Vitamin D derivatives enhance cytotoxic effects of H₂O₂ or cisplatin on human keratinocytes

Abstract

Although the skin production of vitamin D is initiated by ultraviolet radiation type B (UVB), the role vitamin D plays in antioxidative or pro-oxidative responses remains to be elucidated.. We have used immortalized human HaCaT keratinocytes as a model of proliferating epidermal cells to test the influence of vitamin D on cellular response to H₂O₂ or the anti-cancer drug, cisplatin. Incubation of keratinocytes with 1,25(OH)₂D₃ or its low calcemic analogues, 20(OH)D₃, 21(OH)pD or calcipotriol, sensitized cells to ROS resulting in more potent inhibition of keratinocyte proliferation by H₂O₂ in the presence of vitamin D compounds. These results were supported by cell cycle and apoptosis analyses, and measurement of the mitochondrial transmembrane potentials (MMP), however some unique properties of individual secosteroids were observed. Furthermore, in HaCaT keratinocytes treated with H₂O₂, 1,25(OH)₂D₃, 21(OH)pD and calcipotriol stimulated the expression of SOD1 and CAT genes, but not SOD2, indicating a possible role of mitochondria in ROS-modulated cell death. 1,25(OH)₂D₃ also showed a short-term, protective effect on HaCaT keratinocytes, as exemplified by the inhibition of apoptosis and the maintenance of MMP. However, with prolonged incubation with H₂O₂ or cisplatin, 1,25(OH)₂D₃ caused an acceleration in the death of the keratinocytes. Therefore, we propose that lead vitamin D derivatives can protect the epidermis against neoplastic transformation secondary to oxidative or UV-induced stress through activation of vitamin D-signaling. Furthermore, our data suggest that treatment with low calcemic vitamin D analogs or the maintenance of optimal level of vitamin D by proper supplementation, can enhance the anticancer efficacy of cisplatin

Vitamin D is a secosteroid produced in the skin via UVB-induced photolysis of 7-dehydrocholesterol (7-DHC), or obtained in limited quantities through the diet [1, 2]. Regardless of the source, vitamin D undergoes activation through two subsequent hydroxylations comprising 25-hydroxylation by CYP2R1 or CYP27A1, predominantly in the liver, followed by 1α-hydroxylation in the kidney by CYP27B1, resulting in the fully active form, 1,25(OH)₂D₃ (also known as calcitriol) [3]. Apart from kidneys, multiple human organs, including the skin, express CYP27B1 and have the capacity to 1α-hydroxylate 25-hydroxyvitamin D3 [4]. Moreover, the skin is the only organ equipped with all elements necessary for the synthesis, activation, and catabolism of vitamin D, as well as responding to it via the vitamin D receptor (VDR) [2, 5].

Recently, novel pathways of vitamin D metabolism and activation have been established that are initiated by CYP11A1, also known as cytochrome P450scc [6–14]. Briefly, CYP11A1 converts 7-DHC to 7-dehydropregnenolone (7-DHP) via sequential hydroxylations of 7-DHC at C-22 and C-20 followed by cleavage of its side chain [7, 12]. 7-DHP is further modified in this pathway by the classical enzymes of steroid metabolism resulting in 5,7-diene-analogues, some of which have been detected in vivo [15, 16]. In vitro studies have shown that these 5,7-dienes can be converted to the corresponding vitamin D analogues following UVB irradiation [9–11]. Several of these new derivatives, which have a short side chain compared to vitamin D₃, display biological activity [6, 10, 11, 15, 17]. In contrast, CYP11A1 acts directly on vitamin D to produce hydroxyvitamin D derivatives which retain a full-length side chain, with these products being detected both in vitro [12–14, 18, 19] and in vivo [20, 21]. The hydroxyvitamin D₃ metabolites produced by these pathways, including the major product, 20(OH)D₃, are biologically active in both in vitro (reviewed in [8]) and in vivo [22] models. Since they are less prone to induce hypercalcemia than 1,25(OH)₂D₃ [23, 24], they deserve special attention as potential therapeutics for the treatment of leukemia [23], melanoma [10, 11, 25, 26] and colorectal cancer [27].

1,25(OH)₂D₃ may exert its effects through both genomic and non-genomic mechanisms [28]. The genomic pathway relies on binding to the intracellular vitamin D receptor (VDR), a member of the nuclear receptor superfamily [29, 30]. The binding of ligand to VDR triggers its heterodimerization with retinoid X receptor and interaction with vitamin D responsive elements (VDREs) in the promoter regions of vitamin D-regulated genes [29, 31]. It is estimated that vitamin D regulates as many as 3000 genes in the human genome [28]. In addition, 1,25(OH)₂D₃ may also elicit rapid responses, independent of the modulation of gene expression, associated with several signal transduction pathways which lead to production of second messengers or to modulation of the intracellular calcium concentration [2, 28]. Rapid responses to 1,25(OH)₂D₃ have been reported to involve plasma-membrane localized VDR [28, 32] or other receptors such as MARRSBP (1α,25(OH)₂D membrane–associated rapid response steroid-binding protein), also described as PDIA3 (protein-disulfide isomerase-associated 3) and ERp57 (endoplasmic reticulum stress protein 57) [32, 33]. Most recently, retinoic acid orphan receptors (RORα and γ) have been identified as alternative receptors for D₃ hydroxy-derivatives which act as reverse agonists [34].

Vitamin D exerts wide spread pleiotropic effects that are additional to its well known involvement in the regulation of calcium homeostasis [3, 35–39]. The noncalcemic effects of vitamin D include direct and indirect regulation of the cell cycle and proliferation, differentiation and apoptosis [40–45]. Therefore, it is not surprising, that vitamin D analogues are being considered for use in cancer prevention and treatment [1]. Numerous epidemiological studies and preclinical data support this proposed use [1, 46–57].

The epidermis is formed predominantly by multiple layers of keratinocytes at different stage of differentiation. This outer most part of the skin provides a protective barrier separating internal organs from the harsh outside environment [5]. Recent data indicate that biologically active forms of vitamin D₃ play an important role in protection against DNA damage [21, 58, 59] and UVB-induced carcinogenesis in the epidermis [60–63]. These protective effects are also seen for the novel, CYP11A1-derived hydroxy-derivatives of vitamin D as demonstrated in human epidermal cells [21], and mouse skin in vivo [64]. Since the predominant effect of UVA irradiation, which has no effect on cutaneous vitamin D production, is to generate reactive oxygen species (ROS), active forms of vitamin D may accelerate elimination of cells with neoplastic potential induced by ROS, which would be consistent with their role as protectors of epidermal integrity [65]. Here we have documented the relationship between vitamin D and oxidative stress in human epidermal keratinocytes, and their interplay with the anticancer drug, cisplatin.

