1,25(OH)₂D₃ was purchased in Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Calcipotriol was a gift from the Pharmaceutical Research Institute (Warsaw, Poland). Cediranib (AZD2171) was purchased from Selleck Chemicals (Houston, TX, USA).

Human melanoma A375 cell line (CRL–1619), RPMI-7951 (HTB-66), MNT-1, and SK-MEL-28 were from the American Type Culture Collection (Manassas, VA, USA). The A375 cell line is derived from a skin melanoma of a 54-year-old female. It should be underlined that these cells carry two mutant genes, B-RAF and CDKN2, both associated with melanoma of sun-damaged skin (46). Since UV radiation is considered the most important environmental risk factor for cutaneous melanoma (47) and it is estimated that 60%–70% of cutaneous malignant melanomas are thought to be caused by ultraviolet (UV) radiation exposure (48), we therefore consider A375 melanoma cells as a particularly good model for our study. A375 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma–Aldrich; Merck KGaA) supplemented with 10% fetal bovine serum (FBS) (Biological Industries, Israel) and 1% penicillin/streptomycin (Sigma–Aldrich; Merck KGaA) in an incubator with 5% CO₂ at 37˚C. RPMI-7951 cells were cultured in Minimum Essential Medium Eagle, with Earle’s salts and non-essential amino acids (MEM, Sigma–Aldrich; Merck KGaA), supplemented with 10% FBS (Biological Industries, Israel), 1% penicillin/streptomycin (Sigma–Aldrich; Merck KGaA), 1 mM sodium pyruvate, and 2 mM L-glutamine (Sigma–Aldrich; Merck KGaA, both). MNT-1 cells were cultured in MEM (Sigma–Aldrich; Merck KGaA) Alpha Modification, supplemented with 20% FBS (Biological Industries, Israel), 1% penicillin/streptomycin, 10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, non-essential amino acids (Sigma–Aldrich; Merck KGaA, all listed before), and 10% AIM-V™ Medium (Thermofisher Scientific, Waltham, USA). SK-MEL-28 cells were cultured in MEM (Sigma–Aldrich; Merck KGaA) Alpha Modification, supplemented with 10% FBS (Biological Industries, Israel), 1% penicillin/streptomycin (Sigma–Aldrich; Merck KGaA), and 2 mM L-glutamine (Sigma–Aldrich; Merck KGaA). Appropriate medium supplemented with 5 times lower concentration of charcoal–stripped FBS was used for all procedures where the effects of vitamin D derivatives were examined (2% for A375, RPMI-7951, and SK-MEL-28 cell lines and 4% for MNT-1 cells).

The sulforhodamine B (SRB) assay was performed according to the procedure previously described (49). Briefly, the human melanoma A375 cells were seeded in 96–well plates (3,000 cells per well), cultured overnight, and then treated simultaneously with serial dilutions of cediranib (0.01–1,000 nM) and vitamin D analogs (calcitriol or calcipotriol) at 100 nM concentration, being tested for an additional 72 h. Cells were fixed with 10% trichloroacetic acid for 1 h at 4˚C. Following washing (5× with distilled water), the staining solution composed of 0.4% SRB (Sigma–Aldrich; Merck KGaA) in acetic acid was added to each well for 15 min, followed by washing with 1% acetic acid. The SRB dye was solubilized using a solution of 10 mM buffered Tris Base (pH 10.5) and the absorbance was measured at 570 nm using an Epoch™ microplate spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA).

