Flow Cytometric Analysis of Nucleoside Transporters Activity in Chemoresistant Prostate Cancer Model

Analýza aktivity nukleosidových transportérů u chemorezistentního nádoru prostaty s využitím průtokové cytometrie

Východiska:
Nukleosidové analogy představují významnou skupinu léčiv zvaných antimetabolity, využívaných pro léčbu různých typů nádorových onemocnění. Nicméně, efektivita léčby je často limitována vývojem lékové rezistence. Schopnost nádorových buněk přenášet nukleosidy a nukleosidová analoga dovnitř buňky je považována za jeden z významných faktorů ovlivňujících odpověď na léčbu antimetabolity. Vzhledem k hydrofilním vlastnostem antimetabolitů je jejich transport skrze plazmatickou membránu zprostředkováván dvěma strukturně odlišnými skupinami transmembránových proteinů hENT (SLC29) a hCNT (SLC28) zprostředkovávající rovnovážný, resp. koncentrativní nukleosidový transport. Ztráta funkčnosti nukleosidových transportérů byla asociována se snížením efektivity léčby antimetabolity a jejich deriváty u řady různých nádorových onemocnění vč. adenokarcinomu slinivky.

Materiál a metody:
Efektivita a kinetika inkorporace antimetabolitů byla analyzována na kontrolních a docetaxel-rezistentních modelech nádoru prostaty. Za tímto účelem bylo využito fluorescenčního nukleosidového analogu uridine-furanu a inhibitoru nukleosidových transportérů S-(4-nitrobenzyl) -6-thioinosinu. Pro analýzu byly využity metodické přístupy zahrnující průtokovou cytometrii, konfokální mikroskopii a kvantitativní real-time polymerázovou řetězovou reakci.

Výsledky:
V této studii jsme s využitím průtokové cytometrie a fluorescenčního nukleosidového analogu, uridine-furanu, aplikovali metodický přístup pro analýzu aktivity nukleosidových transportérů. Zaměřili jsme se na popis dlouhodobé kinetiky a inkorporace uridine-furanu do buňky u kontrolních a chemorezistentních buněčných modelů PC3. Výsledkem naší práce je průkaz asociace aktivity a mRNA exprese nukleosidových transportérů se senzitivitou daných modelů k různým nukleosidovým analogům.

Závěr:
Fluorescenční techniky mohou sloužit jako verifikovaný a efektivní nástroj k analýze aktivity nukleosidových transportérů, který má potenciál aplikace v klinické onkologii.

Klíčová slova:
proteiny přenášející nukleosidy – léková rezistence – rakovina prostaty – chemoterapie

Autoři deklarují, že v souvislosti s předmětem studie nemají žádné komerční zájmy.

Redakční rada potvrzuje, že rukopis práce splnil ICMJE kritéria pro publikace zasílané do biomedicínských časopisů.

Tato práce byla podpořena grantem MZ ČR č. 15- 33999A, všechna práva vyhrazena (K. Sou ček, K. Paruch a M. Svoboda).

Obdrženo:
10. 4. 2018

Přijato:
19. 4. 2018

Authors: Drápela S. ^1–3; R. Fedr ^1,3; P. Khirsariya ^3,4; K. Paruch ^3,4; M. Svoboda ⁵; K. Souček ^1,3
Authors place of work: Department of Cytokinetics, Institute of Biophysics, Academy of Sciences of the Czech Republic, v. v. i., Brno, Czech Republic ¹; Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic ²; International Clinical Research Center, Center for Biomolecular and Cellular Engineering, St. Anne’s University Hospital Brno, Czech Republic ³; Department of Chemistry, CZ-Openscreen, Faculty of Science, Masaryk University, Brno, Czech Republic ⁴; Department of Comprehensive Cancer Care, Masaryk Memorial Cancer Institute, Brno, Czech Republic ⁵
Published in the journal: Klin Onkol 2018; 31(Supplementum1): 140-144
Category: Článek ve sborníku

Summary

Background:
Nucleoside analogues represent a relevant class of antimetabolites used for therapy of various types of cancer. However, their effectivity is limited by drug resistance. The nucleoside transport capability of tumour cells is considered to be a determinant of the clinical outcome of treatment regimens using antimetabolites. Due to hydrophilic properties of antimetabolites, their transport across the plasma membrane is mediated by two families of transmembrane proteins, the SLC28 family of cation-linked concentrative nucleoside transporters (hCNTs) and SLC29 family of energy-independent equilibrative nucleoside transporters (hENTs). Loss of functional nucleoside transporters has been associated with reduced efficacy of antimetabolites and their derivatives and treatment failure in diverse malignancies including solid tumours, such as pancreatic adenocarcinoma.