2. MATERIAL AND METHODS

Chemicals

1,25(OH)₂D₃, hydrogen peroxide (30%) and cisplatin were purchased from Sigma-Aldrich (Poznan, Poland). 21(OH)pD was synthesized according to the procedure described by Żmijewski et al. [11] by ProChimia Surfaces Sp. Z o.o. (Poland). 20(OH)D₃ was synthesized and purified as described previously [13, 18]. Calcipotriol was a kind gift from the Pharmaceutical Research Institute (Warsaw, Poland). Other chemicals were purchased from Sigma-Aldrich (Poznan, Poland) or as indicated.

Cell culture

Human immortalized keratinocytes (HaCaT) were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Charcoal-stripped fetal bovine serum was used for all experimental procedures where the effects of vitamin D compounds were examined in order to eliminate the influence of serum-derived vitamin D or other steroids.

For proliferation assays, cell were seeded in 96 well plates and after 24 h were treated with hydrogen peroxide or vitamin D compounds separately or with hydrogen peroxide and selected vitamin D derivatives in pairs (Scheme 1A). All other experiments were performed according to schema 1B. Briefly, cells were preincubated for 24 h with selected secosteroids and then treated with H₂O₂ for an additional 24 h or as indicated.

Scheme 1.

Overview of experimental protocols for treatment of human immortalized keratinocytes (HaCaT) and their subsequent analysis. (A, SRB analysis; B, flow cytometry and real time PCR).

Proliferation assay

The SRB analysis used relies on the uptake of the negatively charged pink aminoxanthine dye, sulphorhodamine B (SRB), by basic amino acids in the cells and thus measures protein content and indirectly reflects the number of cells in the culture. Experiments were performed as described before [26]. Briefly, HaCaT keratinocytes were seeded in 96-well plates (8,000 per well), cultured for 24 h and then treated with serial dilutions of the compounds being tested for an additional 24 h (Scheme 1A). Cells were fixed with 10% trichloroacetic acid (TCA) for 1 h at 4°C. Plates were washed five times with distilled water and air-dried. Staining solution comprising 0.4% SRB in acetic acid was added to each well and after 15 min plates were washed with 1% acetic acid five times and air-dried. SRB dye was solubilized using a solution of 10 mM buffered Tris Base (pH 10.5) and absorbance measured at 570 nm using an Epoch spectrophotometer (BioTek, Winooski, USA).

Cell cycle analysis

The cell cycle was analyzed by quantification of DNA content by flow cytometry. Cells (trypsinized and cells from culture medium as well) were fixed in 70% ethanol and kept for 24 – 48 h at 4°C. Cells were treated with ribonuclease in order to remove any contaminating RNA and DNA was stained with propidium iodide (PI). Fluorescence of the PI-stained cells was measured by flow cytometry (Ex 536 nm, Em 617 nm, FACSCalibur, Becton Dickinson, Franklin Lakes, USA). Results were analyzed by CellQuest Pro Software (Becton Dickinson, Franklin Lakes, USA) 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.

Apoptosis Assay

A PE Annexin V Apoptosis Detection Kit (Becton Dickinson 559763, Franklin Lakes, USA) was used to quantitatively determine the percentage of cells that were actively undergoing apoptosis. Briefly, after performing the experiment according to scheme B in 6 well-plates, cells were harvested, washed twice with PBS and stained according to the manufacturer’s protocol. The fluorescence of PE Annexin V and 7-AAD was measured by flow cytometry (FACSCalibur, Becton Dickinson, Franklin Lakes, USA) and data analyzed using CellQuest Pro software (Becton Dickinson, Franklin Lakes, USA).

Measurement of changes in mitochondrial potential

The detection of changes in mitochondrial inner membrane electrochemical potential (Δψ) in living cells was performed using the cationic, lipophilic dye, JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′ tetraethylbenzimidazolylcarbocyanine iodide, T-3168, Life Technologies, Carlsbad, USA). In untreated, “healthy” cells the dye accumulates in the mitochondrial matrix forming red fluorescent aggregates. After depletion of the electrochemical potential, the dye disperses throughout the cell and results in a shift from red to green fluorescence (JC-1 monomers). CCCP (carbonyl cyanide 3-chlorophenylhydrazone, C2759 Sigma, Poznan, Poland), a mitochondrial potential disrupter, was used as a control. After treatment with selected compounds (as shown in Scheme 1B and described in Results), cells were harvested and suspended in 1 ml warm PBS. CCCP solution in DMSO was added to the control tube only (2 μM final concentration) and cells incubated at 37°C for 5 min. JC-1 solution (in DMSO, 2 μM final concentration) was added to all tubes and cells were incubated at 37°C for 15 min, then centrifuged and resuspended in 500 μl of PBS. Samples were kept on ice and analyzed on the FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, USA) using CellQuest Pro analysis software (Becton Dickinson, Franklin Lakes, USA). For microscopic observation, cells were seeded in 8-well slide chambers (Nunc Lab-Tek) and incubated as described above except that after the treatment with 1,25(OH)₂D₃ (100 nM, 24 h) and H₂O₂ (2 mM, 5 h), cells were washed with PBS and incubated in DMEM supplemented with JC-1 (2.5 μg/mL) for an additional 30 min at 37°C. Living cells were observed and photographed using the fluorescence microscope, Axiovert 200 (magnification 200x). For control experiments, 1,25(OH)₂D₃ and/or hydrogen peroxide treatments were omitted. For positive controls, cells were treated with CCCP at a final concentration of 2 μM for 5 min at room temperature. Images were acquired from 5–10 randomly chosen fields for each experimental condition showing nuclear cross-sections. Green and red channels were merged and the red/green ratio was shown in blue.

