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Research Articles: Therapeutics, Targets, and Development
1
,25-dihydroxyvitamin D3 (Calcitriol) inhibits hypoxia-inducible factor-1/vascular endothelial growth factor pathway in human cancer cells
1 Department of Pediatrics, Dana Children's Hospital; 2 Prostate Cancer Research Laboratory, Department of Urology; and 3 Bone Disease Unit, Tel-Aviv Sourasky Medical Center, Sackler Faculty of Medicine, Tel Aviv, Israel; and 4 Division of Hematology/Oncology, Department of Internal Medicine, University of Michigan Medical Center, University of Michigan Comprehensive Cancer Center, Ann Arbor, Michigan
Requests for reprints: Nicola J. Mabjeesh, Prostate Cancer Research Laboratory, Department of Urology, Tel-Aviv Sourasky Medical Center, 6 Weizmann Street, Tel Aviv 64239, Israel. Phone: 972-3-6974544; Fax: 972-3-6973183. E-mail: nicolam{at}tasmc.health.gov.il
Abstract
In vitro and in vivo studies have shown that 1
,25-dihydroxyvitamin D3 [1,25(OH)2D3] inhibits angiogenesis in cancer. We now examined whether the antiangiogenic effects of 1,25(OH)2D3 are mediated by the hypoxia-inducible factor (HIF)-1 pathway. Our results showed that 1,25(OH)2D3 reduces the protein expression of both the regulated HIF-1 subunit and the vascular endothelial growth factor (VEGF) in various human cancer cells. 1,25(OH)2D3 also inhibited HIF-1 transcriptional activity (measured by reporter gene assay) as well as HIF-1 target genes, including VEGF, ET-1, and Glut-1. We also showed that 1,25(OH)2D3 inhibits cell proliferation under hypoxia. Using HIF-1 knockout colon cancer cells, we show that the inhibition of the hypoxia-induced VEGF by 1,25(OH)2D3 is mediated through a HIF-dependent pathway. Because HIF-1 is a major positive contributor in human tumorigenesis and angiogenesis, we believe that its inhibition by 1,25(OH)2D3 strengthens the rationale to use vitamin D and its low-calcemic analogues in cancer chemoprevention and therapy. [Mol Cancer Ther 2007;6(4):1433–9]
Introduction
Vitamin D has traditionally been associated with systemic calcium hemostasis (1), but it also has important noncalcemic biological action. It is now recognized that 25-hydroxyvitamin D-1-hydroxylase, which synthesizes 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the active form of the vitamin D, and vitamin D receptor are present in many normal and cancer tissues (2–5). There are increasing lines of evidence that 1,25(OH)2D3 regulates cell proliferation, differentiation, apoptosis, immune responses, and angiogenesis in an autocrine/paracrine fashion (1, 4, 6, 7). Indeed, studies on prostate, colon, breast, lung, and other cancers indicate that 1,25(OH)2D3 prevents cancer progression by reducing cell proliferation, by increasing cell differentiation and apoptosis, and by inhibiting angiogenesis (1, 4–7).
Hypoxia is the major pathophysiologic condition that regulates angiogenesis. Increased angiogenesis in response to hypoxia is part of the cellular adaptation mediated by the key transcription factor, hypoxia-inducible factor (HIF)-1 (8). HIF-1 is composed of the oxygen-regulated subunit HIF-1 and the constitutively expressed HIF-1ß subunit. HIF-1 is produced and rapidly degraded under normoxic conditions due to posttranslational oxygen-dependent hydroxylation on specific proline residues (402 and 564) (review in ref. 8). The hydroxylated protein is ubiquitinated by the von Hippel Lindau protein E3 ligase complex and targeted to proteasomal degradation. Under hypoxia, HIF-1 is stabilized and heterodimerizes with HIF-1ß to bind to an enhancer element called the 'hypoxia response element' (HRE) in target genes. HIF-1 drives the transcription of >70 survival genes, including the glycolytic enzymes, glucose transporter-1 (Glut-1), endothelin-1 (ET-1), vascular endothelial growth factor (VEGF), VEGF receptor-1 (Flt-1), carbonic anhydrase 9 (CA9), and erythropoietin (see ref. 8 for complete list). The hypoxic response pathway has also been recognized as an important contributor to a wide range of human cancers, including breast, prostate, brain, lung, colon, and head and neck (9). Increased levels of HIF-1 activity are often associated with increased tumor aggressiveness, therapeutic resistance, and mortality (9). Therefore, we tested the hypothesis that the antiangiogenic effects of 1,25(OH)2D3 in cancer are mediated through the HIF pathway. Our data showed that 1,25(OH)2D3 inhibits HIF-1 protein expression and HIF-1 target genes. Furthermore, we showed that 1,25(OH)2D3 failed to suppress VEGF expression in HIF-1 knockout human cancer cells. These observations emphasize the role of the HIF pathway in 1,25(OH)2D3 inhibition of angiogenesis in cancer.
