Changes of intracellular calcium, fatty acids and phospholipids during miltefosine-induced apoptosis monitored by fluorescence- and 13C NMR-spectroscopy.

The alkylphosphocholine Miltefosine (hexadecylphosphocholine, HePC) induces apoptosis in human epithelial KB cells, whereas no such effect can be observed in a resistant clone (KBres). Its mode of action is mediated via the cell membrane, whereas the mechanism is still widely unknown. The use of various spectroscopic methods (fluorescence spectroscopy with Fura-2/AM on viable cells, 13C NMR spectroscopy on lipid extracts) reveals osmotic and metabolic changes in HePC treated sensitive cells. Intracellular free Ca(2+)-concentration increased over 300% of control in apoptotic cells, whereas KBres cells showed only a minor increase and no morphological response typical for apoptosis. The Ca(2+)-influx was mediated via calcium channels in the cell membrane. The HePC-induced influx is prevented by Gd3+, which blocks those calcium channels. Cells, grown in Ca(2+)-free medium, showed no apoptotic behaviour after treatment with HePC. If apoptosis was induced, an increased fatty acid and subsequent phospholipid biosynthesis was observed. This effect seems to be a specific marker of apoptosis in KB cells.