Inhibition of lung carcinogenesis by 1α,25-dihydroxyvitamin D3 and 9-cis retinoic acid in the A/J mouse model: Evidence of retinoid mitigation of vitamin D toxicity†
Article first published online: 4 JAN 2007
Copyright © 2006 Wiley-Liss, Inc.
International Journal of Cancer
Volume 120, Issue 7, pages 1402–1409, 1 April 2007
How to Cite
Mernitz, H., Smith, D. E., Wood, R. J., Russell, R. M. and Wang, X.-D. (2007), Inhibition of lung carcinogenesis by 1α,25-dihydroxyvitamin D3 and 9-cis retinoic acid in the A/J mouse model: Evidence of retinoid mitigation of vitamin D toxicity . Int. J. Cancer, 120: 1402–1409. doi: 10.1002/ijc.22462
Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the U.S. Department of Agriculture.
- Issue published online: 30 JAN 2007
- Article first published online: 4 JAN 2007
- Manuscript Accepted: 13 OCT 2006
- Manuscript Received: 14 APR 2006
- NIH. Grant Number: R01CA104932
- U.S. Department of Agriculture. Grant Number: 58-1950-51000-064
- lung cancer;
- 9-cis retinoic acid;
- 1α,25-dihydroxyvitamin D3
9-cis-Retinoic acid (9cRA) and 1α,25-dihydroxyvitamin D3 (1,25D) show promise as potential chemopreventive agents. We examined 9cRA and 1,25D, alone and in combination, for their potential to inhibit carcinogen (NNK)-induced lung carcinogenesis in A/J mice. A/J mice (n = 14/group) were treated with 9cRA (7.5, 15, or 30 mg/kg diet), 1,25D (2.5 or 5.0 μg/kg diet), or a combination of 9cRA (15 mg/kg diet) plus 1,25D (2.5 μg/kg diet) for 3 weeks before and 17 weeks after carcinogen injection. Lung tumor incidence, tumor multiplicity, plasma 1,25D levels and kidney expression of vitamin D 24-hydroxylase (CYP24) were determined. Compared to carcinogen-injected controls, mice receiving 9cRA supplementation had significantly lower tumor multiplicity at all doses (decreased 68–85%), with body weight loss at the higher doses of 9cRA. Mice receiving 1,25D supplementation had significantly lower tumor incidence (decreased 36 and 82%) and tumor multiplicity (decreased 85 and 98%), but experienced significant body weight loss, kidney calcium deposition, elevated kidney CYP24 expression and decreased fasting plasma 1,25D levels. Although, there was no apparent influence on chemopreventive efficacy, addition of 9cRA to 1,25D treatment effectively prevented the weight loss and kidney calcification associated with 1,25D treatment alone. These data demonstrate that 9cRA and 1,25D, alone or combined, can inhibit lung tumor promotion in the A/J mouse model. Combining 1,25D with 9cRA has the potential to mitigate the toxicity of 1,25D, while preserving the significant effect of 1,25D treatment against lung carcinogenesis. The underlying mechanism behind this effect does not appear to be related to retinoid modulation of vitamin D catabolism. © 2006 Wiley-Liss, Inc.
Chemoprevention by diet or drug intervention may offer a realistic and practical means of modifying the risk of lung cancer, the leading cause of cancer deaths in both men and women in the United States.1 Two classes of chemopreventive compounds that have been considered are the retinoids and the active form of vitamin D, 1α,25-dihydroxyvitamin D3 (1,25D, also calcitriol).
Retinoids are required for proper differentiation of lung and upper airway epithelium,2 and both 9-cis retinoic acid (9cRA, also 9-cis tretinoin) and all-trans retinoic acid have been shown to inhibit cell growth and proliferation in lung cancer cell lines.3 Studies in animal models of lung cancer suggest that retinoids may be effective in cancer chemoprevention in vivo, but results have varied with species, dose and timing of treatment in relation to induction of carcinogenesis.4, 5, 6 In human lung cancer, epidemiological evidence supports a role for vitamin A-rich foods and supplements in protection against the development of lung cancer in smokers and nonsmokers alike7; however, clinical trials assessing the efficacy of retinoid supplementation against lung cancer have yielded inconsistent and inconclusive results.8, 9, 10, 11 Difficulties in human trials have included differential responses between smokers, former smokers, and never smokers, and treatment noncompliance, possibly related to the toxic effects of high-dose retinoids.
