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Introduction

Melanocytes are located in various parts of the human body, such as the skin and the eye⁽¹⁾. Their transformation leads to melanoma, an aggressive and deadly neoplasm⁽¹⁾. Cutaneous and uveal melanomas show different characteristics, including significant differences in genetic alterations, metastatic sites and therapeutic response⁽¹⁾. In recent decades, great efforts have been made to obtain a more comprehensive understanding of genetics, genomics and molecular changes, enabling the identification of key cellular processes and signaling pathways in melanomas⁽¹⁾. Major breakthroughs were realized in the treatment of metastatic cutaneous melanoma, but most patients relapse⁽¹⁾. Currently, there is no approved systemic treatment for metastatic uveal melanoma⁽¹⁾. Thus, these two different cancers are in therapeutic need to overcome treatment failure and improve patients’ prognosis⁽¹⁾.

Malignant melanoma, a neoplasm arising from malignant transformation of melanocytes, is predominantly a disease of the skin, but in rare instances it may occur at other sites, including the mucous membranes (hard palate, maxillary gingiva, lip, throat, esophagus, vulva, vagina and perianal region) and the eye (uvea and retina)⁽²⁾.

Melanocytes are particularly susceptible to oxidative stress owing to the prooxidant state generated during melanin synthesis and to the intrinsic antioxidant defenses that may be shattered in pathologic conditions⁽²⁾.

Oxidative stress can be defined as an imbalance between the formation of prooxidants and the ability of the body antioxidant systems to decrease or remove their harmful effects⁽³⁾. This redox state may result in disruption of redox signaling and biomolecules damage⁽³⁾.

Oxidative stress can disrupt the homeostasis of mela­nocytes, causing damage to DNA, protein and cellular components⁽²⁾. Altered reactive oxygen species (ROS) levels could also affect epigenetic mechanisms and promote alterations in gene expression, thus leading to severe impairment of cell survival and to cancer development⁽²⁾. Reactive oxygen species are highly reactive molecules that are constantly produced in all aerobic organisms, mostly as a consequence of aerobic respiration⁽⁴⁾. The term covers several types of chemical species of short-lived molecules with unpaired electrons, including free radicals such as superoxide (O₂-) or hydroxyl (-OH), and nonradicals such as hydrogen peroxide (H₂O₂)⁽⁴⁾. Levels of reactive oxygen species (ROS) are reduced by antioxidant defenses, but increased by transition metals such as iron or copper and by exogenous agents such as ionizing radiation or ozone⁽⁴⁾.

Similarly, nitrogen-derived free radicals are called reactive nitrogen species (RNS) and their utmost representative precursors are nitric oxide (NO) and peroxynitrite (ONOO-)⁽⁴⁾. Nitric oxide (NO) is well known to be a product of the catalytic action of the nitric oxide synthase (NOS) enzyme family on L-arginine⁽⁴⁾.

The main endogenous sources of ROS in mammalian cell include: mitochondria, endoplasmic reticulum, pe­ro­xi­somes, cytosol, plasma membrane, and extracellular space⁽³⁾. The exogenous sources of ROS are related to the physical factors exposure, such as UV solar light, gamma rays radiation exposition, electromagnetic field, alpha particles emitted by radioactive elements decay, environmental chemicals exposure (excessive pollution by organochlorines, xenobiotics, aromatic amines, quinones, polycyclic aromatic hydrocarbons, ozone, singlet oxygen [¹O²], pathogens, chemotherapy, tobacco smoking, iron overload, trauma, drugs and physical exercise)⁽³⁾.

A cell functioning is dependent on its redox state, that is the ratio of the reversible oxidized molecules and the reduced form of a specific redox couples in a cell, such as oxidized/reduced glutathione (GSSG/GSH), nicotinamide adenine dinucleotide (NAD) cation/reduced NAD (NAD⁺/NADH) or nicotinamide adenine dinucleotide phosphate (NADP) cation/reduced NADP (NADP⁺/NADPH) and the balance between oxidants formation and protection by antioxidants systems⁽³⁾.

