Beneficial Regulation of Cellular Oxidative Stress Effects, and Expression of Inflammatory, Angiogenic, and the Extracellular Matrix Remodeling Proteins by 1α,25-Dihydroxyvitamin D3 in a Melanoma Cell Line.

The causes of cancer include the cellular accumulation reactive oxygen species (ROS), which overrides the cellular antioxidants such as superoxide dismutase, from intrinsic aging, genetics, and exposure to environmental pollutants and ultraviolet (UV) radiation. The ROS damage biomolecules such as DNA (including p53 gene), RNA, and lipids, and activate inflammatory, angiogenic, and extracellular matrix (ECM) remodeling proteins; which collectively facilitate carcinogenesis. The 1α,25-dihydroxyvitamin D3 (Vitamin D) has anti-carcinogenic potential from its antioxidant, anti-inflammatory, and endocrine properties. We examined the anti-carcinogenic mechanism of vitamin D through the beneficial regulation of oxidative stress effects (oxidative DNA/RNA damage, superoxide dismutase expression, membrane damage, and p53 promoter activity), and expression (at the protein, mRNA and/or promoter levels) of inflammatory mediators (interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α)), angiogenic mediators (transforming growth factor-β (TGF-β), and vascular endothelial growth factor (VEGF)), and the ECM remodeling proteins (matrix metalloproteinases (MMP)-1 and MMP-2) by vitamin D in melanoma cells. Vitamin D inhibited oxidative DNA/RNA damage and membrane damage; and stimulated superoxide dismutase expression and p53 promoter activity in melanoma cells. It inhibited the expression of IL-1, TNF-α, TGF-β, VEGF, MMP-1 and MMP-2 by transcriptional or post-transcriptional mechanisms. We conclude that vitamin D is beneficial to melanoma cells through the inhibition of oxidative DNA/RNA damage, membrane damage, and the expression of inflammatory, angiogenic and ECM remodeling proteins; and the stimulation of superoxide dismutase expression and p53 promoter activity.


Effect of 1,25-Dihydroxyvitamin D3 (vitamin D) on p53 Promoter Activity and Membrane Damage in Melanoma Cells
Vitamin D significantly stimulated p53 promoter activity, and inhibited membrane damage in melanoma cells (Figure 1).

Effect of 1α,25-Dihydroxyvitamin D3 (vitamin D) on p53 Promoter Activity and Membrane Damage in Melanoma Cells
Vitamin D significantly stimulated p53 promoter activity, and inhibited membrane damage in melanoma cells (Figure 1).

Effect of 1,25-Dihydroxyvitamin D3 (vitamin D) on Transforming Growth Factor Beta (TGF-β), and Vascular Endothelial Growth Factor (VEGF) in Melanoma Cells
Vitamin D significantly inhibited the expression of TGF- and VEGF in melanoma cells ( Figure  5, 6).
Vitamin D at 0.02 µM significantly inhibited the mRNA levels of TGF-β and VEGF to 0.62 and 0.69 fold, respectively, of control (p < 0.05) ( Figure 6A,B).

Effect of 1,25-Dihydroxyvitamin D3 (vitamin D) on Transforming Growth Factor Beta (TGF-β), and Vascular Endothelial Growth Factor (VEGF) in Melanoma Cells
Vitamin D significantly inhibited the expression of TGF- and VEGF in melanoma cells ( Figure  5, 6).
Vitamin D at 0.02 μM significantly inhibited the mRNA levels of TGF- and VEGF to 0.62 and 0.69 fold, respectively, of control (p < 0.05) ( Figure 6A

Effect of 1α,25=Dihydroxyvitamin D3 (vitamin D) on Matrixmetalloproteinase (MMP)-1 and MMP-2 in Melanoma Cells
Vitamin D significantly inhibited MMP-1 and MMP-1 protein levels in melanoma cells (Figure 7). It did not significantly inhibit the MMP-1 promoter activity, or the MMP-1 or MMP-2 mRNA levels in these cells (data not shown).

