Vitamin D(3) metabolism in human glioblastoma multiforme: functionality of CYP27B1 splice variants, metabolism of calcidiol, and effect of calcitriol.
OBJECTIVE
A better understanding of the vitamin D(3) metabolism is required to evaluate its potential therapeutic value for cancers. Here, we set out to contribute to the understanding of vitamin D(3) metabolism in glioblastoma multiforme.


METHODS
We did nested touchdown reverse transcription-PCR (RT-PCR) to identify CYP27B1 splice variants and real-time RT-PCR to quantify the expression of CYP27B1. A cell line was treated with calcitriol to determine the effect on the expression of CYP27B1, 1alpha,25-dihydroxyvitamin D(3)-24-hydroxylase (CYP24), and vitamin D(3) receptor (VDR). We generated three antibodies for the specific detection of CYP27B1 and splice variants. High-performance TLC was done to determine the endogenous CYP27B1 activity and the functionality of CYP27B1 splice variants. Using WST-1 assay, we determined the effect of vitamin D(3) metabolites on proliferation.


RESULTS
We report a total of 16 splice variants of CYP27B1 in glioblastoma multiforme and a different expression of CYP27B1 and variants between glioblastoma multiforme and normal tissues. We found preliminary evidence for enzymatic activity of endogenous CYP27B1 in glioblastoma multiforme cell cultures but not for the functionality of the splice variants. By adding calcitriol, we found a proliferative effect for some cell lines depending on the dose of calcitriol. The administration of calcitriol led to an elevated expression of CYP27B1 and CYP24 but left the expression of the VDR unaltered.


CONCLUSIONS
Our findings show that glioblastoma multiforme cell lines metabolize calcidiol. In addition, we show various effects mediated by calcitriol. We found a special vitamin D(3) metabolism and mode of action in glioblastoma multiforme that has to be taken into account in future vitamin D(3)-related therapies.
Diesel
Britta
B
Institut für Humangenetik, Theoretische Medizin, Universität des Saarlandes, Saarbrücken, Germany.
Radermacher
Jens
J
Bureik
Matthias
M
Bernhardt
Rita
R
Seifert
Markus
M
Reichrath
Jörg
J
Fischer
Ulrike
U
Meese
Eckart
E
D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000235:genetics* / Q000378:metabolism; D017398:Alternative Splicing; D000818:Animals; D015153:Blotting, Western; D001932:Brain Neoplasms / Q000378:metabolism; D019556:COS Cells; D002112:Calcifediol / Q000378:metabolism*; D002117:Calcitriol / Q000494:pharmacology*; D002120:Calcium Channel Agonists / Q000494:pharmacology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002762:Cholecalciferol / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D005909:Glioblastoma / Q000378:metabolism*; D006801:Humans; D010957:Plasmids / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D018167:Receptors, Calcitriol / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013250:Steroid Hydroxylases / Q000378:metabolism; D014162:Transfection; D065668:Vitamin D3 24-Hydroxylase
D002120:Calcium Channel Agonists; D018076:DNA, Complementary; D012333:RNA, Messenger; D018167:Receptors, Calcitriol; D011994:Recombinant Proteins; D002762:Cholecalciferol; D012313:RNA; D013250:Steroid Hydroxylases; D065668:Vitamin D3 24-Hydroxylase; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase; D002117:Calcitriol; D002112:Calcifediol