Vitamin D3 stimulates embryonic stem cells but inhibits migration and growth of ovarian cancer and teratocarcinoma cell lines

Both human (NTERA-2 teratocarcinoma, A2780 ovarian cancer) and murine (P19 embryonal teratocarcinoma) germline-derived immortalized cell lines were employed in our studies. All cell lines were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). NTERA-2 cells were cultured in Dulbecco’s modified Eagle medium (DMEM; GE Healthcare) with heat-inactivated 10 % fetal bovine serum (FBS; Seradigm). A2780 ovarian cancer cells were maintained in Roswell Park Memorial Institute (RPMI) medium 1640 containing L-glutamine (GE Healthcare) and 10 % heat-inactivated FBS. P19 embryonal carcinoma cells were cultured in minimum essential medium (MEM)-α (GE Healthcare) supplemented with ribonucleosides, deoxyribonucleosides, 2.5 FBS, and 7.5 % bovine calf serum. To all media, penicillin (100 units/mL; Corning) and streptomycin (10 μg/mL; Corning) were added. All cells were cultured in a humidified atmosphere of 5 % CO₂ at 37 °C, with exchange of medium every 48 h.

The ESD3 cell line was purchased from ATCC and cultured in DMEM (GE Healthcare) with 4 mM L-glutamine, 1.5 g/L sodium bicarbonate, and 4.5 g/L glucose and supplemented with 0.1 mM 2-mercaptoethanol (Sigma-Aldrich), heat-inactivated 15 % FBS, and murine mature leukemia inhibitory factor (LIF, 5 ng/mL; Santa Cruz Biotechnology). Cells were maintained in a humidified atmosphere of 5 % CO₂ at 37 °C, and the medium was exchanged every 48 h.

Bone marrow mononuclear cells (BMMNCs) were obtained from total BM cells flushed from femurs and tibias, lysed in BD lysing buffer (BD Biosciences, San Jose, CA) for 10 min at room temperature, washed twice in RPMI 1640 medium containing L-glutamine (Corning), and supplemented with 2 % heat-inactivated FBS. Next, MNCs were stained with the following antibodies: anti-CD45R/B220 (phycoerythrin [PE], clone RA-6B2), anti-Gr-1 (PE, clone RB6-8 C5), anti-T cell receptor αβ (PE, clone H57-5970), anti-T cell receptor ɤδ (PE, clone GL3), anti-CD11b (PE, clone M1/70), anti-Ter119 (PE, clone TER-119), anti-CD45 (allophycocyanin [APC]–Cy7, clone 30-F11), and anti-Ly-6A/E (also known as Sca-1, PE–Cy5 or Alexa Fluor 647, clone E13–161.7) for 30 min on ice. Cells were washed and resuspended in RPMI medium with 2 % FBS. Hematopoietic stem cells (HSCs; Sca-1⁺Lin⁻CD45⁺) and very small embryonic-like stem cells (VSELs; Sca-1⁺Lin⁻CD45⁻) were purified from MNCs using a multi-parameter Moflo XDP cell sorter (Beckman Coulter) as described [29].

Clinical-grade human umbilical cord blood (hUCB) research units obtained from Cleveland Cord Blood Center were used for isolation of hUCB-HSPCs and hUCB-VSELs as described in detail in our previous studies [30]. In brief, using a cocktail of biotin-conjugated monoclonal antibodies and anti-biotin monoclonal antibodies conjugated to paramagnetic microbeads (Lineage Cell Depletion kit, Miltenyi Biotec), magnetic labeling of retrieved total nucleated cells was performed, and the lineage-negative (Lin⁻) cells were isolated by depletion of mature hematopoietic cells expressing a panel of lineage antigens using an autoMACS separator (Miltenyi Biotec). Afterwards, the Lin⁻ population were stained with the following antibodies: anti-CD45 (PE or V450, clone HI30) and anti-CD34 (APC or PE, clone 581). After washing, the fluorochrome-labelled cells were resuspended and sorted to obtain populations enriched in HSCs (Lin⁻/CD45⁺/CD34⁺) and VSELs (Lin⁻/CD45⁻/CD34⁺).

