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
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).
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