The reagents and chemicals were purchased as follows: MRS 1220 (Tocris Bioscience); 0.9% w/v sodium chloride intravenous infusion (B. Braun Melsungen AG); GlutaMAX™, TrypLE™ Express Enzyme (Thermo Fisher Scientific, Inc.), Geneticin, Zeocin Selection Reagent (Gibco; Thermo Fisher Scientific, Inc.), Pierce™ BCA Protein Assay kit and Ham's F-12K (Kaighn's) medium (Thermo Fisher Scientific, Inc.); Iscove's modified Dulbecco's medium (IMDM), Eagle's minimum essential medium (EMEM) and RPMI-1640, McCoy's 5A modified medium (Lonza Group Ltd.); Ham's F12 and DMEM/F-12 (HyClone; Cytiva); ethanol (VWR International, LLC); Cell wash reagent, Permeabilizing solution 2 and Fc receptor saturation reagent (BD Biosciences); FBS (Capricorn Scientific GmbH); Coelenterazine h (Biotium, Inc.); CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (Promega Corporation); paraformaldehyde (Merck KGaA); glycine (AMSBIO LLC); Amersham™ ECL™ Prime Western blotting detection reagent and Amersham nitrocellulose membrane 0.2 μM (Cytiva); Britelite plus Luminescence Reporter Gene Assay System (PerkinElmer, Inc.); fluorouracil (5-FU) and gemcitabine (Accord Healthcare Ltd.); carboplatin (Fresenius Kabi Asia-Pacific, Ltd.); doxorubicin (Pharmagen GmbH); 5′-N-ethylcarboxamidoadenosine (NECA), 2-Cl-IB-MECA, dimethyl sulfoxide (DMSO), BSA, EDTA, PI, 5-Bromo-2′-deoxyuridine (BrdU), 5-Bromouridine (BrU), sodium tetraborate decahydrate, nonyl phenoxypolyethoxylethanol (NP-40), RNase A, Fraction V, daunorubicin, puromycin and all other chemicals were purchased from Sigma-Aldrich (Merck KGaA). All compounds used for treatments were of ≥98% purity. For all experiments, DMSO was used as vehicle for MRS 1220, NECA and 2-Cl-IB-MECA, while 0.9% w/v sodium chloride intravenous infusion was used as a vehicle for 5-FU, gemcitabine, carboplatin and doxorubicin.

The following primary antibodies were used: A₃AR (cat. no. ab203298; 1:500), phosphorylated pAKT Ser⁴⁷³ (cat. no. ab192623; 1:500), pAKT Thr³⁰⁸ (cat. no. ab38449; 1:500), NF-κB (cat. no. ab16502; 1:1,000), β-catenin (cat. no. ab32572; 1:2,500), Gli1 (cat. no. ab134906; 1:500), Ptch1 (cat. no. ab53715; 1:1,000), c-Myc (cat. no. ab32072; 1:1,000) all from Abcam; ERK1/2 (cat. no. 9102; 1:1,000), pERK1/2 Thr²⁰²/Tyr²⁰⁴ (cat. no. 20G11; 1:1,000), AKT (cat. no. 9272S; 1:1,000), pNF-κB Ser⁵³⁶ (cat. no. 3033; 1:1,000), GSK-3b (cat. no. 27C10; 1:1,000), pGSK-3b Ser⁹ (cat. no. 9336; 1:1,000), cyclin D1 (cat. no. 2978; 1:500), pc-Myc Ser⁶² (cat. no. 13748S; 1:1,000), pc-Myc Thr⁵⁸ (cat. no. 46650S; 1:1,000) from Cell Signaling Technology, Inc.; MRP1 (cat. no. 801-012-C250; 1:250) from Enzo Life Sciences, Inc.; p-histone H3 Ser¹⁰ (cat. no. 06-570; 1:500) from Merck KGaA; β-actin (cat. no. A2228; 1:3,000), IgG1 isotype control unconjugated (cat. no. M5284; 1:140), P-gp (cat. no. P7965; 1:250) from Sigma-Aldrich (Merck KGaA); and anti-BrdU (cat. no. 11-286-C025; 1:250) from EXBIO Praha, a.s. Secondary antibodies conjugated with HRP anti-rabbit IgG (cat. no. A-0545; 1:10,000) and anti-mouse IgG (cat. no. A-2304; 1:10,000), and anti-mouse-IgG-FITC (cat. no. F2883; 1:250) were purchased from Sigma-Aldrich (Merck KGaA). Anti-rabbit-IgG-Alexa Fluor 488 (cat. no. A-11070; 1:500) was purchased from Thermo Fisher Scientific, Inc.

The JoPaca-1 pancreatic carcinoma and Hep-3B HCC cell lines were purchased from German Collection of Microorganisms and Cell Cultures. The A549 lung carcinoma, BJ non-tumor fibroblast, BT-549 and MDA-MB-231 breast cancer, CCRF-CEM leukemia, non-tumor MRC-5 fibroblast, U2OS osteosarcoma, HCT116 colon carcinoma and HT-29 colorectal cancer cell lines were purchased from the American Type Culture Collection. The human ES-012-A A₃AR aequorin cell line was purchased from Perkin Elmer, Inc., while the parental K562 leukemia and the HCT116 p53^−/− cell lines were purchased from Horizon Discovery Ltd. The CEM-DNR (resistant to daunorubicin) and K562-TAX (resistant to paclitaxel) cell lines were established in our laboratory (45).

