Department of Hygienic Sciences, Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan, 1 Institute of Molecular and Cellular Bioscience, University of Tokyo, Japan and 2 Department of Clinical Nutrition, School of Medicine, University of Tokushima, Japan
* To whom correspondence should be addressed. Tel: +81 78 441 7563; Fax: +81 78 441 7565; Email: t-okano{at}kobepharma-u.ac.jp
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Abstract |
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Abbreviations: 1,25(OH)2D3, 1
,25-dihydroxyvitamin D3; DMBA, 7,12-dimethylbenz[a]anthracene; FBS, fetal bovine serum; GFP, green fluorescent protein; LLC, Lewis lung carcinoma; MMP, matrix metalloproteinase; PBS, phosphate-buffered saline; PTH, parathyroid hormone; VDR, vitamin D receptor; VDRE, vitamin D response element; VEGF, vascular endothelial growth factor
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Introduction |
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A biologically active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3 [1
,25(OH)2D3], is involved in essential cell regulatory processes, such as proliferation, differentiation and apoptosis (25). It has been shown that this hormone promotes cellular differentiation and inhibits the proliferation and invasive potential of a number of different cancer cells in vitro (6,7). Studies in vivo demonstrated that 1
,25(OH)2D3 slows the progression of breast, prostate and other carcinomas (811). These findings, in conjunction with the fact that the vitamin D receptor (VDR) is present in normal and tumor cells, suggest that 1
,25(OH)2D3 acts primarily as an intrinsic and preventive factor to stop the growth and metastasis of cancer cells (1214). These properties suggest the possible clinical use of 1
,25(OH)2D3 in the treatment of cancers. However, the potent hypercalcemic activity of 1
,25(OH)2D3 has precluded its application as a pharmacological agent and limited a number of approaches using animal models (15). For this reason, various 1
,25(OH)2D3 analogs with reduced calcemic activity that retain the antiproliferative activity have been synthesized (6,16). In addition, epidemiological studies suggest that vitamin D3 is involved in the pathogenesis of some cancer types. Several lines of evidence indicate an inverse correlation between dietary vitamin D intake or sunlight exposure and the prevalence of colorectal cancer and an association of high vitamin D3 intake with a low incidence of breast and prostate cancer, although similar studies on human lung cancer have yet to be conducted (1719).
In the cascade of biological events that leads to the establishment of systemic metastasis, tumor cells must complete a sequence of interactions with the host homeostasis mechanisms and avoid destruction by the host defenses (2022). Metastasis begins with an invasive process by which tumor cells penetrate the epithelial basement membrane, enter the underlying host stroma surrounding the primary neoplasm and subsequently reach and penetrate the vasculature. Because of the large number of capillary blood vessels in the lungs, tumor cells circulating in the blood are more likely to lodge in these organs, thereby leading to substantial metastatic growth there. Thus, metastatsis of cancer to the lung is a particularly challenging problem in the clinical setting. Once tumors have invaded the lung the response to classical chemotherapeutic agents is low and the prognosis is poor.
In this study we have focused on the direct effects of 1,25(OH)2D3 on the critical process by which metastatic lung cancer cells in the bloodstream migrate to the potential target organ and attach and lodge in the microvasculature there in a model without the calcemic activity and host defense-related effects of 1
,25(OH)2D3. We employed a direct metastasis model in which the entrapment of tumor cells and metastatic growth are quantified following the injection of Lewis lung carcinoma (LLC) cells expressing green fluorescent protein (GFP) (LLC-GFP cells) into the vein and used VDR knockout (VDR/) mice. A major advantage of using LLC-GFP cells is that imaging requires no preparative procedures and, therefore, is uniquely suited for visualizing live tissue during tumor progression (23,24). In addition, GFP labeling is extremely effective for measuring the number and volume of metastasis nodules in target organs. VDR/ mice were generated from C57BL/6 mice by one of the co-authors using homologous gene targeting and present with a skeletal phenotype typical of a complete lack of genomic 1
,25(OH)2D3 effects. VDR/ mice exhibit no vitamin D-dependent calcemic activity and extremely high serum levels of 1
,25(OH)2D3 due to overexpression of the 1
-hydroxylase gene (25).
