Original article Pro-apoptotic effects of Amblyomin-X in murine renal cell carcinoma ‘‘in vitro’’ Erica Mie Akagi a , Paulo Luiz de Sa ´ Ju ´ nior a , Simone Michaela Simons a , Maria Helena Bellini b , Sandra Alves Barreto a , Ana Marisa Chudzinski-Tavassi a, * a Laboratory of Biochemistry and Biophysics, Butantan Institute, Avenue Vital Brazil, 1500, CEP 05503-900, Sa˜o Paulo, SP, Brazil b Institute of Energy and Nuclear Research, IPEN, Sa˜o Paulo, Brazil 1. Introduction Renal cell carcinoma (RCC) is the most common kidney cancer, accounting for nine out of ten cases of kidney cancer. Its incidence has been increasing steadily over the past decades. According to the American Society of Cancer, kidney cancer is among the ten most common cancers in both men and women [1]. It is estimated that over 58,000 new cases occurred in 2010 and approximately 13,040 people died from this disease. RCC is one of the most difficult urological neoplasm to cure, as it is chemotherapy- resistant and nonradiosensitive. Patients with RCC already have metastasis at the time of diagnosis in 25–57% of the cases, and subsequently develop metastasis within 10 years in approximately 60% of all cases after curative nephrectomy [2,3]. Clinical trials have shown that cytokine therapy with interleu- kin 2 (IL-2) and/or interferon-alpha (IFN-a) can induce tumor regression in a subset of patients with metastatic RCC, but the therapy is associated with high toxicity. Novel targeted therapeutic approaches with higher response rates, especially inhibitors of tyrosine kinase receptors such as sunitinib or sorafenib, have been approved for treatment of advanced kidney cancer. Unfortunately, in the meantime, the clinical response to these agents is limited due to the development of tumor resistance by still unknown mechanisms. Therefore, there is a necessity to investigate clinical and pathological factors that can predict overall outcome of these treatments [4–6]. Apoptosis is a tightly controlled mechanism of cell death in which many catabolic enzymes, mainly proteases and nucleases, are activated leading to biochemical and morphological alterations that culminate in cellular collapse. This process of cell death plays an essential role in maintaining tissue homeostasis, and is important in certain pathological conditions, including cancer [7]. The first morphological characteristics of apoptosis include onset of blebbing, cell shrinkage, cytoskeletal rearrangement, chromatin condensation and nuclear fragmentation. Apoptosis occurs when cells prepare themselves to die in a cascade culminating in DNA degradation (via activation of endonucleases), nuclear disintegration and formation of apoptotic bodies. These apoptotic bodies are rapidly removed by tissue macrophages. Signaling for this is the translocation of phosphatidylserine from the inside to the outside of the membrane, marking the cells to be phagocytosed [8]. Many studies performed in recent years have shown that apoptosis is a constitutive way of death presented by almost all cells, if not all, and may be triggered by a large number of stimuli [9]. The recombinant protein Amblyomin-X, characterized as a Kunitz-type protease inhibitor (GenBank accession AAT68575), was obtained from a cDNA library from the salivary glands of the Amblyomma cajennense tick [10]. This protein is able to inhibit the coagulation factor Xa (FXa) and also promotes cytotoxic activity on several tumor cells, among them pancreatic and melanoma cells, with little or no activity on normal cells [11,12]. Furthermore, in Biomedicine & Pharmacotherapy 66 (2012) 64–69 A R T I C L E I N F O Article history: Received 3 August 2011 Accepted 1 November 2011 Keywords: Renal cell carcinoma Amblyomin-X Apoptosis A B S T R A C T Renal cell carcinoma (RCC) is one of the most lethal urologic cancers and is highly resistant to both radiotherapy and chemotherapy. The recombinant protein Amblyomin-X, characterized as a Kunitz-type protease inhibitor, was obtained from a cDNA library from the salivary glands of the Amblyomma cajennense tick. This paper reports the biological effect of Amblyomin-X on inducing cell death by apoptotic process in vitro. For this purpose, the changes in morphological aspects of cells, the phosphatidylserine exposition and DNA degradation were evaluated after treatment with Amblyomin- X. We found that Amblyomin-X was able to induce apoptosis in Renca cells in a dose-dependent manner. So, the results presented here open perspectives for new researches and developing for Amblyomin-X in the treatment of RCC. ß 2012 Elsevier Masson SAS. All rights reserved. * Corresponding author. Tel.: +55 11 37 26 72 22x2043; fax: +55 11 37 26 10 24. E-mail address: amchudzinski@butantan.gov.br (A.M. Chudzinski-Tavassi). Available online at www.sciencedirect.com 0753-3322/$ – see front matter ß 2012 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biopha.2011.11.015