Clinical & Experimental Metastasis

, Volume 29, Issue 8, pp 971–979

Differential effects of vitamin D treatment on inflammatory and non-inflammatory breast cancer cell lines

Authors

  • Rebecca L. Hillyer
    • The Department of Biological ScienceThe University of Delaware
  • Padma Sirinvasin
    • The Department of Biological ScienceThe University of Delaware
  • Madhura Joglekar
    • The Department of Biological ScienceThe University of Delaware
  • Robert A. Sikes
    • The Department of Biological ScienceThe University of Delaware
    • The Center for Translational Cancer ResearchThe University of Delaware
  • Kenneth L. van Golen
    • The Department of Biological ScienceThe University of Delaware
    • The Center for Translational Cancer ResearchThe University of Delaware
    • The Department of Biological ScienceThe University of Delaware
    • The Center for Translational Cancer ResearchThe University of Delaware
Research Paper

DOI: 10.1007/s10585-012-9486-0

Cite this article as:
Hillyer, R.L., Sirinvasin, P., Joglekar, M. et al. Clin Exp Metastasis (2012) 29: 971. doi:10.1007/s10585-012-9486-0

Abstract

Vitamin D is a known regulator of breast cancer cell proliferation, apoptosis, migration, invasion and differentiation in vitro. Recent studies have suggested a preventative role for vitamin D in breast cancer development and suggested a possible therapeutic application of vitamin D for patients with various forms of breast cancer. Inflammatory breast cancer (IBC) is a highly aggressive and phenotypically unique form of breast cancer that has a very poor prognosis. IBC invades the dermal lymphatics of the breast as tumor emboli early in the course of the disease. Because of the invasive nature of IBC, novel therapeutics are needed desperately. In the current study we examined the effect of the active form of vitamin D, calcitriol, treatment on the aggressive IBC phenotype. Herein we demonstrate that although the vitamin D receptor (VDR) is present in both IBC and non-IBC cell lines, the effect of vitamin D treatment is significant only on the IBC cells. SUM149 IBC cells showed increased protein concentration in response to 24 h of calcitriol exposure; likely mediated by an increase in protein synthesis as opposed to increased cellular proliferation. In addition, treatment with 100 nM calcitriol showed a significant decrease in SUM149 migration (67.8 % decrease, P = 0.030), invasion (43.9 % decrease, P = 0.015), and tumor spheroid size (69.4 % decrease, P = 0.018) compared to nontreated control groups. Finally, calcitriol treatment of SUM149 cells led to significantly fewer IBC experimental metastases as compared to control. Our study demonstrates that calcitriol treatment of SUM149 affected several of the processes important for IBC metastasis but had little effect on MDA-MB-231 cells. Therefore, calcitriol treatment may have the potential to decrease the rate and incidence of metastasis in IBC patients.

Keywords

CalcitriolInflammatory breast cancerInvasionMDA-MB-231MigrationSUM149Tumor emboliVitamin DVitamin D receptor

Abbreviations

VDR

Vitamin D receptor

VDREs

Vitamin D response elements

IBC

Inflammatory breast cancer

FBS

Fetal bovine serum

PBS

Phosphate buffered saline

Introduction

Inflammatory breast cancer (IBC) is arguably the deadliest form of breast cancer with mean 5- and 10-year disease-free survival rates of <45 and 20 %, respectively [1, 2]. IBC is molecularly and phenotypically distinct from other forms of breast cancer manifesting as diffuse sheets or cords of tumor cells in the breast [3, 4]. IBC is highly metastatic and is hallmarked by the presence of tumor emboli in the dermal lymphatic vessels of the skin overlying the breast. Infiltration of the breast dermal lymphatics by IBC results in the unique clinical presentation of the disease, which resembles an infection or rash. At the time of diagnosis nearly all women are lymph node positive, stage 3 disease, and approximately 1/3 have gross distant metastasis [1, 5]. Because IBC is so aggressive it is treated by chemotherapy, followed by surgery and then radiation with limited success [6, 7]. Thus, new therapeutic agents that are less aggressive and more efficient are being sought to treat IBC.