The β-catenin reporter cell lines were prepared as follows: The U2OS, HCT 116 and HT-29 cell lines were stably transduced at multiplicity of infection, 5, at 37°C for 16 h with commercially available lentiviral particles [Cignal Lenti T cell factor/lymphoid enhancer factor (TCF/LEF) Reporter Assays; cat. no. CLS-018L; Qiagen GmbH] carrying the TCF/LEF responsive element coupled to firefly luciferase reporter gene. The following three models of the Wnt/β-catenin pathway were established using antibiotic selection in the presence of 1 μg/ml puromycin: Wild-type (derived from the U2OS cell line; β-catenin^WT); constitutively active with deletion of Ser⁴⁵ in β-catenin, where this single nucleotide variant prevents β-catenin proteasomal degradation (derived from the HCT116 cell line; β-catenin^mut); and constitutively active with a loss-of-function mutation in the APC gene (derived from the HT-29 cell line; APC^mut). Monoclonal cell lines were generated from polyclonal population after one-week selection in presence of 1 μg/ml puromycin using single cell sorting (FACSAria II SORP; BD Biosciences) and maintained in the corresponding media containing 1 μg/ml puromycin. Before the experiments, specific reporter activity for each clonal cell line was verified by stimulation with the Wnt3a ligand and small interfering (si)RNA-mediated downregulation of β-catenin. The experiments were conducted within one month from the transduction.

The cell lines were maintained and sub-cultured according to manufacturer's instructions under the following conditions: JoPaca-1 (IMDM; 20% FBS); K562 (IMDM; 10% FBS); Hep-3B (EMEM; 10% FBS; GlutaMAX™); MRC-5 and BJ (EMEM; 10% FBS); K562-TAX, CCRF-CEM, CEM-DNR, BT-549 and MDA-MB-231 (RPMI-1640; 10% FBS); A549 (Ham's F-12K; 10% FBS); HCT116 and HCT116 p53^−/− (RPMI; 10% FBS); U2OS and HT-29 (McCoy's 5A modified medium; 10% FBS); A₃AR reporter ES-012-A (Ham's F-12; 10% FBS; GlutaMAX™; 0.4 mg/ml geneticin; and 0.25 mg/ml zeocin); β-catenin reporter cell lines (McCoy's 5A modified medium; 10% FBS; 1 μg/ml puromycin). The cell lines were authenticated using short tandem repeat DNA profiling analysis (PowerPlex 18D; Promega Corporation), analyzed every two weeks for mycoplasma contamination (reverse transcription-quantitative PCR), and maintained in T-150 flasks at 37°C in a humidified incubator with 5% CO₂ and 100% humidity. For the experiments, the cells were placed in fresh medium, with no antibiotics at densities specified for each method.

The A₃AR functional assay used was performed as previously described (46) with modifications. Briefly, ES-012-A is an AequoScreen reporter cell line stably expressing A₃AR, the photoprotein aequorin, and the promiscuous Gα16 protein subunit. The ES-012-A cell line was non-enzymatically harvested with PBS/EDTA and incubated in DMEM/F12 medium with 5 μM coelenterazine for 4 h at room temperature (RT) in the dark to reconstitute the active form of aequorin. Meanwhile, 384-well microplates with pre-diluted 2-Cl-IB-MECA (50 μM-1 pM) and MRS 1220 (10 μM-0.01 pM) were prepared using the acoustic liquid handler ECHO 550 (LabCyte, Inc.).

Coelenterazine-loaded cells (5×10³ cells per well) were dispensed using a cell suspension system and the FLIPR tetra multimodal reader (Molecular Devices, LLC.). The resulting luminescence was recorded immediately. The cells were incubated within the instrument for another 15 min at RT and the antagonistic response, represented by a drop in luminescence signal, was measured directly after the addition of the reference agonist NECA, at 80% of maximal effective concentration (EC₈₀). Raw data (area under the curve; AUC) were normalized to the percentage of the reference agonist NECA response (arbitrarily set at 100%). Normalized response in percentage was plotted against concentration, and EC₅₀ (for agonist) and IC₅₀ (for antagonist) were calculated. The data used for analysis were the result of four independent experiments.

The cytotoxicity assay was performed as previously described (47). Briefly, the cell lines were seeded into 384-well microplates, at specific densities for each cell line (0.9-2.5×10³ cells per well). After 24 h, all the tested compounds were added to the microplates using the acoustic liquid handler ECHO 550. Either vehicle, 2-Cl-IB-MECA, MRS 1220 or 2-Cl-IB-MECA in combination with 0.1 μM fixed concentration of MRS 1220 were analyzed at a concentration range 100-0.2 μM in the JoPaca-1 and Hep-3B cell lines. The vehicle volume was equal to the maximal concentration of treatment. In the other tumor and non-tumor cell lines, 2-Cl-IB-MECA was analyzed at 0.02-50 μM.

