Both oxidative/nitrosative stress and alterations in DNA methylation are observed during carcinogenesis of different tumor types, but no clear correlation between these events has been demonstrated until now. Melanoma cell lines were previously established after submitting the nontumorigenicmelanocyte lineage, melan-a, to cycles of anchorage blockade. In this work, increased intracellular oxidative species and nitric oxide levels, as well as alterations in the DNA methylation, were observed after melan-a detachment, which were also associated with a decrease in intracellular homocysteine (Hcy), an element in the methionine (universal methyl donor) cycle. This alteration was accompanied by increase in glutathione (GSH) levels and methylated DNA content. Furthermore, a significant increase in dnmt1 and 3b expression was identified along melan-a anchorage blockade. L(G)-Nitro-L-arginine methyl esther (L-NAME), known as a nitric oxide synthase (NOS) inhibitor, and N-acetyl-L-cysteine (NAC) prevented the increase in global DNA methylation, as well as the increase in dnmt1 and 3b expression, observed during melan-a detachment. Interestingly, both L-NAME and NAC did not inhibit nitric oxide (NO) production in these cells, but abrogated superoxide anion production during anchorage blockade. In conclusion, oxidative stress observed during melanocyte anchorage blockade seems to modulate DNA methylation levels and may directly contribute to the acquisition of an anoikis-resistant phenotype through an epigenetic mechanism.

Exclude experimental models of malignant transformation employ chemical and physical carcinogens or genetic manipulations to study tumor progression. In this work, different melanoma cell lines were established after submitting a nontumorigenic melanocyte lineage (melan-a) to sequential cycles of forced anchorage impediment. The great majority of these cells underwent anoikis when maintained in suspension. After one deadhesion cycle, phenotypic alterations were noticeable in the few surviving cells, which became more numerous and showed progressive alterations after each adhesion impediment step. No significant differences in cell surface expression of integrins were detected, but a clear electrophoretic migration shift, compatible with an altered glycosylation pattern, was observed for beta1 chain in transformed cell lines. In parallel, a progressive enrichment of tri- and tetra-antennary N-glycans was apparent, suggesting increased N-acetylglucosaminyltransferase V activity. Alterations both in proteoglycan glycosylation pattern and core protein expression were detected during the transformation process. In conclusion, this model corroborates the role of adhesion state as a promoting agent in transformation process and demonstrates that cell adhesion disturbances may act as carcinogenic stimuli, at least for a nontumorigenic immortalized melanocyte lineage. These findings have intriguing implications for in vivo carcinogenesis, suggesting that anchorage independence may precede, and contribute to, neoplastic conversion.

No abstract

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 BH4, and increased by the inhibitor of BH4 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.

A melanocyte malignant transformation model was developed in our laboratory, in which different melanoma cell lines were obtained after submitting the non-tumorigenic melanocyte lineage melan-a to sequential cycles of anchorage impediment. Our group has already showed that increased superoxide level leads to global DNA hypermemethylation as well increased Dnmt1 expression few hours after melanocyte anchorage blockade. Here, we showed that Ras/Rac1/ERK signaling pathway is activated in melanocytes submitted to anchorage impediment, regulating superoxide levels, global DNA methylation, and Dnmt1 expression. Interestingly, Ras and Rac1 activation is not related to codon mutations, but instead regulated by superoxide. Moreover, the malignant transformation was drastically compromised when melan-a melanocytes were submitted to sequential cycles of anchorage blockage in the presence of a superoxide scavenger. This aberrant signaling pathway associated with a sustained stressful condition, which might be similar to conditions such as UV radiation and inflammation, seems to be an early step in malignant transformation and to contribute to an epigenetic reprogramming and the melanoma development.