In this report we have attempted to examine whether the metastatic growth of LLC-GFP cells is correlated with the serum concentration of 1,25(OH)2D3 in wild-type (VDR+/+) and VDR/ mice. We found that in this model 1
,25(OH)2D3 significantly inhibits the development of lung metastases without a direct influence of calcemic activity and other actions regulated by 1
,25(OH)2D3 in the host. In addition, VDR/ mice in which serum calcium and 1
,25(OH)2D3 levels were corrected were observed to develop tumors on injection of LLC-GFP cells, the same as VDR+/+ mice. Tumorigenesis was reduced by the continuous administration of 1
,25(OH)2D3 in VDR/ mice without hypercalcemia induced by 1
,25(OH)2D3. Our results suggest that VDR/ mice with corrected hypocalcemia and hypervitaminosis D can be used as an experimental model to screen for the anticancer effects of new vitamin D analogs in vivo. Our findings show that 1
,25(OH)2D3 acts on the metastatic growth of lung cancer cells directly and as an intrinsic factor for the prevention or treatment of cancer.
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Materials and methods |
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Animals
VDR/ mice were generated by homologous gene targeting as described previously (19). Null mutant mice were obtained by intercrossing a heterozygous VDR knockout female and a heterozygous male. Mice were weaned at 3 weeks of age and then fed ad libitum either a normal diet (1.2% calcium, 0.6% phosphorus and 108 IU vitamin D3/100 g), a high calcium diet (2% calcium, 1.25% phosphorus, 108 IU vitamin D3/100 g and 20% lactose), a vitamin D-deficient diet (0.6% calcium, 0.3% phosphorus and 0 IU vitamin D3/100 g) or a high calcium and vitamin D-deficient diet (2% calcium, 1.25% phosphorus, 0 IU vitamin D3/100 g and 20% lactose) (all from Clea Japan Inc., Tokyo, Japan). The mice were maintained under specific pathogen-free conditions with a 12 h light/12 h dark cycle and were given free access to the assigned diet during the feeding period. Mice were injected i.v. with a single dose of 106 LLC-GFP cells in a total volume of 0.2 ml in RPMI-1640 medium containing 10% FBS. The lungs were collected 1, 2 or 3 weeks or 18 days after the injection and the weights, metastatic nodule numbers and GFP/ß-actin mRNA ratio were measured. This study was conducted in accordance with the standards established by the Guidelines for the Care and Use of Laboratory Animals of Kobe Pharmaceutical University.
RT-PCR
Total RNA was prepared from LLC-GFP cells using ISOGEN (Nippon Gene, Tokyo Japan). Aliquots of 2 µg RNA were reverse transcribed with AMV reverse transcriptase (TaKaRa, Japan) and PCR amplified at 95°C for 40 s, 62°C for 40 s and 72°C for 1 min for 2035 cycles, using primer sets specific for VDR (GenBank accession no. NM_009504) (forward primer, 107126; reverse primer, 286305) and ß-actin (GenBank accession no. X03672) (forward primer, 250271; reverse primer, 305326). Agarose gels (2%) were stained with ethidium bromide and visualized under UV lights. All assays were performed in triplicate.
Western blot analysis
Whole cell extracts were harvested in lysis buffer [1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 10 mM TrisHCl (pH 7.4), 5 mM EDTA, protease inhibitor mixture (Complete; Roche Molecular Biochemicals)] from LLC or LLC-GFP cells. Extracts containing 30 µg of each protein were then subjected to 7.5% SDSPAGE. After separation by gel electrophoresis, the proteins were transferred to polyvinylidene difluoride membranes (Amersham Pharmacia Biotech). After blocking with Block Ace reagent (Dainippon Pharmaceuticals, Osaka, Japan) the membranes were incubated with the first antibody, a monoclonal anti-VDR antibody (9A7; Affinity Bioreagents Inc., Golden, CO). After incubation with the horseradish peroxidase-linked anti-IgG second antibody, the proteins were visualized using an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech).
