© 2001 by Oxford University Press
Journal of the National Cancer Institute, Vol. 93, No. 10, 745-753,
May 16, 2001
© 2001 Oxford University Press
Action of Low Calcemic 1
,25-Dihydroxyvitamin D3 Analogue EB1089 in Head and Neck Squamous Cell Carcinoma
Affiliations of authors: J. Prudencio, M. J. Black, Department of Otolaryngology–Head and Neck Surgery, Jewish General Hospital, Montreal, PQ, Canada; N. Akutsu, Y. Bastien, R. Lin, Department of Physiology, McGill University, Montreal; N. Benlimame, T. Wang, Montreal Centre for Experimental Therapeutics in Cancer and Lady Davis Institute for Medical Research, Montreal; M. A. Alaoui-Jamali, Department of Medicine, McGill University, Lady Davis Institute for Medical Research, and Montreal Centre for Experimental Therapeutics in Cancer; J. H. White, Departments of Physiology and Medicine, McGill University, and Montreal Centre for Experimental Therapeutics in Cancer.
Correspondence to: John H. White, Ph.D., Department of Physiology, McIntyre Medical Sciences Bldg., McGill University, 3655 Drummond St., Montreal, PQ, H3G 1Y6, Canada (e-mail: jwhite{at}med.mcgill.ca).
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ABSTRACT |
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 TopNotes Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Background: 1
,25-Dihydroxyvitamin D3 [1,25(OH)2D3] and its analogues inhibit growth of various types of cancer cells. Although the therapeutic potential of 1,25(OH)2D3 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)2D3 and EB1089 on cell growth and expression of p21WAF1/CIP1 and p27KIP1, which encode cyclin-dependent kinase inhibitors, and a novel target, gadd45, 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)2D3 and EB1089 arrested proliferation of AT-84 cells in G0/G1 phase, inhibited p21WAF1/CIP1 expression, and induced expression of p27KIP1 protein. 1,25(OH)2D3 also enhanced the expression of gadd45, apparently by a p53-independent mechanism. There was a statistically significant decrease in tumor growth for 1,25(OH)2D3-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)2D3, EB1089 did not induce cachexia or hypercalcemia. The effects of 1,25(OH)2D3 and EB1089 on expression of p21WAF1/CIP1 and GADD45 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.
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INTRODUCTION |
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TopNotesAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Head and neck cancer represents a substantial burden on society. An estimated 42 800 cases of head and neck squamous cell carcinoma (SCC) occurred in the United States in 1992, with 11 600 deaths (1). Worldwide, more than 500 000 cases are predicted annually (1). While early-stage SCC of the head and neck can be treated successfully with surgery and/or radiation therapy, there remains a 3%–7% annual rate of development of second primary carcinomas (1). Tobacco exposure and alcohol consumption are the primary risk factors predisposing to both initial SCC and second primary carcinoma. Carcinogenesis is a multistep process, involving a continuum of genetic and cellular changes leading from premalignancy to invasive carcinoma (2). The high rate of second primary carcinoma has prompted a search for chemopreventive agents, defined as drugs that suppress the development of invasive cancer by inhibiting or reversing the genetic and phenotypic changes accompanying carcinogenesis (3–5).
Retinoids (vitamin A metabolites), the most studied class of chemopreventive agents to date, inhibit growth and stimulate differentiation of SCC (6–13). The primary effectors of retinoid signaling are members of the retinoic acid receptor (RARs , 
, and 
) and retinoid X receptor (RXRs , , and ) families. RARs and RXRs are nuclear receptors, which function as heterodimers by binding to specific sequences in target genes known as retinoic acid response elements (14). Retinoids thus alter the proliferation and differentiation of target cells by regulating the expression of specific genes. Clinical trials (7–9) of high doses of 13-cis-retinoic acid (13-cis-RA), i.e., isotretinoin, provided evidence for a statistically significant reduction in the incidence of second primary carcinomas in treated patients as compared with control subjects. This effect diminished upon discontinuation of treatment when all sites were considered, but not in the upper aerodigestive tract alone (8). Furthermore, treatment was associated with clinically significant side effects, including cheilitis, dermatitis, conjunctivitis, and hypertriglyceridemia.
Given the substantial toxicity of vitamin A and its metabolites, it is important to investigate other agents that exhibit antiproliferative activity and that are potentially less toxic. The active form of vitamin D3, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], has been shown to inhibit proliferation of cultured cells derived from a number of tumors (15,16). Analogues of 1,25(OH)2D3 inhibit cell proliferation in in vitro and/or in vivo models of myeloid leukemia and carcinomas of the breast, prostate, and colon (17–28). Signaling by 1,25(OH)2D3, which is very similar to that of retinoids, occurs through binding to specific nuclear vitamin D receptors (VDRs). The VDR functions as a heterodimer with RXRs and binds to specific vitamin D response elements (VDREs) located in the promoter regions of target genes (3). Relatively few 1,25(OH)2D3 target genes have been identified to date. Transcription of the gene encoding the cyclin-dependent kinase inhibitor p21WAF1/CIP1 in myelomonocytic cells was shown to be strongly stimulated by 1,25(OH)2D3, and its forced overexpression induced differentiation (29,30). However, the magnitude of the effect of 1,25(OH)2D3 on p21WAF1/CIP1 expression varies widely in different cell lines (29–32).
