Exercise modulates redox-sensitive small GTPase activity in the brain microvasculature in a model of brain metastasis formation

Abstract

Tumor cell extravasation into the brain requires passage through the blood-brain barrier (BBB). There is evidence that exercise can alter the oxidation status of the brain microvasculature and protect against tumor cell invasion into the brain, although the mechanisms are not well understood. In the current study, we focused on the role of microenvironment generated by exercise and metastasizing tumor cells at the levels of brain microvessels, influencing oxidative stress-mediated responses and activation of redox-sensitive small GTPases. Mature male mice were exercised for four weeks using a running wheel with the average voluntary running distance 9.0 ± 0.3 km/day. Mice were then infused with 1.0 × 10(6) D122 (murine Lewis lung carcinoma) cells into the brain microvasculature, and euthanized either 48 hours (in short-term studies) or 2-3 weeks (in long-term studies) post tumor cell administration. A significant increase in the level of reactive oxygen species was observed following 48 hours or 3 weeks of tumor cells growth, which was accompanied by a reduction in MnSOD expression in the exercised mice. Activation of the small GTPase Rho was negatively correlated with running distance in the tumor cell infused mice. Together, these data suggest that exercise may play a significant role during aggressive metastatic invasion, especially at higher intensities in pre-trained individuals.

Figure 1. Variations in running activity in exercised mice.

Mice were adapted to exercise and solitary living for one week, followed by voluntary wheel running for four weeks. (A) Running distance for individual mice used in the study in the last four weeks. High runners (black) run more than 10.2 km/day, mid-runners (gray), between 7.8–10.2 km/day, and low runners (white), less than 7.8 km/day. Average running distance over the four weeks of exercise was 9.0±0.3 km/day. (B) Time spent on running activity (hrs/day) and (C) speed of running. Running time and speed increased gradually during the adaptation period, then remained steady for four weeks of exercise. Average time was 9.6±0.2 hrs/day and average speed was 0.9±0.0 km/hr. Values are mean ± SEM; n = 55.

Figure 2. Peroxide and superoxide levels in brain microvessels of tumor cell-infused exercises and sedentary mice.

Mice were exercised as in Figure 1 with sedentary mice housed in cages with locked wheels. Following exercise period, mice were infused with either 1.0×10⁶ D122 lung carcinoma cells (tumor) or cell culture media (vehicle) and euthanized 48 h (short-term studies) or 2–3 weeks (long-term studies) post tumor cell infusion. Tissue peroxides were measured by 2′,7′-dichlorofluorescein (DCF) fluorescence (A and B) and superoxide levels by dihydroethidium (DHE) fluorescence (C, and D) in freshly isolated brain microvessels. Representative DHE florescent images of freshly isolated brain microvessels visualized by staining (C, right panel). (D) Exercise distance negatively correlated (Pearson’s r = −0.9782) with DHE fluorescence in the tumor cell infused mice. Values are mean ± SEM, *compared to the sedentary plus vehicle group, p<0.05; +compared to sedentary plus tumor group, p<0.05; #compared with the exercised plus vehicle group, p<0.05; n = 4–15 per group.

Figure 3. Expression of antioxidative enzymes in brain microvessels of tumor cell-infused exercised and sedentary mice.

Mice were exercised and infused with tumor cells as in Figure 2, followed by evaluation of protein expression of antioxidative enzymes, MnSOD (A, B), Cu/ZnSOD (C), catalase (D), and GPx (E) in isolated brain microvessels by immunoblotting. The images show representative Western blots and the bar graphs reflect quantitative data from n = 6–9 per group. Values are mean ± SEM; *compared to the sedentary plus vehicle group, p<0.05; +compared to the sedentary plus tumor group, p<0.05; #compared to the exercise plus vehicle, p<0.05.

Figure 4. Small GTPase activity in brain microvessels of tumor cell-infused exercised and sedentary mice.

Mice were exercised and infused with tumor cells as in Figure 2, followed by evaluation of active forms of Rac1 (A), Ras (B), and Rho (C) in isolated brain microvessels. Activation was determined using pull down assays for active (GTP-bound) GTPases, followed by immunoblotting for specific protein. In addition, a correlation between running distance and GTPase activity was determined by Pearson’s r (A–C). Images show representative immunoblotting from n = 8–17 and the bar graphs reflect quantitative data from these experiments. Values are mean ± SEM *compared to the sedentary plus vehicle group at p<0.05; #compared to the exercise plus vehicle group at p<0.05.