Cell cycle analysis
The effects of 1,25(OH)2D3 on the proliferation of LLC-GFP cells in vitro were assessed by cell counting and cell cycle analysis. Cells were seeded at a density of 2 x 105 cells/well in 6-well culture plates in RPMI-1640 medium containing 10% FBS for 24 h. After 24 h in serum-deprived RPMI-1640 medium, fresh medium containing 10% charcoal stripped FBS with or without 1
,25(OH)2D3 (109107 M) was added to cultured cells and incubation continued for 3 days. The medium was changed 2 days thereafter. 1
,25(OH)2D3 was dissolved in ethanol and the final concentration of ethanol in all cultures did not exceed 0.1%. Cells were trypsinized at timed intervals and an aliquot was counted and analyzed. For analysis of the cell cycle, each group of cells was collected and washed once with phosphate-buffered saline (PBS) (calcium and magnesium free: ). Then the cells were resuspended in PBS () containing 0.2% Triton X-100 and 100 µg RNase and incubated at 37°C for 30 min. Cells were washed with PBS () and incubated with 0.5 ml of a DNA-staining solution containing propidium iodide (50 µg/ml) at 4°C for 20 min. The cells were analyzed with a flow cytometer equipped with an argon laser (488 nm) (Becton Dickinson FACScan) and the cell cycle distribution was analyzed using ModiFit LT (Verity).
In vitro invasion assay using matrigel
The capacity of tumor cells to traverse a basement membrane matrix-coated filter has been shown to be representative of their invasiveness. Therefore, the capacity of tumor cells to migrate through matrigel-coated membranes was measured (26,27). Nucleopore filters (8 µm pore size) were coated with a 1:20 dilution of matrigel (Becton Dickinson, Bedford, MA). Representative filters were stained with crystal violet. Medium and 1,25(OH)2D3 were added to the upper and lower compartment of each blind well chemotactic chamber. A dried, coated filter was placed over the lower compartment. After reconstituting the matrigel, 5 x 104 cells were added to the upper compartment together with the same additives as were in the medium in the lower compartment of each chamber. Chambers were incubated for 24 h, after which the filters were removed, wiped clean on the upper surface and fixed in 10% formalin. The number of cells on the lower surface of the filter was counted under a fluorescence microscope. Data are presented as the number of cells per low power field enumerated from triplicate chambers.
Real-time quantitative PCR
LLC-GFP cells or lungs were homogenized in ISOGEN and total RNA was isolated. Aliquots of 2 µg RNA was reverse transcribed with AMV reverse transcriptase (TaKaRa, Japan) and PCR amplified at 95°C for 40 s, 62°C for 40 s and 72°C for 1 min for 35 cycles using primer sets specific for VDR, vascular endothelial growth factor (VEGF) (for primers see ref. 28), matrix metalloproteinase-2 (MMP-2) (GenBank accession no. NM_008610) (forward primer, 424443; reverse primer, 558576), MMP-9 (GenBank accession no. NM_013599) (forward primer, 149167; reverse primer, 242263), GFP (GenBank accession no. AF323988) (forward primer, 417438; reverse primer, 490511) and ß-actin. A quantitative analysis of gene expression was performed using a GeneAmp 5700 Sequence Detection System (PE Biosystems, Foster City, CA) and a SYBR Green core reagent kit (PE Biosystems) as described (29).