Material and Methods:
The effectivity and kinetics of antimetabolite uptake were analysed using control and docetaxel-resistant PC3 cells. For this purpose, fluorescent nucleoside analogue probe uridine-furane and inhibitor of nucleoside transporters, S-(4-nitrobenzyl) -6-thioinosine were exploited. Combination of flow cytometry, confocal microscopy and real-time quantitative polymerase chain reaction methodology were used for the analysis.

Results:
Here we utilized flow cytometric assay for analysis of nucleoside transporters activity employing fluorescent nucleoside analogue, uridine-furane. We have determined the long-time kinetics of uridine-furane incorporation and quantified its levels in the parental prostate cancer cell line PC3 and its chemoresistant derivative. Finally, we have shown an association between the activity and mRNA expression of nucleoside transporters and sensitivity to various nucleoside analogues.

Conclusion:
Fluorescent techniques can serve as an effective tool for the detection of nucleoside transporter activity which has the potential for application in clinical oncology.

Key words:
nucleoside transporter proteins – drug resistance – prostatic neoplasm – chemotherapy

Introduction

Nucleoside analogues represent an important class of antimetabolites, used for cancer therapy either as single agents or in combination regimens [1,2]. The therapeutic effectivity of these drugs in tumour cells is severely limited by different mechanisms of resistance. Nucleoside transporters (NTs) are important determinants for salvage of preformed nucleosides and mediated uptake of antimetabolite nucleoside drugs into target cells [3].

The transport of nucleosides across the plasma membrane is mediated by two families of transmembrane proteins, the SLC28 family of cation-linked concentrative NTs (hCNTs) and SLC29 family of energy-independent equilibrative NTs (hENTs) [4]. With the exception of the nucleoside uptake, NTs play also a significant role in the effectivity of many chemotherapeutic drugs. The most prominent NT, hENT1, was shown to be crucial for the uptake of gemcitabine, standard frontline chemotherapy for pancreatic cancer [5]. Patients with a detectable expression of SLC29A1 had significantly higher overall median survival compared to those lacking SLC29A1 expression [6]. Moreover, hENT1-dependent nucleoside uptake was associated with resistance to nucleoside analogue Ara-C (Cytarabine) in (AML) patients with acute myeloid leukaemia [7,8]. More recently, down-regulation of SLC29A1 gene was associated with decreased drug sensitivity, higher proliferation rate, invasion and induction of epithelial-to-mesenchymal transition [9]. Based on these findings, the expression profile of hENT1 and other nucleoside transporters can be considered as a prognostic factor for chemosensitivity to antimetabolites. The analysis of NTs expression and localization using antibody-based approaches including flow cytometry and microscopy is an important parameter, but not relevant for the determination of its activity. Until now, there has not been identified any effective approach for the detection of NTs activity in the clinical routine. The most frequently used experimental approach is exploiting radioactively-labeled nucleoside analogues or another nucleoside transporter ligands [10]. Despite the sensitivity and specificity of radioactive assays, radioactively-labelled probes remain one of the major obstacles of the analysis. Moreover, it also does not allow single cells analysis and real-time monitoring of intracellular trafficking of nucleosides. Fluorescent nucleoside analogues or reporters are suitable for real-time measurement of NTs activity at single cell level using both fluorescence microscopy and flow cytometry [11,12].

Here we utilized the assay for the functional analysis of nucleoside transporters using flow cytometry and confocal microscopy exploiting fluorescent nucleoside analogue probe uridine-furane and inhibitor of equilibrative nucleoside transporter proteins, S-(4-nitrobenzyl) -6-thioinosine (NBTI) [12] in the experimental model of chemoresistant prostate cancer cells [13].