The main barrier to the clinical use of 1,25(OH)2D3 has been its hypercalcemic effects. However, more than 800 analogues of 1,25(OH)2D3 have been developed in an attempt to maintain the inhibitory effect on tumor cell proliferation while reducing the potential for hypercalcemia (33–35). One such analogue is EB1089, a derivative with a side chain modified to render it less susceptible to catabolic degradation. Additions include 26- and 27-dimethyl groups, an extra carbon atom at C24 (24a), and double bonds at C-22,23 and C-24 (24a) (34,35). EB1089 was 60 times more potent than 1,25(OH)2D3 in inhibiting growth of MCF-7 breast cancer cells in vitro and 100-fold more potent than 1,25(OH)2D3 in inhibiting tumor growth by 50% in animal models of breast cancer, with only half the hypercalcemic activity (21). EB1089 did not induce clinically significant hypercalcemia at doses of 0.1–1.0 µg/kg body weight per day in xenograft mouse models of human breast or prostate carcinoma (16), whereas clinically significant hypercalcemia with ensuing cachexia and death was observed in mice treated with 2.5 µg/kg per day (24,36).
The potent growth-inhibitory properties and potentially limited toxicity of EB1089 suggest that it may be an attractive candidate for chemotherapy/chemoprevention of head and neck SCC. This hypothesis was investigated in a series of in vitro and animal studies using the murine head and neck SCC line AT-84 (37,38), derived from C3H mice. We compared the growth-inhibitory effects of 1,25(OH)2D3 and EB1089 in vitro and assessed their antitumor and calcemic activities in C3H mice implanted with AT-84 tumors. In addition, we analyzed the regulation of 1,25(OH)2D3 target genes encoding cyclin-dependent kinase inhibitors p21WAF1/CIP1 and p27KIP1 and a novel target, the growth-arrest and DNA-damage gene gadd45, whose action has been associated with growth arrest and enhanced DNA repair (39–41).
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MATERIALS AND METHODS |
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TopNotesAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Nuclear Receptor Ligands and Reagents
1,25(OH)2D3 and EB1089 were supplied by Leo Laboratories (Ballerup, Denmark). 13-cis-RA was purchased from ICN Biomedicals (Aurora, OH). 1,25(OH)2D3, EB1089, and 13-cis-RA were dissolved in dimethyl sulfoxide (DMSO), and stock solutions were stored in the dark at –20 °C. Doxorubicin and cisplatin were obtained from Sigma Chemical Co. (St. Louis, MO) and David Bull Laboratories (Melbourne, Australia), respectively, and were resuspended in distilled water. Cycloheximide was purchased from Sigma Chemical Co. (Oakville, ON, Canada) and resuspended in DMSO.
Tissue Culture
The mouse SCC cell line AT-84 (37,38,42) was provided by Dr. Stephen E. Karp (Massey Cancer Center, Virginia Commonwealth University, Richmond, VA). The cells were cultured in RPMI-1640 medium (Life Technologies, Inc. [GIBCO BRL], Burlington, ON, Canada) plus 10% fetal bovine serum supplemented with 100 µM nonessential amino acids (Life Technologies, Inc.), 1 mM sodium pyruvate (Life Technologies, Inc.), 2 mM glutamine (Life Technologies, Inc.), and 50 U/mL penicillin–streptomycin (Life Technologies, Inc.). For the analysis of the effects of 1,25(OH)2D3, EB1089, and 13-cis-RA on cell growth, cells were seeded in six-well plates at 15 000 cells per well in 2 mL of culture medium containing charcoal-stripped serum for 24 hours. The cells were then propagated in charcoal-stripped medium containing vehicle (DMSO) or ligand at the indicated concentrations. Medium was changed every 2 days, and fresh ligand was added as necessary. On the designated day, the cells were washed with 2 mL of phosphate-buffered saline (PBS) and removed from the plate by incubation with 0.5 mL of 0.5% trypsin–EDTA. Cell numbers were counted with the use of a hemocytometer. All treatment conditions were performed in triplicate wells. For subcutaneous injection, the cells were trypsinized and washed three times in Hanks' balanced salt solution (HBSS) (Sigma Chemical Co., Oakville, ON). Two million cells in 0.2 mL of HBSS were injected subcutaneously.
