Differential effects of vitamin D treatment on inflammatory and non-inflammatory breast cancer cell lines
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- Hillyer, R.L., Sirinvasin, P., Joglekar, M. et al. Clin Exp Metastasis (2012) 29: 971. doi:10.1007/s10585-012-9486-0
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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.
KeywordsCalcitriolInflammatory breast cancerInvasionMDA-MB-231MigrationSUM149Tumor emboliVitamin DVitamin D receptor
Vitamin D receptor
Vitamin D response elements
Inflammatory breast cancer
Fetal bovine serum
Phosphate buffered saline
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 . 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 [9–11] 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 . Induction of apoptosis in MCF-7 cells after exposure to 100 nM calcitriol over 48-72 h has been demonstrated . The induction of apoptosis in MCF-7 and SUM159 cells was reported to be through downregulation of Bcl-2 family proteins . 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
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.
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.
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.
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.
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.
Calcitriol treatment does not affect IBC cell proliferation but leads to increased protein synthesis
Calcitriol treatment inhibits IBC cell migration and invasion
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
IBC experimental metastases are decreased with calcitriol treatment
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 . 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 [9–11]. 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 .
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 . 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 . 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.
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.