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Updated: Oct 20, 2025
A 3D Organotypic Melanoma Spheroid Skin Model
Published on: May 18, 2018
Metastatic Melanoma Progression Is Associated with Endothelial Nitric Oxide Synthase Uncoupling Induced by Loss of
Fabiana Henriques Machado de Melo1,2, Diego Assis Gonçalves3,4, Ricardo Xisto de Sousa5
1Pharmacology Department, Universidade Federal de São Paulo, São Paulo 05508-090, Brazil.
Abstract:
Melanoma is the most aggressive type of skin cancer due to its high capability of developing metastasis and acquiring chemoresistance. Altered redox homeostasis induced by increased reactive oxygen species is associated with melanomagenesis through modulation of redox signaling pathways. Dysfunctional endothelial nitric oxide synthase (eNOS) produces superoxide anion (O2-•) and contributes to the establishment of a pro-oxidant environment in melanoma. Although decreased tetrahydrobiopterin (BH4) bioavailability is associated with eNOS uncoupling in endothelial and human melanoma cells, in the present work we show that eNOS uncoupling in metastatic melanoma cells expressing the genes from de novo biopterin synthesis pathway Gch1, Pts, and Spr, and high BH4 concentration and BH4:BH2 ratio. Western blot analysis showed increased expression of Nos3, altering the stoichiometry balance between eNOS and BH4, contributing to NOS uncoupling. Both treatment with L-sepiapterin and eNOS downregulation induced increased nitric oxide (NO) and decreased O2• levels, triggering NOS coupling and reducing cell growth and resistance to anoikis and dacarbazine chemotherapy. Moreover, restoration of eNOS activity impaired tumor growth in vivo. Finally, NOS3 expression was found to be increased in human metastatic melanoma samples compared with the primary site. eNOS dysfunction may be an important mechanism supporting metastatic melanoma growth and hence a potential target for therapy.
Insights
Endothelial nitric oxide synthase (eNOS) dysfunction contributes to aggressive melanoma by increasing reactive oxygen species. Restoring eNOS activity reduced tumor growth and chemoresistance, highlighting eNOS as a potential therapeutic target.
Area of Science:
- Oncology
- Biochemistry
- Molecular Biology
Background:
- Melanoma's aggressiveness stems from metastasis and chemoresistance, often linked to altered redox homeostasis and increased reactive oxygen species (ROS).
- Endothelial nitric oxide synthase (eNOS) dysfunction, producing superoxide anion (O2-•), establishes a pro-oxidant environment crucial for melanoma progression.
- While reduced tetrahydrobiopterin (BH4) is typically linked to eNOS uncoupling, metastatic melanoma cells exhibit high BH4 levels despite eNOS uncoupling.
Purpose of the Study:
- To investigate the role of eNOS dysfunction in metastatic melanoma.
- To explore the relationship between BH4 concentration, eNOS expression, and eNOS uncoupling in melanoma.
- To evaluate the therapeutic potential of restoring eNOS activity in melanoma treatment.
Main Methods:
- Western blot analysis to assess eNOS and BH4 levels.
- Gene expression analysis of biopterin synthesis pathway genes (Gch1, Pts, Spr).
- In vitro treatments with L-sepiapterin and eNOS downregulation; in vivo tumor growth studies.
Main Results:
- Metastatic melanoma cells showed increased NOS3 expression and high BH4 levels, leading to eNOS uncoupling and increased O2-•.
- L-sepiapterin treatment and eNOS downregulation restored NOS coupling, increased nitric oxide (NO), decreased O2•, and reduced cell growth, anoikis resistance, and dacarbazine chemoresistance.
- Restoration of eNOS activity inhibited tumor growth in vivo, and NOS3 expression was elevated in human metastatic melanoma samples.
Conclusions:
- eNOS uncoupling, despite high BH4, is a key mechanism in metastatic melanoma, driven by increased NOS3 expression.
- Targeting eNOS activity by restoring its function presents a promising therapeutic strategy for melanoma, impacting tumor growth and chemoresistance.
- eNOS dysfunction represents a significant contributor to melanoma metastasis and warrants further investigation as a therapeutic target.
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