Imaging and tumor scoring
A Leica fluorescence stereo microscope model MZ FL III (Leica Microsystems Inc., Tokyo, Japan) was used. Selective excitation of GFP was produced through a D480/40 band-pass filter and 510 DCXR dichroic mirror. Emitted fluorescence was collected through a DC300F long-pass filter and digital camera system. The numbers of metastatic tumors on the surface of the lung were counted under the fluorescence stereo microscope. To quantify lung metastasis we counted GFP-expressing spots on the lungs.
Histological analysis
Tissues were fixed in 10% paraformaldehyde and embedded in paraffin. Sections (5 µm thick) were deparaffinized and then stained with hematoxylin and eosin according to standard procedures.
Assay for serum calcium and 1,25(OH)2D3
Serum calcium and 1,25(OH)2D3 levels were determined using a microcolorimetric assay (Wako, Japan) and radio receptor assay using radiolabeled 1
,25(OH)2D3, respectively.
Enzyme immunoassay for mouse VEGF
The mouse VEGF concentration in mouse serum was determined using a VEGF EIA kit (Quantikine M; R&D Systems, Minneapolis, MN, USA), based on a sandwich enzyme immunoassay, according to the manufacturer's instructions. The concentration of VEGF in 100 µl of sample was estimated from a standard curve obtained using standard mouse VEGF serially diluted with a suitable buffer.
Intravenous injection of LLC-GFP cells into VDR+/+ and VDR/ mice fed the high calcium, vitamin D-deficient diet and administered 1,25(OH)2D3
Mice were injected i.v. with a single dose of 106 LLC-GFP cells. Cells were first harvested by trypsinization and washed twice with PBS () and then injected in a total volume of 0.2 ml RPMI-1640 medium containing 10% FBS. A preventative protocol was designed in which 1,25(OH)2D3 was administered continuously using an osmotic minipump (model 2 ML4 Alzet; Alza Corp., Palo Alto, CA) implanted s.c. on the same day as inoculation of LLC-GFP cells. An infusion rate of 1 µg/kg/24 h was chosen and each minipump contained 1
,25(OH)2D3 dissolved in 0.1% Tween-20 and 10% ethanol to deliver a continuous dose of 1
,25(OH)2D3 for up to 18 days at a delivery rate of 2.5 µl/h. Untreated animals were implanted with a minipump containing vehicle alone. Mice with implanted tumors were killed 18 days later. The lungs were removed and the weight, number of lung nodules and level of GFP mRNA expression were measured. Serum calcium and 1
,25(OH)2D3 levels were also measured.
Statistics
Data are presented as means ± SE. Student's t-test was used for the group analysis. Correlation coefficients were calculated for possible interrelations between variables. P values <0.05 were considered statistically significant.
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Results |
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Inhibition of the metastatic activity and angiogenesis-inducing activity of LLC-GFP cells by 1,25(OH)2D3
LLC cells are known to highly express mRNAs of VEGF164 and VEGF120, involved in tumor-induced angiogenesis (32). LLC cells also express the mRNAs of MMP-2 and MMP-9, involved in invasion by tumor cells (33). To further investigate the mechanism of 1,25(OH)2D3 inhibition of metastasis we examined the effects of treatment with 1
,25(OH)2D3 on the down-regulation of these genes. As shown in Figure 2AD, all of these genes were strongly suppressed by treatment with 1
,25(OH)2D3 (109107 M) for 24 h. As shown in Figure 2E, expression of VDR mRNA was unaffected by 1
,25(OH)2D3 treatment in LLC-GFP cells.
