Original articleEtoposide resistance in MCF-7 breast cancer cell line is marked by multiple mechanisms
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)
Etoposide: four decades of development of a topoisomerase II inhibitor
Eur J Cancer
(1998)- et al.
Mechanism of action of eukaryotic topoisomerase II and drugs targeted to the enzyme
Biochim Biophys Acta
(1998) - et al.
Expression analysis of TOP2A, MSH2 and MLH1 genes in MCF-7 cells at different levels of etoposide resistance
Biomed Pharmacother
(2012) - et al.
Reversal of multidrug resistance by small interfering RNA (siRNA) in doxorubicin-resistant MCF-7 breast cancer cells
Biomed Pharmacother
(2011) - et al.
Analysis of relative gene expression data using real-time quantitative PCR and 2–ΔΔCt method
Methods
(2001) Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Anal Biochem
(1976)- et al.
DNA topoisomerase IIβ: a player in regulation of gene expression and cell differentiation
Int J Biochem Cell Biol
(2012) - et al.
Mechanisms of multi drug resistance in cancer
- et al.
Multidrug resistance-associated protein gene overexpression and reduced drug sensitivity of topoisomerase II in a human breast carcinoma MCF-7 cell line selected for etoposide resistance
Cancer Res
(1994) - et al.
Resistance to topoisomerase poisons due to loss of DNA mismatch repair
Int J Cancer
(2001)
Multidrug resistance mediated byMDR-ABC transporters
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