# Vitamin D Regulation of a SOD1-to-SOD2 Antioxidative Switch to Prevent Bone Cancer
## 2 Results
### SOD-to-SOD2 Antioxidative Switch after Vitamin D Treatment of MG-63 Osteosarcoma Cells  
Given the known increase in ROS among MG-63 osteosarcoma cells [39], it was hypothesized that antioxidant defense mechanisms may be involved in vitamin D-dependent suppression of ROS production. Specific SOD activities are correlated with tumorigenicity [30; 43], therefore the transcription of the SOD genes within 1,25D\({}_{3}\)-treated MG-63 cells was monitored over time. A significant and dramatic decrease in the level of SOD1 transcript was observed only after 24 and 48 h of 10 nM 1,25D\({}_{3}\) treatment (Figure 3C). At 100 nM of 1,25D\({}_{3}\) there was no change in SOD1 levels with or without VAZ,  
Figure 3: (**A**) Minimal effects on cytochrome c oxidase subunit 2 (COXII) transcript levels by vitamin D. Real-time PCR analysis of COXII (cytochrome c oxidase subunit 2) transcript levels in MG-63 cells in complete media. (**B**) Minimal effects on subunit 4 isoform 1 (COX4 II) transcript levels by vitamin D. Real-time PCR analysis of COX4 II (subunit 4 isoform 1) transcript levels in MG-63 cells in complete media. (**C**) Dose-dependent suppression of superoxide dismutase 1 (SOD1) transcript levels by vitamin D. Real-time PCR analysis of SOD1 (superoxide dismutase 1) transcript levels in MG-63 cells in complete media. (**D**) Dose- and time-dependent upregulation of superoxide dismutase 2 (SOD2) transcript levels by vitamin D. Real-time PCR analysis of SOD2 (superoxide dismutase 2) transcript levels in MG-63 cells in complete media. (**E**) Dose- and time-dependent suppression of catalase (CAT) transcript levels by vitamin D. Real-time PCR analysis of CAT (catalase) transcript levels in MG-63 cells in complete media. (**F**) Transcript levels presented as arbitrary units normalized to beta actin amounts. \(p\leq 0.05\) *, 0.01 **, 0.001 *** using the 2-way ANOVA with Bonferroni post hoc test (n = 3; error bars represent the SD). VAZ (vitamin D receptor antagonist ZK159222).  
suggesting no direct VDR-dependent regulatory role at higher concentrations of ligand. Interestingly, a dramatic >2.5-8-fold time-dependent persistent increase in SOD2 mRNA levels was observed only with the lower 10 nM 1,25D\({}_{3}\) concentration at both time points (Figure 3D). Once again, at the higher 100 nM 1,25D\({}_{3}\) concentration the regulatory role of VDR toward SOD2 activity was abrogated. Catalase is a peroxisomal enzyme that rapidly degrades hydrogen peroxide (H\({}_{2}\)O\({}_{2}\)) to O\({}_{2}\) and H\({}_{2}\)O. As SODs may yield H\({}_{2}\)O\({}_{2}\), the mRNA levels of the H\({}_{2}\)O\({}_{2}\) antioxidant catalase (CAT) were monitored, and a moderate and statistically significant decrease was observed only after 10 nM 1,25D\({}_{3}\) treatment at the 24-hour time point (Figure 3E). However, a steady increase following another 24 h back toward baseline was also observed (Figure 3E). This suggests the protective role of vitamin D treatment relying less on CAT antioxidant production during the early stages of treatment, and the potential decrease in H\({}_{2}\)O\({}_{2}\) efflux from the mitochondria. Lastly, the real-time PCR results were arranged using arbitrary units in order to compare amounts of transcripts across experiments. In this way, it became more evident that after 1,25D\({}_{3}\) treatment there was a clear shift from SOD1 to SOD2 initiated at the 24-hour time point (Figure 3F). However, by the 48-hour time point SOD2 levels exceeded SOD1 levels to complete the \"switch\" (Figure 3F). The switch from SOD1 to SOD2 may signify the importance of the cellular distribution and actions of each SOD enzyme, cytosol versus mitochondria, respectively. Overall, these findings suggest an active time- and concentration-dependent mitochondrial reparative process involving specific SOD enzymes engaged after vitamin D treatment within MG-63 osteosarcoma cells as part of vitamin D's anti-cancer effects (Figure 4).