Effect of 1,25=Dihydroxyvitamin D3 (vitamin D) on Matrixmetalloproteinase (MMP)-1 and MMP-2 in Melanoma Cells
Vitamin D significantly inhibited MMP-1 and MMP-1 protein levels in melanoma cells ( Figure  7). It did not significantly inhibit the MMP-1 promoter activity, or the MMP-1 or MMP-2 mRNA levels in these cells (data not shown).
The cellular oxidative stress as well as the reduced expression of antioxidant enzymes and tumor suppressor p53 occurs with intrinsic aging, and additionally with exposure to environmental pollutants and UV radiation. Vitamin D significantly inhibited oxidative DNA/RNA damage, and membrane damage; and stimulated superoxide dismutase protein levels, and p53 promoter activity in melanoma cells. It is inferred that vitamin D is effective in directly inhibiting oxidative stress effects and inducing p53 expression that facilitates cell cycle arrest or apoptosis [20].
The key factors that regulate angiogenesis and metastasis are TGF-β, VEGF, and MMPs. TGF-β regulates the cell cycle, angiogenesis, and the extracellular matrix; and has differential effects in different cell types [9][10][11][12]20]. VEGF is key to angiogenesis, and the MMPs to the degradation and remodeling of the ECM. There is coordinate regulation of TGF-β, VEGF, and/or MMPs in cancer cells [9][10][11][12]20,50,52,53]. Vitamin D inhibited TGF-β, and VEGF at the protein and mRNA levels, suggesting transcriptional mechanism, and MMP-1, and MMP-2 at the proteins level, suggesting post-transcriptional mechanism; and its effectiveness in inhibiting angiogenic and metastatic potential in melanoma cells.
Overall, it is concluded that vitamin D is beneficial to melanoma cells through the inhibition of oxidative DNA/RNA damage, membrane damage, and the expression of inflammatory, angiogenic and ECM remodeling proteins; and the stimulation of superoxide dismutase expression and p53 promoter activity. The Vitamin D concentrations of 0.0002 µM, 0.002 µM, 0.02 µM, and 0.2 were effective in significantly regulating all targets examined in a melanoma cell line, except for p53 promoter activity that was upregulated by Vitamin D concentrations of 0.02 µM and 0.2 µM.

Cell Viability
Vitamin D at 0.0002-0.02uM did not alter the viability of melanoma cells, relative to control (data not shown).

Oxidative DNA/RNA Damage
The oxidative DNA/RNA damage was measured with the competitive DNA/RNA Oxidative Damage ELISA Kit (Cayman Chemical) [29]. Aliquots of cells or buffer were incubated with competitive acetylcholinesterase linked to 8-OH-dG (tracer), and antibody to oxidatively damaged guanine for 24 h at 4 • C, washed, incubated with substrate, and the product measured spectrophotometrically at 412 nm. The readings were subtracted from the maximum tracer binding (buffer) to determine the cellular DNA/RNA oxidative damage. Vitamin D at 0.0002-0.02 µM did not alter the total protein content (extracellularly or intracellularly) of melanoma cells, relative to control (data not shown). The total protein content in the media and cells, following experiments, was determined by the Pierce Bicinchoninic Acid (BCA) Protein Assay (Thermo Fischer Scientific, Waltham, MA, USA); by incubating aliquots with the BCA and cupric ion reagent, and measuring the formation of BCA-cuprous ion, proportional to the total protein content, spectrophotometrically at 562 nm.

Membrane Damage
The media were examined for lactate dehydrogenase (LDH) activity, indicative of membrane damage, with the LDH toxicity kit (Sigma, Tox-7) [27,29]. The aliquots of media were incubated with the LDH substrate, cofactor and tertrazolium dye reagents, and the reduction of the tertrazolium dye was measured spectrophotometrically at 490nm. 4.7. RNA Levels: IL-1, TNF-α, TGF-β, VEGF, MMP-1 and MMP-2 mRNA, and 18S RNA The RNA was extracted from cells, following incubation with or without vitamin D, using the RNeasy Plus Mini Kit (Qiagen) [49]. Vitamin D did not significantly alter the total RNA content, relative to control; which was measured by incubating aliquots of the extracted RNA with a fluorescent dye and measuring fluorescence at 490 nm excitation/540 nm emission (QuantiFluor RNA system, Promega, Madison, WI, USA). Vitamin D did not significantly alter the mRNA levels of MMPs, relative to control (data not shown). The aliquots of RNA were reverse transcribed to cDNA with the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA), and analyzed for IL-1, TNF-α, TGF-β, VEGF, MMP-1, MMP-2, and 18S (for normalization, as internal control) cDNA levels by quantitative-polymerase chain reaction (qPCR) using specific primers (Qiagen) and SsoAdvanced Universal SYBR Green Supermix (Bio-Rad). The fold change of mRNA expression was calculated by the 2 (-Delta Delta Ct) method (Qiagen).

Data Analysis
The data were analyzed for significant difference by ANOVA and student t-tests at 95% confidence interval.