Materials and Methods
Reagents and Antibodies
1,25(OH)2D3 was a generous gift from Dr. Zeev Mazor (Teva Pharmaceuticals Industries, Pitach Tikva, Israel). Cycloheximide was purchased from Sigma-Aldrich (St. Louis, MO). Primary antibodies were mouse monoclonal anti-HIF-1 (BD Biosciences, San Diego, CA), goat polyclonal anti-actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and polyclonal human antibody to human topoisomerase I (TopoGEN, Columbus, OH). Secondary antibodies were conjugated to horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA). The chemiluminescence reagent was from Biological Industries Ltd. (Kibbutz Beit Haemek, Israel).
Cell Lines and Hypoxic Treatment
PC-3 and LNCaP cells were cultured in RPMI 1640. CL-1, MCF-7, and SW-480 cells were maintained in DMEM. Parental HCT116 and HCT116HIF-1–/– were maintained in McCoy's 5A medium and passed in parallel to preserve the same passage number (10). All media were supplemented with 10% FCS and antibiotics. All cells were cultured at 37°C in a humidified atmosphere and 5% CO2 in air. For hypoxic exposure, the cells were placed in a sealed modular incubator chamber (Billups-Rothenberg, Del Mar, CA) flushed with 1% O2, 5% CO2, and 94% N2.
1,25(OH)2D3 Treatment and Cell Proliferation Assay
Cells (2,000–5,000 per well) were seeded in 96-well plates in a volume of 200 µL for cell proliferation assay using a 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt kit (Biological Industries). On the next day, the cells were treated with increasing concentrations of 1,25(OH)2D3 (in triplicates) and cultured under either normoxic or hypoxic conditions for 2 to 4 days. 1,25(OH)2D3 was solubilized in 100% ethanol at 1 mmol/L concentrations as a stock solution. All samples, including controls, contained 0.1% ethanol. After the indicated time of treatment, the 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt reagent was added and the cells were processed as described previously (11).
Protein Extraction and Immunoblot Analysis
For Western blotting, the cells were seeded in six-well or 6-cm plates. After 24 h, the cells were treated overnight with 1,25(OH)2D3 under either normoxic or hypoxic conditions. They were then harvested, and whole-cell or nuclear extracts were prepared and processed for Western blotting as described previously (12).
Reporter Gene Assay and Luminescence Measurements
For HIF-1 transcriptional activity, we used a reporter expression plasmid (pBI-GL V6L) containing the luciferase gene under the control of HRE of the VEGF as described previously (12, 13).
Isolation and Analysis of RNA
Total RNA was extracted from cells using RNeasy Mini kit (Qiagen, Inc., Valencia, CA), reverse transcribed into cDNA using Reverse-iT 1st Strand Synthesis kit (ABgene, Epsom, Surrey, United Kingdom), and analyzed using quantitative real-time PCR to determine the expression of VEGF, ET-1, Glut-1, and HIF-1 genes as described previously (11). The expression of the each gene was normalized to Niemann-Pick mRNA expression levels. Sequences of the PCR primers, annealing, elongation, and acquisition temperatures for each gene are outlined in Table 1
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Data Analysis
The experiments presented in the figures are representative of three or more different repetitions. Quantification of band densities was done using the public domain NIH Image (version 1.61). Statistical analysis was done using a one-way ANOVA or t test. P < 0.05 was considered statistically significant.
Results
1,25(OH)2D3 Inhibits the Proliferation of Prostate and Colon Cancer Cells under Normoxia and Hypoxia
We first examined the effects of 1,25(OH)2D3 on cell proliferation under hypoxic conditions in various cancer cells (Fig. 1
). We used androgen-insensitive prostate cancer cells (CL-1 and PC-3), colon cancer cells (SW-480), and breast cancer cells (MCF-7; Fig. 1). CL-1 cells are highly aggressive androgen-insensitive prostate cancer cells originally derived from LNCaP cells (14). Compared with LNCaP, these cells represent a higher proliferative and malignant potential phenotype and express significantly higher levels of VEGF (14). The IC50 of 1,25(OH)2D3 on proliferation was significantly lower (10-fold) under hypoxia compared with normoxia in all cell lines tested (Fig. 1). In the SW-480 cells, the difference in the potency of 1,25(OH)2D3 on proliferation was even more pronounced under hypoxia (IC50, 0.4 nmol/L) than under normoxia (IC50, >10 nmol/L; Fig. 1B). Similar antiproliferative effects of 1,25(OH)2D3 were also observed in the androgen-sensitive LNCaP cells (data not shown). Our data show that the inhibitory effects of 1,25(OH)2D3 on cell proliferation were also apparent under hypoxia as they were under normoxia. Interestingly, the effect of 1,25(OH)2D3 on cell proliferation was even more potent under hypoxic conditions (Fig. 1).