Cell Cycle Analysis
The cells were plated at a density of 1 million per 75-cm2 flask. Twenty-four hours later, the cells were treated continuously with EB1089 at the indicated concentration for 48 hours. The cells were then collected by trypsin treatment, washed twice in PBS, and resuspended at a density of 2 million cells/mL in a solution of PBS, containing 522 mg/L spermidine trihydrochloride (Sigma Chemical Co., St. Louis), 0.5 mM Tris–HCl (pH 8.0), 35 µg/mL ribonuclease A, and 50 mg/mL propidium iodide. Fluorescence-activated cell sorting (FACS) analysis was performed on an Epics-Profile II flow cytometer (Beckman Coulter, Inc., Fullerton, CA), and the cell cycle distribution was determined with the use of the Multicycle Program (Pheonix Flow Systems Inc., San Diego, CA).
RNA Isolation and Northern Blotting
The cells were grown in 100-mm dishes. When appropriate, media were replaced with charcoal-stripped medium containing ligand at the indicated concentrations. Cycloheximide was added to 200 nM 1 hour before the addition of ligand where indicated. After incubation, total RNA was extracted with TRIZOL (Life Technologies, Inc.) according to the manufacturer's instructions. PolyA+ RNAs were isolated with the use of an Oligotex messenger RNA (mRNA) kit (QIAGEN, Valencia, CA) according to the manufacturer's instructions.
Complementary DNA probes used for northern blotting were generated by reverse transcription–polymerase chain reaction (PCR) amplification of polyA+ RNA from AT-84 cells. RNAs were reverse transcribed by oligo dT priming, and PCR amplification was performed with the use of primer sets: p21WAF1/CIP1, 5'-GCGCGGATCCACAGCGATATCCAGACATTC-3' and 5'-GCGGGGATCCCGTGGGCACTTCAGGG-3'; gadd45, 5'-GGGAGGTACCCGCACTTGCAATATGAC-3' and 5'-ATTTGGTACCGTTATTTCCATTCGGATGCC-3'; -actin, 5'-TGTGATGGTGGGAATGGGTCAG-3' and 5'-TTTGATGTCACGCACGCACGATTTCC-3'. Amplified fragments were subcloned into Bluescript SK+ (Stratagene, Aurora, ON, Canada) and verified by dideoxy sequencing.
For northern blotting, 20 µg of total RNA or 1 µg of polyA+ RNA was separated on a 1.0% agarose gel containing 6.3% formaldehyde, 20 mM 3-(N-morpholino)propanesulfonic acid (pH 7.0), 15 mM sodium acetate, and 1 mM EDTA. Separated RNAs were transferred to a nylon membrane (Hybond-N+; Amersham, Baie d'Urfe, Quebec, Canada). The blotted membrane was soaked in 3x standard saline citrate (SSC) (Sigma Chemical Co., St. Louis) and 0.1% sodium dodecyl sulfate (SDS) at 50 °C and prehybridized at 42 °C in 50 mM phosphate buffer (pH 6.5), 50% formamide, 5x SSC, and 10% Denhardt's solution containing 250 µg/mL sheared and denatured salmon sperm DNA. Hybridization was carried out in the same solution by the addition of 32P-labeled complementary DNA probes. After hybridization, the membrane was washed four times in 2x SSC and 0.2% SDS for 5 minutes and three times in 0.1x SSC and 0.2% SDS for 30 minutes at 50 °C, dried, and autoradiographed.
Immunoprecipitation and Western Blotting
AT-84 cell lysates were prepared essentially as described by Hahn et al. (43). After incubation, cells were washed twice with PBS, harvested by scraping in 1 mL of PBS, and centrifuged at 11 750g for 7 minutes at 4 °C. Pellets were resuspended in 800 µL of ice-cold lysis buffer (10 mM Tris–HCl [pH 7.9], 10 mM KCl, 1 mM dithiothreitol, 1.5 mM MgCl2, and 0.5% Nonidet-P40), and cell lysates were collected by centrifugation at 11 750g for 10 minutes at 4 °C after 15 minutes of incubation on ice. Nuclei were pelleted, washed with Buffer A (10 mM Tris–HCl [pH 7.9], 10 mM KCl, 1 mM dithiothreitol, and 1.5 mM MgCl2), resuspended in ice-cold Buffer B (50 mM Tris–HCl [pH 7.5], 500 mM KCl, 2 mM dithiothreitol, 5 mM MgCl2, 0.1 mM EDTA, 10% sucrose, and 20% glycerol), and agitated for 1 hour at 4 °C to extract the nuclear proteins. Following centrifugation at 11 750g for 3 minutes at 4 °C, the supernatants were dialyzed twice with TM-1 buffer (25 mM Tris–HCl [pH 7.6], 100 mM KCl, 0.5 mM dithiothreitol, 5 mM MgCl2, 0.5 mM EDTA, and 10% glycerol). Protease inhibitor cocktail (Boehringer Mannheim GmbH, Mannheim, Germany) was added to the above buffers immediately before use. Samples (200 µg) were immunoprecipitated at 4 °C overnight with 3 µg of anti-GADD45 (4T-27) antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) with 30 µL of 50% slurry protein G-Sepharose (Amersham). Beads were centrifuged at 735g for 3 minutes at 4 °C, and the pellets were washed four times in each lysis buffer and boiled for 3 minutes in 2x SDS–polyacrylamide gel sample buffer. Proteins were resolved by electrophoresis on a 20% polyacrylamide gel containing SDS, transferred to a nitrocellulose membrane (Hybond-C; Amersham), and probed with anti-GADD45 antibody (H-165) (Santa Cruz Biotechnology, Inc.). Western blot analyses were performed with 30 µg of total cell proteins resolved on a 20% polyacrylamide gel containing SDS. Expression of p21WAF1/CIP1 and of p27KIP1 was analyzed with the use of F-5 and F-8 monoclonal antibodies, respectively (Santa Cruz Biotechnology, Inc.), and p53 was detected with the use of the Ab-3 monoclonal antibody (Oncogene Research Products, Boston, MA). VDR and RXR were detected with the use of C-20 and D-20 polyclonal antibodies, respectively (Santa Cruz Biotechnology, Inc.). Specific proteins were detected with the enhanced chemiluminescence system (NEN Life Science Products, Inc., Boston, MA).