Retinoids regulate biological activity through two types of nuclear receptors: retinoic acid receptors (RARs) and retinoid X receptors (RXRs).12 While much research has focused on RARβ as a tumor suppressor and has utilized ligands specific for the RARs (such as all-trans retinoic acid), fewer studies have investigated ligands that also bind RXRs, including 9cRA. A study showing that 3 months of daily oral 9cRA (100 mg), but not 13cRA (1 mg/kg) plus α-tocopherol (1,200 IU), restored RARβ expression in the bronchial epithelium of former smokers suggests that the ability to bind both types of retinoid receptors may instill 9cRA with benefits above and beyond RAR-selective ligands, and that the beneficial effects seen in cell studies may be relevant to the human population.13 We have recently shown that treatment with 9cRA (15 mg/kg diet) induced RARβ expression and decreased lung tumor multiplicity in the A/J mouse model of lung cancer.14
Epidemiological and ecological studies have shown correlations between low sunlight exposure or low serum vitamin D and an increased risk of cancer, suggesting a role for vitamin D in the prevention of many types of cancer.15, 16, 17 Further, target genes regulated by vitamin D and its deltanoid analogs have been identified using oligonucleotide microarrays and include genes that control inter- and intracellular signaling, cell adhesion, extracellular matrix composition, cell cycle progression and immune system function.18, 19 This suggests multiple potential roles for vitamin D in cancer chemoprevention. It may act as an antiproliferative agent and inducer of cellular differentiation, and it may exert genoprotective effects. 1,25D exerts these effects by binding to the nuclear vitamin D receptor (VDR) and modulating transcription of target genes involved in cellular growth and differentiation.20 Recently, researchers demonstrated that 1,25D inhibited the metastatic growth of VDR-positive Lewis lung carcinoma cells in vivo21; others associated the combination of surgery performed during the summer months and high vitamin D intake with improved survival in early stage nonsmall cell lung cancer patients.22 This suggests that lung cancer, in addition to cancers of the colon and prostate, may be susceptible to the differentiating effects of 1,25D.
Retinoid receptors and the vitamin D receptor belong to the same subfamily of nuclear receptors. They are localized predominantly in the nucleus in the absence of ligand, and conformational changes upon ligand binding appear to be a key step in their activation pathway. A protein–protein interaction between the VDR and the RXR is required for binding to vitamin D response elements in the DNA of target genes,23 allowing for the possibility of functional interactions between 1,25D and retinoids. Combinations of retinoids and 1,25D or vitamin D analogs have been shown to induce additive or synergistic growth inhibition in many cell lines, including lung cancer cells.24, 25, 26, 27, 28, 29, 30, 31, 32 However, multiple forms of vitamin A have also been shown to exert antagonistic effects on certain vitamin D actions, including serum calcium elevation and bone resorption.33, 34, 35, 36 The combinatorial effects of 1,25D and 9cRA against lung carcinogenesis in vivo have not been investigated.
In this preliminary investigation, we tested 1,25D and 9cRA for their ability to prevent lung tumor development in an animal model of carcinogen-initiated cancer. We further tested the combination of 1,25D and 9cRA to determine if these compounds have additive, synergistic, or antagonistic effects against lung tumor development in vivo.
Material and methods
9-cis Retinoic acid (9cRA) (Midwest Research Institute, Kansas City, MO) was obtained in powder form and mixed directly into AIN-93M powdered semi-purified diet (Dyets, Bethlehem, PA)37 at concentrations of 7.5, 15, and 30 mg/kg diet (∼1.5, 3.0, and 6.0 mg/kg body weight/day). The lowest dose corresponds to an amount previously demonstrated to reverse the loss of RARβ in the bronchial epithelium of former smokers (100 mg/day ≈ 1.4 mg/kg body weight/day),13 while the higher doses bracket the amount recommended for phase II clinical trials in adults with solid tumors (100 mg/m2/day ≈ 4.4 mg/kg body weight/day).8 1α,25-dihydroxyvitamin D3 (LKT Laboratories, St. Paul, MN) was dissolved in a small volume of ethanol and added directly to the diet at concentrations of 2.5 and 5.0 μg/kg diet (∼0.5 and 1.0 μg/kg body weight/day). These doses were extrapolated from a study of 1,25D in a rat model of carcinogen-induced mammary cancer38 and adjusted based on pilot data from our lab. They are within the dose range given orally in a Phase I study in human patients with androgen-independent prostate cancer, where a dose of 0.5 μg/kg body weight/day was recommended for Phase II studies.39 The combination of 1,25D at a dose of 2.5 μg/kg diet with 9cRA provided at a dose of 15 mg/kg diet was also tested. Diet preparation involving 9cRA was performed under red light to prevent degradation of the retinoid. All diets were prepared in 2 kg batches and stored in 500 g airtight bags under nitrogen blanket at 4°C. Once opened, bags were used to feed animals for 1 week, giving animals fresh portions daily and storing the remainder at 4°C under nitrogen blanket.
