Abstract: The vitamin D3 metabolite 1,25-dihydroxycholecalciferol (DHC) and analogues derived from it are being investigated as potential agents for the treatment of cancer. Combining ketoconazole (KTZ) with DHC has been recommended to enhance the anticancer activity of DHC. DHC exerts its biological activities through the vitamin D receptor (VDR). VDR is recognized to be a regulator of P-glycoprotein (P-gp), a member of the ABC transporter family well known for its role in multidrug resistance in cancer chemotherapy. We have investigated the effect of DHC and adding KTZ together with DHC on P-gp and VDR expression and the functional consequences of P-gp induction in intestinal human colonic adenocarcinoma cells LS174T cells. DHC increased P-gp expression by two times, and the addition of KTZ further increased the expression to four times. The combination of DHC + KTZ also significantly increased VDR expression, consistent with the enhanced increase in P-gp expression by this combination. The increase in P-gp expression was accompanied by increased P-gp function, as measured by decreased Rh123 accumulation in the LS174T cells. In addition, DHC significantly decreased colchicine cytotoxicity in a dose-sensitive manner, and the addition of KTZ further decreased the colchicine cytotoxicity, indicating the chemo-protective effect of DHC is enhanced by KTZ, consistent with the enhanced expression of P-gp. The results of this study raise the possibility that DHC and the addition of KTZ to DHC treatment may decrease the effectiveness of cancer chemotherapy by promoting P-gp-mediated drug resistance.
1,25-Dihydroxycholecalciferol (DHC) is a physiologically active metabolite of vitamin D that maintains calcium homeostasis and phosphate homeostasis . DHC exerts its effects by binding with vitamin D receptor (VDR), a member of the ligand-activated nuclear receptor superfamily . The role of DHC via ubiquitously expressed VDR in inhibition of cell proliferation and stimulation of differentiation of malignant cells in vitro and in vivo is well established [3,4]. Interestingly, epidemiological studies observed lower incidence and mortality rates in several cancers in regions where solar radiation is high [5,6]. In addition, deficiency of vitamin D3 has been associated with a variety of conditions, including cardiovascular disease , hypertension , type 1  and type 2 diabetes  and multiple sclerosis . The classical effects of vitamin D3 deficiency include suboptimal calcium absorption, secondary hyperparathyroidism, increased bone resorption and decreased muscle strength, which result in increased risk of fall injuries and mortality in the elderly .
Several studies suggest that combination of DHC and its analogues with other agents may be beneficial to their anti-tumour activity [13–15]. One such combination recommended in the literature is ketoconazole (KTZ) and DHC . The rationale for this is that KTZ inhibits CYP24A1, an enzyme that metabolizes DHC, thereby enhancing VDR expression by the increased and prolonged occupancy of VDR by DHC [17,18]. In addition, cancer cells tend to develop resistance to DHC by up-regulation of CYP24A1 expression [19–21]. It has thus been suggested that KTZ allows DHC to remain active for longer and exert more anti-tumour activity [16,22].
P-glycoprotein (P-gp) is a transmembrane protein associated with multidrug resistance in cancer therapy [23,24]. P-gp is localized at biological barriers, including the hepatic bile canaliculi, epithelial cells of the proximal tubules of kidney and columnar epithelial cells of colon and jejunum , bronchial cells in the lung, capillary endothelial cells of brain and testes and in placental trophoblasts , all sites where it exerts a major influence on disposition of substrate drugs. Intestinal P-gp has become one of the important targets in drug interaction studies due its capacity to limit oral drug bioavailability by transporting its substrates into the intestinal lumen [24,27–29].
Recently, VDR was identified as one of the important transcription factors involved in the regulation of P-gp [30,31]. In addition, DHC and its analogues were shown to increase P-gp expression and function in Caco2 cells  and LS180 cells . Similarly, DHC treatment showed a tissue-dependent increase in P-gp expression in rats [34,35]. Therefore, available in vitro and in vivo data raised the possibility of involvement of VDR in P-gp-mediated drug interactions. With the growing realization of the importance of adequate levels of vitamin D3 in human health, there is an increasing tendency to raise the dose of vitamin D3 recommended by medical authorities. There is a paucity of knowledge concerning induction of P-gp by vitamin D3 and its functional consequences. A greater understanding of the effect of vitamin D3 on P-gp-mediated drug transport is needed to avoid unwanted drug interactions and optimize concurrent drug therapy. In this study, we investigated the effect of DHC on P-gp induction and function in the intestinal LS174T cell line. We also tested the effect of KTZ on the DHC responses, to determine whether this combination affected VDR-mediated P-gp expression and function, and the effect of the DHC-KTZ combination on colchicine cytotoxicity.
