Cancer Chemother Pharmacol (2015) 75:281–291 DOI 10.1007/s00280-014-2643-1
ORIGINAL ARTICLE
Cellular uptake kinetics of bortezomib in relation to efficacy in myeloma cells and the influence of drug transporters Jannick Clemens · Anja Seckinger · Dirk Hose · Dirk Theile · Magdalena Longo · Walter Emil Haefeli · Jürgen Burhenne · Johanna Weiss
Received: 13 August 2014 / Accepted: 1 December 2014 / Published online: 5 December 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Purpose Despite overall successful application to multiple myeloma patients, clinical efficacy of the proteasome inhibitor bortezomib is typically challenged by primary and secondary resistance of unknown origin. So far, the potential impact of intracellular concentrations on drug efficacy of bortezomib and the influence of drug transporters are unknown. Methods We determined cellular bortezomib kinetics in nine myeloma cell lines using ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry. The potential influence of drug transporters on the uptake kinetics observed in these cell lines was investigated by testing substrate characteristics of bortezomib for several transporters in over-expressing model cells. Additionally, transporter mRNA expression was quantified in myeloma cell lines by real-time polymerase chain reaction (RT-PCR). Results All myeloma cells revealed an extensive intracellular bortezomib accumulation (47.5–183 ng/ml) exceeding extracellular concentrations (0.04–0.17 ng/ml) by more than factor 1,000. Only organic anion-transporting polypeptide 1B1 facilitated the uptake in over-expressing cells, however, to a negligible extent (factor 1.36). Bortezomib efflux via P-glycoprotein was confirmed by demonstrating
J. Clemens · D. Theile · M. Longo · W. E. Haefeli · J. Burhenne · J. Weiss (*) Department of Clinical Pharmacology and Pharmacoepidemiology, University Hospital Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany e-mail: [email protected]‑heidelberg.de A. Seckinger · D. Hose Department of Internal Medicine V, Oncology, Hematology, and Rheumatology, University Hospital Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany
reduced sensitivity (IC50 11.6 vs. 2.8 ng/ml) and intracellular concentrations (−56.1 %) in over-expressing cells compared to controls. RT-PCR revealed a varying but overall weak transporter expression in the studied myeloma cells without any correlation to intracellular concentrations. Although principally valid as demonstrated in the P-glycoprotein over-expressing cell model, there was no significant correlation between intracellular concentrations and bortezomib efficacy in myeloma cell lines. Conclusion Differences in intracellular concentrations in myeloma cell lines neither result from variable transporter expression nor represent the main factor determining bortezomib efficacy in vitro. Keywords Bortezomib · Multiple myeloma · Intracellular concentration · Enrichment · P-glycoprotein · OATP1B1
Introduction The accumulation of clonal plasma cells in the bone marrow of multiple myeloma patients causes a variety of symptoms that usually result from the displacement of normal hematopoiesis, the formation of osteolytic bone lesions, and the production of monoclonal protein [1, 2]. However, the administration of the reversible proteasome inhibitor bortezomib has significantly improved the outcome in a great number of affected myeloma patients [3, 4]. Although plasma pharmacokinetics showed a strong distribution [5–7], which suggests considerable intracellular enrichment, cellular bortezomib kinetics in targeted cells (e.g., myeloma) has not been studied so far. Moreover, it is unclear whether variability in cellular uptake or enrichment modulates bortezomib efficacy and possibly also resistance. This is particularly interesting because multiple myeloma
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still cannot be cured in the majority of patients [8]. So far, the molecular principles of both primary and secondary resistance to bortezomib are poorly understood [9, 10]. Among the different proposed resistance mechanisms such as gene mutations of proteasome subunits [11], altered proteasome turnover [12], over-expression of heat shock proteins [13], and formation of stress granules [14], multidrug resistance (MDR) mechanisms also need to be considered because of the generally high predisposition of myeloma cells to altered gene expression [15, 16]. While over-expression of drug transporters has already been observed in myeloma patients [17], the general impact of both influx and efflux transporters on intracellular bortezomib concentrations in myeloma cells has not been thoroughly investigated. In particular, comprehensive investigation of drug efflux mechanisms should clarify the controversial discussion about the contribution of MDR transporters to bortezomib resistance in leukemic and myeloma cells [11, 18–24]. In this study, we therefore determined intracellular concentration–time profiles of bortezomib in nine different myeloma cell lines and investigated whether differences in intracellular concentrations observed in these cell lines might be determined by variable drug transporter expression and to which extent the divergent intracellular concentrations are related to the efficacy of bortezomib.
