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.

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.

Repair of abdominal wall defects is one of the major clinical challenges in abdominal surgery. Most biomaterials are associated to infection and severe complications, making necessary safer and more biocompatible approaches. In the present work, the adequate mechanical properties of synthetic polymer meshes with tissue-engineered matrices containing stromal mesenchymal cells is combined to generate a novel cell-containing tissue-like artificial stroma (SCTLAS) for use in abdominal wall repair. SCTLAS consisting on fibrin-agarose hydrogels seeded with stromal cells and reinforced with commercial surgical meshes (SM) are evaluated in vitro and in vivo in animal models of abdominal wall defect. Inflammatory cells, collagen, and extracellular matrix (ECM) components are analyzed and compared with grafted SM. Use of SCTLAS results in less inflammation and less fibrosis than SM, with most ECM components being very similar to control abdominal wall tissues. Cell migration and ECM remodeling within SCTLAS is comparable to control tissues. The use of SCTLAS could contribute to reduce the side-effects associated to currently available SM and regenerated tissues are more similar to control abdominal wall tissues. Bioengineered SCTLAS could contribute to a safer treatment of abdominal wall defects with higher biocompatibility than currently available SM.

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