Calcitriol and Calcipotriol Modulate Transport Activity of ABC Transporters and Exhibit Selective Cytotoxicity in MRP1-overexpressing Cells

Abstract

Efflux transporters P-glycoprotein (P-gp/ABCB1), multidrug resistance protein 1 (MRP1/ABCC1), and breast cancer resistance protein (BCRP/ABCG2) can affect the efficacy and toxicity of a wide variety of drugs and are implicated in multidrug resistance (MDR). Eight test compounds, recently identified from an intramolecular FRET-based high throughput screening, were characterized for their interaction with MRP1. We report that the active metabolite of vitamin D₃, calcitriol, and its analog calcipotriol are selectively cytotoxic to MRP1-overexpressing cells, besides inhibiting transport function of P-gp, MRP1, and BCRP. Calcitriol and calcipotriol consistently displayed a potent inhibitory activity on MRP1-mediated doxorubicin and calcein efflux in MRP1-overexpressing H69AR and HEK293/MRP1 cells. Vesicular transport studies confirmed a strong inhibitory effect of calcitriol and calcipotriol on MRP1-mediated uptake of tritium-labeled estradiol glucuronide and leukotriene C₄ In cytotoxicity assays, MRP1-overexpressing cells exhibited hypersensitivity toward calcitriol and calcipotriol. Such collateral sensitivity, however, was not observed in HEK293/P-gp and HEK293/BCRP cells, although the vitamin D₃ analogs inhibited calcein efflux in P-gp-overexpressing cells, and mitoxantrone efflux in BCRP-overexpressing cells. The selective cytotoxicity of calcitriol and calpotriol toward MRP1 over-expressing cells can be eliminated with MRP1 inhibitor MK571. Our data indicate a potential role of calcitriol and its analogs in targeting malignancies in which MRP1 expression is prominent and contributes to MDR.

Fig. 1.

Chemical structures of the test compounds.

Fig. 2.

Effects of test compounds on MRP1 activity in cell-based assays. (A) Immunoblot analysis of whole cell lysates prepared from indicated cell lines was performed as described in the Materials and Methods section. (B and C) Calcein accumulation assay. Control and MRP1-overexpressing cells were pre-treated with 50 μM MK571 or test compounds for 10 minute before incubation with 25 nM calcein-AM at 37°C for 30 minute. Fluorescence intensity of intracellular calcein was detected using flow cytometry, with excitation and emission wavelengths of 480 and 533/30 nm, respectively. Data are combined from three experiments (performed in duplicate) and presented as mean ± S.E.M. *P < 0.05; ***P < 0.001 compared with control of each cell line, calculated using linear mixed model and Sidak post hoc test. (D) Doxorubicin accumulation assay. HEK293T cells transiently transfected with MRP1-GFP (green) were pre-treated with 50 μM test compounds, before incubation with doxorubicin (red) at 37°C for 1 hour. Images were acquired using confocal microscopy. GFP and doxorubicin were excited at 488 nm, and detected at 475/42 and 605/64 nm, respectively.

Fig. 3.

Effects of test compounds on MRP1 activity in membrane vesicle-based assays. (A and B) Inside-out membrane vesicular uptake assay. Membrane vesicles (2 µg protein) prepared from HEK293/pcDNA3.1 (vector control) and HEK293/MRP1 cells were incubated with 10 or 50 μM test compounds and (A) 400 nM/20 nCi [³H]E₂17βG or (B) 50 nM/2 nCi [³H]LTC₄ at 37°C for 1 minute. MRP1 uptake activity was determined by measuring the radioactivity retained on collected membrane vesicles using liquid scintillation counting. Data are combined from ≥3 experiments (performed in triplicate) and presented as mean ± S.E.M. ^#, value below background; **P < 0.01; ***P < 0.001 compared with control, calculated using linear mixed model and Sidak post hoc test. Concentration-dependent inhibition of MRP1 activity by calcitriol (C) and calcipotriol (D). MRP1-mediated uptake of [³H]E₂17βG was performed by incubating HEK293/MRP1 membrane vesicles (2 µg protein) with various concentrations of calcitriol or calcipotriol. Data are representative of three experiments and presented as mean ± S.D. (n = 3).