The link between cancer metabolism and redox homeostasis

It has been shown that melanoma cells regularly reprogram their metabolism to provide an equivalent reduction and support in antioxidant protection⁽⁵⁾. There are different signaling pathways involved in supplying and regulation redox power in melanoma cells⁽⁵⁾. In particular, it has been demonstrated that during melanomagenesis the oxidative pentose phosphate pathway (ox-PPP), serine biosynthesis and 1-CM (one-carbon metabolism) are responsible for adenine dinucleotide phosphate (NADPH) and glutathione (GSH) production⁽⁵⁾. On the other hand, monocarboxylate transporters (MCTs) and glycolytic enzymes, such as pyruvate kinase (PK)-M2 (PKM2), are also associated to redox homeostasis during melanoma initiation and progression⁽⁵⁾.

Pentose phosphate pathway (PPP) is primarily catabolic and serves as an alternative glucose oxidizing pathway for the generation of NADPH that can be involved in redox metabolic adaptation of melanoma⁽⁵⁾. The antioxidant role of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of PPP that catalyzed the first reaction with the production of NADPH, has been studied in an in vitro melanoma model⁽⁵⁾. Indeed, the inhibition of G6PD sensitized malignant melanoma cells A375 to oxidative stress, decreased proliferation and induced apoptosis⁽⁶⁾.

Another study showed that the high expression of G6PD promotes melanoma growth via the signal transducer and activator of transcription 3/5 (STAT3/5) pathway in a human melanoma xenograft model⁽⁷⁾. The role of G6PD in cooperation with NADPH oxidase 4 (NOX4) for the support of redox homeostasis has been related to melanoma cells in vitro, indeed targeting both enzymes suppressed cell proliferation⁽⁸⁾. These studies indicate that PPP represents an essential redox metabolic pathway in melanoma and serves as a pivotal role in survival and adaptation of melanoma cells.

Glycolysis is a metabolic pathway converting glucose into pyruvate and lactate as final metabolites, and releasing energy to form ATP and NADH molecules. Several enzymes belonging to this metabolic pathway have been found to be associated with melanoma genesis⁽⁵⁾.

A unique biochemical feature of the melanocyte is the generation of melanin, which leads to the generation of hydrogen peroxide and the consumption of reduced glutathione (GSH)⁽¹⁰⁾. Successful attempts to reverse or inhibit this process have been initiated⁽⁹⁾, but to date they have not been clinically tested.

Melanoma cells overexpress a redox-depending enzyme, PKM2, an isoform of the pyruvate kinase, that con­verts phosphoenolpyruvate into pyruvate, being the last irreversible reaction of aerobic glycolysis⁽¹¹⁾. Reactive oxy­gen species oxidize a specific cysteine residue in PKM2, thus diverting glucose away from lactate production and towards the oxidative branch of pentose phosphate pathway, leading to increased NADPH production and, thus, to redox homeostasis⁽⁵⁾. These data provide a direct link between cancer meta­bolism and redox homeostasis. Melanoma cell invasion and metastasis levels were positively correlated with high PKM2 activity as well as the glycolytic capability. Knockdown of PKM2 markedly attenuated the malignant phenotypes of melanoma cells, including cell proliferation, invasion and metastasis in vitro and in vivo, suggesting that PKM2 is a potential therapeutic target in melanoma⁽¹¹⁾.

Metastatic pathways in patients with melanoma

Metastasis represents the end-product of an intricate biological process, which necessarily involves dissemination of neoplastic cells to different anatomic sites and adaptation of neoplastic cells to foreign tissue microenvironments⁽¹²⁾.

The process of metastasis is determined by the interplay between metastatic tumor cells, various host factors and homeostatic mechanisms⁽¹³⁾. Metastasis is a multistep process, which includes proliferation, neovascularization, immune system evasion, lymphangio­genesis, invasion, circulation, embolism, extravasation and colonization⁽¹³⁾.

The interactions between the neoplastic cells and the non-neoplastic stromal cells are important in the progression of the invasion-metastasis cascade^(12,14).