Thorough mixing of supplement(s) to diet was confirmed by HPLC analysis of 9cRA in separate aliquots of the mixed diet. Briefly, 100 mg of diet samples were extracted twice using 3 ml of hexane–butyl methyl ether (1:1 v/v). The combined extract was dried by N2 under red light and dissolved in 100 μl of ethanol and analyzed using a gradient reverse phase HPLC system as described previously, with minor modifications.40 The HPLC system consisted of a Waters 2695 Separations Module (Waters Chromatography Division/Millipore Corporation, Medford, MA) and a Waters 2996 photodiode array detector. 9cRA was analyzed on a reverse phase C18 column (3 μm, 4.6 × 85 mm2, Perkin Elmer, CA) with a flow rate of 1 ml/min. The gradient procedure was as follows: (i) 100% solvent A (acetonitrile/tetrahydrofuran/1% ammonium acetate, 50:20:30, v/v/v) was used for 4 min, followed by a 6-min linear gradient to 50% solvent A/50% solvent B (acetonitrile/tetrahydrofuran/1% ammonium acetate, 50:44:6, v/v/v); (ii) a 9-min hold followed by a 2-min linear gradient to 100% solvent B and then 100% solvent B for 10 min. HPLC was performed on a 9cRA reference standard, extracts from the base diet with no added 9cRA, and extracts from several batches of diet mixed with 9cRA, and the 9cRA peak was identified by co-elution with the standard, matching for retention time and spectral properties (λmax = 340 nm).
Animal model and experimental design
The A/J strain of mouse is an established animal model for chemoprevention studies.41, 42 Previous studies, including ours, have shown that a single intraperitoneal injection of 2 mg or 100 mg/kg body weight of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific carcinogen, is sufficient to induce tumors in 100% of A/J mice at a level of 10–12 lung adenomas per mouse at 16 weeks post-injection.14, 43 Male A/J mice (6 weeks old) obtained from Jackson Labs (Bar Harbor, ME) were randomly assigned to 1 of 9 experimental groups (n = 14/group). Each group was fed an AIN-93M powdered semipurified diet (Dyets, Bethlehem, PA) for 14 days, and then begun on study diets as described below:
- 1No agents plus i.p. injection of 100 mg normal saline/kg body weight
- 2No agents plus i.p. injection of 100 mg NNK/kg body weight
- 39cRA (7.5 mg/kg diet) plus i.p. injection of 100 mg NNK/kg body weight
- 49cRA (15 mg/kg diet) plus i.p. injection of 100 mg NNK/kg body weight
- 59cRA (30 mg/kg diet) plus i.p. injection of 100 mg NNK/kg body weight
- 61,25D (2.5 μg/kg diet) plus i.p. injection of 100 mg NNK/kg body weight
- 71,25D (5.0 μg/kg diet) plus i.p. injection of 100 mg NNK/kg body weight
- 81,25D (2.5 μg/kg diet) plus 9cRA (15 mg/kg diet) plus i.p. injection of 100 mg NNK/kg body weight
- 9No agents plus i.p. injection of 100 mg NNK/kg body weight and weight-matched to group 7
NNK (Toronto Research Chemicals, Ontario, Canada) injections were administered after 3 weeks of exposure to study diets. Animals were maintained on study diets for 17 weeks post-injection with daily feeding and observation and weekly weighing. Mice were fasted for at least 12 hr prior to terminal exsanguination under deep anesthesia (99.9% isoflurane, Baxter Healthcare Corp., Deerfield, IL) and blood was collected and stored as plasma. The right lungs were perfused with buffered Formalde-Fresh solution (Fisher Chemicals, Fairlawn, NJ), and lungs and one half of each kidney were maintained in Formalde-Fresh until sectioning and slide preparation. The left lungs and the other half of each kidney were snap frozen in liquid nitrogen and stored at −80°C for subsequent analysis.
