Original articleExpression of MDR1 and MDR3 gene products in paclitaxel-, doxorubicin- and vincristine-resistant cell lines
Introduction
Multidrug resistance (MDR) is a major problem in the chemotherapeutic treatment of cancer patients [1]. Some cancers exhibit significant primary resistance to cytostatic agents, while other cancers acquire the MDR phenotype after prolonged exposure to cytostatic drugs. Drug resistance can be mediated by different mechanisms. However, drug transporters from the ABC family play a central role in this process [2]. The most important ABC protein is P-glycoprotein, which is encoded by the MDR1 (ABCB1) gene [3]. Overexpression of P-gp has been directly implicated in resistance to a broad spectrum of chemotherapeutic agents, including the taxanes, the anthracyclines, and the vinca alkaloids [4], [5], [6]. For various types of cancer, accumulated evidence from numerous studies indicates that MDR1 overexpression may be the predominant factor that limits the efficacy of chemotherapeutic agents. The human MDR1 gene exhibits high nucleotide sequence homology (approximately 80%) with the MDR3 (ABCB4) gene [7], [8]. MDR3 is known as a phosphatidylcholine transporter [9], [10]. However, increasing evidence suggests that MDR3 plays a role in drug resistance. High levels of MDR3 gene expression have been reported in leukaemic cells from patients with prolymphocytic leukaemia (PLL) that did not express the MDR1 gene [11]. Cyclosporin A (CyA), a P-gp inhibitor, increased daunorubicin accumulation in these cells, suggesting that MDR3 plays a role in drug resistance [11]. Herweijer et al. reported that the MDR3 gene is coexpressed with the MDR1 gene in B-cell chronic lymphocytic leukaemia (B-CLL). However, in B-cell prolymphocytic leukaemia (B-PLL), only MDR3 expression has been detected [12]. Higher MDR3 expression levels in the advanced stages of chronic lymphocytic leukaemia (CLL) than in the early stages of CLL have been reported [13]. Arai et al. suggested that MDR3 expression has been negatively correlated with clinical outcome [14]. Furthermore, treatment of MDR3 positive, but MDR1 negative, cells with CyA lead to significant increases in rhodamine 123 (Rh123) at the intracellular level [14]. Expression of MDR3 together with MDR1, or independently of MDR1, has also been reported in patients with soft tissue sarcomas (STS) [15]. Furthermore, MDR3 expression was increased to a greater degree than MDR1 after chemotherapy. The expression of MDR3 has also been reported in ovarian cancer cell lines [16]. In addition, MDR3 has been shown to be able to transport MDR1 substrates in both humans [17] and mice [18]. Smith et al. reported the increased directional transport of several MDR1-targeted chemotherapeutic agents, such as digoxin, paclitaxel, and vinblastine, through the polarised layers of MDR3-transfected cells [17]. Devault and Gros reported overlapping substrate specificities between MDR1 and MDR3 in a mouse model [18]. These results indicate that MDR3 may indeed play a role in drug resistance. However, MDR3 expression has mainly been investigated in different types of leukaemia. The role of MDR3 in cancer drug resistance remains poorly understood.
In the present study, we used a cancer model of drug resistance to determine the role of MDR3 expression in drug resistance during cancer. The objective of our research was to compare the expression of MDR1 and MDR3 in doxorubicin-, paclitaxel- and vincristine-resistant cell lines and determine the correlation between the expression of these genes and levels of resistance to doxorubicin and paclitaxel.
Section snippets
Reagents
Doxorubicin, paclitaxel, vincristine, RPMI 1640 medium, EMEM medium, foetal bovine serum, an antibiotic-antimycotic solution and L-glutamine were purchased from Sigma (Saint-Louis, MO). A Cell Proliferation Kit I (MTT) was purchased from Roche Diagnostics GmbH (Mannheim, Germany).
Cell lines and cell culture
The established human colon adenocarcinoma cell line LoVo, the doxorubicin-resistant subline LoVoDX, and the human ovarian carcinoma cell line A2780 were purchased from ATCC. The primary human ovarian cancer cell line
MTT analyses of drug-resistant and sensitive cell lines
All of the drug-resistant cell lines were established by the stepwise selection of drug sensitive cells that had been cultured in growth media containing increasing drug concentrations. In order to determine the sensitivity of the drug sensitive and drug-resistant cells lines, the cells were treated for 72 h with increasing concentrations of the drugs that were used to derive drug-resistant cell lines. We observed dose-dependent effects of doxorubicin in all of the investigated cell lines (Fig. 1
Discussion
In the present study, we analysed the expression of the MDR1 and MDR3 genes in various drug-resistant cancer cell lines. MDR1 expression in drug-resistant cancers and drug-resistant cancer cell lines is well documented. However, the literature concerning the role of MDR3 gene expression in drug resistance is sparse. Available papers describe MDR3 expression in several types of leukaemia [11], [12], [13], [14], soft tissue sarcomas [15], and paclitaxel-resistant breast and ovarian cancer cell
Conclusion
In summary, our results revealed that MDR3 is expressed with MDR1 in different cancer cell lines that are resistant to doxorubicin, paclitaxel and vincristine. MDR3 expression seemed to play a compensatory role in most of the investigated cell lines. However, in the LoVoDx cell line, MDR3 expression was increased to a higher degree than MDR1, suggesting that MDR3 plays an important role in drug resistance in this cell line.
High correlations between MDR3 expression and doxorubicin and paclitaxel
Disclosure of interest
The authors declare that they have no conflicts of interest concerning this article.
Acknowledgements
This study was supported by grant No. N N401 204139 from the National Science Centre.
References (21)
- et al.
Multidrug resistance in the laboratory and clinic
Adv Clin Chem
(1994) - et al.
Structure of the human MDR3 gene and physical mapping of the human MDR locus
J Biol Chem
(1991) - et al.
The human MDR3 P-glycoprotein promotes translocation of phosphatidylcholine through the plasma membrane of fibroblasts from transgenic mice
FEBS Lett
(1994) - et al.
MDR1 P-glycoprotein is a lipid translocase of broad specificity, while MDR3 P-glycoprotein specifically translocates phosphatidylcholine
Cell
(1996) - et al.
High expression of MDR3 multidrug resistance gene in advanced-stage chronic lymphocytic leukemia
Blood
(1992) - et al.
Expression of the MDR1 and MDR3 gene products in acute and chronic leukemias
Leuk Res
(1997) - et al.
MDR3 P-glycoprotein, a phosphatidylcholine translocase, transports several cytotoxic drugs and directly interacts with drugs as judged by interference with nucleotide trapping
J Biol Chem
(2000) - et al.
In vitro prediction of cytostatic drug resistance in primary cell cultures of solid malignant tumours
Eur J Cancer
(1993) Cellular mechanism of multidrug resistance of tumor cells
Biochemistry
(2000)- et al.
Multidrug resistance in cancer: role of ATP-dependent transporters
Nat Rev Cancer
(2002)
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