Detection of the production of intracellular reactive oxygen species

The intracellular production of reactive oxygen species (ROS) was measured using H₂DCFDA (D399, Life Technologies, Carlsbad, USA). Cells were incubated with 100 nM 1,25(OH)₂D₃ for 24 h followed by exposure to 1 mM hydrogen peroxide for 1 h or 24 h (Scheme 1B). Thirty minutes before the end of the incubation, H₂DCFDA was aded to a final concentration of 10 μM. Cells were washed and suspended in cold PBS. Samples were kept on ice and analyzed using the FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, USA) using CellQuest Pro analysis software (Becton Dickinson, Franklin Lakes, USA).

Measurement of mRNA levels

The relative mRNA levels of particular genes were determined by Real-Time PCR (qPCR). Total RNA was isolated using the Total RNA Prep Plus kit (A&A Biotechnology, Gdynia, Poland), according to the manufacturer’s instructions. The concentration and quality of RNA samples were determined spectrophotometrically (Epoch BioTek, Winooski, USA). One microgram of RNA was used for reverse transcription using a RevertAid™ First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, USA). The qPCR reaction comprised 1 μL cDNA, 150 nM of each primer and Real-Time PCR Mix SYBR B (A&A Biotechnology, Gdynia, Poland) and was performed using Step One Plus (Life Technologies-Applied Biosystems, Grand Island, USA) in total volume of 20 μL. The reactions were run in duplicate and the resulting data were averaged prior to analysis with Step One Plus ver. 2.2.2. software (Life Technologies-Applied Biosystems, Grand Island, USA). The RPL37 gene was used as a control to normalize the values by the ΔΔCt quantification method.

Statistical analyses

Statistical analysis was performed using Microsoft Excel or GraphPad Prism v6.03 (GraphPad Software, San Diego, CA, USA). Data were subjected to Student’s t-test (for two groups) or one-way analysis of variance and appropriate post hoc test (the ANOVA Kruskal–Wallis test for comparison of several groups). Data are expressed as mean ± S.D. Each experiment was repeated at least three times in triplicate. Differences are shown as significant at p<0.05, p<0.01 or p<0.001, as indicated.

3. RESULTS

3.1. Vitamin D analogues enhance the cytotoxic effects of hydrogen peroxide on HaCaT cells

We tested the effect of the vitamin D compounds 1,25(OH)₂D₃, 20(OH)D₃, 21(OH)pD and calcipotriol, on the sensitivity of human HaCaT keratinocytes to reactive oxygen species. As expected, these compounds [8, 19, 66] inhibited the proliferation of HaCaT keratinocytes as determined using the Sulphrodamin B assay (SRB) (Figure 1A–D). IC₅₀ values ranged from 1.44 nM for 21(OH)pD to approximately 0.1 nM for the other secosteroids (Table 1). Hydrogen peroxide (H₂O₂) chosen as the agent to promote oxidative stress, inhibited the growth of HaCaT cells with an IC₅₀ of 186 nM. 1,25(OH)₂D₃, 20(OH)D₃, 21(OH)pD and calcipotriol decreased the IC₅₀ observed with H₂O₂, thus enhancing their sensitivity to H₂O₂ (Figure 1E–H), with the strongest effect being seen for 21(OH)pD where the IC₅₀ decreased 3.8 fold. (Table 1).

Figure 1.

The effect of Vitamin D compounds on the proliferation of HaCaT keratinocytes treated with hydrogen peroxide. HaCaT keratinocytes were treated with serial dilutions (10⁻¹²–10⁻⁶ M) of 1,25(OH)₂D₃ (A), 20(OH)D₃ (B), 21(OH)pD (C) or calcipotriol (D), or were treated with serial dilutions of H₂O₂ (0.0256–5 mM) alone or in combination with 10 nM 1,25(OH)₂D₃ (E), 20(OH)D₃ (F), 21(OH)pD (G) or calcipotriol (H) for 24 h (according to Scheme 1A). Data are shown as mean from three independent experiments ± S.D. Statistical significance was estimated using One–Way ANOVA and presented as *p < 0.05, **p < 0.005, ***p < 0.0005 versus control. Please note that the same curve showing the response to H₂O₂ alone was included into panels E – H in order to underline the effects of co-incubation with vitamin D analogues.

Table 1.

Summary of IC₅₀ values for inhibition of proliferation of HaCaT keratinocytes. IC₅₀ measurements were carried out as described in Figure 1.

Compound	IC50 [nM] Secosteroid	IC50 [nM] H2O2
H2O2	-	186
1,25(OH)2D3	0.089	72
20(OH)D3	0.274	120
21(OH)pD	1.44	49
Calcipotriol	0.081	63

3.2. Preincubation of HaCaT cells with vitamin D compounds sensitizes them to hydrogen peroxide treatment

We investigated whether pretreatment with vitamin D compounds sensitize HaCaT keratinocytes to H₂O₂, using flow cytometry. HaCaT keratinocytes were pretreated with secosteroids (100 nM) for 24 h and then subjected to 1 mM H₂O₂ for an additional 24 h. To ensure that maximal stimulation was achieved, secosteroids were tested at concentrations which approximately corresponded to the concentration of 25(OH)D₃ in the plasma (75–125 nM) [67]. It should be noted, however, that serum level of 1,25(OH)₂D₃ concentration in humans is less than 1 nM [68].

Treatment of HaCaT keratinocytes with H₂O₂, alone, resulted in a dose-dependent decrease in the number of cells at the G0/G1 phase and an increase in the number of SubG1 cells, indicating induction of apoptosis (Figure 2A). Interestingly, an increase in the number of cells in the G2/M phase was observed in HaCaT cells with increasing H₂O₂ concentrations, up to 1 mM, suggesting inhibition of the mitotic process by H₂O₂. 1,25(OH)₂D₃ had only minor effects on the distribution of HaCaT cells in the different phases of cell cycle, with a statistically significant decrease in the number of cells in the subG1 and G1 phases compared to control cells (Figure 2B).

Figure 2.

Effect of vitamin D compounds and hydrogen peroxide on the distribution of HaCaT keratinocytes through the cell cycle. HaCaT cells were treated with hydrogen peroxide (0.1 – 5 mM) (A) or with 1,25(OH)₂D₃ (0.1 – 100 nM) for 24 h. HaCaT keratinocytes were also preincubated with 100 nM 1,25(OH)₂D₃ (C), 20(OH)D₃ (D), 21(OH)pD (E) or calcipotriol (F)) for 24 h, and subsequently exposed to 1 mM hydrogen peroxide for an additional 24 h (according to Scheme 1B). Cells were harvested, stained with propidium iodide and analyzed by Flow Cytometry. Data are presented as mean ± S.D. of three independent experiments carried out in triplicate. *p<0,05; **p<0,01; ***p<0,001 vs. control.