Total RNA was extracted and purified from teratocarcinomas and ESD3 cells using the RNeasy Mini kit (Qiagen Inc.) after treatment with DNase I (Qiagen Inc.) on a column. For VSELs and HSCs, total RNA was extracted using Trizol Reagent (Ambion; Austin, TX, USA) as described earlier [31]. The purified mRNA was afterwards reverse-transcribed into cDNA using Taqman Reverse Transcription Reagents (Applied Biosystems). Amplification of synthesized cDNA fragments was carried out using Amplitaq Gold polymerase (Applied Biosystems). The PCR conditions were: 1 cycle of 8 min at 95 °C; 2 cycles of 2 min at 95 °C, 1 min at 60 °C, and 1 min at 72 °C; 40 cycles of 30 s at 95 °C, 1 min at 60 °C, and 1 min at 72 °C; and 1 cycle of 10 min at 72 °C. Semi-nested PCR was performed only to evaluate expression of the VDR in sorted VSELs. The human and murine sequence-specific primers used for VDR amplification are listed in Additional file 1: Figure S1. Samples without template controls and reverse transcriptase were used in each run. All primers were designed using the NCBI/Primer-Blast program, as at least one primer included an exon–intron boundary. Afterwards, all PCR products were analyzed by 2 % agarose gel electrophoresis.

RT-qPCR experiments were performed to detect and quantify relative levels of VDR mRNA in both human and murine germline-derived immortalized cell lines. The purified RNA was reverse-transcribed with MultiScribe Reverse Transcriptase, oligo(dT), and a random hexamer primer mix (all from Applied Biosystems Life Technologies, CA, USA). Quantitative evaluation of the target gene was then performed by using an ABI Prism 7500 sequence detection system (Applied Biosystems Life Technologies) with Power SYBR-green PCR Master Mix reagent and specific primers (Additional file 1: Figure S1). The PCR cycling conditions were 95 °C (15 s), 40 cycles at 95 °C (15 s), and 60 °C (1 min). According to melting point analysis, only one PCR product was amplified under these conditions. The relative quantity of a target gene, normalized to the β2-microglobulin gene as the endogenous control and relative to a calibrator, was expressed as 2^–ΔΔCt (fold difference), where Ct is the threshold cycle, ΔCt = (Ct of target genes) – (Ct of the endogenous control gene, β2-microglobulin), and ΔΔCt = (ΔCt for target gene in test sample) – (ΔCt for target gene in calibrator sample).

Normal 2-month-old C57Bl6 mice (male) were exposed to a once-daily oral dosage of 8 × 10² IU 1,25-dihydroxyvitamin D3 in sesame oil for five consecutive days. Control mice received vehicle orally. Mice were also exposed to BrdU (1 mg/animal/100 μL; BD Pharmingen) 2 days before starting with 1,25-dihydroxyvitamin D3 treatment and continued to the end of the experiment as described [29]. Control mice were injected with saline and BrdU solution. After 30 days, mice were sacrificed, and a single-cell suspension was obtained and stained for the HSC and VSEL phenotypes as described above. After cell-surface staining of cells, the FITC BrdU Flow kit (BD Pharmingen) staining protocol was employed, including fixation and permeabilization, treatment with DNase to expose incorporated BrdU, and finally staining with anti-BrdU–FITC antibody. After washing, samples were analyzed using a BD LSR II flow cytometer (BD Biosciences). At least one million events were captured and analyzed using BD FACSDiva software.