Animal Experiments
Male syngeneic C3H mice, 6–8 weeks old, were obtained from Charles River Laboratories, St. Zotique, PQ, Canada. The mice weighed 20–24 g. All experiments were approved by the Animal Care Committee, McGill University, Montreal, Quebec. The animals were housed in cages of 10–11 and were fed standard rodent chow and water ad libitum. Experiment 1 was designed to evaluate the effect of EB1089 and 1,25(OH)2D3 on early tumors. Thirty mice were inoculated subcutaneously with 2.0 x 106 AT-84 cells in 0.1 mL of HBSS and were randomly assigned to one of three groups, each group containing 10 animals. The groups received 0.1-mL daily intraperitoneal doses as follows: Group 1 received vehicle, group 2 was given 0.25 µg/kg EB1089, and group 3 received 0.25 µg/kg 1,25(OH)2D3. In experiment 2, the mice were inoculated subcutaneously as in experiment 1. The tumors became palpable at approximately 8–10 days and could be reliably measured at 12 days. On day 12, the animals were randomly assigned to one of four groups; each group contained 11 animals. Starting on day 12, the mice received 0.1-mL daily intraperitoneal injections as follows: Group 1 received placebo vehicle, group 2 was given 0.5 µg/kg EB1089, group 3 was given 1.0 mg/kg 13-cis-RA, and group 4 received 1.0 mg/kg 13-cis-RA and 0.5 µg/kg EB1089.
Mouse weights were measured with a digital balance accurate to 0.05 g, and tumor length (L) and width (W) were measured twice weekly with the use of a caliper accurate to 0.5 mm. Tumor volume (V) was calculated with the use of the formula V = L x W x 1/2W. Growth was calculated as the ratio of tumor volume on a given day to initial volume. The animals that lost more than 25% of their starting weight or that showed substantial distress were killed.
After the mice were anesthetized with 0.5 mL of 2.5% Avertin (Sigma Chemical Co., St. Louis) by intraperitoneal injection, intracardial puncture was carried out to obtain 300–500 µL of blood for determinations of serum calcium and albumin levels. Blood was placed in 600-µL heparinized tubes and centrifuged at 4 °C for 10 minutes at 1500g. Blood serum was then separated and stored at -20 °C until analysis. Serum calcium and albumin levels were determined with the use of a spectrophotometric assay per manufacturer's instructions (Vitros Chemistry Products, Ortho-Clinical Diagnostics, Raritan, NJ). At the time that the animals were killed, their tumors were dissected and weighed. Part of the tumor was snap-frozen in liquid nitrogen, and the rest of the tumor was preserved in buffered formalin for future use.
Immunohistochemistry
Immunoperoxidase staining for p21WAF1/CIP1 and GADD45 in formalin-fixed, paraffin-embedded tumor sections was performed by a labeled avidin–biotin method (Vector Laboratories, Inc., Burlingame, CA). Sections of tumors (6 µm) were deparaffinized in toluene and rehydrated through graded alcohol to water. Endogenous peroxidase activity was quenched by incubation in 1.5% hydrogen peroxide for 30 minutes at room temperature. Sections were immersed in 10 mM sodium citrate buffer (pH 6.0) and subjected to heat-induced antigen retrieval. To block binding of endogenous biotin, sections were incubated with an endogenous avidin–biotin blocking kit (Zymed Laboratories, Inc., San Francisco, CA) according to the manufacturer's instructions. To reduce nonspecific protein binding, sections were incubated with 2% goat normal serum (Jackson Laboratories, West Grove, PA) for 1 hour at room temperature. The sections were then incubated with primary antibodies: anti-p21WAF1/CIP1 monoclonal antibody at a dilution of 1 : 100 and anti-GADD45 polyclonal antibody at a dilution of 1 : 200 (Santa Cruz Biotechnology, Inc.) overnight at 4 °C. After being rinsed extensively with PBS, the sections were incubated with the appropriate biotinylated secondary antibodies for 30 minutes at room temperature (Vector Laboratories, Inc.), followed by a 30-minute incubation with the avidin–biotin–horseradish peroxidase complex (Vector Laboratories, Inc.) in PBS and then by final color development with diaminobenzidine substrate kit peroxidase (Vector Laboratories, Inc.). Negative control experiments were performed by omission of the primary antibody. Sections were then lightly counterstained with Harris' hematoxylin (BDH Inc., Toronto, ON, Canada), dehydrated in graded alcohols, and cleared in toluene. Sections were analyzed by conventional light microscopy and photographed with the use of Kodak color slides.