The cell cycle status was analyzed based on quantification of DNA content using flow cytometry. Melanoma cells were treated for 24 h with vitamin D compounds (calcitriol or calcipotriol) at 100 nM concentration, followed by 72 h incubation with cediranib at 500 or 1,000 nM concentration. Trypsinized human malignant melanoma cells together with cells from culture medium were fixed in 70% ethanol for 24–48 h at 4°C, then treated with ribonuclease to remove any contaminating RNA, and the DNA was stained with propidium iodide (PI; Sigma–Aldrich; Merck KGaA) for 30 min at 37°C. The fluorescence of the PI–stained cells was measured by flow cytometry (FACSCalibur™; Becton, Dickinson and Company, Franklin, Lakes, NJ, USA). The results were analyzed using the CellQuest™ Pro Software version 6.0 (Becton, Dickinson and Company) and expressed as a percentage of cells with DNA content corresponding to apoptotic/necrotic cells (subG1 fraction) or cells in G1, S, and G2/M phases of the cycle. Supplementary Figure 1 presents row cytometrical data.

A375 melanoma cells were seeded on an 8-well chamber slide (3 × 10⁵ cells per well) and were cultured overnight. Melanoma cells were pretreated with vitamin D compounds (calcitriol or calcipotriol) at 100 nM concentration. After 24 h, a mechanical wound was created by physical scraping using a pipette tip in a confluent cell monolayer. Cediranib at 500 or 1,000 nM concentration was diluted in a fresh medium and added to the cells for 72 h and cell migration process was observed. The experiment was carried out as a live imaging with Olympus cellVivo IX83 and cell free area was calculated as a percentage closure relative to original size [(wound area in μm²)*100/(original wound area in μm²)] with the Olympus cellSens software with use of TruAI technology.

A375 melanoma cells were treated for 24 h with vitamin D compounds (calcitriol or calcipotriol) at 100 nM concentration, followed by 24 h incubation with cediranib at 500 or 1,000 nM concentration. Trypsinized human malignant melanoma cells at 1 × 10⁶ density were harvested by centrifugation and rinsed two times in 3 ml of incubation buffer (0.5% bovine serum albumin in PBS). Following 10 min blocking in the incubation buffer, cells were stained for 30 min at room temperature with primary antibody anti-VEGFR2 (Cell Signaling, cat. no. 2479, rabbit monoclonal, 1:200) dissolved in the incubation buffer. Following rinsing 2× in incubation buffer, cells were incubated for 30 min with the secondary antibody (goat anti-rabbit IgG ThermoFisher Scientific A11008, 1:500) diluted in the incubation buffer. Cells were rinsed 2× with incubation buffer, dissolved in 0.5 ml of PBS and analyzed cytometrically on FACSCalibur™ (Becton, Dickinson and Company, Franklin, Lakes, NJ, USA) using the CellQuest™ Pro Software version 6.0 (Becton, Dickinson and Company). The results were expressed as a fluorescence geometric mean.

After preincubation either with 1,25(OH)₂D₃ or with calcipotriol at 100 nM concentration for 24 h, A375, SK-MEL-28, RPMI-7951 or MNT-1 melanoma cells were treated for an additional 24 h with cediranib at 500 or 1,000 nM concentration. Subsequently, cells were scraped and lysed in the presence of ice-cold RIPA buffer (Sigma–Aldrich; Merck KGaA) supplemented with protease inhibitor cocktail. Protein concentrations were determined by the Bradford assay. An equal amount of protein from each sample (40 μg) was loaded per lane, and proteins were resolved by SDS-PAGE (4%–20% Mini-PROTEAN^® TGX Stain-Free™ Protein Gels, Bio-Rad Laboratories, Hercules, CA, USA) and then transferred onto an Immun-Blot™ PVDF membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were incubated with primary antibodies: anti-VDR (mouse monoclonal, 1:1,000; Santa Cruz sc-13133), anti-VEGFR1 (rabbit polyclonal, 1:1,000; Cell Signaling Technology 2893), anti-VEGFR2 (rabbit monoclonal, 1:1,000; Cell Signaling Technology 2479), anti-PDGFR alpha (rabbit monoclonal, 1:1,000; Cell Signaling Technology 3174), anti-PDGFR beta (rabbit monoclonal, 1:1,000; Cell Signaling Technology 3169), or HRP-conjugated anti-β-actin antibody (mouse monoclonal, 1:10,000; Santa Cruz Biotechnology, sc-47778) overnight at 4°C. After three washes in TBST, secondary goat anti-rabbit antibodies conjugated to horseradish peroxidase (1:10,000; Santa Cruz Biotechnology, sc-2004) or secondary bovine anti-mouse antibodies conjugated to horseradish peroxidase (1:20,000; Santa Cruz Biotechnology, sc-2371) were added, and following incubation for 1 h at room temperature, blots were developed with Western Lightning^® Ultra chemiluminescent substrate (PerkinElmer, Inc. Waltham, MA, USA) according to the manufacturer’s protocol. Changes in protein level were assessed by densitometric scanning of the bands and corrected for β-actin loading control.

