Mehellou, YoucefAlzahrani, Faris2023-12-102023-12-102023-12-05https://hdl.handle.net/20.500.14154/70131Background: With-no-lysine (WNK) kinases are crucial in regulating ion homeostasis and blood pressure. Dysregulation of WNKs contributes to cancer progression via phosphorylation of STE20/SPS1 related proline/alanine-rich kinase (SPAK) and Oxidative stress response kinase-1 (OSR1). This study aimed to explore OSR1 expression in various cell lines, assess cytotoxicity of WNK1 OSR1/SPAK-Na-K-2Cl cotransporters (NKCCs) inhibitors on normal and cancer cell lines and determine interactions between OSR1 and myelin basic protein (MBP). Methods and Materials: Throughout these experiments, MDA-MB231, MDA-MB468, MCF-7 and HEK293 cell lines were used. Western blotting assessed the activity of OSR1. MTT assay evaluated cytotoxicity of Closantel, Rafoxanide, WNK463, and Bumetanide on HEK293 and MDA-MB-231 cells. Immunoprecipitation investigated OSR1-MBP interactions. Statistical analysis was used. Results: MDA-MB-231 exhibited pOSR1 overexpression at Ser373 and Thr185, suggesting a role in cancer progression. Closantel, Rafoxanide, and WNK463 resulted in cytotoxicity of HEK293 cells (IC50=39.5352μM, 38.966μM and 32.7368μM, respectively), and Closantel and Rafoxanide caused cytotoxicity in MDA-MB-231 cells (IC50=38.739μM and 40.32μM, respectively). Bumetanide exhibited variable responses. Immunoprecipitation didn’t reveal a direct OSR1 MBP relationship. Conclusion: OSR1 overexpression may drive cancer progression, as shown in MDA-MB-231 at Ser373/Thre185 phosphosites. Closantel, Rafoxanide, and WNK463 show promise as potential anticancer agents. Further studies are needed to reveal their mechanisms and further explore OSR1's role in cancer progression.73enWNK: With-no-Lysine (K)Lys: LysineATP: Adenosine-5’-triphosphateMAPK: Mitogen-activated protein kinaseSTE kinases: Serine/threonine kinasesThr: ThreonineSer: SerineNKCC: Na+ - K+ - 2Cl- cotransportersNCCs: Na+ - Cl- cotransportersKCCs: K+ -Cl− cotransportersSPAK: STE 20-related Proline/Alanine-rich KinaseOSR1: Oxidative Stress-Responsive Kinase 1F524/F526: Phenylalanine residues 524 and 526CCDs: Coiled-coil domainsSH3: Src homology 3 domainsPHA2: Pseudo-hypoaldosteronism type IIROMK1: Renal outer medullary potassium 1PARP: Poly (ADP-ribose) polymeraseRT-PCR: Reverse transcription-polymerase chain reactionSOK1: Ste20/oxidant stress response kinase-1SOK1 Ste20/oxidant stress response kinase-1CCT Conserved carboxy-terminalY2H Yeast two-hybridizationIP ImmunoprecipitationCCCs Cation-chloride cotransportersMO25 Mouse protein-25ClC-3 Chloride Channel 3TRPC6 Transient receptor potential canonical channel 6ENaC Epithelial sodium channelAKT Alpha serine/threonine-protein kinaseTGF-β Transforming growth factor betaGSK3-β Glycogen Synthase kinase 3 betaWnt Wingless-related integration siteERK Extracellular signal-regulated kinaseSMAD Suppressor of Mother against DecapentaplegicEMT Epithelial-mesenchymal transitionsiRNA Small interfering RNAVEGFR Vascular Endothelial Growth Factor ReceptorVEGF-A Vascular Endothelial Growth Factor - AEC Endothelial cellhECs Human endothelial cellsMMPs Matrix MetallopeptidasespOSR1 Phosphorylated OSR1GAPDH Glyceraldehyde 3-phosphate dehydrogenasetOSR1 Total OSR1IC50 Half-maximal inhibitory concentrationMTT 3-(45-dimethylthiazol-2-yl)-25-diphenyl-2H-tetrazolium bromideMBP Myelin basic proteinPKC Protein kinase cBAD Bcl-2-associated death promoterRFxV/I Arginine-Phenylalanine-x-Valine/IsoleucinePxxP Proline-Xaa-Xaa-Proline motifTRPV Transient recetor potential vanilloidSGK1 Serum-and glucocorticoid-inducible protein kinaseTBST Tris-buffered saline and Tween 20 bufferNADP Nicotinamide adenine dinucleotide phosphateThesis