Corresponding author: Petr Mlejnek (
Academic editor: Nikolai Zhelev
A detailed understanding of the molecular cause of cancer enabled “targeted” treatment: an effective treatment with a minimal damage for healthy cells. Targeted therapy utilises rationally designed drugs that interfere with specific molecules (molecular targets) essential for proliferation and survival of malignant cells. Targeted therapy brought about a revolution in cancer treatment in the last decades (
Tyrosine kinases are especially important target because they play a crucial role in the modulation of number specific signal transduction pathways including cell proliferation and differentiation and therefore if mutated they have the potential to induce oncogenic transformation. Tyrosine kinase inhibitors (TKIs), low molecular weight inhibitors that compete with the ATP binding site of the catalytic domain of oncogenic tyrosine kinases, exhibit effective antitumor activity both
Despite the promising results of TKIs in clinical trials and clinical practise, recent
Here, we addressed the question how expression levels of ABCB1 and/or ABCG2 affect the cell resistance to DAS. We observed that the expression level of the studied ABC-transporter is an important factor that affects the cell resistance. While cells expressing high levels of drug transporters, which are often used in laboratory experiments, exhibit a high degree of resistance to DAS, cells with low expression levels of drug transporters, which can occur in clinical samples, exhibit much lower but significant degree of resistance to DAS.
In our study, human leukemia cells with high and low expression levels of ABCB1 or ABCG2 were used. Expression levels of the ABCB1 transporter in K562/Dox and K562/DoxDR1 cells are given in Fig.
The reduction of
Owing to the fact that DAS inhibits cell proliferation and induces apoptosis (
MDR to chemotherapy is a serious obstacle in the treatment of cancer patients. Cancer MDR is defined as the cross-resistance or insensitivity of cancer cells to the cytostatic or cytotoxic actions of various anticancer drugs which are structurally or functionally unrelated and have different molecular targets (
Controversy in results is a common feature of studies evaluating the expression and prognostic role of these proteins and is mostly attributed to methodical factors, largely caused by an insufficient reliability and accuracy of methods used for the assessment of the ABC transporters expression and function (
We believe that contradictory results might be due to oversimplification of evaluation of the MDR phenotype in cancer cells. The approach to this issue must be more complex. Therefore, if we want to evaluate a contribution of a particular ABC transporter to the drug resistance, a clear quantitative relationship among intracellular drug level, particular ABC transporter expression level, and cell sensitivity to this drug must be established. Importantly, the transporter expression level must be relevant to that found in clinical samples (
The later requirement is crucial as the transporter expression level significantly affects the results. The higher transporter expression level effluxes drugs with increased efficiency and thus mediates higher resistance. While some drugs are effluxed effectively even in cells with low transporter expression levels, others are effluxed only poorly or their transport is below detection limits (
Similarly here, observed resistance to DAS depends on ABCB1 and ABCG2 expression levels (Figs
Our results correspond to the finding of other authors who demonstrated that DAS is a substrate of both efflux proteins, ABCB1 and ABCG2 and that these transporters could mediate resistance to this drug (
We think that contradictory results that exist between
In this work we also address another important issue, namely the concentration of the studied drug in the experimental system used. Recent reports suggested that the interaction between TKIs and ABC transporters is more complex. Indeed, some TKIs, including DAS, may serve as substrates of ABCB1 or ABCG2 at low concentrations, and as their inhibitors at high concentrations (
In conclusion, the antiproliferative and pro-apoptotic effects of DAS might be reduced by ABCB1 or ABCG2 overexpression at clinically relevant concentration. However, the actual effect of the studied ABC transporters on DAS efficiency depends on their expression levels. The lower expression levels of ABC transporters mediate lower resistance. Considering the fact that expression levels of ABCB1 and ABCG2 transporters are hardly high in clinical samples, their contribution to the overall resistance to DAS is probably low but significant.
