Insulin-like growth factor binding protein-3 mediates 1 alpha,25-dihydroxyvitamin d(3) growth inhibition in the LNCaP prostate cancer cell line through p21/WAF1.
OBJECTIVE
We determined that insulin-like growth factor binding protein 3 (IGFBP-3) induction by 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) is a necessary component of 1,25-(OH)(2)D(3) mediated growth inhibition of the LNCaP human prostate cancer cell line. In addition, induction of the cyclin dependent kinase inhibitory protein p21/WAF/CIP1 by 1,25-(OH)(2)D(3) is mediated by IGFBP-3.


METHODS
Induction of IGFBP-3 by 1,25-(OH)(2)D(3) was determined by enzyme-linked immunosorbent assay for IGFBP-3 protein and by Northern blot analysis for IGFBP-3 messenger (m) RNA. Growth assays for LNCaP cells were determined by measuring DNA content. The contribution of IGFBP-3 toward 1,25-(OH)(2)D(3) mediated growth inhibition was determined by adding either antisense oligonucleotides or immuno-neutralizing antibodies to culture media of growth assays. Regulation of p21/WAF/CIP1 was determined by Western blot analysis.


RESULTS
Adding 1,25-(OH)(2)D(3) to LNCaP prostate cancer cells demonstrated that 1,25-(OH)(2)D(3) significantly up-regulated IGFBP-3 at the mRNA and protein levels in these cells approximately 3-fold over control levels. Also, adding IGFBP-3 protein to LNCaP cell growth medium inhibited LNCaP cell growth. Interestingly adding IGFBP-3 antisense oligonucleotides or antibodies directed toward IGFBP-3 abolished the growth inhibitory actions of 1,25-(OH)(2)D(3), indicating that this effect is IGFBP-3 dependent. Furthermore, to connect the mechanisms of IGFBP-3 and 1,25-(OH)(2)D(3) mediated growth inhibition we demonstrated that IGFBP-3 up-regulates the expression of p21/WAF1 protein to approximately 2-fold over the control level. Adding an IGFBP-3 immuno-neutralizing antibody completely prevented the 1,25-(OH)(2)D(3) induced up-regulation of p21/WAF1.


CONCLUSIONS
1,25-(OH)(2)D(3) up-regulates IGFBP-3 in the LNCaP cell line at the mRNA and protein levels. The growth inhibitory action of 1,25-(OH)(2)D(3) on LNCaP cells depends on active IGFBP-3, as evidenced by the loss of growth inhibition induced by IGFBP-3 antisense oligonucleotide and immuno-neutralization experiments. A possible connection between IGFBP-3 and 1,25-(OH)(2)D(3) lies in the cyclin dependent kinase inhibitory protein p21/WAF1 since IGFBP-3 and 1,25-(OH)(2)D(3) each up-regulate this protein and both inhibit LNCaP cell growth. Therefore, we hypothesize that the mechanism of action by which IGFBP-3 and 1,25-(OH)(2)D(3) induce growth inhibition is the induction of p21/WAF1 because IGFPB-3 immuno-neutralizing antibodies completely abrogate the 1,25-(OH)(2)D(3) mediated up-regulation of p21/WAF1 and growth inhibition.
Boyle
B J
BJ
Stanford University School of Medicine, Division of Endocrinology, Stanford, California 94305-5103, USA.
Zhao
X Y
XY
Cohen
P
P
Feldman
D
D
D002117:Calcitriol / Q000494:pharmacology*; D002120:Calcium Channel Agonists / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000502:physiology*; D008297:Male; D016376:Oligonucleotides, Antisense; D011471:Prostatic Neoplasms / Q000503:physiopathology*; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D002120:Calcium Channel Agonists; D018972:Insulin-Like Growth Factor Binding Protein 3; D016376:Oligonucleotides, Antisense; D002117:Calcitriol