All vitamin D compounds tested had only a slight effect on the distribution of HaCaT keratinocytes in the cell cycle with a decrease in the number of cells in the G1/G0 phase and an increase in the S and G2/M phases. Interestingly, all the secosteroids except 21(OH)pD protected cells against apoptosis as illustrated by the decrease in the subG1 fraction. Furthermore, preincubation of HaCaT keratinocytes for 24 h with 1,25(OH)₂D₃ or calcipotriol, prior to H₂O₂ treatment for 24 h, resulted in an increase in the percentage of cells in the G0/G1 phase in comparison to cells without pretreatment (7% and 3.5%, respectively; P <0.01; Figure 2, panels C and F). Curiously, pretreatment with 20(OH)D₃ or 21(OH)pD had the opposite effect with the fraction of cells in the G0/G1 phase being elevated by 5% and 7.5%, respectively, compared to cells treated with hydrogen peroxide only (p <0.05; Figure 2, panels D and E). Furthermore, pretreatment with the secosteroids was accompanied by a proportional decrease in the number of cells in S and G2/M phases for 1,25(OH)₂D₃ and calcipotriol, and an increase for 20(OH)D₃ and 21(OH)pD (Figure 2C and F). For all the secosteroids used for pretreatment, there was a trend to increase the percentage of cells in the SubG1 phase following H₂O₂ treatment (p <0.001 for 1,25(OH)₂D₃ p <0.01 for 20(OH)D₃), although for 21(OH)pD and calcipotriol the effect was not statistically significant.

3.3. Preincubation with 1,25(OH)₂D₃ amplifies apoptotic and necrotic events in HaCaT cells exposed to H₂O₂

Preincubation of HaCaT keratinocytes with vitamin D compounds led to an increase in the percentage of cells in the SubG1 fraction following H₂O₂ treatment (Fig. 2). To define whether these effects are related to either necrosis or apoptosis, HaCaT keratinocytes were preincubated with secosteroids and subsequently with H₂O₂ (Schema 1B). Then presence of apoptotic or necrotic cells were evaluated by simultaneous staining with Annexin V and 7-AAD and flow cytometry (Figure 3). Incubation of HaCaT keratinocytes with H₂O₂, alone, for 24 h resulted in a dose-dependent induction of both apoptosis (Annexin V+ and 7-AAD-cells) and necrosis (Annexin V+ and 7-AAD+ cells). Apoptosis was induced at lower concentrations of H₂O₂ (≤1 mM) than for necrosis (2 to 5 mM). Interestingly, an amplifying effect of 1,25(OH)₂D₃ on the pro-apoptotic activity of hydrogen peroxide was only observed for 1 mM H₂O₂. While 1 mM H₂O₂ alone had only a limited effect on the induction of apoptosis (Annexin V+ and 7-AAD-cells), pretreatment with 100 nM 1,25(OH)₂D₃ effectively enhanced both apoptosis and necrosis of keratinocytes (Figure 3B). Pretreatment with 1,25(OH)₂D₃ stimulated necrosis, but not apoptosis, in keratinocytes treated with 2 mM H₂O₂ (Figure 3C) and had no effect at 5 mM H₂O₂ (Figure 3D). The lack of effect of 1,25(OH)₂D₃ on keratinocytes treated with the highest concentration of H₂O₂ might be explained by the fact that 5 mM H₂O₂, alone, was sufficient to induce cell death in the majority of keratinocytes.

Figure 3.

The effect of hydrogen peroxide on the induction of apoptosis in HaCaT keratinocytes pretreated with 1,25(OH)₂D₃. HaCaT cells were treated with 100 nM 1,25(OH)₂D₃ for 24 h and subsequently exposed to 0.5 mM (A), 1 mM (B), 2 mM (C) or 5 mM (D) hydrogen peroxide for an additional 24 h (according to Scheme 1B). Cells were harvested, stained with AnnexinV/7-Aminoactinomycin and analyzed by Flow Cytometry. Data are presented as mean ± S.D. of three independent experiments carried out in triplicate. *p<0,05; **p<0,01; ***p<0,001 vs. control.

3.4. Pretreatment with vitamin D compounds affects H₂O₂-induced production of reactive oxygen species and changes in the mitochondrial membrane potential

Pretreatment of keratinocytes with 1,25(OH)₂D₃ did not significantly influence the production of reactive oxygen species (ROS), as measured by H₂DCFDA (Figure 4A and B). However, preincubation of cells with the secosteroids affected ROS production after treatment with H₂O₂ and this effect was time dependent. Pretreatment with 1,25(OH)₂D₃ had no statistically significant effect on ROS production in keratinocytes treated with H₂O₂ for one hour. However, prolongated exposition to H₂O₂ (24 h treatment) resulted in a significant decrease of the ROS levels in keratinocytes pretreated with 1,25(OH)₂D₃ in comparison to untreated controls (Figure 4B).

Figure 4.

Effect of secosteroid preteatment on reactive oxygen levels and on mitochondrial transmembrane potential. HaCaT cells were treated with 100 nM 1,25(OH)₂D₃ for 24 h, and subsequently exposed to 1 mM hydrogen peroxide for 1 h (A) or for 24 h (B) (according to Scheme 1B). Cells were stained with H₂DCFDA and analyzed by Flow Cytometry. HaCaT cells were also preincubated with 1,25(OH)₂D₃ (C), 20(OH)D₃ (D), 21(OH)pD (E) or with calcipotriol (F) for 24 h followed by exposure to 1 mM hydrogen peroxide for 1 h or 3 h, then stained with JC-1 and analyzed by Flow Cytometry (according to Scheme 1B). Data are presented as mean ± S.D. of three independent experiments carried out in triplicate. *p<0,05; **p<0,01; ***p<0,001 vs. control. The image (G) shows the relative changes in mitochondrial membrane potentials which are expressed as shifts from red to green fluorescence (I) or the red to green ratio that produces blue fluorescence (II). Cells were incubated with 100 nM 1,25(OH)₂D₃ for 24 h and subsequently exposed to 2 mM hydrogen peroxide for 5 h (according to Scheme 1B). The positive control (cccp) was exposed to cccp for 5 min before staining with JC-1.