All cell lines were rendered quiescent by incubation in their basic medium supplemented with 0.5 % bovine serum albumin (BSA, Sigma-Aldrich) at 37 °C and then seeded at a density of 6 × 10⁴ cells/100 μL/insert into the upper chambers of Transwell inserts with 8-μm polycarbonate membranes (Corning). The lower Boyden chambers received 1,25-dihydroxyvitamin D3 (Sigma-Aldrich) at both physiological (10⁻¹⁰ M) and supra-physiological (10⁻⁹–10⁻⁷ M) concentrations in culture medium with 0.5 % BSA. The lower chambers containing 10 FBS and 0.5 % BSA (plus vehicle) in RPMI 1640 medium served as a positive and negative control, respectively. After 24-h stimulation at 37 °C, the upper chambers were carefully removed, the cells that had not migrated were removed with a cotton applicator swab from the upper side, and the cells that had transmigrated to the lower side of the membrane were fixed and stained with HEMA-3 reagent (Protocol, Fisher Scientific, Pittsburgh, PA) and then counted using an inverted microscope.

All cell lines were made quiescent for 3 h with 0.5 % BSA in basic medium in a humidified atmosphere of 5 % CO₂ at 37 °C. Next, the cells were stimulated with 1,25-dihydroxyvitamin D3 (at concentrations ranging from 10⁻¹⁰–10⁻⁷ M), SDF-1 (300 ng/mL), or 0.5 % BSA (plus vehicle) in their respective basic culture media. Cells were then added directly and allowed to adhere to the fibronectin-coated wells (3000 cells/well) in 96-well plates at 37 °C. The wells were first coated with 70 μL of fibronectin (Sigma-Aldrich; 10 μg/mL) overnight at 4 °C and blocked before the experiment with BSA for 2 h at 37 °C. After a 5-min incubation at 37 °C, the wells were vigorously washed three times with PBS, and the remaining adherent cells were counted directly under an inverted microscope.

To evaluate the functionality of the vitamin D receptor (VDR), quiescent cells were stimulated with 0.5 % BSA medium (containing vehicle) or 1,25-dihydroxyvitamin D3 (10⁻¹⁰, 10⁻⁹, 10⁻⁸, or 10⁻⁷ M) for 5 min or 12 h at 37 °C. The harvested cells were washed with PBS, lysed with RIPA lysis buffer supplemented with protease and phosphatase inhibitors (Santa Cruz Biotech) for 30 min on ice, and centrifuged at 15,000 rpm at −4 °C for 15 min. The protein concentration was measured using the Pierce BCA Protein Assay Kit (Pierce, Rockford, IL). The adjusted extracted proteins (20 μg/each sample) were then separated on a 4–12 % SDS-PAGE gel, and the fractionated proteins were transferred to a PVDF membrane (Bio-Rad). All membranes were blocked with 2.5 % BSA in Tris-buffered saline containing 0.1 % Tween (TBST) for 1 h at room temperature. After washing with TBST, phosphorylation of the intracellular kinase p44⁄42 mitogen-activated protein kinase (p42/44 MAPK) and AKT was detected by incubating the membranes with phosphospecific anti-p-p42/44 MAPK (clone no. 9101, diluted 1:1000) and anti-p-AKT (Ser473; clone no. 9271, diluted 1:1000) rabbit polyclonal antibodies (Cell Signaling), respectively, overnight at 4 °C. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG was used as a secondary antibody (Santa Cruz Biotech, 1:5000) and was incubated with the PVDF membranes for 2 h at RT. To confirm equal protein loading in all lanes, blots were stripped using stripping buffer (Thermo Scientific) and then reprobed with appropriate anti-rabbit p42/44 MAPK (clone no. 9102) and anti-rabbit AKT (clone no. 9272) monoclonal antibodies (both from Cell Signaling). All membranes were treated with an enhanced chemiluminescence (ECL) reagent (Amersham Life Sciences) and subsequently exposed to film (Hyperfilm, Amersham Life Sciences). For band visualization, the automatic machine supplied with fresh warm developer and fixer solutions was used.