In the β-catenin reporter cell lines, 2-Cl-IB-MECA was analyzed at a single concentration of 50 μM. In the synergy combination experiments, 2-Cl-IB-MECA was analyzed at a concentration range from 1.56 to 50 μM in the JoPaca-1 cell line and from 0.78 to 25 μM in the Hep-3B cell line. The chemotherapeutic agents were used at concentrations as follows: 5-FU and carboplatin (0.78-200 μM), doxorubicin (0.1-25 μM), gemcitabine (0.2-50 μM). For the 2-Cl-IB-MECA and MRS 1220 combination experiments, MRS 1220 was added to the cells 30 min prior to 2-Cl-IB-MECA.

The treated cells were incubated at 37°C with 5% CO₂ and 100% humidity for 24 and 48 h (β-catenin reporters) or 72 h (other cell lines). After incubation, MTS solution and phenazine methosulfate (PMS), an electron coupling reagent, was prepared according to manufacturer's instructions and dispensed to the microplates using a Multidrop Combi reagent dispenser (Thermo Fisher Scientific, Inc.). The final concentration of MTS/PMS in the wells was 235 μg/ml. All the cell lines were allowed to metabolize the MTS/PMS reagent into formazan for 1.5-3 h at 37°C with 5% CO₂ and 100% humidity. Absorbance of the formazan product was measured at 490 nm using an EnVision plate reader (Perkin Elmer, Inc.) and, after subtracting the blank, the cytotoxic (anti-proliferative) effects were expressed as percentage of inhibition. The data used for analysis were the result of three independent experiments.

For the enhanced effect of combined treatments in synergy experiments, the effects were expressed as the fraction affected (FA) normalized to cells treated with vehicle (DMSO for 2-Cl-IB-MECA and MRS 1220; 0.9% w/v sodium chloride intravenous infusion for chemotherapeutic agents), and was calculated using the following equation: FA=1-(sample-blank/control-blank), where the sample is the value from a single well treated with a compound, the control is the average absorbance of the cells treated with vehicle and the blank is the average absorbance of the background without cells.

The effects of two drug treatments were assessed using the CalcuSyn software (version 2.0; Biosoft), as a combination index (CI) and the Chou-Talalay method (48,49). The FA was used as data input, where CI <0.9 was quantitatively defined as synergism, CI >0.9 and <1.1 as an additive effect, and CI >1.1 as antagonism.

Cell cycle analysis was performed as previously described (50). The JoPaca-1 and Hep-3B cell lines were seeded 24 h before treatment in 6-well multi dishes, at 2.7- and 2.2×10⁵ cells per well, respectively. The JoPaca-1 and Hep-3B cells were treated for 24 h with either vehicle, 20 nM or 20 μM 2-Cl-IB-MECA using the ECHO 550 dispenser. Both adherent and detached cell fractions were subsequently harvested using TrypLE reagent on ice, washed with PBS, fixed with 70% ethanol at −20°C added drop by drop, and immediately transferred to −20°C for another 24 h. The fixed cells were washed with citrate buffer to enhance extraction of the fragmented DNA (the subG₁ population), stained with 600 μl PI (50 μg/ml) for 30 min at 37°C in the dark, followed by incubation of samples with added 500 μl RNAse (2 mg/ml) for another 15 min under the same conditions. Before adding PI, half of each sample was separated and incubated for 1 h with an antibody against p-histone H3 Ser¹⁰ (pH3) at RT in the dark. The samples were washed with PBS containing 1% FBS, then incubated with fluorescent secondary antibody, Alexa Fluor 488 for another 30 min at RT in the dark, washed with PBS containing 1% FBS and stained with PI as aforementioned.

PI and Alexa Fluor 488 fluorescence intensities in relative fluorescence units (RFU) were then measured using a flow cytometer (FACS Calibur; BD Imunocytometry Systems) with a 488 nm single beam argon laser, with two types of settings for fluorescence measurement of PI (linear for cell cycle and pH3 and logarithmic for subG₁). Analysis of the cell cycle phases was performed using ModFit LT (version 2.0; Verity Software House, Inc.) and quantifications of the subG₁ and pH3-marked mitotic cells were performed using CellQuest software (version 3.3; BD Immunocytometry Systems). Each sample consisted of at least 1×10⁴ cells in compliance with the required parameters from three independent experiments.

The level of DNA synthesis was quantified by incorporating BrdU into the DNA from the JoPaca-1 and Hep-3B cell lines according to a previous study (50). Briefly, the cells were seeded 24 h before treatment in 6-well multi dishes at 2.7×10⁵ and 2.2×10⁵ cells per well for the JoPaca-1 and Hep-3B cells, respectively, to achieve an approximate density of 60% at the time of treatment. The cell lines were treated with either vehicle, 20 nM or 20 μM 2-Cl-IB-MECA, or with the combination of 2-Cl-IB-MECA (20 nM or 20 μM) and MRS 1220 (0.1 μM) for 24 h, for the JoPaca-1 and Hep-3B cells, respectively. For the combination experiments, MRS 1220 was added to the cells 30 min before 2-Cl-IB-MECA. The cells were then labelled with 1 μM BrdU solution at 37°C with 5% CO₂ and 100% humidity for 60 min before harvesting.

