Balance of human choline kinase isoforms is critical for cell cycle regulation: Implications for the development of choline kinase-targeted cancer therapy.

The enzyme choline kinase (CK), which catalyzes the phosphorylation of choline to phosphorylcholine in the presence of ATP, has an essential role in the biosynthesis of phosphatidylcholine, the major constituent of all mammalian cell membranes. CK is encoded by two separate genes expressing the three isoforms CKα1, CKα2 and CKβ that are active as homodimeric or heterodimeric species. Metabolic changes observed in various cancer cell lines and tumors have been associated with differential and marked up-regulation of the CKα genes, and specific inhibition of CKα activity has been proposed as a potential anti-cancer strategy. As a result, less attention has been given to CKβ and its interaction with CKα. With the aim of profiling the intracellular roles of CKα and CKβ, we used RNA interference (RNAi) as a molecular approach to down-regulate the expression of CK in HeLa cells. Individual and simultaneous RNAi-based silencing of the CK α and β isoforms was achieved using different combinations of knockdown strategies. Efficient knockdown was confirmed by immunodetection using our isoform-specific antibodies and by quantitative real-time PCR. Our analyses of the phenotypic consequences of CK depletion showed the expected lethal effect of CKα knockdown. However, CKβ- and CKα + CKβ-silenced cells had no aberrant phenotype. Therefore, our results support the hypothesis that the balance of the α and β isoforms is critical for cancer cell survival. The suppression of the cancer cell killing effect of CKα silencing by simultaneous knockdown of both isoforms implies that a more effective CK-based anti-cancer strategy can be achieved by reducing cross-reactivity with CKβ

Novel 4-amino bis-pyridinium and bis-quinolinium derivatives as choline kinase inhibitors with antiproliferative activity against the human breast cancer SKBR-3 cell line.

Choline kinase (ChoK) is the first enzyme in the CDP-choline pathway that synthesizes phosphatidylcholine, the major phospholipid in eukaryotic cell membranes. Human ChoK has three isoforms: ChoKa1, a2, and b. Specific inhibition of ChoKa has been reported to selectively kill tumor cells. In this study, ten new symmetrical bis-pyridinium and bis-quinolinium derivatives were synthesized and tested for their ability to inhibit human ChoKa2. These compounds have electron-releasing groups at position 4 of the pyridinium or quinolinium rings. 1,1’-[(Butane-1,3-diylbis(benzene-1,4-diylmethylene)]bis[4-(4- bromo-N-methylanilino)pyridinium)] dibromide and 1,1’-(biphenyl- 3,3’-diylmethylene)bis[7-chloro-4-(perhydroazepine-1- yl)quinolinium] dibromide were identified as highly potent ChoK inhibitors with IC50 values of 80 nm. Kinetic enzymatic assays indicated a mixed and predominantly competitive mechanism of inhibition for these compounds, which exhibited strong antiproliferative activity (EC50 1 mm) against the human breast cancer SKBR3 cell line