Ingenol Mebutate-Induced Cell Death Patterns in Normal and Cancer Epithelial Cells
October 2012 | Volume 11 | Issue 10 | Original Article | 1181 | Copyright © October 2012
Martin Stahlhut PhD,a* Malene Bertelsen PhD,a* Maria Høyer-Hansen PhD,a Nannette Svendsen,a Andre H. Eriksson PhD,a Janet M. Lord PhD,b Dagmar Scheel-Toellner PhD,b Stephen P. Young PhD,b and John R. Zibert PhDa
*These authors contributed equally to the work. aLEO Pharma A/S, Biological Research, Ballerup, Denmark bSchool of Immunity and Infection, Birmingham University Medical School, Birmingham, UK
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 μM 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 μM 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.
J Drugs Dermatol. 2012;11(10):1181-1192.
INTRODUCTION
Ingenol mebutate (ingenol-3-angelate), derived from the sap of the plant Euphorbia peplus, is a highly bioactive diterpene ester with cytotoxic and immune-stimulatory activity.1-3 Numerous
studies have evaluated the biological effects of ingenol mebutate in cancer-related indications.4 Ingenol mebutate has been FDA-approved for the treatment of actinic keratosis. Actinic keratosis is confined to the epidermis, caused by UV-induced DNA-damage to the skin following prolonged sun exposure, and can develop into potentially life-threatening squamous cell carcinoma.
5,6 Due to this risk of developing severe forms of non-melanoma
skin cancer from untreated actinic keratoses, treatment of actinic keratosis is now considered an important step towards the prevention of skin cancer forms with greater morbidity.7
Cell death induced by ingenol mebutate is observed at high micromolar concentrations and has been reported to bear
characteristics of necrotic cell death presenting with unchanged or dilated nuclei and swollen mitochondria.2 Only a subset of cancer cell lines is particularly sensitive to ingenol mebutate, probably mediated by activation of the PKC-delta isoform.8,9 Currently, the clinical efficacy of ingenol mebutate in actinic keratosis is attributed to a combination of direct cytotoxic and PKC-dependent accessory effects, sometimes called a dual mechanism of action.10
We sought to better define the molecular basis of ingenol mebutate-mediated cell death. Our results suggest that the
cytotoxic effects of ingenol mebutate in cancer cells and normal keratinocytes are caused by different molecular mechanisms, are subsequent to changes in mitochondrial structure, and
depend on the cellular differentiation state.
MATERIALS AND METHODS
Treatment And Analysis Of Full Thickness Skin Equivalents
Ingenol mebutate 0.05% (1.15 mM) gel was applied to human full thickness (epidermis and dermis) skin equivalents EpidermFT
(MatTek Corporation, Ashland, MA) in a volume of 10 μl