Previously, we reported that H₂O₂ treatment of human HaCaT keratinocytes resulted in a decrease in the mitochondrial membrane potential, Δψ [69]. Our initial studies on JC-1 stained cells (Δψ indicator) showed that incubation of cells with 1,25(OH)₂D₃ alone resulted in an increase in Δψ and, furthermore, pretreatment of cells with 1,25(OH)₂D₃ prevented a H₂O₂-triggered decrease in Δψ (Figure 4G). Next, JC1-stained cells were analysed by flow cytometry (Figure 4C–F). A protective effect of pretreatment with 1,25(OH)₂D₃ as relates to Δψ was observed only after a short (1 h) incubation of cells with 1 mM H₂O₂ (Figure 4B). However prolonged (3 h) incubation with H₂O₂ of 1,25(OH)₂D₃-pretreated keratinocytes (Figure 4C) resulted in a statistically significant decrease in the mitochondrial membrane potential, Δψ, in comparison to cells treated solely with H₂O₂. The 24-h preincubation with the low-calcemic vitamin D analogues, 20(OH)D₃, 21(OH)pD or calcipotriol (all at 100 nM concentrations) resulted in a trend towards an elevation of Δψ, however the data did not reached statistical significance. Similar to 1,25(OH)₂D₃, pretreatment of cells with 20(OH)D₃ or 21(OH)pD for 24 h prior to a 3 h incubation with 1 mM H₂O₂, decreased Δψ, whereas calcipotriol did not have a significant effect (Figure 4F).

3.5. Vitamin D compounds modulate the sensitivity of keratinocytes to cisplatin

Cisplatin is a well-known anticancer drug, which in addition to its ability to intercalate with DNA, can induce production of ROS [70]. It is considered in combinational therapy of skin cancer including metastatic basal cell carcinoma [71]. Cisplatin can induce apoptosis of HaCaT cells by a hydroxyl-radical mediated mechanism [72]. Simultaneous treatment with 1,25(OH)₂D₃ (100 nM) and cisplatin (0.00384 to 300 μM) decreased the IC₅₀ for the antiproliferative effect of cisplatin almost three fold (17.1 μM for cisplatin + 1,25(OH)₂D₃ vs 46.4 μM for cisplatin alone), as measured by the SRB assay (Figure 5A). Interestingly, cisplatin treatment did not cause a rapid induction of apoptosis. Instead, as shown by cell cycle analysis, treatment of HaCaT keratinocytes with both 12 and 120 μM cisplatin resulted in a significant decrease in the number of cells in the G1/G0 phase, which was coincidental with an increase in the proportion in the G2/M phase (Tabele 2 and 3). In addition, treatment of HaCaT keratinocytes with a high concentration of cisplatin (120 μM) resulted in an increase in the percentage of cells in the S phase of the cell cycle. Pretreatment of HaCaT keratinocytes with 1,25(OH)₂D₃ or calcipotriol amplified the effects of cisplatin with a significantly higher proportions of cells in the G2/M phase, and in the S phase for calcipotriol. However, the effects on the G2/M phase were not seen with the short side-chain analog, 21(OH)pD, for which only a small protective effect was observed in the S phase with the higher concentration of cisplatin (120 nM) (Table 3).

Figure 5.

The effect of vitamin D compounds on the sensitivity of HaCaT keratinocytes to cisplatin. Panel A, HaCaT keratinocytes were treated with serial dilutions of cisplatin (0.004 – 300 μM) or were exposed to 10 nM cisplatin simultaneously with 100 nM 1,25(OH)₂D₃ for 24 h. Cell proliferation was measured using the SRB assay. Data are represented as mean normalized absorbance (at 570 nm) from three independent experiments ± S.D. Panels B – E, cells were treated with 100 nM 1,25(OH)₂D₃ (B and C), 21(OH)pD (D) or calcipotriol (E) for 24 h and subsequently with 2.4 or 12 μM (B), or 120 μM cisplatin (C – E) for an additional 3 h. Cells were harvested, stained with JC-1 and Δψ analyzed by Flow Cytometry. Data are presented as mean ± S.D. of three independent experiments performed in triplicate. *p<0,05; **p<0,01 vs. control.

Tabele 3.

Effect of vitamin D compounds and cisplatin on the distribution of HaCaT keratinocytes through the cell cycle. HaCaT cells were treated with 100 nM 1,25(OH)₂D₃, 21(OH)pD or calcipotriol for 24 h, and subsequently exposed to 120 μM cisplatin for an additional 24 h. Cells were harvested, stained with propidium iodide and the distribution of cells in the cell cycle analyzed by Flow Cytometry. Data are presented as mean ± S.D. of three independent experiments carried out in triplicate.

	Sub G1	G1	S	G2/M
Control	7.16 ± 0.68	64.38 ± 1.32	9.13 ± 0.55	18.11 ± 1.31
Cisplatin	3.75 ± 0.46**	46.55 ± 1.72***	25.33 ± 0.43***	23.70 ± 1.06**
1,25(OH)2D3	3.51 ± 0.15**	70.41 ± 0.37**	7.47 ± 0.29**	17.80 ± 0.43
1,25(OH)2D3 + cisplatin	8.29 ± 0.78##	21.62 ± 1.16**; ###	15.97 ± 1.61**; ##	51.26 ± 3.39***; ##
21(OH)pD	8.10 ± 1.88	64.17 ± 2.09	9.03 ± 0.13	18.08 ± 1.02
21(OH)pD + cisplatin	3.56 ± 0.56**	54.19 ± 0.35**; ###	20.42 ± 0.20***; ###	22.25 ± 1.11**
Calcipotriol	3.54 ± 0.27**	69.48 ± 0.87**	8.02 ± 0.38*	17.95 ± 0.19
Calcipotriol + cisplatin	3.27 ± 0.09**	42.76 ± 1.03***; ##	28.53 ± 0.72***; ##	25.84 ± 0.95***; #

^*p<0,05;

^**p<0,01;

^***p<0,001 vs. control;

^#- cisplatin versus cisplatin + vitamin D.