Materials and methods Materials Bortezomib was purchased from Absource Diagnostics (München, Germany) while the internal standard D8-bortezomib was obtained from Toronto Research Chemicals (Toronto, Canada). Olmesartan was purchased from Sequoia Research Products (Pangbourne, UK). Dulbecco’s modified Eagle medium (DMEM), M199, RPMI-1640, glutamine, penicillin, streptomycin sulfate, trypsin, ethylenediaminetetraacetic acid (EDTA), phosphate-buffered saline (PBS), 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazoliumbromide (MTT), probenecid, aprotinin, and anti-β-actin (Clone AC-74) were purchased from SigmaAldrich (Taufkirchen, Germany). Fetal calf serum (FCS) and geneticin (G418) were supplied by PAA (Pasching, Austria). Iscove’s modified Dulbecco’s medium (IMDM) and 2-mercaptoethanol (2-ME) were purchased from Invitrogen (Karlsruhe, Germany). DMEM/Ham’s F12 was purchased from Biochrom (Berlin, Germany). Zosuquidar (LY335979) was kindly provided by Eli Lilly (Bad Homburg, Germany), while 5-(3-(2-(7-chloroquinolin-2-yl) ethenyl)phenyl)-8-dimethyl-carbamyl-4,6-dithiaoctanoic acid sodium salt hydrate (MK571) was purchased from
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Enzo Life Science (Lörrach, Germany). Crystal violet, dimethyl sulfoxide (DMSO), rifampicin, Triton® X-100, sodium dodecyl sulfate (SDS), glycerol, 2-amino-2(hydroxymethyl)-propan-1,3-diol (TRIS), dithiothreitol (DTT), and Tween®20 were purchased from AppliChem (Darmstadt, Germany). Pefabloc and Collagen R were obtained from Serva (Heidelberg, Germany), and vincristine and the antibody against human P-glycoprotein (P-gp) clone C219 from Calbiochem (Darmstadt, Germany). Bromphenol blue, leupeptin, and pepstatin were from Biomol (Hamburg, Germany). The BCA® Protein Assay Kit and the SuperSignal® West Pico Chemiluminescent Substrate Kit were purchased from Pierce (Rockford, USA), Slim-Fast® from Allpharm (Messel, Germany), and the nitrocellulose membranes (Optitran BA-S 85) from Schleicher & Schuell BioScience (Dassel, Germany). The secondary anti-mouse antibody was obtained from Amersham (Freiburg, Germany), the RNeasy Mini-Kit from Qiagen (Hilden, Germany), and the RevertAid™ H Minus First Strand cDNA Synthesis Kit from Fermentas (St. Leon-Rot, Germany). Methanol, ethanol, 2-propanol, formic acid, acetic acid, sodium hydroxide solution (NaOH), acetonitrile (ACN), and Rotiphorese® gel 30 were obtained from Carl Roth GmbH (Karlsruhe, Germany). Methyl-tert-butylether (MTBE) and hydrochloric acid (HCl) were from Merck KGaA (Darmstadt, Germany). Casy® ton, Casy® clean, and the Cytotoxicity Detection Kit (LDH) were obtained from Roche Diagnostics and Roche Applied Science (Mannheim, Germany). Primers were synthesized by Eurofins MWG Operon (Ebersberg, Germany). The Absolute QPCR SYBR Green Mix was from Abgene (Hamburg, Germany), and the 96-well plates for PCR were obtained from Biozym (Hessisch Oldendorf, Germany). Cell culturing bottles, reaction tubes, and 6-well cell culture plates were purchased from Greiner (Frickenhausen, Germany). Ninety-six-well (300 µl) microtiter plates were supplied by Nunc (Wiesbaden, Germany) and glass vials by VWR International (Darmstadt, Germany). Cell lines Myeloma cell lines Cellular uptake kinetics of bortezomib were investigated in nine myeloma cell lines. Briefly, four non-adherently (Karpas-620, KMS-12-BM, L363, OPM-2) and five partly adherently (EJM, KMM-1, LP-1, RPMI-8226, U266) growing myeloma cell lines were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) and cultured as previously described [25]. The culture medium consisted of IMDM for EJM and LP-1 and RPMI-1640 medium for all other myeloma cell lines, respectively. Each culture medium was
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supplemented with 10 % FCS, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate. HEK‑293 cells In order to investigate human organic anion-transporting polypeptides (OATPs = solute carriers of organic anions (SLCOs)), HEK-293 cells over-expressing SLCO1B1 (HEK-OATP1B1), SLCO1B3 (HEK-OATP1B3), or an empty control vector (HEK-293-VC G418) were used for bortezomib uptake studies. The cell lines were kindly provided by D. Keppler (German Cancer Research Centre, Heidelberg, Germany). Following standard cell culture conditions, cells were cultured in DMEM supplemented with 10 % FCS, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin sulfate, and 800 µg/ml G418. CHO cells Similarly, organic anion transporter 1 (OAT1) was tested for bortezomib uptake in over-expressing CHO-hOAT cells and control CHOpIRES cells, transfected only with the empty vector. Both cell lines were cultured in DMEM/ Ham’s F12 supplemented with 10 % FCS, 100 U/ml penicillin, 100 µg/ml streptomycin sulfate, and 600 µg/ml G418. The cells were kindly provided by T. Cihlar (Gilead Sciences, Foster City, USA). LLC cells Possible transport of bortezomib by P-gp was investigated in LLC-MDR1 (over-expressing P-gp (MDR1/ABCB1)) cells in comparison with the parental cell line LLC-PK1 (available at ATCC, Manassas, USA). The over-expressing cells were kindly provided by A. H. Schinkel (The Netherlands Cancer Institute, Amsterdam, the Netherlands). Both LLC cell lines were cultured in M199 medium supplemented with 10 % FCS, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate. Vincristine (0.64 µM) was only added to the culture medium of LLC-MDR1 cells to maintain P-gp over-expression. For the experiments, both cell lines were either directly seeded in (see growth inhibition assay) or fed with (see cellular uptake) vincristine-free medium 1 day prior to the respective assay. MDCKII cells Potential transport of bortezomib by other common multidrug resistance transporters [26] was investigated in the respective over-expressing MDCKII cell lines. MDCKIIMRP1, MDCKII-MRP2, MDCKII-MRP3 (over-expressing multidrug resistance-associated protein 1 (MRP1/ABCC1),