Dasatinib monohydrate (DAS, Spyracel, BMS-354825), IUPAC name N-(2-chloro-6-methylphenyl)-2-(6-(4-(2-hydroxyethyl)piperazin-1-yl)-2-methylpyrimidin-4-ylamino)thiazole-5-carboxamide, was obtained from Selleckchem (Munich, Germany). Zosuquidar trihydrochloride (ZSQ; LY335979), IUPAC name (αR)-4-[(1aα,6α,10bα)-1,1-difluoro-1,1a,6,10b-tetrahydrodibenzo[a,e]cyclopropa[c]cyclohepten-6-yl]-α-[(5-quinolinyloxy)methyl]-1-piperazineethanol trihydrochloride, was purchased from Selleckchem (Huston, TX, USA). Ko143 (3S,6S,12aS)-1,2,3,4,6,7,12,12a-Octahydro-9-methoxy-6-(2-methylpropyl)-1,4-dioxopyrazino-[1’,2’:1,6] pyrido [3,4-b]indole-3-propanoic acid 1,1-dimethylethyl ester were obtained from Enzo Life Sciences AG (Lausen, Switzerland).
Human chronic myelogenous leukemia K562 cells, obtained from ECACC, were cultured in the RPMI-1640 medium supplemented with a 10% calf foetal serum and antibiotics in 5% CO2 atmosphere at 37°C. K562/Dox cells, which overexpress P-gp (ABCB1, MDR1), were kindly provided by Prof J.P. Marie (University of Paris 6, France). K562/Dox cells were cultured under the same conditions. More detailed characterisation of K562/Dox cell line is given elsewhere (
K562/DoxDR1 cells with down-regulated expression of P-gp were established by stable transfection of K562/Dox cells with a plasmid vector expressing shRNA targeting the ABCB1 gene (
K562/ABCG2 cells, which overexpress wild type ABCG2 (BCRP1), were kindly provided by Prof B. Sarkadi (National Blood Center and Semmelweis University, Budapest, Hungary). K562/ABCG2 cells were cultured under the same conditions. Detailed characterisation of K562/ABCG2 cell line is given elsewhere (
Cells expressing different levels of ABCG2 were established by a single cell cloning by limiting dilution of K562/ABCG2 cells (
ABCB1 expression was studied by using UIC2 (Beckman Coulter, USA) monoclonal antibody conjugated with phycoerythrin (UIC2-PE) according to the manufacturer’s instruction. Phycoerythrin conjugated isotype IgG2a was used as a control. ABCG2 expression was studied by using mouse anti-human CD 338 (BD Biosciences, USA) monoclonal antibody conjugated with phycoerythrin (CD338-PE) according to the manufacturer’s instruction. Phycoerythrin conjugated isotype IgG2b was used as a control. The fluorescence of the cells was analysed by flow cytometry (Cytomics FC500, Beckman Coulter, USA). ABCB1 expression was determined by the ratio of the mean fluorescence intensity (MFI) shift of UIC2-PE antibody to isotype control (UIC2-PE/IgG2a-PE). Similarly, ABCG2 expression was determined by the ratio of the MFI shift of CD338-PE antibody to isotype control (CD338- PE/IgG2b-PE). For each sample 10 000 events were collected. All the experiments were performed in triplicate.
The MTT assay was used for estimation of cell viability and growth as originally described by Mosmann (
Cells were fixed and stained with Hoechst 33342 (Fluka), as described previously (
The method is based on an optimised extraction of cells with formic acid after their separation from the growth medium by centrifugation through a layer of silicone oil (
The HPLC system consisted of UltiMate 3000 RS pump, degasser, autosampler and column compartment (Dionex, Germering, Germany). Separations were performed at ambient temperature on a Polaris C18-A 150 x 2.0mm (i.d.), 5µm particle size column (Varian Inc., Lake Forest, CA, USA) connected with a guard C18 4.0 x 2.0mm (i.d.) precolumn (Phenomenex, Torrance, CA, USA). Solvents used for separation were A (95% methanol in 0.25% FA, v/v) and B (0.5% FA, v/v). The flow rate was 250 μl/min with linear gradient elution from 0 to 3 min (60 to 95% of solvent A), from 3 to 4 min (95% of solvent A), from 4 to 5 min (95 to 60% of solvent A) and from 5 to 8 min (60% of solvent A). Sample injection volume was set at 10 µl. The effluent was introduced into the API 3200 triple quadrupole mass spectrometer (MDS SCIEX, Ontario, Canada) and electrospray ionization in positive ion mode was used for detection. The mass spectrometer was operated in the multiple-reaction monitoring (MRM) mode. DAS was monitored by MRM transition 488 > 401 (dwell-time = 150 miliseconds). Ion spray probe parameters were set to the following values: needle voltage 5500 V, temperature 400ºC, curtain gas (nitrogen) 1.38bar, nebulizer gas (zero air quality) 3.45 bar, turbo V-gas (zero air quality) 3.45 bar. The nitrogen pressure in the second quadrupole was measured at 4.0 x10-8 bar. The declustering potential, the collision energy and the entrance potential were set at 61 V, 33 V and 7.5 V, respectively. The instrument was operated in unit resolution. Data were collected and processed using Analyst® software (version 1.5.1).