Cells were cultured in 96-well plates (Cell Star; Greiner Bio-One) at an initial density of 3 × 10³ cells/mL with 0.5 % BSA in DMEM (NTERA2), RPMI 1640 (A2780), or αMEM (P19) medium in the presence or absence of 1,25-dihydroxyvitamin D3 (10⁻¹⁰ M, 10⁻⁹ M, 10⁻⁸ M, or 10⁻⁷ M). The medium containing 0.5 % BSA with vehicle was used as a negative control, while full medium containing 10 % FBS was treated as a positive control. The cell number was calculated directly after cell seeding (0 h) as well as at 24, 48, and 72 h after adding 1,25-dihydroxyvitamin D3. At these time points, cells were harvested from the wells after trypsinization and counted using FACS.

Prior to in vivo transplantation, human germline-derived immortalized cells were pretreated ex vivo with 1,25-dihydroxyvitamin D3 (10⁻⁹ M) or vehicle alone for 12 h. The cells were then washed and transplanted (10 × 10⁵ per mouse) into severe combined immunodeficient (SCID)-beige inbred mice (n = 3 per group), which were irradiated with 750 cGy 24 h before transplantation. At 48 h post transplantation, bone marrows, livers, and lungs were collected, and the presence of metastasized cancer cells (i.e., murine–human chimerism) was evaluated as described [32]. Briefly, genomic DNA was purified from organs using the QIAamp DNA Mini kit (Qiagen). Next, detection of human α-satellite and murine β-actin DNA levels was carried out using real-time PCR and the ABI Prism Fast 7500 Sequence Detection System (Applied Biosystems). A 25-μL reaction mixture containing 12.5 μL SYBR Green PCR Master Mix, 300 ng DNA template, and specific primers (5′-accactctgtgtccttcgttcg-3′ [sense] and 5′-actgcgctctcaaaaggagtgt-3′ [antisense] primers for α-satellite DNA; 5′-ttcaattccaacactgtcctgtct-3′ [sense] and 5′ ctgtggagtgactaaatggaaacc-3′ [antisense] primers for β-actin DNA) were used. Real-time PCR conditions for the amplification process were as follows: 95 °C (15 s); 40 cycles at 95 °C (15 s), and 60 °C (1 min). Samples without template controls were used in each run. The ΔCt values were determined, where Ct is the threshold cycle. For each cell line, the number of human cells present in the murine organs (the degree of chimerism) was calculated according to the standard curve generated by mixing different concentrations of human cells with a constant number of murine cells in a linear manner.

Human carcinoma cells were treated in RPMI/0.5 % BSA medium containing either 1,25-dihydroxyvitamin D3 (10⁻¹⁰ M, 10⁻⁹ M, or 10⁻⁸ M) or vehicle only. Cells subjected to culture medium with 10 % DMSO were used as a positive control. After a 12-h treatment period, the cells were gently detached using non-enzymatic cell dissociation buffer (Sigma-Aldrich), pelleted, and washed twice with cold PBS. Next, to define the levels of early and late apoptotic cells, the FITC Annexin V Apoptosis Detection kit (#556547; BD Pharmingen) was employed according to the manufacturer’s protocol. In brief, 1 × 10⁵ cells/100 μL Annexin V Binding Buffer were incubated in the dark with FITC–annexin V and propidium iodide (PI), as a vital dye, for 15 min at 25 °C. The cells that were left unstained or stained with either FITC–annexin V or PI served as compensation staining controls. Afterwards, cells were analyzed by flow cytometry after adding Annexin V Binding Buffer (400 μL) to all tubes. The percentage of cells that had undergone apoptosis was then determined by calculating the percentage of apoptotic cells in the untreated population (vehicle) from the percentage of apoptotic cells in the 1,25-dihydroxyvitamin D3-treated population.

Statistical analysis was carried out using GraphPad Prism 6 (GraphPad Software Inc) and Sigma software (Sigma Software Inc). All data are presented as mean ± SD. Statistical analysis of the data was done using one-way ANOVA and Tukey’s test for post hoc pairwise multiple comparison. In all analyses, p ≤ .05, p ≤ .01, and p ≤ .001 were considered significant.