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2 (MRP2/ABCC2), and 3 (MRP3/ABCC3)), and MDCKIIBCRP (over-expressing breast cancer resistance protein (BCRP/ABCG2)) cell lines were kindly provided by P. Borst and A. H. Schinkel (The Netherlands Cancer Institute, Amsterdam, the Netherlands). Following standard cell culture conditions, all MDCKII cells were cultured in DMEM supplemented with 10 % FCS, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate. Determination of cellular uptake of bortezomib Cellular uptake of bortezomib was determined with ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry (UPLC/MS/MS) after liquid–liquid extraction according to our previously described method [27]. Because of its instability in vitro and the potential differences between the used cell lines, it is not sufficient to only determine absolute intracellular bortezomib concentrations [27]. Thus, concentrations in the extracellular compartment (medium) were also quantified, because intracellular bortezomib concentrations can only be reliably interpreted in relation to the actual extracellular exposure. However, since the process of decay did not substantially influence the interpretation of the results (correlations or significances), we limited the discussion to absolute intracellular concentrations and derived areas under the intracellular concentration–time curves (AUCs) to facilitate the comprehensibility of the study results. Both for incubation in experiments and UPLC/MS/MS calibration, we prepared a single stock solution (1.32 mM) by dissolving bortezomib in acetonitrile/H2O (1/1, v/v +0.01 % formic acid). Uptake kinetics including intracellular and extracellular concentration–time profiles were determined in nine different myeloma cell lines over 48 h of incubation with 0.2, 1, 5, and 25 nM bortezomib (0.08, 0.38, 1.92, and 9.61 ng/ml), which covers a concentration range around determined IC50 values (Table 1). To investigate whether bortezomib is a substrate of OATP1B1 or OATP1B3, intracellular concentrations of bortezomib were quantified after 10 and 60 min of incubation with 1 and 5 nM (0.38 and 1.92 ng/ml) both in parental HEK-293 cells and corresponding cells over-expressing the respective transporter. For control, experiments were performed with or without rifampicin (20 µM), a well-known OATP inhibitor [28]. Similar experiments were conducted in CHO cells over-expressing OAT1 with the use of probenecid (500 µM) or olmesartan (10 µM) as inhibitor. To verify the P-gp substrate characteristics of bortezomib, its uptake was assessed in over-expressing LLC cells and compared to the respective parental cell line after 24 h of incubation with 1 and 5 nM (0.38 and 1.92 ng/ml). For control, experiments were performed with or without the specific P-gp inhibitor LY335979 (2 µM).
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Table 1 Relationship between sensitivity (IC50) and intracellular uptake (AUC) in the tested myeloma cells after 48 h of incubation Sensitivity of myeloma cells
AUC of intracellular uptake [(ng/ml) × 48 h]
Cell line
Exposure concentration (nM)
IC50 (nM)
0.2
1
5
25
EJM LP-1 KMM-1 Karpas-620 RPMI-8226 KMS-12-BM L-363 U266
1.6 ± 0.1 5.1 ± 0.1 5.4 ± 0.4 6.0 ± 0.3 6.4 ± 0.1 6.5 ± 0.3 7.2 ± 0.6 7.3 ± 0.3
1,826 1,094 818 590 977 769 730 822
7,010 4,266 4,229 2,127 3,814 4,244 3,032 3,252
11,190 15,780 14,120 8,502 14,440 13,050 9,485 9,883
17,760 15,940 12,940 10,310 10,920 13,750 11,800 12,920
OPM-2
8.8 ± 0.5
957
3,155
10,840
13,180
Data consist of the mean ± SD of n = 32 samples for the IC50 values and the mean AUC0–48h for the intracellular uptake, respectively
Growth inhibition assays Using growth inhibition assays with crystal violet staining, we screened for possible involvement of efflux transporters (P-gp, BCRP, and MRP1-3) in the disposition of bortezomib; by lowering intracellular concentrations, active efflux would yield higher IC50 values in transporter over-expressing cell lines compared to parental cells [29]. Additionally, the specific inhibitors LY335979 (for P-gp), FTC (for BCRP), and MK571 (for MRP1-3) were used to further confirm involvement of the respective transporter in the observed efflux changes. Moreover, growth inhibition assays using the tetrazolium dye procedure were used to assess the cellular sensitivity to bortezomib in myeloma cell lines and relate it to both transporter expression and cellular uptake. Both growth inhibition assays were conducted according to previously established methods [30]. For quantification, mean absorption of cell-free wells (background, equal to 0 % proliferation) was first subtracted from each cell-containing well. The final proliferation rate was then calculated from the ratio of absorption in compound-containing wells to drug-free wells (corresponding to 100 % proliferation). Each experiment investigated an octuplet of each concentration and was performed four times. Sigmoid concentration–response curves and IC50 values were calculated by GraphPad Prism version 5.01 (GraphPad Software Inc., La Jolla, USA).
