Sphingosine kinase assay system with fluorescent detection in high performance liquid chromatography

Area of Science:

• Biochemistry
• Cell Biology
• Pharmacology

Background:

• Sphingosine kinase (Sphk) activation elevates sphingosine 1-phosphate (S1P), a lipid mediator implicated in various cancers.
• Sphk inhibition is a promising strategy for developing novel anticancer therapeutics.
• A robust assay is crucial for identifying effective Sphk inhibitors.

Purpose of the Study:

• To establish a convenient, non-radioactive assay for Sphingosine kinase (Sphk) activity.
• To utilize this assay for screening potential Sphk inhibitors.
• To optimize assay conditions using specific cell lines and analytical techniques.

Main Methods:

• Development of a fluorescent Sphk activity assay coupled with High-Performance Liquid Chromatography (HPLC).
• Utilization of murine teraticarcinoma mutant F9-12 cells, engineered to lack S1P lyase and express Sphk1.
• Optimization of assay parameters including substrate concentration, cell lysate protein amount, and incubation time.

Main Results:

• Optimal conditions determined: 100 µM C17-Sph, 30 µg F9-12 cell lysate protein, 20 min incubation.
• Sphingosine analog C17-Sph efficiently phosphorylated by Sphk (Km: 67.08 µM, Vmax: 1507.5 pmol/min/mg).
• Phorbol 12-myristate 13-acetate (PMA) doubled Sphk activity, while N,N-dimethylsphingosine (DMS) reduced it by 70% under optimized conditions.

Conclusions:

• A non-radioactive and convenient Sphk assay system was successfully established using HPLC and F9-12 cells.
• This assay provides a valuable tool for the discovery of Sphk inhibitors.
• The optimized method facilitates efficient screening for potential anticancer drug candidates targeting Sphk.