Cutaneous melanoma can metastasize hemato­genously or lymphogenously⁽¹⁵⁾. The three predominant models that endeavor to explain the patterns of melanoma progression are the stepwise spread model, the simultaneous spread model and the model of differential spread⁽¹⁵⁾. The stepwise spread model posits that melanoma metastasizes initially via the lymphatic system towards regional lymph nodes and, subsequently, systemic dissemination occurs^(16,17).

The second predominant model is the simultaneous spread model, which involves that primary cutaneous melanoma metastasizes simultaneously by hemato­genous and lymphatic pathways⁽¹⁸⁾.

The third model, which attempts to explain the patterns of progression of cutaneous melanoma, has been coined the model of differential spread⁽¹⁷⁾. This model proposes that there are multiple independent dissemination pathways⁽¹⁷⁾.

The time course to the development of metastases differs between the different metastatic routes⁽¹⁵⁾. There are several clinical and histopathological risk factors for the different metastatic pathways⁽¹⁵⁾. In particular, patient’s sex and the anatomical location of the primary tumor influence the patterns of disease progression⁽¹⁵⁾.

Metastatic disease is detectable at diagnosis in less than 4% of uveal melanoma cases⁽¹⁹⁾. Uveal melanoma metastasizes exclusively through hematogenous dissemination of tumor cells preferentially and almost exclusively to the liver, with up to 90% of metastatic uveal melanoma associated with hepatic lesions, and in most cases the liver is the only affected organ^(20,21). Other sites, such as the lungs (24%), bones (16%), skin/subcutaneous tissues (11%) and lymph nodes (10%), can also be affected⁽²¹⁾, while the involvement of brain and fellow eye is rare.

In this paper, we undertook a comparative analysis of the evolution of markers of oxidative stress (lipid peroxides, albumin thiols, total antioxidants) in patients with cutaneous malignant melanoma compared to those with uveal melanoma. The aim is to describe possible differences in oxygen metabolism between the two diseases, differences that may be clinically useful or may justify the relatively different biological behaviors of these two locations or, potentially, the different response to systemic treatments.

Materials and method

In this study, 45 patients, respectively 19 women and 26 men, aged between 30 and 84 years old, with cutaneous malignant melanoma, as well as a cohort of 44 patients diagnosed with uveal malignant melanoma were enrolled. Due to the heterogeneity of the patients’ group, in terms of age, sex and the extent of the disease at initial presentation, the patients with uveal melanoma were separated into three distinct subgroups, depending on the therapeutic method applied, respectively:

• the first group was treated with radiotherapy (n=14);

• the second group was treated strictly by enucleation (n=18);

• the third group was also treated by enucleation, but with systemic treatment applied postoperatively (mainly chemotherapy, only one case with interferon immunotherapy) (n=12).

All patients were followed-up clinically and biologically for two years after the diagnosis, respectively before and after the initial medical intervention, and subsequently at regular intervals of 3-6 months. Seven determinations of biochemical parameters of oxidative stress were obtained:

• markers of lipid peroxidation by measuring the reaction of malondialdehyde (MDA) as a final product;

• total thiol-albumin groups (by the Shoshinsky method), as a measure of the oxidative degradation of circulating proteins.

Results

For all patients, a series of biochemical tests were performed that followed oxygen metabolism and the installation of oxidative stress in the dynamics of the treatment. The therapeutic approach was chosen in accordance with established protocols that targeted optimal tumor regression, patient’s quality of life and overall health. Lipid oxidation parameters were initially analyzed in order to identify changes that could lead to demonstrable differences in the mechanisms of development of the two types of melanoma, cutaneous versus uveal. The average values obtained by measuring the final concentration of MDA, a product of lipid degradation under the action of reactive oxygen species, are presented in Figure 1. For reference, the baseline value was 4 µmol/100 ml.
 