possible differences in their relative expression. Similarly, basal mRNA expression of ABCB1 was compared between LLC-MDR1 cells and the corresponding parental cell line. RNA was extracted from harvested cells using the RNeasy Mini-Kit. Before storage at −80 °C until analysis, purity and concentration of the isolated RNA were determined spectrophotometrically. According to the manufacturer’s instructions, cDNA was synthesized with the RevertAid™ H Minus First Strand cDNA Synthesis Kit. mRNA expression was quantified by RT-PCR with the LightCycler® 480 (Roche Applied Science, Mannheim, Germany). PCR amplification was carried out in 20 µl reaction volume containing 5 µl 1:10 diluted cDNA and 1 × Absolute QPCR SYBR Green Mix. The utilized primer sequences for both target and housekeeping genes were published previously [29, 31]. Since the comparison of gene expression between different cell lines can be hampered by different expression levels of housekeeping genes among the cell lines, basal mRNA expressions in the myeloma cell lines were normalized to the following three housekeeping genes which were expressed at a similar level in all investigated cell lines: glucose-6-phosphate dehydrogenase (G6PDH), β2microglobulin (β2 mg), and hypoxanthine-phosphoribosyl‑ transferase 1 (hPRT). As a calibrator, a mixture (in equal parts) of the cDNA of all tested cell lines was used. Data were evaluated by calibrator-normalized relative quantification with efficiency correction using the LightCycler® 480 software version 1.5 (Roche Applied Science, Mannheim, Germany). This software calculates the relative amount of the target gene and the housekeeping gene based on the crossing points (Cp) and the underlying calibration curve. The results were expressed as the target/reference ratio divided by the target/reference ratio of the calibrator. The results are therefore corrected for sample inhomogeneities and variance caused by detection. All samples were amplified at least in duplicate. Whenever mRNA expression was below the detection limit (Cp value >35), the respective cell line was excluded from analysis and marked (#) in the figure. Western blot analysis of P‑gp Protein expression of P-gp in LLC-MDR1 cells and the respective parental cell line were compared by SDS-polyacrylamide gel electrophoresis followed by Western blotting as published previously [29]. Blots were semiquantified by ImageJ 1.43u (NIH, USA).
Quantification of transporter mRNA expression by RT‑PCR
Statistical analysis
The mRNA expression of the transporter genes ABCB1 and SLCO1B1 was investigated in all myeloma cell lines to assess their overall expression level and to determine
Data were analyzed using GraphPad Prism version 5.01 and GraphPad InStat version 3.05 (GraphPad Software Inc., La Jolla, USA). Intracellular concentrations of
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Fig. 1 Cellular uptake of bortezomib in nine myeloma cell lines after up to 48 h of incubation. Time-dependent intracellular and extracellular concentration profiles over 48 h are exemplarily depicted for incubation with 1 nM (a). Each data point represents the mean concentration ± SD of three samples at the respective point in time.
Box-and-whisker plots depict the range of the calculated AUC0–48 h of intracellular and extracellular bortezomib concentrations after incubation with 0.2, 1, 5, and 25 nM (b). Given numbers represent the median AUC
bortezomib per ml cell volume were calculated by dividing the quantified amount in each cell pellet by the pellet’s mean volume (product of mean individual cell volume and number of cells). AUCs of the intracellular (cell samples) and extracellular (supernatant samples) concentration–time curves were calculated for the entire incubation period of 48 h (AUC0–48 h). Intracellular concentrations of myeloma, HEK-293, and LLC cells were compared and tested using ANOVA with Tukey’s post hoc test. All IC50 values were calculated from sigmoid concentration–response curves (nonlinear regression) and tested using ANOVA with Dunnett’s post hoc test. Correlation analysis of transporter expression with AUC0–48 h of the intracellular concentration–time curves was performed using the Pearson’s correlation coefficient. Spearman’s rank correlation coefficient was utilized for the correlation with cellular sensitivities because of the exceptionally lower IC50 value of EJM cells (Table 1). p ≤ 0.05 was considered significant.