Fig. 4.

Collateral sensitivity of MRP1-overexpressing cells toward calcitriol and calcipotriol. H69 and H69AR cells were treated with increasing concentrations of calcitriol or calcipotriol in the absence or presence of MK571 for 96 hour (A). Similar experiments were performed using calcitriol or calcipotriol in HEK293/pcDNA3.1 and HEK293/MRP1 cells in the absence or presence of MK571 for 72 hour (B). Cell viability was evaluated with MTT assay. Data are representative of three experiments and expressed as mean ± S.D. (n = 3).

Fig. 5.

Effects of calcitriol and calcipotriol on the mRNA and protein expression of MRP1. Cells were treated with DMSO, calcitriol, or calcipotriol (10 µM) for 72 hour. (A) mRNA levels of parental (HEK293 and H69) cells and MRP1-overexpressing cells (HEK293/MRP1 and H69AR) were quantified using qPCR as described in the Materials and Methods section. Data are combined from three experiments (each performed in triplicate) and presented as mean ± S.E.M. *P < 0.05; ***P < 0.001 compared with control, calculated using linear mixed model and Sidak post hoc test. (B) Immunoblot analysis of whole cell lysates prepared from indicated cell lines with indicated treatments was performed as described in the Materials and Methods section. Data are representative of three experiments. Mean MRP1 protein expression (±S.E.M., n = 3) shown as a fold change of the DMSO-treated control is shown in (C). Protein band density was analyzed using the Image Studio Lite (LI-COR Biotechnology) software and corrected for uneven sample loading and transfer using α-tubulin as the loading control. *P < 0.05; ***P < 0.001 compared with control, calculated using linear mixed model and Sidak post hoc test.

Fig. 6.

Calcitriol and calcipotriol inhibit P-gp- and BCRP-mediated efflux. Control, P-gp-overexpressing HEK293 (A) or MDCKII (B), and BCRP-overexpressing HEK293 (C) or MDCKII (D) cells were pre-treated with calcitriol and calcipotriol at 37°C for 10 minute before incubation with 25 nM calcein-AM (A and B) or 5 µM mitoxantrone (C and D) for 30 minute. Verapamil and Ko143 were used as positive controls for P-gp and BCRP inhibition, respectively. Intracellular accumulation of calcein and mitoxantrone was quantified by flow cytometry. Data are combined from three experiments (performed in duplicate) and presented as mean ± S.E.M. *P < 0.05; **P < 0.01; ***P < 0.001 compared with control, calculated using linear mixed model and Sidak post hoc test.

Fig. 7.

P-gp- and BCRP-overexpressing cells do not show collateral sensitivity toward calcitriol and calcipotriol. HEK293/pcDNA3.1, HEK293/P-gp, and HEK293/BCRP cells were treated with increasing concentrations of calcitriol (A) or calcipotriol (B) for 72 hour. Cell viability was evaluated with MTT assay. Data are representative of three experiments and expressed as mean ± S.D. (n = 3).

Fig. 8.

Effects of calcitriol and calcipotriol on drug sensitivity of HEK293/P-gp and HEK293/BCRP cells. HEK293/P-gp (A) and HEK293/BCRP (B) cells were treated with increasing concentrations of vincristine and mitoxantrone, respectively, in the absence and presence of calcitriol or calcipotriol (10 µM) for 72 hour. Verapamil (25 µM) and Ko143 (1 µM) were used as positive controls in HEK293/P-gp and HEK293/BCRP cells, respectively. HEK293/pcDNA cells were included as negative control for drug resistance. Cell viability was evaluated with MTT assay. Data are representative of three experiments and expressed as mean ± S.D. (n = 3).