Novel vitamin D hydroxyderivatives inhibit melanoma growth and show differential effects on normal melanocytes

Abstract

Background/aims: To test the activity of novel hydroxyvitamin D(3) analogs (20(OH)D(3), 20,23(OH)(2)D and 1,20(OH)(2)D(3)) on normal and malignant melanocytes in comparison to 1,25(OH)(2)D(3).

Materials and methods: Human epidermal melanocytes and human and hamster melanoma cells were used to measure effects on proliferation and colony formation in monolayer and soft agar. Cell morphology and melanogenesis were also analyzed. QPCR was used to measure gene expression.

Results: Novel secosteroids inhibited proliferation and colony formation by melanoma cells in a similar fashion to 1,25(OH)(2)D(3), having no effect on melanogenesis. These effects were accompanied by ligand-induced translocation of VDR to the nucleus. In normal melanocytes 1α-hydroxyderivatives (1,25(OH)(2)D(3) and 1,20(OH)(2)D(3)) had stronger anti-proliferative effects than 20(OH)D(3) and 20,23(OH)(2)D(3), and inhibited dendrite formation. The cells tested expressed genes encoding VDR and enzymes that activate or inactivate vitamin D(3).

Conclusion: Novel secosteroids show potent anti-melanoma activity in vitro with 20(OH)D(3) and 20,23(OH)(2)D(3) being excellent candidates for pre-clinical testing.

Figure 1

Structures and enzymatic methods of production of novel vitamin D₃ hydroxy-derivatives.

Figure 2

Comparison of the anti-proliferative activity of the vitamin D derivatives 1,25(OH)₂D₃, 20(OH)D₃, 20,23(OH)₂D₃ and 1,20(OH)₂D₃ between cultured normal human epidermal melanocytes (A) and human SKMel-188 melanoma cells (B). The cells were treated with the secosteroids (10^–7 M) for 7 days and their numbers were counted. Data are shown as mean±SD (n=3); *p<0.05; **p<0.01; ***p<0.001. 1,25(OH)₂D₃ and 1,20(OH)₂D₃, but not 20(OH)D₃ or 20,23(OH)₂D₃ inhibited dendrite formation (C). The differences between ethanol-treated control and treatments were analyzed by student's t-test

Figure 3

Novel vitamin hydroxyderivatives inhibit DNA synthesis in human melanoma cells. YUROB cells were treated for 48 h with 1,25(OH)₂D₃, 20(OH)D₃, 20,23(OH)₂D₃, or 1,20(OH)₂D₃ (10^–7 M) and the rate of ³H-thymidine incorporation into DNA served as a measure of proliferative activity. Data are presented as mean±SD (n=4). Statistical significance was estimated using one-way ANOVA Incorporation into DNA is shown as a percentage (%) of control (ethanol-vehicle treated cells). *p<0.05 and ***p<0.001.

Figure 4

Novel vitamin D hydroxyderivatives inhibit the ability of human melanoma cells to form colonies in monolayer (platting efficiency). SBCE2 cells were plated at a density 20 cells/cm², grown in the presence or absence of 1,25(OH)₂D₃, 20,23(OH)₂D₃ or 1,20(OH)₂D₃, and after 10 days formation of colonies larger than 0.2 mm (A) or 0.5 mm (B) in diameter was determined. Data are shown as mean±SD (n = 4); statistical significance was estimated using one-way ANOVA and presented as *p<0.05, **p<0.01 and ***p<0.001. Insert shows Western blot detection of VDR in SBCE2 human melanoma cells. The whole extracts from cells were subjected to immunoblotting with anti- VDR, and anti- β-actin (internal control) as described before (3). The numbers on the left in the insert represent molecular weight in kD.

Figure 5

Novel vitamin hydroxyderivatives inhibit the anchorage independent growth (ability to form colonies in soft agar) of hamster melanoma cells. AbC1 melanoma cells were plated in soft agar at 1,000 cells/well and grown in the presence or absence of 1,25(OH)₂D₃, 20(OH)D₃ or 20,23(OH)₂D₃. After two weeks colonies with a diameter larger than 0.2 mm (A) or 0.5 mm (B) were counted. Data are shown as mean±SD (n=4); statistical significance was estimated using one-way ANOVA and presented as *p<0.05, **p<0.01 and ***p<0.001.

Figure 6

Novel vitamin hydroxyderivatives inhibit the anchorage independent growth (ability to form colonies in soft agar) of human melanoma cells. SKMel-188 human melanoma cells were grown in soft agar in the presence or absence of 1,25(OH)₂D₃ (A), 20(OH)D₃ (B) or 20,23(OH)₂D₃ (C). Panels A and B are from from the same whilke panel C for separate experiment. After two weeks colonies with a diameter larger than 0.5 mm were counted. Data are shown as mean±SD (n=4); statistical significance was estimated using one-way ANOVA and presented as *p<0.05 and **p<0.001. Insert: representative plates incubated with solvent (ethanol) or 10^–7 M secosteroids.

Figure 7

The effect of vitamin D₃ derivatives on the translocation of VDR from the cytoplasm to the nucleus. The panel on the right shows photographs of the cells with fluorescent VDR-EGFP fusion protein in the nucleus. The left pannel shows the percentage of cells with fluorescent nuclei. Data are presented as means±SEM (n≥6). The differences between ethanol-treated control and treatment were analyzed by student's t-test: p<0.05 (*), p<0.01 (**).