incubation with 1 nM bortezomib, bortezomib accumulated 43-fold into myeloma cell lines compared to extracellular concentrations. Steadily increasing intracellular concentrations reached their maximum after 24 h in the majority of myeloma cell lines. However, this maximum concentration was between 47.5 ng/ml (Karpas-620) and 183 ng/ml (EJM) after incubation with 1 nM bortezomib and thus differed significantly (p
ORIGINAL ARTICLE
Cellular uptake kinetics of bortezomib in relation to efficacy in myeloma cells and the influence of drug transporters Jannick Clemens · Anja Seckinger · Dirk Hose · Dirk Theile · Magdalena Longo · Walter Emil Haefeli · Jürgen Burhenne · Johanna Weiss
Received: 13 August 2014 / Accepted: 1 December 2014 / Published online: 5 December 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Purpose Despite overall successful application to multiple myeloma patients, clinical efficacy of the proteasome inhibitor bortezomib is typically challenged by primary and secondary resistance of unknown origin. So far, the potential impact of intracellular concentrations on drug efficacy of bortezomib and the influence of drug transporters are unknown. Methods We determined cellular bortezomib kinetics in nine myeloma cell lines using ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry. The potential influence of drug transporters on the uptake kinetics observed in these cell lines was investigated by testing substrate characteristics of bortezomib for several transporters in over-expressing model cells. Additionally, transporter mRNA expression was quantified in myeloma cell lines by real-time polymerase chain reaction (RT-PCR). Results All myeloma cells revealed an extensive intracellular bortezomib accumulation (47.5–183 ng/ml) exceeding extracellular concentrations (0.04–0.17 ng/ml) by more than factor 1,000. Only organic anion-transporting polypeptide 1B1 facilitated the uptake in over-expressing cells, however, to a negligible extent (factor 1.36). Bortezomib efflux via P-glycoprotein was confirmed by demonstrating
J. Clemens · D. Theile · M. Longo · W. E. Haefeli · J. Burhenne · J. Weiss (*) Department of Clinical Pharmacology and Pharmacoepidemiology, University Hospital Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany e-mail: [email protected]‑heidelberg.de A. Seckinger · D. Hose Department of Internal Medicine V, Oncology, Hematology, and Rheumatology, University Hospital Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany
reduced sensitivity (IC50 11.6 vs. 2.8 ng/ml) and intracellular concentrations (−56.1 %) in over-expressing cells compared to controls. RT-PCR revealed a varying but overall weak transporter expression in the studied myeloma cells without any correlation to intracellular concentrations. Although principally valid as demonstrated in the P-glycoprotein over-expressing cell model, there was no significant correlation between intracellular concentrations and bortezomib efficacy in myeloma cell lines. Conclusion Differences in intracellular concentrations in myeloma cell lines neither result from variable transporter expression nor represent the main factor determining bortezomib efficacy in vitro. Keywords Bortezomib · Multiple myeloma · Intracellular concentration · Enrichment · P-glycoprotein · OATP1B1
Introduction The accumulation of clonal plasma cells in the bone marrow of multiple myeloma patients causes a variety of symptoms that usually result from the displacement of normal hematopoiesis, the formation of osteolytic bone lesions, and the production of monoclonal protein [1, 2]. However, the administration of the reversible proteasome inhibitor bortezomib has significantly improved the outcome in a great number of affected myeloma patients [3, 4]. Although plasma pharmacokinetics showed a strong distribution [5–7], which suggests considerable intracellular enrichment, cellular bortezomib kinetics in targeted cells (e.g., myeloma) has not been studied so far. Moreover, it is unclear whether variability in cellular uptake or enrichment modulates bortezomib efficacy and possibly also resistance. This is particularly interesting because multiple myeloma
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still cannot be cured in the majority of patients [8]. So far, the molecular principles of both primary and secondary resistance to bortezomib are poorly understood [9, 10]. Among the different proposed resistance mechanisms such as gene mutations of proteasome subunits [11], altered proteasome turnover [12], over-expression of heat shock proteins [13], and formation of stress granules [14], multidrug resistance (MDR) mechanisms also need to be considered because of the generally high predisposition of myeloma cells to altered gene expression [15, 16]. While over-expression of drug transporters has already been observed in myeloma patients [17], the general impact of both influx and efflux transporters on intracellular bortezomib concentrations in myeloma cells has not been thoroughly investigated. In particular, comprehensive investigation of drug efflux mechanisms should clarify the controversial discussion about the contribution of MDR transporters to bortezomib resistance in leukemic and myeloma cells [11, 18–24]. In this study, we therefore determined intracellular concentration–time profiles of bortezomib in nine different myeloma cell lines and investigated whether differences in intracellular concentrations observed in these cell lines might be determined by variable drug transporter expression and to which extent the divergent intracellular concentrations are related to the efficacy of bortezomib.
