New uracil analogs as downregulators of ABC transporters in 5-fluorouracil-resistant human leukemia HL-60 cell line
Overexpression of ATP-binding cassette (ABC) transporters causing multidrug resistance (MDR) in cancer cells is one of the major obstacles in cancer chemotherapy. The 5-FU resistant subclone (HL-60/5FU) of the human HL-60 promyelocytic leukemia cell line was selected by the conventional method of continuous exposure of the cells to the drug up to 0.08 mmol/L concentration. HL-60/5FU cells exhibited six-fold enhanced resistance to 5-FU than HL-60 cells. RT-PCR and ELISA assay showed significant overexpression of MDR-related ABC transporters, ABCB1, ABCG2 but especially ABCC1 in the HL-60/5FU as compared with the parental cell line. Three novel synthetic 5-methylidenedihydrouracil analogs, U-236, U-332 and U-359, selected as highly cytotoxic for HL-60 cells in MTT test, showed similar cytotoxicity in the resistant cell line. When co-incubated with 5-FU, these analogs were found to down-regulate the expression of all three transporters. However, the most pronounced effect was caused by U-332 which almost completely abolished ABCC1 expression in the resistant HL-60/5FU cells. Additionally, U-332 inhibited the activity of ATPase, an enzyme which catalyzes hydrolysis of ATP, providing energy to efflux drugs from the cells through the cellular membranes. Taken together, the obtained data suggest that acquired 5-FU resistance in HL-60/5FU cells results from overexpression of ABCC1 and that targeting ABCC1 expression could be a potential approach to re-sensitize resistant leukemia cells to 5-FU. The synthetic uracil analog U-332, which can potently down-regulate ABC transporter expression and therefore disturb drug efflux, can be considered an efficient ABCC1 regulator in cancer cells.
KeywordsMDR phenotype ABCB1 ABCC1 ABCG2 ATP-ase activity
Acute myeloid leukemia (AML), characterized as a heterogeneous clonal disorder of hematopoietic progenitor cells, is known to be a frequent cause of cancer-related deaths . Therapies offered for patients with AML are not very successful and survival after relapse remains poor. The main reason of this poor therapeutic outcome is the resistance to anticancer drugs . Neoplastic cells can develop several mechanisms of multidrug resistance (MDR), such as DNA mutations, metabolic changes leading to drug degradation or drug target alteration, inhibition of cell death and, quite often, overexpression of ATP-binding cassette (ABC) transporters [3, 4, 5].
The ABC transporter family consists of transmembrane proteins that use the energy from ATP hydrolysis to efflux various potentially dangerous compounds of diverse structure across a cell membrane [3, 4, 5]. While such efflux is a normal physiological process, it is also a known mechanism of drug resistance in cancer cells. Up to now 49 ABC transporters have been identified in human cells . Among them P-glycoprotein (ABCB1), multidrug resistance-associated protein 1 (ABCC1) and breast cancer resistance protein (ABCG2) are three best known transmembrane proteins from the ABC family that in many cases reduce the cellular uptake of drugs into cancer cells, defending them from medical interventions [7, 8].
Materials and methods
Synthetic uracil analogs
Synthesis of 5-methylidenedihydrouracils U-236, U-332 and U-359 was performed using Horner–Wadsworth–Emmons methodology according to the described procedure. The purity of compounds established by NMR and analytical HPLC was over 96% .
The promyelocytic leukemia cell line (HL-60) was purchased from the European Collection of Cell Cultures (ECACC). HL-60 cells were cultured in RPMI 1640 plus GlutaMax I medium (Invitrogen, Grand Island, NY, USA), supplemented with antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) and 10% fetal bovine serum (FBS). Cells were maintained at 37 °C in a 5% CO2 atmosphere and were grown until 80% confluence. Cells not treated with the tested compounds were used as control. Both, untreated cells and cells incubated with the analogs were cultured in the same way.
Selection of 5-FU-resistant HL-60 cells (HL-60/5FU)
Selection of 5-FU-resistant cells was performed by the 3 months exposure of HL-60 cells to increasing 5-FU concentrations (0.001–0.08 mmol/L).
MTT-based toxicity assay was used to analyze the metabolically active cells. When the proliferation was similar to parental HL-60 cells, the concentration of 5-FU was increased. The procedure was repeated until the cells were able to tolerate up to 0.08 mmol/L of 5-FU. 5-FU-resistant HL-60 cells, designated HL-60/5FU, were than evaluated in other tests.
MTT-based toxicity assay
The cytotoxicity of novel analogs was determined by MTT colorimetric assay which measures the activity of cellular dehydrogenases .