While vitamin D is an important dietary component, the majority of vitamin D within the body is obtained through exposure to sunlight with supplementation through the consumption of natural and fortified foods [8]. Vitamin D obtained through diet or exposure to sunlight is inert originally and must be activated within the body to form 1,25-dihydroxyvitamin D3 also known as calcitriol.

The effect of calcitriol on cancer cells is well studied [911] and previous reports demonstrated the anti-proliferative effects of calcitriol on MCF-7 and SUM159 breast cancer cells. Arrest in the G1 phase of the cell cycle in response to calcitriol was associated with alterations in the expression of cell cycle regulators, such as increased cyclin-dependent kinase inhibitors [12]. Induction of apoptosis in MCF-7 cells after exposure to 100 nM calcitriol over 48-72 h has been demonstrated [13]. The induction of apoptosis in MCF-7 and SUM159 cells was reported to be through downregulation of Bcl-2 family proteins [14]. In addition, it has been suggested that modulation of growth factor signaling may be responsible for the decreased MCF-7 proliferation. Specifically, calcitriol has been shown to block the mitogenic effects of insulin-like growth factor I (IGF-I) through the down-regulation of its receptor, leading to a decrease in proliferation and an increase in apoptosis [15, 16]. Although multiple epidemiological studies showed an inverse relationship between Vitamin D3 and breast cancer risk, current randomized clinical trial results have not confirmed this link. The discrepancy may be due to the variable and insufficient dosage of Vitamin D3 used in these trials. For example, a randomized, double blind, seven-year study revealed no correlation between breast cancer risk and Vitamin D3 supplementation. The dose of Vitamin D3 given in this study was 400 IU/day, far below the required 1,700–2,000 IU needed to produce a 25(OH)D3 serum level of 75 nmol/L which had been linked to low breast cancer risk [17, 18]. The dose of Vitamin D or calcitriol used is crucial not only in a preventative application but especially in a therapeutic application.

In the current study, we compared the effects of 1,25-dihydroxyvitamin D3 (calcitriol) treatment on the SUM149 IBC cell line compared with the highly invasive, and cell-type-of-origin matched MDA-MB-231 non-IBC cell line. A large number of inflammatory breast cancers are triple-negative and fall within the basal subtype of origin category. SUM149 cells are triple-negative and basal subtype of origin; and, they have proven to be highly representative of IBC patient samples. The MDA-MB-231 non-inflammatory breast cancer cell line is also triple negative and basal subtype of origin. For comparing IBC with non-IBC cells, the MDA-MB-231 cell line is an appropriate comparator with SUM149. We did not use MCF-7 cells in this study since they are ER+, have low invasive and metastatic capabilities and are of the luminal A subtype of origin. We found that calcitriol treatment did not affect cell proliferation. The IBC cells, however, had a marked decrease in their ability to migrate and invade; whereas, the MDA-MB-231 cells essentially were unaffected after 12 h treatment. Additionally, calcitriol treatment affected the ability of the IBC cells to form tumor emboli in a novel 3D growth system. Finally, calcitriol treatment of SUM149 cells led to significantly fewer experimental metastases by IBC cells as compared to controls. These results suggest a potentially significant effect of calcitriol on IBC cells.

