Elsevier

Steroids

Volume 66, Issues 3–5, 1 March–1 May 2001, Pages 327-337
Steroids

Differential activities of 1α,25-dihydroxy-16-ene-vitamin D3 analogs and their 3-epimers on human promyelocytic leukemia (HL-60) cell differentiation and apoptosis

https://doi.org/10.1016/S0039-128X(00)00142-2Get rights and content

Abstract

To clarify physiological role of the carbon 3 (C-3) epimerization of 1α,25(OH)2D3 and biologic significance of a 3-epi metabolite of 1α,25(OH)2D3, we examined biologic activities of the 3-epimers of 1α,25(OH)2D3 and 1α,25(OH)2-16-ene-D3 analogs in terms of modulation of cell cycle phase distribution and cell-surface CD11b antigen expression of HL-60 cells, transactivation of vitamin D target genes in transfected cells, stimulation of VDR/RXRα heterodimer formation in a rabbit reticulocyte lysates transcription/translation system, stimulation of VDR/RXRα/VDRE complex formation, and induction of HL-60 cell apoptosis. The analogs tested here were 1) 1α,25(OH)2D3, 2) 1α,25(OH)2-3-epi-D3, 3) 1α,25(OH)2-16-ene-D3, 4) 1α,25(OH)2-16-ene-3-epi-D3, 5) 1α,25(OH)2-16-ene-23-yne-hexafluoro(F6)-D3, 6) 1α,25(OH)2-16-ene-23-yne-hexafluoro(F6)-3-epi-D3, 7) 1α,25-(OH)2-16-ene-20-epi-23-yne-D3, and 8) 1α,25(OH)2-16-ene-20-epi-23-yne-3-epi-D3. When compared to the 3-natural (β) analogs, the 3-epi (α) analogs were biologically significantly less active. The findings support the hypothesis that the C-3 epimerization is an inactivation pathway of 1α,25(OH)2D3 and its analogs in vitamin D target tissues. We also found that the 3-epi analogs, but not the 3-natural (β) analogs, were the potent inducers of apoptosis of HL-60 cells. These results suggest that the analogs could be divided into two groups, in which the 3-epi analogs were the potent inducers of apoptosis of HL-60 cells, and the 3-natural analogs were the potent modulators of HL-60 cell growth and differentiation. This is the first report demonstrating that the 3-epimerization of the hydroxyl group at C-3 of the A-ring of 1α,25(OH)2D3 plays an important role to modulate HL-60 cell differentiation and apoptosis.

Introduction

It is well established that the hormonally active form of vitamin D3, 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] is metabolized in the target tissues via two oxidation pathways. The carbon 24 (C-24) oxidation pathway initiated by hydroxylation at C-24 is the major pathway, which leads to the formation of the final metabolite, calcitroic acid [1], [2], [3]. The carbon 23 (C-23) and carbon 26 (C-26) oxidation pathways, initiated by hydroxylations at C-23 and C-26, respectively, are the minor pathways, which lead to the formation of 1α,25(OH)2D3-23,26-lactone [4], [5]. Recently, Reddy et al. reported that 1α,25(OH)2D3 is also metabolized into 1α,25(OH)2-3-epi-D3 through the carbon 3 (C-3) epimerization pathway in human keratinocytes [6], human colon carcinoma cells (Caco-2) [7], bovine parathyroid cells [8], rat osteosarcoma cells (UMR 106 and ROS 17/2.8) [9] and in vivo in rats [10]. It is demonstrated that 1α,25(OH)2-3-epi-D3 when compared to the natural hormone, is less active in stimulating intestinal calcium absorption, calcium mobilization from bone, and induction of calbindin D28K, suggesting that the C-3 epimerization pathway seems to be an alternative pathway for inactivation of 1α,25(OH)2D3 in target tissues. It is, however, reported that 1α,25(OH)2-3-epi-D3 retains 24% binding affinity to the VDR when compared to 1α,25(OH)2D3 [11], [12] and furthermore, we found that 22-oxa-1α,25(OH)2-3-epi-D3 generated from 22-oxa-1α,25(OH)2D3 by UMR 106 had still 30% activity of the parent compound in transactivating human osteocalcin gene promoter in transfected MG-63 cells (unpublished data). Therefore, it still remains to be determined whether the C-3 epimerization is an inactivation pathway of 1α,25(OH)2D3 and its analogs. To address this issue and to explore new biologic activities, we examined biologic activities of the 3-epimers of 1α,25(OH)2D3 and 1α,25(OH)2-16-ene-D3 analogs at the molecular and cellular levels.

Section snippets

Vitamin D3 compounds

1α,25(OH)2D3 was purchased from Solvay–Duphar Co., Weesp, The Netherlands. [26,27-methyl-3H]-1α,25(OH)2D3 (specific activity: 6.7 TBq/mmol) were purchased from Amersham Co. (Buckinghamshire, UK). 3-Epimers of 1α,25(OH)2D3 and 1α,25(OH)2-16-ene-D3 analogs used in this study were synthesized by Roche Research Center, Hoffmann–La Roche Inc., Nutley, NJ, USA, and are depicted in Fig. 1. All analogs were dissolved in aldehyde-free absolute ethanol as stock solutions at 10−5 M and stored at −35°C

Binding affinity for calf-thymus VDR

Fig. 2 shows the calf-thymus VDR binding assay with the analogs. Inversion of the orientation of the hydroxyl group at C-3 of the A-ring induced significant reduction in VDR binding. 1α,25(OH)2-16-ene-D3 was the highest-affinity ligand of the tested compounds. 1α,25(OH)2-16-ene-20-epi-23-yne-D3 and its 3-epimer were low- or the lowest-affinity ligands of the tested compounds, respectively.

Cell-differentiation activity

Fig. 3 shows the effects of the analogs on the differentiation of HL-60 cells. At a concentration of 10−8

Discussion

In 1994, Reddy et al. reported that 1α,25(OH)2D3 is metabolized into 1α,25(OH)2-3-epi-D3 in neonatal human keratinocyte cell culture [6]. Since then, this 3-epimerization of the hydroxyl group at C-3 of the A-ring has been well recognized as a new metabolic pathway of 1α,25(OH)2D3 and a variety of mammalian cells including either 24-oxidation pathway positive cells, such as rat osteosarcoma cells (UMR 106), human colon carcinoma cells (Caco-2), bovine parathyroid cells and so forth, or

Acknowledgements

This work was supported in part by a Grants-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan, a Grant for Cooperative Research administered by the Japan Private School Promotion Foundation, and a Grant-in-aid from the Ministry of Health and Welfare of Japan.

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