Boyle B J, Zhao X Y, Cohen P, Feldman D
Stanford University School of Medicine, Division of Endocrinology, Stanford, California 94305-5103, USA.
J Urol. 2001 Apr;165(4):1319-24.
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.
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.
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.
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.
我们确定1,25 - 二羟基维生素D3（1,25-(OH)2D3）诱导胰岛素样生长因子结合蛋白3（IGFBP - 3）是1,25-(OH)2D3介导的LNCaP人前列腺癌细胞系生长抑制的必要组成部分。此外，1,25-(OH)2D3诱导细胞周期蛋白依赖性激酶抑制蛋白p21/WAF/CIP1是由IGFBP - 3介导的。
通过酶联免疫吸附测定法检测IGFBP - 3蛋白，并用Northern印迹分析法检测IGFBP - 3信使核糖核酸（mRNA），以确定1,25-(OH)2D3对IGFBP - 3的诱导作用。通过测量DNA含量来确定LNCaP细胞的生长测定。通过向生长测定的培养基中添加反义寡核苷酸或免疫中和抗体，来确定IGFBP - 3对1,25-(OH)2D3介导的生长抑制的作用。通过蛋白质印迹分析来确定p21/WAF/CIP1的调节情况。
向LNCaP前列腺癌细胞中添加1,25-(OH)2D3表明，1,25-(OH)2D3在mRNA和蛋白质水平上显著上调这些细胞中的IGFBP - 3，比对照水平高约3倍。此外，向LNCaP细胞生长培养基中添加IGFBP - 3蛋白可抑制LNCaP细胞生长。有趣的是，添加IGFBP - 3反义寡核苷酸或针对IGFBP - 3的抗体可消除1,25-(OH)2D3的生长抑制作用，表明这种作用依赖于IGFBP - 3。此外，为了连接IGFBP - 3和1,25-(OH)2D3介导的生长抑制机制，我们证明IGFBP - 3将p21/WAF1蛋白的表达上调至比对照水平高约2倍。添加IGFBP - 3免疫中和抗体完全阻止了1,25-(OH)2D3诱导的p21/WAF1上调。
1,25-(OH)2D3在mRNA和蛋白质水平上上调LNCaP细胞系中的IGFBP - 3。1,25-(OH)2D3对LNCaP细胞的生长抑制作用取决于活性IGFBP - 3，IGFBP - 3反义寡核苷酸和免疫中和实验诱导的生长抑制丧失证明了这一点。IGFBP - 3和1,25-(OH)2D3之间的可能联系在于细胞周期蛋白依赖性激酶抑制蛋白p21/WAF1，因为IGFBP - 3和1,25-(OH)2D3均上调该蛋白且两者均抑制LNCaP细胞生长。因此，我们假设IGFBP - 3和1,25-(OH)2D3诱导生长抑制的作用机制是诱导p21/WAF1，因为IGFBP - 3免疫中和抗体完全消除了1,25-(OH)2D3介导的p21/WAF1上调和生长抑制。
