The tricyclic antidepressant amitriptyline is cytotoxic to HTB114 human leiomyosarcoma and induces p75 NTR -dependent apoptosis Grazia Pula * , Alessandra Pistilli * , Claudia Montagnoli, Anna M. Stabile, Maria G. Rambotti and Mario Rende Nerve growth factor (NGF) receptors, TrKA and p75 NTR , are being investigated in cancer therapy. Our previous data show that, in HTB114 uterine leiomyosarcoma cells, p75 NTR -dependent apoptosis is inducible by cytotoxic drugs and can suppress nerve growth factor-dependent growth. Although amitriptyline can kill cancer cells and bind TrKA/B, its effects on p75 NTR -dependent apoptosis are unknown. The aim of this paper was to evaluate the antineoplastic potential of amitriptyline, and the role of p75 NTR -dependent apoptosis in the chemoresistant uterine HTB114 leiomyosarcoma. Using proliferation assays and fluorescence-activated cell sorting analysis, we found that amitriptyline caused a marked reduction in HTB114 cell viability, associated with the parallel upregulation of p75 NTR expression. This converted the TrKA + -proliferating cells into TrKA + /p75 NTR + , leading to downregulation of TrKA-prosurvival signaling (AKT) and activation of p75 NTR -dependent apoptosis (through caspase-3). Overall, we provide novel evidence that HTB114 uterine leiomyosarcoma cells are highly sensitive to amitriptyline, supporting the role of p75 NTR -dependent apoptosis as a novel cytotoxic mechanism of this drug and of p75 NTR as an inducible stress receptor and a novel target in clinical oncology. Anti-Cancer Drugs 24:899–910  c 2013 Wolters Kluwer Health | Lippincott Williams & Wilkins. Anti-Cancer Drugs 2013, 24:899–910 Keywords: amitriptyline, apoptosis, cancer, cancer therapy, neurotrophins, nerve growth factor, p75 NTR , TrKA, uterine leiomyosarcoma Anatomy Section, Department of Medico-Surgical Specialties and Public Health, School of Medicine, University of Perugia, Perugia, Italy Correspondence to Grazia Pula, MD, PhD, Anatomy Section, Department of Medico-Surgical Specialties and Public Health, School of Medicine, University of Perugia, Via del Giochetto, 06126 Perugia, Italy Tel: +39 075 585 7449; fax: +39 075 5857454; e-mail: pula@unipg.it *Grazia Pula and Alessandra Pistilli contributed equally to this work. Received 15 January 2013 Revised 6 June 2013 Revised form accepted 12 June 2013 Introduction Uterine leiomyosarcoma is an aggressive tumor, which is radioresistant and chemoresistant. Generally, resistance to chemotherapy is linked to the ability of cancer cells to repair DNA damage and develop adaptations, which promote survival in toxic environments. Thus, finding alternative strategies to ‘switch off ’ the survival pathways of these cells may provide a clinical benefit. Furthermore, investigation of existing drugs that are currently pre- scribed for other indications can prove cost-effective. Like other developmental factors, neurotrophins (NTs) have been increasingly linked to cancer [1–6]. In fact, accumulating evidences show that tumors can develop autonomous secretion of NTs, especially nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF). This triggers an autocrine loop, leading to constitutive activation of their cognate receptors, unrest- ricted growth, and resistance to apoptosis. Thus, neurotrophin receptors (NTRs) have become promising targets in cancer therapy [7,8]. There are two type of NTRs, namely, the NT-selective tropomyosin receptor kinases (TrKA, TrKB, and TrKC) and the nonselective p75 NTR , which binds all NTs [9]. The TrKs are tyrosine- kinase receptors. They are encoded by proto-oncogenes and activate the classic signaling pathways of growth factor receptors, including (a) the mitogenic p38 mito- gen-activated protein kinase pathway and (b) the prosurvival phosphatidylinositol 3-kinase (PI3K)/AKT pathway, which also inhibits apoptosis [10]. They can also trigger Ca 2+ release through the phospholipase C-g/ phosphatidylinositol bisphosphate/inositol trisphosphate/ diacylglycerol pathway [9]. In contrast, p75 NTR is a death receptor of the tumor necrosis factor (TNF) receptor superfamily, with a functional death domain [5,11,12]. However, this receptor is also a stem cell marker (CD271) [13], associated with cancer growth [13,14], and can form complexes with different coreceptors activating multiple pathways. Therefore, whereas the TrKs are generally prosurvival, the role of p75 NTR is more complex and enigmatic [11–15] and it can induce either (a) apoptosis, through c-jun N-terminal kinase (JNK)/ caspase-3, -6, and -9, or (b) survival, through NF-kB (NF- kB), depending on crosstalk with the TrKs and other coreceptors [13,15,16]. This ambiguity has been de- scribed in other death receptors [17] and reflects the different signaling molecules expressed by differentiated Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website (www.anti-cancerdrugs.com). Preclinical report 899 0959-4973  c 2013 Wolters Kluwer Health | Lippincott Williams & Wilkins DOI: 10.1097/CAD.0b013e328364312f Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.