Low concentrations of cisplatin (2.4 μM and 12 μM) induced an increase in Δψ for HaCaT keratinocytes after 3 h of incubation (Figure 5B). However, treatment of HaCaT keratinocytes with a higher concentration of cisplatin (120 μM) had no effect on Δψ. Pretreatment of the cells with the secosteroids did not significantly alter Δψ following treatment with cisplatin under the experimental conditions used (Figure 5C–E).

3.6. Vitamin D compounds modify the expression of genes involved in removal of ROS, following H₂O₂ treatment

We tested whether vitamin D compounds can modulate expression of ROS related genes including superoxide dismutase types I and II (SOD1 and SOD2), and catalase (CAT). Pretreatment of HaCaT keratinocytes with 1,25(OH)₂D₃, 20(OH)D₃, 21(OH)pD or calcipotriol had limited effects on the mRNA levels of selected ROS related genes (SOD1, SOD2, CAT). Pretreatment with 21(OH)pD and calcipotriol resulted in a small but statistically significant decrease in catalase (CAT) gene expression (Figure 6C). As expected, treatment of HaCaT keratinocytes with 1 mM H₂O₂ alone resulted in an increase of SOD1, SOD2 and CAT mRNA levels (p<0.01 for SOD1, p<0,001 for SOD2, p<0,05 for CAT). Pretreatment of HaCaT keratinocytes with the vitamin D analogues (10 nM) for 24 h, prior to treatment with 1 mM H₂O₂, had variable effects on mRNA level of the genes under investigation. These secosteroids, except 20(OH)D₃, significantly stimulated the H₂O₂-mediated expression of SOD1 and CAT, while 20(OH)D₃ pretreatment resulted in a significant decrease in mRNA levels for these genes. Pretreatment of HaCaT keratinocytes with calcipotriol resulted in a decrease in the mRNA level for SOD2.

Figure 6.

Effects of hydrogen peroxide on SOD1(A), SOD2(B), CAT (C), VDR (D), PDIA3 (E), CYP3A4 (F), CYP27B1 (G) and CYP24A1 (H) gene expression in HaCaT keratinocytes pretreated with vitamin D compounds. HaCaT cells were incubated with 10 nM 1,25(OH)₂D₃ (I), 20(OH)D₃ (II), 21(OH)pD (III) or calcipotriol (IV) for 24 h followed by exposure to 1 mM hydrogen peroxide for an additional 24 h. mRNA levels were masured by qPCR. Data are shown as means ± S.D of three independent experiments carried out in duplicate. *p<0,05; **p<0,01; ***p<0,001 vs. control; # - H₂O₂ versus H₂O₂ + vitamin D; & - vitamin D versus control.

3.7. The effect of preincubation of HaCaT keratinocytes with vitamin D compounds on the expression of genes related to vitamin D action or metabolism, prior to H₂O₂ treatment

Pretreatment of HaCaT keratinocytes with the secosteroids under study increased VDR mRNA levels, with the strongest effect (approximately 10-fold induction) being observed in cells treated with 20(OH)D₃ and 21(OH)pD (Figure 6D). H₂O₂ treatment alone did not have a significant effect on the expression of the VDR. However, in HaCaT keratinocytes pretreated with vitamin D compounds, H₂O₂ caused a significant attenuation of the stimulation of VDR expression caused by the secosteroids, with the exception being for 1,25(OH)₂D₃.

The pretreatment of HaCaT keratinocytes with the vitamin D compounds, or treatment with 1 mM H₂O₂, had a marginal effect on the expression of PDIA3. However, when pretreatment with the secosteroids was followed by treatment with H₂O₂, statistically significant increases in mRNA levels for PDIA3 were observed. The strongest induction (4-fold increase vs controls) was observed in HaCaT keratinocytes pretreated with 21(OH)pD (Figure 6E).

Genes involved in vitamin D₃ activation or metabolism (CYP27B1, CYP24A1 and CYP3A4,) were also examined [73, 74]. The preincubation with secosteroids had minimal effects on the mRNA level for CYP3A4, with only 21(OH)pD resulting in a statistically significant decrease (Figure 6F). Treatment of HaCaT keratinocytes with H₂O₂ (1 mM) alone resulted in a decrease in the expression of CYP3A4 (p<0.05). However, pretreatment with secosteroids other than calcipotriol, with subsequent treatment with H₂O₂ resulted in a marked increase in CYP3A4 expression (2 – 7 fold). Calcipotriol caused a significant decrease in the mRNA level for CYP3A4.

Preincubation with vitamin D compounds had no effect on the mRNA level for CYP27B1 (Figure 6G). However, expression of CYP27B1 was stimulated with subsequent incubation with H₂O₂ for 1,25(OH)₂D₃, 21(OH)pD and calcipotriol. In contrast, cells pretreated with 20(OH)D₃ and treated with H₂O₂ showed a statistically significant decrease in the CYP27B1 mRNA level (Figure 6G). As expected, 1,25(OH)₂D₃ pretreatment alone caused a strong induction of CYP24A1 expression (Figure 6H). Subsequent treatment with H₂O₂ markedly attenuated this induction. The low calcemic analogues of vitamin D₃, 20(OH)D₃, 21(OH)pD and calcipotriol, produced a relatively small to moderate induction of CYP24A1 expression (1.5, 4 and 250 fold respectively), but as for 1,25(OH)₂D₃ the effect was blunted by H₂O₂.

4. Discussion

Current pre-clinical and clinical data strongly suggest that vitamin D can function as an anticancer agent. This is exemplified by a strong correlation between higher serum 25(OH)D₃ level and decreased incidence of breast [51, 52], colon [53], lung [75], prostate [54] and skin cancer [76]. Moreover, it has been reported that active forms of vitamin D used in combined therapy enhance the effectiveness of anticancer drugs such as cisplatin [77–81], doxorubicin [82, 83], cyclophosphamide [82] or gemcitabine [83]. Interestingly, the mechanism of action of these compounds at least partially relies on generation of reactive oxygen species [70, 84–89]. However, the nature of the action of vitamin D on ROS homeostasis requires clarifications, because published papers frequently describe opposite effects [59, 90–95]. Also, while UVR induces production of both vitamin D and ROS, the effects are wavelengths-dependent with UVB on one hand transforming 7DHC to vitamin D and on the other inducing DNA damage [96–98], with UVA inducing oxidative damage [99, 100].

