Inhibition of MG-63 cell cycle progression by synthetic vitamin D3 analogs mediated by p27, Cdk2, cyclin E, and the retinoblastoma protein
Introduction
Most of the biological actions of 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3, 1,25D) are mediated through the nuclear VDR, which is a member of structurally related steroid/thyroid hormone superfamily of ligand-dependent transcription factors [1]. VDR forms heterodimers with another member of this family, the retinoid X receptor, and regulates gene expression positively or negatively through binding to the vitamin D response elements (VDREs) in promoter regions of target genes. In addition to its role in mineral homeostasis, the active 1α,25(OH)2D3 affects growth and differentiation of different cell types, e.g., human breast, brain, colon, prostate, and skin cells [2], [3], [4], [5], [6], [7]. The potentially beneficial use of the hormone in treatment of hyperproliferative diseases is, however, compromised by hypercalcemia and hypercalciuria developing at therapeutic doses. This has evoked an increased interest in synthesis and evaluation of new 1α,25(OH)2D3 analogs that would retain the properties of the parent compound on cellular proliferation and differentiation while having reduced calcemic activity [2], [8], [9]. New analogs could be potentially useful in the treatment of, e.g., several types of cancer and skin disorders [2], [8]. One interesting group among the side chain analogs of 1α,25(OH)2D3 is the 20-epi analogs. The prolonged effects of these analogs quite likely result from their resistance against cell metabolism further leading to increased stabilization of the analog–receptor complex [10], [11].
Inhibition of cell proliferation is tightly linked to mechanisms that regulate cell cycle progression. Knowledge of mechanisms by which cell growth is arrested after exposure to 1α,25(OH)2D3 and its analogs is quite fragmentary and partly unclear. In most cases, the inhibition of human cell growth in response to 1α,25(OH)2D3 involves cell cycle arrest in the G1 phase [12], [13], [14]. In dividing mammalian cells, cell cycle progression is regulated by sequential formation, activation, and subsequent inactivation of a series of cyclin dependent kinase (Cdk)–cyclin complexes. Cdk activities are regulated by phosphorylations and cyclin binding, as well as by the action of specific Cdk inhibitor (CKI) proteins ([15], [16], [17], [18] and references therein). In mammalian cells, complexes such as cyclin D/Cdk4,6 and cyclin E-Cdk2 are catalytically active during the G1 phase and the main function of these complexes is phosphorylation of the retinoblastoma (Rb) protein. Indeed, many of these events have been identified during 1α,25(OH)2D3-induced inhibition of human cell proliferation. In a variety of human cells, the 1α,25(OH)2D3-induced cell cycle arrest is mediated by CKIs including p21 (Cip1/Waf1) and p27 (Kip1) [14], [19], [20], [21]. There are also studies showing that 1α,25(OH)2D3 signaling is mediated by the cell cycle regulatory proteins Rb and p57 (Kip2) and by the induction of apoptosis [22], [23], [24]. In addition to the inhibitory role of the Cip/Kip family proteins on cell cycle progression p21, and especially p27 can also be found in a stimulatory role in promoting activation of cyclin D1-Cdk4,6 in proliferative cells [18], [25].
The 20-epi analogs, especially KH1060 and CB1093, as well as EB1089, an analog with a natural side chain orientation at carbon-20, have already shown their therapeutical potencies as antileukemic compounds and inhibitors of tumor cell growth as well as differentiation-inducing agents [23], [26], [27], [28], [29]. The present study was undertaken, first, to analyze several candidates of cell cycle controlling proteins possibly responsible for the G1 arrest caused by 1α,25(OH)2D3 in human bone cells. Second, we were also interested in comparing these effects with those of selected 1α,25(OH)2D3 analogs. We used human MG-63 osteosarcoma cells which share many features of normal human osteoblasts and, therefore, should provide useful information relevant to osteoblastic proliferative events. Our results indicated that, in the osteoblastic cells, 1α,25(OH)2D3 signaling appears to be mediated through the Rb pathway, as cells treated with 1α,25(OH)2D3 showed decreased amounts of hyperphosphorylated Rb protein. Consistent with its effects on Rb protein, 1α,25(OH)2D3 reduced Cdk2 activity through increased p27, but not p21 protein levels. The selected analogs, KH1060, EB1089, and CB1093, were clearly more potent than the parent compound in their effects on these cell cycle control mechanisms.
Section snippets
Chemicals and antibodies
1α,25-Dihydroxyvitamin D3 and the synthetic analogs 1α,25-dihydroxy-20-epi-22-oxa-24a,26a,27a-trihomo-vitamin D3 (KH1060), 1α,25-dihydroxy-22,24-diene-24a,26a,27a-trihomo-vitamin D3 (EB1089), and 1α,25-dihydroxy-20-epi-22-ethoxy-23-yne-24a,26a,27a-trihomo-vitamin D3 (CB1093) (Fig. 1) were a kind gift from Leo Pharmaceutical Products Ltd. The vitamin D3 compounds were dissolved in isopropanol at 4 mM concentration and diluted with ethanol. Radioactive labels [α-]dCTP and [γ-]dATP were from
Effects of 1α,25(OH)2D3 and its analogs on MG-63 cell growth and on cell cycle phase distribution
To establish conditions to investigate the mechanism of action of the vitamin D3 compounds on inhibition of MG-63 cell growth, the cells were treated with the compounds (concentrations from 1 pM to 0.1 μM) and cell numbers were assessed after 72 hr. All the compounds studied reduced MG-63 cell numbers dose-dependently (Fig. 2). The concentration needed for 50% inhibition of proliferation in our study was calculated to be 120 pM (1α,25(OH)2D3), 3.5 pM (KH1060), 17 pM (EB1089) and 3.8 pM (CB1093),
Discussion
Progression of cell cycle from the G1 phase to the S phase is one of the most critical steps in the control of cell proliferation and also the step most often altered in cancer cells. In cancer therapy, much attention has been focused on the inhibition of cell proliferation and regulation of the G1 phase cyclin-Cdk functions [17], [37]. The antiproliferative effects of 1α,25(OH)2D3 and its synthetic analogs may be useful, e.g., in cancer treatment, and therefore a more detailed understanding of
Acknowledgements
We thank Dr. Lise Binderup from Leo Pharmaceutical Products Ltd, for kindly providing the synthetic vitamin D3 analogs and by Dr. Joan Massague (Memorial Sloan Kettering Cancer Center and Howard Hughes Medical Institute) for the p27 probe. We also thank Mrs. Maija Hiltunen for skilful technical assistance. This work has been supported in part by Grant 30566 from the Academy of Finland and by Grant 40949/98 from the Technology Development Centre of Finland, Finland. We also thank the Finnish
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