Abstract
The modes of action of the novel anti-skin tumor agent ingenol-3-angelate (PEP005) are incompletely understood. Crucially, the cytotoxic functions of neutrophils recruited to the tumor in response to topical application of PEP005 are necessary for effective ablation of the treated lesion. Here, we investigated the hypothesis that the phorbol ester-like properties of PEP005 and its ability to activate PKC could directly activate endothelial cells (EC) so that they support the recruitment of neutrophils. Exposure of EC to PEP005 induced mRNA and/or protein for E-selectin, ICAM-1 and IL-8 in a dose dependent manner, while in a flow based adhesion assay, PEP005 treated EC supported the recruitment of neutrophils at levels comparable to EC stimulated with TNF-α. Neutrophil adhesion was inhibited by antibody against E-selectin but not P-selectin. Activation of EC was inhibited by the PKC inhibitor bisindolylmaleimide-1 and confocal immuno-fluorescent studies demonstrated translocation of PKC-δ from the cytosol to the peri-nuclear membrane in response to PEP005. Importantly, the knock down of PKC-δ using siRNA completely abolished neutrophil recruitment to EC subsequently treated with PEP005. Thus, we describe a novel route by which the anti-tumor agent PEP005 regulates the recruitment of cytotoxic leukocytes by directly activating EC in a PKC-δ dependent manner.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Weedon D, Chick J (1976) Home treatment of basal cell carcinoma. Med J Aust 1:928
Ogbourne SM, Suhrbier A, Jones B et al (2004) Antitumor activity of 3-ingenyl angelate: plasma membrane and mitochondrial disruption and necrotic cell death. Cancer Res 64:2833–2839
Challacombe JM, Suhrbier A, Parsons PG et al (2006) Neutrophils are key component of the anti-tumor efficacy of topical chemotherapy with ingenol-3-angelate. J Immunol 177:8123–8132
Zimmerman GA, Prescott SM, McIntyre TM (1992) Leukocyte–endothelial cell interactions. Immunol Today 13:93–100
Imhof BA, Dunon D (1995) Leukocyte migration and adhesion. Adv Immunol 58:345–416
Springer TA (1995) Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration. Ann Rev Physiol 57:827–872
Kedei N, Lundberg DJ, Toth A et al (2004) Characterization of the interaction of ingenol 3-angelate with protein kinase C. Cancer Res 64:3243–3255
Buckley CD, Ross EA, McGettrick HM et al (2006) Identification of a phenotypically and functionally distinct population of long lived neutrophils in a model of reverse endothelial migration. J Leuk Biol 79:303–311
Lally F, Smith E, Filer A et al (2005) A novel mechanism of neutrophil recruitment in a coculture model of the rheumatoid synovium. Arth Rheum 52:3460–3469
Cooke BM, Usami S, Perry I et al (1993) A simplified method for culture of endothelial cells and analysis of blood cells under conditions of flow. Microvasc Res 45:33–45
Luu NT, Rainger GE, Nash GB (1999) Kinetics of the different steps during neutrophil migration through cultured endothelial monolayers treated with tumour necrosis factor-α (TNF). J Vasc Res 36:477–485
Shimamura K, Takashiro Y, Akiyama N et al (2004) Expression of adhesion molecules by sphingosine 1-phosphate and histamine in endothelial cells. Eur J Pharmacol 486:141–150
Mason JC, Yarwood H, Sugars K et al (1997) Human umbilical vein and dermal microvascular endothelial cells show heterogeneity in response to PKC activation. Am J Physiol 273:C1233–1240
Rahman A, Anwar KN, Uddin S et al (2001) Protein kinase C-δ regulates thrombin-induced ICAM-1 gene expression in endothelial cells via activation of p38 mitogen activated protein kinase. Mol Cell Biol 21:5554–5565
Minami T, Abid MR, Zhang J et al (2003) Thrombin stimulation of vascular adhesion molecule-1 in endothelial cells is mediated by protin kinase C (PKC)-δ-NF-κB and PKC-ζ-GATA signalling pathways. J Biol Chem 278:6976–6984