A375 melanoma cells were seeded in 8-well chambers. Cells were preincubated for 24 h with vitamin D derivatives at 100 nM concentration and subsequently incubated for an additional 24 h with cediranib at 500 or 1,000 nM concentration. Following fixing with 4% paraformaldehyde (PFA) for 10 min at room temperature (RT), cells were permeabilized in 0.2% Triton X-100 solution in PBS for 10 min. Blocking was performed with 1% BSA in PBS for 30 min at RT. Following washing 3 × 5 min in PBS, primary antibodies were applied to the cells (VEGFR2 rabbit monoclonal, 1:200; Cell Signaling Technology 2479; EEA1 mouse monoclonal, 1:250, BD Biosciences 610457) and incubated at 4°C overnight. Following rinsing 3 × 5 min in PBS, slides were incubated with an appropriate secondary antibody (A11008 goat anti-rabbit IgG Alexa Fluor 488, 1:500; A11008 donkey anti-mouse IgG Alexa Fluor 594, 1:500, Life Technologies) for 1 h at RT. Following rinsing, cultures were counterstained with DAPI (Sigma–Aldrich; Merck KGaA). Images were collected with Olympus cellVivo IX83 and analyzed with Olympus cellSens software.

After preincubation either with 1,25(OH)₂D₃ or with calcipotriol at 100 nM concentration for 24 h, A375 melanoma cells were treated for an additional 24 h with cediranib at 500 or 1,000 nM concentration. Subsequently, total RNA was extracted by using the ExtractME^®Total RNA Kit (Blirt, Poland, EM09.1-250), according to the manufacturer’s instructions. The concentration and purity of isolated RNA were measured by an EpochMicroplate Spectrophotometer (BioTek, USA). Extracted RNA was reverse transcribed and cDNA synthesized using RevertAid™ First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Inc., USA). Real-Time PCR was performed using a StepOnePlus™ Real-Time PCR System (LifeTechnologies-Applied Biosystems, Grand Island, NY, USA) with RealTime AMPLIFYME SYBR™ Green No-ROX Mix (Blirt, Poland, AM01). All primers were purchased from Sigma-Aldrich (Merck KGaA). The expression of the genes was normalized by comparative -ΔΔCt method, using RPL37A as a housekeeping gene, followed by calibration (fold change) to normalized expression data of samples from control (ratio = 1). To ensure specificity of the PCR amplification, dynamic melting curve analysis was performed for all reactions. Primer sequences are summarized in Table 1 .

Statistical analysis was performed using GraphPad Prism v 7.05 (GraphPad Software, San Diego, CA, USA) or Microsoft Excel. Data were subjected to Student’s t-test (for two groups), one-way or two-way analysis of variance and appropriate post-hoc test (the ANOVA Tukey’s or Sidak’s multiple comparison test). Data are expressed as mean of 3 to 5 independent experiments ± S.D (n = 2–6 in each). Differences are shown as significant at *p < 0.05,**p < 0.01, ***p < 0.001, or ****p < 0.0001 as indicated.