Methods and materials

Cell culture

The MDA-MB-231 cell line was obtained from ATCC (no. HTB-26), and the SUM149 cell line was received from Dr. Steve Ethier, the cell line originator. MDA-MB-231 human breast cancer cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, CellGro, Manassas, VA) supplemented with 5 % FBS (HyClone), 1 % penicillin–streptomycin solution (10,000 IU/mL penicillin, 10,000 μg/mL streptomycin, CellGro), 1 % l-glutamine (200 mM solution, CellGro), and 0.2 % insulin stock solution (0.75 mg/mL insulin in PBS, Sigma-Aldrich, St. Louis, MO). SUM149 human inflammatory breast cancer cells were maintained in Ham’s F-12 Medium (CellGro) supplemented with 5 % FBS, 1 % penicillin–streptomycin solution, 1 % antibiotic–antimycotic solution (10,000 IU/mL penicillin, 10,000 μg/mL streptomycin sulfate, 25 μg/mL amphotericin, CellGro), 1 % l-glutamine, 0.1 % hydrocortisone stock solution (1 mg/mL, Sigma-Aldrich) and 1 % insulin–transferrin–selenium stock solution (1 mg/mL insulin, 1 mg/mL transferrin, 3.4 μM sodium selenite, ScienCell, san Diego, CA). Both cell lines were incubated in a humidified atmosphere maintained at 5 % CO2 and 37 °C.

Prior to all experimental procedures, both MDA-MB-231 and SUM149 cell lines were grown to approximately 75 % confluence in a T-75 flask. Cells were detached from the bottom of the flask using trypsin (0.25 % trypsin in HBSS without EDTA, CellGro, Vanassas, VA). The cell suspension was transferred to a 15 mL tube and media with 5 % FBS was added to the cell suspension. The cells were centrifuged at 2,500 rpm for 5 min to form a pellet. The supernatant was removed and the cell pellet was re-suspended in the appropriate volume of media. The concentration of cells in the solution was determined using a hemocytometer, and the cell suspension was diluted to obtain the necessary cell concentration for each procedure.

Reverse transcription polymerase chain reaction (RT-PCR)

Prior to RT-PCR, RNA was extracted from the MDA-MB-231 and SUM149 cell lines with Trizol Reagent (Invitrogen, Carlsbad, CA) per the manufacturer’s recommendations. The final RNA pellet was resuspended in 40 μL RNAase-free water (Invitrogen, Carlsbad, CA) and the concentration and quality of the RNA was measured. Complementary DNA (cDNA) was then made using the SuperScript 2 First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA) per the manufacturer’s directions using 35 cycles.

RT-PCR was performed by combining 1 μL of the forward primer (100 nM), 1 μL of the reverse primer (100 nM), 1 μL cDNA, and 7 μL GoTaq Green Master Mix (Promega, Madison, Wi). The GoTaq Green Master Mix was made using the components of the GoTaq Hot Start Polymerase kit (Promega, Madison, Wi) and contained the following: 10 μL of 5× Green GoTaq Flexi Buffer, 4 μL of 25 mM MgCl2, 1 μL of 10 mM dNTP mix, 0.25 μL GoTaq Hot Start Polymerase, and 23.75 μL nuclease free water. The base sequence of the forward primer for the VDR was 5′-ATGGCCATCTGCATCGTCTC-3′ and the base sequence of the reverse primer for the VDR was 5′-GCACCGCCACAGGCTGTCCTA-3′. The positive control contained the β-actin forward primer (5′-TGTGATGGTGGGAATGGGTCAG-3′) and reverse primer (5′-TTTGATGTCACGCACGATTTCC-3′); the negative control contained no primer.

Proliferation assay

MDA-MB-231 and SUM149 cell lines were plated in 6-well tissue culture dishes at a concentration of 1.0 × 106 cells/mL. Each well was then treated with increasing concentrations, 0, 10 and 100 nM, of calcitriol (Alexis Biochemicals, Plymouth Meeting, PA). The cells were incubated at 37 °C for 24 h. The media was removed from each well, cells trypsinized and counted by hemocytometer.

