Elsevier

Toxicology in Vitro

Volume 22, Issue 1, February 2008, Pages 96-105
Toxicology in Vitro

Combined treatment of P-gp-positive L1210/VCR cells by verapamil and all-trans retinoic acid induces down-regulation of P-glycoprotein expression and transport activity

https://doi.org/10.1016/j.tiv.2007.08.011Get rights and content

Abstract

The development of the most common multidrug resistance (MDR) phenotype associated with a massive overexpression of P-glycoprotein (P-gp) in neoplastic cells may result in more than one hundred fold higher resistance of these cells to several drugs. L1210/VCR is a P-gp-positive drug resistant cell line in which P-gp overexpression was achieved by repeated cultivation of parental cells with a stepwise increasing concentration of vincristine. Relatively little is known about regulation of P-gp expression. Therefore, serious efforts have been made to recognize all aspects involved in regulation of P-gp expression. Retinoic acid nuclear receptors are involved in regulating expression of a large number of different proteins. Several authors have described that all-trans retinoic acid (ATRA, ligand of retinoic acid receptors, RARs) may induce alterations in P-gp expression and/or activity in drug resistant malignant cell lines. There are also other nuclear receptors for retinoidsretinoid X receptors (RXRs) – that may be involved in the development of the P-gp-mediated MDR phenotype. The topic of the present paper is a study of the relationship, if any, between the regulatory pathways of nuclear receptors for retinoids and P-glycoprotein expression.

Increased levels of mRNAs encoding the retinoic acid nuclear receptors RARα and γ, as well as decreased levels of the mRNAs encoding RARβ and the retinoid X receptor RXRγ or slightly decreased levels of RXRβ mRNA, were observed in L1210/VCR cells in comparison with parental L1210 cells. Neither L1210 cells nor L1210/VCR cells contained measurable amounts of mRNA encoding the RXRα receptor. ATRA did not influence the viability of L1210/VCR cells differently from L1210 cells. A combined treatment of L1210/VCR cells with vincristine (1.08 μmol/l) and ATRA induced slightly higher cell death than that observed with ATRA alone. When applied alone, ATRA did not influence P-gp expression (monitored by anti P-gp antibody c219 using western blot analysis) or transport activity (monitored by use of calcein/AM as a P-gp substrate by FACS) in L1210/VCR cells. In contrast, when ATRA was applied together with verapamil (an often used P-gp inhibitor), a significant decrease in P-gp expression and transport activity were observed. However, no significant differences in [11, 12-3H]-ATRA uptake were observed in either sensitive or resistant cells, in the latter case in the absence or presence of vincristine. Moreover, verapamil did not influence ATRA uptake under any conditions.

Thus, we can conclude that the combined treatment of L1210/VCR cells with ATRA and verapamil is able to depress P-gp expression, and consequently its activity. ATRA is not a P-gp-transportable substance, and thus this effect could not be attributed to verapamil-induced inhibition of P-gp that would allow ATRA to reach retinoic acid nuclear receptors and activate them.

Introduction

Multidrug resistance of cancer tissue represents a real obstacle in effective pharmacotherapy of neoplastic diseases (for review see Perez-Tomas, 2006). Several mechanisms of MDR have been identified recently, with overexpression of the drug-transporting ATP-dependent pump P-glycoprotein (P-gp) known to occur most frequently (Gottesman and Ling, 2006). P-gp is a member of ABC (ATP Binding Cassette, ABCB1) transporter superfamily and is known as a transporter with broad substrate specificity (Breier et al., 2000, Chen and Simon, 2000). The presence of this transporter in the plasma membrane causes elimination of several drugs (like vincristine, doxorubicin, actinomycin D, mitomycin C, etc.) from intracellular retention, which reduces their pharmacological efficacies dramatically (for review Breier et al., 2005). Therefore, an important task for scientists who are dealing with molecular oncology is to find procedures to suppress P-glycoprotein expression and/or its transport activity (Modok et al., 2006). Recently, several authors have shown that down-regulation of P-gp expression may be achieved in several P-gp-positive cell lines by treatment with barnidipine and verapamil (Takara et al., 2002), pentoxifylline and its derivatives (Drobná et al., 2002, Kupsáková et al., 2004), staurosporine (Wartenberg et al., 2002) and trifluoperazine (Shin et al., 2006).

