Exploring the role of WNK-SPAK/OSR1 kinases in cancer
Date
2023-12-05
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Publisher
Saudi Digital Library
Abstract
Background: 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.
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Keywords
WNK: With-no-Lysine (K), Lys: Lysine, ATP: Adenosine-5’-triphosphate, MAPK: Mitogen-activated protein kinase, STE kinases: Serine/threonine kinases, Thr: Threonine, Ser: Serine, NKCC: Na+ - K+ - 2Cl- cotransporters, NCCs: Na+ - Cl- cotransporters, KCCs: K+ -Cl− cotransporters, SPAK: STE 20-related Proline/Alanine-rich Kinase, OSR1: Oxidative Stress-Responsive Kinase 1, F524/F526: Phenylalanine residues 524 and 526, CCDs: Coiled-coil domains, SH3: Src homology 3 domains, PHA2: Pseudo-hypoaldosteronism type II, ROMK1: Renal outer medullary potassium 1, PARP: Poly (ADP-ribose) polymerase, RT-PCR: Reverse transcription-polymerase chain reaction, SOK1: Ste20/oxidant stress response kinase-1, SOK1 Ste20/oxidant stress response kinase-1, CCT Conserved carboxy-terminal, Y2H Yeast two-hybridization, IP Immunoprecipitation, CCCs Cation-chloride cotransporters, MO25 Mouse protein-25, ClC-3 Chloride Channel 3, TRPC6 Transient receptor potential canonical channel 6, ENaC Epithelial sodium channel, AKT Alpha serine/threonine-protein kinase, TGF-β Transforming growth factor beta, GSK3-β Glycogen Synthase kinase 3 beta, Wnt Wingless-related integration site, ERK Extracellular signal-regulated kinase, SMAD Suppressor of Mother against Decapentaplegic, EMT Epithelial-mesenchymal transition, siRNA Small interfering RNA, VEGFR Vascular Endothelial Growth Factor Receptor, VEGF-A Vascular Endothelial Growth Factor - A, EC Endothelial cell, hECs Human endothelial cells, MMPs Matrix Metallopeptidases, pOSR1 Phosphorylated OSR1, GAPDH Glyceraldehyde 3-phosphate dehydrogenase, tOSR1 Total OSR1, IC50 Half-maximal inhibitory concentration, MTT 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide, MBP Myelin basic protein, PKC Protein kinase c, BAD Bcl-2-associated death promoter, RFxV/I Arginine-Phenylalanine-x-Valine/Isoleucine, PxxP Proline-Xaa-Xaa-Proline motif, TRPV Transient recetor potential vanilloid, SGK1 Serum-and glucocorticoid-inducible protein kinase, TBST Tris-buffered saline and Tween 20 buffer, NADP Nicotinamide adenine dinucleotide phosphate