The enzymatic component of the nitric oxide system in mitochondria of melanoma and skin: What changes occur?

Abstract

e21537 Background: Malignant melanoma has a multifactorial etiology and a heterogeneous course. Modern concepts about the role of mitochondrial processes in oncogenesis are expanding, and the involvement of the nitric oxide (NO) system in the regulation of mitochondrial function and dynamics has been demonstrated. However, the bimodality of NO effects in carcinogenesis and tumor progression requires close study. The aim of the work was to study the dynamics of inducible nitric oxide synthase (NOS2) and endothelial nitric oxide synthase (NOS3) in mitochondria of tumor and skin cells in female mice of the C57BL/6 line at the growth stages of experimental melanoma B16/F10. Methods: Female C57BL/6 mice (n=84) were divided into two groups: intact controls (n=21) and the main group (n=63), which underwent subcutaneous transplantation of B16/F10 melanoma. Animals were euthanized at 1, 2, and 3 weeks post-implantation. Mitochondria were isolated from melanoma cells and from distal skin (taken at the maximum distance from the tumor) using differential centrifugation. Concentrations of NOS2 and NOS3 in the mitochondrial fraction were determined by enzyme-linked immunosorbent assay (ELISA). All reported data were statistically significant (p<0.05) as determined by nonparametric statistical tests. Results: NOS2 levels in melanoma cell mitochondria progressively decreased throughout tumor growth: by 5.5-fold at week 1, 127.6-fold at week 2, and 192.6-fold at week 3 compared to levels in intact skin mitochondria. Similarly, NOS3 levels in tumor mitochondria declined by 24.4-fold at week 1, 62.5-fold at week 2, and 508.5-fold at week 3. In contrast, mitochondrial NOS2 levels in the skin of tumor-bearing mice showed a significant change only at week 3, decreasing 9.2-fold relative to intact controls and were 7.9-fold lower on average than levels at weeks 1 and 2. Mitochondrial NOS3 content in the skin decreased at all observed time points: by 2.5-fold at week 1, 3.0-fold at week 2, and 20.3-fold at week 3 compared to intact values. At week 3, the skin NOS3 level was 8.0-fold and 6.7-fold lower than at weeks 1 and 2, respectively. Conclusions: Thus, mitochondrial nitric oxide (NO) system dysfunction in melanoma is multifaceted: a pronounced local suppression of NOS2 and NOS3 in tumor mitochondria is accompanied by significant, albeit less marked, alterations in the mitochondria of distant skin. The suppression of NOS3 observed in both tumor and distant skin mitochondria may indicate the involvement of the vascular component in melanoma progression. These findings underscore the significant role of the mitochondrial NO synthase (NOS) pool not only in the autonomous behavior of the tumor but also in the target organ's response. The identified patterns may facilitate the search for potential targets for novel therapeutic strategies.