Inhibitory effect of 22-oxa-1,25-dihydroxyvitamin D3, maxacalcitol, on the proliferation of pancreatic cancer cell lines

Effective chemotherapy for pancreatic cancer is urgently needed. The aim of this study was to compare the anti-proliferative activity on pancreatic cancer cell lines of the vitamin D(3) analog, 22-oxa-1,25-dihydroxyvitamin D(3), maxacalcitol, with that of 1,25-dihydroxyvitamin D(3), calcitriol, with analysis of vitamin D receptor status and the G(1)-phase cell cycle-regulating factors. Antiproliferative effects of both agents were compared using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method and by measuring the tumor size of xenografts inoculated into athymic mice. Scatchard analysis of vitamin D receptor contents, and mutational analysis of receptor complementary DNA were performed. Levels of expression of cyclins, cyclin-dependent kinases and cyclin-dependent kinase inhibitors, p21 and p27, were analysed by western blotting. In vitro, maxacalcitol and calcitriol markedly inhibited the proliferation and caused a G(1) phase cell cycle arrest with the appearance of numerous domes. In vivo, maxacalcitol inhibited the growth of BxPC-3 xenografts more significantly than calcitriol, without inducing hypercalcemia. Responsive cells had abundant functional vitamin D receptors. However, Hs 766T, showing no response to either agent, had the second highest receptor contents with no abnormalities in its primary structure deduced by receptor complementary DNA. In the responsive cells, p21 and p27 were markedly up-regulated after 24h of treatment with both agents. In non-responsive cells, no such changes were observed. In conclusion, maxacalcitol and calcitriol up-regulate p21 and p27 as an early event, which in turn could block the G(1)/S transition and induce growth inhibition in responsive cells, and maxacalcitol may provide a more useful tool for the chemotherapy of pancreatic cancer than calcitriol because of its low toxicity.     

Insulin-like growth factor binding protein-5 interacts with the vitamin D receptor and modulates the vitamin D response in osteoblasts

The 1,25 dihydroxyvitamin D3 [1,25(OH)2D3]-induced differentiation of osteoblasts comprises the sequential induction of cell cycle arrest at G0/G1 and the expression of bone matrix proteins. Reports differ on the effects of IGF binding protein (IGFBP)-5 on bone cell growth and osteoblastic function. IGFBP-5 can be growth stimulatory or inhibitory and can enhance or impair osteoblast function. In previous studies, we have shown that IGFBP-5 localizes to the nucleus and interacts with the retinoid receptors. We now show that IGFBP-5 interacts with nuclear vitamin D receptor (VDR) and blocks retinoid X receptor (RXR):VDR heterodimerization. VDR and IGFBP-5 were shown to colocalize to the nuclei of MG-63 and U2-OS cells and coimmunoprecipitate in nuclear extracts from these cells. Induction of osteocalcin promoter activity and alkaline phosphatase activity by 1,25(OH)2D3 were significantly enhanced when IGFBP-5 was down-regulated in U2-OS cells. Moreover, we found IGFBP-5 increased basal alkaline phosphatase activity and collagen alpha1 type 1 expression, and that 1,25(OH)2D3 was unable to further induce the expression of these bone differentiation markers in MG-63 cells. Expression of IGFBP-5 inhibited MG-63 cell growth and caused cell cycle arrest at G0/G1 and G2/M. Furthermore, IGFBP-5 reduced the effects of 1,25(OH)2D3 in blocking cell cycle progression at G0/G1 and decreased the expression of cyclin D1. These results demonstrate that IGFBP-5 can interact with VDR to prevent RXR:VDR heterodimerization and suggest that IGFBP-5 may attenuate the 1,25(OH)2D3-induced expression of bone differentiation markers while having a modest effect on the 1,25(OH)2D3-mediated inhibition of cell cycle progression in bone cells.     