Data are reported as the mean ± S.D. Statistical significance of differences was determined by Student’s t-test. Only the P values less than 0.05 were considered significant.
This work was supported by grant No: IGA_LF_2016_035 (Internal grant of Palacky University).
Analysis of ABCB1 and ABCG2 expression.
Flow cytometric analysis of ABCB1 expression. Isotype control (grey histogram); K562 parental cell line (solid line); K562/Dox cells (dash-dot line); K562/DoxDR1 cells (dot line).
Quantitative analysis of ABCB1 expression. ABCB1 expression was quantified as the mean fluorescence intensity (MFI) shift (ratio of MFI of UIC2-PE antibody and isotype control). The experimental points represent mean values from three replicate experiments, with standard deviations. * denotes significant change in ABCB1 expression (P<0.05) between K562 cells and cells expressing various levels of ABCB1 (K562/Dox, K562/DoxDR1).
Flow cytometric analysis of ABCG2 expression. Isotype control (grey histogram); K562 parental cell line (solid line); K562/ABCG2CL10 cells (dash-dot line); K562/ABCGCL1 cells (dot line).
Quantitative analysis of ABCG2 expression. ABCG2 expression was quantified as the mean fluorescence intensity (MFI) shift (ratio of MFI of CD338-PE antibody and isotype control). The experimental points represent mean values from three replicate experiments, with standard deviations. * denotes significant change in ABCG2 expression (P<0.05) between K562 cells and cells expressing various levels of ABCG2 (K562/ABCGCL10, K562/ABCGCL1).
Intracellular levels of DAS in cells expressing ABCB1 or ABCG2. Parental cell line K562, which does not express any of transporter was used as a control. Cells were incubated with 300nM DAS or with 300nM DAS + appropriate inhibitor (100nM ZSQ or 300nM Ko143), as indicated. Intracellular level of DAS was determined after 3h incubation at 37°C. The experimental points represent mean values from three replicate experiments, with standard deviations.
A relative intracellular levels of DAS in cells overexpressing ABCB1 transporter. * denotes significant change in intracellular level of DAS (P<0.05) between K562 cells and cells expressing various levels of ABCB1, K562/Dox and K562/DoxDR1.
A relative intracellular levels of DAS in cells overexpressing ABCG2 transporter. * denotes significant change in intracellular level of DAS (P<0.05) between K562 cells and cells expressing various levels of ABCG2, K562/ABCG2CL10 and K562/ABCG2CL1.
Pro-apoptotic effects of DAS in cells expressing ABCB1 or ABCG2. Cells were treated with DAS or with DAS + appropriate inhibitor, as indicated. After 48h the number of apoptotic cells was determined using fluorescence microscopy. The experimental points represent mean values from three replicate experiments, with standard deviations.
Induction of apoptosis by DAS treatment in parental cell line K562, which does not express any of transporters.
Induction of apoptosis by DAS treatment in cells overexpressing ABCB1 transporter. * denotes significant change in the number of apoptotic cells (P<0.05) between resistant K562/Dox and K562/DoxDR1 cells treated with DAS and DAS+ZSQ.
Induction of apoptosis by DAS treatment in cells overexpressing ABCG2 transporter. * denotes significant change in the number of apoptotic cells (P<0.05) between resistant ABCG2CL10 and ABCG2CL1 cells treated with DAS and DAS+Ko143.
Effect of DAS on cell survival and viability in cells with high and low expression levels of ABCB1 and ABCG2. Cells were treated with DAS for 48h prior to MTT assay.
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K562 | 0,38±0,13 (nM) | 0,43±0,15 (nM) |
K562/Dox | 7,66±0,84 (nM) | 0,41±0,21 (nM) |
K562/DoxDR1 | 0,82±0,17 (nM) | 0,45±0,14 (nM) |
K562/ABCG2CL10 | 5,34±0,71 (nM) | 0,54±0,23 (nM) |
K562/ABCG2CL1 | 0,89±0,18 (nM) | 0,52±0,14 (nM) |