INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN-3 MEDIATES 1α,25-DIHYDROXYVITAMIN D 3 GROWTH INHIBITION IN THE LNCaP PROSTATE CANCER CELL LINE THROUGH P21/WAF1

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Abstract
We determined that insulin-like growth factor binding protein 3 (IGFBP-3) induction by 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) is a necessary component of 1,25-(OH)2D3 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)2D3 is mediated by IGFBP-3. Induction of IGFBP-3 by 1,25-(OH)2D3 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)2D3 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. Adding 1,25-(OH)2D3 to LNCaP prostate cancer cells demonstrated that 1,25-(OH)2D3 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)2D3, indicating that this effect is IGFBP-3 dependent. Furthermore, to connect the mechanisms of IGFBP-3 and 1,25-(OH)2D3 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)2D3 induced up-regulation of p21/WAF1. 1,25-(OH)2D3 up-regulates IGFBP-3 in the LNCaP cell line at the mRNA and protein levels. The growth inhibitory action of 1,25-(OH)2D3 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)2D3 lies in the cyclin dependent kinase inhibitory protein p21/WAF1 since IGFBP-3 and 1,25-(OH)2D3 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)2D3 induce growth inhibition is the induction of p21/WAF1 because IGFPB-3 immuno-neutralizing antibodies completely abrogate the 1,25-(OH)2D3 mediated up-regulation of p21/WAF1 and growth inhibition.