HCQ was purchased from the Tokyo Chemical Industry Co., Ltd. Adriamycin (ADM) was obtained from the Kangbaotai Biochemical Industry Company. Newborn bovine serum was obtained from the Rongye Biotech Company (http://royabio.800400.net). RPMI-1640 medium was acquired from Gibco (Thermo Fisher Scientific, Inc.), MTT from Sigma-Aldrich (Merck KGaA); and TRIzol^® was from Invitrogen (Thermo Fisher Scientific, Inc.). Specific primers for mdr1 and β-actin were synthesised by the Takara Bio, Inc. Antibodies against P62 (cat. no. 88588) and light chain (LC)3 (cat. no. 4108) were obtained from Cell Signaling Technology, Inc.; anti-β-actin antibody (cat. no. A1813) was from BioVision, Inc.; antibodies against Bax (cat. no. TA346891), Bcl-2 (cat. no. TA803003) and cleaved caspase-3 (cat. no. TA336455) were purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.; anti-P-gp antibody (cat. no. BM4508), horseradish peroxidase (HRP)-linked anti-rabbit (cat. no. BA1054) and anti-mouse (cat. no. BA1050) IgG antibodies were from the Wuhan Boster Biological Technology, Ltd.

The human ADM-resistant leukaemia cell line (K562/ADM cells) and its parental subline (K562 cells) were both provided by the Medical Experimental Center of Lanzhou University. K562/ADM and K562 cells were maintained in RPMI-1640 medium, supplemented with 10% inactivated newborn bovine serum and 2 mmol/l L-glutamine, at 37°C in a cell incubator with 5% CO2. Experiments were performed when the cells reached the mid-log phase.

K562/ADM and K562 cells were seeded at a density of 1×10⁵ cells/ml in 96-well plates. The cells were treated with 0, 2, 4, 8, 16, 20 and 40 µmol/l of HCQ for 24 h at 37°C. MTT solution (10 µl; 5 g/l) was added to each well before the cells were incubated at 37°C for a further 4 h. Then, 100 µl of 10% acidulated sodium dodecyl sulfate (SDS) were added to each well, which was incubated for 12 h at 37°C to dissolve the formazan crystals. Cell proliferation was then detected by MTT colorimetric assay. The optical density (OD) was measured at 570 nm using a Powerwave X plate reader (BioTek Instruments, Inc.). Cell proliferation inhibition rates were calculated using the following formula: Cell proliferation inhibition rate = [(ODcontrol-ODexperiment)/ODcontrol] × 100%. 4 and 16 µmol/l of HCQ were selected as the maximum non-toxic concentration to treat with K562 and K562/ADM cells in the following experiments. Cells were seeded in 96-well plates at a density of 1×10⁵ cells/ml in triplicate and were cultured with ADM or/and HCQ at the indicated concentrations at 37°C for 12, 24 or 48 h. K562 cells were treated with 0, 0.09, 0.18, 0.375, 0.75, 1.5 µmol/l of ADM or pretreated with 4 µmol/l of HCQ for 3 h before exposure to ADM. K562/ADM cells were treated with 0, 1.5, 3, 6, 12, 24 µmol/l of ADM or pretreated with 16 µmol/l of HCQ for 3 h before exposure to ADM, and then MTT assay was executed as above. The half-maximal inhibitory concentration (IC50) was calculated using cell proliferation inhibition rate by SPSS 17.0 software (SPSS, Inc.). All samples were prepared in triplicate.

AO and EB staining have been used to discriminate normal cells from those undergoing apoptosis and death (28). K562/ADM and K562 cells were treated with 35 and 0.75 µmol/l of ADM, respectively, for 24 h or pretreated with HCQ (16 and 4 µmol/l, respectively) for 3 h prior to exposure to ADM. Cells were harvested and washed twice with PBS. After being suspended in 100 µl of PBS, the cells were stained with 5 µl of EB (200 mg/l) and 5 µl of AO (200 mg/l) for 30 min at 37°C in the dark. Subsequently, the cells were rinsed with PBS three times, and a drop of suspension was placed on a glass slide. Cells were observed under a fluorescence microscope (Olympus Corporation) with a blue filter in fields with ×400 magnification.