Briefly, cancer cells (104/well) were seeded into 24-well plates (in 100 µL) and left for 24 h. Various concentrations of the tested compounds were added and after another 24 h the MTT solution was added to each well. The plates were incubated for 2 h at 37 °C, then the medium was removed and 100 µL of dimethyl sulfoxide (DMSO) was added to each well. The absorbance of the blue formazone product was measured at 560 nm using the iMark Bio-Rad microplate reader (Hercules, CA, USA).
Quantitative real-time PCR assay
The mRNA levels of ABC transporter genes were analyzed by quantitative RT-PCR. Briefly, the HL-60 and HL-60/5FU cells were seeded on the 6-well plates at the appropriate cell density (4.0 × 105 cells/well) and left to grow. Then, the cells were treated with the tested compounds or co-incubated with the tested compounds and 5-FU (at IC50 concentration each) for 24 h. The effects of the combination treatment were compared with those produced by the tested compounds alone.
Total RNA was extracted using the Total RNA Mini Kit (A&A Biotechnology, Gdynia, Poland) according to the manufacturer protocol. The concentration of RNA was measured using sensitive single-tube fluorimeter for fluorescence-based quantitation of nucleic acids and proteins. The concentration used for further experiments was always 150 ng/µL. cDNA was synthesized using Transcriba Kit (A&A Biotechnology, Gdynia, Poland).
Primer sequences for RT-PCR reaction
Assessment of ABCB1, ABCC1 and ABCG2 protein levels by ELISA-based method
The ABCB1, ABCC1 and ABCG2 protein levels in HL-60 and HL-60/5FU cells incubated with the tested compounds were measured by the ELISA-based method using ABCB1, ABCC1 and ABCG2 ELISA Kits. Briefly, cells were seeded on 6-well plates (4 x 105 cells/well) and incubated with the tested compounds at IC50 concentration each for 24 h. The cells were washed with phosphate-buffered saline (PBS) and collected by centrifugation (200×g, 5 min). Cellular protein extracts were prepared using Protein Extract Kit (Active Motif, Carlsbad, CA, USA). Properly diluted cellular protein extracts (25 μg) and standards were added into each well of 96-well plates pre-coated with ABCB1, ABCC1 and ABCG2 specific antibodies. ABC transporter proteins present in the tested samples specifically bound to the wells by immobilized antibodies. Addition of a secondary antibody conjugated to horseradish peroxidase (HRP) provided sensitive colorimetric readout. Finally, the stop solution was added to each well. The optical density (OD) of the yellow solution was measured spectrometrically at the wavelength of 450 nm.
The ATPase activity was measured using the ATP/ADP Luminescent Detection Assay, according to the manufacturer guidelines. Briefly, HL-60 and HL-60/5FU cells were seeded on 96-well plates at the density 1.0 × 104/mL in 100 μL of standard growth medium and incubated with the tested compounds (at IC50 concentration) for 24 h. To assess ATPase inhibition, HL-60/5FU cells were co-incubated with the tested compounds and 5-FU (at IC50 concentration each).
After incubation, 100 μL of ADP-Glo day reagent was added. Each plate was shaken for 2 min on an orbital shaker and left at room temperature for 40 min for stabilization. Then, the detection reagent was added to each well. The luminescence signal was quantified using Flexstation 3. The effects of the combination treatment were compared with those produced by the tested compounds alone.
Establishment of 5FU-resistant HL-60 cell line
HL-60 cells were selected for 5-FU-resistant phenotype by a long-time exposure (3 months) of cells to increasing 5-FU concentrations, until the cells were able to tolerate the drug up to 0.08 mmol/L and proliferate normally.
Sensitivity of HL-60/5FU cells to 5FU
Sensitivity of HL-60/5FU cells to uracil analogs
Cytotoxic activity of 1, 3-disubstituted 5-methylidenedihydrouracil analogs and 5-FU in HL-60/5FU cell line
Cancer cell line
IC50 values (µM)
7.21 ± 0.11
0.94 ± 0.01
3.81 ± 0.81
82.54 ± 0.91
10.12 ± 0.21
1.11 ± 0.01
4.31 ± 0.91
505.12 ± 9.01
Gene expression alterations in 5FU resistant cells treated with uracil analogs
The expression levels and the −∆∆Ct log-fold-change of ABC transporters were analyzed by quantitative RT-PCR.