As established by SRB proliferation assay, cediranib alone inhibited A375 melanoma cell proliferation maximally about 6% at 1,000 nM concentration during 72 h of incubation ( Figures 1A, B ). However, simultaneous treatment with cediranib and 1,25(OH)₂D₃ or calcipotriol, at 100 nM concentration, resulted in a profound decrease in the proliferation of melanoma cells. The effect of vitamin D derivatives varied as to the level of maximal inhibition of melanoma cell proliferation, which ranged from approximately 30% for cediranib and 1,25(OH)₂D₃ ( Figure 1A ) to 43% for cediranib and calcipotriol ( Figure 1B ), p < 0.0001 both. Similar effects were observed in SK-MEL-28 melanoma cells. Cediranib alone inhibited proliferation of the cells maximally about 12% at 1,000 nM under experimental conditions used ( Figures 1C, D ). Simultaneous treatment with cediranib and 1,25(OH)₂D₃ or calcipotriol, at 100 nM concentration, resulted in further decrease in the proliferation of melanoma cells. The effect of vitamin D derivatives varied as to the level of maximal inhibition of melanoma cell proliferation, which ranged from approximately 26% for cediranib and 1,25(OH)₂D₃ ( Figure 1C ) to 22% for cediranib and calcipotriol ( Figure 1D ), p < .05 both. On the other hand, treatment of MNT-1 and RPMI-7951 melanoma cells with cediranib in the presence of 1,25(OH)₂D₃ ( Figures 1E–H ) or calcipotriol did not show additive effect of co-treatment. Cediranib alone inhibited proliferation of these melanoma cells maximally about 11% or 18%, respectively, at 1,000 nM concentration during 72 h of incubation.

Since the most profound effect of vitamin D analogs to the inhibition of melanoma cell proliferation treated with cediranib was observed in A375 cells, this line was used as a model for further detailed analysis. In agreement with our previous studies (15, 44), treatment of A375 melanoma cells with vitamin D resulted in G0/G1 (G0/G1—stationary/growth phase) cell cycle arrest ( Figures 2A, B ). G0/G1 arrest was observed also in melanoma cells treated with cediranib, p < 0.0001 ( Figure 2 ). Additionally, we noticed an increase in the number of SubG1 cells, indicating induction of apoptosis by cediranib in melanoma cells, p < 0.0001 ( Figure 2 ). To investigate the mechanism of proliferation inhibition of melanoma A375 cells by the combination of vitamin D analogs with cediranib, melanoma cells were pretreated either with 1,25(OH)₂D₃ ( Figure 2A ) or with calcipotriol ( Figure 2B ) at 100 nM concentration for 24 h and then incubated with cediranib at 500 or 1,000 nM for an additional 72 h. Preincubation of melanoma cells with 1,25(OH)₂D₃ ( Figure 2A ) prior to cediranib treatment for 72 h resulted in an increase in the percentage of cells in the G2/M phase (preparation for mitosis/mitosis) in comparison to cells without pretreatment, p < 0.001 for cediranib at 500 nM concentration and p < 0.05 for cediranib at 1,000 nM concentration ( Figure 2A ), which was accompanied by a proportional decrease in the number of SubG1 cells (SubG1—apoptotic/necrotic cells). Similar results were observed for calcipotriol ( Figure 2B ); however, we noticed an increase in the percentage of cells not only in the G2/M phase, but also in the S phase, in comparison to cells without pretreatment.