Protein analysis

MDA-MB-231 and SUM149 cell lines were plated in 6-well culture dishes at a concentration of 1.0 × 106 cells/mL, treated with increasing concentrations, 0, 10 and 100 nM, of calcitriol and incubated at 37 °C for 24 h or 7 days. Cycloheximide, to inhibit protein synthesis, was added at 10 μg/ml. The media was removed from the wells, and each well was rinsed twice with phosphate buffered saline (PBS). Cells were lysed by addition of 500 μL lysis buffer [(0.5 mL of 1 % Triton (Fisher Scientific), 0.1 mL of 1 mM EDTA (Fisher Scientific), and 0.5 mL of 10 mM Tris (Fisher Scientific), 10 μL of protease inhibitor mix (BD Biosciences, San Jose, CA) and 10 μL of PMSF (Gold Biotechnology, St Louis, MO) for 5 min on ice. Cells were scraped and transferred to microfuge tubes. A 10 μL aliquot of each sample was added to a 96-well plate and protein concentrations determined using BCA Kit (Thermo Scientific Pierce, Waltham, MA). The MTT assay was performed according to manufacturer’s protocol.

Scratch assay

MDA-MB-231 and SUM149 cell lines were seeded in 6-well culture dishes at concentrations of 3.0 × 105 cells/mL and incubated at 37 °C for 24 h until confluent. After incubation, the medium was removed and new medium added with 0, 10, or 100 nM calcitriol. A scratch was made down the middle of each well with a silicon coated pipette tip and 4 pictures were taken of each scratch. The cells were incubated for 48 h and final pictures were taken of each scratch. Using ImageJ 1.42q, the width of each scratch was calculated. For each well, the average width of the initial scratch was compared to the average width of the final scratch, and the calculated difference was considered the migration distance of the cells over 48 h.

Invasion assay

Precoated 24-well Matrigel invasion chambers (BD Biosciences) brought to RT and rehydrated with serum-free medium for 2 h at 37 °C. Cells were harvested and suspended in serum-free media at a concentration of 1.0 × 105 cells/mL and 0.25 mL added to each insert (2.5 × 104 cells/well). Then, 0.25 mL of serum-free media containing 0, 20, 100, or 200 nM 1,25-dihyrdoxyvitamin D3 was added to each insert. Functioning as the chemoattractant for cellular invasion, 0.75 mL of serum-containing growth medium was added to the bottom of each well. The invasion chambers were incubated at 37 °C for 22 h in a humidified tissue culture incubator.

After incubation, the media was removed from the inserts and the bottom of the wells. A cotton swab was used to remove non-invaded cells from the top of the inserts. 0.5 mL of crystal violet stain (Invitrogen) was added to each insert for 45 min, washed in water to remove excess stain and allowed to dry for 48 h. Photomicrographs at 2.5× were taken of each insert using a confocal microscope. The number of invaded cells in each picture was counted using the Volocity Software (Improvision/Perkin Elmer, Waltham, MA). Results were validated by manual counting.

In vitro IBC spheroid growth

SUM149 cells were suspended in media supplemented with 0.5 % low melting point agar, analytical grade (Thermo Scientific, Waltham, MA) in 25 cm3 suspension flasks (Greiner BioOne, Monroe, NC). Cells were treated with 0 or 100 nM calcitriol and incubated on an orbital shaker at approximately 40 rpm for 72 h to allow for emboli formation. Six representative pictures were taken per flask of the formed emboli. The ImageJ 1.42q computer program was used to calculate the diameter of the representative emboli from each flask.

Experimental metastasis assay

SUM149 cells were grown in T150 flasks, treated with, 0, or 100 nM, of calcitriol and incubated at 37 °C for 24 h. Cells harvested by brief trypsinization followed by washing and resuspended at a final concentration of 1 × 106 cells/100 μl. Female athymic nude mice, 8 weeks old, were injected via tail vein with 100 μl tumor cells and monitored for 10 weeks. The two groups consisted of 5 mice each as determined by power analysis. Mice were euthanized, lungs harvested, fixed in neutral buffered formalin and stained with hemotoxylin and eosin. Lungs were assessed by a pathologist and one investigator and reported as mean and range of metastases.