Materials and methods Materials Bortezomib was purchased from Absource Diagnostics (München, Germany) while the internal standard D8-bortezomib was obtained from Toronto Research Chemicals (Toronto, Canada). Olmesartan was purchased from Sequoia Research Products (Pangbourne, UK). Dulbecco’s modified Eagle medium (DMEM), M199, RPMI-1640, glutamine, penicillin, streptomycin sulfate, trypsin, ethylenediaminetetraacetic acid (EDTA), phosphate-buffered saline (PBS), 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazoliumbromide (MTT), probenecid, aprotinin, and anti-β-actin (Clone AC-74) were purchased from SigmaAldrich (Taufkirchen, Germany). Fetal calf serum (FCS) and geneticin (G418) were supplied by PAA (Pasching, Austria). Iscove’s modified Dulbecco’s medium (IMDM) and 2-mercaptoethanol (2-ME) were purchased from Invitrogen (Karlsruhe, Germany). DMEM/Ham’s F12 was purchased from Biochrom (Berlin, Germany). Zosuquidar (LY335979) was kindly provided by Eli Lilly (Bad Homburg, Germany), while 5-(3-(2-(7-chloroquinolin-2-yl) ethenyl)phenyl)-8-dimethyl-carbamyl-4,6-dithiaoctanoic acid sodium salt hydrate (MK571) was purchased from
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Enzo Life Science (Lörrach, Germany). Crystal violet, dimethyl sulfoxide (DMSO), rifampicin, Triton® X-100, sodium dodecyl sulfate (SDS), glycerol, 2-amino-2(hydroxymethyl)-propan-1,3-diol (TRIS), dithiothreitol (DTT), and Tween®20 were purchased from AppliChem (Darmstadt, Germany). Pefabloc and Collagen R were obtained from Serva (Heidelberg, Germany), and vincristine and the antibody against human P-glycoprotein (P-gp) clone C219 from Calbiochem (Darmstadt, Germany). Bromphenol blue, leupeptin, and pepstatin were from Biomol (Hamburg, Germany). The BCA® Protein Assay Kit and the SuperSignal® West Pico Chemiluminescent Substrate Kit were purchased from Pierce (Rockford, USA), Slim-Fast® from Allpharm (Messel, Germany), and the nitrocellulose membranes (Optitran BA-S 85) from Schleicher & Schuell BioScience (Dassel, Germany). The secondary anti-mouse antibody was obtained from Amersham (Freiburg, Germany), the RNeasy Mini-Kit from Qiagen (Hilden, Germany), and the RevertAid™ H Minus First Strand cDNA Synthesis Kit from Fermentas (St. Leon-Rot, Germany). Methanol, ethanol, 2-propanol, formic acid, acetic acid, sodium hydroxide solution (NaOH), acetonitrile (ACN), and Rotiphorese® gel 30 were obtained from Carl Roth GmbH (Karlsruhe, Germany). Methyl-tert-butylether (MTBE) and hydrochloric acid (HCl) were from Merck KGaA (Darmstadt, Germany). Casy® ton, Casy® clean, and the Cytotoxicity Detection Kit (LDH) were obtained from Roche Diagnostics and Roche Applied Science (Mannheim, Germany). Primers were synthesized by Eurofins MWG Operon (Ebersberg, Germany). The Absolute QPCR SYBR Green Mix was from Abgene (Hamburg, Germany), and the 96-well plates for PCR were obtained from Biozym (Hessisch Oldendorf, Germany). Cell culturing bottles, reaction tubes, and 6-well cell culture plates were purchased from Greiner (Frickenhausen, Germany). Ninety-six-well (300 µl) microtiter plates were supplied by Nunc (Wiesbaden, Germany) and glass vials by VWR International (Darmstadt, Germany). Cell lines Myeloma cell lines Cellular uptake kinetics of bortezomib were investigated in nine myeloma cell lines. Briefly, four non-adherently (Karpas-620, KMS-12-BM, L363, OPM-2) and five partly adherently (EJM, KMM-1, LP-1, RPMI-8226, U266) growing myeloma cell lines were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) and cultured as previously described [25]. The culture medium consisted of IMDM for EJM and LP-1 and RPMI-1640 medium for all other myeloma cell lines, respectively. Each culture medium was
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supplemented with 10 % FCS, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate. HEK‑293 cells In order to investigate human organic anion-transporting polypeptides (OATPs = solute carriers of organic anions (SLCOs)), HEK-293 cells over-expressing SLCO1B1 (HEK-OATP1B1), SLCO1B3 (HEK-OATP1B3), or an empty control vector (HEK-293-VC G418) were used for bortezomib uptake studies. The cell lines were kindly provided by D. Keppler (German Cancer Research Centre, Heidelberg, Germany). Following standard cell culture conditions, cells were cultured in DMEM supplemented with 10 % FCS, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin sulfate, and 800 µg/ml G418. CHO cells Similarly, organic anion transporter 1 (OAT1) was tested for bortezomib uptake in over-expressing CHO-hOAT cells and control CHOpIRES cells, transfected only with the empty vector. Both cell lines were cultured in DMEM/ Ham’s F12 supplemented with 10 % FCS, 100 U/ml penicillin, 100 µg/ml streptomycin sulfate, and 600 µg/ml G418. The cells were kindly provided by T. Cihlar (Gilead Sciences, Foster City, USA). LLC cells Possible transport of bortezomib by P-gp was investigated in LLC-MDR1 (over-expressing P-gp (MDR1/ABCB1)) cells in comparison with the parental cell line LLC-PK1 (available at ATCC, Manassas, USA). The over-expressing cells were kindly provided by A. H. Schinkel (The Netherlands Cancer Institute, Amsterdam, the Netherlands). Both LLC cell lines were cultured in M199 medium supplemented with 10 % FCS, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate. Vincristine (0.64 µM) was only added to the culture medium of LLC-MDR1 cells to maintain P-gp over-expression. For the experiments, both cell lines were either directly seeded in (see growth inhibition assay) or fed with (see cellular uptake) vincristine-free medium 1 day prior to the respective assay. MDCKII cells Potential transport of bortezomib by other common multidrug resistance transporters [26] was investigated in the respective over-expressing MDCKII cell lines. MDCKIIMRP1, MDCKII-MRP2, MDCKII-MRP3 (over-expressing multidrug resistance-associated protein 1 (MRP1/ABCC1),
