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Research ArticleArticle
Open Access

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

Kee W. Tan, Angelina Sampson, Bremansu Osa-Andrews and Surtaj H. Iram
Drug Metabolism and Disposition December 2018, 46 (12) 1856-1866; DOI: https://doi.org/10.1124/dmd.118.081612
Kee W. Tan
Department of Chemistry and Biochemistry, College of Natural Sciences, South Dakota State University, Brookings, South Dakota
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Angelina Sampson
Department of Chemistry and Biochemistry, College of Natural Sciences, South Dakota State University, Brookings, South Dakota
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Bremansu Osa-Andrews
Department of Chemistry and Biochemistry, College of Natural Sciences, South Dakota State University, Brookings, South Dakota
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Surtaj H. Iram
Department of Chemistry and Biochemistry, College of Natural Sciences, South Dakota State University, Brookings, South Dakota
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    Fig. 1.

    Chemical structures of the test compounds.

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    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.

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    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).

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    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).

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    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.

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    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.

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    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).

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    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).

Tables

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    TABLE 1

    Primer sequences

    GeneForward primerReverse primer
    MRP1AGGACACGTCGGAACAAGTCGGAAGTAGGGCCCAAAGGTC
    BΒ-ActinCATGTACGTTGCTATCCAGGCCTCCTTAATGTCACGCACGAT
    Ubiquitin CEATGCGGGACTTCAAGAGGAGAAAATGACCGCGTTCCACAC
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    TABLE 2

    Reaction parameters derived from the cytotoxicity and membrane vesicular transport assays

    AssayIC50 (µM)a
    CytotoxicityCalcitriolCalcipotriol
    H6929.0 ± 1.2*23.9 ± 0.5*
    H69AR (control)11.0 ± 0.78.5 ± 1.0
    H69AR + 10 μM MK57116.4 ± 1.915.0 ± 1.1*
    H69AR + 50 μM MK57161.6 ± 1.2**42.4 ± 1.0**
    HEK293/pcDNA3.133.4 ± 2.5**23.3 ± 3.7
    HEK293/MRP1 (control)8.9 ± 1.36.0 ± 1.8
    HEK293/MRP1+ 10 μM MK57111.7 ± 1.116.5 ± 0.4*
    HEK293/MRP1+ 50 μM MK57135.3 ± 4.0**27.5 ± 0.2**
    HEK293/P-gp43.1 ± 4.3**37.9 ± 5.1**
    HEK293/BCRP31.3 ± 5.3**20.8 ± 2.7*
    Membrane vesicular transport
    E217βG9.0 ± 1.312.3 ± 0.1
    • ↵a Mean ± S.E.M. of n = 3 independent experiments.

    • ↵* P < 0.05; **P < 0.01 significantly different then the control in each cell line group, calculated using linear mixed model with Sidak post hoc test.

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    TABLE 3

    Effect of calcitriol and calcipotriol (10 µM) on IC50 of vincristine and mitoxantrone in HEK293/P-gp and HEK293/BCRP, respectively

    Verapamil (25 µM) and Ko143 (1 µM) are used as positive controls.

    Cell line/TreatmentVincristine, IC50 (nM)aMitoxantrone, IC50 (nM)a
    HEK293/pcDNA3.13.70 ± 1.26***4.57 ± 1.48***
    HEK293/P-gp (control)1394.00 ± 174.46
    HEK293/P-gp + verapamil2.95 ± 1.16***
    HEK293/P-gp + calcitriol758.93 ± 323.48*
    HEK293/P-gp + calcipotriol982.60 ± 118.54
    HEK293/BCRP (control)30.88 ± 4.18
    HEK293/BCRP + Ko1431.33 ± 0.14***
    HEK293/BCRP + calcitriol12.84 ± 0.26**
    HEK293/BCRP + calcipotrioll29.66 ± 3.21
    • ↵a Mean ± S.E.M. of n = 3 independent experiments.

    • ↵* P < 0.05; **P < 0.01; ***P < 0.001 significantly different then control in each treatment group, calculated using linear mixed model with Sidak post hoc test.

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Drug Metabolism and Disposition: 46 (12)
Drug Metabolism and Disposition
Vol. 46, Issue 12
1 Dec 2018
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Research ArticleArticle

Collateral Sensitivity of MRP1-Overexpressing Cells

Kee W. Tan, Angelina Sampson, Bremansu Osa-Andrews and Surtaj H. Iram
Drug Metabolism and Disposition December 1, 2018, 46 (12) 1856-1866; DOI: https://doi.org/10.1124/dmd.118.081612

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Research ArticleArticle

Collateral Sensitivity of MRP1-Overexpressing Cells

Kee W. Tan, Angelina Sampson, Bremansu Osa-Andrews and Surtaj H. Iram
Drug Metabolism and Disposition December 1, 2018, 46 (12) 1856-1866; DOI: https://doi.org/10.1124/dmd.118.081612
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