Results

Calcitriol treatment does not affect IBC cell proliferation but leads to increased protein synthesis

Calcitriol activates gene transcription trough binding to the Vitamin D receptor (VDR). Therefore in order for cancer cells to respond to calcitriol by gene activation or suppression, the VDR has to be expressed. A review of microarray data on Oncomine (www.oncomine.org) demonstrates that the VDR mRNA is present in nearly all forms of breast cancer including IBC. It was also shown that the MDA-MB-231 breast cancer cell line expresses the VDR mRNA; however, it is unknown if the SUM149 IBC cell line expresses VDR. Detection of VDR protein is notoriously difficult therefore we performed RT-PCR using 35 cycles to determine the presence of VDR mRNA in MDA-MB-231 and SUM149 cell lines. Figure 1 demonstrates that the mRNA for VDR is present in both breast cancer cell lines. These data suggest that SUM149 cells should respond to calcitriol treatment.
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Fig. 1

Presence of vitamin D receptor mRNA. a Expression of the vitamin D receptor mRNA in the MDA-MB-231 and SUM149 cell lines was assayed by RT-PCR (35 cycles). Primers and reaction conditions are listed in the “Materials and methods” section. Shown is a representative gel electrophoresis of RT-PCR products. The experiment was performed in triplicate

Protein concentration is a measure of both individual cell size and total cell number. Any changes in protein content can be attributed to growth of individual cells, an increase in cell number, or both. As shown in Fig. 2, neither MDA-MB-231 nor SUM-149 cell lines exhibit any significant changes in cell number with calcitriol treatment. Therefore changes in protein content are due to increased cell size or protein content per cell. BCA protein analysis was used to determine the protein content of both cells lines. Cells were plated in a 6-well dish at equal cell numbers and treated with 10 or 100 nM of calcitriol. Control groups were untreated MDA-MB-231 and SUM149 cells. Following 24 h of treatment protein was extracted. Since 100 nM calcitriol treatment in SUM149 cells increased protein concentration after 24 h we investigated if it increases protein synthesis at longer time points. For this cells were stimulated with calcitriol for 7 days. As controls, cells were pretreated with cycloheximide to inhibit de novo protein synthesis. BCA protein analysis then was used to determine the concentration of total cellular protein within each sample as shown in Fig. 2. Additionally, a MTT assay was performed with cells treated with calcitriol for 7 days to determine metabolic activity of the cells. As determined by ANOVA analysis, the protein content obtained from MDA-MB-231 cells and SUM149 cells treated with calcitriol was significantly different compared to control (P < 0.05) while the metabolic activity was unchanged at day 7 compared to the respective control groups for the SUM149 cells.
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Fig. 2

Effect of 1,25-dihydroxyvitamin D3 on proliferation and protein concentration. a Effect of calcitriol on proliferation. Cells were treated for 24 h followed by cell counting. The effect of 1,25-dihydroxyvitamin D3 treatment had no effect on cell number as determined by ANOVA followed by Tukey’s test. Error bars shown are one standard deviation. b Effect of calcitriol on protein content. Cells were treated with calcitriol at time of plating. Twenty four hours after plating, the cells were used in BCA protein analysis. c Effect of calcitriol on SUM149 protein content after 7 days. Cycloheximide (10 μg/ml) was added as an inhibitor of protein synthesis. d MTT assay of SUM149 cells after 7 days of calcitriol treatment. The protein concentrations were compared to the control (0 nM 1,25-dihydroxyvitamin D3) within each experiment to calculate the percent change. Error bars shown are one standard deviation. *Significant compared to control, p < 0.05 as determined by ANOVA followed by Tukey’s test. The experiment was performed in triplicate

Calcitriol treatment inhibits IBC cell migration and invasion

Since calcitriol treatment had no effect on breast cancer cell growth, we set out to determine if linear migration and invasion were affected. A monolayer scratch assay was performed on calcitriol treated SUM149 and MDA-MB-231 cell lines. As shown in Fig. 3a, SUM149 cells had a dose-dependent decrease in migration in response to calcitriol treatment. Compared to untreated cells SUM149 cells treated with 100 nM calcitriol showed a significant decrease (67.8 %) in migration. In contrast the MDA-MB-231 cells did not show any significant change in migration when Calcitriol treated cells were compared to controls.
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Fig. 3