Material and Methods

Docetaxel-resistant PC3 prostate cancer cell line was derived as previously reported [13] and kindly provided by prof. Zoran Culig (Medical University, Innsbruck, Austria). Docetaxel resistance was maintained by a continuous supply of docetaxel (Cell Signaling Technology, Ins., 9886, USA) in the final concentration 12.5 nM. Cell lines were maintained at 37 °C (5% CO₂) in RPMI 1640 (ThermoFisher Scientific, 72400-021, USA) media supplemented with 10% fetal bovine serum and 100 U/mL penicillin/streptomycin. Uridine-furane (UF) was prepared in six steps from uridine by slightly modified published procedure [12]. The compound was purified by flash column chromatography on silica gel (eluent – dichloromethane/methanol 3: 2) followed by trituration with diethyl ether. The ¹H and ¹³C nuclear magnetic resonance (NMR) spectra of the prepared material matched those published. The kinetics and activity of uridine-furane incorporation were determined by flow cytometry analysis (BD FACSAria II SORP, Beckton Dickinson) and confocal microscopy (Leica TCS SP5 X) using UV laser. Acquired data were evaluated within FlowJo software TreeStar, USA). Dead cells were excluded from analysis based on their positivity to propidium iodide stain. Cell aggregates and debris were excluded based on a dual-parameter dot plot in which the pulse ratio (signal height/y-axis vs. signal area/x-axis) was displayed. The relative mRNA levels of nucleoside transporters were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) (LightCycler 480, Roche). Dose-response analysis of tested drugs was performed as described previously [14].

Results and discussion

Kinetics of uridine-furane incorporation

To explore the impact of nucleoside transporter activity we employed the prostate cancer cell line PC3 and its docetaxel-resistant deriative [13]. Using in vitro live cell microscopic analysis, we investigated the incorporation potential of fluorescent nucleoside analogue UF as a promising probe for the analysis of nucleoside transporter activity. Microscopic data nicely reflected the formation of UF foci in a early stage of exposure located mostly in the plasmatic membrane with the subsequent signal extension to the nucleus (Fig. 1A). Moreover, the small molecule inhibitor NBTI was fully capable to avoid the incorporation of UF in both models, confirming the role of hENT’s in the transportation of nucleoside analogues into the cells (Fig. 1B). Fluorescence increase and UF incorporation were detected continuously throughout the whole measurement. Obtained data are in the favour of previously published study introducing uridine-furane as useful fluorescent nucleoside derivative for the analysis of nucleoside analogue uptake and nucleoside transporters function [12].

Representative live cell images A) corresponding to the UV channel after the exposure to 500 μM uridine-furane at different time points. Fluorescence signal was measured in the presence and absence of 50 μM NBTI. Magnification and arrowheads represent the formation of UF foci in the membrane. As a control, PBS was used to detect autofluorescence of the cells. Bar size = 50 μm.
B) Quantification of the intracellular fluorescence after uridine-furan addition. The background subtraction was used to normalise the data. — Fig. 1. Representative live cell images A) corresponding to the UV channel after the exposure to 500 μM uridine-furane at different time points. Fluorescence signal was measured in the presence and absence of 50 μM NBTI. Magnification and arrowheads represent the formation of UF foci in the membrane. As a control, PBS was used to detect autofluorescence of the cells. Bar size = 50 μm. B) Quantification of the intracellular fluorescence after uridine-furan addition. The background subtraction was used to normalise the data.

Docetaxel-resistant model possess the lower expression of nucleoside transporters

Our next aim was to determine the expression profile of nucleoside transporter families SLC29 and SLC28. We hypothesized that docetaxel-resistant cells possess decreased gene levels of both concentrative and equlibrative nucleoside transporters. We have employed qRT-PCR and identified three nucleoside transporters, hENT1, hENT3 and hCNT2 down-regulated in the docetaxel-resistant model compared to controls (Fig. 2). These findings are consistent with other study suggesting an association between decreased levels of NTs and resistance to various chemotherapy drugs [15].