The cells were harvested with TrypLE reagent, washed with PBS, fixed with 70% ethanol at −20°C, added dropwise and immediately transferred to −20°C for 24 h. The samples were then incubated with 2 M HCl/Triton X-100 solution for 30 min at RT. The DNA denaturing reaction was neutralized with 0.1 M sodium tetraborate decahydrate. The cells were then washed with PBS containing 1% BSA and 0.5% Tween-20 and stained with primary anti-BrdU antibody for 30 min at RT in the dark. After washing with PBS, the cells were incubated with secondary anti-mouse-IgG-FITC antibody for 30 min at RT in the dark. The stained samples were washed with PBS one last time and incubated with 0.5 mg/m RNAse A for 15 min, followed by 0.1 mg/ml PI for another 15 min both at RT in the dark.

The samples were measured using a FACSCalibur flow cytometer and a 488 nm argon laser. Each sample included at least 1×10⁴ cells in compliance with the required parameters from three independent experiments. The rate of DNA synthesis was analyzed using CellQuest software (version 3.3; BD Immunocytometry Systems).

RNA synthesis was quantified by incorporating BrU into RNA in the JoPaca-1 and Hep-3B cell lines according to a previous study (50). In short, the cells were seeded 24 h before treatment in 6-well multi dishes at 2.7×10⁵ and 2.2×10⁵ cells per well for the JoPaca-1 and Hep-3B cell lines, respectively, to achieve ~60% density at the time of treatment. The concentrations and combinations of the compounds added to the cells were identical to the DNA synthesis experiment, as aforementioned. The cells were incubated for 24 h. Before harvesting, the cells were labelled with 1.5 μM BrU solution for 90 min at 37°C with 5% CO₂ and 100% humidity. The cells were harvested using TrypLE reagent followed by fixation in 1% paraformaldehyde, with 0.05% NP-40 in PBS for 15 min on a rotating wheel at RT. The samples were stored overnight at 4°C. Subsequently, the cells were washed with PBS, containing 1% glycine, with PBS alone, then stained with unconjugated anti-BrdU antibody for 45 min at RT, which cross-reacts with the BrU incorporated into the cells. After washing again with PBS containing 0.1% NP-40 and 0.1% BSA, the cell suspension was fluorescently labelled by incubation with secondary anti-mouse-IgG-FITC antibody for 30 min at RT in the dark. The labelled samples were washed with PBS containing 0.1% NP-40 and 0.1% BSA and fixed with 1% paraformaldehyde and 0.05% NP-40 in PBS for 15 min at RT in the dark and, subsequently, incubated for 1 h at 4°C. After the last wash with PBS, the cells were incubated with 0.5 mg/ml RNAse A for 15 min at RT, followed by 0.1 mg/ml PI for another 15 min at RT in the dark. BrU-labelled samples were analyzed as previously described for DNA staining and the data used was from three independent experiments.

The JoPaca-1 and Hep-3B cell lines were seeded in 6-well multi dishes, at densities of 2.7×10⁵ and 2.2×10⁵ cells, respectively, and treated with either vehicle (24 h), 20 μM 2-Cl-IB-MECA, or 0.1 μM MRS 1220 for 30 min prior to 2-Cl-IB-MECA, for 24 and 48 h. The cells were harvested on ice using a scraper and lysed with ice-cold RIPA buffer (150 μM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 μM Tris-HCl pH 8.0 and 1 μM EDTA) for 30 min, then sonicated at 35 kHz for 1 min in a 4°C ultrasonic water bath. Next, the cell debris were discarded using centrifugation at 14,000 × g for 15 min at 4°C. The protein concentrations were determined using a BCA protein assay. After 5X concentrated Laemmli buffer was added, the protein samples were incubated at 70°C for 10 min. Equal concentrations (20 μg) of each sample were separated using 7.5 or 12% SDS-PAGE. The resolved proteins were transferred to nitrocellulose membranes using the semi-dry blot method (Trans-Blot Turbo Transfer System; Bio-Rad Laboratories, Inc.). The membranes were blocked with 5% BSA in TBS, containing 0.1% Tween-20 for 1 h at RT, then incubated overnight at 4°C with the primary antibodies. Subsequently, the membranes were washed in TBS, containing 0.1% Tween-20 and incubated for 1 h at RT with HRP anti-rabbit IgG or anti-mouse IgG secondary anti-bodies. Next, the proteins were visualized using ECL substrate and a ChemiDoc MP Imaging System (Bio-Rad Laboratories, Inc.). The immunoblot images were processed using Image Lab v6.1 (Bio-Rad Laboratories, Inc.) and, if relevant, band densities were analyzed using ImageJ software (51). Relative intensities were calculated using β-actin as the loading control and expressed as fold change. In the same manner, A₃AR endogenous protein expression was analyzed using western blot analysis in the MRC-5, JoPaca-1 and Hep-3B cell lines. The data used for analysis were the result of three independent experiments.

