Endothelial nitric oxide synthase uncoupling as a key mediator of melanocyte malignant transformation associated with sustained stress conditions

Melanoma cell lines and cells corresponding to premalignant melanocytes were established by our group after subjecting a nontumorigenic murine melanocyte lineage, melan-a, to sequential cycles of anchorage blockade. Previous results showed that in melan-a cells the superoxide level increases after such procedure. Superoxide production during melanocyte de-adhesion was inhibited by L-sepiapterin, the precursor of eNOS cofactor BH(4), and increased by the inhibitor of BH(4) synthesis, DAHP, hence indicating a partial uncoupling state of eNOS. the eNOS uncoupling seems to be maintained in cells derived from melan-a, because they present decreased nitric oxide and increased superoxide levels. the inhibition of superoxide production in Tm5 melanoma cells with L-sepiapterin reinforces their eNOS-uncoupled state. the maintenance of oxidative stress seems to be important in melanoma apoptosis resistance because Mn(III)TBAP, a superoxide scavenger, or L-sepiapterin renders Tm5 cells more sensitive to anoikis and chemotherapy. More importantly, eNOS uncoupling seems to play a pivotal role in melanocyte malignant transformation induced by sustained anchorage impediment, because no malignant transformation was observed when L-NAME-treated melanocytes were subjected to sequential cycles of de-adhesion. Our results show that uncoupled eNOS contributes to superoxide production during melanocyte anchorage impediment, contributing to anoikis resistance and malignant transformation. (C) 2011 Elsevier Inc. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal de São Paulo, Disciplina Imunol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Disciplina Farmacol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Disciplina Nefrol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Disciplina Imunol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Disciplina Farmacol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Disciplina Nefrol, BR-04023900 São Paulo, BrazilFAPESP: 06/61293-1FAPESP: 05/60334-3FAPESP: 08/50366FAPESP: 09/03335-8Web of Scienc