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Tumorigenesis in VDR+/+ mice injected with an LLC-GFP cell suspension (LLC-GFP CSI VDR+/+ mice)
We i.v. injected VDR+/+ C57BL/6 mice (8 weeks of age) with a single dose of 106 LLC-GFP cells and collected the lungs 18 days after injection. The LLC-GFP cells metastasized to the lungs from the circulation and generated numerous metastatic nodules, as shown by bright field stereo microscopy (Figure 3A) and fluorescence stereo microscopy (Figure 3B). Metastatic tumors expressing GFP were only detectable in the lungs, demonstrating organ-specific metastasis and stable expression of GFP by the cells. In order to investigate the metastatic potential of LLC-GFP cells, we i.v. injected VDR+/+ mice (8 weeks of age) with a single dose of 106 cells and collected the lungs 1, 2 and 3 weeks after injection. Weights, metastatic nodule numbers and GFP/ß-actin mRNA ratios of the lungs were measured. All of the animals (15/15 in each group) had highly fluorescent lung tumors and all values for the metastatic parameters increased with time (Figure 3C), demonstrating the appropriateness of using LLC-GFP cells for the evaluation of metastatic growth in VDR+/+ mice. All the animals injected with LLC-GFP cells survived for 2 weeks and only 3 of 15 had died by 3 weeks post-injection. Based on these in vivo observations, it seemed that lung metastatic tumors on day 18 after the injection provided the most appropriate material for evaluating the anti-metastatic activity of 1,25(OH)2D3.
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To exclude the above possibility and show that 1,25(OH)2D3 plays an essential role in inhibition of the metastatic growth of LLC-GFP cells, we fed VDR+/+ and VDR/ mice (3 weeks of age) either a normal diet, a high calcium diet, a vitamin D-deficient diet or a high calcium and vitamin D-deficient diet for 7 weeks. Then, we injected the mice with LLC-GFP cells and continued to feed the assigned diets before collecting the lungs on day 18 after injection. In the VDR+/+ and VDR/ mice fed the normal diet injection of LLC-GFP cells did not affect the serum calcium levels (Figure 5A) and induced a small, but insignificant, decline in serum 1
,25(OH)2D3 levels of both groups of mice (Figure 5B and C). In the VDR+/+ and VDR/ mice fed the high calcium diet the serum calcium levels of both groups of mice were within the normal range (Figure 5A). The high calcium diet induced a significant decline in the serum 1
,25(OH)2D3 levels of VDR+/+ mice, leading to a moderate hypovitaminosis D, and induced a small, but insignificant, decline in the serum 1
,25(OH)2D3 levels of VDR/ mice, although the mice still had extremely severe hypervitaminosis D (Figure 5B and C). These findings clearly indicate that in VDR/ mice, irrespective of tumor burden, a high calcium diet is able to normalize the serum calcium level but fails to normalize the serum 1
,25(OH)2D3 level, as reported previously (36). Feeding of the vitamin D-deficient and the high calcium and vitamin D-deficient diets resulted in complete elimination of 1
,25(OH)2D3 from the serum of both VDR+/+ and VDR/ mice (Figure 5B and C). Intriguingly, metastatic growth of LLC-GFP cells was remarkably reduced in VDR/ mice fed either the normal diet or the high calcium diet, while it was not reduced or even enhanced in VDR/ mice fed the vitamin D-deficient and the high calcium and vitamin D-deficient diets (Figure 6AC). Moreover, VDR+/+ mice fed the manipulated diets displayed an apparent inverse relationship between serum levels of 1
,25(OH)2D3 and GFP mRNA expression (Figures 5C and 6C). These results indicate that physiological levels of 1
,25(OH)2D3 have a preventive effect against tumorigenesis. The vitamin D-deficient diet appeared to increase serum calcium levels in VDR+/+ and VDR/ mice with tumors (Figure 5A). This is probably due to LLC-GFP cells inducing hypercalcemia during the process of tumorigenesis (39,40).
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Discussion |
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VDR/ mice are an animal model lacking vitamin D activity, which is involved in not only calcium metabolism in the intestine, kidney and bone, but also a broad range of essential cell regulatory processes, including cellular proliferation, differentiation and apoptosis. Also, VDR/ mice display important defects in macrophage function and cellular immunity in vitro and in vivo (45). However, irrespective of these important functions of 1,25(OH)2D3, almost all the other abnormalities observed in VDR/ mice except for high serum levels of 1
,25(OH)2D3 can be reversed by feeding a high calcium diet (35,4547). Through dietary manipulation this animal model has helped us to more precisely define the direct effects of 1
,25(OH)2D3 on cancer cells and tumor growth and metastasis under conditions without the calcemic activity and other actions of 1
,25(OH)2D3 in the host.