Data Analysis
For in vitro studies, we calculated the relative growth by dividing each of the triplicate cell counts for a given concentration and day by the mean of the counts for the control wells on that day. Regression analysis was used to compare relative growth curves over time and to fit and to compare slopes for individual concentrations between groups. For in vivo studies in animal experiment 1, in which 1,25(OH)2D3 or EB1089 treatment was initiated immediately after AT-84 cell injection, the animal weight and the corrected serum calcium measurements were compared between groups with the use of the nonparametric Kruskal–Wallis test. To analyze tumor growth, we performed an analysis of variance (ANOVA) comparing all groups, with group as an independent variable and volume as a repeated measure as a function of time. This comparison was followed up with comparisons between two groups at a time. The Greenhouse–Geisser correction factor was used to address the heterogeneity of covariances. The analysis was repeated with the use of the log of volume to reduce heterogeneity of variances and to increase the linearity. In animal experiment 2, in which EB1089 or 13-cis-RA treatment was initiated 12 days after the injection of AT-84 cells, we derived a relative growth measurement by dividing the volumes at each time by the initial tumor volume measured. This standardization was done to correct for any differences in tumor growth between groups before treatment was initiated. Statistical analyses were performed as in animal experiment 1. All statistical tests were two-sided and were considered to be significant at P<.05.
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RESULTS |
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Antiproliferative Effects In Vitro of 1,25(OH)2D3 and EB1089 in Murine SCC Line AT-84
The effects of 1,25(OH)2D3 and of its analogue EB1089 on proliferation of the AT-84 line in vitro were determined by incubation of the cells with a range of ligand concentrations over a 10-day period. Both 1,25(OH)2D3 and EB1089 completely inhibited AT-84 cell growth at low nanomolar concentrations (Fig. 1
, A and B). While, at concentrations of 10 nM or higher, there were no statistically significant differences in growth suppression between 1,25(OH)2D3 and EB1089, at 1 nM, EB1089 showed a statistically significantly greater reduction in cell growth (P for difference in slopes was <.001), as shown by regression analysis of the slope of log relative growth versus time. Cell cycle analyses indicated that EB1089-treated cells were arrested in G0/G1 (Table 1). Similar G0/G1 arrest was observed with 1,25(OH)2D3-treated cells, and no evidence was found for apoptosis in cells treated with either ligand (data not shown). These results are consistent with the cytostatic effects of 1,25(OH)2D3 and EB1089 in the human head and neck SCC line SCC25 (our unpublished results) and in cell lines derived from a range of other malignancies (17,18,25,28,30). It is interesting that 13-cis-RA had no effect on AT-84 proliferation under these conditions (Fig. 1, C). This result is noteworthy because ligand-bound VDR and RARs form heterodimers with common RXR partners, and VDR/RXR and RAR/RXR heterodimers recruit common coregulatory proteins (3,14).
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Effects of 1,25(OH)2D3 on Expression of p21WAF1/CIP1, p27KIP1, and gadd45
Genes in AT-84 Cells
We were interested in probing the molecular mechanisms underlying the growth-inhibitory effects of 1,25(OH)2D3 in AT-84 cells. Western blotting studies revealed that the VDR and RXR were present in AT-84 cells (Fig. 2, A). These levels were similar to those detected in the human SCC line SCC25 (data not shown). Northern blot analysis showed that treatment with 1,25(OH)2D3 or with EB1089 induced expression of the endogenous 24-hydroxylase gene (Fig. 2, B), indicating that AT-84 cells express functional receptors. Previous work (29–32) has revealed that the gene encoding the cyclin-dependent kinase inhibitor p21WAF1/CIP1 is a target of 1,25(OH)2D3 signaling, although the magnitude of its induction varies widely among different cell types. Remarkably, we found that 1,25(OH)2D3 decreases the expression of p21WAF1/CIP1 mRNA in AT-84 cells (Fig. 2, C), representing a 50% reduction in p21 WAF1/CIP1 mRNA levels over a 48-hour period when normalized to those of actin (data not shown). Consistent with these findings, treatment with 1,25(OH)2D3 also reduced p21WAF1/CIP1 protein levels (Fig. 2, D). These results are important because they indicate that induction of p21WAF1/CIP1 expression does not contribute to the antiproliferative effects of 1,25(OH)2D3 in AT-84 cells and thus does not play an essential role in 1,25(OH)2D3-dependent growth inhibition in all cell types. Our findings are also in agreement with the observation of a similar inhibitory effect of 1,25(OH)2D3 on p21WAF1/CIP1 expression in another mouse head and neck SCC model (44). Treatment with 1,25(OH)2D3 had no effect on the expression of the p53 protein in AT-84 cells (Fig. 2, D), in agreement with results obtained in SCC25 cells (data not shown). However, consistent with its effects on other cell types (29–32,44), 1,25(OH)2D3 treatment did lead to increased p27KIP1 protein levels (Fig. 2, D).
