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 degrees 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.

Melatonin is an indoleamine that is synthesised from tryptophan under the control of the enzymes arylalkylamine N-acetyltransferase (AA-NAT) and acetylserotonin methyltransferase (ASMT). Melatonin inhibits colon cancer growth in both in vivo and in vitro models; however, a precise mechanism responsible for inhibiting tumour growth has not been clearly described. Endothelin-1 (ET-1) is a peptide that acts as a survival factor in colon cancer, inducing cell proliferation, protecting carcinoma cells from apoptosis and promoting angiogenesis. The data presented show that melatonin inhibits edn-1 mRNA expression (the first step in ET-1 synthesis), ECE-1 protein expression and the release of ET-1 from colorectal cancer cells in vitro. ET-1 levels in cultured media present a similar inhibition pattern to that of edn-1 mRNA expression despite the inhibition of ECE-1 protein after melatonin treatment, which suggests that an endopeptidase other than ECE-1 could be mainly responsible for ET-1 synthesis. The inhibition of edn-1 expression is due to an inactivation of FoxO1 and NF-κβ transcription factors. FoxO1 inactivation is associated with an increased Src phosphorylation, due to elevated cAMP content and PKA activity, whereas NF-κβ inactivation is associated with the blockade of Akt and ERK phosphorylation due to the inhibition of PKC activity after melatonin treatment. Melatonin also inhibits edn-1 promoter activity regulated by FoxO1 and NF-κβ. Finally, a significant correlation was observed between AA-NAT and edn-1 expression downregulation in human colorectal cancer tissues. In conclusion, melatonin may be useful in treating colon carcinoma in which the activation of ET-1 plays a role in tumour growth and progression.

Some data suggest that cholecystokinin (CCK) receptor agonists stimulate the growth of colon cancer. Melatonin, an endogenous indoleamine with strong antioxidant properties, displays antiproliferative and proapoptotic properties both in vivo or in vitro in several types of tumors. We used HT-29 human colon cancer cells, expressing CCK receptors, to test the antiproliferative effects of several antagonists of CCK-A and/or CCK-B and their possible synergism with melatonin. HT-29 cells were cultured in RPMI 1640 medium supplemented with fetal bovine serum at 37 degrees C. Cell proliferation was assessed by the incorporation of [3H]-thymidine into DNA. Annexin V-FITC plus propidium iodine were used for flow cytometry apoptosis/necrosis evaluation. The following drugs were tested: gastrin (CCK-B agonist); CCK-8s (CCK-A agonist); proglumide (CCK-A plus CCK-B antagonist); lorglumide (CCK-A antagonist); PD 135,158 (CCK-B antagonist and weak CCK-A agonist); devazepide or L 364,718 (CCK-A antagonist); L 365,260 (CCK-B antagonist), and melatonin. The results shown a lack of effects of gastrin on HT-29 cell proliferation, whereas CCK-8s induced proliferation at high doses. The order of the antiproliferative effect of the other drugs was devazepide > lorglumide > proglumide. These drugs produce cell death mainly inducing apoptosis. Melatonin showed strong antiproliferative effect at millimolar concentrations, and it induced apoptotic cell death. Melatonin generally enhanced the antiproliferative effects of devazepide, lorglumide and proglumide and increased the proglumide-induced apoptosis. These results suggest that melatonin and CCK-A antagonists are useful for controlling human colon cancer cell growth in culture and in combined therapy significantly increases their efficiency.

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The characterization of colon cancer stem cells (CSCs) may help to develop novel diagnostic and therapeutic procedures. p53 loss increases the pool of CSCs in colorectal cancer (CRC). Recent reports suggest that the oncostatic effects of melatonin could be related to its ability to kill CSCs. Although there are no data linking the loss of p53 function and melatonin synthesis or signaling in cancer, melatonin does activate the p53 tumor-suppressor pathway in this disease. In this work, we analyze whether the expression of melatonin synthesis and signaling genes are related to the expression of CSC markers and the implication of p53 status in samples from patients with CRC. Arylalkylamine N-acetyltransferase (AA-NAT), MT1, and MT2 expression decreased in tumor samples versus normal mucosa samples in mutated p53 (mtp53) tumors versus those with wild-type p53 (wtp53). Further, AA-NAT and MT2 expression were lower in advanced stages of the disease in wtp53 tumors. On the contrary, CD44 and CD66c expression was higher in tumor versus normal mucosa in wtp53 tumors. Additionally, CD44 expression was higher in advanced stages of the disease regardless of the p53 status. Patients with CD44highCD66chigh and wtp53 tumors in advanced stages showed low expression of AA-NAT and MT2 in wtp53 tumors. These results could indicate a possible interaction of these pathways in CRC.