Quantitation of lung lesions and kidney calcification
Lung lesions were quantified by determining the incidence and multiplicity of pulmonary pleural surface tumors, counting both the right and left lungs of each mouse. Tumor incidence was defined as the number of lung sets (right and left lungs) in each group containing 1 or more tumors divided by the total number of lung sets examined. Tumor multiplicity was defined as the total number of pulmonary tumors in each group divided by the total number of lung sets examined. Mean tumor number in tumor-bearing mice (total number of pulmonary tumors in each group divided by the total number of lung sets positive for tumors) is also reported. Lung tumors were visually counted by two researchers blinded to the treatment group, and lung sections were microscopically examined to confirm presence of bronchioalveolar adenoma. Three independent researchers blinded to the treatment group examined both kidneys for discoloration or irregular surface indicative of calcium deposits in the kidneys, and results are reported as percentage of animals in each group with evidence of gross kidney calcification. Selected kidneys from each group were processed histologically and stained using hematoxylin-eosin and von Kossa techniques to confirm presence or absence of kidney calcium deposits for validation of the gross kidney calcification measure.
Fasting plasma 1α,25-dihydroxyvitamin D3 levels
Plasma 1α,25-dihydroxyvitamin D3 was measured using a 125I-based RIA kit (DiaSorin, Stillwater, MN) according to manufacturer's instructions. Because of the volume demands of the assay and limited plasma, samples were batched in groups of 2 or 3 within treatment groups.
Kidney vitamin D 24-hydroxylase mRNA levels
Total RNA was isolated from kidney tissue (∼80 mg wet weight) using TriPure Isolation Reagent (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's protocol. cDNA was prepared from the RNA samples using M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA) and an automated thermal cycler (MJ Research PTC-200, Bio-Rad Laboratories, Hercules, CA), and quantified using a fluorescence-based real-time detection method (ABI PRISM 7000 Sequence Detection System, Perkin-Elmer Applied Biosystems, Foster City, CA) using SYBR Green reagents (Invitrogen, Carlsbad, CA). The PCR reaction was carried out in 20 μl of reaction mixture containing 10 μl of SYBR Green Supermix, 1 μl of 10 μM primer mix (including forward and reverse primers for vitamin D 24-hydroxylase (CYP24) or an internal reference gene, β-actin), and 9 μl of 20 ng cDNA diluted in RNase-free water. Cycling conditions were 50°C for 2 min and 95°C for 2 min, followed by 40 cycles at 95°C for 15 sec and 60°C for 30 sec. Melting curves were run following the cycling program to confirm the specificity of the reaction for the amplicon. Sequences for primers are as follows (Sigma-Genosys, The Woodlands, TX):
CYP24 forward primer: 5′-GCGGCCATCAAAACAATGA,
CYP24 reverse primer: 5′-TCACAAAGGAAATCCGCACC,
β-actin forward primer: 5′-TAGACTTCGAGCAGGAGATGGC,
β-actin reverse primer: 5′-CCACAGGATTCCATACCCAAGA.
Relative standard curves and a relative efficiency plot were constructed and verified over the working range of mRNA levels to confirm that amplification efficiencies of the genes were approximately equal. Quantification of gene expression was calculated relative to average values for the Sham control group using the comparative Ct method. For each sample and each gene, PCR reactions were carried out in triplicate.
Group means were compared using ANOVA analysis with Tukey's honestly significantly different (HSD) posthoc procedure applied for comparisons across multiple groups (SPSS software for Windows, version 11.5.0). All group values are expressed as means ± standard deviation, and differences between groups were considered significant if p < 0.05. Significantly different group means are expressed using distinguishing letter superscripts in tables and figures. Groups that do not share the same superscript letter are significantly different from each other (p < 0.05).
