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.

Cutaneous melanoma emerges from the malignant transformation of melanocytes and is the most aggressive type of skin cancer. The progression can occur in different stages: radial growth phase (RGP), vertical growth phase (VGP), and metastasis. Reactive oxygen species contribute to all phases of melanomagenesis through the modulation of oncogenic signaling pathways. Tetrahydrobiopterin (BH4) is an important cofactor for NOS coupling, and an uncoupled enzyme is a source of superoxide anion (O2•−) rather than nitric oxide (NO), altering the redox homeostasis and contributing to melanoma progression. In the present work, we showed that the BH4 amount varies between different cell lines corresponding to distinct stages of melanoma progression; however, they all presented higher O2•− levels and lower NO levels compared to melanocytes. Our results showed increased NOS expression in melanoma cells, contributing to NOS uncoupling. BH4 supplementation of RGP cells, and the DAHP treatment of metastatic melanoma cells reduced cell growth. Finally, Western blot analysis indicated that both treatments act on the PI3K/AKT and MAPK pathways of these melanoma cells in different ways. Disruption of cellular redox homeostasis by the altered BH4 concentration can be explored as a therapeutic strategy according to the stage of melanoma.

PURPOSE:To describe a technique to obtain guinea pigs cochlear fluids and measure nitric oxide (NO) concentration. METHOdS

Problem Ototoxicity continues to be the most common side effect of gentamicin, and its physiopathology is still unclear. Currently the main accepted theory relates the ototoxicity with over production of free radicals, like nitric oxide (NO). Objective: To analyze the NO levels in the cochlear fluids of guinea pigs exposes to gentamicin and its relation with ototoxicity and propose an experimental model to study ototoxicity and drugs that may prevent it. Methods Six guinea pigs were used, divided in two groups: a) Study – submitted to intraperitoneal injections of gentamicin, (135mg/kg/day) for 14days; b) Control - submitted to intraperitoneal injections of distilled water, daily, for 14days; the hearing loss was evaluated with Preyer's reflex. On 14 day the guinea pigs were sacrificed and the cochlear fluids were collected for dosage of the nitric oxide through the chemiluminescence method (NOA-TM 280). Results The Preyer's reflex was absent at the end of the experiment in the Study group, what did not happen in the control group. The average of the nitric oxide levels of the guinea pigs of the study group was 26.2μ and the Control group 11.26μM. Conclusion It was possible to measure nitric oxide levels in guinea pigs cochlear fluids. The results reinforce the role of nitric oxide in ototoxicity induced by gentamicin. Significance Experimental study (Level 2).