The recorded values show fluctuations due to the treatment applied, with overall increase in the level of lipid peroxidation. Oxidative stress can affect all the organic molecules of the cell, which become targets for the following damage: deoxyribonucleic acid (DNA), proteins, lipids, carbohydrates. For effectively following the therapeutic intervention, the initial target of oxidative stress must be known. For example, DNA is known to be the initial target of damage produced by the addition of H₂O₂ in mammalian cell cultures, such that DNA strand breaks occur before “detectable” lipid peroxides or “detectable” oxidized proteins. The method of detecting lesions produced on some target molecules can give incomplete information; thus, highlighting protein damage through the detection of carbonyl radicals in the initial stages of the damage can be negative, but the determination of the oxidation of -SH groups, which occurs earlier, is positive. Trying to apply the results from the experimental model to the patients enrolled in the study, we investigated the biochemical parameters of oxidative stress in the dynamics of the applied treatment, considering that they can provide additional data related to the metabolism of the malignantly transformed cell, data that correlate with the angiogenesis process and, last but not least, data related to the destructive evolution of malignant cells, following equally the effectiveness of the treatment. Following the dynamics, it is observed in all investigated patients the increase in the intensity of the reaction measured, suggesting, in accordance with literature data, that the presence of a tumor is associated with an increase in oxidative stress, and this is why surgical excision can decrease oxidative stress. It is important to take into consideration that lipid peroxidation is a reaction with a chain mechanism; the measured effects are total, even those at a distance, thus suggesting an increase in the concentration of the final product measured.

For providing a more comprehensive picture, albumin thiols were also measured, reflecting the intensity of protein degradation under the attack of reactive oxygen radical species (baseline value: 450 µmol/L). The results (Figure 2) show the increase of the oxidative attack on the proteins as well, suggesting the installation of oxidative stress.
 

Comparing the oxidative degradation of proteins by measuring the end-product, namely albumin thiols, changes are observed between the two cohorts. The values are higher in the case of patients with uveal melanoma, once again confirming the hypothesis that in uveal melanoma there is a higher oxidative stress than the one which occurs after the treatment of cutaneous melanoma.

Next, the values of total antioxidants were measured, which, through the values recorded and presented in Figure 3, suggest the activation of natural antioxidant protection systems in response to an installed oxidative stress (baseline value: 1.2 µmol/L). It is difficult to say whether this increase is due only to endogenous antioxidants, because no study or questionnaire on the intake of exogenous antioxidants has been done.
 

We consider that each patient is his own witness, and the recorded values are related to the initial value for each patient. Furthermore, the study assumed that there were no essential lifestyle changes during the investigations (no changes were reported by the patients). We could draw the conclusion that the tumor is an oxidative stress factor to which the body reacts at the cellular level. The investigated parameters show values consistent with the state of health and could be used in patient monitoring and treatment management.

In cancer, the tumor presence itself is an oxidative stress inductor; however, anti-tumor treatments also have the ability to induce oxidative stress, by destroying tumor cells through degradative processes initiated at the molecular level.

Changes between the two groups indicate different molecular signaling mechanisms of endogenous antioxidant activity. They are continuously increasing in cutaneous melanoma; however, in uveal melanoma, at the end of the treatment, there is a decrease of recorded serum levels of oxidative parameters. It is thus suggested that the uveal melanoma leads to the installation of a more pronounced oxidative stress compared to cutaneous melanoma, with an intensification of oxygen metabolism and a lower response capacity of the body, possibly related to the increased aggressiveness of this malignant cell type. This could explain the increased resistance to immunotherapy of uveal melanoma when compared to cutaneous melanoma.

Discussion

Reactive oxygen species behave as a double-edged sword at the cellular level. When reactive species of oxygen are in a low concentration, they have a role in cell signaling, converting the cell phenotype to angiogenesis and neovascularization, which contributes to tumor growth and proliferation. On the other hand, when ROS production is in excess, it becomes destructive and damages tumor cells. The generation of oxidative stress during the process of tumor growth and development is particularly highlighted by the stimulation of lipid peroxidation, a process that generates numerous electrophilic aldehydes, stable compounds that can diffuse inside the cell, exerting several cytotoxic effects. In addition, lipid peroxidation products could be responsible for blocking cell cycle and/or apoptotic mechanisms, leading to decreased therapeutic efficacy and the establishment of treatment resistance. Excess lipid peroxidation products can inhibit cell cycle progression by blocking cyclin-dependent kinases, keeping tumor cells in the G0 phase, prolonging the G1 phase, and delaying entry into the S phase, reducing the rate of tumor cell proliferation.

