Ingenol mebutate is a diterpene ester derived from the plant and is FDA approved for the topical treatment of actinic keratoses (AK). in killing melanoma cells.3-5 How it works is still speculative, but this natural product, derived from the sap of the tree has been used extensively for its medicinal properties. 2 1n this issue of the Journal, Stahlhut et al. reveal for the first time evidence for the role of apoptosis and mitochondrial permeability as a possible Neurod1 mechanism of ingenol mebutate-mediated cytotoxicity. The authors demonstrate that ingenol mebutate elicits a strong and sustained increase in intracellular calcium that involves both ER-associated and mitochondrial-associated calcium stores. Interestingly, cancer cells take up ingenol mebutate and have a more robust ca lcium release promoting cell death upon treatment as compared to the same dose of ingenol mebutate in cultured donor keratinocytes. These findings are consistent with previous reports showing that ingenol mebutate specifically targets rapidly dividing cells in the basal cell layer (ie, dysplastic keratinocytes). The findings in this study are the first to evaluate the intracellular Ezogabine pontent inhibitor mechanisms involved in how ingenol mebutate may promote dysplastic and neoplastic keratinocyte cell death and elimination while sparing normal keratinocytes. This study employed an in vitro method and utilized a model of reconstituted skin to demonstrate the localization of this novel compound. Future studies are needed to evaluate how this novel compound functions in vivo, especially considering how this compound implicates apoptosis and alternative death pathways in promoting cell death. Ingenol mebutate treatment was shown to promote death of dysplastic and neoplastic skin cel ls and subsequently promoted keratinocyte proliferation in a mouse model of UVB-induced actinic keratosis and skin cancer.6 The authors of this earlier study suggest that early inflammation and neutrophil infiltration were the initiating events for regenerating keratinocytes at the basal layer. Additionally, Le et al. have further shown an immunostimulatory effect of ingenol mebu-tate resulting in an increase in anti-tumor CD8+ cells.7 Ingenol mebutate was also shown to differentially regulate apoptosis and TNF- related apoptosis ligand (TRAIL) induced apoptosis in melanoma cancer cells.4 However, the apoptosis regulators are yet to be determined. Cozzi et al., showed that ingenol mebutate specifically targeted cells with mutant regulation of p53.6 Indeed, p53 expression and function are altered in cancer cells and thus ingenol mebutate may be acting on regulators of apoptosis. For instance, novel regu lators such as the anti-apoptotic protein Fortilin are known to regulate p53, yet little else is known of this protein in relation to how apoptosis is modulated based on Forti lin-mediated Ezogabine pontent inhibitor regulation of p53.8 B-cell lymphoma-extra large (Bcl-xl) has further been shown to differentially regulate mitochondrial and ER-calcium stores.9 Other studies suggest a role for apoptotic regulators including Bel proteins and the Protein Kinase C regulated M itogen Activated Protein Kinase pathways (PKC-MAPK) in ingenol mebutate-mediated cell death.8-10 Additionally, Ezogabine pontent inhibitor the authors of the current study show a differential regulation of mitochondrial and ER stores of calcium. Apoptosis regulators in the Bcl-2 family, including Bcl-xL and Bax, are known to regulate calcium-mediated cell death yet the mechanisms are still to be worked out.11 Based on these published findings and the known role of the immune system in tumor surveillance, one possible mechanism of ingenol mebutate is through antigen presentation of apoptotic keratinocytes.7-12 The findings from this work strongly suggest such a mechanism. Several lines of evidence suggest cross-presentation as a possible mechanism specifically targeting dying neoplastic cells. Similar mechanisms have been shown in an infectious model of where dying infected macrophages enhance cross-presentation of antigenic apoptotic bodies to CD8+ cytotoxic T-cells.13,14 In humans, BDCA3+ dendritic cells are considered to be responsible for cross-presentation to CD8+ T-cells.15 Stahlhut’s present study suggests a role of both mitochondrial and ER-associated calcium efflux as.