Expression of ABC transporter genes involved in multidrug resistance in HL-60 and HL-60/5FU cells treated with uracil analogs or 5-FU
1.0 ± 0.011
1.0 ± 0.01
1.0 ± 0.01
1.2 ± 0.03
1.7 ± 0.05***
3.7 ± 0.01***
1.0 ± 0.02
1.0 ± 0.01
1.0 ± 0.01
0.7 ± 0.01**
0.2 ± 0.01
0.8 ± 0.08
1.0 ± 0.25
1.0 ± 0.33
1.0 ± 0.22
0.2 ± 0.002***
0.1 ± 0.001***
0.4 ± 0.001**
1.0 ± 0.011
1.0 ± 0.01
1.0 ± 0.01
0.2 ± 0.008*
0.6 ± 0.009**
0.3 ± 0.009**
1.0 ± 0.22
1.0 ± 0.03
1.0 ± 0.003
6.68 ± 1.04**,##
31.02 ± 4.23***,###
5.09 ± 0.01***
1.0 ± 0.001
1.0 ± 0.01
1.0 ± 0.01
0.8 ± 0.01*,+++
0.1 ± 0.02***,+++
0.8 ± 0.02*,+++
1.0 ± 0.01
1.0 ± 0.01
1.0 ± 0.01
0.2 ± 0.01**,+++
0.1 ± 0.01***,+++
0.3 ± 0.01** +++
1.0 ± 0.02
1.0 ± 0.01
1.0 ± 0.07
0.2 ± 0.003**,+++
0.6 ± 0.001***,+++
0.3 ± 0.002**,+++
HL-60/5FU (co-incubation with 5-FU)
1.0 ± 0.02
1.0 ± 0.03
1.0 ± 0.02
1.1 ± 0.01
0.7 ± 0.01
0.9 ± 0.01***
1.0 ± 0.01
1.0 ± 0.08
1.0 ± 0.23
0.4 ± 0.01**
0.1 ± 0.01***
0.6 ± 0.01**
1.0 ± 0.02
1.0 ± 0.22
1.0 ± 0.07
0.5 ± 0.03**
0.4 ± 0.01***
0.9 ± 0.01
In the sensitive and resistant cell lines uracil analogs U-236, U-332, 359 (used at IC50 concentration each) moderately and similarly (1.2- to 5-fold) down-regulated transporter gene levels. Then, resistant cells were co-incubated with 5-FU and each of the analogs. U-332 in combination with 5-FU drastically down-regulated mRNA expression of ABCC1, while the effect produced by the other two analogs was less pronounced.
Assessment of ABCB1, ABCC1 and ABCG2 protein level in HL-60 and HL-60/5FU cell lines
To measure ABCB1, ABCC1 and ABCG2 protein concentration in cancer cell lysates, human ABC ELISA kits were used. Cancer cell lysates were prepared after 24 h exposure of cells to uracil analogs alone or with 5-FU (at IC50 concentration each). The effects of the concomitant treatments were compared with those produced by 5-FU or with the analogs alone.
Modulation of ABC transporter-mediated ATP hydrolysis by uracil analogs
ABC transporters mediate the transport of substrates against a concentration gradient using energy derived from ATP hydrolysis, which is proportional to the transporter activity and could easily be detected by a luminescence method. Measuring ATPase activity allows for the assessment of ABC transporter levels [31, 32, 33].
The ATP/ADP Luminescent Detection Assay was used to determine the level of ATPase activity in HL-60 and HL-60/5FU cells incubated with uracil analogs alone or in combination with 5-FU.
Co-incubation of cells with U-236, U-332 or U-359 and 5-FU powerfully decreased the relative activity of ATPase, to 29% (for U-236 and U-359) and 24% (for U-332), compared with the effects produced by 5-FU alone.
The appearance of the MDR phenotype is a major and still unresolved problem in the therapy of leukemia. Cancer cells may either possess inherent resistance to some drugs or can become resistant after cycles of chemotherapy (acquired resistance). Numerous studies have shown that drug-resistance can be linked to enhanced efflux of various drugs from cancer cells by ABC transporters . The major anticancer drugs such as doxorubicin, mitoxantrone, etoposide, topotecan, 5-FU are all substrates for the ABC transporters, usually overexpressed in tumor cells and the delivery of these drugs to the site of action is often very limited if not impossible [35, 36, 37].
For this reason, a lot of research have been focused on identifying inhibitors/downregulators of ABC transporters [38, 39, 40]. Such compounds may affect drug transport in several ways. They can directly interact with ABC transporter proteins, decrease the level of intracellular ATP which is the source of energy for the ABC pumps, they can influence membrane phospholipids, increasing membrane permeability for ions that reduce activity of these transporters . Such inhibitors/downregulators, used in combination with known anticancer drugs can improve drugs’ efficacy and suppress resistance.