The β-catenin^WT, β-catenin^mut, and APC^mut reporter cell lines were seeded onto 384-well microplates, at a density of 2.5×10³ cells per well. After incubation for 24 h at 37°C with 5% CO₂ and 100% humidity, all analyzed compounds were added to the microplates, at the indicated concentrations as aforementioned using an acoustic liquid handler ECHO 550 and the treated cells were incubated for specific durations (1, 3, 6, 12, 24 and 48 h).

Immediately after the addition of the Britelite plus reagent, the luminescence signal proportional to the transcriptional activity of the reporters was measured using an EnVision plate reader. After subtracting the blank, β-catenin transcriptional activity was calculated as a percentage of inhibition, defined similarly to the MTS assay. The data used for analysis were from three independent experiments.

he JoPaca-1 and Hep-3B cell lines were seeded in 6-well multi dishes, at 2.7×10⁵ and 2.2×10⁵ cells per well, respectively. The cells at ~60% density were treated with either vehicle or 20 μM 2-Cl-IB-MECA 24 h later. After another 24 h, the cells were collected using TrypLE reagent, centrifuged at 500 × g for 5 min, at RT and the pellet was washed with PBS. The cells were fixed with cold methanol (−20°C; 100%), added dropwise, then the cells were stored at −20°C for a minimum of 24 h before analysis.

Subsequently, the cells were washed with rinsing buffer, composed of Cell wash reagent, 0.5% BSA, 0.1% NP-40, then permeabilized with Permeabilizing solution 2 for 10 min, and washed again with rinsing buffer. A saturation reagent that blocks Fc receptors to prevent unspecific binding of the antibody (Cell wash reagent, 0.5% BSA and Fc receptor saturation reagent) was added to the cells, then incubated for 10 min at RT, and washed twice more with Cell wash reagent containing 0.5% BSA and 0.1% NP-40. The samples were divided into two tubes: One for P-gp expression analysis (monoclonal anti-P-gp antibody) and one for the isotype control (IgG1 isotype control; unconjugated); both tubes were incubated with the antibodies for 30 min at RT in the dark. Following which, all the samples were washed with rinsing buffer and incubated with FITC-conjugated secondary antibody anti-mouse-IgG for 30 min at RT, washed once more, then stored at 4°C for 15 min.

Median fluorescence intensities (MFI) for both samples and isotype controls were measured with a 488 nm argon laser and a FACS Calibur flow cytometer, where the isotype controls defined the intensity threshold at a median of 10 relative fluorescence units. MFI raw data were the result of three independent experiments and analyzed using CellQuest (version 3.3; BD Immunocytometry Systems).

The K562-TAX cell line, derived from K562 chronic myelogenous leukemia cell line, was developed at the Institute of Molecular and Translational Medicine, as a cell line that is resistant to paclitaxel, with high expression of the P-gp transporter and low expression levels of MRP1 and lung resistance protein (LRP) transporters (45).

The P-gp efflux assay was conducted as previously described (52). Briefly, the K562-TAX cells were seeded in 6-well multi dishes at 2.5×10⁵ and allowed to proliferate for 24 h. The next day, the cells were treated with 1 μM daunorubicin (fluorescent) for 60 min at 37°C in the dark. The cells were washed twice with complete medium and incubated with 2-Cl-IB-MECA (0.78-100 μM), either alone or following 0.1 μM MRS 1220 pre-treatment for 30 min at 37°C, for another 60 min under the same conditions as aforementioned. Following which, the K562-TAX cells were harvested using a scraper and fluorescence intensity was measured immediately using a FACS Calibur flow cytometer and a 488 nm argon laser.

Each sample comprised at least 1×10⁴ cells, that were compliant with the required parameters. The raw data were the result of three independent experiments and were measured as MFI. MFI for the vehicle was set to 10 relative fluorescence units. MFI comparisons were analyzed using CellQuest (version 3.3; BD Immunocytometry Systems).

The results are expressed as the mean ± SD, with the number of independent replicates (n) indicated for each experiment. Quantification of the results was performed using GraphPad Prism v9 for Windows (GraphPad Software, Inc.), including IC₅₀ values for the JoPaca-1 and Hep-3B cell lines. The IC₅₀ values in the MTS experiment using the other tumor and non-tumor cell lines were calculated using Dotmatics software (version 5.5; Dotmatics, Ltd.). Statistical analyses were performed using R statistical software, v3.5.0 (R Foundation for Statistical Computing; www.r-project.org). The data was checked for normality using the Shapiro-Wilk test. Statistical differences between treatment group and the reference group (vehicle) were analyzed using a Student's one-sample t-test. An unpaired Student's two-sample t-test was used for comparison of treatments. ANOVA followed by Dunn's post hoc test was used to analyze 2-Cl-IB-MECA at 24 and 48 h and the reference group (MRS 1220 at 24 h). A one-way ANOVA test, followed by Dunn's post hoc test was used to analyze the 2-Cl-IB-MECA group at 24 and 48 h and the vehicle as the reference group, for phosphorylated/total protein. In case of comparison of more than two treatment conditions, ANOVA followed by Tukey's post hoc test was used. P<0.05 was considered to indicate a statistically significant difference.

