Mitochondrial dysfunction under the growth of B16/F10 melanoma in C57Bl/6 mice

National Medical Research Centre for Oncology

Russia, Rostov-on-Don

Mitochondria are present in all organs and tissues of the organism being responsible for the control of various aspects of the cellular function, providing the necessary supply of ATP, regulating Ca2+ signaling, controlling levels of reactive oxygen species (ROS) etc. Mitochondria perform specialized functions unique to certain tissues. Abnormalities in mitochondria disorder the key physiological functions such as the ATP production, oxidative phosphorylation, the reactive oxygen species production, and the Ca2+ regulation. This is considered as the mitochondrial dysfunction [5]. It is known that tumor mitochondria undergo some adaptive changes to further accelerate the rapid reproduction of tumor cells in an acidic and hypoxic microenvironment [2].

Aims. The aims in our study were to investigate dysfunctional signs of mitochondria in tumor cells and the internal organs in mice under the growth of B16/F10 melanoma.

Materials and methods . We used mice of both sexes of the C57BL/6 line (n=336). We composed the following groups of the C57BL/6 mice: the intact animal group (n=42); the reference group (n=42) with a model reproducing chronic neurogenic pain (CNP); the comparison group (n=126) with subcutaneous transplantation of B16/F10 melanoma; the main test group (CNP+B16/F10) (n=126). The male and female mice of the comparison group and the main test group were injected under the skin of the back below the angle of the left shoulder blade with 0.5 ml of a suspension of B16/F10 mouse melanoma tumor cells in saline solution at a dilution of 1:20. Chronic neurogenic pain was reproduced by ligation of the sciatic nerve on 2 sides under xylazolethyl anesthesia. B16/F10 melanoma was transplanted into the animals of the main test group 3 weeks after the wound had healed. Mitochondria were isolated according to the standard method using differential centrifugation with a high-speed refrigerated centrifuge. In mitochondrial samples of the tumor, the skin, the heart, the brain, and the liver cells, the following concentrations were determined by ELISA: cytochrome C (ng/mg protein), caspase 9 (ng/mg of protein), Bcl-2 (ng/mg of protein), AIF (ng/mg protein), calcium (Ca2+) (mmol/g of protein), and the protein concentration in mg/ml was determined by the Biuret method (Olvex Diagnosticum, Russia). The statistical analysis of the data was completed with software package Statistica 10.0.

Results . An analysis of the system of apoptosis in the mitochondria of the internal organs in the C57BL/6 mice with an independent growth of melanoma and that combined with CNP demonstrated some dysfunctional changes in mitochondria both in the tumor and the tumor-bearing organ, namely, the skin, and also in all organs, both under the independent tumor growth and that in combination with CNP [3]. The nature of the dysfunctional changes in mitochondria was attributed to the functional specificity of the organ; considering all the internal organs, the heart’s reaction was the most intensive: that was evidenced by the morphologically confirmed infarctions under the growth of melanoma with CNP and suppression of the apoptosis factors [1,4].

Apoptosis processes differed in tumor mitochondria under the independent and CNP-combined tumor growth. In mitochondria of the tumor cells in the females during all 3 weeks of the independent tumor growth, we observed the higher level of Ca2+, on average 60.0 times higher than that in the intact skin, and the higher Bcl-2 value on average by 3.8 times, while AIF was found to be 7.5 times lower, cytochrome C was recorded to be 3.9 times lower, and caspase 9 was reported to be 2.0 times lower.

In the males with the independent tumor growth, only the level of Ca2+ demonstrated an increase in its value by a factor of 78.5, while the Bcl-2 and cytochrome C values were recorded to be 2.9 times and 3.7 times lower, respectively. The CNP-linked tumor growth in the females led to a sharp increase in the Ca2+ level in tumor mitochondria at the initial stage, and then, from the logarithmic stage to the terminal stage, to its multiple drop. In the males, the level of Ca2+ in the tumor mitochondria was reduced throughout the tumor growth period with CNP on average by a factor of 3.2.

Conclusion. We believe that the results obtained may indicate the presence of a deep stress response to the growth of melanoma in its independent variant and with CNP, which begins to manifest itself from the subcellular level with the "involvement" and "subordination" of the tumor disease of all organs. While the dysfunctional mitochondrial “pathway” shows its own features depending on the sex of the animal and the variant of the pathological process.