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2-fold) after exposure to hypoxia in all cells and significantly was inhibited by 1,25(OH)2D3 under normoxia as well as under hypoxia with various extents (between 40–60%) among the different cell lines (Fig. 2). These results show that 1,25(OH)2D3 inhibits VEGF secretion in cancer cells under normoxic and hypoxic conditions.
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Protein Expression and HIF-1 Transcriptional Activity protein in a dose-dependent manner (Fig. 3A) between 60% to 90% among the various cells (Fig. 3B).
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50% significant inhibition of the expression of all genes tested especially under hypoxia (Fig. 4C). Taken together, our results show that 1,25(OH)2D3 inhibits HIF-1 protein expression as well as HIF-1–mediated transcriptional activation of VEGF and other HIF target genes.
1,25(OH)2D3 Affects HIF-1 Protein Expression on the Translational Level
To clarify the mechanism by which 1,25(OH)2D3 involves HIF-1, we examined the effects of 1,25(OH)2D3 on HIF-1 transcriptional and posttranscriptional levels. Analysis of RNA prepared from untreated and treated CL-1 cells with increasing doses of 1,25(OH)2D3 under normoxia or hypoxia revealed that HIF-1 mRNA expression was not significantly (P > 0.05) affected by any concentration of 1,25(OH)2D3 (Fig. 5A
). HIF-1 mRNA levels were relatively decreased under hypoxia compared with normoxia (Fig. 5A).
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protein stability. HIF-1 was induced by exposing the cells to hypoxia and subsequent exposure to room air (reoxygenation) in the presence of the protein translation inhibitor, cycloheximide (Fig. 5B, top). Reoxygenation causes a rapid HIF-1 degradation and, in the presence of cycloheximide, the new protein synthesis is inhibited; thus, HIF-1 levels predominantly reflect the degradation process of the protein. CL-1 cells were treated with either 0.1% ethanol or 100 nmol/L 1,25(OH)2D3 under hypoxia and then they were exposed to room air in the presence of cycloheximide for various times and analyzed by Western blotting for HIF-1 protein levels. Within 5 min of exposure to O2 in the presence of cycloheximide, HIF-1 protein levels from untreated and treated cells were decreased by 50% (Fig. 5B, bottom). Although the levels of the HIF-1 signal were reduced by 40% after 1,25(OH)2D3 treatment (different signals at the zero time point), the degradation rate of HIF-1 protein was almost the same under both conditions. Taken together, these results indicate that 1,25(OH)2D3 does not affect HIF-1 either on the transcriptional or on the posttranslational levels. It is most likely that 1,25(OH)2D3 affects HIF-1 new protein synthesis.
1,25(OH)2D3 Does Not Inhibit VEGF Expression in HIF-1 Knockout HCT116 Cells
Our results showed that 1,25(OH)2D3 treatment inhibits both the HIF-1 and the VEGF protein levels. To determine whether the decrease in VEGF levels by 1,25(OH)2D3 was mediated directly by the HIF-1 pathway, we used HIF-1 knockout colon cancer cells (HCT116HIF-1–/–). These cells do not exhibit any transcriptional activity of HIF as measured by reporter gene assay, which indicates that, although they may express HIF-2, it is transcriptionally inactive (data not shown). We measured the proliferation and VEGF secretion of these cells before and after treatment with 1,25(OH)2D3. As a control, we used the parental HCT116 cells, which express HIF-1 protein and exhibit induction of HIF-1 transcriptional activity under hypoxia unlike HCT116HIF-1–/– cells (data not shown). 1,25(OH)2D3 inhibited the proliferation of both HCT116 and HCT116HIF-1–/– cells under normoxic and hypoxic conditions (Fig. 6A and B
). Of note, HCT116 cells were more sensitive to 1,25(OH)2D3 under hypoxic than normoxic conditions similar to the observed results from SW-480 colon cancer cells (Fig. 1B).