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Induction or overexpression of the growth-arrest and DNA-damage gene gadd45
has been widely associated with growth arrest (39–41). The effect of 1,25(OH)2D3 on gadd45 expression was analyzed by northern blotting, which revealed a gradual increase in gadd45 mRNA levels over a 48-hour period (Fig. 2
, C). This effect was direct, since treatment with the protein synthesis inhibitor cycloheximide for 48 or 72 hours did not block induction (Fig. 2, C). The enhanced expression of gadd45 mRNA led to increased levels of both cytoplasmic and nuclear GADD45 proteins after 48 hours (Fig. 2, E). This result suggests that 1,25(OH)2D3-dependent growth inhibition is mediated, at least in part, by enhancing the expression of GADD45. The gadd45 gene is also a target of p53, whose activity is induced by DNA-damaging agents (45). Peak levels of GADD45 induced by 1,25(OH)2D3 were similar to, or somewhat less than, those induced by treatment of AT-84 cells with the DNA-damaging agents cisplatin and doxorubicin, respectively (Fig. 2, F). Unlike 1,25(OH)2D3 treatment, both cisplatin and doxorubicin treatments increased the p53 protein levels in AT-84 cells (Fig. 2, F). Doxorubicin treatment also induced expression of p21WAF1/CIP1 (data not shown). The lack of induction of p53 or p21WAF1/CIP1 by 1,25(OH)2D3 suggests that the observed increase in GADD45 expression occurs independently of p53. However, we cannot rule out the possibility that 1,25(OH)2D3 may enhance p53 activity through post-translational mechanisms, leading to a selective effect on GADD45 expression. Effects of EB1089 on AT-84 Tumor Growth and Serum Calcium Levels in C3H Mice
The in vitro experiments presented above suggest that the AT-84 cell line provides an ideal model for testing the antiproliferative effects of 1,25(OH)2D3 and EB1089 in vivo in the syngeneic C3H mouse. Previous experiments (38,42) have shown that AT-84 cells form rapidly growing tumors in C3H mice. We have assessed the effects of 1,25(OH)2D3 and EB1089 on tumor growth in two experimental models conducted in C3H mice inoculated with 2 x 106 AT-84 cells. Experiment 1 represents an early tumor model, in which treatment is initiated on the day of inoculation; in contrast, in experiment 2, treatment was not started until 12 days after inoculation so that tumors could become established and vascularized. We also followed animal weights and corrected serum calcium levels in experiment 1.
In experiment 1, tumor growth was followed over a 29-day period in mice treated daily with vehicle (control), 1,25(OH)2D3 (0.25 µg/kg per day), or EB1089 (0.25 µg/kg per day) (Fig. 3). ANOVA with repeated measures revealed a statistically significant time effect [F(6,19) = 59.895; P<.001] and time-by-group interaction [F(12,40) = 4.594; P<.001]. This analysis was followed by comparisons between two groups at a time with the use of the Greenhouse–Geisser correction factor. Both 1,25(OH)2D3 (F = 16.5; P<.001) and EB1089 (F = 60.051; P<.001) had a statistically significant effect on tumor volume when individually compared with the control, and EB1089 showed a statistically significant greater reduction in tumor volume than 1,25(OH)2D3 (F = 16.177; P<.001). By day 9, the tumor volume in the control group was more than double that in either treatment group. By day 29, the mean volume in the control group had reached 1312 mm3 (95% confidence interval [CI] = 1204 to 1420 mm3), which was twice the mean volume in the 1,25(OH)2D3-treated group (mean volume = 613 mm3; 95% CI = 486 to 740 mm3) and four times the mean volume in the EB1089-treated group (mean volume = 333 mm3; 95% CI = 270 to 396 mm3).
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width). Points represent the mean tumor volume of surviving animals. Statistical significance was determined with the use of repeated measures analysis of variance with Greenhouse–Geisser correction factor for sphericity; all statistical tests were two-sided. Error bars represent 95% confidence intervals.While 1,25(OH)2D3 did result in statistically significant growth inhibition compared with the control, there was toxicity associated with its action. Four animals in this group had to be killed because of cachexia. Of the remaining animals at the completion of the experiment, the mean weights were 24.1 g, 17.0 g, and 21.6 g for the control, 1,25(OH)2D3-treated, and EB1089-treated groups, respectively (P<.001, Kruskal–Wallis test). Corrected serum calcium levels (Fig. 4
) revealed statistically significant hypercalcemia in the 1,25(OH)2D3-treated group (P = .01, Kruskal–Wallis test), with a mean corrected serum calcium level of 3.35 mmol/L. However, there was no detectable hypercalcemia in the EB1089-treated group, with a mean corrected serum calcium level of 2.45 mmol/L, which is nearly identical to that in the control group (mean corrected serum calcium of 2.41 mmol/L).