We observed that simultaneous addition of H₂O₂ and the secosteroids 1,25(OH)₂D₃, 20(OH)D₃, 21(OH)pD or calcipotriol to HaCaT keratinocytes resulted in more potent inhibition of keratinocytes proliferation by H₂O₂ than seen with H₂O₂ alone. Similar enhancement of H₂O₂ cytotoxicity by 1,25(OH)₂D₃ in HaCaT keratinocytes has been reported previously, but at 100 nM rather than the 10 nM concentration used in our study [101]. A similar response has also been reported for 1,25(OH)₂D₃ and H₂O₂ for the breast cancer cell line, MCF-7 [102], and a colon cancer cell line [103]. Moreover, using similar experimental conditions we show that the vitamin D₃ compounds caused a 3 fold reduction in the IC₅₀ value for inhibition of cell growth by cisplatin (Figure 5A), consistent with data described for the murine leukemia cell line, WEHI-3, and the human breast cancer cell lines, MCF-7 and T47D [83]. However, the reduction in IC₅₀ value was not as dramatic in our study (3 fold) compared to these previous studies (55 fold) which might be explained by the relatively strong resistance of HaCaT cells to cisplatin. This resistance is further reflected in the lack of stimulation of the proportion of HaCaT cells in the subG1 fraction by cisplatin (Tables 2 and 3). This is likely due to the mutation in both alleles of p53 in HaCaT cells [104, 105], since it has been shown that cells with p53 mutation or p53 silencing are resistant to cisplatin-induced apoptosis [106, 107]. Cell cycle distribution analysis showed that 24 h pretreatment of HaCaT keratinocytes with 100 nM 1,25(OH)₂D₃ or calcipotriol resulted in an increase in the proportion of cells in the G2/M phase of the cell cycle following incubation with 120 μM cisplatin for 24 h (Table 3). A similar effect was previously reported for 1,25(OH)₂D₃ with other platinum drug, carboplatin, for the prostate cancer cell line, LNCaP [108]. In the case of cisplatin we did not observe an increase in the proportion of HaCaT keratinocytes in the subG1 fraction, as observed for H₂O₂ treatment (Figure 2). The pretreatment of HaCaT keratinocytes with 1,25(OH)₂D₃ or 20(OH)D₃ resulted in a further increase in the proportion of cells in the subG1 phase after subsequent treatment with H₂O₂ (Figure 2C and D).

Tabele 2.

Effect of vitamin D compounds and cisplatin on the distribution of HaCaT keratinocytes through the cell cycle. HaCaT cells were treated with 100 nM 1,25(OH)₂D₃, 21(OH)pD or calcipotriol for 24 h, and subsequently exposed to 12 μM cisplatin for an additional 24 h. Cells were harvested, stained with propidium iodide and the distribution of cells in the cell cycle analyzed by Flow Cytometry. Data are presented as mean ± S.D. of three independent experiments carried out in triplicate.

	Sub G1	G1	S	G2/M
Control	3.58 ± 0.80	56.78 ± 0.95	14.88 ±0.22	23.19 ±1.51
Cisplatin	3.38 ±0.39	35.98 ± 1.59***	15.76 ± 0.13*	41.16 ± 1.25***
1,25(OH)2D3	2.00 ±0.40*	56.94 ± 0.99	14.43 ± 0.21*	25.02 ± 0.83
1,25(OH)2D3 + cisplatin	2.83 ± 0.30	32.30 ± 0.76***; #	18.03 ± 0.28***; ###	44.41 ± 2.06***; ###
21(OH)pD	4.25 ± 0.80	53.30 ± 1.01**	16.37 ± 0.06**	25.07 ± 0.28
21(OH)pD + cisplatin	4.32 ± 0.05	38.85 ± 1.44**	17.18 ± 0.26**; #	38.04 ± 1.59**
Calcipotriol	3.51 ± 0.55	55.96 ± 0.54	14.31 ± 0.08*	25.08 ± 0.45
Calcipotriol + cisplatin	2.62 ± 0.28#	30.57 ± 1.13***; ##	17.53 ± 0.38***; ##	47.73 ± 1.80***; ##

^*p<0,05;

^**p<0,01;

^***p<0,001 vs. control;

^#- cisplatin versus cisplatin + vitamin D.

Interestingly, our data reveal that incubation of HaCaT keratinocytes with 1,25(OH)₂D₃ prevented the loss of the transmembrane mitochondrial potential, which is a sensitive indicator of the well-being of the cells, following a 1 h incubation with H₂O₂ (Figure 4C). Similar results were reported for cortical neurons, where pretreatment with 1,25(OH)₂D₃ protected them from the loss of mitochondrial transmembrane potential induced by cyanide [109]. It seems, however, that the protective effect is time-dependent, since a 3 h incubation with hydrogen peroxide resulted in a greater decrease in the mitochondrial transmembrane potential after pretreatment with 1,25(OH)₂D₃, 20(OH)D₃ or 21(OH)pD, compared to cells without pretreatment (Figure 4C – E).

The bimodal action of secosteroids was also confirmed by flow cytometry results. 1,25(OH)₂D₃, 20(OH)D₃ or calcipotriol treatment for 24 h decreased the proportion of HaCaT keratinocytes in the subG1 fraction, while after subsequent treatment with H₂O₂ there was an increase in the number of apoptotic and necrotic cells in comparison to HaCaT keratinocytes treated with H₂O₂ only (Figure 2). Interestingly, increased viability of cells and protection from apoptosis after treatment with secosteroids alone, was also observed in HaCaT keratinocytes by others [101, 110], as well as in human umbilical vein endothelial cells (HUVECs) [111]. Furthermore, active forms of vitamin D were shown to protect keratinocytes against UVB-induced DNA damage [112] and decrease formation of ROS in keratinocytes and melanocytes subjected to UVB irradiation [21]. Of note, in our study there was a slight decrease in the level of ROS formation in HaCaT keratinocytes after treatment with 1,25(OH)₂D₃ but the data did not reach statistical significance (Figure 4A and B). A similar inhibition of ROS formation by vitamin D was observed in U937 monocytes [113] and leukemia WEHI-3B cells [114]. Taken together, the results suggest a complex mechanism for the protective actions of of low calcemic vitamin D compounds in the human epidermis. Once the level of ROS exceeds the capacity of cellular defense system, active forms of vitamin D enhance the effect of prooxidants leading to cell death/apoptosis. This could eliminate cells with malignant potential.