Relative expression levels of <i>SLC28</i> and <i>SLC29</i> genes. The plot represents gene expression of three selected nucleoside transporters downregulated in docetaxel-resistant cells, compared to the control derivatives. The analysis was performed in two biological and technical replicates. Acquired data were normalized to housekeeping gene <i>GAPDH</i>. — Fig. 2. Relative expression levels of *SLC28* and *SLC29* genes. The plot represents gene expression of three selected nucleoside transporters downregulated in docetaxel-resistant cells, compared to the control derivatives. The analysis was performed in two biological and technical replicates. Acquired data were normalized to housekeeping gene *GAPDH*.

Flow cytometric analysis of nucleoside transporter activity

As expression levels are not the relevant parameter for the evaluation of the nucleoside transporter function we employed flow cytometric analysis of UF incorporation via NTs. We have utilized an approach for the determination of nucleoside transporter activity exploiting NTs inhibition using NBTI, reflecting the phenotype of drug uptake suppression. Preliminary analysis of the kinetics showed more robust incorporation of UF into the control cells compared to the docetaxel-resistant derivatives, disabled by NBTI pre-treatment in both models (Fig 3A). Although endpoint data showed higher incorporation in docetaxel-resistant cells based on fluorescence intensity, normalization and the ratio between the mean of fluorescence intensity of UF alone and in combination with NBTI revealed higher NTs activity in the control cells (Fig. 3B). Finally, we also performed dose-response analysis of cytotoxicity for three clinically used nucleoside analogues – gemcitabine, cytarabine and fludarabine. Determined IC50 values were then associated it with the activity of NTs. The results showed that decreased sensitivity of docetaxel-resistant PC3 cells to all three nucleoside analogues is related to the decreased activity of NTs (Fig. 3C). This observation is in agreement with the recent study demonstrating hENT1 expression as a marker of better prognostic for a patient with pancreatic cancer treated with adjuvant gemcitabine chemotherapy [16]. Collectively, the results showed a decrease in the NTs activity in docetaxel-resistant model, predicting worse response to the treatment by various nucleoside analogues. These findings correspond with the studies considering decreased expression and function of hENT as factors causatively responsible for the acquisition of Ara-C or trifluorothymidine resistance, resp. [15,17].

Fig. 3. A) Flow cytometric analysis of fluorescence kinetics of 500 μM uridine-furane with or without 50 μM NBTI pre-treatment. B) Endpoint analysis corresponding to the 30 min, 500 μM uridine-furane exposure with or without 50 μM NBTI pre-treatment. NT activity (%) = 100–100/(MFI (UF)/MFI (NBTI). For MFI calculation, unstained controls were used. C) Association between the activity of nucleoside transporters and sensitivity to various nucleoside analogues in the model of drug sensitive and drug-resistant cells. Four-parameter dose-response model – Y = Bottom + (Top-Bottom)/(1 + 10 ^ (LogIC50–X) * HillSlope) was used to calculate IC50 as the concentration of agonist that gave a response that was halfway between Bottom and Top.

Conclusions

Our experimental results suggest that analysis of nucleoside transporters activity, particularly hENT1, might serve as a reliable parameter for the prediction of treatment response in chemoresistant prostate cancer. Fluorescent techniques are useful tools for analysis of NTs activity with the potential for utilization in clinical practice.

Acknowledgement

Authors would like to thank Iva Liskova, Martina Urbankova and Katerina Svobodova for technical assistance; prof. Zoran Culig for providing PC3 AG and PC3 DR cell lines.

The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.

The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.

Submitted: 10. 4. 2018

Accepted: 19. 4. 2018

Mgr. Karel Soucek, Ph.D.

Department of Cytokinetics Institute of Biophysics Academy of Sciences of the Czech Republic, v. v. i.

Kralovopolska 135 612 65 Brno Czech Republic

e-mail: ksoucek@ibp.cz

Zdroje

1. Neoptolemos JP, Palmer DH, Ghaneh P et al. Comparison of adjuvant gemcitabine and capecitabine with gemcitabine monotherapy in patients with resected pancreatic cancer (ESPAC-4): a multicentre, open-label, randomised, phase 3 trial. Lancet 2017; 389 (10073): 1011–1024. doi: 10.1016/S0140-6736 (16) 32409-6.