In the present study, to fully visualize and quantify metastasis in vivo, we developed GFP-expressing Lewis lung carcinoma cells as a far more powerful model with which to study the mechanisms of tumor progression, including regional and distant metastasis representative of lung cancer. LLC-GFP cells express the VDR and their proliferative and invasive activities are significantly inhibited by 1,25(OH)2D3 treatment. The mechanism of this inhibition in response to 1
,25(OH)2D3 has not been made clear. However, our observations in vitro suggest that the mRNA levels of MMP-2, MMP-9 and VEGF, which are the most important factors for tumor invasion and tumor-induced angiogenesis, were decreased by 1
,25(OH)2D3 treatment in a dose-dependent manner (Figure 2). Also, we observed secretion of MMP-2, MMP-9 and VEGF into the culture medium by gelatin zymography and ELISA. All of these secretions were inhibited by 1
,25(OH)2D3 treatment for 6, 12 and 24 h (unpublished data). These findings suggest that 1
,25(OH)2D3 may play a critical role in the expression and secretion of MMP-2, MMP-9 and VEGF in metastatic cancer cells. Previously the molecular mechanisms of this effect were unclear, but it has been reported that 1
,25(OH)2D3 decreases the production of granulocytemacrophage colony-stimulating factor, reduces protein kinase A activity and increases levels of polymerized actin in LLC cells (26,27). In human MDA-MB-231 breast cancer cells 1
,25(OH)2D3 diminished the activity of MMP-9 and two serine proteases, urokinase-type plasminogen activator and tissue-type plasminogen activator, concomitant with a reduced invasiveness of the cells (4852). The molecular mechanisms behind our in vitro results in response to 1
,25(OH)2D3 are presently unknown. Through its interaction with the vitamin D response element (VDRE), the VDR is essential for hormone action on target genes (53). Since there are no known VDRE nucleotide sequences in the promoter region of the MMP and VEGF genes, these effects must occur through other means. One possibility may be 1
,25(OH)2D3-dependent transcriptional repression through a negative regulatory VDRE, as proposed for the vitamin D 1
-hydroxylase gene by Murayama et al. (38). Another possibility is that 1
,25(OH)2D3 mediates its inhibitory action on MMP and VEGF secretion via an autocrine inhibitory loop.
Here we have focused on the direct effects of 1,25(OH)2D3 on the critical processes of metastatic lung cancer cells without an association with its calcemic activity and regulation of the host defenses. Therefore, we employed VDR/ mice and a direct metastasis model, involving the entrapment of VDR-positive and 1
,25(OH)2D3-responsive LLC-GFP cells in a vein. When VDR/ mice were fed the normal and high calcium diets they displayed a high serum level of 1
,25(OH)2D3 and the metastatic growth of LLC-GFP cells was remarkably decreased. However, no such effect was observed in VDR/ mice fed a vitamin D-deficient diet. These results suggest that serum 1
,25(OH)2D3 directly inhibits the metastatic and angiogenesis-inducing activity of LLC-GFP cells irrespective of the serum concentration of calcium. Furthermore, administration of 1
,25(OH)2D3 prevented tumorigenesis in VDR+/+ and VDR/ mice. The hypercalcemia observed when high doses of 1
,25(OH)2D3 are administered appears to be mediated through the nuclear receptor, because VDR/ mice could be treated with extremely high doses of 1
,25(OH)2D3 without any side-effects, whereas VDR+/+ mice showed lethal hypercalcemia on administration of 1 µg/kg/day 1
,25(OH)2D3. These results clearly suggest that the inhibitory effect of 1
,25(OH)2D3 on the metastasis of lung cancer cells is not related to the calcemic activity and other actions of 1
,25(OH)2D3 in the host. The beneficial effects of 1
,25(OH)2D3 in cancer treatment have been supported by many studies with 1
,25(OH)2D3 and synthetic analogs. However, despite a few encouraging results, clinical applications have been limited due to an extremely narrow therapeutic window, i.e. effective doses cannot be administered without inducing hypercalcemia (15). Therefore, whether the calcemic activity of and regulation of the host defenses by 1
,25(OH)2D3 contribute to the effects on cancer cells has not been elucidated. In this paper we report for the first time that 1
,25(OH)2D3 inhibits the metastasis and angiogenesis of lung cancer cells without an association with the calcemic activity and other actions of 1
,25(OH)2D3 in the host.
