Evaluation of anticancer activity of potential ribosomal S6 kinase (RSK) inhibitors in coloumn cancer cell model

RSK is a multifunctional protein that plays critical roles in various cellular pathways, including metastasis, proliferation, and cell growth and development. Numerous studies have highlighted that RSK is expressed at higher levels in cancer cells compared to normal cells. In this thesis, the aim was to investigate the in vitro effects of commercially available potential RSK inhibitors, identified via in silico analyses, on colorectal cancer cell lines and a healthy control cell line (HUVEC). Initially, the total RSK expression levels in SW480 and HT-29 cell lines were demonstrated at the protein level using the western blotting technique. The cytotoxic properties of the selected commercial RSK inhibitor candidates were determined through MTT assays on SW480 and HT-29 cells, and their IC₅₀ values were calculated. Based on these results, compounds Y040-4759, S322-0083, and C592-3214 were particularly notable for their low IC₅₀ values in both cell lines. Apoptosis was evaluated using both Annexin V/PI assay and western blotting. For HT-29 cells, compounds Y040-4759, 6190-0389, V030-2239, S322-0083, C592-3214, and V031- 1748 were effective in inducing apoptosis, while F688-0005, Y040-4759, and S322-0083 were effective in SW480 cells. Colony formation and wound healing (scratch) assays were performed to assess the colony-forming ability and migratory potential of the cells. Accordingly, F688-0005, C592-3214, V031-1748, V030-2239, and Y040-3184 inhibited wound healing in HT-29 cells, while F688-0005, C592-3214, and S322-0083 showed similar effects in SW480 cells. Regarding colony formation, F688-0005 and Y040-4759 significantly reduced colony formation in SW480 cells, whereas F688-0005, C592-3214, V031-1748, V030-2239, 6190-0389, and Y040-3184 suppressed colony formation in HT-29 cells. At the protein level, compound Y040-4759 was found to reduce total and phosphorylated RSK levels in SW480 cells, while F688-0005 and C592-3214 decreased these levels in HT- 29 cells. In conclusion, especially F688-0005 and C592-3214 show promising potential as anticancer agents targeting RSK inhibition in colorectal cancer cells.