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. 2015;2015:673512.
doi: 10.1155/2015/673512. Epub 2015 Nov 4.

Doxorubicin Differentially Induces Apoptosis, Expression of Mitochondrial Apoptosis-Related Genes, and Mitochondrial Potential in BCR-ABL1-Expressing Cells Sensitive and Resistant to Imatinib

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Doxorubicin Differentially Induces Apoptosis, Expression of Mitochondrial Apoptosis-Related Genes, and Mitochondrial Potential in BCR-ABL1-Expressing Cells Sensitive and Resistant to Imatinib

Ewelina Synowiec et al. Biomed Res Int. .
Free PMC article

Abstract

Imatinib resistance is an emerging problem in the therapy of chronic myeloid leukemia (CML). Because imatinib induces apoptosis, which may be coupled with mitochondria and DNA damage is a prototype apoptosis-inducing factor, we hypothesized that imatinib-sensitive and -resistant CML cells might differentially express apoptosis-related mitochondrially encoded genes in response to genotoxic stress. We investigated the effect of doxorubicin (DOX), a DNA-damaging anticancer drug, on apoptosis and the expression of the mitochondrial NADH dehydrogenase 3 (MT-ND3) and cytochrome b (MT-CYB) in model CML cells showing imatinib resistance caused by Y253H mutation in the BCR-ABL1 gene (253) or culturing imatinib-sensitive (S) cells in increasing concentrations of imatinib (AR). The imatinib-resistant 253 cells displayed higher sensitivity to apoptosis induced by 1 μM DOX and this was confirmed by an increased activity of executioner caspases 3 and 7 in those cells. Native mitochondrial potential was lower in imatinib-resistant cells than in their sensitive counterparts and DOX lowered it. MT-CYB mRNA expression in 253 cells was lower than that in S cells and 0.1 μM DOX kept this relationship. In conclusion, imatinib resistance may be associated with altered mitochondrial response to genotoxic stress, which may be further exploited in CML therapy in patients with imatinib resistance.

Figures

Figure 1
Figure 1
Cell viability of mouse-derived 32D cells transfected with the BCR-ABL1 oncogene sensitive to imatinib (filled circles) or cells with primary resistance to imatinib caused by the Y253H mutation in BCR-ABL1 (filed squares) or acquired imatinib resistance (empty squares). Viability was evaluated by MTT assay. Each point is mean of six independent experiments; error bars represent SEM but in some points they were smaller than the symbol radius.
Figure 2
Figure 2
Basal mRNA expression of mitochondrial cytochrome b (MT-CYB) and NADH dehydrogenase 3 (MT-ND3) genes determined by real-time PCR in mouse-derived 32D BCR-ABL1+ cells sensitive to imatinib (S) and cells with resistance caused by the Y253H mutation in the BCR-ABL1 gene (253) or acquired imatinib resistance (AR). The expression of either gene was normalized to the mouseβ-actin gene; n = 3 for each cell line; number above bars shows significant p values for comparison between pairs connected by brackets.
Figure 3
Figure 3
Relative mRNA expression of mitochondrial cytochrome b (MT-CYB) and NADH dehydrogenase 3 (MT-ND3) genes determined by real-time PCR in mouse-derived 32D BCR-ABL1+ cells sensitive to imatinib (S) or cells with imatinib resistance caused by the Y253H mutation in the BCR-ABL1 gene (253) or acquired imatinib resistance (AR). The cells were exposed at 37°C for 24 h to doxorubicin (DOX) at 1.0 µM. Presented is a ratio of expression for DOX-exposed and DOX-nonexposed cells. The expression of either gene was normalized to the mouseβ-actin gene; n = 3 for each cell line; the numbers above bars indicate significant p values for comparison between pairs connected by brackets.
Figure 4
Figure 4
Apoptosis in mouse-derived 32D BCR-ABL1+ cells sensitive to imatinib (S) and cells with imatinib resistance caused by the Y253H mutation in the BCR-ABL1 gene (253) or acquired imatinib resistance (AR). The cells were exposed at 37°C for 24 h to doxorubicin (DOX) at 1 µM. Apoptosis was evaluated by flow cytometry with annexin V APC and SYTOX Green dyes. Apoptotic index was calculated as the percentage of apoptotic cells in 5 × 104 cells measured in each of three independent experiments. The dot plots show results of one representative experiment for each kind of cells treated with DOX. The number in the corner of Q3 quadrant presents a fraction of early and late apoptotic cells. The activity of caspases 3 and 7 was determined fluorometrically with CellEvent Caspase-3/7 Green Detection Reagent and presented in relative fluorescent units (RFU). p < 0.05 and ∗∗∗ p < 0.001 as compared with imatinib-sensitive cells.
Figure 5
Figure 5
Mitochondrial membrane potential expressed as JC-1 aggregate to monomer ratio in mouse-derived 32D BCR-ABL1+ cells sensitive to imatinib (S) and cells with imatinib resistance resulted from the Y253H mutation in the BCR-ABL1 gene (253) or acquired imatinib resistance (AR) (a). The cells were exposed at 37°C for 24 h to doxorubicin (DOX) at 1 µM (black bars) or unexposed (control, empty bars). Each experiment was repeated four times and error bars denote SD. Numbers indicate significant p values for comparison between pairs connected by brackets. Fluorescent microscopy images of control (untreated) cells and cells treated with 1 µM DOX (b). Red fluorescence of JC-1 dimers is present in the cell areas with high mitochondrial membrane potential, while green fluorescence of JC-monomers is prevalent in the cell areas with low mitochondrial membrane potential. The JC-1 stained cells were visualized under an Olympus inverted fluorescence microscope, model IX70 (Olympus, Tokyo, Japan) with 400x magnification.

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