Betulinic acid induces a novel cell death pathway that depends on cardiolipin modification.

Cancer is associated with strong changes in lipid metabolism. For instance, normal cells take up fatty acids (FAs) from the circulation, while tumour cells generate their own and become dependent on de novo FA synthesis, which could provide a vulnerability to target tumour cells. Betulinic acid (BetA) is a natural compound that selectively kills tumour cells through an ill-defined mechanism that is independent of BAX and BAK, but depends on mitochondrial permeability transition-pore opening. Here we unravel this pathway and show that BetA inhibits the activity of steroyl-CoA-desaturase (SCD-1). This enzyme is overexpressed in tumour cells and critically important for cells that utilize de novo FA synthesis as it converts newly synthesized saturated FAs to unsaturated FAs. Intriguingly, we find that inhibition of SCD-1 by BetA or, alternatively, with a specific SCD-1 inhibitor directly and rapidly impacts on the saturation level of cardiolipin (CL), a mitochondrial lipid that has important structural and metabolic functions and at the same time regulates mitochondria-dependent cell death. As a result of the enhanced CL saturation mitochondria of cancer cells, but not normal cells that do not depend on de novo FA synthesis, undergo ultrastructural changes, release cytochrome c and quickly induce cell death. Importantly, addition of unsaturated FAs circumvented the need for SCD-1 activity and thereby prevented BetA-induced CL saturation and subsequent cytotoxicity, supporting the importance of this novel pathway in the cytotoxicity induced by BetA.