Original article
Etoposide resistance in MCF-7 breast cancer cell line is marked by multiple mechanisms

https://doi.org/10.1016/j.biopha.2013.09.007Get rights and content

Abstract

Purpose

Acquired or intrinsic drug resistance is one of the major handicaps in the success of chemotherapy. Etoposide is a topoisomerase II poison widely used in chemotherapy. Similar to other topoisomerase inhibitors and DNA damaging agents, resistance to etoposide may arise as a result of alterations in target expression and activity, increased drug efflux and alterations in DNA damage response mechanisms. Here, we tested the involvement of such mechanisms in etoposide-resistant MCF-7 breast cancer cells.

Methods

Relative etoposide resistance was determined by XTT cell proliferation assay. For gene expression analysis, total RNA was extracted from each cell line and gene expression was quantified by real-time PCR following reverse transcription. Topoisomerase II activities of each cell line were compared by using in vitro topoisomerase II activity assay.

Results

Etoposide-resistant sublines MCF-7/1E and MCF-7/4E are 2.6- and 4.6-fold more resistant to etoposide compared to parental cell line MCF-7/S. TOP2A, the gene encoding the topoisomerase II alpha, is significantly downregulated in drug resistant sublines while topoisomerase II activity seemed similar among cell lines. MRP1, which encodes an etoposide efflux pump, is significantly upregulated in etoposide-resistant sublines. Two DNA damage response proteins TOPBP1 and EDD were found to be downregulated in etoposide-resistant sublines.

Conclusions

This study sheds light into the etoposide resistance in breast cancer by investigating previously proposed and novel factors that may have a role in development or progression of etoposide resistance which can be considered as diagnostic markers and therapy targets.

Introduction

Acquired or intrinsic drug resistance is the most severe problem in chemotherapy. Due to common resistance mechanisms, cancer cells can be simultaneously resistant to several structurally and functionally unrelated drugs, a concept known as multidrug resistance. Enhanced drug efflux, suppressed apoptosis, increased drug detoxification, altered expression or location of target, enhanced DNA repair is of common multidrug resistance mechanisms [1]. Multidrug resistant cells can accommodate more than one of these mechanisms, making it crucial to understand the particular causes of drug resistance.

Etoposide is a semi-synthetic anticancer agent derived from podophyllotoxin produced by Podophyllum peltatum [2]. Classified under the group of “topoisomerase II poisons”, etoposide targets topoisomerase II enzyme, which is essential for events, such as DNA replication and transcription. Etoposide exerts its toxicity by stabilizing the temporary double strand breaks created by the topoisomerase II during its catalytic cycle, thereby initiating DNA damage response and apoptosis [3]. Unrepaired strand breaks, on the other hand, initiate DNA damage response and ultimately cause apoptosis. Still, the cells can evade apoptosis and continue proliferating under etoposide-induced stress and become resistant to the drug. Previous studies reported that altered topoisomerase II expression, decreased sensitivity of topoisomerase II to etoposide [4], decreased expression of genes involved in DNA mismatch repair [5], [6], increased expression of ABC transporters, such as MDR1 and MRP1 [7] can be responsible for etoposide resistance. In addition, there are plenty of studies implicating the role of deregulated DNA damage signalling in chemoresistance. Topoisomerase IIβ-binding protein 1 (TOPBP1) is a protein functioning in DNA replication, proliferation, DNA damage signalling [8], [9]. One of its interacting partners, E3 ubiquitin-protein ligase EDD, is regarded as a tumor supressor protein. It has a role in checkpoint response, regulation of DNA damage signalling [10]. Recent reports suggested that both proteins are abnormally expressed in breast cancer cases [8], [9], [10]. Indirectly, they are also implicated in resistance to certain chemotherapy drugs [10], [11]. Here, using two etoposide-resistant breast adenocarcinoma cell line (MCF-7) as model system, we checked the association of those mechanisms with etoposide resistance.

Section snippets

Chemicals and reagents

Etoposide was obtained from Ebewe. RPMI 1640 media is purchased from HyClone, USA. Heat-inactivated fetal bovine serum, trypsin-EDTA, gentamycin, XTT cell proliferation assay were obtained from Biological Industries, Israel. Phosphate buffered saline tablets and TRI reagents were from Sigma–Aldrich, USA. Random hexamer primer, RNase-free DNase I and RevertAid reverse transcriptase, proteinase K was from Fermentas, Lithuania. FastStart Universal SYBR Master Mix was obtained from Roche

Etoposide resistance

IC50 is a parameter defined as “inhibitory concentration of a drug leading to 50% survival with respect to untreated controls”. That value informs about the relative resistance against a drug. IC50 values of parental MCF-7 cell line and two etoposide-resistant sublines indicated that MCF-7 cells have an intrinsic resistance to etoposide (IC50 = 49.2 μM). MCF-7/1E and MCF-7/4E sublines are 2.6- and 4.6-fold more resistant to etoposide relative to parental MCF-7/S cell line, respectively (Table 2).

Etoposide resistance is accompanied by decreased TOP2A expression but not topoisomerase II activity

Discussion

This study investigated the potential ways and consequences of etoposide resistance in MCF-7 breast cancer cells. Etoposide-resistant sublines of MCF-7 developed in our laboratory have been investigated earlier for expression of TOP2A encoding the topoisomerase II isoform and two mismatch repair genes, MLH1 and MSH2. Here, a more resistant subline, MCF-7/4E, was obtained and investigated further for other resistance mechanisms.

XTT cell proliferation assay suggested that MCF-7/4E and MCF-7/1E

Conclusion

Our study demonstrated the alterations and invariabilities in the mechanisms that were regarded crucial in etoposide resistance. However, in order to fully understand mechanistic details of resistance, one should elucidate the events occurring at levels of chromatin and protein–protein interactions.

Disclosure of interest

The authors declare that they have no conflicts of interest concerning this article.

Acknowledgements

This study was supported by the Research Fund of METU Grant Number BAP-01-08-2012-010.

References (23)

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    Multidrug resistance mediated byMDR-ABC transporters

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