The pregnane X receptor (PXR) was described to modulate P-gp expression in the human colon carcinoma cell line LS174T via nuclear receptor-responsive elements in the mdr1 gene promoter (Geick et al., 2001). In normal tissue, a positive correlation between levels of RNA encoding P-gp and PXR was found in peripheral blood (Owen et al., 2004), but not in liver (Wolbold et al., 2003). PXR is activated by a broad spectrum of hydrophobic xenobiotics, including prescription drugs, herbs, pesticides, endocrine disruptors and other environmental contaminants (Kliewer, 2003). This nuclear receptor binds to DNA as a heterodimer with retinoid X nuclear receptors (Krüger et al., 2005). This fact indicates that an interplay between P-glycoprotein expression and vitamin A metabolites may exist. However, information about the effectiveness of 9-cis-retinoic acid, a ligand of RXR receptors (Brtko and Thalhamer, 2003), on P-gp expression is lacking. Retinoic acid receptors (RAR) bind not only 9-cis-retinoic acid, but also all-trans retinoic acid (ATRA, Brtko and Thalhamer, 2003). In contrast to 9-cis retinoic acid, ATRA was described to modulate P-gp expression positively in acute myeloid leukemia cells (Kasumi-1 and Kasumi-6, Tokura et al., 2002, Tabe et al., 2006) and negatively in human colon carcinoma cells (LoVo, Bartolini et al., 2006). Stromskaya et al. (2005), using four stably transfected leukemia cell lines with RARα, have described the following results: only one line (H9 cells) showed a slight elevation of mRNA encoding P-gp; quantities of mRNA encoding P-gp were not affected in two cell lines (KG-1 and NB4); the level of mRNA encoding P-gp was slightly decreased in the K562 line. These trends were not found to reflect changes in P-gp transport activity, as measured by rhodamine 123 retention assays in FACS. ATRA was able to induce a more pronounced elevation of P-gp activity in two transfected cell lines as compared with their parental counterparts. Thus, it could be expected that retinoid nuclear receptors and their ligands would affect P-glycoprotein expression differently in different cells.

Overexpression of P-gp was achieved by long-term adaptation of L1210 cells to vincristine, which yielded our MDR cell model, the L1210/VCR cell line (Poleková et al., 1992). Overexpression of P-gp represents a dominant feature that is responsible for the MDR characteristics of these cells (Bohacova et al., 2000). Doxorubicin, similarly to vincristine, induced a similar MDR phenotype in L1210 cells based on P-gp overexpression (Boháčová et al., 2006). Overexpression of P-gp in these cells is stable and was found to endure through fifteen rounds of cultivation without vincristine as a selection agent (Fiala et al., 2003). The MDR phenotype of L1210/VCR cells is regulated at least indirectly by PKC (Barančík et al., 1995), p38-MAPK (Barančík et al., 2001), ERK (Kišucká et al., 2001) and PI3K/Akt kinase (Barančík et al., 2006). Development of the MDR phenotype is associated with several changes in cell architecture (Uhrík et al., 2006).

We did not have any information about the possible role of nuclear receptors for retinoids in regulation of the expression and/or activity of P-gp in L1210/VCR cells. Therefore, we aimed to investigate if overexpression of P-gp is associated with changes in expression of nuclear receptors for retinoids. Another question that we had to solve was whether ATRA is able to alter the MDR phenotype, as well as P-gp expression and/or activity, in L1210/VCR cells.

Section snippets

Cell cultivation conditions

Sensitive L1210 (S) and vincristine-resistant L1210/VCR mouse leukemia cell lines were used and were cultivated in RPMI medium containing 4% fetal bovine serum (Gibco, USA) in a humidified atmosphere with 5% CO2 in air at 37 °C for 48 h. The last cultivation of resistant cells prior to the experiment was done in the absence (R) or presence (V) of vincristine (108 nmol/l, Gedeon Richter Co., Hungary).

Estimation of mRNA levels encoding retinoic acid nuclear receptors (RARα, RARβ and RARγ) and retinoic X receptor (RXRβ)

Total RNA was isolated using Trizol reagent according to the manufacturer’s instructions. Reverse

Expression of RARα, β and γ as well as RXRα, β and γ in S, R and V cells

Overexpression of P-glycoprotein is associated with increases in levels of mRNAs encoding RARα and RARγ, as is documented for R and V cells in Fig. 1. In contrast, mRNAs encoding RARβ and the retinoid X receptor RXRγ were decreased in R and V cells as compared with S cells. Levels of mRNA for RXRβ were found to be slightly decreased in R and V cells. Neither L1210 (S) nor L1210/VCR (R) and (V) cells contained measurable amounts of mRNA encoding RXRα receptor. Any significant changes in the

