Inhibition of insulin- and insulin-like growth factor-I-stimulated growth of human breast cancer cells by 1,25-dihydroxyvitamin D3 and the vitamin D3 analogue EB1089

1, 25 Dihydroxyvitamin D3 (1,25-(OH)2D3) and a number of synthetic vitamin D3 analogues with low calcaemic activity, have been shown to inhibit breast cancer cell growth in vitro as well as in vivo. The purpose of the present study was to investigate a possible interaction of 1, 25-(OH)2D3 and the vitamin D3 analogue EB1089 with the insulin-IGF-I regulatory system. The oestrogen receptor-positive MCF-7 human breast cancer cells used in this study are able to grow autonomously and their growth is stimulated by insulin. In order to avoid interference of IGF-binding proteins (IGF-BPs), we used an analogue of IGF-I, long R3 IGF-I, which stimulated MCF-7 cell growth similar to insulin. The growth stimulation by insulin and by long R3 IGF-I was completely inhibited by 1,25-(OH)2D3 and EB1089. Autonomous growth was also inhibited by 1,25-(OH)2D3 and EB1089. The analogue EB1089 was active at 50 times lower concentrations than 1,25-(OH)2D3. It was shown that growth inhibition was not achieved through downregulation of insulin and IGF-I binding after 48 h. Paradoxically, after prolonged treatment (8 days), an upregulation of insulin and IGF-I binding was observed. Two possible intracellular mediators of the insulin-IGF mitogenic signal are C-FOS and mitogen-activated protein (MAP) kinase. Insulin-induced C-FOS mRNA was inhibited by 1,25-(OH)2D3, suggesting that it could be involved in the growth inhibition by 1,25-(OH)2D3. MAP kinase activation appeared not to be involved in growth stimulation by both insulin and IGF-I. Together, the present study demonstrates that vitamin D3 compounds can block the mitogenic activity of insulin and IGF-I, which may contribute to their tumour suppressive activity observed in vivo. Copyrigh

Inhibition of Breast Cancer Cell Growth by Combined Treatment with Vitamin D3 Analogues and Tamoxifen

The steroid hormone 1,25-dihydroxyvitamin D3 [l,25-(OH)2D3] has potential to be used as an antitumor agent, but its clinical application is restricted by the strong calcemic activity. Therefore, new vitamin D3 analogues are developed with increased growth inhibitory and reduced calcemic activity. In the present study, we have examined the antiproliferative effects of four novel vitamin D3 analogues (CB966, EB1089, KH1060, and 22-oxa-calcitriol) on breast cancer cells, either alone or in combination with the antiestrogen tamoxifen. The estrogen-dependent ZR-75-1 and estrogen-responsive MCF-7 cell lines were used as a model. It was shown that, with EB1089 and KH1060, the same growth inhibitory effect as l,25-(OH)2D3 could be reached at up to 100-fold lower concentrations, whereas CB966 and 22-oxa-calcitriol were nearly equipotent with 1,25-(OH)2D3. The growth inhibition by the vitamin D3 compounds could be augmented by combined treatment with tamoxifen. At the maximal effective concentrations of the vitamin D3 compounds, the effect of combined treatment was additive (MCF-7 cells) or less than additive (ZR-75-1 cells). Tamoxifen increased the sensitivity of the cells to the vitamin D3 compounds 2- to 4000-fold, which was expressed by a shift to lower median effective concentration values. Thereby, the vitamin D3 compounds may be used at even lower dosages in combination therapy with tamoxifen. A major problem of tamoxifen therapy is the development of tamoxifen resistance. We have observed that tamoxifen-resistant clones of ZR-75-1 cells retain their response to the vitamin D3 compounds. Regulation of the growth-related oncogene c-myc (mRNA level) and the estrogen receptor (protein level) were studied but appeared not to be related to the antiproliferative action of the vitamin D3 compounds. Together, our data point to a potential benefit of combination therapy with 1,25-(OH)2D3 or vitamin D3 analogues and tamoxifen for the treatment of breast cancer.</p

Inhibition of Breast Cancer Cell Growth by Combined Treatment with Vitamin D3 Analogues and Tamoxifen

The steroid hormone 1,25-dihydroxyvitamin D3 [l,25-(OH)2D3] has potential to be used as an antitumor agent, but its clinical application is restricted by the strong calcemic activity. Therefore, new vitamin D3 analogues are developed with increased growth inhibitory and reduced calcemic activity. In the present study, we have examined the antiproliferative effects of four novel vitamin D3 analogues (CB966, EB1089, KH1060, and 22-oxa-calcitriol) on breast cancer cells, either alone or in combination with the antiestrogen tamoxifen. The estrogen-dependent ZR-75-1 and estrogen-responsive MCF-7 cell lines were used as a model. It was shown that, with EB1089 and KH1060, the same growth inhibitory effect as l,25-(OH)2D3 could be reached at up to 100-fold lower concentrations, whereas CB966 and 22-oxa-calcitriol were nearly equipotent with 1,25-(OH)2D3. The growth inhibition by the vitamin D3 compounds could be augmented by combined treatment with tamoxifen. At the maximal effective concentrations of the vitamin D3 compounds, the effect of combined treatment was additive (MCF-7 cells) or less than additive (ZR-75-1 cells). Tamoxifen increased the sensitivity of the cells to the vitamin D3 compounds 2- to 4000-fold, which was expressed by a shift to lower median effective concentration values. Thereby, the vitamin D3 compounds may be used at even lower dosages in combination therapy with tamoxifen. A major problem of tamoxifen therapy is the development of tamoxifen resistance. We have observed that tamoxifen-resistant clones of ZR-75-1 cells retain their response to the vitamin D3 compounds. Regulation of the growth-related oncogene c-myc (mRNA level) and the estrogen receptor (protein level) were studied but appeared not to be related to the antiproliferative action of the vitamin D3 compounds. Together, our data point to a potential benefit of combination therapy with 1,25-(OH)2D3 or vitamin D3 analogues and tamoxifen for the treatment of breast cancer

Evidence for auto/paracrine actions of vitamin D in bone: 1α-Hydroxylase expression and activity in human bone cells

Vitamin D is an important regulator of mineral homeostasis and bone metabolism. 1α-Hydroxylation of 25-(OH)D3 to form the bioactive vitamin D hormone, 1α,25-(OH)2D3, is classically considered to take place in the kidney. However, 1α-hydroxylase has been reported at extrarenal sites. Whether bone is a 1α,25-(OH) 2D3 synthesizing tissue is not univocal. The aim of this study was to investigate an autocrine/paracrine function for 1α,25-(OH)2D3 in bone. We show that 1α-hydroxlase is expressed in human osteoblasts, as well as the vitamin D binding protein receptors megalin and cubilin. Functional analyses demonstrate that after incubation with the 1α-hydoxylase substrate 25-(OH)D 3, the osteoblasts can produce sufficient 1α,25-(OH) 2D3 to modulate osteoblast activity, resulting in induced alkaline phosphatase (ALP) activity, osteocalcin (OC) and CYP24 mRNA expression, and mineralization. The classical renal regulators of 1α-hydroxylase, parathyroid hormone, and ambient calcium do not regulate 1α-hydroxylase in osteoblasts. In contrast, interleukin (IL)-1β strongly induces 1α-hydroxylase. Besides the bone-forming cells, we demonstrate 1α-hydroxylase activity in the bone resorbing cells, the osteoclasts. This is strongly dependent on osteoclast inducer RANKL. This study showing expression, activity, and functionality of 1α-hydoxylase unequivocally demonstrates that vitamin D can act in an auto/paracrine manner in bone