Author's personal copy World J Urol DOI 10.1007/s00345-014-1469-0
ORIGINAL ARTICLE
Differential expression of the multidrug resistance 1 (MDR1) protein in prostate cancer cells is independent from anticancer drug treatment and Y box binding protein 1 (YB-1) activity Madeleine Saupe · Lisa Rauschenberger · Melanie Preuß · Stefan Oswald · Sebastian Fussek · Uwe Zimmermann · Reinhard Walther · Cornelius Knabbe · Martin Burchardt · Matthias B. Stope
Received: 27 November 2014 / Accepted: 18 December 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Purpose The development of a drug-resistant phenotype is the major challenge during treatment of castration-resistant prostate cancer (PC). In solid cancer entities, one of the major contributors to chemoresistance is the multidrug resistance 1 (MDR1) protein. Believed to be involved in the induction of MDR1 expression is the presence of anticancer drugs as well as the Y box binding protein 1 (YB-1). Methods Basal as well as drug-induced expression of MDR1 in established PC cell lines was assessed by Western blotting and mass spectrometry. Subsequently, the influence of YB-1 on MDR1 expression was examined via transient overexpression of YB-1. Results While LNCaP and PC-3 cells showed no detectable amounts of MDR1, the resistance factor was found to be expressed in 22Rv1 cells. Despite this difference, all three cell lines demonstrated similar growth behavior in the
M. Saupe · L. Rauschenberger · M. Preuß · S. Fussek · U. Zimmermann · M. Burchardt · M. B. Stope (*) Department of Urology, University Medicine Greifswald, Ferdinand-Sauerbruch-Straße, 17475 Greifswald, Germany e-mail:
[email protected] S. Oswald Department of Pharmacology, Center of Drug Absorption and Transport (C_DAT), University Medicine Greifswald, Felix-Hausdorf-Straße 3, 17487 Greifswald, Germany R. Walther Department of Medical Biochemistry and Molecular Biology, University Medicine Greifswald, Ferdinand-Sauerbruch-Straße, 17475 Greifswald, Germany C. Knabbe Institute for Laboratory and Transfusion Medicine, Heart and Diabetes Center North Rhine-Westphalia, Ruhr University Bochum, Georgstraße 11, 32545 Bad Oeynhausen, Germany
presence of the first-line chemotherapeutic agent docetaxel. Incubation of 22Rv1 cells with docetaxel, cabazitaxel, and abiraterone did not significantly alter MDR1 expression levels. Furthermore, overexpression of the MDR1 controlling factor YB-1 showed no impact on MDR1 expression levels. Conclusions MDR1 was detectable in the PC cell line 22Rv1. However, this study suggests that MDR1 is of less importance for drug resistance in PC cells than in other types of solid cancer. Furthermore, in contrast to YB-1 properties in other malignancies, MDR1 regulation through YB-1 seems to be unlikely. Keywords Prostate cancer · Chemoresistance · Multidrug resistance protein 1 · Y box binding protein 1 Abbreviations PC Prostate cancer MDR1 Multidrug resistance 1 YB-1 Y box binding protein 1 CYP17A1 Cytochrome P450 17A1 GAPDH Glyceraldehyde 3-phosphate dehydrogenase LC–MS/MS Liquid chromatography–tandem mass spectrometry ABC ATP-binding cassette transporter MRP1 Multidrug resistance-associated protein 1
Introduction The development of a multidrug-resistant phenotype of tumor cells represents one of the major obstacles regarding the treatment of castration-resistant prostate cancer (PC). Patients with advanced PC are treated with cytotoxic agents all characterized by limited response duration. Allowing a
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median survival of 19.2 months, the first-line chemotherapy of advanced PC with docetaxel inevitably ends in relapse and tumor progression [1]. Believed to be implicated in chemoresistance is the multidrug resistance 1 (MDR1) protein. MDR1 represents an efflux pump which is capable of lowering the intracellular concentration of a vast range of anticancer compounds [2]. MDR1 gene expression is primarily controlled by the transcription and translation factor Y box binding protein 1 (YB-1), which is known to be a predictor of tumor progression and poor prognosis [3, 4]. Experimental data concerning MDR1 regulation and expression in PC cells are, however, far from conclusion. While some studies describe a higher MDR1 expression in healthy prostate tissue and benign prostate hyperplasia than in PC tissue [5], opposite findings have been reported as well [6]. Furthermore, control of MDR1 expression in distinct phases of PC progression remains unanswered. With the hope of being able to overcome tumor resistance one day, the objective of this study was to advance the knowledge of the role and regulation of MDR1 in PC. For this purpose, we primarily focused on examinations of the established PC cell lines 22Rv1, LNCaP, and PC-3. For anticancer drug incubation approaches, the approved PC therapeutics docetaxel, cabazitaxel, and abiraterone were applied. Docetaxel and cabazitaxel are both members of the taxane group; however, docetaxel has been described as a substrate of MDR1, whereas the second-generation taxane cabazitaxel demonstrated a low affinity toward the MDR1 transporter [7, 8]. Abiraterone efficacy is in contrast based on the inhibition of the steroid synthesis enzyme cytochrome P450 17A1 (CYP17A1), reflecting a completely different molecular mode of action [9]. Finally, a putative regulation of MDR1 expression by the multifunctional factor YB-1 was assessed by modulating the YB-1 expression.