Effect of 1,25-dihydroxyvitamin D3 treatment on IBC cell migration and invasion. a Cells were grown to confluence and a scratch were made. The cells were treated with1,25-dihydroxyvitamin D3 24 h after plating. The migration distance was calculated from the difference in scratch width at 24 h after plating compared to the scratch width at 72 h after plating. The migration distances were then compared to the control (0 nM calcitriol) within each experiment to get the percent change. *Significant compared to control, P < 0.05 as determined by ANOVA followed by Tukey’s test. (B Cells were treated with 1,25-dihydroxyvitamin D3 at time of seeding into a Matrigel invasion assay. Invaded cells were stained and counted 24 h after plating and invasion was measured as the percent change in number of invaded cells compared to the control for each experiment. *Significant compared to control (0 nM 1,25-dihydroxyvitamin D3), P < 0.05 as determined by ANOVA followed by Tukey’s test. Error bars shown are one standard deviation. The experiments were performed four separate times

Next we set out to determine if invasion was affected by calcitriol treatment. Treatment with calcitriol had a significant effect on SUM149 cellular invasion (P = 0.015). As shown in Fig. 2b, SUM149 cells demonstrated a dose-dependent decrease in invasion in response to 10, 50 and 100 nM calcitriol treatment, respectively 10.7, 33.3 and 43.9 %. However, MDA-MB-231 cells did not show any significant change in invasion following calcitriol treatment as compared to the controls.

Calcitriol treatment reduces formation of IBC spheroids in vitro

A hallmark of IBC is the formation of tumor emboli in the dermal lymphatic vessels, thus, we wanted to determine if SUM149 cells grown in conditions to promote in vitro spheroid formation would be affected by calcitriol treatment. SUM149 cells were plated in suspension flasks with medium containing 0.5 % low melting point agar to increase the viscosity to approximate that of lymphatic fluid. The MDA-MB-231 cells do not form spheroids under these conditions. At the time of plating, cells were left untreated or treated with 100 nM calcitriol. As shown in Fig. 4, the SUM149 cells treated with 100 nM calcitriol for 72 h formed emboli that were 69.4 % smaller (P = 0.018) than control SUM149 cells.
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Fig. 4

Changes in SUM149 Spheroid Growth. Cells were treated with 1,25-dihydroxyvitamin D3 at the time of plating. At 72 h after plating the diameter of the spheroids was measured. a, b representative images of nontreated and treated emboli. c Quantification of emboli size. *Significant compared to control (0 nM Calcitriol), P < 0.05 as determined by ANOVA. Error bars shown are one standard deviation. Shown are the results of three separate experiments

IBC experimental metastases are decreased with calcitriol treatment

To determine if calcitriol treatment affected metastasis, we performed an experimental metastasis assay. SUM149 cells were either left untreated or treated with calcitriol ex vivo for 24 h and injected via tail vein into female athymic nude mice. Figure 5 demonstrates that calcitriol pretreatment led to significantly fewer experimental metastases (P = 0.0025). All mice (5/5) injected with untreated SUM149 cells had evidence of lung metastases. Two of these tumors (2/5 or 40 %) had pathological necrosis due to rapid growth. By contrast, 40 % (2/5) mice injected with calcitriol treated SUM149 cells had evidence of experimental metastasis. This is a 60 % reduction as compared to untreated SUM149 cells.
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Fig. 5

Effect of 1,25-dihydroxyvitamin D3 on metastasis. SUM149 cells were pretreated with 1,25-dihydroxyvitamin D3 or vehicle control 24 h prior to injection. Athymic female nude mice were injected via tail vein with 1 × 106 cells (n = 5 for each group) and kept for 10 weeks. Lungs were harvested, fixed, H&E stained and examined for gross and micrometastases. *Significant compared to untreated control, P = 0.0025 as determined by a paired t test. Shown are representative lung sections along with median and range of lung metastases