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In experiment 2, we also analyzed the effects of 13-cis-RA on AT-84 tumor growth to see if they were consistent with the lack of antiproliferative activity in vitro. Mice were randomly assigned to either treatment or control groups after 12 days of tumor growth. To correct for the differences in the mean tumor volume at this time, we present the results as growth relative to the initial measurement after randomization; the results are shown in Fig. 5
. A repeated measures ANOVA showed a statistically significant time effect [F(5,47) = 250.342; P<.001] and time-by-group interaction [F(15,147) = 4.364; P<.001]. This analysis was followed by comparisons between two groups at a time with the use of the Greenhouse–Geisser correction factor. The EB1089-treated group (F = 7.419; P = .001) and the group treated with EB1089 plus 13-cis-RA (F = 6.082; P = .003) showed a statistically significant reduction in tumor growth as compared with the control group. 13-cis-RA alone did not produce a statistically significant reduction in tumor growth (F = 1.767; P = .179). No statistically significant differences were found between treatment groups. After 18 days of treatment, the tumor volume in the EB1089-treated group had increased 10.1-fold, whereas the tumor volume increased 25.2-fold in the controls. Strikingly, similarly reduced tumor growth (9.8-fold) was observed in the group treated with 13-cis-RA (1 mg/kg per day), in spite of our observations that AT-84 cells are resistant to 13-cis-RA in vitro (Fig. 1, C). The combination of EB1089 and 13-cis-RA was more effective than EB1089 alone, with only a 6.3-fold increase in tumor volume. However, these differences failed to reach statistical significance. It is possible that a longer observation period may reveal statistically significant differences among treatment groups; however, large tumor volumes in the control group precluded continuation of the experiment.
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Effects of EB1089 and 1,25(OH)2D3 on GADD45
and p21WAF1/CIP1 Expression in Tumors
Northern and western blot analyses showed that 1,25(OH)2D3 and EB1089 directly stimulated expression of GADD45 and inhibited expression of p21WAF1/CIP1 in AT-84 cells in vitro (Fig. 2). However, it is important to determine if the molecular events underlying the action of 1,25(OH)2D3 and EB1089 in vivo are consistent with molecular analyses of their function in vitro. Expression levels of GADD45 and p21WAF1/CIP1 in vivo were determined by immunohistochemical analyses of tumor sections from controls and animals treated with 1,25(OH)2D3 or EB1089 for 29 days. These experiments revealed that GADD45 levels were elevated in tumors from 1,25(OH)2D3- or EB1089-treated animals relative to controls, whereas levels of p21WAF1/CIP1 were decreased (Fig. 6), consistent with in vitro studies. Taken together, these results suggest that induction of GADD45 expression, but not of p21WAF1/CIP1, contributes to the antiproliferative effects of 1,25(OH)2D3 and EB1089 both in vitro and in vivo.
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DISCUSSION |
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TopNotesAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have been interested in both investigating the therapeutic potential of 1,25(OH)2D3 analogues for treatment of head and neck SCC and understanding their underlying mechanisms of action. Early-stage primary SCCs of the head and neck (stages I and II) can be successfully treated in 60%–80% of patients. In these patients, death is more likely to be due to subsequent primary carcinomas than to recurrence of the initial lesion. Patients with more advanced disease have a poorer prognosis (5,46,47). These observations highlight the need for identification of chemopreventive and chemotherapeutic agents as alternatives and/or adjuncts to the established treatment modalities. Retinoids have been well studied to this end; however, their main limitation remains the substantial toxicity associated with their use and consequent poor patient compliance. In addition, their effectiveness at preventing second primary carcinoma is reversible upon discontinuation of treatment (7,8). While 1,25(OH)2D3 also has associated toxicity due to its physiologic effect on calcium metabolism, analogues have been developed that have potent antiproliferative and differentiating effects but reduced calcemic activity. This study shows that one such analogue, EB1089, is able to inhibit growth of AT-84 murine squamous cells in vitro and can suppress AT-84 tumor growth in vivo without inducing hypercalcemia.