While the vitamin D₃ compounds tested had little effect by themselves on the expression of genes encoding proteins that remove ROS (SOD1, SOD2 and catalase), 1,25(OH)₂D₃ 20(OH)pD and calcipotriol, but not 20(OH)D₃, all enhanced their expression induced by H₂O₂. This suggests a protective role of these secosteroids against oxidative stress. On the other hand, H₂O₂ treatment attenuated the stimulation of VDR expression caused by 20(OH)D₃, 21(OH)pD or calcipotriol (but not 1,25(OH)₂D₃) and stimulated the expression of PDIA3 in the presence of these secosteroids. Furthermore, pretreatment of HaCaT keratinocytes with 1,25(OH)₂D3, 20(OH)D₃ or 21(OH)pD (but not calcipotriol) and subsequent exposure to H₂O₂, resulted in elevated expression of CYP3A4 mRNA. CYP3A4 is the major cytochrome P450 isoform involved in drug metabolism, including that of some forms of vitamin D [73, 74]. Thus, the combination of active forms of vitamin D and oxidative stress would appear to enhance the ability of the cells to metabolize a range of drugs, at least in the HaCaT keratinocytes.

Our data indicate that oxidative stress and active forms of vitamin D₃ (other than 20(OH)D₃) together stimulate the expression of CYP27B1, the enzyme required for the formation of 1,25(OH)₂D₃ [73] and inhibit the expression CYP24A1, the major enzyme involved in the degradation of 1,25(OH)₂D₃. These effects therefore promote an increase in the level of 1,25(OH)₂D₃. Oxidative stress and active forms of vitamin D₃ together tend to decrease the expression of VDR, but enhance the expression of PDIA3, suggesting that there is a shift away from genomic pathways of vitamin D action towards nongenomic pathways under these conditions. It appears that VDR expression may influence ROS regulation in cells as well. For instance, VDR downregulation in HIV-infected cells contributes to HIV-induced ROS generation in kidney cells and, interestingly, vitamin D administration attenuates this phenomenon [115]. This implies that the increased VDR mRNA level we observed after a 24 h incubation of HaCaT cells with all four of the secosteroids tested (Figure 6D) might be considered as an indirect antioxidative effect of these compounds.

To our knowledge this is the first report describing the interaction of low calcemic analogs (20(OH)D₃, 21(OH)pD and calcipotriol), with hydrogen peroxide or cisplatin, and reveals the unique properties of the individual secosteroids tested.. Importantly, 20(OH)D₃, 21(OH)pD and calcipotriol have less or no influence on calcium levels compared to 1,25(OH)₂D₃ [8, 23, 24, 26, 116, 117], indicating biased effects on the VDR as proposed previously [8]. For instance, of the secosteroids tested 20(OH)D₃ displayed the strongest stimulation of VDR expression (Figure 6D). These differential effects might be related to the conformational change induced by specific analogues that may modify the interaction of the VDR with coactivators. Also, the nature of ligand receptor interaction can influence whether the action is primarily genomic or non-genomic. Furthermore, vitamin D analogues may bind to alternative receptors to the VDR, such as RORα or RORγ [34]. The lower activity of 21(OH)pD compared to the other secosteroids in inhibiting the growth of HaCaT keratinocytes might be attributed to its short 2C side chain, which influenced its docking to VDR [118]. The relative inability of 20(OH)D₃ compared to 1,25(OH)₂D₃ to stimulate the expression of CYP24A1, as observed in this study, has been well documented before [8, 119]. Another example of the variation between the secosteroids tested in the present study is the inhibition of the H₂O₂-induced expression of catalase by 20(OH)D₃, in contrast to the stimulation seen for 1,25(OH)₂D₃, 21(OH)pD and calcipotriol. It is therefore apparent that specific analogues may be developed for specific therapeutic applications on particular types of cells or pathways.

We used HaCaT keratinocytes as a model cell in this study because of their highly proliferative phenotype, which is characteristic for a variety of skin pathologies including psoriasis and skin cancers of epidermal origin. The low calcemic vitamin D analogues were used at concentrations corresponding to the optimal serum 25(OH)D₃ level (75–125 nM) [67]. While the plasma concentration of 1,25(OH)₂D₃ is at least 100 times lower than that of 25(OH)D₃ [120], the calculated IC₅₀ values for secosteroids tested were actually similar to this concentration, between 0.081 and 1.44 nM for inhibition of cell proliferation. The influence of the secosteroids on the sensitivity of keratinocytes to the ROS generating drug, cisplatin, that we observed highlights the importance of maintaining an adequate serum level of 25(OH)D₃ during treatment of hyperproliferative cutaneous diseases. Vitamin D deficiency is extremely common in patients with psoriasis [121] and skin cancers (see [50] for recent review). Our data indicating the enhancement of the cytotoxic activity of cisplatin by the low-calcemic vitamin D analogues, should be of practical application for cancer therapy.

Vitamin D analogs show both prooxidative and antioxidative properties Noncalcemic vitamin D derivatives enhance cytotoxic effects of H2O2 Noncalcemic vitamin D derivatives sensitize human immortalized keratinocytes to cisplatin.

Abbreviations

1α,25(OH)₂D₃

1α,25-dihydroxyvitamin D₃ (calcitriol)

20(OH)D₃

20S-hydroxyvitamin D₃

21(OH)pD

21-hydroxypregnacalciferol

25(OH)D₃

25-hydroxyvitamin D₃ (calcifediol)

7-DHC

7-dehydrocholesterol (provitamin D₃, cholesta-5,7-dien-3β-ol)

7-DHP

7-dehydropregnenolone

CAT

catalase

CCCP

carbonyl cyanide 3-chlorophenylhydrazone, a protonophore, a chemical inhibitor of oxidative phosphorylation

MARRS receptor

Membrane-Associated Rapid Response to Steroid binding protein (other names: ERp57, GRp58, Pdia3)

PDIA3

Protein disulfide-isomerase A3

ROS

reactive oxygen species

SOD1

superoxide dismutase 1

SOD2

superoxide dismutase 2

UVA/B

ultraviolet radiation A and B

VDR

vitamin D receptor

VDRE

vitamin D response elements