Nongenotoxic ABCB1 activator tetraphenylphosphonium can contribute to doxorubicin resistance in MX-1 breast cancer cell line

Abstract

Hyperactivation of ABC transporter ABCB1 and induction of epithelial-mesenchymal transition (EMT) are the most common mechanism of acquired cancer chemoresistance. This study describes possible mechanisms, that might contribute to upregulation of ABCB1 and synergistically boost the acquisition of doxorubicin (DOX) resistance in breast cancer MX-1 cell line. DOX resistance in MX-1 cell line was induced by a stepwise increase of drug concentration or by pretreatment of cells with an ABCB1 transporter activator tetraphenylphosphonium (TPP⁺) followed by DOX exposure. Transcriptome analysis of derived cells was performed by human gene expression microarrays and by quantitative PCR. Genetic and epigenetic mechanisms of ABCB1 regulation were evaluated by pyrosequencing and gene copy number variation analysis. Gradual activation of canonical EMT transcription factors with later activation of ABCB1 at the transcript level was observed in DOX-only treated cells, while TPP⁺ exposure induced considerable activation of ABCB1 at both, mRNA and protein level. The changes in ABCB1 mRNA and protein level were related to the promoter DNA hypomethylation and the increase in gene copy number. ABCB1-active cells were highly resistant to DOX and showed morphological and molecular features of EMT. The study suggests that nongenotoxic ABCB1 inducer can possibly accelerate development of DOX resistance.

Figure 1

Characterization of chemoresistant MX-1 cell sublines. (A) Representative phase-contrast microscopic images demonstrating morphology of MX-1 cells and morphological changes in MX-1/D and MX-1/T cells cultured in media with doxorubicin and TPP⁺ respectively. Magnification—× 200. (B) Cytotoxicity of doxorubicin measurements with the MTT assay in the parental MX-1 cells and chemoresistant MX-1/D (cultured with 80 nM of doxorubicin), MX-1/T (cultured with ABCB1 transporter activator TPP⁺ (128 nM)) and MX-1/TD (MX-1/T cells cultured in media supplemented with up to 1280 nM of doxorubicin for a week). The indicated P-value was calculated at the end point of the experiment, compared the viability of each chemoresistant cell sublines with wild-type cells. Resistance index was calculated IC50 values of chemoresistant cells dividing by IC50 of parental MX-1 cells. (C) Heatmap showing the gene expression profile of the parental MX-1 and chemoresistant MX-1/D, MX-1/T and MX-1/TD cells. The results were obtained by Human Gene Expression (v2) 8 × 60 K microarrays (design ID 039494). (D) Venn diagram, showing the numbers of overlapping deregulated genes among MX-1/D, MX-1/T and MX-1/TD sublines compared to untreated MX-1 cells. Listed genes are common in the all investigated cells.

Figure 2

Cellular responses/pathways induced in chemoresistant MX-1 cell sublines. Heatmaps of differentially expressed genes (adjusted p < 0.05) from gene expression microarrays of chemoresistant MX-1 cell sublines: (A) epithelial–mesenchymal transition-related, (B) immune response-related, (C) cell adhesion and motility-related, (D) stem cells-related, (E) chemoresistance-related, (F) ion channels and (G) epigenetic regulators. The scale-bars indicate the normalized mRNA expression level, where the zero represents the mean expression level of the particular gene set. (H) The two hypothetical models of chemoresistance development were made according the human gene expression microarrays (Additional file 1, Table S1) and quantitative PCR results. The different coloring and directions of the arrows reflect the possibly different sequence of the activation of particular response pathway. In DOX-only exposed cells (red arrows) the deregulated expression of canonical EMT related genes and CSC markers was observed, what possibly caused cell migration and chemoresistance with an upregulated expression of drug metabolism-related enzymes (e.g. DOX metabolism-related AKR1B10) and later activation of ABCB1 mRNA expression. In TPP⁺-exposed cells (green arrows) marked activation of ABCB1 and immune response related genes were observed what supposedly caused cell migration, chemoresistance and possibly later activation of non-canonical EMT and CSC-related genes. The blue arrows indicate the pathways activated in both DOX-only and TPP⁺ treated cells. This figure was created using images from Servier Medical Art Commons Attribution 3.0 Unported License (http://smart.servier.com). Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License. EMT epithelial–mesenchymal transition, CSC cancer stem cell, PCDHs protocadherins, MMPs matrix metalloproteinases.

Figure 3

Differential gene expressions of the chemoresistant MX-1 cell sublines. (A–D) Results from quantitative PCR (qPCR) analysis showing the comparative mRNA expression of Epithelial–mesenchymal transition related genes in the parental MX-1 and chemoresistant cells, cultured with increasing concentrations (10 nM, 20 nM, 40 nM and 80 nM) of doxorubicin (MX-1/D), ABCB1 transporter activator TPP⁺ (MX-1/T) or both (MX-1/TD). (E) Western blot analysis of the protein expression of CDH1 and CDH2 in MX-1 and MX-1/D80 cells. (F–H) Relative expression level of cancer stem cells related genes. The graphs show the mean (± SEM) ratio of the indicated gene expression compared to endogenous control HPRT1 expression. Indicated P values were calculated based on paired t-tests.

Figure 4

Upregulation of ABCB1 transporter in chemoresistant MX-1 cells. (A) mRNA levels of ABCB1 in the MX-1 and chemoresistant cells, cultured with increasing concentrations (10 nM, 20 nM, 40 nM and 80 nM) of doxorubicin (MX-1/D10-80), ABCB1 transporter activator TPP⁺ (MX-1/T) or both (MX-1/TD) obtained by qPCR. The graph shows the mean (± SEM) ratio of the ABCB1 expression compared to endogenous control HPRT1 expression. (B) P-glycoprotein expression level in the parental MX-1 and chemoresistant MX-1/D80, MX-1/T and MX-1/TD cells obtained by Western blot analysis and the representative images of the experiment. The graphs show the mean (± SEM) ratio of the protein expression compared to actin β expression. P values were calculated based on paired t-tests. (C) Schematic figure detailing the CpG dinucleotides analysed by pyrosequencing in the downstream promoter of ABCB1 gene and the methylation level of the selected CpGs in MX-1 and chemoresistant MX-1/D80, MX-1/T and MX-1/TD cells, depicted in the colours and expressed by percentage. (D) The copy number of ABCB1 in non-treated MX-1 and chemoresistant MX-1/D80, MX-1/T and MX-1/TD cells evaluated by qPCR. The number of copies was calculated with Copy Caller Software v2.0 using RNaseP as an internal control.