Previous studies have shown both pro- and antitumor effects of 2-Cl-IB-MECA in vitro and in vivo at either sub-micromolar or micromolar concentrations (reviewed in 53). To determine the overall cytotoxic ability of 2-Cl-IB-MECA (Fig. 1A), the effects of 2-Cl-IB-MECA against a panel of tumor and non-tumor cell lines was performed. Using a MTS assay, the cells were incubated with 2-Cl-IB-MECA for 72 h and the cellular response was analyzed using different concentrations (0.2-100 μM for the JoPaca-1 and Hep-3B cell lines and 0.02-50 μM for the other cell lines). Cytotoxicity against 2-Cl-IB-MECA was found in micromolar concentrations across various tumor cell lines, of different histogenetic origin, including JoPaca-1 and the most sensitive cell line, Hep-3B, with their respective IC₅₀ values of 25.26±1.6 and 10.68±1.1 μM (Table I). Furthermore, the non-tumor fibroblast cell lines, MRC-5 and BJ cells remained metabolically active despite increasing 2-Cl-IB-MECA concentrations, with IC₅₀ values >50 μM.

Next, the compounds, 2-Cl-IB-MECA and MRS 1220 were both analyzed on reporter cell lines expressing A₃AR. The functional assay revealed A₃AR activation by 2-Cl-IB-MECA (EC₅₀, 32.28±11.2 nM), and its inhibition by MRS 1220 (IC₅₀, 1.41±0.9 nM) at nanomolar concentrations (Fig. 1B).

To identify whether any possible effects could be A₃AR related, western blot analysis was performed to analyze A₃AR protein expression (Fig. 1C). By contrast to the non-tumor MRC-5 fibroblasts, the results showed the presence of A₃AR in both the JoPaca-1 and Hep-3B tumor cell lines analyzed.

To address the nature of the effects of 2-Cl-IB-MECA on human pancreatic cancer and HCC cells more precisely, the JoPaca-1 and Hep-3B cell lines were treated with increasing concentrations of 2-Cl-IB-MECA and MRS 1220 alone, and in combination for 72 h. MRS 1220 is a specific A₃AR antagonist (54,55) and MRS 1220 intrinsic cytotoxicity as well as its ability to counteract the effect of the A₃AR agonist 2-Cl-IB-MECA was analyzed in the cancer cells. Notably, pre-treatment of the cells with 0.1 μM MRS 1220 led to significantly decreased 2-Cl-IB-MECA cytotoxicity in both cell lines. MRS 1220 alone at sub-micromolar concentrations were not cytotoxic for either cell line (Fig. 1D and E). Taken together, 2-Cl-IB-MECA inhibited the proliferation of the cancer cell lines, including JoPaca-1 and HEP-3B cancer cells at micromolar concentrations and its effect in the JoPaca-1 and HEP-3B cell lines was reduced by MRS 1220. Therefore, 20 nM (the A₃AR-activating concentration) and 20 μM (considerable cytotoxicity-inducing concentration) of 2-Cl-IB-MECA was used for further experiments.

Previous studies showed that the N6-adenosine analogue 2-Cl-IB-MECA reduces the cell cycle in other cancer cell types, such as NPA thyroid carcinoma and A375 melanoma cells (12,56). Flow cytometry analysis was performed to analyze cell cycle progression, including the subG₁ population, histone H3 phosphorylation, and changes in RNA and DNA synthesis, to further assess a possible anticancer mechanism of 2-Cl-IB-MECA. Based on the cytotoxicity experiments, the JoPaca-1 and Hep-3B cell lines were each treated with either vehicle, 20 nM or 20 μM of 2-Cl-IB-MECA for 24 h. In DNA and RNA synthesis experiments, the cells were pre-treated with 0.1 μM MRS 1220 for 30 min prior to the addition of 2-Cl-IB-MECA.

Only 20 μM 2-Cl-IB-MECA affected the cell cycle, showing an increase in the number of cells in the G₁ phase. In addition, there was a simultaneous increase in the subG₁ population, representing cells undergoing apoptosis (Fig. 2A-C). The Hep-3B cell line was more responsive compared with that in the JoPaca-1 cell line (Fig. 2B). In concordance with the block at the G₁ transition point, there was a reduction in the number of cells in S phase. Indeed, the reduction in the number of cells in the S phase was further accentuated by the drop in the cell population actively incorporating BrdU and BrU, indicating an inhibition of DNA (Fig. 2E), and RNA synthesis (Fig. 2F), respectively. Notably, pre-treatment with 0.1 μM MRS 1220 significantly reversed the effects of 2-Cl-IB-MECA on BrdU (in the JoPaca-1 cell line at 20 nM and 20 μM of 2-Cl-IB-MECA) and BrU incorporation (in the Hep-3B cell line at 20 nM and 20 μM of 2-Cl-IB-MECA). There was also decrease in RNA synthesis in the JoPaca-1 cell line following treatment with 0.1 μM MRS 1220, suggesting a possible intrinsic effect of MRS 1220 alone.

With respect to the JoPaca-1 cell line, the minor changes in the number of cells in the G₂/M phase were mirrored by the decrease in the mitosis marker histone H3 phosphorylated at Ser¹⁰ (Fig. 2D).