Discussion

The main finding of the current paper is that combined treatment of P-gp-positive R and V cells with verapamil and ATRA induced a depression of P-gp expression and/or transport function more markedly as verapamil alone (Fig. 4, Fig. 5). Verapamil is the best known P-gp inhibitor that is transported by this efflux pump through the membrane. P-gp binds verapamil competitively with respect to its other substrates, such as rhodamine 123, vinblastine, quinidine or talinol (Doppenschmitt et al., 1999

Acknowledgements

This study was supported by Slovak Grant Agency for Science VEGA (Grant No. 2/6080 and 2/7122) and by the Slovak Science and Technology Assistance Agency (Grant No. APVT-51-027-404).

References (42)

  • I. Kupsáková et al.

    Reversal of P-glycoprotein mediated vincristine resistance of L1210/VCR cells by analogues of pentoxifylline. A QSAR study

    Eur. J. Pharm. Sci.

    (2004)
  • D. Macejova et al.

    MNU-induced carcinogenesis of rat mammary gland: effect of thyroid hormone on expression of retinoic acid receptors in tumours of mammary gland

    Mol. Cell. Endocrinol.

    (2005)
  • S. Modok et al.

    Modulation of multidrug resistance efflux pump activity to overcome chemoresistance in cancer

    Curr. Opin. Pharmacol.

    (2006)
  • M. Ohata et al.

    RAR and RXR expression by Kupffer cells

    Exp. Mol. Pathol.

    (2000)
  • S.Y. Shin et al.

    Suppression of P-glycoprotein expression by antipsychotics trifluoperazine in adriamycin-resistant L1210 mouse leukemia cells 1

    Eur. J. Pharm. Sci.

    (2006)
  • Z. Sulova et al.

    Expression of P-glycoprotein in L1210 cells is linked with rise in sensitivity to Ca2+

    Biochem. Biophys. Res. Commun.

    (2005)
  • Y. Tabe et al.

    Up-regulation of MDR1 and induction of doxorubicin resistance by histone deacetylase inhibitor depsipeptide (FK228) and ATRA in acute promyelocytic leukemia cells

    Blood

    (2006)
  • K. Takara et al.

    Effects of 12 Ca2+ antagonists on multidrug resistance, MDR1-mediated transport and MDR1 mRNA expression

    Eur. J. Pharm. Sci.

    (2002)
  • Y. Tokura et al.

    Augmented expression of P-gp/multi-drug resistance gene by all-trans retinoic acid in monocytic leukemic cells

    Leuk. Res.

    (2002)
  • M. Wartenberg et al.

    Modulation of intrinsic P-glycoprotein expression in multicellular prostate tumor spheroids by cell cycle inhibitors

    Biochim. Biophys. Acta

    (2002)
  • R. Wolbold et al.

    Sex is a major determinant of CYP3A4 expression in human liver

    Hepatology

    (2003)
  • Cited by (27)

    • Acute myeloid leukemia cells MOLM-13 and SKM-1 established for resistance by azacytidine are crossresistant to P-glycoprotein substrates

      2015, Toxicology in Vitro
      Citation Excerpt :

      The statistical significance was analyzed by the unpaired Student’s t-test. P-gp transport activity was measured using a previously described calcein retention assay (Sulova et al., 2008). To remove AzaC after cultivation, cells were centrifuged (500g, 5 min) and washed three times with PBS containing 0.2% BSA.

    • A new nanostructured carrier design including oil to enhance the pharmaceutical properties of retinoid therapy and its therapeutic effects on chemo-resistant ovarian cancer

      2014, European Journal of Pharmaceutics and Biopharmaceutics
      Citation Excerpt :

      As shown in Fig. 4A, ABCB1 (P-glycoprotein) expression in SKOV-3PR cells exposed up to 300 nM paclitaxel was substantially elevated when compared with the chemo-naive cell line SKOV-3 and cells exposed to lower paclitaxel level (30 nM). To confirm if this translated into increased drug efflux, a functional assay with the rhodamine 123 as drug transporter substrate as described was performed [22]. Significantly less fluorescent drug was retained by SKOV-3PR cells than the chemo-naive SKOV-3 cells (Fig. 4B), confirming the elevated drug efflux activity in the chemo-exposed SKOV-3PR subline.

    View all citing articles on Scopus
    View full text