calcipotriene and Osteosarcoma

We have compared the actions of calcitriol and its three synthetic analogs, 20-epi-22-oxa-24a,26a,27a-trihomo-1 alpha,25-dihydroxyvitamin D3 (KH 1060), 1 alpha,24S-(OH)2-22-ene-26,27-cyclopropyl vitamin D3 (MC 903) and 20-epi-1 alpha,25-dihydroxyvitamin D3 (MC 1288), on the expression of two marker genes of differentiated osteoblasts, namely alkaline phosphatase and osteocalcin, using human MG-63 osteosarcoma cells. Calcitriol and the analogs had qualitatively similar stimulatory effects on target-gene activation. Quantitatively, MC 903 behaved in most experiments essentially as the parent compound calcitriol. In vitamin D receptor/DNA complex formation MC 903, however, was more potent than calcitriol. In contrast, the 20-epi analogs, KH 1060 and MC 1288, were much more potent even at lower concentrations, than calcitriol and MC 903 in stimulating alkaline phosphatase activity, osteocalcin mRNA synthesis and osteocalcin secretion. The stimulation occurred to a greater degree and for a longer period than with calcitriol. This effect was apparently mediated by stronger and longer lasting binding of the vitamin D receptor to the osteocalcin vitamin D responsive element by the 20-epi analogs. After a 6-h treatment and during subsequent culture without hormone, the effects of the 20-epi analogs were also stronger and lasted longer than those with calcitriol or MC 903. Collectively, at comparable and lower concentrations, the 20-epi analogs, KH 1060 and MC 1288, mediate much stronger and longer lasting stimulatory effects than calcitriol or its analog MC 903 on target-gene expression associated with the differentiated phenotype of the MG-63 human osteosarcoma cells.

Topics: Alkaline Phosphatase; Base Sequence; Binding Sites; Calcitriol; Cell Differentiation; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Humans; Molecular Sequence Data; Osteocalcin; Osteosarcoma; Promoter Regions, Genetic; Receptors, Calcitriol; RNA, Messenger; Stereoisomerism; Time Factors; Transcriptional Activation; Tumor Cells, Cultured

Although numerous studies have shown potent antiproliferative and differentiation-inducing effects of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) and its analogs on cells not directly related to bone metabolism, only few reports focussed on the effects of these analogs on bone. We compared the action of several recently developed analogs with that of 1,25-(OH)2D3 on human (MG-63) and rat (ROS 17/2.8) osteoblast-like cells and on in vitro bone resorption. In MG-63 cells the analogs EB1089 and KH1060 were about 166,000 and 14,000 times more potent than 1,25-(OH)2D3 in stimulating type I procollagen and 100 and 6,000 times more potent in stimulating osteocalcin production, respectively. Also in ROS 17/2.8 cells EB1089 and KH1060 were most potent in inducing osteocalcin synthesis. In vitro bone resorption was 2.3 and 17.5 times more potently stimulated by EB1089 and KH1060, respectively. In MG-63 cells, 1,25-(OH)2D3 and the analogs inhibited cell proliferation, whereas both 1,25-(OH)2D3 and the analogs stimulated the growth of ROS 17/2.8 cells. Differences in potency could neither be explained by affinity for the vitamin D receptor nor by a differential involvement of protein kinase C in the action of the analogs. Together, these data show that also in bone the analogs EB1089 and KH1060 are more potent than 1,25-(OH)2D3 but that the potency of the analogs compared to 1,25-(OH)2D3 is dependent on the biological response. On the basis of these observations it can be concluded that the reported reduced calcemic effect in vivo is not the result of a decreased responsiveness of bone to these analogs. Lastly, in view of eventual clinical application of 1,25-(OH)2D3-analogs, the observed stimulation of in vitro bone resorption and growth of an osteosarcoma cell line warrant in vivo studies to further examine these effects.

Topics: Animals; Antineoplastic Agents; Binding, Competitive; Bone Resorption; Calcitriol; Cell Division; Cells, Cultured; Cholecalciferol; Enzyme Inhibitors; Glyceryl Ethers; Humans; In Vitro Techniques; Osteoblasts; Osteocalcin; Osteosarcoma; Procollagen; Protein Kinase C; Rats; Receptors, Calcitriol; Structure-Activity Relationship; Tumor Cells, Cultured

The effects of steroid hormone calcitriol (Cal) and its analogue calcipotriol on human osteosarcoma cell line HOS-8603 were determined. When cells grew in monolayer culture in the presence of hormones, their proliferations were inhibited both in dose- and time-dependent manners. The cells showed marked morphologic changes after a 4-d treatment to apparently less transformed fibroblast-like ones. Anchorage-independent growth studies indicated that both Cal and calcipotriol at 10 nmol.L-1 inhibited colony formation by HOS-8603 cells. As a marker enzyme of the osteoblastic phenotype, alkaline phosphatase activity was induced in response to Cal or calcipotriol 100 nmol.L-1. These results suggested that Cal and calcipotriol play an important role in regulating growth and differentiation of HOS-8603 cells.

Topics: Alkaline Phosphatase; Antineoplastic Agents; Calcitriol; Cell Differentiation; Cell Division; Humans; Osteosarcoma; Tumor Cells, Cultured

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] is the active hormonal form of vitamin D3 and has potent effects on bone and calcium regulation. Over the past decade it has become apparent that 1,25-(OH)2D3 has other effects on cellular proliferation that potentially could be developed for therapy in human malignancy. Since the hypercalcemic effects of 1,25-(OH)2D3 have limited that use in the human, novel nonhypercalcemic analogs of 1,25-(OH)2D3 have been synthesized. The molecular mechanism of this divergence in these antiproliferative and calcium-regulating actions is unexplained. We have previously examined the human bone-specific gene osteocalcin as a model of the molecular mechanisms of vitamin D action in bone and have shown that induction of the osteocalcin gene by 1,25-(OH)2D3 is mediated through an unique and complex palindromic region of the promoter similar to but distinct from those of other steroid hormone-responsive elements. Using an osteosarcoma cell line permanently transfected with the vitamin D-responsive promoter of the human osteocalcin gene linked to a "reporter" gene, we have shown that there is a dose-dependent induction of CAT activity by 1,25-(OH)2D3 and that the potencies of vitamin D metabolites and analogs are comparable to those found in other vitamin D bioassays. Furthermore, vitamin D analogs, including MC-903, 22-oxa-1,25-(OH)2D3, and delta 22-1,25S,26-trihydroxyvitamin D3, which effect cellular differentiation but lack hypercalcemic activity in vivo, exhibit osteocalcin promoter inductive actions virtually identical to those of 1,25-(OH)2D3. Consideration of these and other data support the hypothesis that the divergent effects of such analogs on differentiation and calcium homeostasis reflect pharmacokinetic differences in vivo rather than distinct 1,25-(OH)2D3-sensitive pathways.

Topics: Animals; Calcitriol; Calcium; Hydroxycholecalciferols; Osteocalcin; Osteosarcoma; Promoter Regions, Genetic; Rats; Transfection; Tumor Cells, Cultured