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2 (MRP2/ABCC2), and 3 (MRP3/ABCC3)), and MDCKIIBCRP (over-expressing breast cancer resistance protein (BCRP/ABCG2)) cell lines were kindly provided by P. Borst and A. H. Schinkel (The Netherlands Cancer Institute, Amsterdam, the Netherlands). Following standard cell culture conditions, all MDCKII cells were cultured in DMEM supplemented with 10 % FCS, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate. Determination of cellular uptake of bortezomib Cellular uptake of bortezomib was determined with ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry (UPLC/MS/MS) after liquid–liquid extraction according to our previously described method [27]. Because of its instability in vitro and the potential differences between the used cell lines, it is not sufficient to only determine absolute intracellular bortezomib concentrations [27]. Thus, concentrations in the extracellular compartment (medium) were also quantified, because intracellular bortezomib concentrations can only be reliably interpreted in relation to the actual extracellular exposure. However, since the process of decay did not substantially influence the interpretation of the results (correlations or significances), we limited the discussion to absolute intracellular concentrations and derived areas under the intracellular concentration–time curves (AUCs) to facilitate the comprehensibility of the study results. Both for incubation in experiments and UPLC/MS/MS calibration, we prepared a single stock solution (1.32 mM) by dissolving bortezomib in acetonitrile/H2O (1/1, v/v +0.01 % formic acid). Uptake kinetics including intracellular and extracellular concentration–time profiles were determined in nine different myeloma cell lines over 48 h of incubation with 0.2, 1, 5, and 25 nM bortezomib (0.08, 0.38, 1.92, and 9.61 ng/ml), which covers a concentration range around determined IC50 values (Table 1). To investigate whether bortezomib is a substrate of OATP1B1 or OATP1B3, intracellular concentrations of bortezomib were quantified after 10 and 60 min of incubation with 1 and 5 nM (0.38 and 1.92 ng/ml) both in parental HEK-293 cells and corresponding cells over-expressing the respective transporter. For control, experiments were performed with or without rifampicin (20 µM), a well-known OATP inhibitor [28]. Similar experiments were conducted in CHO cells over-expressing OAT1 with the use of probenecid (500 µM) or olmesartan (10 µM) as inhibitor. To verify the P-gp substrate characteristics of bortezomib, its uptake was assessed in over-expressing LLC cells and compared to the respective parental cell line after 24 h of incubation with 1 and 5 nM (0.38 and 1.92 ng/ml). For control, experiments were performed with or without the specific P-gp inhibitor LY335979 (2 µM).
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Table 1 Relationship between sensitivity (IC50) and intracellular uptake (AUC) in the tested myeloma cells after 48 h of incubation Sensitivity of myeloma cells
AUC of intracellular uptake [(ng/ml) × 48 h]
Cell line
Exposure concentration (nM)
IC50 (nM)
0.2
1
5
25
EJM LP-1 KMM-1 Karpas-620 RPMI-8226 KMS-12-BM L-363 U266
1.6 ± 0.1 5.1 ± 0.1 5.4 ± 0.4 6.0 ± 0.3 6.4 ± 0.1 6.5 ± 0.3 7.2 ± 0.6 7.3 ± 0.3
1,826 1,094 818 590 977 769 730 822
7,010 4,266 4,229 2,127 3,814 4,244 3,032 3,252
11,190 15,780 14,120 8,502 14,440 13,050 9,485 9,883
17,760 15,940 12,940 10,310 10,920 13,750 11,800 12,920
OPM-2
8.8 ± 0.5
957
3,155
10,840
13,180
Data consist of the mean ± SD of n = 32 samples for the IC50 values and the mean AUC0–48h for the intracellular uptake, respectively
Growth inhibition assays Using growth inhibition assays with crystal violet staining, we screened for possible involvement of efflux transporters (P-gp, BCRP, and MRP1-3) in the disposition of bortezomib; by lowering intracellular concentrations, active efflux would yield higher IC50 values in transporter over-expressing cell lines compared to parental cells [29]. Additionally, the specific inhibitors LY335979 (for P-gp), FTC (for BCRP), and MK571 (for MRP1-3) were used to further confirm involvement of the respective transporter in the observed efflux changes. Moreover, growth inhibition assays using the tetrazolium dye procedure were used to assess the cellular sensitivity to bortezomib in myeloma cell lines and relate it to both transporter expression and cellular uptake. Both growth inhibition assays were conducted according to previously established methods [30]. For quantification, mean absorption of cell-free wells (background, equal to 0 % proliferation) was first subtracted from each cell-containing well. The final proliferation rate was then calculated from the ratio of absorption in compound-containing wells to drug-free wells (corresponding to 100 % proliferation). Each experiment investigated an octuplet of each concentration and was performed four times. Sigmoid concentration–response curves and IC50 values were calculated by GraphPad Prism version 5.01 (GraphPad Software Inc., La Jolla, USA).