for YB-1 overexpression [10] using pcDNA3.1 (Invitrogen, Karlsruhe, Germany) as empty control vector. Cell culture All cell lines were acquired from Cell Lines Service (CLS, Heidelberg, Germany) and cultured in 1640 RPMI medium supplemented with 5 % pyruvate, 10 % fetal bovine serum, and 5 % penicillin/streptomycin (all PAN Biotech, Aidenbach, Germany) at 37 °C and 5 % CO2. Western blotting Protein preparation was done using TRIzol reagent (Life Technologies, Darmstadt, Germany) according to the manufacturer’s protocol. A total amount of 200 µg of protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Whatman, Dassel, Germany). Immobilized protein was specifically detected by antibodies and visualized by LumiGLO reagent (Cell Signaling Technology) in a ChemiDoc XRS System (Bio-Rad). Protein signals were compared to GAPDH signals as internal reference. Protein quantification by liquid chromatography–tandem mass spectrometry (LC–MS/MS) Sample preparation and MDR1 quantification was done by MS-based targeted proteomics using a validated LC– MS/MS method as recently described [11]. Final protein
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Antibodies, chemicals, and plasmids Primary antibodies directed against MDR1 (Santa Cruz Biotechnology, Heidelberg, Germany), YB-1 (Abcam, Cambridge, UK), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Abfrontier, Seoul, Korea) and mouse-specific and rabbit-specific secondary antibodies (Cell Signaling Technology, Danvers, MA, USA) were used. Cabazitaxel (3.0 × 10−10 M) was kindly provided by Sanofi-Aventis (Frankfurt/M., Germany), abiraterone (3.0 × 10−5 M) was kindly provided by Janssen-Cilag (Neuss, Germany), and docetaxel (1.0 × 10−8 M) was obtained from Sigma-Aldrich (Deisenhofen, Germany). pcDNA3.1(+)-YB-1 was applied
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Fig. 1 Basal expression of MDR1 in PC cell lines 22Rv1, LNCaP, and PC-3. a Detection of MDR1 via Western blotting using an MDR1-specific antibody. GAPDH was used as loading control. b Detection of MDR1 by LC–MS/MS analysis. A cell lysate of the cell line HEK-293 was used as a positive control. The measurements in the LNCaP and PC-3 cells were found to be under the lower limit of quantification
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expression data were calculated by normalization to total protein content. Cell viability assay 1.0 × 104 cells in a 96-well plate were incubated after indicated time points with 15 µl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; SigmaAldrich) solution for 2 h at 37 °C. After removal of the supernatant, cells were lysed with 120 µl of a lysis reagent and analyzed in a microplate reader (Infinite M200 Pro, Tecan, Switzerland) at 550 nm. Transfection experiments
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Statistical analysis was performed by using GraphPad Prism 5.0 software. Data shown in the figures are either representative experiments or expressed as mean ± SD of at least three independent experiments.