Identification of a functional domain in a GADD45-mediated G2/M checkpoint

Cell cycle checkpoints are essential for the maintenance of genomic stability in response to DNA damage. We demonstrated recently that GADD45, a DNA damage-inducible protein, activates a G(2)/M checkpoint induced by either UV radiation or alkylating agents. GADD45 can interact in vivo with the G(2) cell cycle-specific kinase, Cdc2, proliferating cell nuclear antigen (PCNA), and the cell cycle kinase inhibitor p21(waf1). The ability of GADD45 to induce a G(2)/M arrest may be caused in part by the inhibition of Cdc2 kinase activity. Here, we report the identification of a region of GADD45 that is involved in this G(2)/M checkpoint. Mutants of GADD45 that lacked either the first 35 or the last 80 residues still retained an ability to induce G(2)/M arrest. A mutant with a deletion of the central region (residues 50-76), which is conserved in the family members GADD45beta and GADD45gamma, lacked such activity. This mutant also lacked an ability to bind to Cdc2, PCNA, and p21(waf1) in vivo. Consistently, either GADD45beta or GADD45gamma bind to Cdc2 in vivo. However, unlike GADD45, neither GADD45beta nor GADD45gamma inhibited the Cdc2 kinase or induced G(2)/M arrest. The unique effect of GADD45 may be caused by the presence of a region containing DEDDDR residues. Alanine substitutions in the region abolished GADD45 induction of a G(2)/M arrest and its inactivation of the Cdc2 kinase but not its binding to Cdc2, PCNA, or p21(waf1). Therefore, the binding of GADD45 to Cdc2 was insufficient to induce a G(2)/M arrest, and additional activity contributed by the DEDDDR residues may be necessary to regulate the G(2)/M checkpoint.     

Vitamin D and prostate cancer

Vitamin D and its metabolites are best known for their actions in calcium and bone metabolism. However, epidemiological studies have suggested that an increased prostate cancer risk is associated with decreased production of vitamin D. In vitro and in vivo studies have shown that the biologically active form of vitamin D, 1alpha,25-dihydroxyvitamin D3 (1,25D), inhibits proliferation of cancer cells derived from multiple tissues, including the prostate. Although the mechanisms underlying the growth inhibitory effects of 1,25D have not been fully elucidated, in prostate cancer cells 1,25D reduces cell growth via a number of cellular pathways, including cell cycle arrest, induction of apoptosis, and altered activation of growth factor signaling. The hypercalcemia induced by 1,25D in vivo limits its use clinically as a therapeutic agent. However, several 1,25D analogs have been developed that reduce prostate tumor growth in rodent xenograft models without causing hypercalcemia. Additional studies are required in order to determine whether these 1,25D analogs will be useful therapeutic agents for the treatment of prostate cancer.     

Inhibition of prostate cancer growth by vitamin D: Regulation of target gene expression

Prostate cancer (PCa) cells express vitamin D receptors (VDR) and 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) inhibits the growth of epithelial cells derived from normal, benign prostate hyperplasia, and PCa as well as established PCa cell lines. The growth inhibitory effects of 1,25(OH)(2)D(3) in cell cultures are modulated tissue by the presence and activities of the enzymes 25-hydroxyvitamin D(3) 24-hydroxylase which initiates the inactivation of 1,25(OH)(2)D(3) and 25-hydroxyvitamin D(3) 1alpha-hydroxylase which catalyses its synthesis. In LNCaP human PCa cells 1,25(OH)(2)D(3) exerts antiproliferative activity predominantly by cell cycle arrest through the induction of IGF binding protein-3 (IGFBP-3) expression which in turn increases the levels of the cell cycle inhibitor p21 leading to growth arrest. cDNA microarray analyses of primary prostatic epithelial and PCa cells reveal that 1,25(OH)(2)D(3) regulates many target genes expanding the possible mechanisms of its anticancer activity and raising new potential therapeutic targets. Some of these target genes are involved in growth regulation, protection from oxidative stress, and cell-cell and cell-matrix interactions. A small clinical trial has shown that 1,25(OH)(2)D(3) can slow the rate of prostate specific antigen (PSA) rise in PCa patients demonstrating proof of concept that 1,25(OH)(2)D(3) exhibits therapeutic activity in men with PCa. Further investigation of the role of calcitriol and its analogs for the therapy or chemoprevention of PCa is currently being pursued.     

Opposite effect of NF-kappa B and c-Jun N-terminal kinase on p53-independent GADD45 induction by arsenite

Cell cycle checkpoint, a major genomic surveillance mechanism, is an important step in maintaining genomic stability and integrity in response to environmental stresses. Using cells derived from human bronchial epithelial cells, we demonstrate that NF-kappaB and c-Jun N-terminal kinase (JNK) reciprocally regulate arsenic trioxide (arsenite)-induced, p53-independent expression of GADD45 protein, a cell cycle checkpoint protein that arrests cells at the G(2)/M phase transition. Inhibition of NF-kappaB activation by stable expression of a kinase-mutated form of IkappaB kinase caused increased and prolonged induction of GADD45 by arsenite. In contrast, the induction of GADD45 by arsenite was transient and less potent in cells where the NF-kappaB activation pathway was normal. Analysis of the cell cycle profile by flow cytometry indicated that NF-kappaB inhibition potentiates arsenite-induced G(2)/M cell cycle arrest. Abrogation of JNK activation, on the other hand, decreased GADD45 expression induced by arsenite, suggesting a role for JNK activation in GADD45 induction. These results indicate a molecular mechanism by which NF-kappaB and JNK may differentially contribute to cell cycle regulation in response to arsenite.     

The Effect of GADD45a on Furazolidone‐Induced S‐Phase Cell‐Cycle Arrest in Human Hepatoma G2 Cells

In this study, overexpression of GADD45a induced by furazolidone in HepG2 cells could arouse S‐phase cell cycle arrest, suppress cell proliferation, and increase the activities of cyclin D1, cyclin D3, and cyclin‐dependent kinase 6 (CDK6). To the opposite, GADD45a knockdown cells by RNAi could reduce furazolidone‐induced S‐phase cell cycle arrest, increase the cell viability, decrease the activities of cyclin D1, cyclin D3, and CDK6; however, cyclin‐dependent kinase 4 (CDK4) showed no change. Moreover, data from our current studies show that cyclin D1, cyclin D3, and CDK6 are target genes functioning at the downstream of the GADD45a pathway induced by furazolidone. These results demonstrate that the GADD45a pathway is partially responsible for the furazolidone‐induced S‐phase cell cycle arrest. GADD45a influences furazolidone‐induced S‐phase cell cycle arrest in human hepatoma G2 cells via cyclin D1, cyclin D3, and CDK6, but not CDK4.     

1,25-Dihydroxyvitamin D3 and the regulation of human cancer cell replication

Several human and animal cancer cell lines have been shown to possess specific high affinity receptors for 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). The replication of several of these cell types has also been shown to be regulated by this hormone, both in vitro and in vivo. To further understand the mechanisms of these actions, we have examined cancer cells in vitro and in vivo. The in vitro studies extend our previous reports on the treatment of human breast cancer cells (T 47D) with 10(-9) to 10(-6) M 1,25-(OH)2D3, which resulted in a dose- and time-dependent decrease in cell numbers over 6 days. Treatment with 10(-8) M 1,25-(OH)2D3, which reduced cell numbers to approximately one half of those found in control cultures at 6 days, was associated with a doubling of the proportion of cells in the G2 + M phase of the cell cycle and was accompanied by a significant decline in the proportion of G0/G1 cells. At higher concentrations there was a significant decline in S phase cells with accumulation of cells in both G0/G1 and G2 + M phases. The antiestrogen, tamoxifen, at a concentration which caused similar effects on cell number, resulted in proportional decreases in both S and G2 + M phase cells and accumulation of G0/G1 cells. The effects of 1,25-(OH)2D3 on T 47D cell proliferation were associated with time- and concentration-dependent reductions in epidermal growth factor receptor levels to a minimum level of about half that seen in control cultures. The in vivo experiments extend our previous studies, which demonstrated marked inhibition of the growth of human cancer xenografts in immunosuppressed mice by 1,25-(OH)2D3. Xenograft growth was inhibited with 1,25-(OH)2D3 (0.1 microgram ip three times per week) but growth was rapidly restored when the 1,25-(OH)2D3 was withdrawn. Thus, there are clear-cut time- and dose-dependent, yet reversible, effects of 1,25-(OH)2D3 on the replication of human cancer cells in vitro and in vivo, which are possibly mediated through changes in growth factor receptor levels. Further study of these effects may advance understanding of the hormonal control of cellular replication in human cancers.     

Cell cycle arrest induced by the vitamin D(3) analog EB1089 in NCI-H929 myeloma cells is associated with induction of the cyclin-dependent kinase inhibitor p27

EB1089, a 1,25-dihydroxyvitamin D(3) analog, has been known to have potent antiproliferative properties in a variety of malignant cells in vitro and in vivo. In the present study, we analyzed the effect of EB1089 on human myeloma cell lines. EB1089 inhibited the proliferation of NCI-H929 cells and RPMI8226 cells in a dose-dependent manner among three myeloma cell lines tested. The antiproliferative effect of EB1089 on myeloma cells was related to the expression level of vitamin D receptor. To investigate the mechanism of the antiproliferative effect of EB1089, cell cycle analysis was attempted in EB1089-sensitive NCI-H929 cells. EB1089 (1 x 10(-8) M) efficiently induced G(1) arrest of the cell cycle. Analysis of G(1) regulatory proteins demonstrated that protein levels of CDK2, CDK4, cyclin D1, and cyclin A were decreased in a time-dependent manner, but not those of CDK6 and cyclin E, by EB1089. In addition, EB1089 (1 x 10(-8) M, 72 h) increased the protein level of the CDKI p27 and markedly enhanced the binding of p27 with CDK2 compared to EB1089-untreated cells. Furthermore, the activity of CDK2-associated cyclin kinase was decreased, which was accompanied by the reduction of cyclin-D1-, cyclin-E-, and cyclin-A-associated kinase activities, resulting in the hypophosphorylation of Rb protein. These results suggest that EB1089 can inhibit the proliferation of human myeloma cells, especially NCI-H929 cells, via a G(1) block in association with the induction of p27 and the reduction of CDK2 activity.     

1,25-Dihydroxyvitamin D3 inhibits the clonogenic growth of transformed cells via its receptor

Anchorage-independent growth in soft agar is a unique property of transformed cells which is known to be correlated with tumorigenicity. We report here that 1,25-dihydroxyvitamin D3 suppresses colony formation by a number of cultured cancer cell lines in soft agar in a dose dependent manner with an ID50 of 5-7 X 10(-10) M. This effect is also achieved with analogues of 1,25-dihydroxyvitamin D3 in accordance with their binding affinity for the hormone's receptor. Only cells with 1,25-dihydroxyvitamin D3 receptor protein are inhibited in their colony formation by vitamin D analogs indicating that the hormone receptor complex may be integrally involved in the in vitro suppression of the anchorage-independent phenotype.     

Role of c-Abl kinase in DNA mismatch repair-dependent G2 cell cycle checkpoint arrest responses

Current published data suggest that DNA mismatch repair (MMR) triggers prolonged G(2) cell cycle checkpoint arrest after alkylation damage from N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) by activating ATR (ataxia telangiectasia-Rad3-related kinase). However, analyses of isogenic MMR-proficient and MMR-deficient human RKO colon cancer cells revealed that although ATR/Chk1 signaling controlled G(2) arrest in MMR-deficient cells, ATR/Chk1 activation was not involved in MMR-dependent G(2) arrest. Instead, we discovered that disrupting c-Abl activity using STI571 (Gleevec, a c-Abl inhibitor) or stable c-Abl knockdown abolished MMR-dependent p73alpha stabilization, induction of GADD45alpha protein expression, and G(2) arrest. In addition, inhibition of c-Abl also increased the survival of MNNG-exposed MMR-proficient cells to a level comparable with MMR-deficient cells. Furthermore, knocking down GADD45alpha (but not p73alpha) protein levels affected MMR-dependent G(2) arrest responses. Thus, MMR-dependent G(2) arrest responses triggered by MNNG are dependent on a human MLH1/c-Abl/GADD45alpha signaling pathway and activity. Furthermore, our data suggest that caution should be taken with therapies targeting c-Abl kinase because increased survival of mutator phenotypes may be an unwanted consequence.     