Effects of 1α,25-dihydroxyvitamin D3 and 9-cis retinoic acid on body weight and kidney calcification
There were no significant differences in mean body weight at baseline between any of the study groups, and no weight differences at the conclusion of the study between the groups receiving Sham treatment, NNK alone, the 2 lower doses of 9cRA, and the combination of 9cRA+1,25D (Table I). However, mean body weight at sacrifice was 20% lower in the NNK+9cRA (30 mg/kg diet) and NNK+1,25D (2.5 μg/kg diet) groups, 34% lower in the NNK+1,25D (5.0 μg/kg diet) group, and 32% lower in the weight-matched NNK group (all compared to carcinogen-injected controls, p < 0.001). Gross kidney calcification was noted in 29% of the animals in the NNK+1,25D (2.5 μg/kg diet) group and 82% of the animals in the NNK+1,25D (5.0 μg/kg diet) group [p = 0.007 and p < 0.001, respectively, compared to carcinogen-injected controls, Figures 1a and 1b show data for NNK alone group and NNK+1,25D (5.0 μg/kg diet)]. In addition, there was 1 premature death in the NNK+9cRA (30 mg/kg diet) group (after 18 weeks of 9cRA exposure) and 3 early deaths in the NNK+1,25D (5.0 μg/kg diet) group (after 6–15 weeks of 1,25D exposure), compared to 1 early death in the Sham control group and no early deaths in the other groups. The addition of 15 mg 9cRA/kg diet to 1,25D treatment (2.5 μg/kg diet) prevented the significant weight loss and gross kidney calcification caused by 1,25D treatment alone (Table I). Staining of select kidney sections using hemotoxylin and eosin, and the von Kossa method for calcium showed that sections from the 1,25D (2.5 μg/kg diet) group displayed mineralization in the kidney medulla and sections from the 1,25D (5.0 μg/kg diet) group displayed extensive and diffuse mineralization, while sections from the NNK alone group had no discernable mineralization [Figs. 1c–1f, data for NNK alone group and NNK+1,25D (5.0 μg/kg diet) are shown]. Sections from the Sham control group and from the combination treatment group (NNK+1,25D+9cRA) also appeared normal, with no discernable mineralization (data not shown).
|Treatment group||Body weight at baseline (g)||Body weight at sacrifice (g)||Presence of kidney calcium deposits|
|Sham||19.86 ± 2.27||23.04 ± 2.71a||0/13a|
|NNK||19.84 ± 1.39||23.36 ± 1.98a||0/14a|
|NNK+9cRA (7.5 mg/kg diet)||19.89 ± 1.80||23.01 ± 3.31a||0/14a|
|NNK+9cRA (15 mg/kg diet)||19.92 ± 1.39||21.24 ± 2.64a,b||0/14a|
|NNK+9cRA (30 mg/kg diet)||19.87 ± 1.57||18.61 ± 2.13b,c||0/13a|
|NNK+1,25D (2.5 μg/kg diet)||19.87 ± 1.27||18.80 ± 2.76b||4/14 (29%)b|
|NNK+1,25D (5.0 μg/kg diet)||19.84 ± 1.36||15.36 ± 2.29d||9/11 (82%)c|
|NNK+1,25D+9cRA||19.88 ± 1.29||22.14 ± 2.55a||0/14a|
|Weight-matched NNK||20.59 ± 1.39||15.84 ± 0.97c,d||0/14a|
Effects of 9-cis retinoic acid on lung tumorigenesis
There were no tumors present in any of the lungs examined from the Sham control group. Compared to this group, the group receiving NNK alone had significantly higher tumor incidence and tumor multiplicity (p < 0.001, Table II, Fig. 2). Treatment with 9cRA had no effect on tumor incidence at any of the doses tested; however, all 3 doses had significant effects on tumor multiplicity, compared to the NNK alone group (Table II). 9cRA decreased tumor multiplicity by 68% at a dose of 7.5 mg/kg diet, decreased tumor multiplicity by 70% at a dose of 15 mg/kg diet, and decreased tumor multiplicity by 85% at a dose of 30 mg/kg diet (p < 0.001 for all). There was a trend toward a dose-dependent effect, with the NNK + 9cRA (30 mg/kg diet) group showing a 15% greater reduction in tumor multiplicity than the NNK+9cRA (15 mg/kg diet) group (p = 0.082 for group comparison). Considering only tumor-bearing mice, the results for tumor multiplicity were similar but the trend towards a dose-dependent effect did not approach statistical significance (Table II).
|Treatment group||Tumor incidence||Tumors per mouse||Tumors per tumor-bearing mouse|
|NNK||14/14 (100%)b||15.50 ± 2.47c||15.50 ± 2.47a|
|NNK+9cRA (7.5 mg/kg diet)||13/14 (93%)b||4.93 ± 3.17b||5.31 ± 2.95b|
|NNK+9cRA (15 mg/kg diet)||14/14 (100%)b||4.64 ± 3.59b||4.64 ± 3.59b|
|NNK+9cRA (30 mg/kg diet)||11/13 (85%)b||2.31 ± 2.10a,b||2.73 ± 2.00b|
Effects of 1α,25-dihydroxyvitamin D3 on lung tumorigenesis
Treatment with 1,25D had a significant and dose-dependent effect on tumor incidence and a highly significant effect on tumor multiplicity at the 2 doses tested (Table III). Compared to the NNK alone group, treatment with 1,25D at a dose of 2.5 μg/kg diet decreased tumor incidence by 36% (p = 0.01) and tumor multiplicity by 85% (p < 0.001). Treatment with 1,25D at a dose of 5.0 μg/kg diet decreased tumor incidence by 82% (p < 0.001) and tumor multiplicity by 98% (p < 0.001). The dose-dependent effect on tumor incidence between the two 1,25D doses was highly significant (p = 0.001), and the highest dose of 1,25D reduced both tumor incidence and tumor multiplicity to levels that were not statistically different from the Sham group that received no carcinogen injection. Considering only tumor-bearing mice, the magnitude of the changes in tumor multiplicity decreased slightly, but significant effects on tumor multiplicity at both doses of 1,25D remained (Table III).
