Urokinase gene knockout and melanoma mitochondria: Association with free radical oxidation processes.

Abstract

e21535 Background: Mitochondria play a key role in carcinogenesis, and an imbalance in the oxidant-antioxidant system influences tumor development. The growth rate of malignant neoplasms varies, and knockout animals are used to model this process. Deficiency of the urokinase gene (uPA) allows for the reproduction of a slower tumor progression. The aim of the research was to study the intensity of free radical processes and antioxidant defense in the mitochondria of tumor and skin cells in female uPA-knockout mice during the growth stages of B16/F10 melanoma. Methods: The study was conducted on female mice (n=126): intact and experimental group (n=21+42) — genetically modified C57BL/6-Plau<tm1.1Bug> ThisPlau<GFDhu/GFDhu> mice (uPA–/–); intact and control group (n=21+42) — C57Bl/6 mice (uPA+/+). B16/F10 melanoma was transplanted subcutaneously, and animals were sacrificed at 1 and 3 weeks post-transplantation. Mitochondria were isolated from tumor and skin cells at the maximum distance from the melanoma focus by differential centrifugation. The concentrations of superoxide dismutase (SOD-2) (pg/mg), glutathione peroxidase-1 (GPX-1) (ng/mg), malondialdehyde (MDA) (μmol/g protein), and advanced oxidation protein products (AOPP) (μmol/g) were determined in the mitochondria by ELISA. Statistical analysis was performed using Statistica 10.0. Results: Tumor volume was smaller in uPA–/– animals compared to uPA+/+ mice. In tumor mitochondria of uPA–/– mice at 1 week, SOD-2 and AOPP levels were 2.5-fold and 11.3-fold higher, respectively, while GPX-1 and MDA levels were 4.7-fold and 9.6-fold lower than in uPA+/+ mice. At 3 weeks of B16/F10 growth, SOD-2 and AOPP levels in tumor mitochondria of uPA–/– females remained higher (1.9-fold, p<0.05 and 8.6-fold, respectively), and GPX-1 and MDA levels were lower (9.3-fold and 3.6-fold) compared to uPA+/+ females. In skin mitochondria of intact uPA–/– females, SOD-2 and GPX-1 levels were 1.8-fold (p<0.05) and 5.1-fold lower, respectively, while MDA and AOPP levels were 3.1-fold and 3.2-fold higher than in intact uPA+/+ mice. At 1 week of B16/F10 growth in uPA–/– mice, skin mitochondrial SOD-2 was 2.7-fold lower, GPX-1 was 15-fold lower, MDA was 5.8-fold lower, while AOPP was 2.6-fold higher. At 3 weeks, uPA–/– females showed increased skin mitochondrial SOD-2 (15.4-fold) and MDA (3-fold), and decreased AOPP (7.2-fold) and GPX-1 (6-fold). Conclusions: Slowed melanoma growth in uPA–/– mice is associated with a restructuring of mitochondrial oxidative balance. In the tumor: increased AOPP with decreased MDA suggests a shift in stress targets. The combination of elevated SOD-2 and reduced GPX-1 indicates activation of primary antioxidant defense against a possible depletion of the glutathione system. A similar but dynamic imbalance in skin mitochondria confirms the systemic influence of genotype and tumor on the redox homeostasis of the target organ.