K562/ADM and K562 cells were treated with 35 and 0.75 µmol/l of ADM, respectively, for 24 h or pretreated with HCQ (16 and 4 µmol/l, respectively) for 3 h prior to exposure to ADM. Cells were fixed with glutaraldehyde at 4°C overnight. The next day, after rinsing with PBS three times for 5 min, cells were fixed with 1% OsO4 at room temperature for 1.5 h. After dehydration, cells were embedded in epoxy resin and then solidified for 12 h at 45°C. After being sliced into 70 nm-thick sections, the cells were stained with 2% uranyl acetate for 30 min and 2% lead citrate for 15 min at 37°C. Finally, the ultrastructure of the cells was observed under a JEM1230 transmission electron microscope in fields with ×8,000 magnification (JEOL, Ltd.).

K562/ADM and K562 cells were treated with 35 and 0.75 µmol/l of ADM, respectively, for 24 h or pretreated with HCQ (16 and 4 µmol/l, respectively) for 3 h prior to exposure to ADM. For the determination of Bax and Bcl-2 levels, 1×10⁶ cells were fixed with 200 µl of an acetone and glutaraldehyde mixture (1:1) for 20 min at 4°C. After rinsing with PBS, each sample was incubated with anti-Bax FITC (1:20, cat. no. DB1010, DB Biotech) or anti-Bcl-2 FITC (1:20, cat. no. DB1011) for 15 min in 100 µl of PBS in the dark at room temperature, and then suspended in 500 µl of PBS. For detection of P-gp expression, cells were collected and washed with PBS, and then incubated with antibodies against P-gp (1:20, cat. no. ab93590, Abcam) for 15 min in the dark. After rinsing with PBS, cells were suspended in 500 µl of PBS. To analyse caspase-3 activity, 1 µl of FITC-DEVD-FMK (1:100, cat. no. QIA911KIT, Sigma-Aldrich; Merck KGaA) was added to cell suspensions in 100 µl of washing buffer. After incubation for 30 min at 37°C, the cells were washed and suspended in 500 µl of washing buffer. After treatment, these samples were analysed using a flow cytometer (Beckman Coulter, Inc.) and analysed using Windows Multiple Document Interface for Flow Cytometry software (version 2.8; The Scripps Research Institute, La Jolla, CA, USA).

K562/ADM and K562 cells were treated with 35 and 0.75 µmol/l of ADM, respectively, for 24 h or pretreated with HCQ (16 and 4 µmol/l, respectively) for 3 h prior to exposure to ADM. A total of 1×10⁶ cells were collected, and then total RNA was extracted from cells with a TRIzol kit according to the manufacturer's protocols. Both the concentrations and purity of the extracted RNA samples were determined by spectrophotometry. Subsequently, 500 ng of each RNA sample was converted into cDNA using a Prime Script RT Master Mix (Takara Bio, Inc.). Temperature protocol: 70°C for 30 min, 37°C for 15 min, 95°C for 5 min. qPCR assays were performed on a Rotor-Gene 3000 quantitative PCR amplifier (Corbett, Australia) with a SYBR Premix Taq II kit (Takara) and primers. The following primers were used: mdr1 forward, 5′-CTCATAATTCCATTAGGACG-3′; mdr1 reverse, 5′-GCTCACGCTACAGGTCCTGT-3′; β-actin forward, 5′-TGCTCCTCCTGAGCGCAAGTA-3′; and β-actin reverse, 5′-CCACATCTGCTGGAAGGTGGA-3′. The conditions included an initial denaturing step at 95°C for 10 sec, then 40 cycles of denaturing at 95°C for 5 sec and annealing at 60°C for 30 sec. The relative expression of each mRNA was calculated by comparison to β-actin mRNA using the 2^−ΔΔCq method (29). All samples were prepared in triplicate.