Discussion

Inflammatory breast cancer is arguably the most deadly form of breast cancer and this is thought to be due to its propensity to disseminate through the body via the dermal lymphatics as tumor emboli. IBC is phenotypically and molecularly distinct from other types of breast cancer. Typically, IBC has alterations in protein expression that are different from that of other forms of breast cancer [4]. For example, E-cadherin and caveolin-1 levels increase in IBC; whereas, their expression tends to be lost in non-IBC [1, 19, 20]. Because IBC is so aggressive current treatment begins with chemotherapy, involving both taxanes and anthracyclines, followed by radical mastectomy and finally localized radiation therapy. Despite aggressive intervention, the 5- and 10-year disease-free survival rates are <45 and 20 %, respectively. Therefore, new therapies are aggressively being sought to treat IBC.

Vitamin D is an important dietary component that is converted to calcitriol and has shown promise in decreasing breast cancer cell growth and inducing apoptosis [911]. In the current study, we compared calcitriol treatment of the SUM149 IBC cell line with a similar, highly invasive, cell-type-of-origin matched non-IBC line, MDA-MB-231 and found a difference in the effect on IBC migration and invasion.

In our study we found that both cell lines express the mRNA for the Vitamin D receptor, suggesting that both should respond to calcitriol treatment. We found that calcitriol treatment of the cell lines did not affect growth or survival of either highly metastatic breast cancer cell line SUM149 or MDA-MB-231 within 24 h or 7 days of treatment. Interestingly, stimulation of SUM149 cells with calcitriol led to a significant increase in protein concentration after 24 h and 7 days. An MTT assay after 7 days of calcitriol treatment, however, showed no increased metabolic activity of SUM149 cells. These data suggest that the cells do not undergo apoptosis in response to calcitriol treatment. Previous studies using MCF-7 and SUM159, cells with low to moderate metastatic potential, demonstrated G1 arrest and down-regulation of the anti-apoptotic protein Bcl-2 in response to calcitriol. Even at the highest dose of 100 nM, SUM149 and MDA-MB-231 had no significant change in cell numbers after 7 days (% change in cell number: SUM149 2.8 ± 7.6 %, MDA-MB-231 1.1 ± 2.4 %). A similar finding was reported where the MDA-MB-231 cell line had little to no growth inhibition when treated with up to 1 mM of calcitriol [21].

The motile capabilities of the SUM149 IBC cells were significantly decreased (68 % for migration and 42 % of invasion) at 100 nM of calcitriol treatment but not at 10 nM. No significant effect of calcitriol on the invasive and migratory potential of the MDA-MB-231 cells in response to 24–48 h of exposure was observed. However, recent research showed a dose-dependent decrease in invasion of MDA-MB-231 cells when treated for 4 days or longer with a concentration of 3.2 nM 1,25-dihydroxyvitamin D3, potentially suggesting a difference in the response of genes induced by vitamin D [22]. Upon Calcitriol stimulation, tumor spheroids were about 5 fold smaller. This reduction in tumor spheroid size could potentially impact upon the metastatic spread of IBC. Calcitriol treated emboli injected into mice showed reduced ability to form lung metastases.

The use of vitamin D as a potential adjuvant therapy for IBC is very attractive as it is well tolerated with minimal and manageable side-effects. In adults, vitamin D intoxication has been reported only at very high doses, greater than 50,000 IU [23]. Since IBC readily disseminates throughout the body, a treatment that specifically targets the motile phenotype of IBC would be ideal. Currently, we are testing a preclinical model of vitamin D therapy for IBC.

Acknowledgments

We would like to thank Dr. Kirk Czymmek and the Delaware Biotechnology Institute for their assistance with imaging and computer analysis. We would also like to thank Vimal Gangadharan and Heather Unger for their help with experimental procedures We thank Jomnarong Lertsuwan and Adam Aguiar, for their help with RT-PCR and scratch assay methods.

Copyright information

© Springer Science+Business Media B.V. 2012