Di Carlo E, Forni G, Lollini P et al (2001) The intriguing role of polymorphonuclear neutrophils in antitumor reactions. Blood 97:339–345
Di Carlo E, Forni G, Musiani P (2003) Neutrophils in the antitumoral immune response. Chem Immunol Allergy 83:182–203
Kemp TJ, Ludwig AT, Earel JK et al (2005) Neutrophil stimulation with Mycobacterium bovis bacillus Calmette-Guerin (BCG) results in the release of functional soluble TRAIL/Apo-2L. Blood 106:3474–3482
Sanford MA, Yan Y, Canfield SE et al (2001) Independent contributions of GR-1+ leukocytes and Fas/FasL interactions to induce apoptosis following interleukin-12 gene therapy in a metastatic model of prostate cancer. Hum Gene Ther 12:1485–1498
Stockmeyer B, Beyer T, Neuhuber W et al (2003) Polymorphonuclear granulocytes induce antibody-dependent apoptosis in human breast cancer cells. J Immunol 171:5124–5129
Hernandez-Ilizaliturri FJ, Jupudy V, Ostberg J et al (2003) Neutrophils contribute to the biological antitumor activity of rituximab in a non-Hodgkin’s lymphoma severe combined immunodeficiency mouse model. Clin Cancer Res 9:5866–5873
Niitsu N, Khori M, Hayama M et al (2005) Phase I/II study of the rituximab-EPOCT regimen in combination with granulocyte colony-stimulating factor in patients with relapsed or refractory follicular lymphoma including evaluation of its cardiotoxicity using B-type natriuretic peptide and troponin T levels. Clin Cancer Res 11:697–702
Otten MA, Rudolph E, Dechant M et al (2005) Immature neutrophils mediate tumor cell killing via IgA but not IgG Fc receptors. J Immunol 174:5472–5480
Middleton J, Neil S, Wintle J et al (1997) Transcytosis and surface presentation of IL-8 by venular endothelial cells. Cell 91:385–395
May MJ, Pearson JD (1994) The role of protein kinase C (PKC) in Eselectin expression by human umbilical vein endothelial cels (HUVEC): comparison of the effects of IL-1 and PMA. J Physiol 475P
Tamaru M, Narumi S (1999) E-selectin gene expression is induced synergistically with rthe cocexistence of activated classic protein kinase C and signals elicited by interleukin-1β but not tumour necrosis factor-α. J Biol Chem 274:3753–3763
Quagliaro L, Piconi L, Assaloni R et al (2005) Intermittent high glucose enhances ICAM-1, VCAM-1 and E-selectin expression in human umbilical vein endothelial cells in culture: the distinct role of protein kinase C and mitochondrial superoxide production. Atherosclerosis 183:259–267
Hendy B, Zhu CL, Greenstein S (2002) Fas activation opposes PMA-stimulated changes in the localisation of PKC delta: a mechanism for reducing neutrophil adhesion to endothelial cells. J Leuk Biol 71:863–870
Umei T, Ohhara N, Okamura S et al (1993) Activation of neutrophils NADPH oxidase by PMA—cytosol activity is translocated in phorbol-primed neutrophils. Int J Biochem 25:631–633
El Benna J, Dang PMC, Andrieu V et al (1999) P40(phox) associates with neutrophil Triton X-100-insoluble cytoskeletal fraction and PMA-activated membrane skeleton: a comparative study with P67(phox) and P47(phox). J Leuk Biol 66:1014–1020
Acknowledgments
The work reported here was supported by funding from the European Commission (Integrated project LSHB-CT-2004-503467).
Conflict of interest statement
The authors have no conflict of interest to declare.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Hampson, P., Kavanagh, D., Smith, E. et al. The anti-tumor agent, ingenol-3-angelate (PEP005), promotes the recruitment of cytotoxic neutrophils by activation of vascular endothelial cells in a PKC-δ dependent manner. Cancer Immunol Immunother 57, 1241–1251 (2008). https://doi.org/10.1007/s00262-008-0458-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00262-008-0458-9