Results MDR1 basal expression is detectable only in the PC cell line 22Rv1 MDR1 expression in the established PC cell lines 22Rv1, LNCaP, and PC-3 was determined via Western blotting. Detectable amounts of MDR1 could be found only in 22Rv1 cells, whereas Western blotting analysis of LNCaP and PC-3 cells failed to show MDR1 signals (Fig. 1a). These data were evaluated by MS analysis again validating
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Fig. 2 Assessment of sensitivity toward docetaxel in 22Rv1 cells, LNCaP cells, and PC-3 cells. Cells were treated with three different concentrations of docetaxel as indicated and cell viability was assessed after 24, 48, 72, and 96 h applying a MTT viability assay in a 22Rv1 cells, b LNCaP cells, and c PC-3 cells. Despite the presence of MDR1 in 22Rv1 cells, no advantage of survival could be detected
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MDR1 protein expression exclusively in 22Rv1 cells (0.230 pmol MDR1/mg total protein) and HEK-293 control cell lysate (0.268 pmol MDR1/mg total protein) with MDR1 concentrations under the lower limit of quantification in LNCaP (0.003 pmol MDR1/mg total protein) and PC-3 (0.041 pmol MDR1/mg total protein) cells (Fig. 1b).
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MDR1 expression in 22Rv1 cells confers no survival advantage in the presence of docetaxel compared to MDR1 negative LNCaP and PC-3 cells Using the MTT viability assay, cell survival of 22Rv1, LNCaP, and PC-3 cells were examined in the presence of 1.0 × 10−11, 1.0 × 10−9, and 1.0 × 10−7 M docetaxel, respectively, over a period of 96 h. Notably, cellular viability of 22Rv1 cells (Fig. 2a), LNCaP cells (Fig. 2b), and PC-3 cells (Fig. 2c) revealed a similar pattern of growth inhibition. For instance, the incubation with 1.0 × 10−7 M docetaxel for 96 h led to a reduction of living 22Rv1 cells of 12.3 ± 1.7 % compared with untreated control cells, while 26.4 ± 3.0 % LNCaP cells and 19.4 ± 2.2 % PC-3 cells survived docetaxel incubation. Despite the presence of MDR1, 22Rv1 cells showed an equal and by trend a slightly increased sensitivity toward docetaxel compared with the other PC cell lines.
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The development of an MDR1 phenotype is believed to be the consequence of tumor cell stimulation with anticancer drugs. Hence, 22Rv1 cells were incubated with docetaxel (Fig. 3a), cabazitaxel (Fig. 3b), and abiraterone (Fig. 3c) for 24 and 72 h, after which intracellular MDR1 protein was determined. The treatment of 22Rv1 cells with chemotherapeutics did not lead to a significant modulation of MDR1 protein compared to vehicle treated cells for any of the three tested compounds.
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Fig. 3 MDR1 expression modulation in the presence of drugs. Western blot analysis of MDR1 expression in 22Rv1 cells after incubation with a docetaxel, b cabazitaxel, and c abiraterone for 24 and 72 h, respectively. DMSO served used as a vehicle control and GAPDH as a loading control
The transcription and translation factor YB-1 does not control MDR1 expression in 22Rv1 cells Discussion Aside from having been reported to be up-regulated during PC progression, YB-1 is discussed as a drug-inducible putative regulator of the MDR1 gene expression. Performing drug incubation approaches as aforementioned, YB-1 protein expression appeared to not be linked to the presence of docetaxel, cabazitaxel, and abiraterone, respectively, for 24 h (Fig. 4a) and 72 h (Fig. 4b). Furthermore, overexpression of YB-1 as shown in Fig. 4c did not lead to a modulation of MDR1 after 24 and 72 h (Fig. 4d), suggesting that YB-1 is not responsible for the regulation of MDR1 expression.