The first generation of inhibitors, also referred to as chemosensitizers, such as verapamil, calmodulin antagonists or indol alkaloids, was characterized by low activity which required the use of high doses and therefore resulted in elevated toxicity [38, 42]. Agents of the second generation, such as cyclosporin A, GF120918 (elacridar) and dexverapamil had less side effects but still low affinity for ABC transporters, as they were also substrates for cytochrome P450 and were quickly metabolized by this enzyme . Inhibitors of the third-generation were designed to specifically inhibit activity of only one transporter, in most cases ABCB1, and some of them are currently in various stages of clinical trials [44, 45]. Examples of such agents are laniquidar, anthranilamide derivative tariquidar and pipecolinate analog biricodar . Despite their diverse chemical structures they all have high potency and specificity for ABCB1 transporter. The fourth-generation inhibitors include various classes of natural compounds belonging to many chemical families, such as alkaloids, flavonoids, coumarins, terpenoids. These compounds offer a potential for semi-synthetic modifications to produce new scaffolds which may evade the toxicities shown by currently used inhibitors [46, 47]. Some of these compounds are inhibitors of ABCC1 and ABCG2 (hydrophobic flavones, acridones, chromanones) but they are still in the phase of in vitro studies .
In this report we tested three novel synthetic 5-methylidenedihydrouracil analogs which significantly inhibited proliferation in HL-60 cells, as potential expression modulators of ABCB1, ABCC1 and ABCG2 proteins, considered responsible for anticancer drug insensitivity in leukemic cells.
The 5-FU resistant subline was obtained from the HL-60 cell line by the conventional method of intermittent and continuous exposure of the cells to 5-FU. This subclone was reproducible and the cells were sixfold more resistant to 5-FU in the MTT assay compared with the parental cells.
In the resistant cells treated with 5-FU expression of the three mentioned above transporters was up-regulated but the highest increase (over 30-fold) was observed for ABCC1. We have also shown that in the HL-60/5FU cells relative ABCB1, ABCC1 and ABCG2 protein levels were twofold higher than in the sensitive HL-60 cells, indicating that 5-FU was probably a substrate for these transporters.
Three new synthetic 5-methylidenedihydrouracil analogs, U-236, U-332 and U-359, highly cytotoxic for HL-60 cells in MTT test, showed similar cytotoxicity also in the resistant cell line. These analogs were then evaluated as possible inhibitors/downregulators of ABCB1, ABCC1 and ABCG2 transporters in HL-60/5FU cell line. All three compounds, but most significantly U-332, were able to reverse resistance of HL-60/5FU cells to 5-FU treatment.
To confirm the substrate or inhibitor nature of 5-FU and the analogs, their influence on the ATPase activity was then investigated. As opposed to 5-FU which stimulated ATPase activity, being therefore a substrate for the transporters, U-332 significantly inhibited the ATP hydrolysis in both tested cell lines. Co-incubation of HL-60/5FU cells with U-332 and 5-FU powerfully down-regulated the relative activity of ATPase, confirming that this analog could decrease the activity of ABC transporters in HL-6-/5FU cells.
In conclusion, in the present study the leukemia 5-FU-selected HL-60 cell line with multidrug resistance characteristics was established. These characteristics included overexpression of ABCB1, ABCG2 and ABCC1 transporters, indicating that their high level is responsible for drug resistance. Out of these three transporters, ABCC1 was found to be the most overexpressed. The synthetic uracil analog U-332 potently decreased the level of ABCC1 protein in the resistant cells and can be considered an efficient inhibitor/downregulator of this transporter in cancer cells.
The present study was supported by grant Preludium No DEC-2017/25/N/NZ3/01039 to A.D-P from the National Science Center (NCN).
Research design and conducting experiments: AD-P. Designing, synthesis and analytical characterization of new analogs: MP and TJ. Writing the manuscript: AD-P and AJ.
Compliance with ethical standards
Conflict of interests
The author(s) declare no competing interests.