|Treatment group||Tumor incidence||Tumors per mouse||Tumors per tumor-bearing mouse|
|NNK||14/14 (100%)c||15.50 ± 2.47c||15.50 ± 2.47a|
|NNK+1,25D (2.5 μg/kg diet)||9/14 (64%)b||2.36 ± 2.76b||3.67 ± 2.65b|
|NNK+1,25D (5.0 μg/kg diet)||2/11 (18%)a||0.27 ± 0.65a,b||1.50 ± 0.71b|
|Weight-matched NNK||14/14 (100%)c||14.13 ± 2.55c||14.13 ± 2.55a|
The chemopreventive effects seen in the 1,25D groups were accompanied by increased mortality in the group receiving the highest dose of 1,25D and significantly lower body weight in both 1,25D treatment groups (Table I). To separate out the effects of 1,25D treatment from the effects of weight loss in these groups, the 1,25D-treated mice were compared to a group of animals that were given an NNK injection, maintained on the base semipurified diet, and group weight-matched to the mean weekly body weight of the lowest weight group (NNK+1,25D 5.0 μg/kg diet). This was accomplished by weekly food intake assessment and caloric restriction. The mean body weight at sacrifice for this weight-matched group was not significantly different from the NNK+1,25D (5.0 μg/kg diet) group (Table I); however, tumor incidence and tumor multiplicity remained statistically similar to the NNK alone group (Table III).
Effects of combined 9-cis retinoic acid and 1α,25-dihydroxyvitamin D3 on lung tumorigenesis
While treatment with 2.5 μg 1,25D/kg diet caused a significant reduction in mean body weight at sacrifice and led to kidney calcium deposits in 29% of the animals, the group receiving the combination of 2.5 μg 1,25D/kg diet plus 15 mg 9cRA/kg diet had a mean body weight that was not significantly different from the Sham or NNK alone groups and no evidence of kidney calcification (Table I). The combination of NNK+1,25D+9cRA resulted in significantly lower tumor incidence and tumor multiplicity than the NNK+9cRA (15 mg/kg diet) group (p = 0.007 and p = 0.043, respectively), but results were not significantly different from the NNK+1,25D (2.5 μg/kg diet) group (Table IV). Considering only tumor-bearing mice, the tumor multiplicity in the combination group was not statistically different from either of the single-treatment groups.
|Treatment group||Tumor incidence||Tumors per mouse||Tumors per tumor-bearing mouse|
|NNK||14/14 (100%)c||15.50 ± 2.47c||15.50 ± 2.47a|
|NNK+9cRA (15 mg/kg diet)||14/14 (100%)c||4.64 ± 3.59b||4.64 ± 3.59b|
|NNK+1,25D (2.5 μg/kg diet)||9/14 (64%)b||2.36 ± 2.76a,b||3.67 ± 2.65b|
|NNK+1,25D+9cRA||8/14 (57%)b||1.93 ± 2.09a||3.38 ± 1.60b|
Effects of 1α,25-dihydroxyvitamin D3 and 9-cis retinoic acid on fasting plasma 1,25D levels and kidney CYP24 mRNA expression
Fasting plasma 1,25D levels averaged 29.40 pg/ml in the Sham group and 24.33 pg/ml in the NNK alone group (Fig. 3a). 9cRA treatment (15 mg/kg diet) did not significantly alter fasting plasma 1,25D (mean value 33.50 pg/ml). 1,25D treatment (2.5 μg/kg diet) resulted in a trend toward decreased plasma 1,25D (mean value 13.17 pg/ml), as compared to the Sham control group (p = 0.053), but this difference was not significant compared to the NNK alone group. The combination of 1,25D and 9cRA treatment resulted in an average fasting plasma 1,25D level of 10.17 pg/ml, significantly lower than that in the Sham group (p = 0.016), but not significantly different from that in the NNK alone group or the NNK+1,25D (2.5 μg/kg diet) group.