LS174T cells (ATCC, Manassas, VA, USA) were grown in DMEM supplemented with 5% FBS (Invitrogen, Mulgrave, Australia) in a humidified incubator at 37°C and 5% CO2. Cells were regularly subcultured once they reached confluence. To determine the effect of DHC and KTZ on P-gp expression and function, LS174T cells were trypsinized using TrypLE™ Express reagent (Invitrogen) and seeded at different cell densities in 6-well plates. Medium was changed on alternative days when fresh drug/vehicle was added. Cells were harvested at specified times.
LS174T cells (ATCC) were seeded in 6-well plates at 0.5 × 106 per well and treated with 50 nM DHC (Sigma-Aldrich, Castle Hill, Australia), 10 μM KTZ (Sigma-Aldrich) (added 1 hr before DHC when combined with it), or vehicle (ethanol and/or DMSO) for 48 hr. To determine the involvement of VDR in P-gp regulation, LS174T cells were incubated with VDR antagonist 0.1 μM ZK159222 (a gift from Bayer Schering Pharma AG, Berlin, Germany) for 1 hr before adding DHC and KTZ. After the drug treatment, protein cell lysates were prepared using 100 μl per 1 million cells of RIPA lysis buffer (50 mM Tris–HCl pH 8, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) containing 2 μl mammalian protease inhibitor cocktail (Sigma-Aldrich). Protein concentrations were estimated using the micro BCA protein estimation kit (Thermo Fischer Scientific, Scoresby, Australia). Proteins (20 μg per lane) were separated using 4–12% NuPAGE® Bis-Tris Gels (Invitrogen). After electrophoresis, protein was transferred onto PVDF membrane (Roche Diagnostics, Mannheim, Germany). The blots were blocked with blocking buffer (5% skim milk in Tris-buffered saline + 0.1% Tween 20) for 1 hr at room temperature. After blocking, membranes were exposed to monoclonal anti-P-gp antibody (F4 clone; Sigma-Aldrich) at 1:5000 dilution in blocking buffer or mouse monoclonal anti-VDR (D6) at 1:1000 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-tubulin (Santa Cruz Biotechnology) at 1:10,000 for 1 hr at room temperature, washed three times for 15 min. with Tris-buffered saline, followed by secondary anti-mouse Ig-horseradish peroxidase (Promega, Madison, WI, USA) at 1:10,000 in blocking buffer for 1 hr with three washes as above. Bound secondary antibody was detected with enhanced chemiluminescence (Roche Diagnostics), recorded on film.
Rhodamine 123 assay.
LS174T cells were seeded in 6-well plates 1 day prior to the experiment at 1 × 105 cells/well. On the next day, cells were treated with DHC (5, 15 or 50 nM), 10 μM KTZ (added 1 hr before DHC when combined with it), or vehicle for 48 hr. After the drug treatment, cells were gently washed with PBS, and 10 μM rhodamine 123 (Rh123; Sigma-Aldrich) was added in the presence or absence of 10 μM verapamil (P-gp inhibitor) to each well. After 15-min. incubation at 37°C, cells were washed with cold PBS, trypsinized, and kept on ice until analysis. The accumulation of Rh123 in control and DHC-treated cells was measured using a Coulter (EPICS® XL-MCL) flow cytometer. Propidium iodide (PI) (Sigma-Aldrich) (2 μg/ml) was added to each sample just before the flow analysis to exclude dead cells. Mean fluorescence of Rh123 in cell samples was calculated using Flow Jo 4.6.2 (TreeStar, Ashland, OR, USA).
