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. 2012 Mar 13;106(6):1224-33.
doi: 10.1038/bjc.2012.40. Epub 2012 Feb 21.

Detection and characterisation of multi-drug resistance protein 1 (MRP-1) in human mitochondria

E A Roundhill  1 , S A Burchill
Affiliations

Detection and characterisation of multi-drug resistance protein 1 (MRP-1) in human mitochondria

E A Roundhill et al. Br J Cancer. .

Abstract

Background: Overexpression of plasma membrane multi-drug resistance protein 1 (MRP-1) can lead to multidrug resistance. In this study, we describe for the first time the expression of mitochondrial MRP-1 in untreated human normal and cancer cells and tissues.

Methods: MRP-1 expression and subcellular localisation in normal and cancer cells and tissues was examined by differential centrifugation and western blotting, and immunofluorescence microscopy. Viable mitochondria were isolated and MRP-1 efflux activity measured using the calcein-AM functional assay. MRP-1 expression was increased using retroviral infection and specific overexpression confirmed by RNA array. Cell viability was determined by trypan blue exclusion and annexin V-propidium iodide labelling of cells.

Results: MRP-1 was detected in the mitochondria of cancer and normal cells and tissues. The efflux activity of mitochondrial MRP-1 was more efficient (55-64%) than that of plasma membrane MRP-1 (11-22%; P<0.001). Induced MRP-1 expression resulted in a preferential increase in mitochondrial MRP-1, suggesting selective targeting to this organelle. Treatment with a non-lethal concentration of doxorubicin (0.85 nM, 8 h) increased mitochondrial and plasma membrane MRP-1, increasing resistance to MRP-1 substrates. For the first time, we have identified MRP-1 with efflux activity in human mitochondria.

Conclusion: Mitochondrial MRP-1 may be an exciting new therapeutic target where historically MRP-1 inhibitor strategies have limited clinical success.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
MRP-1 protein expression and activity in cancer and normal cell lines. Expression of MRP-1 in. (A) Total cell fraction and (B) plasma membrane fractions on western blot are shown. T98G cell extract is included on all blots as a positive control. TC-32 cell extracts were included on every original blot to allow comparisons between expression levels on different western blotting membranes. (C) The efflux of calcein-F from whole ESFT cells by plasma membrane MRP-1 following a pre-incubation with calcein-AM for 30 min. The results are expressed as percentage efflux of calcein-F compared with initial fluorescence following loading with calcein-AM. Results are shown as mean ±s.e.m. (n=9). Results of three independent experiments. Abbreviation: MW=molecular weight marker.
Figure 2
Figure 2
Mitochondrial MRP-1 expression and activity in cancer and normal cells. Expression of MRP-1 in (A) mitochondrial fractions on western blot are shown. T98G cell extract is included on all blots as a positive control. TC-32 cell extracts were included on every original blot to allow comparisons between expression levels on different western blotting membranes. (B) Expression of MRP-1 in the mitochondria isolated from ESFT cells. Equal loading was confirmed by expression of the mitochondrial marker Grp75. (C) The efflux of calcein-F from the mitochondria of ESFT cell lines following a pre-incubation with calcein-AM for 30 min. The results are expressed as percentage efflux of calcein-F compared with initial fluorescence following loading with calcein-AM. Results are shown as mean ±s.e.m. (n=9). Results of three independent experiments. (D and E) Co-localisation of MRP-1 and the mitochondrial-specific MitoTrackerCMXRos dye in cell lines. Cells have been labelled with MitoTrackerCMXRos (red; mitochondria), fixed and stained with DAPI (blue; nuclei) and MRP-1 (green). For each cell line the three different fluorescent stains and the merged image are shown; images are representative of each cell population analysed. Arrows indicate regions of co-localisation. Positive staining by multiple fluorescent compounds was compared with the staining with each individual compound alone to confirm the staining was not a result of interactions between the fluorescent compounds. (F) Z-stack images of SKES-1 cells created and rendered using the Nikon NIS-elements software. Scale bar=10 μm. Abbreviation: MW= molecular weight marker.
Figure 3
Figure 3
Co-localisation of MRP-1 expression and the mitochondrial-specific Grp75 in tissue sections. (A–D) Images of fixed tissue sections stained with DAPI (blue; nuclei), Grp75 (red; mitochondria) and for MRP-1 (green) are shown, in addition to a merged image. Images are representative of each tissue. Arrows indicate regions of co-localisation. Positive staining by multiple fluorescent compounds was compared with the staining with each individual compound alone, to confirm the staining was not a result of interactions between the fluorescent compounds. (E) Z-stack images created and rendered using the Nikon NIS-elements software. Images are representative of the tissue population analysed. Scale bar=10 μm.
Figure 4
Figure 4
Expression of MRP-1 in TC-32MRP-1.Fb-neo cells. (A) Subcellular expression of MRP-1 in TC-32.Fb-neo control and TC-32MRP-1.Fb-neo cells. α-tubulin, sodium potassium ATPase, Grp75 and TATA TBP were included as loading controls for total, plasma membrane, mitochondrial and nuclear fractions, respectively. Results are representative of three independent sets of extracts. MW=molecular weight marker. (B) Percentage of viable TC-32MRP-1.Fb-neo and TC-32.Fb-neo cells, determined by the trypan blue exclusion assay, following incubation with doxorubicin (0.85–28), etoposide (7–240 nM), vincristine (2–60 nM) and actinomycin D (0.1–7.2 nM) for 48 h. Viable cell number expressed as mean percentage of vehicle control (±s.e.m.). Results of three independent experiments. (C) ABC transporter gene expression in TC-32.Fb-neo and TC-32MRP-1.Fb-neo cells. Gene expression calculated using the comparative Ct method, normalising expression to the endogenous control gene PPIA and expressed relative to ABC transporter expression in the TC-32.Fb-neo cells.
Figure 5
Figure 5
Subcellular expression of MRP-1 in TC-32 cells after incubation with (A) doxorubicin (0.85 nM) and (B) actinomycin D (0.1 nM). α-tubulin, sodium potassium ATPase, Grp75 and TATA TBP were included as loading controls for total, plasma membrane, mitochondrial and nuclear fractions, respectively. Results are representative of two independent sets of extracts. (C) Percentage increase in resistance to doxorubicin and etoposide after doxorubicin-dependent upregulation of mitochondrial and membrane MRP-1 expression. Percentage of viable, unlabelled TC-32 cells after pre-treatment with a non-toxic concentration of doxorubicin (0.85 nM) followed by a 16 h treatment with 3.5 nM doxorubicin, 8.6 nM vincristine, 61.8 nM etoposide, 0.2 nM actinomycin D and 2.56 μM fenretinide, determined by annexin V/PI labelling of cells and flow cytometry. Results are shown as mean percentage increase in resistance±s.e.m. (n=9). Any significant differences in the percentage of unlabelled cells were determined by ANOVA and Bonferroni's post hoc test. Abbreviation: MW=molecular weight marker.
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