Vitamin D 24-hydroxylase (cytochrome P450 enzyme 24, CYP24) is the major metabolic enzyme for vitamin D and a key vitamin D-inducible protein. There were no significant changes in CYP24 mRNA levels with NNK treatment or NNK+9cRA (15 mg/kg diet), but 1,25D (2.5 μg/kg diet), either alone or in combination with 9cRA, resulted in a significant induction in the expression of this enzyme (38- and 46-fold induction, respectively, p < 0.001 for both, Fig. 3b). The difference in the mean CYP24 mRNA level between the 1,25D treatment group and the group receiving the combination of 1,25D with 9cRA was not statistically significant.
The present exploratory study has demonstrated that supplementation with 1,25D dose-dependently decreased tumor incidence and significantly decreased tumor multiplicity in a mouse model of lung cancer. However, these effects were accompanied by weight loss and kidney histology suggestive of systemic hypercalcemia. While this makes it difficult to attribute the chemopreventive effects seen in this study to vitamin D directly, it is unlikely that reductions in caloric intake or weight loss played a major role since a group of control mice weight-matched to the 1,25D treatment group showed no evidence of tumor inhibition. Further, a recent study investigating the effects of 1,25D in an implanted tumor model of metastatic growth showed that a high calcium diet alone had no significant effect on lung tumor nodules.21 In that study, the authors concluded that 1,25D was able to inhibit metastasis and angiogenesis of tumor cells in vivo without an association with its calcium regulatory function.21 In our study, we provide evidence that 1,25D may also be able to act earlier in the process of carcinogenesis, inhibiting the development of NNK-induced lung tumors.
The chemopreventive effect of 9cRA has been previously reported in lung cancer cell studies3, 44 and appears to be particularly relevant to former smokers, where 9cRA supplementation reversed the loss of RARβ expression in the bronchial epithelium.13 In this study, treatment with 9cRA at doses of 7.5 and 15 mg/kg diet was shown to effectively decrease lung tumor multiplicity without appreciable weight loss, confirming earlier findings from our lab.14 The ability of 9cRA to alter tumor multiplicity suggests that this treatment is acting to inhibit tumorigenesis at an early stage in the process of tumor promotion. This observation complements existing literature documenting the chemopreventive effects of high-dose retinoids in the prevention of new primary tumors in patients curatively resected for stage I lung cancer.11 There did appear to be a non-significant trend toward a dose-dependent decrease in weight with increasing dose of 9cRA, suggesting that increasing doses of this treatment may be accompanied by increasing toxicity.
The most important observation in this study relates to the effect of combination treatment with 9cRA and 1,25D. While earlier studies suggested a possible synergistic interaction in cancer chemoprevention,24, 25, 26, 27, 38 we found no evidence of cooperative effects in our study. Instead, our results suggest retinoid mitigation of vitamin D toxicity with no antagonism of chemoprevention. Addition of 9cRA to 1,25D treatment completely prevented the weight loss and kidney calcification seen with 1,25D treatment alone, without decreasing the effectiveness of 1,25D treatment against lung tumor incidence and multiplicity. An effect of retinoids on calcium homeostasis has been shown more clearly in studies investigating the association between vitamin A and fracture risk,45, 46, 47, 48 and the influence of retinoids on vitamin D regulation of serum calcium and bone mineral homeostasis.35, 49, 50 Unfortunately, we were unable to obtain sufficient data to report serum or urine calcium levels in our study. Metabolic cages were not used in this study, and urine collections did not provide sufficient volume to perform urine calcium measurements in all pertinent groups. Further, due to sample limitations, we were only able to analyze fasting plasma calcium levels, in duplicate, in a single batched sample per group using direct current plasma emission spectrophotometry (data not shown). While this provided some sense of plasma calcium levels, it did not allow for any statistical analysis of group differences. Further, blood samples were drawn after a 12–15 hr fast, after 4 months of chronic exposure to 1,25D. Therefore, we believe that the combination of gross kidney calcification data along with the histological analysis of calcium deposits using von Kossa staining in selected kidneys more accurately reflects the physical ramifications of chronic exposure to 1,25D, alone and in combination with 9cRA.
