Prediction of nitric oxide concentrations in melanomas
Introduction
Nitric oxide may play a role in the growth and metastasis of melanomas, the most malignant of skin cancers [1], [2]. The presence of inducible nitric oxide synthase (iNOS) and nitrotyrosine provides evidence of NO production within some human melanomas, and these two markers for NO are correlated with poor survival rates of patients [3]. Studies with melanoma cells suggest that NO derived from iNOS may stimulate proliferation as well as promote resistance to apoptosis [4]. However, efforts to establish mechanistic links have been hampered by the absence of information on NO concentrations. Our objective was to provide concentration estimates by combining cell kinetic data with a mathematical model designed to describe reactions and diffusion of NO in cutaneous melanomas.
Metastatic melanomas are typically in the vertical-growth phase and can penetrate more than 4 mm into the dermis, sometimes reaching the subcutaneous tissue before spreading to regional lymph nodes [5]. A network of blood vessels extends upwards from the subcutaneous tissue into the dermis. The tumor stroma that is interposed between the malignant melanoma cells and normal dermal tissue consists largely of extracellular matrix components. It is an interstitial region which accommodates the expansion of the tumor as it invades the surrounding tissue. In cutaneous melanomas, the stroma typically contains inflammatory cells such as macrophages, and the infiltration of macrophages has also been linked to tumor growth [6]. Thus, synthesis and consumption of NO by macrophages as well as melanoma cells must be taken into account when estimating NO concentrations.
Nitric oxide synthesized within a cell can readily cross its plasma membrane and diffuse to neighboring cells. The permeation of NO through lipid bilayers and its diffusion through water are both very similar to that of O2 [7], [8]. Nitric oxide is consumed by autoxidation, intracellular pathways which are both enzymatic and non-enzymatic, and oxidation by oxyhemoglobin. Each of these rate processes requires consideration when simulating NO levels in melanomas.
The uncatalyzed reaction of NO with O2 (autoxidation) yields as the end-product, with NO2 and N2O3 formed as intermediates [9]. The overall stoichiometry of this multi-step oxidation isThis reaction will occur both within cells and in extracellular spaces. However, although it is important in culture media and was taken into account in the experiments reported here, it is too slow to have a significant effect on NO concentrations within a melanoma, as will be shown. One pathway for the intracellular consumption of NO involves its rapid reaction with to form peroxynitrite (ONOO−) and [10], [11]:Because is a byproduct of respiration and Reaction (2) is fast enough to compete with superoxide dismutase, it is expected to occur in virtually all cells which are exposed to NO. Other pathways for intracellular consumption of NO involve nitrogen dioxygenases [12], and reactions with oxygen-ligated reduced metals [13]. Measurements of NO consumption by rat hepatocytes indicate that the overall intracellular NO consumption rate is first-order in NO concentration [14], which is consistent also with data from our laboratory for various cell types [15], [16]. Lastly, any NO that reaches blood vessels will react rapidly with oxyhemoglobin (HbO2) to form methemoglobin (metHb) and [13]:Accordingly, capillaries act as very effective sinks for NO in tissues.
It should be mentioned that Reaction (2) can also lead to NO consumption outside cells, because activated macrophages synthesize extracellular via a membrane-bound NADPH oxidase [17], [18]. However, the diffusion-limited kinetics of Reaction (2), together with the fact that NO production exceeds extracellular synthesis, makes this an extremely localized process. It has been estimated that the produced by a macrophage is fully exhausted within 1 μm of the cell [19]. Moreover, the rate of sustained release into the surrounding media by a macrophage has been found to be only 15% of its rate of NO production [16]. For these reasons, extracellular consumption of NO by can be neglected when modeling NO concentrations in tissues.
To provide the cell kinetic data needed for a reaction–diffusion model, we studied NO synthesis and consumption in a melanoma cell line, A375. Previous work with A375 cells suggests that endogenous NO affects melanoma growth; they were shown to constitutively express iNOS [20] and treatment of A375 cells with a NO scavenger (c-PTIO) resulted in decreased cell growth [1]. Complementing the A375 data were previous measurements of NO synthesis and consumption in a macrophage cell line and in primary mouse macrophages [16]. We combined this cellular information with data on the rates of NO diffusion and reaction in aqueous media and with anatomical length scales to simulate steady-state NO concentrations within a melanoma.
Section snippets
Cell culture
A375 melanoma cells were obtained from American Type Culture Collection and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing l-glutamine supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% (v/v) heat-inactivated fetal bovine serum (FBS) (BioWhittaker, Walkersville, MD). All cells were maintained at 37 °C in a humidified 5% CO2 atmosphere.
NO synthesis by A375 cells
To assess the synthesis of NO by A375 cells under simulated inflammatory conditions, the supernatant of cells incubated in
Results
Nitric oxide production by A375 melanoma cells was not detected using Griess assays, despite the use of systemic inflammatory factors (TNF-α and IFN-γ) which lead to iNOS expression in other cell types [27], [28]. This negative result is unlikely to have been due to analytical limitations, because such measurements of accumulation have been used successfully by us and others with various NO-producing cells [16], [29], [30]. Thus, melanoma cells appear to generate NO at rates low enough to
Discussion
We developed a mathematical model to estimate spatially varying NO concentrations in a cutaneous melanoma. By using differential equations to describe rates of diffusion and reaction of NO, concentrations were obtained as a function of position in an idealized tumor. Key inputs in the model were the rates of NO synthesis and consumption by macrophages [16] and melanoma cells. Nitric oxide synthesis by A375 melanoma cells was not detected using Griess assays, which is generally consistent with
Acknowledgments
We thank Laura J. Trudel for the instruction that she provided in cell culture techniques and Dr. Gerald Wogan for helpful discussions of melanoma pathophysiology.
Grants
This work was supported by a grant from the National Cancer Institute (PO1 CA26731). M.P.C. was supported in part by a graduate student fellowship from the National Science Foundation.
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