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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.

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.

Background: 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., Methods: 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., Results: 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., Conclusions: 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.

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., (Copyright 2001 Academic Press.)