Antiproliferative Activity of Non-Calcemic Vitamin D Analogs on Human Melanoma Lines in Relation to VDR and PDIA3 Receptors

Abstract

Vitamin D is a precursor for secosteroidal hormones, which demonstrate pleiotropic biological activities, including the regulation of growth and the differentiation of normal and malignant cells. Our previous studies have indicated that the inhibition of melanoma proliferation by a short side-chain, low calcemic analog of vitamin D-21(OH)pD is not fully dependent on the expression of vitamin D receptor (VDR). We have examined the effects of classic vitamin D metabolites, 1,25(OH)₂D₃ and 25(OH)D₃, and two low calcemic vitamin D analogs, (21(OH)pD and calcipotriol), on proliferation, mRNA expression and vitamin D receptor (VDR) translocation in three human melanoma cell lines: WM98, A375 and SK-MEL-188b (subline b of SK-MEL-188, which lost responsiveness to 1,25(OH)₂D₃ and became VDR^-/-CYP27B1^-/-). All tested compounds efficiently inhibited the proliferation of WM98 and A375 melanoma cells except SK-MEL-188b, in which only the short side-chain vitamin D analog-21(OH)pD was effective. Overall, 21(OH)pD was the most potent compound in all three melanoma cell lines in the study. The lack of responsiveness of SK-MEL-188b to 1,25(OH)₂D₃, 25(OH)D₃ and calcipotriol is explained by a lack of characteristic transcripts for the VDR, its splicing variants as well as for vitamin D-activating enzyme CYP27B1. On the other hand, the expression of VDR and its splicing variants and other vitamin D related genes (RXR, PDIA3, CYP3A4, CYP2R1, CYP27B1, CYP24A1 and CYP11A1) was detected in WM98 and A375 melanomas with the transcript levels being modulated by vitamin D analogs. The expression of VDR isoforms in WM98 cells was stimulated strongly by calcipotriol. The antiproliferative activities of 21(OH)pD appear not to require VDR translocation to the nucleus, which explains the high efficacy of this noncalcemic pregnacalciferol analog in SK-MEL-188b melanoma, that is, VDR^-/-. Therefore, we propose that 21(OH)pD is a good candidate for melanoma therapy, although the mechanism of its action remains to be defined.

Keywords: 1,25(OH)2D3; 21(OH)pD; VDR translocation; anti-melanoma activity; calcipotriol; human melanoma cell lines; melanoma; vitamin D; vitamin D analogs.

Figure 1

Chemical structure of vitamin D₃ analogs: 1,25(OH)₂D₃ (A), 25(OH)D₃ (B), calcipotriol (C) and 21(OH)pD (D).

Figure 2

Effects of 1,25(OH)₂D₃, 25(OH)D₃, calcipotriol and 21(OH)pD on growth of human SK-MEL-188b, A375 and WM98 melanoma cells. Cells were seeded into 96-well plates and incubated in a medium supplemented with serial dilution of vitamin D analogs from 0.01 nM to 1000 nM concentration (as described in Material and Methods). The statistical significance of results has been analyzed using one-way ANOVA (GraphPad Software, San Diego, CA, USA) and data are presented as means ± SEM for at least three independent measurements. The cutoff point of significance was defined as p < 0.05 (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Figure 3

Differences in mRNA level of VDR, PDIA3, CYP27A1 and CYP27B1 genes in SK-MEL-188b cells in comparison with A375 and WM98 cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a reporter gene to normalize all samples. See Table S1 for primer sequences and the predicted length of PCR fragments.

Figure 4

Vitamin D analogs treatment modulate the expression of (A) retinoid X receptor (RXR), (E) disulfide isomerase (PDIA3), 25-hydoxylases ((B) CYP27A1, (C) CYP2R1 or (F) CYP3A4, (G) 1α-hydroxylase (CYP27B1)) and 24-hydroxylases ((D) CYP24A1 or (H) CYP24SV) in WM98 melanoma cell line. Cells were stimulated with 1 µM 1,25(OH)₂D₃, 25(OH)D₃ or calcipotriol for 24 h. Quantitative PCR analyses were performed as described in Materials and Methods. Statistical significance was estimated using t-test and data are presented as means ± SD (n = 3). The cutoff point for significance is defined as p < 0.05 (* p < 0.05, ** p < 0.01).

Figure 5

Effects of vitamin D compounds (1,25(OH)₂D₃, 21(OH)pD or calcipotriol) on RXR (A), VDR (B), PDIA3 (C), CYP3A4 (D), CYP2R1 (E), CYP27B1 (F), CYP24A1 (G) and CYP11A1 (H) genes expression in A375 melanoma cells. A375 melanoma cells were incubated with 100 nM of 1,25(OH)₂D₃, 21(OH)pD or calcipotriol for 24 h. mRNA levels were measured by qPCR. Data are shown as means ± S.D of three independent experiments carried out in duplicate. The cutoff point for significance is defined as p < 0.05 (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 6

(A) Five different PCR primers sets were designed (Pr 1–Pr 5) in order to detect the expression of VDR splicing variants in WM98, A375 and SK-MEL-188b cell lines. The most common isoforms are a b and c and the rarely identified isoforms are d, e, f and g. The positions of sets of primers designed to differentiate VDR isoforms is also shown. Product 1 (Pr 1) of 132 b.p. is characteristic for isoforms a, b, c and f; Product 2 (Pr 2) of 180 b.p. is universal for all isoforms accept f; Product 3 (Pr 3) of 386 b.p. is unique for isoform a; Product 4 (Pr 4), depending on length of the PCR fragment, indicates isoform “b” 532 b.p. or “c” 410 b.p.; Product 5 (Pr 5) of 384 b.p. is characteristic for isoforms a, b, c, d and e, but not g and f (diagram of VDR splicing variants taken from AceView https://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/). (B) Semiquntitative PCR was used to differentiate the VDR isoforms (see Material and Methods for details); molecular weight marker (M) as described in. (C) Effects of vitamin D analogs on the expression of VDR isoforms were analyzed in WM98 melanoma cells. Melanoma cells were treated with 25(OH)D₃ or calcipotriol at 1 µM concentration for 24 h. Statistical significance was estimated using t-test and data are presented as means ± SD (n = 3). The cutoff point for significance is defined as p < 0.05 (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 7

Effects of vitamin D analogs: (A) 1,25(OH)₂D₃, (B) calcipotriol or (C) 21(OH)pD₃ at 0 (C—control), 0.01, 0.1 or 1 μM concentrations on VDR translocation to the nucleus (green fluorescence shown in a gray scale). Melanoma A375 cells were transduced using pLenti-CMV-VDR-GFP-pgkpuro construct: see Materials and Methods. Vitamin D analogs except 21(OH)pD induce VDR translocation to the nucleus after 1 h of incubation in a concentration-dependent manner. Each panel shows four random micrographs for each concentration taken by fluorescent microscope under 100× magnification.