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Microarray-based detection and expression analysis of ABC and SLC transporters in drug-resistant ovarian cancer cell lines

https://doi.org/10.1016/j.biopha.2012.11.011Get rights and content

Abstract

Multiple drug resistance of cancer cells is multifactorial. A microarray technique may provide information about new candidate genes playing a role in drug resistance. Drug membrane transporters from ABC and SLC families play a main role in this phenomenon. This study demonstrates alterations in ABC and SLC gene expression levels in methotrexate, cisplatin, doxorubicin, vincristine, topotecan and paclitaxel-resistant variant of W1 ovarian cancer cell line. Resistant W1 cell lines were derived by stepwise selection of cells in increasing concentration of drugs. Affymetrix GeneChip® Human Genome U219 Array Strip was used for hybridizations. Statistical significance was determined by independent sample t-test. The genes having altered expression levels in drug-resistant sublines were selected and filtered by scater plot. Genes up/downregulated more than threefolds were selected and listed. Among ABC genes, seven were upregulated and three were downregulated. Three genes: ABCB1, ABCB4 and ABCG2 were upregulated very significantly (over tenfold). One ABCA8 was significantly downregulated. Among 38 SLC genes, 18 were upregulated, 16 were downregulated and four were up- or downregulated dependent on the cell line. Expression of 10 SLC genes was changed very significantly (over tenfold). Four genes were significantly increased: SLC6A1, SLC9A2, SLC12A1, SLC16A6 and six genes were significantly decreased: SLC2A14, SLC7A3, SLC7A8, SLC7A11, SLC16A14, SLC38A9. Based on the expression profiles, our results provide a preliminary insight into the relationship between drug resistance and expression of membrane transporters involved in drug resistance. Correlation of specific drug transporter with drug resistance requires further analysis.

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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