In an effort to determine the mechanism(s) behind this retinoid mitigation of vitamin D toxicity, we measured fasting plasma 1,25D and kidney mRNA expression of CYP24, the major enzyme responsible for metabolizing 1,25D to an inactive form. While treatment with 1,25D resulted in slight declines in fasting plasma 1,25D and dramatic increases in kidney CYP24 expression (possibly responsible for the decreased fasting plasma 1,25D levels in these groups), 9cRA had no effect on these two measures of vitamin D metabolism. 9cRA did not alter either measure when given alone, nor did it attenuate the effects of 1,25D when given in combination. We attempted to measure CYP24 mRNA levels in the lung using real-time PCR, as increased CYP24 expression has been noted in human lung tumors and modification of CYP24 expression in the lung could help explain the chemopreventive effects of our treatments.51 There were no statistically significant differences in lung CYP24 mRNA levels between groups; however, lung CYP24 levels were correlated with kidney CYP24 (data not shown). Expression of this enzyme in lung tissue samples was very low, with only 46% of lung samples tested showing detectable levels of CYP24 mRNA. Coupled with high inter-animal variability in the groups receiving 1,25D, this resulted in inadequate statistical power to show any treatment effects. Future studies should be done to examine the effects of retinoid and 1,25D treatment, alone and in combination, on serum calcium levels and expression of lung CYP24 in areas likely to give rise to tumors.52
Our results provide strong evidence that retinoid antagonism of 1,25D is not occurring through alteration of vitamin D metabolism and are in agreement with recent data showing that all-trans retinoic acid can antagonize the in vivo action of a 24-fluoridated analog of 1α,25-dihydroxyvitamin D3, a form that is not susceptible to metabolism by CYP24.49 Although these results appear contrary to earlier findings demonstrating an influence of similar doses of retinoids on modulation of renal vitamin D metabolism and induction of CYP24, there are several differences between our study and the earlier reports. The retinoid modulation of vitamin D metabolism observed by Trechsel and Fleisch was in a kidney cell culture model53 and, to our knowledge, has not been confirmed in vivo. The subtle changes in CYP24 expression (3.1-fold induction with 10 mg/kg 9cRA and 2.1-fold induction with 1 mg/kg 9cRA) reported by Allegretto et al. were measured using Northern blotting analysis in the kidneys of BALB/c strain mice,54 while our study utilizes the more sensitive tool of real-time PCR analysis of mRNA expression in A/J mice. Allegretto et al. were able to show that a synthetic rexinoid (RXR-selective agonist) could stimulate transcription from CYP24 promoter sequences in vitro when cotransfected with RXRγ or RARγ.54 While interestingly, this does not adequately explain the retinoid antagonism of vitamin D effects observed in this study or in the recent work by Rohde and DeLuca.49 Further research needs to be done to confirm results and hypotheses generated from in vitro work with retinoids and vitamin D, and to test their validity in complex living systems. We are currently investigating the mechanisms underlying the chemopreventive effects of 9cRA and 1,25D in vivo, and continue to investigate other mechanisms to explain the retinoid antagonism of vitamin D observed in this study, including nuclear receptor “cross-talk” and retinoid antagonism of vitamin D receptor (VDR) binding to the vitamin D response element.
In conclusion, the present in vivo study demonstrates that treatment with 9cRA can decrease tumor multiplicity and treatment with 1,25D can decrease both tumor incidence and tumor multiplicity, albeit with significant toxicity, in a carcinogen-induced lung cancer model. While not immediately translatable to human trials, this work forms a basis for future study into lower doses of 1,25D and combinations of deltanoids (vitamin D and its synthetic analogs) and retinoids in cancer prevention and therapy. However, this study also raises important questions about the possible interaction of retinoids and deltanoids in vivo. Our data show that 9cRA has the potential to mitigate the toxicity of a pharmacologic dose of 1,25D with respect to body weight loss and kidney calcium deposition. The lack of antagonism with regard to tumor prevention seen in our combination group suggests that, at certain doses, retinoids may be able to mitigate vitamin D toxicity without reducing chemopreventive efficacy. Further studies into the mechanism underlying this antagonism will help us clarify which roles of vitamin D may be affected by retinoids and how we should best modulate retinoid levels in the use of vitamin D-based cancer therapies.
The authors thank Ms. Senait Assefa, Ms. Shahin Sarkarati, Ms.Stephanie Valliere and Roderick Bronson for technical assistance. This project was performed, in part, using compounds provided by the National Cancer Institute's Chemical Carcinogen Reference Standards Repository operated under contract by Midwest Research Institute, No. N02-CB-07008.
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- 33Inhibition of the 1,25-dihydroxyvitamin D3-induced increase in vitamin D receptor (VDR) levels and binding of VDR-retinoid X receptor (RXR) to a direct repeat (DR)-3 type response element by an RXR-specific ligand in human keratinocyte cultures. Biochem Pharmacol 1998; 55: 767–73., , , .