Background
Ingenol-3-angelate is a hydrophobic diterpene ester that can be isolated from the euphorb E. peplus. It rapidly induces cell death in proliferating keratinocytes through plasma membrane and mitochondrial disruption, although it can also initiate apoptosis in some cancer cell lines (LD90 = ~190 ?M).1 Secondarily, ingenol-3-angelate causes inflammation due, at least in part, to activation of PKC, leading to antibody-dependent cellular cytotoxicity.2,3,4 These actions have relevance in diminishing actinic keratosis and, possibly, other cancers of the skin.5,6
1.Ogbourne, S.M., Suhrbier, A., Jones, B., et al.Antitumor activity of 3-ingenyl angelate: Plasma membrane and mitochondrial disruption and necrotic cell deathCancer Res.64(8)2833-2839(2004)
2.Gillespie, S.K., Zhang, X.D., and Hersey, P.Ingenol 3-angelate induces dual modes of cell death and differentially regulates tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in melanoma cellsMol. Cancer Ther.3(12)1651-1658(2004)
3.Li, L., Shukla, S., Lee, A., et al.The skin cancer chemotherapeutic agent ingenol-3-angelate (PEP005) is a substrate for the epidermal multidrug transporter (ABCB1) and targets tumor vasculatureCancer Res.70(11)4509-4519(2010)
4.Hasler, C.M., Acs, G., and Blumberg, P.M.Specific binding to protein kinase C by ingenol and its induction of biological responsesCancer Res.52(1)202-208(1992)
5.Lebwohl, M., Swanson, N., Anderson, L.L., et al.Ingenol mebutate gel for actinic keratosisN. Engl. J. Med.366(11)1010-1019(2012)
6.Micali, G., Lacarrubba, F., Nasca, M.R., et al.Topical pharmacotherapy for skin cancer. Part I. PharmacologyJ. Am. Acad. Dermatol.70(6)965.e961-956.e912(2014)
Protocol
Cell experiment: | KG1a cells are transiently transfected with EGFP-tagged mouse PKC-δ subcloned into pEGFP-N1 plasmid using an Amaxa nucleofection apparatus. Cells are treated with Ingenol Mebutate (0.2 μM-20 μM) 24 hours after transfection. Cell viability in EGFP-positive cells is assessed and loss of viability confirmed in the total cell culture by MTT assay after 3 days. Briefly, 24 hours after transfection, 2 × 104 cells are plated in 5 wells in 96-well plates and exposed to 0, 0.2, 2, and 20 μM Ingenol Mebutate. At 72 hours, 20 μL MTT substrate at 5 mg/mL is added and plates are incubated at 37°C. After 3 hours, 150 μL media is removed and replaced with 200 μL dimethyl sulfoxide (DMSO). Absorbance at an optical density (OD) of 550 nm is read on a plate reader and corrected for absorbance obtained from blank media controls[2]. |
参考文献: [1]. Kedei N, et al. Characterization of the interaction of ingenol 3-angelate with protein kinase C. Cancer Res. 2004 May 1;64(9):3243-55.
[2]. Hampson P, et al. PEP005, a selective small-molecule activator of protein kinase C, has potent antileukemic activity mediated via the delta isoform of PKC. Blood. 2005 Aug 15;106(4):1362-8.
[3]. Ghoul A, et al. Epithelial-to-mesenchymal transition and resistance to ingenol 3-angelate, a novel protein kinase C modulator, in colon cancer cells. Cancer Res. 2009 May 15;69(10):4260-9. |
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