Cellular mechanisms involved in the melatonin inhibition of HT-29 human colon cancer cell proliferation in culture Introduction A series of studies have shown the oncostatic activity of melatonin both in vivo and in vitro in different type of tumors [1–6]. Major mechanisms involved in the antitu- moral effect of melatonin were related to its influence on the neuroendocrine system [7] and its immunoenhancing prop- erties [8–10]. However, due to the broad spectrum of melatonin actions, other mechanisms, including a direct effect of melatonin on cancer cell proliferation [11], cannot be discarded. Melatonin exerts some of its physiological effects through specific membrane receptors. These receptors, associated with protein G i /G 0 , have been classified according to their pharmacological characteristics, in Mel 1A , Mel 1B and Mel 1C subtypes [12, 13]. Two of these, Mel 1A and Mel 1B , now named mt 1 and MT 2 receptor subtypes, respectively, have been identified in humans [14, 15]. The third subtype, MT 3 , seems to be related to quinone reductase, although its function remains unclear. Recent data support a role for MT 2 in the antiproliferative effect of melatonin on human choriocarcinoma cells [16]. Melatonin is a highly lipophilic molecule [17] that easily crosses cell membranes reaching intracellular organelles including nucleus and mitochon- dria. It was reported that the accumulation of melatonin in the nucleus and its interaction with specific nuclear recep- tors [18, 19]. Nuclear receptors for melatonin were identi- fied [20] and some studies related them with the melatonin control of cell growth and differentiation [21]. Increasing evidence for a melatonin-mitochondria rela- tionship includes the antiapoptotic properties of the indoleamine through its interaction with the mitochondrial permeability transition pore [22, 23]. Moreover, melatonin is soluble in aqueous solutions [24], suggesting that the indoleamine acts in hydrosoluble environments such as the cytosol without the need for specific receptors. One of the receptor-independent effects of melatonin involved in the control of cell proliferation is its interaction with calmodulin. Melatonin binds with high affinity to Ca 2+ - calmodulin (CaCaM) [25], inhibiting the CaCaM-dependent processes including cAMP-dependent phosphodiesterase [26] and nitric oxide synthase (NOS) [27, 28] activities Abstract: The antiproliferative and proapoptotic properties of melatonin in human colon cancer cells in culture were recently reported. To address the mechanisms involved in these actions, HT-29 human colon cancer cells were cultured in RPMI 1640 medium supplemented with fetal bovine serum at 37°C. Cell proliferation was assessed by the incorporation of [ 3 H]-thymidine into DNA. Cyclic nucleotide levels, nitrite concentration, glutathione peroxidase and reductase activities, and glutathione levels were assessed after the incubation of these cells with the following drugs: melatonin membrane receptor agonists 2-iodo-melatonin, 2-iodo-N-butanoyl- 5-methoxytryptamine, 5-methoxycarbonylamino-N-acetyltryptamine (GR- 135,531), and the antagonists luzindole, 4-phenyl-2-propionamidotetralin, and prazosin; the melatonin nuclear receptor agonist CGP 52608, and four synthetic kynurenines analogs to melatonin 2-acetamide- 4-(3-methoxyphenyl)-4-oxobutyric acid, 2-acetamide-4-(2-amino- 5-methoxyphenyl)-4-oxobutyric acid, 2-butyramide-4-(3-methoxyphenyl)- 4-oxobutyric acid and 2-butyramide-4-(2-amino-5-methoxyphenyl)- 4-oxobutyric acid. The results show that the membrane receptors are not necessary for the antiproliferative effect of melatonin and the participation of the nuclear receptor in this effect is suggested. Moreover, the antioxidative and anti-inflammatory actions of melatonin, counteracting the oxidative status and reducing the production of nitric oxide by cultured HT-29 cells seem to be directly involved in the oncostatic properties of melatonin. Some of the synthetic kynurenines exert higher antiproliferative effects than melatonin. The results reinforce the clinical interest of melatonin due to the different mechanisms involved in its oncostatic role, and suggest a new synthetic pathway to obtain melatonin agonists with clinical applications to oncology. Ana Garcı ´a-Navarro 1 , Cristina Gonza ´ lez-Puga 1 , Germaine Escames 2 , Luis C. Lo ´ pez 2 , Ana Lo ´ pez 2 , Manuel Lo ´ pez- Cantarero 2 , Encarnacio ´n Camacho 3 , Antonio Espinosa 3 , Miguel Angel Gallo 3 and Darı ´o Acun ˜ a-Castroviejo 1,4 1 Departamento de Cirugı ´a, Hospital Universitario San Cecilio; 2 Departamento de Fisiologı ´a, Instituto de Biotecnologı ´a, Universidad de Granada; 3 Departamento de Quı ´mica Farmace ´utica y Orga ´ nica, Facultad de Farmacia, Universidad de Granada; 4 Servicio de Ana ´ lisis Clı ´nicos, Hospital Universitario San Cecilio, Granada, Spain Key words: antioxidant, HT-29 cancer cells, melatonin, melatonin receptors, nitric oxide, oncostatic therapy, oxidative stress Address reprint requests to Darı ´o Acun ˜a- Castroviejo, Departamento de Fisiologı ´a, Facultad de Medicina; Avda. de Madrid 11, E-18012 Granada, Spain. E-mail: dacuna@ugr.es Received April 21, 2007; accepted May 01, 2007. J. Pineal Res. 2007; 43:195–205 Doi:10.1111/j.1600-079X.2007.00463.x Ó 2007 The Authors Journal compilation Ó 2007 Blackwell Munksgaard Journal of Pineal Research 195