possible differences in their relative expression. Similarly, basal mRNA expression of ABCB1 was compared between LLC-MDR1 cells and the corresponding parental cell line. RNA was extracted from harvested cells using the RNeasy Mini-Kit. Before storage at −80 °C until analysis, purity and concentration of the isolated RNA were determined spectrophotometrically. According to the manufacturer’s instructions, cDNA was synthesized with the RevertAid™ H Minus First Strand cDNA Synthesis Kit. mRNA expression was quantified by RT-PCR with the LightCycler® 480 (Roche Applied Science, Mannheim, Germany). PCR amplification was carried out in 20 µl reaction volume containing 5 µl 1:10 diluted cDNA and 1 × Absolute QPCR SYBR Green Mix. The utilized primer sequences for both target and housekeeping genes were published previously [29, 31]. Since the comparison of gene expression between different cell lines can be hampered by different expression levels of housekeeping genes among the cell lines, basal mRNA expressions in the myeloma cell lines were normalized to the following three housekeeping genes which were expressed at a similar level in all investigated cell lines: glucose-6-phosphate dehydrogenase (G6PDH), β2microglobulin (β2 mg), and hypoxanthine-phosphoribosyl‑ transferase 1 (hPRT). As a calibrator, a mixture (in equal parts) of the cDNA of all tested cell lines was used. Data were evaluated by calibrator-normalized relative quantification with efficiency correction using the LightCycler® 480 software version 1.5 (Roche Applied Science, Mannheim, Germany). This software calculates the relative amount of the target gene and the housekeeping gene based on the crossing points (Cp) and the underlying calibration curve. The results were expressed as the target/reference ratio divided by the target/reference ratio of the calibrator. The results are therefore corrected for sample inhomogeneities and variance caused by detection. All samples were amplified at least in duplicate. Whenever mRNA expression was below the detection limit (Cp value >35), the respective cell line was excluded from analysis and marked (#) in the figure. Western blot analysis of P‑gp Protein expression of P-gp in LLC-MDR1 cells and the respective parental cell line were compared by SDS-polyacrylamide gel electrophoresis followed by Western blotting as published previously [29]. Blots were semiquantified by ImageJ 1.43u (NIH, USA).
Quantification of transporter mRNA expression by RT‑PCR
Statistical analysis
The mRNA expression of the transporter genes ABCB1 and SLCO1B1 was investigated in all myeloma cell lines to assess their overall expression level and to determine
Data were analyzed using GraphPad Prism version 5.01 and GraphPad InStat version 3.05 (GraphPad Software Inc., La Jolla, USA). Intracellular concentrations of
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Fig. 1 Cellular uptake of bortezomib in nine myeloma cell lines after up to 48 h of incubation. Time-dependent intracellular and extracellular concentration profiles over 48 h are exemplarily depicted for incubation with 1 nM (a). Each data point represents the mean concentration ± SD of three samples at the respective point in time.
Box-and-whisker plots depict the range of the calculated AUC0–48 h of intracellular and extracellular bortezomib concentrations after incubation with 0.2, 1, 5, and 25 nM (b). Given numbers represent the median AUC
bortezomib per ml cell volume were calculated by dividing the quantified amount in each cell pellet by the pellet’s mean volume (product of mean individual cell volume and number of cells). AUCs of the intracellular (cell samples) and extracellular (supernatant samples) concentration–time curves were calculated for the entire incubation period of 48 h (AUC0–48 h). Intracellular concentrations of myeloma, HEK-293, and LLC cells were compared and tested using ANOVA with Tukey’s post hoc test. All IC50 values were calculated from sigmoid concentration–response curves (nonlinear regression) and tested using ANOVA with Dunnett’s post hoc test. Correlation analysis of transporter expression with AUC0–48 h of the intracellular concentration–time curves was performed using the Pearson’s correlation coefficient. Spearman’s rank correlation coefficient was utilized for the correlation with cellular sensitivities because of the exceptionally lower IC50 value of EJM cells (Table 1). p ≤ 0.05 was considered significant.
incubation with 1 nM bortezomib, bortezomib accumulated 43-fold into myeloma cell lines compared to extracellular concentrations. Steadily increasing intracellular concentrations reached their maximum after 24 h in the majority of myeloma cell lines. However, this maximum concentration was between 47.5 ng/ml (Karpas-620) and 183 ng/ml (EJM) after incubation with 1 nM bortezomib and thus differed significantly (p