To further understand the mechanism of 2-Cl-IB-MECA in the pancreatic and HCC cell lines, the protein expression level of three essential pathways, previously indicated to be affected, either directly or indirectly, by AdoRs and their modulators (57), was analyzed. The JoPaca-1 and Hep-3B cell lines were treated with vehicle or 20 μM 2-Cl-IB-MECA for 24 and 48 h. The cells were pre-treated with 0.1 μM MRS 1220 for 30 min prior to the addition of 2-Cl-IB-MECA, to confirm whether the effects were A₃AR mediated.

First, A₃AR protein expression level was analyzed after 24 and 48 h of 2-Cl-IB-MECA treatment. There was a decrease in A₃AR protein expression level in the Hep-3B cell line, while there were no changes in the JoPaca-1 cell line (Fig. 3). Then, the expression level of proteins in the PI3K/AKT/NF-κB pathway were analyzed (53). It was found in both cell lines that the proliferation-related proteins, AKT and NF-κB, were phosphorylated, at Ser⁴⁷³ and Ser⁵³⁶, respectively, in a time-dependent manner, after 2-Cl-IB-MECA treatment alone. Surprisingly, AKT kinase was dephosphorylated at Thr³⁰⁸ following 2-Cl-IB-MECA treatment for 24 and 48 h in the JoPaca-1 cell line, and after 48 h in the Hep-3B cell. line. Furthermore, the results demonstrated that MRS 1220 pre-treatment did not prevent 2-Cl-IB-MECA from most of its effects at 24 and 48 h, with the exception of AKT at Thr³⁰⁸ and pNF-κB at Ser⁵³⁶ (Fig. 3). Notably, there were differences between the JoPaca-1 and Hep-3B cell lines in the expression and phosphorylation of ERK1/2. The Hep-3B cell line showed a decrease in the expression level of ERK1/2 and pERK1/2 following treatment for 48 h with 2-Cl-IB-MECA and in combination with MRS 1220; however, total and phosphorylated ERK1/2 protein expression level in the JoPaca-1 cell line was increased in the same experimental groups in a time-dependent manner.

Another signaling pathway that has been associated with several types of cancer, including pancreatic and HCC, is the Wnt/β-catenin pathway (58,59). Therefore, the effects of 2-Cl-IB-MECA on the protein expression level of β-catenin and GSK-3β kinase, two main molecules in the Wnt signaling pathway were analyzed (Fig. 4). The β-catenin protein expression levels were decreased in both cell lines following 2-Cl-IB-MECA treatment for 24 and 48 h. Surprisingly, GSK-3β kinase expression did not change distinctly in either cell line within the same experimental group as β-catenin. In addition, its phosphorylated form, pGSK-3β, remained unaltered in the JoPaca-1 cell line, but was increased in the Hep-3B cell line after 2-Cl-IB-MECA treatment at 48 h, suggesting that kinase inactivation only occurs in the Hep-3B cell line. Notably, these effects on the components of the Wnt/β-catenin signaling pathway were partially abrogated by the A₃AR antagonist, MRS 1220.

The Shh/Ptch/Gli axis was identified as another relevant pathway for bypassing cell death for malignant pancreatic and liver cells (59-61). The essential component of the Shh pathway, the Gli1 transcription factor, further regulates the expression level of cyclin D1, c-Myc, and Ptch1 receptor, another key molecule of the Shh pathway. In the JoPaca-1 and Hep-3B cell lines, 2-Cl-IB-MECA-mediated the decrease in the protein expression level of both Gli1 and Ptch1 in a time-dependent manner. Furthermore, the protein expression level of cyclin D1 and c-Myc, together with the c-Myc phosphorylated forms, was reduced following treatment with 2-Cl-IB-MECA alone for 24 and 48 h. These effects were, however, partially reversed by MRS 1220 (Fig. 4).

Furthermore, β-catenin transcriptional activity upon 2-Cl-IB-MECA treatment was analyzed using a reporter system at various time points (Fig. 5). β-catenin can bind to the TCF/LEF transcription factor, triggering subsequent transcriptional activity of Wnt/β-catenin pathway target genes, e.g. c-Myc and cyclin D1 (62,63). Therefore, three reporter systems were designed to assess the transcriptional activity of: The WT Wnt/β-catenin pathway (β-catenin^WT); a β-catenin mutation that prevents its proteasomal degradation (β-catenin^mut); and a loss of function of the APC gene, which is critical for binding β-catenin in a complex with AXIN and, thus, regulates β-catenin availability for transcription (APC^mut). The β-catenin^WT (Fig. 5A), β-catenin^mut and APC^mut (Fig. 5B) reporter cell lines were treated with either vehicle, 50 μM 2-Cl-IB-MECA or 0.1 μM MRS 1220 for 30 min followed by 50 μM 2-Cl-IB-MECA. MTS assays were performed in parallel for 24 and 48 h to distinguish any decline in reporter activities from simple cytotoxic effects. The results indicated early inhibition of β-catenin transcription. WT transcriptional activity was inhibited by 75% after 6 h (Fig. 5A). Furthermore, 2-Cl-IB-MECA was more effective in the mutated reporter systems, where 2-Cl-IB-MECA completely abrogated β-catenin transcription within 12 h (Fig. 5B). This early decline in transcriptional activity was not primarily caused by inhibited proliferation and the degree of MRS 1220 antagonism against 2-Cl-IB-MECA was insignificant in this case.

