# Vitamin D Regulation of a SOD1-to-SOD2 Antioxidative Switch to Prevent Bone Cancer
## 2 Results
Characterization of 1,25D\({}_{3}\)-Mediated Cell Cycle and Vitamin D Catabolic and Osteogenic Genes in MG-63 Osteosarcoma Cells  
To acquire further insight into the molecular mechanisms underlying the increased inhibition of MG-63 osteosarcoma cell viability by 1,25D\({}_{3}\), gene expression analysis was performed of cell cycle, vitamin D signaling and osteogenic-related genes. For this part, MG-63 cells were treated with 10 nM and 100 nM 1,25D\({}_{3}\), or vehicle (equi-volume ethanol) for 24 and 48 h in complete media. First, proliferating cell nuclear antigen (PCNA) mRNA was appraised by real-time PCR to assess the level of DNA replication in the samples. After 1,25D\({}_{3}\) treatment at 10 nM, a statistically significant \(>\)2-fold suppression of PCNA transcripts was observed compared to vehicle-treated samples for both the 24- and 48-hour time points (Figure 2A). However, no large difference in PCNA transcript levels was observed at the higher 100 nM of 1,25D\({}_{3}\) at both time points, which was also confirmed upon vitamin D antagonist ZK159222 (VAZ) treatment that blocks VDR genomic actions [45] (Figure 2A). These results suggest that 1,25D\({}_{3}\) suppresses DNA replication (i.e., cell division) only at 10 nM in the system. Figure 2: (**A**) Dose-dependent suppression of proliferating cell nuclear antigen (PCNA) transcript levels by vitamin D. Real-time PCR analysis of PCNA (proliferating cell nuclear antigen) transcript levels in MG-63 cells in complete media. (**B**) Dose- and time-dependent suppression of cytochrome P450 family 24 subfamily A member 1 (CYP24A1) transcript levels by vitamin D. Real-time PCR analysis of CYP24A1  
(cytochrome P450 family 24 subfamily A member 1) transcript levels in MG-63 cells in complete media. (**C**) Dose- and time-dependent suppression of vitamin D receptor (VDR) transcript levels by vitamin D. Real-time PCR analysis of VDR (vitamin D receptor) transcript levels in MG-63 cells in complete media. (**D**) Dose- and time-dependent suppression of bone Gamma-Carboxylutamate Protein, or osteoclastin (BGLAP) transcript levels by vitamin D. Real-time PCR analysis of BGLAP (bone Gamma-Carboxylutamate Protein, or osteoclastin) transcript levels in MG-63 cells in complete media. \(p\leq 0.05\) *, 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). In order to monitor the control of vitamin D signaling, cytochrome P450 family 24 subfamily A member 1 (CYP24A1) and VDR transcript levels were monitored. CYP24A1 is a vitamin D\({}_{3}\) 24-hydroxylase and a VDR target gene that is involved in the catabolism of calcitriol. After treatment of MG-63 cells with 10 nM 1,25D\({}_{3}\) for both 24 and 48 h, there was a significant reduction in the level of CYP24A1 compared to vehicle-treated controls (Figure 2B). However, after 100 nM 1,25D\({}_{3}\) treatment, there was a significant increase in CYP24A1 mRNA compared to 10 nM-treated samples. This response was mediated by genomic VDR signaling, as a significant decrease in CYP24A1 mRNA was observed after VAZ co-treatment. This observation suggests a vitamin D-dependency at the 100 nM concentration, and an overall suppression of vitamin D signaling through 1,25D\({}_{3}\) catabolism. VDR mRNA level was measured and shown to be significantly decreased after 10 nM 1,25D\({}_{3}\) treatment that was more striking after 48 h (Figure 2C). However, there was no change in VDR mRNA levels after 100 nM 1,25D\({}_{3}\) treatment at both time points. Furthermore, the VDR mRNA level was not altered after VAZ treatment confirming the presence of receptorin the system and limited autoregulation [7]. Although CYP24A1 is commonly upregulated in cancers and thought to be one mode for vitamin D resistance [46], this appears not to be the case for MG-63 cells treated with 100 nM 1,25D\({}_{3}\) based on the MTT results (Figure 1A).