Cellular motility and migration are well-established hallmarks of malignant tumors spreading their metastases (50). We recorded therefore migration of A375 melanoma cells and wound closure live with Olympus cellVivo IX83 every 30 min for 72 h and cell free area was calculated as a percentage of closure relative to its original size. The wound closure curves ( Figure 3 ) revealed that non-treated malignant melanoma A375 cells approached up to 38% closure and migrate faster than cells from any treatment groups (p < 0.0001 for any treatment group vs. control, not marked in Figure 3 ). In agreement with our previous study (15), we observed that 1,25(OH)₂D₃ efficiently inhibited melanoma cell migration (p < 0.0001 vs. control, not marked in Figure 3 ) during 72 h, leaving approximately 70% of the wound original size. Interestingly, calcipotriol was even more efficient than 1,25(OH)₂D₃, leaving as much as 81.5% of the wound original size. Curiously, cediranib at both tested concentrations, 500 and 1,000 nM, inhibited melanoma cell migration to a similar extent, leaving approximately 74%–75% of the wound original size. It should be emphasized, however, that vitamin D pretreatment profoundly diminished cellular mobility in melanoma cells treated with cediranib. The most efficient reduction of melanoma cells mobility was observed in cells 24 h pretreated with 1,25(OH)₂D₃ and incubated subsequently for 72 h with cebiranib at 500 nM, in which the wound area was reduced by only 15% [p < 0.0001 for melanoma cells 1,25(OH)₂D₃ pretreated and incubated with cediranib at 500 nM concentration vs. 500 nM cediranib alone; Figure 3 ], giving a further significant 10% reduction in cellular mobility as compared to monotreatment with cediranib. Substantial 8% reduction in cellular mobility was observed also in melanoma cells pretreated with calcipotriol compared to monotreatment with cediranib.

Since cediranib is a small-molecule inhibitor of several tyrosine kinases, including VEGFR1 and VEGFR2, of which the latter seems to play a predominant role (51), we investigated therefore whether vitamin D preincubation will affect the extracellular expression of VEGFR2 in A375 melanoma cells exposed to cediranib. We noticed that cediranib alone did not influence the extracellular expression of VEGFR2 in A375 melanoma cells during 24-h incubation ( Figure 4 ). However, the extracellular expression of VEGFR2 increased significantly in melanoma cells pretreated either with 1,25(OH)₂D₃ ( Figure 4A ) or with calcipotriol ( Figure 4B ) for 24 h as compared to monotreatment with cediranib or to control cells.

Next, we checked whether the preincubation of A375 melanoma cells with vitamin D derivatives affected the protein level of VEGFR1, VEGFR2, PDGFRa, PDGFRb, or VDR after subsequent treatment with cediranib ( Figure 5 ). No significant effect was observed as to the VEGFR1 or PDGFRb protein level neither by 1,25(OH)₂D₃, nor by cediranib under the experimental conditions used ( Figures 5A, E , respectively). We also noticed that the expression of VEGFR2 at the protein level was not changed by cediranib alone ( Figure 5B ). However, we observed a significant increase in VEGFR2 protein level in melanoma cells pretreated with vitamin D (although in case of calcipotriol, only with cediranib at 500 nM concentration). Both vitamin D derivatives increased the VDR protein level ( Figure 5C ). Cediranib alone increased the protein level of PDGFRa (p < 0.05), while preincubation with 1,25(OH)₂D₃ reversed that effect for cediranib at 1,000 nM concentration, p < 0.01 ( Figure 5D ).

Since we observed the upregulation of VEGFR2, at both protein level and its extracellular expression, under experimental conditions, concurrently with a very desirable decrease in A375 melanoma cell proliferation and mobility, we hypothesized whether the presence of VEGFR2 protein or its level could potentially influence the extent to which 1,25(OH)₂D₃ may enhance the cytotoxic effect of cediranib in MNT-1, RPMI-7951, and SK-MEL-28 melanoma cells. We checked, therefore, whether the preincubation of aforementioned melanoma cell lines with 1,25(OH)₂D₃ affected the protein level of VEGFR2, PDGFRa, or VDR, after subsequent treatment with cediranib ( Figure 6 ). No significant effect was observed as to the VDR protein level neither by 1,25(OH)₂D₃, nor by cediranib under the experimental conditions used in MNT-1 melanoma cells ( Figure 6 ). However, we observed a significant increase in VDR protein level in RPMI-7951 and SK-MEL-28 melanoma cells treated with 1,25(OH)₂D₃ ( Figures 6C, I ). No significant effect was observed as to the PDGFRa protein level neither by 1,25(OH)₂D₃, nor by cediranib under the experimental conditions used in RPMI-7951 and SK-MEL-28 melanoma cells ( Figures 6B, H ); what is more, we did not detect any PDGFRa protein product in MNT-1 melanoma cells ( Figure 6E ). Interestingly, we did not detect any VEGFR2 protein product neither in MNT-1, nor in RPMI-7951 melanoma cells ( Figures 6D, G ). However, we observed a significant increase in VEGFR2 protein level in SK-MEL-28 melanoma cells treated with cediranib alone or cediranib with 1,25(OH)₂D₃ ( Figure 6A ), which underlines the key role of VEGFR2 in an interaction between vitamin D and cediranib.