Multidrug resistance (MDR) can seriously hinder anticancer treatment. Recently, some studies have reported an association between AdoRs and MDR (37,43). Therefore, MRP1 and P-gp protein expression level was analyzed in both the pancreatic and HCC cell lines. In addition to analyzing the protein expression levels, P-gp activity was also analyzed using a functional test of K562-TAX overexpressing P-gp protein.

To analyze the protein expression level, the cells were treated with either vehicle or 20 μM 2-Cl-IB-MECA for 24 h. The results indicated the reduction of MRP1 protein expression level after treatment with 2-Cl-IB-MECA, with a more significant effect in the Hep-3B cell line (Fig. 6A and B). P-gp expression was analyzed using flow cytometry under the same conditions. Similarly, P-gp overall expression was significantly attenuated by 20 μM 2-Cl-IB-MECA after 24 h (Fig. 6C and D). Of note, the Hep-3B cell line had higher endogenous P-gp levels.

Reducing P-gp expression could be a powerful tool against chemoresistance in cancer cells. Nonetheless, it was also investigated whether 2-Cl-IB-MECA could diminish the P-gp efflux function to any extent. Chemotherapeutic agent daunorubicin serves as the P-gp substrate and its intracellular uptake reflects P-gp ability to actively transport this xenobiotic from the cells (64). Therefore, P-gp functional ability to actively effuse daunorubicin when treated with 2-Cl-IB-MECA was analyzed. For this purpose, the K562-TAX cell line, with high P-gp expression, was used (45). The K562-TAX cells were incubated with 1 μM daunorubicin for 1 h prior to treatment with either vehicle or 2-Cl-IB-MECA (0.78-100 μM) for an additional 1 h. For pre-treatment with MRS 1220, 0.1 μM MRS 1220 was added 30 min before the 1 h incubation with 2-Cl-IB-MECA. Within 1 h of treatment, 2-Cl-IB-MECA, at high micromolar concentrations was able to increase P-gp efflux activity (Fig. 6E). This functional interference suggests that 2-Cl-IB-MECA is a direct substrate of P-gp. Surprisingly, the negative effects of 2-Cl-IB-MECA on P-gp function were partially abolished by A₃AR antagonist MRS 1220.

The aforementioned results showed that 2-Cl-IB-MECA mediated the inhibition of key components of the Wnt/β-catenin and Shh/Ptch/Gli pathways, together with disruption of the xenobiotic efflux system. These are two interlinked mechanisms in which cancer cells can resist pharmacotherapy (60,61,65). Therefore, we hypothesized the beneficial effects of 2-Cl-IB-MECA could be part of a combined cancer treatment. First, cellular proliferation after treatment was normalized to vehicle as FA, then the derived analysis of the combination effect of two compounds was calculated. The resulting CI values for respective concentration ratios are shown in Fig. 7. The outcomes of treatment combinations were analyzed: 2-Cl-IB-MECA (1.56-50 μM for the JoPaca-1 cell line and 0.78-25 μM for the Hep-3B cell line) and each of the following four current conventional chemotherapeutic agents: 5-FU (0.78-200 μM), carboplatin (0.78-200 μM), doxorubicin (0.1-25 μM) and gemcitabine (0.2-50 μM).

The JoPaca-1 and Hep-3B cell lines were treated with each compound alone and in either a concurrent or sequential combination comprising 2-Cl-IB-MECA and one of the four chemotherapeutics for 72 h. For sequential combinations, 2-Cl-IB-MECA was added to the cells first, followed 16 h later by one of the chemotherapeutics, or vice versa. Possible synergistic or additive effects of 2-Cl-IB-MECA on cancer cell sensitivity to the aforementioned drugs were analyzed using a MTS assay. Endpoint analysis of cytotoxicity was performed 72 h after concurrent or sequential treatment and the CI was calculated. The degree of synergism, additive effect or antagonism was evaluated based on the CI method (48) and color coded (green indicates the highest synergy, while pink indicates no synergy).

In the JoPaca-1 cell line, synergistic effects for 2-Cl-IB-MECA concurrent combinations with carboplatin, doxorubicin and 5-FU were found at several concentration ratios (green color). For gemcitabine, synergy was found only if cells were concurrently treated with 2-Cl-IB-MECA at concentrations ≥IC₅₀ (Fig. 7A). The Hep-3B cell line was more sensitive to 2-Cl-IB-MECA alone; therefore, the highest concentration of 2-Cl-IB-MECA was 25 μM (Fig. 7B). Similar to the JoPaca-1 cell line, synergy was found in the Hep-3B cell line treated with multiple, independent, concomitant combinations of 2-Cl-IB-MECA with either carboplatin or doxorubicin, whereas 5-FU and gemcitabine showed synergistic effects exclusively at high 2-Cl-IB-MECA concentrations (~IC₇₅) and low 2-Cl-IB-MECA concentrations with 5-FU and gemcitabine at ~IC₅₀.