Molecular mechanisms of drug resistance in single-step and multi-step drug-selected cancer cells

Abstract

Multidrug resistance (MDR) remains one of the key determinants in chemotherapeutic success of cancer patients. Often, acquired resistance is mediated by the overexpression of ATP-binding cassette (ABC) drug transporters. To study the mechanisms involved in the MDR phenotype, investigators have generated a variety of in vitro cell culture models using both multi-step and single-step drug selections. Sublines produced from multi-step selections have led to the discovery of several crucial drug transporters including ABCB1, ABCC1, and ABCG2. Additionally, a number of mechanisms causing gene overexpression have been elucidated. To more closely mimic in vivo conditions, investigators have also established MDR sublines with single-step drug selections. Here, we examine some of the multi-step and single-step selected cell lines generated to elucidate the mechanisms involved in the development of MDR in cancer cells.

Figure 1. Analysis of ABC transporter expression and function in the multi-step doxorubicin-selected MCF7/ADR cell line

A) The delta-delta CT method was used to determine the fold change of ABC transporter gene expression in the multi-step doxorubicin-selected cells, MCF7/ADR (17), compared to their parental line, MCF-7. The values represent the mean and standard deviation (n=2). The overexpression of ABCB1 is highlighted by the black circle. B) Using C219, the ABCB1-specific monoclonal antibody, the relative quantities of ABCB1 were determined for MCF7/ADR and MCF-7 in whole-cell lysates. Lane 1, MCF-7 control and lane 2, MCF7/ADR, (100,000 cells for all samples). C) Calcein-AM efflux assays were performed using flow cytometry. Assays compared MCF-7 and MCF7/ADR. Charcoal grey histogram, MCF7/ADR; dark grey histogram, MCF7/ADR cells in the presence of 10 μM cyclosporine A (CSA); black histogram, MCF-7, and light grey histogram, MCF-7 in the presence of cyclosporine A. The schematic on the far left side depicts the multi-step selection with doxorubicin in 0.025 μg/ml doxorubicin and increasing the selection pressure by 2-fold until the cells grew in the presence of 2 μg/ml doxorubicin.

Figure 2. Single-step doxorubicin-selected clones overexpress ABCG2

A) The schematic of the single-step selection for the MCF-7 cells with doxorubicin. B) Characterization of selected ABC transporter gene expression levels in several single-step clones. Doxorubicin-resistant MCF-7 clones were established employing a single step selection with either 14 or 21 nM treatment for 10 days followed by culturing continuously in drug-free medium. The average fold change compared to parental MCF-7 cells ± SD (n=4) was calculated using delta delta Ct method from real-time RT-PCR data. Reference gene is PMCA4 (64). The key for selected five ABC transporters is given in the figure. C) Western blotting analysis of ABCG2 protein using BXP-21 antibody following no treatment (lane 1), 50 nM negative siRNA treatment (lane 2) and 50 nM G2-2 siRNA treatment (lane 3). D) Cytotoxicity assays with mitoxantrone evaluating the effect of silencing ABCG2 in the 21 nM single-step clone. Dose response curves were derived from three independent experiments using the CCK-8 assay. White box, 21 nM cells with 12.5 nM G2-2 siRNA and black triangle, 21 nM cells with 12.5 nM negative siRNA. Error bars indicate standard deviation (n=3).