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While it is widely acknowledged that MDR1 is capable of actively extruding toxic compounds out of cancer cells, little is known about the role of MDR1 in PC chemotherapy. Even though Henrique et al. [5] have reported low MDR1 expression levels in untreated PC-3 and LNCaP cells, numerous studies demonstrated a lack of basal MDR1 expression on mRNA and protein level [6, 12–15], confirming the data of our recent study. A possible explanation for the inconsistencies in demonstrating the presence of MDR1 in PC cell lines might be the fact that MDR1
Author's personal copy World J Urol Fig. 4 YB-1 expression modulation in the presence of drugs and impact of YB-1 overexpression on the expression levels of MDR1. Detection of MDR1 and YB-1 in 22Rv1 cells after incubation with docetaxel, cabazitaxel and abiraterone for a 24 h and b 72 h. DMSO was used as a vehicle control and GAPDH as a loading control. c YB-1 was transiently overexpressed in 22Rv1 cells with the empty vector pcDNA3.1(−) as control. d MDR1 expression levels in 22Rv1 cells overexpressing YB-1, compared to control transfected cells. The experiment was conducted after an incubation time of 24 and 72 h. GAPDH served as a loading control
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is a stress-responsive gene. Aside from cytostatic agents, MDR1 expression can be induced by various other stimuli such as environmental changes, infections, and ultraviolet radiation [2]. Additionally, Corcoran and co-workers have specified an exosomal MDR1 protein transfer from doxorubicin-resistant PC cells to drug-sensitive maternal cells, remarkably, pointing to MDR1 regulation accomplished independently of cellular gene expression machinery [16]. However, the PC cell line 22Rv1, which is derived from a xenograft model that relapsed during antihormonal treatment [17], weakly exhibited basal MDR1 protein expression as demonstrated for the first time by Western blotting as well as MS analysis. Notably, even though 22Rv1 cells clearly express detectable amounts of MDR1, incubation experiments with docetaxel as a model compound for chemotherapy manifested no differences in growth inhibition compared to MDR1-negative LNCaP and PC-3 cells. This is especially interesting, considering the fact that docetaxel is a well-known substrate of MDR1 [7]. On the other hand, there are no data available yet on the MDR1 gene sequence in 22Rv1 tumor cells. Consequently, we cannot exclude that a dominant negative mutation may circumvent an effective detoxification in the presence of anticancer compounds. A growing body of the literature has shown that members of the ATP-binding cassette (ABC) transporter superfamily are regulated at the expression level
and are frequently induced by anticancer drugs, and that the presence of these transporters is linked to poor prognosis and chemoresistance in a wide variety of malignancies [2]. MDR1 expression rates in drug-resistance PC cell lines appear to be regulated by promoter methylation [18]. Since former studies have reported a correlation between YB-1 and drug resistance in PC [19–21] and overexpression of YB-1 has been described to increase the efflux of the MDR1 substrate vinblastine [3], we hypothesized YB-1 to be an inductor of MDR1 expression in 22Rv1 cells. However, experiments applying the PC drugs docetaxel, cabazitaxel, and abiraterone to 22Rv1 cells demonstrated no significant alterations of MDR1 as well as YB-1 in none of the three incubation approaches. Subsequent transfection experiments with the goal of overexpressing YB-1 in 22Rv1 cells served as proof-of-principle; however, elevated levels of YB-1 did not modulate intracellular MDR1 protein concentrations. Since experimental evidence for direct binding of YB-1 to the promoter of the MDR1 gene is still missing, some authors discussed a parallel induction/activation of YB-1 and MDR1 as part of a general stress response [22, 23]. Moreover, Kaszubiak et al. [24] demonstrated that down-regulation of YB-1 in drug-resistant gastrointestinal cells did not lead to a shift of the MDR1 expression pattern. While YB-1 has been shown to play an important part in the progression from androgen-dependent PC into
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castration-resistant PC, our data suggest that this is not caused through up-regulation of MDR1. In fact, this study supports the increasing evidence that MDR1 expression is independent from YB-1. While we conclude that MDR1 regulation is one of the lesser important genetic alterations taking place during PC progression, the role of YB-1 in this type of cancer is subject of further studies. Moreover, our recent results support the hypothesis that MDR1 is unlikely to be a main actor to cause PC cells to acquire a druginduced chemoresistant phenotype and that other factors, for instance the multidrug resistance-associated protein 1 (MRP1) [14], seem more likely to be responsible for drug resistance in PC.
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13. Acknowledgments The authors thank Anne Brandenburg and Katja Wittig for excellent technical assistance. Conflict of interest By way of disclosure of conflict of interest, the compound abiraterone acetate was kindly provided by Janssen-Cilag GmbH, Neuss, Germany, and the compound cabazitaxel was kindly provided by Sanofi-Aventis, Frankfurt/M., Germany.
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Ethical standard The manuscript does not contain clinical studies or patient data. 16.
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