title: Ingenol mebutate: induced cell death patterns in normal and cancer epithelial cells.
abstract: 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.
authors.affiliation: LEO Pharma A/S, Biological Research, Ballerup, Denmark.
authors.forename: Martin
authors.initials: M
authors.lastname: Stahlhut
authors.forename: Malene
authors.initials: M
authors.lastname: Bertelsen
authors.forename: Maria
authors.initials: M
authors.lastname: Hoyer-Hansen
authors.forename: Nannette
authors.initials: N
authors.lastname: Svendsen
authors.forename: Andre H
authors.initials: AH
authors.lastname: Eriksson
authors.forename: Janet M
authors.initials: JM
authors.lastname: Lord
authors.forename: Dagmar
authors.initials: D
authors.lastname: Scheel-Toellner
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Young
authors.forename: John R
authors.initials: JR
authors.lastname: Zibert
mesh_terms: D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D002454:Cell Differentiation; D002462:Cell Membrane / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003902:Detergents / Q000494:pharmacology; D004224:Diterpenes / Q000494:pharmacology*; D006367:HeLa Cells; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects; D009336:Necrosis; D017830:Octoxynol / Q000494:pharmacology; D012867:Skin / Q000187:drug effects / Q000473:pathology*
chemical_list: C486592:3-ingenyl angelate; D000972:Antineoplastic Agents, Phytogenic; D003902:Detergents; D004224:Diterpenes; D017830:Octoxynol; D007770:L-Lactate Dehydrogenase; D002118:Calcium