Although intense oxidative stress could be effective in inducing tumor cytotoxicity, moderately induced oxidative stress could have opposite effects, contributing to the development of resistance mechanisms, decreasing therapeutic efficacy. Moreover, numerous in vivo and in vitro studies^(22,23) claim that tumor cells and tissues suffer an intense, chronic, sublethal oxidative stress, compared to normal cells, even in the absence of prior exogenous stimulation, being absolutely necessary for tumor viability and growth. Intense intratumorally oxidative stress may play an important role in the process of tumor invasion and metastasis.

The action of active oxygen metabolites at the level of structural or enzymatic proteins causes their denaturation^(3,10). A critical factor in this oxidation reaction of protein SH groups by ROS is the steric factor. Exposure of thiol groups in proteins is essential. This explains the increased sensitivity to oxidation of denatured proteins (this property is used as an effective method for identifying the content of thiol groups, as well as for identifying the secondary and tertiary structure of proteins). By oxidative degradation, enzymatic activity is inhibited, as a result of the modification of the active catalytic center (due to the oxidation of thiol groups or hemeprotein, but also by oxidative degradation of the aromatic cycles in the structure of some amino acids). Structural proteins are inactivated due to changes induced by polymerization reactions, branching or induced cleavage of polypeptide chains by ROS.

In many human conditions, oxidative stress is only a consequence, not a cause of the pathological disease process. Tissue damage by aggressive agents, such as infections, trauma, toxins, extreme temperatures etc., leads to the overproduction of “injury mediators”, such as prostaglandins, leukotrienes, interleukins and cytokines (such as tumor necrosis factor – TNF), which play an important role in the production of tissue lesions. In the neoplastic cell, the establishment of an imbalance between the production of reactive oxygen species and the elimination of free radicals leads to a state of oxidative stress and to the destruction of some essential components of the cell. Several studies^(4,10,23) concluded that oxidative stress resulting from this imbalance has a causative role but is also a consequence of carcinogenesis, interfering with all phases of this process, such as initiation, promotion, progression, invasion and metastasis.

In the patients enrolled in the study, we investigated the biochemical parameters of oxidative stress in the dynamics of the applied treatment, considering that they can provide additional data related to the metabolism of the malignantly transformed cell, data to correlate with the angiogenesis process and, finally, data related to the destructive evolution of malignant cells, equally aiming at the efficiency of the treatment. Lipid peroxidation reaction is a reaction with a chain mechanism, the measured effects are total, even those at a distance, thus suggesting an increase in the concentration of the final product measured. Since this parameter alone cannot provide a complete picture, we also measured albumin thiols, a measure of the intensity of protein degradation under the attack of reactive oxygen radical species. Following the dynamics, an increase in the intensity of the measured reaction is observed in all investigated patients, suggesting, as the data are also presented in the specialized literature^(24,25), that the presence of a tumor is associated with an increase in oxidative stress, while surgical resection could induce its decrease. Finally, it is worth noting that proteins are not the primary target of oxidative attack.

Conclusions

The results indicate an increase of lipid peroxidation reaction, serum albumin thiols and serum total antioxidants in correlation with tumor evolution, both in uveal and cutaneous melanoma, in comparison with baseline values. As such, the presence of the tumor is an oxidative stress inductor, with anti-tumor treatment also having the ability to induce oxidative stress through the destruction of tumor cells. Changes between the two groups indicate different molecular signaling mechanisms of endogenous antioxidant activity. They are continuously increasing in cutaneous melanoma; however, in uveal melanoma, at the end of the treatment, there is a decrease of recorded serum levels of oxidative parameters. It is thus suggested that uveal melanoma leads to the installation of a more pronounced oxidative stress compared to cutaneous melanoma, with an intensification of oxygen metabolism and a lower response capacity of the body, possibly related to increased aggressiveness of this malignant cell type. This could explain the increased resistance to immunotherapy of uveal melanoma when compared to cutaneous melanoma. Our results can be useful in monitoring the tumor evolution under the treatment.  

Conflicts of interests: The authors declare no conflict of interests.