The C-terminal decapeptide of prothymosin α is responsible for its stimulatory effect on the functions of human neutrophils in vitro Pinelopi Samara a , Kyriaki Ioannou a , Monica Neagu b , Niki Arnogiannaki c , Alexandros Ardavanis d , Wolfgang Voelter e , Ourania Tsitsilonis a, ⁎ a Department of Animal and Human Physiology, Faculty of Biology, University of Athens, Greece b “Victor Babes” National Institute of Pathology, Bucharest, Romania c Department of Pathology, St. Savas Cancer Hospital, Athens, Greece d First Department of Medical Oncology, St. Savas Cancer Hospital, Athens, Greece e Biochemistry Institute, University of Tübingen, Germany abstract article info Article history: Received 27 September 2012 Received in revised form 15 November 2012 Accepted 16 November 2012 Available online 29 November 2012 Keywords: Human neutrophils Antimicrobial functions Prothymosin alpha Immunoreactive decapeptide Breast cancer Neutrophils are short-lived leukocytes and major components of the innate immune system. They are key players in the body's defense against pathogens, but their contribution to tumor growth and metastasis is controversial. Nevertheless, improving the functions of neutrophils in cancer patients, particularly in those undergoing chemotherapy, is of clinical significance. In this study, we investigated the ability of the immuno- reactive fragment of the polypeptide prothymosin alpha (proTα), i.e., the decapeptide proTα(100–109), to enhance the functions of neutrophils isolated from the peripheral blood of breast cancer patients in compar- ison with those from healthy donors. Activation of neutrophils from both groups with proTα(100–109) sig- nificantly increased phagocytosis and the production of intracellular reactive oxygen species (ROS) compared to controls. The release of extracellular ROS and oxidative burst of proTα(100–109)-stimulated neutrophils were less improved. Most importantly, upon activation with proTα(100–109), neutrophils from breast can- cer patients showed significantly enhanced cytotoxicity against tumor cell targets. Using a scrambled peptide as a control, we showed that the proTα(100–109)-induced effects were sequence-specific and comparable to those exerted by the parental molecule proTα. The responsiveness of neutrophils to proTα(100–109) or intact proTα did not correlate with the tumor grade or other established tumor characteristics. Our results suggest that proTα(100–109) activates neutrophils, particularly those derived from breast cancer patients, and these effects could potentially be used to improve some functions of neutrophils in the clinical setting. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Neutrophils have long been considered as short-lived effector cells of the innate immune system with a main role in resistance against extracellular pathogens. These cells are classically characterized as phagocytic cells that produce reactive oxygen intermediates and re- lease lytic enzymes with antimicrobial activity from their granules. Recent evidence has extended the functions of neutrophils to the regu- lation of innate and adaptive immunity, infections caused by intracellu- lar pathogens, autoimmunity, chronic inflammation, and cancer [1]. The essential role of neutrophils in innate immunity is illustrated by the debilitating and life-threatening conditions associated with congenital or acquired abnormalities in the life-cycle or function of neutrophils. Cancer patients represent a typical case of such disorders as their neutrophils demonstrate reduced activity compared to those from healthy individuals and their total number, particularly upon drug-induced myelosuppression, can be dramatically decreased. Neu- trophils from patients with gastrointestinal, breast, and gynecological cancers showed reduced migratory capacity, phagocytosis, and the production of reactive oxygen species (ROS), respectively [2–4]. In patients with oral cavity and head and neck cancers, neutrophils expressed low levels of inducible nitric oxide synthase and a signifi- cant number were immature [5,6]. The functions of human neutro- phils can be stimulated by growth factors (e.g., granulocyte and granulocyte-macrophage colony-stimulating factor) and a variety of peptides, such as N-formyl-methionine-leucine-phenylalanine (fMLP), bradykinin, β-amyloid peptide and synthetic hexapeptides [7,8]. Recent evidence suggests that neutrophils, via their cytokine secre- tion profile (interleukin (IL)-2, interferon-γ, and IL-5) and expression International Immunopharmacology 15 (2013) 50–57 Abbreviations: proTα, prothymosin alpha; proTα(100–109), prothymosin alpha immunoactive decapeptide residues 100–109; ROS, reactive oxygen species; NBT, nitroblue tetrazolium; fMLP, N-formyl-methionine-leucine-phenylalanine; HBSS-g, Hank's Balanced Salt Solution containing 0.1% gelatin; DHR 123, dihydrorhodamine 123; R 123, rhodamine 123. ⁎ Corresponding author at: Department of Animal and Human Physiology, Faculty of Biology, University of Athens, Panepistimiopolis, Ilissia 15784, Athens, Greece. Tel.: +30 210 727 4215; fax: +30 210 727 4635. E-mail address: rtsitsil@biol.uoa.gr (O. Tsitsilonis). 1567-5769/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.intimp.2012.11.011 Contents lists available at SciVerse ScienceDirect International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp