seocalcitol and Carcinoma--Squamous-Cell

The objective of this study is to evaluate the role of the cyclin-dependent kinase inhibitor p57 in EB1089-inhibited proliferation of human laryngeal squamous carcinoma cells (HEp-2). HEp-2 cells were treated with the vitamin D3 analogue EB1089 for 48 h and total RNA was extracted for reverse transcription-PCR amplification using primers for the p57 coding sequence. Proteins were detected by Western blot analysis. For interference using silencing RNA (siRNA), HEp-2 cells were transfected with siRNA specific for p57 (siRNA-p57) or a negative control sequence (siRNA-con) followed by treatment with 10 nmol/L EB1089. The effects of EB1089 on cell proliferation were evaluated by 5-bromo-2'-deoxyuridine incorporation and '3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide assay. Cell death and cell cycle dynamics were monitored using flow cytometry. EB1089 significantly inhibited HEp-2 cell proliferation and increased p57 mRNA and protein levels; this was blocked by siRNA-p57 but not by siRNA-con. The EB1089-induced suppression of HEp-2 cell proliferation recovered to near-normal levels with siRNA-p57 transfection. EB1089 inhibits the proliferation of HEp-2 cells and p57 plays an important role in this.

Topics: Animals; Antineoplastic Agents; Calcitriol; Carcinoma, Squamous Cell; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p57; Humans; Laryngeal Neoplasms; Mice; Mice, Nude; RNA, Small Interfering; Time Factors; Tumor Cells, Cultured

The biologically active 1,25(OH)2 vitamin D3 and its analogs have been shown to have antiproliferative and differentiating effects in a variety of malignant and non-malignant cells. For squamous carcinoma cell lines of the head and neck (SCCHN) we could show that this antiproliferative activity of 1,25(OH)2 vitamin D3 is due to induced expression of the cell-cycle inhibitory proteins p21 and p27, causing an arrest in the G0/G1 cell-cycle phase.. In this work we investigated the effects of three vitamin D3 analogs, EB1089, MC1288 and CB1093, on proliferation behavior and cell-cycle status in a laryngeal carcinoma cell line (JPPA) as well as in control human immortalized keratinocytes (HaCaT). To study the molecular mechanism the functional activity of the promoter region of p21, a potential target gene of vitamin D3 transcriptional regulation, was investigated. For this reason a 2.7-kb fragment of the p21 promoter was isolated by polymerase chain reaction from HaCaT, JPPA and SCC9 (tongue carcinoma) cells and directionally cloned into an enhanced green fluorescence protein (EGFP) reporter gene vector system. A construct was used to stably transfect HaCaT cells and to monitor the expression of the EGFP gene by confocal microscopy.. Analysis of proliferation and cell-cycle status revealed decreased growth rates and G0/G1I cell-cycle arrest in cells treated with 1,25(OH)2 vitamin D3 and its analogs The EGFP reporter gene-transfected cells showed distinct fluorescence under the influence of 1,25(OH)2 vitamin D3 and its analogs compared to control cells.. These results demonstrate that the cell-cycle inhibitor protein p21 is a direct target gene of biologically active 1,25(OH)2 vitamin D3, inducing G0/G1 cell-cycle arrest. The ability of vitamin D analogs to act via the same molecular mechanism as the natural hormone but with less hypercalcemic activity may have therapeutic implications for patients with SCCHN malignancy.

Topics: Antineoplastic Agents; Calcitriol; Carcinoma, Squamous Cell; Cell Cycle; Cell Division; Cell Survival; G1 Phase; Gene Expression Regulation, Neoplastic; Genes, Reporter; Green Fluorescent Proteins; Humans; Laryngeal Neoplasms; Luminescent Proteins; Promoter Regions, Genetic; Proto-Oncogene Proteins p21(ras); Resting Phase, Cell Cycle; Transcription, Genetic; Transfection; Tumor Cells, Cultured

Treatment of cancer cells with 1,25-dihydroxyvitamin D3 [1,25(OH)(2)D(3)] or its analogs induces growth arrest and expression of the cyclin-dependent kinase inhibitor p27(KIP1). Although 1,25(OH)(2)D(3) transiently enhances p27(kip1) gene transcription in some cells, its effects on p27(KIP1) protein levels are generally more gradual and sustained. This suggests that 1,25(OH)(2)D(3) treatment may be stabilizing p27(KIP1) protein, which is sensitive to modification by the SCF(SKP2) protein ubiquitin ligase and proteosomal degradation. Here, we show that treatment of AT-84 head and neck squamous carcinoma cells with the 1,25(OH)(2)D(3) analog EB1089 increases p27(KIP1) protein levels without significantly affecting expression of its mRNA. EB1089 treatment repressed expression of mRNAs encoding the F-box protein p45(SKP2), a marker of poor head and neck cancer prognosis, and the cyclin kinase subunit CKS1, which is essential for targeting p45(SKP2) to p27(KIP1). This coincided with a reduction of total p45(SKP2) protein, and p45(SKP2) associated with p27(KIP1). Consistent with these findings, turnover of p27(KIP1) protein was strongly inhibited in the presence of EB1089. A similar reduction in p45(SKP2) expression and stabilization of p27(KIP1) protein was observed in 1,25(OH)(2)D(3)-sensitive UF-1 promyelocytic leukemia cells, which also respond by transiently increasing p27(kip1) gene transcription. Our results reveal that 1,25(OH)(2)D(3) analogs increase levels of p27(KIP1) in different cell types by inhibiting expression of SCF(SKP2) subunits and reducing turnover of p27(KIP1) protein.

Topics: Animals; Antineoplastic Agents; Blotting, Northern; Calcitriol; Carcinoma, Squamous Cell; Cell Cycle Proteins; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Drug Stability; Enzyme Inhibitors; G1 Phase; Gene Expression; Head and Neck Neoplasms; Humans; Immunosorbent Techniques; Mice; Neoplasms; Resting Phase, Cell Cycle; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; S-Phase Kinase-Associated Proteins; Tumor Cells, Cultured; Tumor Suppressor Proteins

The active form of vitamin D3, 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] is key mediator of calcium homeostasis and is a component of the complex homeostatic system of the skin. 1,25-(OH)2D3 regulates cellular differentiation and proliferation and has broad potential as an anticancer agent. Oligonucleotide microarrays were used to assess profiles of target gene regulation at several points over a 48 h period by the low calcemic 1,25-(OH)2D3 analog EB1089 in human SCC25 head and neck squamous carcinoma cells. One hundred fifty-two targets were identified, composed of 89 up- and 63 down-regulated genes distributed in multiple profiles of regulation. Results are consistent with EB1089 driving SCC25 cells toward a less malignant phenotype, similar to that of basal keratinocytes. Targets identified control inter- and intra-cellular signaling, G protein-coupled receptor function, intracellular redox balance, cell adhesion, and extracellular matrix composition, cell cycle progression, steroid metabolism, and more than 20 genes modulating immune system function. The data indicate that EB1089 performs three key functions of a cancer chemoprevention agent; it is antiproliferative, it induces cellular differentiation, and has potential genoprotective effects. While no evidence was found for gene-specific differences in efficacy of 1,25-(OH)2D3 and EB1089, gene regulation by 1,25-(OH)2D3 was generally more transient. Treatment of cells with 1,25-(OH)2D3 and the cytochrome P450 inhibitor ketoconazole produced profiles of regulation essentially identical to those observed with EB1089 alone, indicating that the more sustained regulation by EB1089 was due to its resistance to inactivation by induced 24-hydroxylase activity. This suggests that differences in action of the two compounds arise more from their relative sensitivities to metabolism than from differing effects on VDR function.

Topics: Biomarkers, Tumor; Calcitriol; Carcinoma, Squamous Cell; Cell Adhesion; Cell Differentiation; Cell Division; Cholecalciferol; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Immune System; Ketoconazole; Oligonucleotide Array Sequence Analysis; RNA, Messenger; Signal Transduction; Tumor Cells, Cultured

1alpha,25-Dihydroxyvitamin D(3) [1,25(OH)2D3] inhibits growth of cells derived from a variety of tumors in vitro and in vivo. Proliferation in vitro of human SCC25 cells, derived from a primary squamous cell carcinoma (SCC) of the tongue, was blocked by 1,25(OH)2D3 and its analog EB1089. A similar effect was observed with 13-cis retinoic acid (RA), which has been used in chemoprevention of SCC. We identified amphiregulin, a member of the epidermal growth factor family, as a 1,25(OH)2D3 target gene in SCC25 cells. Induction of amphiregulin mRNA by 1,25(OH)2D3 was rapid and sustained over 48 h, and was unaffected by cycloheximide. 1,25(OH)2D3 also induced amphiregulin mRNA in estrogen receptor-positive and -negative human breast cancer cell lines, but not in LNCaP human prostate cancer cells. RAR- or RXR-specific retinoids did not affect amphiregulin mRNA levels in SCC25 cells; however, 13-cis RA partially blocked the response to 1,25(OH)2D3. Amphiregulin partially inhibited growth of SCC25 cells in culture. Our data show that amphiregulin is a 1,25(OH)2D3 target gene, and suggest that its induction may contribute to the growth inhibitory effects of 1,25(OH)2D3.

Topics: Amphiregulin; Antineoplastic Agents; Breast Neoplasms; Calcitriol; Carcinoma, Squamous Cell; Cell Division; Cholecalciferol; Dose-Response Relationship, Drug; EGF Family of Proteins; Gene Expression Regulation, Neoplastic; Glycoproteins; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Isotretinoin; RNA, Messenger; Tumor Cells, Cultured

1alpha,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and its analogues inhibit growth of various types of cancer cells. Although the therapeutic potential of 1,25(OH)(2)D(3) is limited by its tendency to induce hypercalcemia, analogues such as EB1089 are potent inhibitors of cell growth and exhibit reduced calcemic effects. We analyzed the antiproliferative and calcemic effects of EB1089 in tissue culture and animal models of head and neck squamous cell carcinoma (SCC) to investigate its potential as a chemotherapeutic/chemopreventive agent.. The effects of 1,25(OH)(2)D(3) and EB1089 on cell growth and expression of p21(WAF1/CIP1) and p27(KIP1), which encode cyclin-dependent kinase inhibitors, and a novel target, gadd45alpha, a growth-arrest and DNA-damage gene, were monitored in cultured murine AT-84 SCC cells. The effects of these agents on AT-84 cell growth in vitro and on growth of AT-84 tumors in syngeneic C3H mice were monitored; treatment started at the time of tumor implantation (early tumor model) or after 12 days (late tumor model). Weight and serum calcium levels were also monitored in these animals. All P values were two-sided.. Both 1,25(OH)(2)D(3) and EB1089 arrested proliferation of AT-84 cells in G(0)/G(1) phase, inhibited p21(WAF1/CIP1) expression, and induced expression of p27(KIP1) protein. 1,25(OH)(2)D(3) also enhanced the expression of gadd45alpha, apparently by a p53-independent mechanism. There was a statistically significant decrease in tumor growth for 1,25(OH)(2)D(3)-treated mice (P<.001 for early tumor model) and EB1089-treated mice (P<.001 and P =.001 for early and late tumor models, respectively). Unlike 1,25(OH)(2)D(3), EB1089 did not induce cachexia or hypercalcemia. The effects of 1,25(OH)(2)D(3) and EB1089 on expression of p21(WAF1/CIP1) and GADD45alpha were similar in tumors and in vitro.. EB1089 completely inhibited growth of AT-84 SCC cells at nanomolar concentrations, reduced tumor growth, and did not have calcemic effects. Our results support continued investigation of EB1089 as a chemopreventive/chemotherapeutic agent for head and neck SCC.

Topics: Animals; Antineoplastic Agents; Blotting, Northern; Blotting, Western; Calcitriol; Carcinoma, Squamous Cell; Cell Cycle; Cell Cycle Proteins; Cell Division; Cell Nucleus; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Cytoplasm; DNA Damage; GADD45 Proteins; Genes, p53; Head and Neck Neoplasms; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; Male; Mice; Mice, Inbred C3H; Microtubule-Associated Proteins; Neoplasm Transplantation; Precipitin Tests; Proteins; RNA; Time Factors; Tumor Cells, Cultured; Tumor Suppressor Proteins

Analogs of 1alpha,25-dihydroxyvitamin D(3) (1alpha, 25(OH)2D3) inhibit growth in vitro and in vivo of cells derived from a variety of tumors. Here, we examined the effects of 1alpha,25(OH)2D3 and its analog EB1089 on proliferation and target gene regulation of human head and neck squamous cell carcinoma (SCC) lines SCC4, SCC9, SCC15, and SCC25. A range of sensitivities to 1alpha,25(OH)2D3 and EB1089 was observed, from complete G0/G1 arrest of SCC25 cells to only 50% inhibition of SCC9 cell growth. All lines expressed similar levels of vitamin D3 receptor (VDR) mRNA and protein, and no significant variation was observed in 1alpha,25(OH)2D3-dependent induction of the endogenous 24-hydroxylase gene, or of a transiently transfected 1alpha,25(OH)2D3-sensitive reporter gene. The antiproliferative effects of 1alpha,25(OH)2D3 and EB1089 in SCC25 cells were analyzed by screening more than 4,500 genes on two cDNA microarrays, yielding 38 up-regulated targets, including adhesion molecules, growth factors, kinases, and transcription factors. Genes encoding factors implicated in cell cycle regulation were induced, including the growth arrest and DNA damage gene, gadd45alpha, and the serum- and glucocorticoid-inducible kinase gene, sgk. Induction of GADD45alpha protein in EB1089-treated cells was confirmed by Western blotting. Moreover, while expression of proliferating cell nuclear antigen (PCNA) was reduced in EB1089-treated cells, coimmunoprecipitation studies revealed increased association between GADD45alpha and PCNA in treated cells, consistent with the capacity of GADD45alpha to stimulate DNA repair. While 1alpha,25(OH)2D3 and EB1089 modestly induced transcripts encoding the cyclin-dependent kinase inhibitor p21(waf1/cip1), no changes in protein levels were observed, indicating that p21(waf1/cip1) induction does not contribute to the antiproliferative effects of 1alpha,25(OH)2D3 and EB1089 in SCC cells. Finally, in partially resistant SCC9 cells, there was extensive loss of target gene regulation (10 of 10 genes tested), indicating that resistance arises from widespread loss of 1alpha,25(OH)2D3-dependent gene regulation in the presence of normal levels of functional VDRs.

Topics: Blotting, Northern; Calcitriol; Carcinoma, Squamous Cell; Cell Cycle Proteins; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cyclins; Drug Resistance; GADD45 Proteins; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Intracellular Signaling Peptides and Proteins; Oligonucleotide Array Sequence Analysis; Proliferating Cell Nuclear Antigen; Proteins; Reverse Transcriptase Polymerase Chain Reaction; Transfection; Tumor Cells, Cultured; Tumor Suppressor Proteins

EB1089, an analogue of 1,25 dihydroxyvitamin D with low calcemic activity is a potent inhibitor of parathyroid hormone-related peptide (PTHRP) production in vitro. The purpose of the present study was to determine whether EB1089 could reverse established hypercalcemia in BALB C nude mice implanted s.c. with a human epithelial cancer previously shown to produce high levels of PTHRP in vitro. Total plasma calcium was monitored before and after tumor development and increased steadily when the tumor reached > or =0.5 cm3. When total calcium was 22.85 mmol/liter, animals were treated with a constant infusion of EB1089 or vehicle alone for a period of 2 weeks. A significant and sustained reduction of plasma calcium from 3.2+/-0.1 to 2.7+/-0.08 (P < 0.01) mmol/liter was observed during infusion with EB1089. In contrast, calcium levels in vehicle-treated animals continued to rise during the infusion period. Tumor growth velocity also slowed significantly after the administration of EB1089 as compared with vehicle-treated animals. Plasma PTHRP levels measured at the end of the 2 weeks' infusion period were significantly lower in animals treated with EB1089 as compared with animals treated with vehicle alone (44+/-8 pg/ml versus 194+/-35 pg/ml, P < 0.001). These results, therefore, demonstrate that EB1089 can reverse established hypercalcemia in a human model of squamous cancer.

Topics: Animals; Antineoplastic Agents; Calcitriol; Carcinoma, Squamous Cell; Cell Division; Cell Line, Transformed; Cell Transformation, Viral; Drug Screening Assays, Antitumor; Genes, ras; Humans; Hypercalcemia; Keratinocytes; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Proteins; Neoplasm Transplantation; Papillomaviridae; Parathyroid Hormone-Related Protein; Peptide Fragments; Skin Neoplasms

Humoral hypercalcemia of malignancy, a frequent complication of squamous cell carcinomas of the lung, is mediated by the parathyroid hormone-related peptide (PTHrP). This study was undertaken to determine whether 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and two nonhypercalcemic analogues. EB1089 and 22-oxa-1,25(OH)(2)D(3) (OCT), suppress PTHrP gene expression in a human lung squamous cancer cell line, NCI H520. All three compounds (1) decreased steady-state PTHrP mRNA and secreted peptide levels via a transcriptional mechanism; (2) modulated promoter activity of 1,25(OH)(2)D(3)-responsive DNA sequences; and (3) activated the vitamin D receptor (VDR) both in vitro and in vivo. Thus, EB1089 and OCT inhibit PTHrP gene expression in NCI H520 cells and modulate gene expression through the same mechanism as 1,25(OH)(2)D(3), namely, activation of the VDR. 1,25(OH)(2)D(3) is hypercalcemic in vivo. However, the noncalcemic analogues EB1089 and OCT have a therapeutic potential through suppression of PTHrP gene transcription.

Topics: Antineoplastic Agents; Calcitriol; Carcinoma, Squamous Cell; Gene Expression; Humans; Hypercalcemia; Lung Neoplasms; Parathyroid Hormone-Related Protein; Proteins; Receptors, Calcitriol; RNA, Messenger; Transcription, Genetic; Tumor Cells, Cultured

We have examined the in vitro effects of 1,25 dihydroxy-vitamin D3 [1,25(OH)2D3] and of two side-chain modified analogs of 1,25(OH)2D3 (EB1089 and MC903) on cell growth and parathyroid hormone related peptide (PTHRP) production in immortalized (HPK1A) and neoplastic (HPK1A-ras) keratinocytes. Cell proliferation was strongly inhibited by 1,25(OH)2D3 and its analogs in HPK1A cells, and in this system EB1089 was 10-100 times more potent than 1,25(OH)2D3 or MC903. A similar effect on cell proliferation was observed in HPK1A-ras cells; however, 10-fold higher concentrations of 1,25(OH)2D3 or its analogs were required. We also observed a strong and dose-dependent inhibitory effect of these compounds on PTHRP secretion and gene expression. In both immortalized and neoplastic keratinocytes, EB1089 was 10-100 times more potent than 1,25(OH)2D3 or MC903 on inhibiting PTHRP production. However, although effective in HPK1A-ras cells, 10-fold higher concentrations of 1,25(OH)2D3 or its analogs were required to produce similar actions in this neoplastic model. These studies therefore demonstrate that a 1,25(OH)2D3 analog with low calcemic potency in vivo (EB1089) can inhibit keratinocyte proliferation and PTHRP production by such cells with greater potency than 1,25(OH)2D3. The observed effects of such analogs in neoplastic keratinocytes predicts their potential usefulness in vivo in inhibiting squamous cancer growth and its associated hypercalcemia.

Topics: Animals; Antineoplastic Agents; Calcitriol; Carcinoma, Squamous Cell; Cell Division; Cell Line, Transformed; Cell Transformation, Viral; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Gene Expression Regulation, Neoplastic; Humans; Hypercalcemia; Keratinocytes; Mice; Mice, Nude; Neoplasm Proteins; Neoplasm Transplantation; Parathyroid Hormone-Related Protein; Protein Biosynthesis; Skin Neoplasms; Tumor Cells, Cultured