Tucatinib (purity 99.38%) was purchased from Selleck Chemicals. Topotecan, Hoechst 33342, mitoxantrone, cisplatin and fumitremorgin C (FTC) were purchased from Sigma-Aldrich (Merck KGaA). RPMI-1640, DMEM, BSA, FBS, penicillin/streptomycin and 0.25% trypsin were purchased from Hyclone (GE Healthcare Life Sciences). [³H]-mitoxantrone (4 Ci/mmol) was purchased from Moravek Biochemicals, Inc. The monoclonal antibody against ABCG2 was purchased from Santa Cruz Biotechnology, Inc., while the AlexaFluor488-conjugated goat anti-mouse IgG secondary antibody was purchased from Signet Laboratories Inc. HRP-conjugated rabbit anti-sheep IgG secondary antibody was purchased from Sigma-Aldrich (Merck KGaA), while the monoclonal antibody against GAPDH was purchased from Kangcheng BioTech Co., Ltd. Dimethylsulfoxide (DMSO), MTT and paraformaldehyde were purchased from Sigma-Aldrich (Merck KGaA). Mitoxantrone, topotecan and FTC were used as positive controls to confirm the mechanism of drug resistance in LSC models. Cisplatin (a non-substrate of ABCG2) was used as a negative control.

The human HL60 and K562 leukemia cell lines were purchased from the Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College (Tianjin, China). The HL60/ABCG2 and K562/ABCG2 cell lines (which overexpress ABCG2) were established, in the present study, by the transduction of the HL60 and K562 cell lines, respectively, with a HaMyBCRP retrovirus that contains Myc-tagged human BCRP (ABCG2) cDNA in Ha retrovirus vector (23–25). Subsequently, the transfected cells were selected using 4.0 µM mitoxantrone for 7 days. The cell lines were cultured in RPMI-1640 containing 10% FBS, 100 U/ml penicillin and 100 U/ml streptomycin, at 37°C in a humidified atmosphere with 5% CO₂, as previously described (23).

MTT (purity 99.59%) reagent was used to determine the cell sensitivity to different chemotherapeutic agents (mitoxantrone, topotecan, cisplatin and tucatinib) with minor modifications (26). The IC₅₀, which is defined as the drug concentration resulting in 50% cell death, was calculated from the cell survival curves using the Bliss method (27–29). The cells were collected and seeded in 96-well plates at 5×10³ cells/well in 160 µl medium. After culturing for 24 h, the cells were pre-incubated with 0.1, 0.2 and 0.4 µM tucatinib for a further 72 h at 37°C. The cells were then treated with various concentrations of the chemotherapeutic agents by 2-fold dilution (mitoxantrone, 0.5–50 µM; topotecan, 0.5–50 µM; and cisplatin, 0.5–50 µM) for a further 68 h. Subsequently, 20 µl MTT solution (5 mg/ml) was added to the cells, followed by further incubation for 4 h at 37°C then, the medium was discarded, and 200 µl DMSO was added to dissolve the formazan product formed from the metabolism of MTT. The absorbance was determined at 540 nm, with a background subtraction at 670 nm, using a Model550 microplate reader (Bio-Rad Laboratories, Inc.) (30). The resistance fold change was calculated by dividing the IC₅₀ values of the substrates, in the presence or absence of tucatinib, by the IC₅₀ of the parental cells without the inhibitor treatment (23).

The present study was approved by the Ethics Review Committee at Sun Yat-Sen University. A total of 15 patients with leukemia were recruited into the study between June 2017 and April 2018; however, the bone marrow samples were obtained from 6 patients, at diagnosis, and randomly selected from a study pool of blast samples and examined. The patient population consisted of 2 males and 4 females, with a median age of 27 years. According to the French-American-British classification (31), three of the patients were diagnosed with acute lymphocytic leukemia, two of the patients were diagnosed with acute myeloid leukemia, and one patient was diagnosed with T lymphoblastic lymphoma. After the patients provided written informed consent, leukemia blasts were isolated using Ficoll-Hypaque density gradient centrifugation (at 300 × g for 25 min at room temperature) and cultured in RPMI-1640, containing 20% FBS, with penicillin (100 U/ml) and streptomycin (100 U/ml) at 37°C. Western blot analysis was performed to detect the protein expression level of ABCG2 in the patient samples. The patient characteristics are summarized in Table S1.

