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

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

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

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

Antitumor Effects of Vitamin D Analogs on Hamster and Mouse Melanoma Cell Lines in Relation to Melanin Pigmentation

Deregulated melanogenesis is involved in melanomagenesis and melanoma progression and resistance to therapy. Vitamin D analogs have anti-melanoma activity. While the hypercalcaemic effect of the active form of Vitamin D (1,25(OH)2D3) limits its therapeutic use, novel Vitamin D analogs with a modified side chain demonstrate low calcaemic activity. We therefore examined the effect of secosteroidal analogs, both classic (1,25(OH)2D3 and 25(OH)D3), and novel relatively non-calcemic ones (20(OH)D3, calcipotriol, 21(OH)pD, pD and 20(OH)pL), on proliferation, colony formation in monolayer and soft-agar, and mRNA and protein expression by melanoma cells. Murine B16-F10 and hamster Bomirski Ab cell lines were shown to be effective models to study how melanogenesis affects anti-melanoma treatment. Novel Vitamin D analogs with a short side-chain and lumisterol-like 20(OH)pL efficiently inhibited rodent melanoma growth. Moderate pigmentation sensitized rodent melanoma cells towards Vitamin D analogs, and altered expression of key genes involved in Vitamin D signaling, which was opposite to the effect on heavily pigmented cells. Interestingly, melanogenesis inhibited ligand-induced Vitamin D receptor translocation and ligand-induced expression of VDR and CYP24A1 genes. These findings indicate that melanogenesis can affect the anti-melanoma activity of Vitamin D analogs in a complex manner

Polish Adaptation of the Modified Tampa Scale of Kinesiophobia for Fatigue (TSK-F) and the Revision of the Tampa Scale in Terms of Pain for Cancer Patients

The aim of this study was to create a Polish adaptation of the Tampa Scale of Kinesiophobia considering fatigue, and to verify the usefulness of the scale in the context of pain in cancer patients. The study was conducted at the Breast Cancer Unit, operating at the Greater Poland Cancer Centre, and at the Poznan Centre for Specialist Medical Services in Poznan. After considering the exclusion criteria, 100 people qualified for the interviews for the final study: 50 breast cancer patients and 50 healthy respondents (without cancer). Statistical analysis of the CFA score showed that the chi-square test was not significant (χ2 = 10.243, p = 0.332), indicating an acceptable fit of items across scales. The reliability of the internal consistency of the scales was tested by examining the Cronbach’s alpha scores for each question/statement. The mean values for this indicator were 0.74 for the pain-related scale and 0.84 for the fatigue-related scale. Construct validity was confirmed for the scales; AVE for the pain-related scale was 0.64 and for the fatigue-related scale was 0.68. The results suggest the validity of examining kinesiophobia in the context of pain- and fatigue-related mobility anxiety among breast cancer patients in Poland, and that the Tampa Scale of Kinesiophobia can be adapted for different dimensions of the condition. Both versions of the scale demonstrated adequately prepared parametric constructs, and all correlations showed a statistically significant relationship (p < 0.05). The use of the Tampa Scale of Kinesiophobia in oncology patient studies in Poland may ultimately improve rehabilitation programs and enable the development of strategies to assist patients in supporting treatment to reduce movement anxiety

Effect of GA on localization and protein level of HSPs in OS 143B cells.

<p>OS 143B cells were treated with 4 µM GA for 6 h; the levels of Hsp60, Hsp70, total cellular Hsp90 protein were determined by immunofluorescence. <b>A</b>. GA did not change the mitochondrial localization of Hsp60, however, it decreased the mitochondrial pool of the protein. Cell nuclei were shown in blue while Hsp60, AIF immunoreactivities in green and red, respectively. Merged image of all kinds of staining (orange) was also presented. <b>B</b>. GA upregulated Hsp70 protein level. Cell nuclei were shown in blue, while Hsp70 immunoreactivity in green. Merged image of both was also presented. <b>C</b>. GA upregulated Hsp90 protein level. Cell nuclei were shown in blue while Hsp90 immunoreactivity in red. Merged image of both was also presented. Original magnification ×40. Each experiment was performed at least three times. The representative data were shown.</p

The effect of GA on proliferation and induction of cell death of OS 143B cells.

<p><b>A</b>. GA inhibits OS 143B cell growth. OS 143B cells were treated for 24 h with serial GA dilutions (within the range of 0.8 µM–50 µM). The cell viability was then determined by means of MTT assay. Data from at least three independent experiments are presented as mean ± SE. The absence of error bar denotes a line thickness greater than the error. Data were analyzed by GraphPad Prism Software version 6.02 performing One-way ANOVA combined with Dunett's Multiple Comparison Test. *P<0.01 vs. control. <b>B–D</b>. GA induced cell death of OS 143B cells. 143B OS cells were treated with 4 µM GA for 24 h, the cells were then harvested and the percentage of apoptotic and necrotic cells was determined performing double PI-Annexin V staining. <b>B</b>. Annexin V, PI-live/dead dot plots showing apoptosis and necrosis before and after treatment with GA. Plots are representative of five individual experiments. <b>C–D</b>. Total apoptotic (C) and necrotic (D) cell number before and after treatment with GA. Data from at least three independent experiments are presented as mean ± SE. Data were analyzed using GraphPad Prism (GraphPad Software, Inc., version 6.02, USA). Significant differences between groups were determined by Student's t-test. *P<0.01, ***P<0.0001 vs. control.</p

GA affected HSPs gene expression in OS 143B cells.

<p>OS 143B cells were treated with GA (4 µM) for 6 h; the levels of Hsp60, Hsp70, Hsp90AA1, Hsp90AB1, Hsp90B1 transcript were then determined by means of Real Time PCR. Treatment with GA resulted in upregulation of Hsp60 (A), Hsp70 (B), Hsp90AA1 (C), Hsp90AB1 (D) transcript, however it did not impact Hsp90B1 gene expression (E). Values are mean ± SE of three independent experiments, relative mRNA levels of HSP/beta-actin are presented. The data were analyzed by Student's t-test using GraphPad Prism Software version 6.02. *P<0.01, **P<0.001 vs. control.</p

GA affected HSPs protein levels in OS 143B cells.

<p>OS 143B cells were treated with 4 µM GA for 6 h; the levels of Hsp60, Hsp70, total cellular Hsp90, Hsp90AA1, Hsp90AB1proteins were determined by Western blotting. <b>A</b>. GA decreased the level of Hsp60 and induced post-translational modification of Hsp60 partially derived from the hyperacetylated isoform. <b>B–E</b>. GA upregulated Hsp70 (B), total cellular Hsp90 (C), Hsp90AA1 (D), Hsp90AB1 (E) protein levels. Each experiment was performed at least three times. The representative data are shown. Densitometric analysis of HSP/beta-actin was performed using Quantity one 4.5.2 software.</p