172 Original article A recombinant protein based on Trypanosoma cruzi surface molecule gp82 induces apoptotic cell death in melanoma cells Vanessa D. Atayde a , Miriam G. Jasiulionis b , Mauro Cortez a and Nobuko Yoshida a Trypanosoma cruzi infection is known to confer resistance to tumor development in mice, and in-vitro studies have shown the toxic effects of parasite extracts on cancer cell cultures. Investigations in which T. cruzi molecules exhibit antitumor activity have just begun. Here, we used a tumorigenic cell line Tm5, derived from mouse melanocytes melan-a, to test the effect of J18, a recombinant protein based on T. cruzi surface molecule gp82 fused to glutathione-S-transferase (GST). J18 induced actin cytoskeleton disruption in Tm5 but not in melan-a cells. Several changes indicative of apoptosis were detected in Tm5 melanoma cells but not in melan-a cells treated with J18, such as the flipping of phosphatidylserine from the inner to the external side of the plasma membrane, altered nuclear morphology, DNA fragmentation, increase in mitochondria depolarization, and in caspase-3 activity. Retention of NF-jB in the cytoplasm was another alteration observed specifically in J18-treated Tm5 cells. No such alterations were found in Tm5 cells treated with GST. In-vivo experiments showed that C57BL/6 mice inoculated with Tm5 cells, treated at the site of tumor cell inoculation with J18, developed tumors of smaller size than mice treated with phosphate-buffered saline or GST and survived longer. Melanoma Res 18:172–183  c 2008 Wolters Kluwer Health | Lippincott Williams & Wilkins. Melanoma Research 2008, 18:172–183 Keywords: surface molecule gp82, melanoma cells, Trypanosoma cruzi a Department of Microbiology, Immunology and Parasitology and b Department of Pharmacology, Federal University of Sa ˜o Paulo, Sa ˜ o Paulo, Brasil Correspondence to Nobuko Yoshida, Escola Paulista de Medicina, Universidade Federal de Sa ˜ o Paulo, Rua Botucatu, 862-61 Andar, 04023-062, Sa ˜o Paulo, SP, Brasil Tel: +55 115 576 4532; fax: +55 115 571 1095; e-mail: nyoshida@unifesp.br Received 9 October 2007 Accepted 27 February 2008 Introduction The incidence of cutaneous malignant melanoma, the most aggressive form of skin cancer, has increased in recent decades in most fair-skinned people throughout the world [1,2]. Melanoma arises from the malignant transformation of melanocytes, the pigment-producing cells that reside in the basal layer of the epidermis, through a complex process involving genetic as well as environmental factors. Failure to undergo apoptosis is among the essential alterations that dictate malignant growth, which include insensitivity to growth-inhibitory signals, limitless replicative potential, sustained angio- genesis, tissue invasion, and metastasis [3]. Strategies to counterbalance this failure may therefore contribute to anticancer therapies. Apoptosis, a physiological process of cell death, is triggered by signals from either the intracellular or extracellular milieu and is orchestrated by a suicide machinery conserved through evolution, in which a proteolytic system involving a family of proteases known as caspases is a core component [4]. DNA fragmentation, exposure of phosphatidylserine in the outer leaflet of the plasma membrane, and loss of mitochondrial membrane permeability are among the alterations that accompany apoptosis. From a number of studies in recent years, the actin cytoskeleton has emerged as a key regulator of apoptosis [5]. By preventing actin filament elongation with cytochalasin D, apoptosis was induced in adherent epithelial cells [6]. Treatment of cells with cytochalasin D enhanced apoptosis in human leukemia CMK-7 cells by greatly accelerating caspase-3 activation [7]. Jasplakinolide, a drug that stabilizes and aggregates actin filaments, was shown to increase apoptosis induced by cytokine deprivation in CTLL-20 cells, the murine interleukin 2-dependent T cell line, apparently by acting upstream of the induction of capsase-3-like activity [8]. As regards the cell death induced by jasplakinolide, through a caspase-3-like protease pathway, an important finding was that Jurkat cells, derived from human acute lymphoblastic T cell leukemia, were more susceptible than normal nontransformed cells [9]. Resistance to tumors in infections by microbial patho- gens, including the protozoan parasites Toxoplasma gondii and Trypanosoma cruzi, has been reported [10,11]. In-vitro experiments have shown the direct inhibitory effect of T. cruzi lysates on cultured human breast cancer cells [12] and sarcoma-180 cells [13]. These antitumor effects may be because of the proapoptotic activity of T. cruzi 0960-8931  c 2008 Wolters Kluwer Health | Lippincott Williams & Wilkins Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.