dose-independent manner. Although BIX 02565 exhibited strong anti-angiogenic effects, it also displayed irritant properties, while LJH685 showed marked anti-angiogenic effects without causing embryotoxic or irritant effects. In conclusion, this thesis study demonstrated that the p90RSK inhibitors BI-D1870, LJH685, and BIX 02565 strongly and consistently suppressed angiogenesis. In particular, the effects of LJH685 and BIX 02565 on angiogenesis were detailed for the first time in this study. The data obtained suggest that p90RSK inhibitors may be promising potential therapeutic agents for angiogenesis-related diseases and cancer treatment, and provide a solid foundation for further molecular studies and research using vertebrate animal models.