The sorting and analysis of the SP cells was performed according to the methods described by Vieyra et al (32). In brief, the cells (1×10⁶ cells/ml) were cultured in prepared DMEM, containing 2% FBS and 10 mmol/I HEPES. Subsequently, 5 µg/ml Hoechst 33342 dye was added to the cells in the presence or absence of 10 µmol/l FTC. Following incubation for 90 min, with intermittent shaking at 37°C, the cells were washed twice with ice-cold PBS. The Hoechst dye was excited at 355 nm, and the fluorescence profile was measured during the analysis (blue, 402–446 nm; red, 650–670 nm; MoFlo™ XDP; Beckman Coulter). The Summit v5.2 Software (Beckman Coulter) was used for the analysis.

The HL60/ABCG2 cell lines (1×10⁵) were cultured in 6-well plates, containing various concentrations of tucatinib (0.1, 0.2 and 0.4 µM) for 24 h. Subsequently, 0.5 µg/ml Hoechst 33342 dye was added, followed by a further incubation for 30 min at 37°C. Finally, the cells were washed with ice-cold PBS, three times and re-suspended in PBS for flow cytometric analysis (MoFlo™ XDP; Beckman Coulter). The Summit v5.2 Software (Beckman Coulter) was used for the analysis.

The [³H]-mitoxantrone intracellular accumulation assay was used for the assessment of the reversal effects of tucatinib. In brief, the HL60, K562, HL60/ABCG2 and K562/ABCG2 cells (5×10⁵) were suspended in RPMI 1640 medium, then incubated with or without 0.1 and 0.4 µM tucatinib or 2.5 µM FTC for 1 h at 37°C. Subsequently, the cells were cultured in medium containing 0.1 µM [³H]-mitoxantrone for a further 2 h at 37°C. After washing twice with 10 ml ice-cold PBS and lysed with 1% SDS (Ph 7.4), the radioactivity of the cells was measured. Each sample was placed in scintillation fluid (5 ml), then the radioactivity was analyzed using a Packard TRI-CARB 1900CA liquid scintillation analyzer (PerkinElmer, Inc.), as described previously (33).

Following the accumulation assay, the HL60, K562, HL60/ABCG2 and K562/ABCG2 cells (5×10⁵) were suspended in RPMI 1640 medium, pre-incubated with or without 0.1 and 0.4 µM tucatinib or 2.5 µM FTC for 2 h at 37°C. Subsequently, 0.1 µM [³H]-mitoxantrone was added to each sample for 2 h at 37°C, and following washing with ice-cold PBS, the cells were lysed at various time points (0, 30, 60 and 120 min). Each sample was placed in scintillation fluid and the radioactivity was analyzed (Packard TRI-CARB 1900CA liquid scintillation analyzer; PerkinElmer, Inc.), as previously described (33). The Spectra Works2™ v2.0 STD and Quanta-Smart v5.2 Software (PerkinElmer, Inc.) were used for the analysis.

The ABCG2-associated ATPase activity assay was determined using a SB-BRCP-M-PREDEASY-ATPase kit (TEBU-BIO nv,), with modified protocols (34). Cell membranes that overexpressed ABCG2 were incubated with an assay buffer containing 5 mM sodium azide, 1 mM ouabain, 2 mM dithiothreitol, 10 mM MgCl₂, 50 mM potassium chloride, 2 mM ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetra acetic acid, 50 mM pH 6.8 2-(N-morpholino) ethanesulfonic acid MES and 0.3 mM sodium orthovanadate (Na₃VO₄) at 37°C for 5 min. Na₃VO₄ was used as an ATPase inhibitor. Various concentrations of tucatinib (0–40 µM) were incubated with the membranes for 5 min at 37°C. The ATPase reaction was initiated by the addition of 5 mM Mg²⁺ ATP. Luminescent signals of free phosphate group (Pi) were initiated and measured at 880 nm using a spectrophotometer (Bio Rad Laboratories, Inc.) following incubation at 37°C for 40 min with brief mixing. The changes in relative light units were determined by comparing Na₃VO₄-treated samples with the tucatinib-treated groups, using the colorimetric method (35).