- 2.Zhang J, Gu Y, Chen B (2019) Mechanisms of drug resistance in acute myeloid leukemia. OncoTargets Ther 12:1937Google Scholar
- 3.Sonneveld P, Suciu S, Weijermans P, Beksaç M, Neuwirtova R, Solbu G, Segeren CM (2001) Cyclosporin A combined with vincristine, doxorubicin and dexamethasone (VAD) compared with VAD alone in patients with advanced refractory multiple myeloma: an EORTC–HOVON randomized phase III study (06914). Brit J Haematol 115:895–902Google Scholar
- 6.Długosz A, Janecka A (2016) ABC transporters in the development of multidrug resistance in cancer therapy. Curr Pharm Design 22:4705–4716Google Scholar
- 8.Dean M, Annilo T (2005) Evolution of the ATP-binding cassette (ABC) transporter superfamily in vertebrates. Annu Rev Genom Hum Genet 6:123–142Google Scholar
- 9.de Figueiredo-Pontes LL, Pintão MCT, Oliveira LC, Dalmazzo LF, Jácomo RH, Garcia AB, Rego EM (2008) Determination of P-glycoprotein, MDR-related protein 1, breast cancer resistance protein, and lung-resistance protein expression in leukemic stem cells of acute myeloid leukemia. Cytom B 74:163–168Google Scholar
- 10.Chen J, Tian W, Cai H, He H, Deng Y (2009) Down-regulation of microRNA-200c is associated with drug resistance in human breast cancer. Med Oncol 29:2527–2534Google Scholar
- 11.Nakanishi T, Ross DD (2012) Breast cancer resistance protein (BCRP/ABCG2): its role in multidrug resistance and regulation of its gene expression. Chin J Cancer Res 31:73–99Google Scholar
- 15.Paszel-Jaworska A, Rubiś B, Bednarczyk-Cwynar B, Zaprutko L, Rybczyńska M (2015) Proapoptotic activity and ABCC1-related multidrug resistance reduction ability of semisynthetic oleanolic acid derivatives DIOXOL and HIMOXOL in human acute promyelocytic leukemia cells. Chem Biol Interact 242:1–12PubMedGoogle Scholar
- 19.de la Cueva A, de Molina AR, Álvarez-Ayerza N, Ramos MA, Cebrián A, del Pulgar TG, Lacal JC (2013) Combined 5-FU and ChoKα inhibitors as a new alternative therapy of colorectal cancer: evidence in human tumor-derived cell lines and mouse xenografts. PLoS ONE 8:e64961–e64974PubMedPubMedCentralGoogle Scholar
- 21.Gao K, Liang Q, Zhao ZH, Li YF, Wang SF (2016) Synergistic anticancer properties of docosahexaenoic acid and 5-fluorouracil through interference with energy metabolism and cell cycle arrest in human gastric cancer cell line AGS cells. World J Gastroenterol 22:2971–2980PubMedPubMedCentralGoogle Scholar
- 23.Gomtsyan A (2012) Heterocycles in drugs and drug discovery. Chem Heterocycl Compd 48:7–10Google Scholar
- 27.Drozd E, Gruber B, Marczewska J, Drozd J, Anuszewska E (2016) Intracellular glutathione level and efflux in human melanoma and cervical cancer cells differing in doxorubicin resistance. Postepy Hig Med Dosw 70:319–328Google Scholar
- 28.Pięta M, Kędzia J, Kowalczyk D, Wojciechowski J, Wolf WM, Janecki T (2019) Enantioselective synthesis of 5-methylidenedihydrouracils as potential anticancer agents. Tetrahedron 75:2495–2505Google Scholar
- 29.Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63Google Scholar
- 30.Winer J, Jung CK, Shackel I, Williams P (1999) Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro. Anal Biochem 15:41–49Google Scholar
- 34.Lopes-Rodrigues V, Di Luca A, Mleczko J, Meleady P, Henry M, Pesi M, Falcon-Perez JM (2017) Identification of the metabolic alterations associated with the multidrug resistant phenotype in cancer and their intercellular transfer mediated by extracellular vesicles. Sci Rep-UK 7:44541–44580Google Scholar
- 40.Li L, Xiao-Dong L (2014) Alterations in function and expression of ABC transporters at blood-brain barrier under diabetes and the clinical significances. Front Pharmacol 5:273–282Google Scholar
- 47.Genovese I, Ilari A, Assaraf YG, Fazi F, Colotti G (2017) Not only P-glycoprotein: amplification of the ABCB1-containing chromosome region 7q21 confers multidrug resistance upon cancer cells by coordinated overexpression of an assortment of resistance-related proteins. Drug Resist Update 32:23–46Google Scholar
- 48.Gozzi GJ, Bouaziz Z, Winter E, Daflon-Yunes N, Honorat M, Guragossian N, Pinaud N (2015) Phenolic indeno [1, 2-b] indoles as ABCG2-selective potent and non-toxic inhibitors stimulating basal ATPase activity. Drug Des Dev Ther 9:3481–3493Google Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.