Disruption of Redox Homeostasis by Alterations in Nitric Oxide Synthase Activity and Tetrahydrobiopterin along with Melanoma Progression.

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.

The Role of the BH4 Cofactor in Nitric Oxide Synthase Activity and Cancer Progression: Two Sides of the Same Coin.

Cancer development is associated with abnormal proliferation, genetic instability, cell death resistance, metabolic reprogramming, immunity evasion, and metastasis. These alterations are triggered by genetic and epigenetic alterations in genes that control cell homeostasis. Increased reactive oxygen and nitrogen species (ROS, RNS) induced by different enzymes and reactions with distinct molecules contribute to malignant transformation and tumor progression by modifying DNA, proteins, and lipids, altering their activities. Nitric oxide synthase plays a central role in oncogenic signaling modulation and redox landscape. Overexpression of the three NOS isoforms has been found in innumerous types of cancer contributing to tumor growth and development. Although the main function of NOS is the production of nitric oxide (NO), it can be a source of ROS in some pathological conditions. Decreased tetrahydrobiopterin (BH4) cofactor availability is involved in NOS dysfunction, leading to ROS production and reduced levels of NO. The regulation of NOSs by BH4 in cancer is controversial since BH4 has been reported as a pro-tumoral or an antitumoral molecule. Therefore, in this review, the role of BH4 in the control of NOS activity and its involvement in the capabilities acquired along tumor progression of different cancers was described.

Metastatic Melanoma Progression Is Associated with Endothelial Nitric Oxide Synthase Uncoupling Induced by Loss of eNOS:BH4 Stoichiometry.

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.

Imbalance between nitric oxide and superoxide anion induced by uncoupled nitric oxide synthase contributes to human melanoma development.

Melanoma is the most aggressive type of cutaneous tumors due to its metastatic potential and high mortality. Increased levels of reactive oxygen species, including superoxide anion (O2-), and the consequent installation of a pro-oxidant environment are associated with melanoma development. The enzyme nitric oxide synthase (NOS), responsible for the production of nitric oxide (NO), when uncoupled is as a source of O2-, for example by the absence of its cofactor tetrahydrobiopterin (BH4). Western blot analysis showed increased expression of endothelial and inducible NOS in human melanoma cells, altering the stoichiometry between NOS levels and BH4 concentration and together with decreased BH4:BH2 ratio are contributing to NOS uncoupling. The treatment of melanoma cells with exogenous BH4 increased NO concentration and decreased O2- levels, leading to NOS coupling, which in turn reduced cell viability, cell proliferation and the ability of melanoma cells to form melanoma spheroids. Moreover, BH4 level restoration rendered melanoma cells more sensitive to apoptosis, demonstrating the role of dysfunctional NOS in melanoma genesis.

Receptor tyrosine kinases and downstream pathways as druggable targets for cancer treatment: the current arsenal of inhibitors.

Searching for targets that allow pharmacological inhibition of cell proliferation in over-proliferative states, such as cancer, leads us to finely understand the complex mechanisms orchestrating the perfect control of mitosis number, frequency and pace as well as the molecular arrangements that induce cells to enter functional quiescence and brings them back to cycling in specific conditions. Although the mechanisms regulating cell proliferation have been described several years ago, never before has so much light been shed over this machinery as during the last decade when therapy targets have been explored and molecules, either synthetic or in the form of antibodies with the potential of becoming cancer drugs were produced and adjusted for specific binding and function. Proteins containing tyrosine kinase domains, either membrane receptors or cytoplasmic molecules, plus the ones activated by those in downstream pathways, having tyrosine kinase domains or not, such as RAS which is a GTPase and serine/threonine kinases such as RAF, play crucial role in conducting proliferation information from cell surroundings to the nucleus where gene expression takes place. Tyrosine kinases phosphorylate tyrosine residues in an activating mode and are found in important growth factor receptors, such as for ligands from families collectively known as VEGF, PDGF and EGF, to name a few and in intracellular downstream molecules. They all play important roles in normal physiology and are commonly found mutated or overexpressed in neoplastic states. Our objective here is to present such kinases as druggable targets for cancer therapy, highlighting the ones for which the pharmacological arsenal is available, discussing specificity, resistance mechanisms and treatment alternatives in cases of resistance, plus listing potential targets that have not been successfully worked yet.

Exploração funcional do processo de glicosilação aberrante em tumores: mecanismos envolvidos na atividade pró-migratória de galectina-3 / Exploiting the functional significance of aberrant glycosylation in tumors: mechanisms involved in the activity of galectin-3

Com o objetivo de analisar a expressão dos ligantes de galectina-3 associados à transformação maligna, desenvolvemos uma proteína quimérica, a Gal-3/FA. Observamos que a Gal-3/FA pode ser usada como sonda em ensaios de overlay e de imunoistoquímica. Entre os ligantes de galectina-3 identificamos a 1 integrina, mediador da migração celular. A galectina-3 age como modulador positivo do processo de migração em superfícies de laminina-1. Na presença de galectina-3 observamos diminuição de FAK fosforilado e recrutamento da fosfatase SHP-2 para os complexos focais, levando ao turnover desses complexos / Exploiting the functional significance of aberrant glycosylation in tumors: mechanisms involved in the promigratory activity of galectin-3 / We have generated a novel tool for exploiting the clinical and biological significance of a common hallmark of cancer cells, namely the process of aberrant glycosylation. This tool is a chimeric galectin-3 fused to alkaline phosphatase which allowed for in situ identification of galectin-3 ligands in both human and murine tissues...