Melanin as a Target for Melanoma Chemotherapy: Pro-oxidant Effect of Oxygen and Metals on Melanoma Viability

Melanoma cells have a poor ability to mediate oxidative stress, which may be attributed to constitutive abnormalities in their melanosomes. We hypothesize that disorganization of the melanosomes will allow chemical targeting of the melanin within. Chemical studies show that under oxidative conditions, synthetic melanins demonstrate increased metal affinity and a susceptibility to redox cycling with oxygen to form reactive oxygen species. The electron paramagnetic resonance (EPR)-active 5,5'-dimethyl-pyrollidine N-oxide spin adduct was used to show that binding of divalent Zn or Cu to melanin induces a pro-oxidant response under oxygen, generating superoxide and hydroxyl radicals. A similar pro-oxidant behaviour is seen in melanoma cell lines under external peroxide stress. Melanoma cultures grown under 95% O2/5% CO2 atmospheres show markedly reduced viability as compared with normal melanocytes. Cu- and Zn-dithiocarbamate complexes, which induce passive uptake of the metal ions into cells, show significant antimelanoma activity. The antimelanoma effect of metal- and oxygen-induced stress appears additive rather than synergistic; both treatments are shown to be significantly less toxic to melanocytes

Multiple scanning electrochemical microscopy mapping of tyrosinase in micro-contact printed fruit samples on polyvinylidene fluoride membrane

Herein, we introduce three orthogonal and compatible methods for detecting tyrosinase, a key factor in fruit browning and skin disorders, with high spatial resolution by means of scanning electrochemical microscopy (SECM). All methods are performed subsequently on the same substrate area providing a wide range of relevant information. The first SECM approach that relies on the mapping of a differential pore oxygen concentration induced by the local hydrophobic changes that the adsorption of tyrosinase generates on a porous polyvinylidene fluoride (PVDF) membrane. The second approach is based on the direct monitoring of the enzymatic activity of tyrosinase by detecting amperometrically the reaction products from the enzymatic conversion of L-3,4-dihydroxyphenylalanine (L-DOPA). Finally, tyrosinase was visualized implementing a tyrosinase sandwich immunoassay readout by SECM. The multiple SECM detection strategies were successfully applied to map unequivocally the tyrosinase enzymatic activity of a micro-contact printed banana sample. Furthermore, differential pulse voltammetry and mass spectrometry analyses were employed to elucidate the nature of the electrochemical response obtained during the tyrosinase enzymatic activity experiments

Continuing to illuminate the mechanisms underlying UV-mediated melanomagenesis

The incidence of melanoma is one of the fastest growing of all tumor types in the United States and the number of cases worldwide has doubled in the past 30 years. Melanoma, which arises from melanocytes, is an extremely aggressive tumor that invades the vascular and lymphatic systems to establish tumors elsewhere in the body. Melanoma is a particularly resilient cancer and systemic therapy approaches have achieved minimal success against metastatic melanoma resulting in only a few FDA-approved treatments with limited benefit. Leading treatments offer minimal efficacy with response rates generally under 15% in the long term with no clear effect on melanoma-related mortality. Even the recent success of the specific BRAF mutant inhibitor vemurafenib has been tempered somewhat since acquired resistance is rapidly observed. Thus, understanding the mechanism(s) of melanoma carcinogenesis is paramount to combating this deadly disease. Not only for the treatment of melanoma but, ultimately, for prevention. In this report, we will summarize our work to date regarding the characterization of ultraviolet radiation (UVR)-mediated melanomagenesis and highlight several promising avenues of ongoing research

Pro-Oxidant Properties of Melanosomal Melanin from Melanoma Origin

Melanoma cells, in comparison to normal melanocytes, contain higher concentrations of redox active species and yet are inefficient in mediating oxidative stress. We attribute these features to changes of intracellular melanosomes and melanin reactivity during the pathogenic process. Eumelanins are redox active pigments containing hydroquinone, semiquinone, quinone and quinone-imine functionalities within their structure. We have previously shown that pro-oxidant properties of synthetic melanin are enhanced by stabilization of the oxidized form of melanin as a result of metal chelation. Chemical studies using DNA clipping activity of isolated melanosomes from different sources indicated enhanced pro-oxidant properties of melanosomes from melanoma origin compared to melanin from normal melanocytes and sepia origin. Electron paramagnetic resonance spin trapping studies confirm these results both in electrochemically polymerized dihydroquinones and in intact human melanoma cells and suggested generation of superoxide and hydroxyl radicals. Ultrastructural investigations of melanosomes by electron microscopy revealed abnormalities in melanin deposition and membrane of the melanoma melanosomes compared to melanocyte melanosomes, which may play a part in higher reactivity of melanoma melanosomes. in related experiments exposure of normal human melanocytes to ultraviolet light radiation –B plus copper and cadmium (but not zinc) salts led to cells that phenotypically resembled dysplastic nevus cells in culture

Pro-Oxidant Properties of Melanosomal Melanin from Melanoma Origin

Melanoma cells, in comparison to normal melanocytes, contain higher concentrations of redox active species and yet are inefficient in mediating oxidative stress. We attribute these features to changes of intracellular melanosomes and melanin reactivity during the pathogenic process. Eumelanins are redox active pigments containing hydroquinone, semiquinone, quinone and quinone-imine functionalities within their structure. We have previously shown that pro-oxidant properties of synthetic melanin are enhanced by stabilization of the oxidized form of melanin as a result of metal chelation. Chemical studies using DNA clipping activity of isolated melanosomes from different sources indicated enhanced pro-oxidant properties of melanosomes from melanoma origin compared to melanin from normal melanocytes and sepia origin. Electron paramagnetic resonance spin trapping studies confirm these results both in electrochemically polymerized dihydroquinones and in intact human melanoma cells and suggested generation of superoxide and hydroxyl radicals. Ultrastructural investigations of melanosomes by electron microscopy revealed abnormalities in melanin deposition and membrane of the melanoma melanosomes compared to melanocyte melanosomes, which may play a part in higher reactivity of melanoma melanosomes. in related experiments exposure of normal human melanocytes to ultraviolet light radiation –B plus copper and cadmium (but not zinc) salts led to cells that phenotypically resembled dysplastic nevus cells in culture