With regard to VDR ablation and immune defects and tumorigenesis, Mathieu et al. reported that VDR/ mice display important defects in macrophage function and cellular immunity in vitro and in vivo. However, these defects are an indirect consequence of disruption of the VDR because they can be restored through normalization of calcium levels (45). Thus, in our experimental model VDR/ mice with corrected serum calcium levels had normal immune functions. Zinser et al. have investigated the roles of the VDR in host defense activity and 7,12-dimethylbenz[a]anthracene (DMBA)-induced skin carcinogenesis in vivo using VDR/ mice (54). In their experiments mice were repeatedly exposed to DMBA to induce the formation of skin tumors. Chemically induced carcinomatous growth and the progression of tumorigenesis occurred more rapidly in VDR/than VDR+/+ mice. The authors suggested that VDR ablation is associated with enhanced sensitivity to tumor formation. The development of skin tumors in response to the chemical carcinogen DMBA in VDR/, but not VDR+/+, mice suggests that the VDR acts as a tumor suppressor in the epidermis. Also, Kallay et al. suggested that loss of the VDR may provide a link to the complex process of multi-step carcinogenesis by causing direct DNA damage when 1,25(OH)2D3-mediated growth control is diminished (55). Their report indicated that the anti-tumorigenic effects of 1
,25(OH)2D3 are mediated through the VDR.
From our study it is now clear that the metastatic growth of lung cancer cells is remarkably reduced in response to increased serum levels of 1,25(OH)2D3, but not serum calcium levels and the host defenses. Furthermore, we found that metastatic growth of LLC-GFP cells was increased in VDR+/+ mice fed a vitamin D-deficient diet or a high calcium and vitamin D-deficient diet compared with VDR+/+ mice fed a normal diet (Figure 6AC). VDR+/+ mice fed the manipulated diets displayed an apparent inverse relationship between the serum levels of 1
,25(OH)2D3 and GFP mRNA expression (Figures 5C and 6C). Thus, these results in VDR+/+ mice support the hypothesis that physiological levels of 1
,25(OH)2D3 have a preventive effect against tumorigenesis. Renal and extra-renal production of 1
,25(OH)2D3 may also be a part of important mechanisms in the anti-tumor effects of dietary vitamin D or circulating 1
,25(OH)2D3. However, serum 1
,25(OH)2D3 levels in VDR/ mice are not physiological or feasible for use in human patients. In this study we would like to emphasize that VDR/ mice with corrected hypocalcemia and hypervitaminosis D can be used as an experimental model for screening the anti-cancer effects of new vitamin D analogs in vivo.
In conclusion, we have shown that 1,25(OH)2D3 acts on the metastatic growth of lung cancer cells directly and is an intrinsic factor for the prevention or treatment of cancer. Moreover, we suggest that serum levels of 1
,25(OH)2D3 vary inversely with tumorigenesis and that 1
,25(OH)2D3 may work as an intrinsic factor for the prevention of metastasis in intact animals. These findings should encourage the further development of nutritionally based models for the chemoprevention of metastatic cancer using vitamin D. Current research is aimed at exploring the potential use of 1
,25(OH)2D3 as a preventive factor and anti-proliferative agent against cancer.
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Acknowledgments |
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References |
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