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–/– cells expressed basal levels of VEGF protein similar to those of HCT116 cells (Fig. 6C and D). Whereas VEGF was significantly induced under hypoxia in HCT116 cells (Fig. 6C), its levels were not affected by hypoxia in HCT116HIF-1–/– cells (Fig. 6D). Moreover, 1,25(OH)2D3 inhibited the basal levels of VEGF and the hypoxia-induced VEGF in HCT116 cells (Fig. 6C), whereas 1,25(OH)2D3 did not affect VEGF protein levels under either normoxia or hypoxia in HCT116HIF-1–/– cells (Fig. 6D). These findings showed that the inhibition of VEGF levels by 1,25(OH)2D3 in this cancer cell model is dependent on the presence of HIF-1. Discussion
Inhibition of angiogenesis is one of the mechanisms that contribute to the antitumoral activity of 1,25(OH)2D3. 1,25(OH)2D3 inhibits the proliferation of tumor-derived endothelial cells in vitro (15) and also inhibits angiogenesis in vivo (6, 16). In the current study, we show that a significant part of the antiangiogenic effects of 1,25(OH)2D3 are mediated through the HIF pathway.
1,25(OH)2D3 reduces the expression of HIF-1 protein in various human cancer cells, leading to attenuation of HIF-1 transcriptional activity (Figs. 3 and 4). It seems that 1,25(OH)2D3 affects HIF-1 protein translationally rather than transcriptionally or posttranslationally (Fig. 5), similar to growth factors (8) and androgens (17).
The effects of 1,25(OH)2D3 on the HIF/VEGF pathway were confirmed when we used the HIF-1 knockout colon cancer cells (HCT116HIF-1–/–). 1,25(OH)2D3 failed to suppress VEGF expression levels under either normoxia or hypoxia in HCT116HIF-1–/– cells (Fig. 6). These results further emphasize the importance of the HIF pathway in 1,25(OH)2D3 inhibition of angiogenesis. It should be pointed out that 1,25(OH)2D3 concentrations used were above the physiologic range (0.1–1 µmol/L), which are usually applied for in vitro studies. It is therefore warranted to confirm the effects of 1,25(OH)2D3 on HIF in vivo using physiologic concentrations.
Interestingly, 1,25(OH)2D3 inhibited the proliferation of cancer cells also under hypoxia (Figs. 1 and 6). The results may indicate on the potency of 1,25(OH)2D3 to inhibit tumor growth under hypoxia, which exist in most solid tumors (18).
HIF-1 has become an acceptable target for cancer therapeutics over the past few years, with studies on cancer patients showing a positive role of HIF in tumor progression (19). HIF-1 is clearly overexpressed in the majority of human cancers and is associated with patient mortality and poor response to treatment (19). Thus, inhibiting the HIF pathway, whether selectively or nonselectively, would be useful for potential therapeutic implications. Our in vitro studies support the clinical observations that when 1,25(OH)2D3 was administrated in combination with docetaxel to treat androgen-independent prostate cancer patients, it significantly enhanced the effects of docetaxel (20). This further supports the rationale to use multiple targeted strategies, including 1,25(OH)2D3 analogues, in cancer therapy.
In this study, we found that HIF-1 is downstream to the vitamin D receptor. Interestingly, Banach-Petrosky et al. (21) very recently have shown that the development of high-grade prostate intraepithelial neoplasia can be prevented in Nkx3.1;Pten mutant mice by the early administration of 1,25(OH)2D3. In this model, AKT is constitutively activated (22). Because HIF-1 is downstream of AKT, it might be possible that a part of the preventive activity of 1,25(OH)2D3 in this model is attributed to HIF-1 inhibition. HIF-1 activation is an early event in prostate and other cancers (23, 24). It therefore would be more warranted to use 1,25(OH)2D3 and its low-calcemic analogues in cancer chemoprevention and treatment.
To the best of our knowledge, this is the first documentation that a significant part of the antiangiogenic effects of 1,25(OH)2D3 in hypoxic cancer cells are mediated via the HIF-1 pathway. Our results further support the rationale to use vitamin D as an antineoplastic agent. In addition, we believe that newly developed low-calcemic analogues of vitamin D could be initially tested in HRE-based high-throughput screening assays to evaluate their antiangiogenic potencies.
Acknowledgments
We thank Esther Eshkol for editorial assistance.
Footnotes
Grant support: M.K. Humanitarian Association, PCF Israel, Bat Sheva De Rothschild Foundation Grant of the Israel Ministry of Health and the Israel Academy of Science in memory of the late Professors A. Eldor and Y. Matzner, and the Israel Cancer Association through the Ben-Lehmsdorf Memorial Fund in memory of the late John Furman.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 11/ 3/06; revised 2/12/07; accepted 2/21/07.
References
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