K562/ADM and K562 cells were treated with 35 and 0.75 µmol/l of ADM, respectively, for 24 h or pretreated with HCQ (16 and 4 µmol/l, respectively) for 3 h prior to exposure to ADM. Cells were lysed in RIPA lysis buffer with phenylmethanesulfonyl fluoride (PMSF; PMSF/RIPA=1:100) for 30 min on ice and then centrifuged at 16,000 × g and 4°C for 15 min. The protein concentration of the supernatant was measured using a bicinchoninic acid protein assay kit. Equal amounts of protein (40 µg) from cell extracts were separated via 10% SDS-PAGE and transferred onto PVDF membranes. After blocking with 5% defatted milk in PBST at 24°C for 1 h, the membranes were incubated with primary antibodies including anti-P62 (1:500), anti-LC3 (1:2,000), anti-Bax (1:1,000), anti-Bcl-2 (1:1,000), anti-cleaved Caspase-3 (1:1,000), anti-P-gp (1:500) and anti-β-actin (1:1,000) at 4°C overnight and then with HRP-conjugated secondary antibodies (1:10,000). Finally, the protein bands of the immunoblot were determined using a chemiluminescent approach in a dark room. Protein bands were visualized using enhanced chemiluminescence reagents (EMD Millipore). Western blots were scanned using an Infrared Imaging System (LI-COR Biosciences) and the bands were quantified using ImageJ software (version 1.45S; National Institutes of Health).

Student's t-test or one-way ANOVA was conducted to compare the differences in continuous data. Bonferroni correction was employed to determine the significance of differences between two groups. All statistical analyses were conducted by using SPSS 17.0 software (SPSS, Inc.). All statistical analyses were two-sided, and the results are all presented as the mean ± the standard deviation of at least three independent experiments. P<0.05 was considered to indicate a statistically significant difference.

To investigate the differences in P-gp levels between K562/ADM and K562 cells, P-gp protein expression levels were detected via flow cytometric analysis. As shown in Fig. 1A, the positive rate and mean fluorescence intensity of P-gp were both markedly elevated in K562/ADM cells compared with K562 cells. Similar results were observed with a western blotting assay (Fig. 1B), which indicated that K562/ADM cells exhibited notably higher P-gp protein expression levels compared with K562 cells. Furthermore, the basic autophagic activities of K562/ADM and K562 cells were measured. After observation of autophagosomes in cells under a transmission electron microscope, more cytosolic contents-packaged autophagic vacuoles were identified in K562/ADM cells compared with K562 cells (Fig. 2A). To further verify the results described above, other autophagic indicators were examined, such as LC3 and P62 (also known as sequestosome-1). As we know, the ratio of LC3-II to LC3-I will become larger when the quantity of autophagosomes increases, and P62, a ubiquitination substrate, has a contrary relationship with autophagic activity (27). As presented in Fig. 2B and C, K562/ADM cells demonstrated a notably increased LC3-II/LC3-I ratio and decreased P62 levels compared with K562 cells, indicating that K562/ADM cells exhibited a higher level of autophagic flux.

Different concentrations of HCQ were used to investigate its cytotoxic effects on K562/ADM and K562 cells using an MTT assay. As presented in Fig. 1C, HCQ inhibited the viability of K562/ADM and K562 cells in a dose-dependent manner. K562 and K562/ADM cells demonstrated almost no cytotoxicity (cell viability >80%) following treatment with 4 and 16 µmol/l of HCQ, respectively, which were selected as the maximum non-toxic concentrations in the following experiments. Then, K562/ADM and K562 cells were treated with different concentrations of ADM alone or following pre-treatment with HCQ (16 and 4 µmol/l, respectively) for 3 h prior to exposure to ADM for 12, 24 or 48 h. It was identified that the IC50 values for ADM in K562/ADM cells following HCQ treatment for 12, 24 and 48 h were 45.66±5.08, 21.44±0.59 and 3.26±0.86 µmol/l, respectively, which were 0.84-, 0.46- and 0.21-fold less than those values after only ADM treatment (Fig. 1D). By contrast, HCQ slightly decreased the IC50 values for ADM in K562 cells (Fig. 1E). These results indicated that HCQ could effectively reverse the ADM resistance of K562/ADM cells.