One day prior the experiment, LS174T cells were seeded in a 96-well plate at 1 × 104/well. These cells were treated with DHC (100 nM), 1 μM KTZ (added 1 hr before DHC when combined with it), or vehicle for 48 hr and then colchicine (a P-gp substrate and cytotoxic agent) was added for 48 hr at 8 nM. Cell viability was quantified using the MTS assay following the manufacturer’s instructions. In brief, 20 μl of CellTiter 96® AQueous One Solution reagent (Promega) was added to each well and cells were incubated for 1 hr at 37°C. The absorbance was measured at 490 nm using a 96-well plate reader, corrected for background, to measure the per cent viability.
Data were analysed using GraphPad Prism. One sample t-tests and unpaired t-tests were employed to identify statistically significant differences between the groups. p Values < 0.05 were considered to be statistically significant.
Effect of DHC and KTZ on P-gp and VDR protein expression.
The effect of DHC and KTZ on P-gp protein expression was investigated by treating LS174T cells with these agents for 2 days followed by Western blotting. DHC at the concentration 50 nM increased P-gp expression by two times. KTZ at the concentration 10 μM decreased P-gp expression to a small but significant degree, but the addition of KTZ together with DHC doubled the P-gp expression (from 2 to 4 times compared to control) (fig. 1A). Thus, KTZ enhances DHC-stimulated P-gp expression in LS174T cells. To investigate the mechanism of induction of P-gp by combined DHC and KTZ, we tested their effect on VDR protein expression. Whereas DHC and KTZ alone did not increase VDR expression significantly, the combination did, by two times (fig. 1B). Furthermore, ZK159222, an antagonist of VDR, reduced the induction of P-gp by DHC + KTZ (fig. 1A), as expected if it was up-regulated through VDR.
DHC decreases Rh123 accumulation.
To relate the increased expression of P-gp with its transport function, various concentrations of DHC were tested for their effects on cellular accumulation of P-gp substrate Rh123. Accumulation of Rh123 in LS174T cells was decreased after 2 days of incubation with DHC (5–50 nM), in a dose-dependent manner (fig. 2). In agreement with the enhanced effect of KTZ plus DHC on P-gp expression, the combination decreased Rh123 accumulation to a significantly greater extent than DHC alone (fig. 3). The effect of DHC and the DHC + KTZ combination on Rh123 accumulation was reversed in the presence of verapamil (10 μM), a P-gp inhibitor.
DHC reduces colchicine-mediated cytotoxicity.
To investigate whether DHC also affected the transport of a cytotoxic agent by inducing P-gp, its effect on colchicine cytotoxicity was determined in LS174T cells. Prior exposure to DHC significantly increased the cell viability, from 56% in the control to 71% after 2 days in the presence of 8 nM colchicine. The increase in cell viability by the combination of KTZ with DHC was significantly higher (83%) than DHC alone (71%) (fig. 4).
One of the obstacles in cancer treatment is development of multidrug resistance through the over-expression of P-gp . Moreover, the pharmacological importance of P-gp in the regulation of drug disposition has become well recognized. Thus, identifying and understanding the factors responsible for the regulation of P-gp is important from the clinical and pharmacological points of view. There is growing interest in developing the vitamin D metabolite DHC and its analogues as anticancer agents. To enhance the anti-tumour activity of DHC, combination with KTZ was recommended . However, the combination is known to up-regulate VDR expression . As VDR is an important regulator of P-gp, it is possible that DHC and the combination of KTZ + DHC up-regulate P-gp expression and increase P-gp-mediated drug transport. Therefore, we evaluated the effect of DHC and the combination of DHC and KTZ on P-gp expression and function in intestinal adenocarcinoma LS174T cells.
Our immunoblotting data show that DHC significantly increased P-gp expression in LS174T cells (fig. 1A). Intestinal P-gp is known to regulate the bioavailability of orally administered drugs and is responsible for several drug–drug interactions . Hence, it is important to identify and study factors that regulate intestinal P-gp to anticipate and limit such drug interactions. It is known that nuclear receptors PXR  and CAR  are important regulators of P-gp expression. Recent studies have shown that another DHC receptor, VDR, also regulates P-gp expression [30,31]. Ligands that activate VDR may thus affect intestinal P-gp-mediated drug transport.