Western blot analysis was performed to determine the protein expression levels of ABCG2, following treatment with 0.1, 0.2, 0.4 and 1 µM tucatinib for 48 h or with 0.4 µM tucatinib for 0, 24, 36, 48 and 72 h. The HL60/ABCG2 cells were incubated with 0, 0.1, 0.2, 0.4 and 1 µM tucatinib for 12 h, then harvested and washed twice with ice-cold PBS. The cell extracts were collected using a cell lysis buffer and protein concentration was determined using a BCA Protein assay kit (both from Thermo Fisher Scientific, Inc.) (36). Equal amounts of total cell lysates (30 µg protein) were resolved using a 10% SDS-PAGE and electrophoretically transferred onto PVDF membranes (EMD Millipore). The membranes were blocked with 5% skimmed milk dissolved in TBS-Tween-20 (TBST) buffer (10 mmol/l Tris-HCl, 150 mmol/l NaCl and 0.1% Tween-20, pH 8.0) for 2 h at room temperature. The membranes were then incubated with the primary monoclonal antibodies against GAPDH (cat. no. KC-5G4; 1:1,000) or ABCG2 (cat. no. MAB4145; clone BXP-34; 1:200) at 4°C overnight, then with HRP-conjugated secondary antibody (cat. no. AP147P; 1:1,000) at room temperature for 2 h. Subsequently, the protein-antibody complexes were washed three times with TBST and protein bands were visualized using an enhanced chemiluminescence detection system (Phototope TM-HRP Detection kit; Cell Signaling Technology, Inc.) and the protein bands were analyzed using Scion Image v4.0.3 software (Scion Corporation). GAPDH was used as a loading control.

The HL60/ABCG2 cells were cultured overnight in 24-well plates, then 0.4 µM tucatinib was added to each well for further incubation for 72 h. After washing with PBS, the treated cells were fixed with 4% paraformaldehyde for 15 min and permeabilized using 0.1% Triton X-100 for 10 min, both at room temperature. Subsequently, the cells were incubated with a monoclonal antibody against ABCG2 (cat. no. MAB4145; clone BXP-34; (1:500), overnight at 4°C. Following incubation, the cells were washed with ice-cold PBS and incubated with an Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibody (cat. no. A-10684; 1:1,000) for 1 h, at 4°C. Immunofluorescence images were obtained using an inverted confocal microscope, and 6–8 random microscopic fields (magnification, ×400; model IX70; Olympus Corporation) with IX-FLA fluorescence and a Charge Coupled Devices camera.

Statistical analysis was performed using SPSS v16.0 software (SPSS, Inc.) and data are presented as the mean ± SD, from 3–5 independent experiments. Statistical differences between 2 groups were determined using an unpaired Student's t-test. One-way ANOVA was used to determine differences between the means of multiple samples, with a Dunnett's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Prior to investigating the cytotoxicity of tucatinib, the protein expression levels of ABCG2 in the transfected cell lines were confirmed using western blot analysis. The protein expression levels of ABCG2 were overexpressed in the HL60/ABCG2 and K562/ABCG2 cell lines compared with that in the HL60 and K562 parental cell lines, respectively (Fig. 1A).

To investigate the effects of tucatinib on leukemia cells, MTT assays were subsequently performed to determine the cytotoxicity of tucatinib on 2 leukemia cell lines. As shown in Fig. 1B and C, >80% of the HL60/ABCG2 and K562/ABCG2 ABCG2-overexpressing cell lines, and their parental cell lines, HL60 and K562, survived 0.4 µM tucatinib treatment. Therefore, 0.4 µM tucatinib was selected as the maximum working concentration for further experiments. Subsequently, whether tucatinib, at various concentrations, could increase the sensitivity of ABCG2-overexpressing leukemia drug resistant cells to mitoxantrone and topotecan was investigated. As shown in Tables I and II, the ABCG2-overexpressing HL60/ABCG2 and K562/ABCG2 cell lines showed higher IC₅₀ values to the ABCG2 substrates, mitoxantrone and topotecan compared with that in their parental cell lines, respectively. In the presence of 0.1 and 0.2 µM tucatinib, there was a significant increase in sensitivity of the cell lines to the two drugs. Tucatinib (0.4 µM) further increased the sensitivity of leukemia cells to the two drugs in both the ABCG2-overexpressing HL60/ABCG2 and K562/ABCG2 cell lines, and its efficacy was comparable to that of the known ABCG2 inhibitor, FTC (2.5 µM). Conversely, tucatinib did not significantly alter the IC₅₀ value of cisplatin in all the leukemia cell lines, which is a non-ABCG2 substrate. Taken together, these results suggested that tucatinib may significantly sensitize ABCG2-overexpressing leukemia cells to become anti-neoplastic.

ABCG2 is abundantly expressed in patients with acute leukemia and in LSCs (9). Thus, the present study examined the protein expression levels of ABCG2 and analyzed the cytotoxicity of mitoxantrone, with or without tucatinib treatment in leukemia blast cells derived from patients with leukemia (Fig. 2). The results demonstrated that 2/6 patient samples exhibited detectable expression levels of ABCG2 (Fig. 2A). Notably, treatment of the leukemia blast cells, with tucatinib, effectively increased their sensitivity to the cytotoxicity of mitoxantrone in two patient samples (Fig. 2B and C). These results suggested that the combined use of tucatinib and mitoxantrone may achieve positive clinical effects.