EB1089 is one of the most widely studied analogues of 1,25(OH)2D3, with demonstrated efficacy on a wide variety of cancer cells. Our results showed that EB1089 completely inhibited growth in vitro of the murine head and neck SCC line AT-84 at nanomolar concentrations, inducing cell cycle arrest in G0/G1. Under the same conditions, 13-cis-RA did not statistically significantly affect proliferation of these cells. While the antiproliferative effects of 1,25(OH)2D3 and its analogues are well established, the target genes mediating their effects have not yet been extensively characterized. Several investigators (48–53) have shown that p21WAF1/CIP1 expression is stimulated to varying degrees during growth inhibition in cells treated with 1,25(OH)2D3 or EB1089. Remarkably, we found that p21WAF1/CIP1 mRNA and protein levels decreased under growth-inhibitory conditions in AT-84 cells. Moreover, decreased p21WAF1/CIP1 protein levels relative to controls were observed in vivo in tumor sections from both 1,25(OH)2D3- and EB1089-treated animals. Taken together, these results show that the antiproliferative signals induced by 1,25(OH)2D3 are cell specific. While induction of p21WAF1/CIP1 expression may be an important component of 1,25(OH)2D3 signaling in some cells, our results indicate that it is not essential for 1,25(OH)2D3 or its analogues to act as potent growth inhibitors.
Conversely, we found that both gadd45 mRNA and protein were induced in 1,25(OH)2D3- and EB1089-treated AT-84 cells in vitro and that GADD45 protein levels were elevated in vivo in sections of tumors from 1,25(OH)2D3- or EB1089-treated animals. The induction of GADD45 by 1,25(OH)2D3 was comparable to that seen in AT-84 cells treated with cisplatin and doxorubucin, agents that induce GADD45 by a p53-dependent mechanism. Expression of gadd45 mRNA and protein was also enhanced in 1,25(OH)2D3- and EB1089-treated human SCC25 cells in vitro (data not shown). The antiproliferative effects of gadd45 overexpression are well established (39–41). Experiments (41) have shown that gadd45 expression is rapidly induced in breast cancer cells overexpressing BRCA1. In addition, gene ablation studies in mice (54) have highlighted the role of gadd45 in maintenance of genomic stability. This finding suggests that 1,25(OH)2D3 analogues may act to prevent genomic instability associated with cancer progression, as well as to block cell proliferation, supporting the notion that long-term treatment with analogues would provide a chemopreventive effect.
Inhibition of AT-84 cell proliferation was studied in vivo with the use of both early and late tumor models to test whether EB1089 inhibits the growth of newly injected cells while tumor burden is low, as well as established and vascularized tumors. Tumors in EB1089-treated animals were 25%, on average, of the volume of controls in the early tumor model after 29 days and 40%, on average, of the volume of controls in the late tumor model after 18 days. There was no discernible difference in the effects of EB1089 on the two models, since tumors in treated animals were approximately 40% the volume of controls in the early model after 18 days (Fig. 3). These results are comparable to those obtained by others in breast and prostate cell lines (17,21,23,24,27). In addition, there was no statistically significant hypercalcemia or cachexia associated with the use of EB1089 at doses of 0.25 µg/kg per day. In contrast, 0.25 µg/kg per day of 1,25(OH)2D3 resulted in the premature death of four of 10 mice and in a statistically significant weight loss in the remaining animals (P = .001, Kruskal–Wallis test).
It is noteworthy that 13-cis-RA suppressed growth of tumors in vivo despite resistance of AT-84 cells to its antiproliferative effects in vitro. This result suggests that 13-cis-RA may act on the tumor environment rather than on the AT-84 cells themselves. For example, Lingen et al. (55) have provided evidence that 13-cis-RA may act in an indirect fashion by inhibiting angiogenesis. It will be of interest to study the combined effects of other agents and EB1089 in the AT-84/C3H model. Other studies using EB1089 in combination with agents such as retinoids, cisplatin, paclitaxel, and tamoxifen (56–59) have provided evidence for combined effects.
While a full understanding of the underlying mechanisms of EB1089 action remains to be achieved, it is clearly a promising prospect as a single agent or in combination with retinoids for the treatment of head and neck SCC. A phase I trial using EB1089 in patients with advanced breast and colon cancers has been published (60). Maximum tolerated doses of 7 µg/m2 (0.2 µg/kg) were estimated for prolonged treatment. In view of the mounting in vitro and in vivo evidence for the efficacy of EB1089 and the fact that there is a clear lack of alternatives for head and neck cancer patients who have failed to respond to standard treatments, a phase II trial for head and neck cancer patients represents a logical next step.
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NOTES |
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Supported by a grant MT-15160 from the Canadian Institutes of Health Research to J. H. White and in part by a grant from the National Cancer Institute of Canada (#010254) to M. A. Alaoui-Jamali and by funds from the Department of Otolaryngology of the Jewish General Hospital, Montreal. N. Akutsu is supported by a postdoctoral fellowship from the Royal Victoria Research Institute. J. H.White and M. A. Alaoui-Jamali are chercheurs-boursier of the Fonds de Recherche en Santé du Québec.
We thank Dr. Stephen Karp (Virginia Commonwealth University, Richmond) for the AT-84 cells, Drs. Lise Binderup and Karin Hamberg (Leo Laboratories, Ballerup, Denmark) for 1,25(OH)2D3 and EB1089, and Dr. James Hanley (Department of Epidemiology and Biostatistics, McGill University, Montreal, PQ, Canada) for help with statistical analysis.
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