1,25-Dihydroxyvitamin D3 stimulates 45Ca2+-uptake by cultured vascular smooth muscle cells derived from rat aorta

We have recently shown the presence of receptors for 1,25-dihydroxyvitamin D3 and that 1,25-dihydroxyvitamin D3 stimulates Ca-ATPase in vascular smooth muscle cells presumably via receptor mediated mechanism. These data suggest that the sterol may directly be involved in the regulation of cellular calcium homeostasis. To further define action of vitamin D in smooth muscle cells, we studied effect of the sterol on cellular uptake of calcium. 1,25-dihydroxyvitamin D3 stimulated 45Ca2+ uptake by cultured cells, A7r5, derived from fetal rat aorta, when the cells were incubated with the sterol for 18 hr. The effect was dose-dependent at 10(-10) to 10(-9) M, and three orders of magnitude higher concentration of 25-hydroxyvitamin D3 or 24,25-dihydroxyvitamin D3 was needed to obtain similar effects. Furthermore, the effect of 1,25-dihydroxyvitamin D3 was abolished by cycloheximide (10(-5) M), a protein synthesis inhibitor. These data clearly suggest that 1,25-dihydroxyvitamin D3 may directly regulate cellular calcium homeostasis in vascular smooth muscle cells presumably via receptor mediated mechanism.     

Vitamin D receptor agonists induce prostatic acid phosphatase to reduce cell growth and HER-2 signaling in LNCaP-derived human prostate cancer cells

We have previously shown that concentrations of 1alpha,25-dihydroxyvitamin D(3) (1,25D) that induce G(0)/G(1) cell cycle arrest in androgen-dependent LNCaP prostate cancer cells also decrease expression of c-Myc, a proto-oncogene that stimulates progression from G(1) to S phase of the cell cycle. Since both c-Myc expression and cell cycle progression are regulated by tyrosine kinase activation, we examined the ability of 1,25D to alter tyrosine kinase signaling in LNCaP cells and the androgen-independent LNCaP C81 (C81 LN) cell line. 1,25D selectively reduced protein tyrosine phosphorylation within both the LNCaP and C81 LN cells. This reduction in tyrosine kinase signaling appears to result from elevated levels of cellular prostatic acid phosphatase (PAcP). Western blots and biochemical assays revealed 1,25D increases the level of active PAcP in both cell lines. In addition, 1,25D decreased tyrosine phosphorylation of HER-2, an EGFR family member inactivated by PAcP, and the HER-2 downstream adaptor protein p52 Shc in C81 LN cells. Inhibition of HER-2 signaling by AG825 reduces growth of C81 LN cells and the parental LNCaP cells. These data therefore suggest that 1,25D-mediated decreases in LNCaP and C81 LN cell growth are in part due to decreases in tyrosine kinase signaling that result from up-regulation of PAcP.     

Induction of monocytic differentiation of HL-60 cells by 1,25-dihydroxyvitamin D analogs

1,25-Dihydroxyvitamin D3, the hormonal form of vitamin D, induces differentiation of HL-60 human promyelocytes into monocyte-like cells in vitro. We assessed the relative activity of 30 analogs of 1,25-dihydroxyvitamin D3 in inducing development of monocytic markers in HL-60 cells. The three differentiation markers assayed were nonspecific acid esterase activity, nitro blue tetrazolium reducing activity, and phagocytic capacity. Of the known metabolites of vitamin D, 1,25-dihydroxyvitamin D3 is the most active; 50% of the cells exhibit the mature phenotype following a 4-day treatment with 10(-8) M 1,25-dihydroxyvitamin D3. Removal of either the C-1 or C-25-hydroxyl group reduces activity by 2 orders of magnitude, while epimerization of the 1 alpha- to 1 beta-hydroxyl group virtually abolishes activity. Elongation of the steroidal side chain of 1,25-dihydroxyvitamin D3 by addition of one carbon at C-24 or C-26 improves the potency by an order of magnitude. Truncation of the steroidal side chain leads to a 10-fold reduction in activity for each carbon removed. Elimination of the C-26 and C-27 methyl groups reduces activity 100-fold. Analogs with short aliphatic side chains as 1 alpha-hydroxyhomo- and bishomopregnacholecalciferol have surprisingly high activity, being only 20-fold less potent than the natural hormone. The activity of most analogs in the HL-60 system parallels their known relative affinities for the well characterized 1,25-dihydroxyvitamin D3 receptor in chick intestine, providing further evidence that this function of 1,25-dihydroxyvitamin D3 is receptor mediated.