Phenotypic changes caused by melatonin increased sensitivity of prostate cancer cells to cytokine-induced apoptosis Introduction N-Acetyl-5-methoxy-tryptamine, melatonin, is an indole that is produced endogenously. Mainly, circulatory mela- tonin comes from pineal gland but it is synthesized in other locations including retina, Harderian gland, gut mucosa, cerebellum, airway epithelium, liver, kidney, adrenals, thymus, thyroid, pancreas, ovary, carotid body, placenta, endometrium, or skin. In all the species studied thus far, its synthesis from tryptophan in the pineal gland occurs during darkness [1, 2]. Melatonin was initially only associated with mammals but subsequently has been found in a variety of species including all nonvertebrate taxa and plants [1, 3–5]. Considering its nocturnal synthesis, melatonin is linked to sleep promotion [6], a chemical signal of light/dark cycle [7], and a regulator of reproductive physiology in seasonal breeding mammals [8]. Besides regulating circadian and circannual rhythms, melatonin is a major endogenous antioxidant and a free radical scavenger [9] that plays a major role in cell defense against toxins that increase oxidative damage both in vitro and in vivo [10–13]. The indole has demonstrated to be especially efficient in protecting neurons from oxidative damage in neurodegenerative models [14, 15] and also is a good therapeutic tool to prevent ischemia/reperfusion in numerous tissues [16, 17]. Melatonin functions as a direct scavenging molecule and stimulates gene expression and activities of antioxidant enzymes. As a direct scavenger, melatonin reacts with different free radicals including • OH, O  2 , NO • , and alkyl-peroxyl radicals and it stimulates glutathione, glutathione peroxidase, and superoxide dismu- tases [18, 19]. Additionally, melatonin acts as a receptor- independent autocrine and paracrine antioxidant that modulates basic processes such as growth, differentiation, and cell death by mechanisms still not completely understood. There is an inverse relationship between melatonin levels and tumor growth, in terms of initiation as well as progression and metastasis [20]. The first evidence came from studies showing that breast and prostate cancer patients displayed decreased night serum levels of melato- nin when compared to control patients without malignant tumors [21]. Also in animal models, an increased tumor growth was observed when animals were pinealectomized [22]. This evidence has been reinforced by prospective studies, showing that disturbing normal light/dark cycles by shift work enhances tumor incidence while altering serum melatonin levels [23, 24]. Mechanistically, cell culture studies have demonstrated that melatonin directly inhibits cancer cellsÕ growth, either Abstract: Melatonin has antiproliferative properties in prostate cancer cells. Melatonin reduces proliferation without increasing apoptosis, and it promotes cell differentiation into a neuroendocrine phenotype. Because neuroendocrine cells displayed an androgen-independent growth and high resistance to radiotherapy and chemotherapy, the role of molecules that induce neuroendocrine differentiation was questioned in terms of their usefulness as oncostatic agents. By using human epithelial androgen- dependent and androgen-independent prostate cancer cells, the role of melatonin in drug-induced apoptosis was studied after acute treatments. In addition to cytokines such as hrTNF-alpha and TRAIL, chemotherapeutic compounds, including doxorubicin, docetaxel, or etoposide, were employed in combination with melatonin to promote cell death. Melatonin promotes cell toxicity caused by cytokines without influencing the actions of chemotherapeutic agents. In addition, antioxidant properties of melatonin were confirmed in prostate cancer cells. However, its ability to increase cell death caused by cytokines was independent of the redox changes. Finally, phenotypic changes caused by chronic treatment with the indole, that is, neuroendocrine differentiation, make cells significantly more sensitive to cytokines and slightly more sensitive to some chemotherapeutic compounds. Thus, melatonin is a good inhibitor of the proliferation of prostate cancer cells, promoting phenotypic changes that do not increase survival mechanisms and make cells more sensitive to cytokines such as TNF-alpha or TRAIL. Aida Rodriguez-Garcia 1,2 , Juan C. Mayo 1,2 , David Hevia 3 , Isabel Quiros-Gonzalez 1,2 , Maria Navarro 1 and Rosa M. Sainz 1,2 1 Departamento de Morfologia y Biologia Celular, Universidad de Oviedo, Oviedo, Spain; 2 Instituto Universitario Oncologico del Principado de Asturias (IUOPA), Universidad de Oviedo, Oviedo, Spain; 3 Instituto de Ciencia y Tecnologı ´a de los Alimentos y Nutricio ´n, ICTAN, CSIC, Madrid, Spain Key words: apoptosis, chemotherapy, melatonin, prostate cancer, TNF-alpha, TNF- related apoptosis-inducing ligand Address reprint requests to Rosa M. Sainz, Departamento de Morfologia y Biologia Celular. Facultad de Medicina. Universidad de Oviedo. Julian Claveria, 6. 33006 Oviedo, Spain. E-mail: sainzrosa@uniovi.es Received January 24, 2012; Accepted May 25, 2012. J. Pineal Res. 2012 Doi:10.1111/j.1600-079X.2012.01017.x Ó 2012 John Wiley & Sons A/S Journal of Pineal Research 1 Molecular, Biological, Physiological and Clinical Aspects of Melatonin