To further explore the mechanism underlying the observed increase of VEGFR2 protein level by vitamin D in A375 melanoma cells treated with cediranib, we checked whether this protein is sequestered in early endosomes for potential recycling or degradation, as suggested recently (52). We did not observe, however, any co-localization of VEGFR2 and EEA1, which is a marker of early endosomes ( Figure 7 ).

In order to verify the aforementioned changes in protein level, the impact of 1,25(OH)₂D₃ on the expression of selected VEGF-associated genes was tested in melanoma cells treated with cediranib ( Figure 8 ). Although cediranib alone did not influence mRNA level for VEGFR1 ( Figure 8A ), we observed an increase in mRNA level in melanoma cells treated with the combination of 1,25(OH)₂D₃ and cediranib at both tested concentrations (p < 0.05 vs. control). No significant effect of cediranib alone was observed on the mRNA level of VEGFR2; however, consistent with immunoblotting described above, we observed a marked increase in VEGFR2 mRNA level in melanoma cells pretreated with 1,25(OH)₂D₃ (p < 0.01, Figure 8B ). Both 1,25(OH)₂D₃ and cediranib resulted in an increase in mRNA level for VEGFR3 (p < 0.05, Figure 8C ), with an increasing trend in melanoma cells pretreated with vitamin D subsequently exposed to cediranib, yet without statistical significance in the latter. Interestingly, cediranib alone at 500 nM concentration decreased the mRNA level for VEGF-A (p < 0.01, Figure 8D ), while pretreatment of A375 melanoma cells with 1,25(OH)₂D₃ resulted in an increase in the relevant mRNA in melanoma cells incubated subsequently with cediranib at 1,000 nM concentration (p < 0.05). No significant effect was observed in the expression of VEGF-C under the experimental conditions used ( Figure 8E ). mRNA level for VEGF-D was elevated by both 1,25(OH)₂D₃ and cediranib (p < 0.05, Figure 8F ), and it was elevated also in melanoma cells pretreated with vitamin D. Finally, we observed an increase in mRNA level for PDGFRa and PDGFRb in melanoma cells treated with cediranib at 1,000 nM concentration (p < 0.05, Figures 8G, H , respectively); the effect was further exacerbated by vitamin D pretreatment (p < 0.05 for PDGFRa and p < 0.01 for PDGFRb vs. monotreatment).

Then, we also analyzed the expression of several vitamin D-related genes in the experimental conditions used. We observed that VDR mRNA level was elevated in A375 melanoma cells pretreated with vitamin D and subsequently incubated with cediranib at 1,000 nM concentration (p < 0.05, Figure 9A ). No significant effect was observed in the expression of CYP27B1 under the experimental conditions used ( Figure 9B ). Cediranib treatment resulted, however, in an increase in mRNA level for CYP3A4 and CYP2R1 in melanoma cells pretreated with 1,25(OH)₂D₃ (p < 0.05 vs. control and vs. monotreatment, Figures 9C, D , respectively). Lastly, consistent with our previous results (30) and literature data (53), we observed a marked increase in mRNA level for CYP24A1 (p < 0.01, Figure 9E ) in melanoma cells treated with 1,25(OH)₂D₃. Interestingly, the effect was invariably observed in cells treated subsequently with cediranib. In fact, the mRNA level of CYP24A1 was the highest in melanoma cells treated with cediranib following vitamin D pretreatment (p < 0.01 vs. monotreatment).