In the current study, we investigated the functional impact of DHC-mediated P-gp induction on the accumulation of P-gp substrate Rh123 and the cytotoxicity of the P-gp substrate colchicine. It has been shown that there is a reciprocal relationship between P-gp expression and Rh123 accumulation in human cancer cell lines because of P-gp-mediated efflux of this substrate dye . Colchicine is also a P-gp substrate; P-gp was originally discovered when Chinese hamster ovary cells developed resistance to colchicine . Exposure to DHC resulted in a dose-dependent decrease in the accumulation of Rh123 in LS174T cells (fig. 2), which was sensitive to a P-gp inhibitor (fig. 3) and correlates with the induction of P-gp expression (fig. 1). The reduced cytotoxic effect of colchicine in LS174T cells after DHC treatment (fig. 4) confirms that the P-gp induction has consequences for the sensitivity of cells to substrate cytotoxic drugs, increasing their resistance substantially. Although DHC itself has sometimes been observed to be toxic for other types of cells, there was no evidence of it with LS174T cells. In fact, DHC protected them to a significant degree from colchicines toxicity. As DHC and analogues derived from it are increasingly of interest for development as anticancer agents, it is prudent to screen them for P-gp-mediated drug interactions in vitro and in vivo to avoid unwanted consequences during therapy. Here, we have used the LS174T intestinal cell line as a useful model to study the regulation of P-gp by VDR, taking into consideration that commonly used Caco-2 cells are less amenable to this purpose [41,42]. Our data also suggest that LS174T cells can serve as an excellent model to test the effect of novel VDR inducers/inhibitors on P-gp expression and function.
Our findings that combination of KTZ with DHC enhances P-gp expression and its substrate efflux function are of particular interest for drug interactions and cancer chemotherapy. In vitro and in vivo studies have shown that KTZ enhances the anti-proliferative effects of DHC by inhibiting CYP24A1, an enzyme responsible for the metabolism of DHC. It is also known that KTZ increases DHC-induced VDR expression and prolongs the occupancy of the receptor by DHC [17,18]. Therefore, it was suggested that the use of CYP24A1 inhibitors such as KTZ in combination with DHC may offer advantages in cancer treatment [16,22]. In accordance with this hypothesis, a recent study on the human prostate cancer cell line PC3 and PC3 xenografts in nude mice showed that the administration of KTZ in combination with DHC enhanced its anti-proliferative effects, increased systemic DHC exposure and promoted the activation of the caspase-independent apoptosis pathway . However, as VDR also up-regulates P-gp, the combination of KTZ and DHC in cancer treatment may increase the threat of multidrug resistance.
Ketoconazole, an inhibitor of PXR-mediated P-gp induction, lowered the expression P-gp as expected when used alone. The contrasting enhanced increase in P-gp expression when it was combined with DHC appears to have occurred via stimulation of VDR expression (fig. 1B). This view is consistent with inhibition of the P-gp induction by the VDR inhibitor ZK159222. Inhibition of CYP24A1 by KTZ may have increased and prolonged occupancy of VDR by DHC, as reported previously [17,18], although we have no data on that. Given the demonstrated consequences for Rh123 accumulation (fig. 3) and colchicine toxicity (fig. 4), it is plausible that the combination of KTZ with DHC will exacerbate P-gp-mediated drug resistance in cancer cells. Such concerns may not be limited to KTZ but could also apply to other drugs that increase the half-life of DHC by inhibiting CYP24A1 activity (e.g.: genestein , tetralones ). Further studies in animals and human beings are required to investigate the potential interactions between CYP24A1 inhibitors and DHC in relation to P-gp-mediated drug transport.
In summary, we demonstrated that DHC up-regulated P-gp expression and decreased P-gp-mediated drug accumulation in LS174T cells and also reduced cytotoxicity of a P-gp substrate. We also showed that the combination of KTZ + DHC significantly increased VDR-mediated P-gp expression and function. These findings may have clinical ramifications as DHC and its analogues are being widely studied for development as therapeutic agents to treat cancer. Our study adds a cautionary note on combining KTZ and other CYP24A1 inhibitors to DHC in enhancing drug resistance and warrants further pre-clinical and clinical studies to explore unwanted P-gp-mediated drug interactions.