In the present study, to investigate whether tucatinib has the potential to inhibit the proportion of SP cells, the population of SP cells in the HL60 cell line, treated with various concentrations of tucatinib were analyzed using flow cytometry. As shown in Fig. 3A and B, following treatment with 0.4 µM tucatinib, the proportion of SP cells in the HL60 cell line was significantly decreased from 2.6 to 0.8% (P=0.041) compared with that in the control cells, respectively. These results indicated that tucatinib significantly decreased the SP cell fraction in the HL60 cell line, in a dose-dependent manner.

The potentiation of antitumor activity by transporter inhibitor is typically mediated by inhibiting transporter-mediated drugs, resulting in increased intracellular drug accumulation (37). To determine whether tucatinib may potentiate the chemotherapeutic efficacy in SP cells, the intracellular accumulation levels of Hoechst 33342 were determined (known as fluorescent substrates of ABCG2) using flow cytometry. The results revealed that in the presence of 0.1, 0.2 and 0.4 µM tucatinib, the relative values of Hoechst 33342 in SP cells were significantly increased by 0.85 (P=0.035)-, 0.92 (P=0.028)- and 1.195 (P=0.024)-fold, respectively (Fig. 4A and C). These results indicated that tucatinib may significantly elevated the intracellular accumulation of Hoechst 33342 in a concentration-dependent manner in SP cells. Conversely, there was no significant difference in the intracellular levels of Hoechst 33342 in non-SP (NSP) cells treated with tucatinib and FTC.

To investigate the potential traverse mechanism by which tucatinib sensitized ABCG2-overexpressing leukemia cell lines, the intracellular levels of [³H]-mitoxantrone were analyzed in the presence or absence of tucatinib. In the presence of 0.1 and 0.4 µM, tucatinib significantly increased the intracellular levels of [³H]-mitoxantrone in the HL60/ABCG2 cell lines, but the accumulative effect of [³H]-mitoxantrone following 0.4 µM tucatinib was lower compared with that of FTC, at 2.5 µM, respectively (Fig. 5E). In addition, tucatinib, at 0.4 µM, significantly reduced the efflux of [³H]-mitoxantronein close to the effect of FTC at 2.5 µM in the HL60/ABCG2 cell lines (Fig. 5B). Similarly, pretreatment of tucatinib also effectively improved the intracellular levels of [³H]-mitoxantrone and decreased its efflux in K562/ABCG2 cells (Fig. 5D and F). Neither tucatinib nor FTC significantly affected the intracellular levels of [³H]-mitoxantrone in the parental HL60 and K562 cells (Fig. 5A and C). These results indicated that tucatinib increased the intracellular levels of the antitumor drugs in a dose-dependent manner.

The drug efflux function of ABCG2 has been associated with ATP hydrolysis, which is mediated by the presence of its substrates or inhibitors (34). To further investigate the mechanisms involved for the potential of tucatinib to overcome MDR, the efficacy of tucatinib on the ATPase activity of ABCG2 transporters was determined, by measuring BCRP-mediated ATP hydrolysis in the presence or absence of 0–40 µM tucatinib (Fig. 6A). As shown in Fig. 6B, using the colorimetric method, the maximum ATPase activities of ABCG2 increased to 114.28±8.91 nmoles P_i/mg protein/min in the presence of tucatinib (from 0–10 µM), the maximum stimulation was 4.28-fold greater compared with that at the basal level. The concentration of tucatinib required to obtain 50% stimulation was 2.7 µM. These results suggested that tucatinib stimulated the ATPase activity of ABCG2 by interacting with the drug substrate-binding site, thereby restricting the efflux function of ABCG2.

To determine whether the antagonistic effects of tucatinib on MDR were achieved by altering ABCG2 protein expression levels or changing its subcellular localization, western blot and immunofluorescence analyses were performed. There were no notable changes in the cellular localization of ABCG2 transporters, following incubation with 0.4 µM tucatinib for 72 h in the HL60/ABCG2 cell line (Fig. 7A). In addition, the ABCG2 protein expression level was not altered following 72 h of treatment with 0.4 µM tucatinib or following treatment with 0.1, 0.2, 0.4 or 1 µM tucatinib for 48 h in the HL60/ABCG2 cell line. (Fig. 7B). These results indicated that the reversal effects of tucatinib were neither accomplished by altering the expression levels nor by affecting the intracellular localization of ABCG2 in the HL60/ABCG2 cell line.