ReviewMicroarray-based detection and expression analysis of ABC and SLC transporters in drug-resistant ovarian cancer cell lines
Introduction
Multidrug resistance (MDR) is the main cause of chemotherapy effectiveness lack in most of the cancers [1]. Some cancers exhibit significant primary resistance to cytostatics, others acquire MDR phenotype after prolonged exposure to cytostatic drugs. The development of MDR makes further treatment ineffectual [2]. Drug resistance can be mediated by different cellular mechanisms, however, expression of membrane transporters from ABC family plays the leading role in this process [3].
The ABC transporter superfamily, in humans, encloses at least 49 genes grouped into seven families (from A to G) with different functions (ABC transporter web page: http://nutrigene.4t.com/humanabc.htm). These transporters mediate energy-dependent drug efflux and are frequently associated with decreased cellular accumulation of anticancer drugs and MDR phenotype of cancers [3]. Many types of cancers with primary resistance to chemotherapy exhibit high expression of proteins from ABC family. Increased expression of ABC proteins is associated with development of chemotherapy resistance in vivo and in vitro [2], [3].
The most important ABC protein is glycoprotein P (P-gp) encoded by ABCB1 (multidrug resistance protein 1 - MDR1) gene [4]. This protein actively removes nearly 20 cytostatic drugs from the cell. Other well-known genes responsible for MDR are MRP1 (MDR related protein 1, ABCC1) [5]; MRP2 (ABCC2) [6] and breast cancer resistance protein–BCRP (ABCG2) [7]. It is expected that at least 10 other ABC proteins can be involved in drug resistance [8].
Another group of membrane transporters involved in drug resistance is the solute carrier (SLC) transporters, which function mainly as influx transporters [9]. SLC transporters include over 300 members organized into 51 families [9] (http://www.bioparadigms.org/slc/menu.asp). SLC carriers utilize facilitated and secondary active transport to specific substrates, such as metals, organic cations, anions, phosphates, monocarboxylic acid, sugars, amino acids, oligopeptides, nucleosides, and water-soluble vitamins [10], [11], [12]. These transporters mediate the cellular uptake of hydrophilic drugs and could be useful tools for delivery drugs to cancer cells. Some SLC transporters like nutrient transporters can be upregulated in drug-resistant cells, in response to higher energy and nutrition requirement [10], [11], [12]. On the other hand, transporters involved in drug transport are often downregulated [10], [11], [12]. So far nearly 30 SLC transporters involved in chemoresistance are known, but probably many others can also play a role in this process.
Genome wide expression analysis by oligonucleotide microarray is a powerful molecular tool for the discovery of new genes involved in molecular processes including drug resistance. This study shows alterations in gene expression levels of ABC and SLC transporters in methotrexate (W1MR), cisplatin (W1CR), doxorubicin (W1DR), vincristine (W1VR), topotecan (W1TR) and paclitaxel-resistant (W1PR) variant of W1 primary ovarian cancer cell line.
Section snippets
Cell lines and cell culture
Human ovarian cancer cell line W1 was established from ovarian cancer tissue obtained from an untreated patient. Sublines resistant to MTX - W1MR (W1 methotrexate resistant), CIS - W1CR (W1 cisplatin resistant), DOX - W1DR (W1 doxorubicin resistant), VIN - W1VR (W1 vincristine resistant), TOP - W1TR (W1 topotecan resistant) and PAC - W1PR (W1 paclitaxel-resistant) were obtained by exposure of the W1 line to stepwise increasing drug concentration. Final concentration of each drug was twofold
Gene chip scanning and preliminary analysis
The quality of all GeneChip expression data were in “good sample” limits according to preliminary data analysis parameters such as background and noise averages, percentage of present calls, presence of internal hybridization controls in increasing signals, presence of poly-A controls as decreasing signals and GAPDH to beta actin 3′/5′ signal ratios.
Data analysis, gene lists and evaluation
We have analyzed expression of 45 genes from ABC superfamily and 350 genes from SLC superfamily in six drug-resistant sublines. Table 1, Table 2
Discussion
This report presents expression of genes encoding membrane transporters from two superfamilies: ABC and SLC in six drug-resistant ovarian cancer cell lines. To the best of our knowledge, this is the first report, which analyzed six cell lines resistant to six different cytostatic drugs together. Since genes having fold change values between 3 and 0.33 were not considered as a significant, their relation to drug-resistant will not be discussed.
The microarray data was confirmed with the qPCR
Conclusion
In summary, our results confirmed a predominant role of ABCB1 in resistance to Dox, Vin and PAC and BCRP in resistance to Top. It seems that ABCB4 can play a complementary role in Dox and Vin resistance. Our results also confirm that downregulation of SLC19A1 plays a role in MTX resistance especially if expression of other genes responsible for MTX resistance is not changed.
We also have found changes in expression of other genes from ABC and SLC families, not described so far. However, the role
Disclosure of interest
The authors declare that they have no conflict of interests including employment, consultancies, stock ownership, honoraria, paid expert testimony, patent applications/registrations, and grants or other funding.
Acknowledgements
We thank Dr. Michał W. Łuczak from Department of Biochemistry and Molecular Biology, Poznań University of Medical Sciences and Dr. Bartosz Kempisty from Department of Histology and Embryology, Poznań University of Medical Sciences for helpful discussions, and insight into this manuscript.
Grant support: This study was supported by grant No. N N401 204139 from National Science Centre.
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