To investigate the effect of ADM on autophagy, K562/ADM and K562 cells were treated with 35 and 0.75 µmol/l of ADM, respectively, for 24 h or pretreated with HCQ (16 and 4 µmol/l, respectively) for 3 h prior to exposure to ADM. Then, they were observed under a transmission electron microscope for autophagic vacuoles. As presented in Fig. 2A, more autophagic vacuoles (indicated by black arrows) were found in ADM-treated cells than in the controls, and HCQ increased the number of autophagic vacuoles in K562 and K562/ADM cells treated with ADM. To further validate these results, the levels of the autophagy markers LC3 and P62 were analysed using a western blotting assay. It was found that ADM induced autophagy in K562 and K562/ADM cells, as indicated by the increased LC3-II/LC3-I ratios (P=0.001 and 0.023, respectively; Fig. 2B and D) and a decrease in the P62 level (P=0.023 and 0.001, respectively; Fig. 2C and E). Furthermore, HCQ increased the accumulation of LC3-II (P=0.009 and 0.012, respectively) and P62 (P=0.012 and 0.022, respectively) in both K562 and K562/ADM cells exposed to ADM, indicating that HCQ reduced autophagy activity induced by ADM. Collectively, these findings indicated that HCQ can inhibit the ADM-induced autophagy response in K562 and K562/ADM cells by preventing the degradation of autophagic vacuoles.

To explore the apoptosis response in two cell lines following ADM exposure, K562/ADM and K562 cells were treated with 35 and 0.75 µmol/l of ADM separately for 24 h and stained with AO/EB. Observed under a fluorescence microscope, the controls exhibited green-stained integral nuclei, whereas yellow or orange nuclei with condensed and fragmentary chromatin were found in cells treated with ADM, suggesting that ADM induced apoptosis in the two cell lines. Furthermore, when cells were pretreated with HCQ for 3 h prior to exposure to ADM, the proportion of apoptotic K562/ADM cells was notably increased compared with those treated only with ADM; however, the effects of HCQ pre-treatment on the apoptosis of K562 cells were not pronounced (Fig. 3A). Morphological changes indicated that HCQ upregulated ADM-induced apoptosis in K562/ADM.

To confirm these apparent changes in apoptosis, caspase-3 activity was evaluated using flow cytometry, and the expression of the cleaved forms of caspase-3 was detected via western blotting. As presented in Fig. 3B and C, ADM led to an increased mean fluorescence intensity for activated caspase-3 in K562 (P=0.032) and K562/ADM (P=0.001) cells. K562/ADM cells pre-treated with HCQ prior to exposure to ADM exhibited a significantly greater mean fluorescence intensity for activated caspase-3 compared with cells exposed to only ADM (P=0.007), whereas K562 cells were not significantly affected by HCQ pre-treatment. Similar results were also obtained from the western blot assay (Fig. 3D and E).

These results were verified by the determination of the protein expression levels of Bax/Bcl-2. Flow cytometry demonstrated that the ratio of Bax/Bcl-2 was upregulated in K562/ADM cells by ADM (P=0.001), which could be further enhanced following 3 h of HCQ pre-treatment (P=0.001; Fig. 4A-C). However, there were no notable effects on K562 cells. The results of the western blot assay of K562/ADM cells were also consistent with these observations, but the situations of K562 cells were incompatible between the flow cytometry and western blotting results (Fig. 4D and E). The change of K562 cells may be induced by other causes in addition to apoptosis. Collectively, these findings indicate that HCQ could sensitise K562/ADM cells to caspase-dependent apoptosis induced by ADM, thereby reversing multidrug resistance.

To further clarify the role of autophagic activity in multidrug resistance, the present study modulated autophagy and observed alterations in the expression of the drug resistance-associated protein P-gp during the autophagy process. ADM (35 µmol/l) was used to induce the autophagic response, and 16 µmol/l HCQ was used to block autophagy in K562/ADM cells. As demonstrated by a western blot assay, P-gp expression in K562/ADM cells was increased in the presence of ADM (P=0.004), and 3 h HCQ treatment decreased ADM-induced P-gp expression to a level similar to that of control treatment (P=0.011; Fig. 5A and B). A flow cytometric assay validated this observation, as a slight increase in the mean fluorescence intensity for P-gp was found in K562/ADM cells following ADM incubation, which fell with HCQ treatment (Fig. 5C). In addition, the mRNA expression profile of the mdr1 gene in K562/ADM cells following the aforementioned treatments, as determined via RT-qPCR analysis, was consistent with P-gp expression (Fig. 5D). Collectively, these findings indicated that the inhibition of autophagy diminished the expression of P-gp, which may be involved in autophagic regulation in K562/ADM cells.