2. Navid F, Willert JR, McCarville MB et al. Combination of gemcitabine and docetaxel in the treatment of children and young adults with refractory bone sarcoma. Cancer 2008; 113 (2): 419–425. doi: 10.1002/cncr.23586.

3. Damaraju VL, Damaraju S, Young JD et al. Nucleoside anticancer drugs: the role of nucleoside transporters in resistance to cancer chemotherapy. Oncogene 2003; 22 (47): 7524–7536. doi: 10.1038/sj.onc.1206952.

4. Young JD, Yao SY, Baldwin JM et al. The human concentrative and equilibrative nucleoside transporter families, SLC28 and SLC29. Mol Aspects Med 2013; 34: 529–547. doi: 10.1016/j.mam.2012.05.007.

5. Mackey JR, Mani RS, Selner M et al. Functional nucleoside transporters are required for gemcitabine influx and manifestation of toxicity in cancer cell lines. Cancer Res 1998; 58 (19): 4349–4357.

6. Giovannetti E, Del Tacca M, Mey V et al. Transcription analysis of human equilibrative nucleoside transporter-1 predicts survival in pancreas cancer patients treated with gemcitabine. Cancer Res 2006; 66 (7): 3928–3935. doi: 10.1158/0008-5472.CAN-05-4203.

7. Zimmerman EI, Huang M, Leisewitz AV et al. Identification of a novel point mutation in ENT1 that confers resistance to Ara-C in human T cell leukemia CCRF-CEM cells. FEBS Lett 2009; 583 (2): 425–429. doi: 10.1016/j.febslet.2008.12.041.

8. Wiley JS, Jones SP, Sawyer WH et al. Cytosine arabinoside influx and nucleoside transport sites in acute leukemia. J Clin Invest 1982; 69 (2): 479–489.

9. Gao PT, Cheng JW, Gong ZJ et al. Low SLC29A1 expression is associated with poor prognosis in patients with hepatocellular carcinoma. Am J Cancer Res 2017; 7 (12): 2465–2477.

10. Santo Bd, Valdés R, Mata J et al. Differential expression and regulation of nucleoside transport systems in rat liver parenchymal and hepatoma cells. Hepatology 1998; 28 (6): 1504–1511. doi: 10.1002/hep.510280609.

11. Johnson DE, Young JD, Campbell REet al. Real-time measurement of transport activity of the human concentrative nucleoside transporter, hCNT3, using a fluorescent reporter. FASEB J 2009; 23 (Suppl 1): 722–796. 722.

12. Claudio-Montero A, Pinilla-Macua I, Fernandez-Calotti P et al. Fluorescent nucleoside derivatives as a tool for the detection of concentrative nucleoside transporter activity using confocal microscopy and flow cytometry. Mol Pharm 2015; 12 (6): 2158–2166. doi: 10.1021/acs.molpharmaceut.5b00142.

13. Puhr M, Hoefer J, Schafer G et al. Epithelial-to-mesenchymal transition leads to docetaxel resistance in prostate cancer and is mediated by reduced expression of miR-200c and miR-205. Am J Pathol 2012; 181 (6): 2188–2201. doi: 10.1016/j.ajpath.2012.08.011.

14. Samadder P, Suchankova T, Hylse O et al. Synthesis and profiling of a novel potent Selective inhibitor of CHK1 kinase possessing unusual N-trifluoromethylpyrazole pharmacophore resistant to metabolic N-dealkylation. Mol Cancer Ther 2017; 16 (9): 1831–1842. doi: 10.1158/1535-7163.MCT-17-0018.

15. Temmink OH, Bijnsdorp IV, Prins HJ et al. Trifluorothymidine resistance is associated with decreased thymidine kinase and equilibrative nucleoside transporter expression or increased secretory phospholipase A2. Mol Cancer Ther 2010; 9 (4): 1047–1057. doi: 10.1158/1535-7163.MCT-09-0932.