We investigated the proposed necrotic mechanism of
ingenol mebutate, a natural compound with anti-
cancer properties in human keratinocytes, the human
squamous cell carcinoma cell line HSC-5, and HeLa cervix
carcinoma cells. Topical application of a clinical dose of
ingenol mebutate 0.05% (1.15 mM) gel to human reconstituted full-thickness skin equivalents strongly reduced epidermal, but not dermal viability.
Ingenol mebutate showed cytotoxic potency between 200-300 M on normal and
cancer cells. When keratinocytes were induced to differentiate, they became significantly less sensitive to
ingenol mebutate and half-maximal induction of cell death required more than 300 M
ingenol mebutate. Cytotoxic concentrations of
ingenol mebutate caused
rupture of the mitochondrial network within minutes paralleled by cytosolic
calcium release in all cells. Subsequently, plasma membrane integrity was lost as seen by
propidium uptake into the cells. This was in sharp contrast to lysis of cells with low concentrations of the
detergent Triton X-100 that permeabilized the plasma membrane within minutes without affecting organelle morphology. Buffering of intracellular
calcium and inhibition of the
mitochondrial permeability transition pore reduced the cytotoxic effect of
ingenol mebutate in
cancer cells, but not in normal keratinocytes. However, these inhibitors could not prevent cell death subsequent to prolonged incubation. Our findings reveal that
ingenol mebutate does not mediate cytotoxicity by a simple lytic, necrotic mechanism, but activates distinct processes involving multiple cell organelles in a cell-type and differentiation-dependent manner. These data improve our understanding of
ingenol mebutate-target cell interactions and offer new insights relevant to the removal of aberrant cells in human skin.