Bone marrow samples of three patients with chemoresistant leukemia from The Third Xiangya Hospital of Central South University (Hunan, China) were collected once written informed consent was obtained (Table I). Primary refractory or resistant disease was defined as not achieving complete remission (CR; i.e., a remaining blast count of ≥5% following 1 to 2 cycles of intense induction therapy) (17) The characteristics of patients with leukemia are presented in Table I. The present study was approved by the Central South University, and the approved protocol was in accordance with the Declaration of Helsinki. Mononuclear cells were isolated using a lymphocyte separation medium (Mediatech, Manassas, VA, USA) (18).

The K562/ADR and K562 human leukemic cell lines were a generous gift from the Cell Center of Xiangya School of Medicine (Changsha, Hunan, China). K562 cell lines were grown in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (both Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). K562/ADR cells were grown in culture medium containing 1 µM ADR (Nanjing KeyGen Biotech, Co., Ltd., Nanjing, China) to maintain drug resistance. Cells were grown at 37°C in an atmosphere of 95% O₂ and 5% CO₂.

The stock solution of Olaparib (Selleck Chemicals, Houston, TX, USA) was prepared by dissolving 10 mg of the drug in 50 µl of dimethyl sulfoxide (Amresco, LLC, Solon, OH, USA). Then it was diluted with RPMI to a concentration of 5 µmol. The aliquots were stored at −80°C. For each experiment a new aliquot was thawed and used.

Cell viability was measured using Cell Counting kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) according to the manufacturer's instructions. Cells were seeded at 1×10⁵ cells/ml in duplicate for each time point (24 h, 48 h and 72 h) in a 96-well culture plate. K562 and K562/ADR cells were exposed to 0, 2, 4, 8, 16 and 32 µmol/l ADR at 37°C for 24, 48 and 72 h respectively. K562/ADR cells at 1×10⁵ cells/ml in a 96-well culture plate were exposed to 0, 1.25, 2.5, 5 and 10 µmol/l Olaparib at 37°C for 72 h. Then 10 µl CCK-8 solution was added to each well and plates were incubated at 37°C for 4 h. The absorbance at 570 nm was then measured using a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). All experiments were performed in triplicate, and three independent experiments were conducted.

A Total RNA Extractor (Omega Bio-Tek, Inc., Norcross, GA, USA) and RT-PCR kit (Toyobo Life Science, Osaka, Japan) were used for cDNA synthesis (25°C for 10 min and 42°C for 50 min, followed by 70°C for 15 min). SYBR-Green reagents (Toyobo Life Science, Osaka, Japan) were used to determine PARP1 and γ-H2A histone family member X (H2AX) expression. The thermocycling conditions consisted of the following: Initial denaturation for 2 min 30 sec at 95°C, followed by 40 cycles of denaturation for 30 sec at 95°C, annealing for 30 sec at 58°C and extension for 30 sec at 72°C, then a final extension at 72°C for 10 min. The gene expression level was normalized using the endogenous control gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). RT-qPCR reactions were performed using a Master Cycler Ep Realplex (Eppendorf, Hamburg, Germany). Relative expression was evaluated using the 2^−ΔΔCq method (17). All PCR assays were performed three times. The primers for the individual genes were as follows: PARP1 forward, 5′-TACCATCCAGGCTGCTTTGTCA-3′ and reverse, 5′-CTTCGCCACTTCATCCACTCCA-3′; γ-H2AX forward, 5′-GGCCTCCAGTTCCCAGTG-3′ and reverse, 5′-TCAGCGGTGAGGTACTCCAG-3′; and GAPDH forward, 5′-GACTCATGACCACAGTCCATGC-3′ and reverse, 5′-AGAGGCAGGGATGATGTTCTG-3′.

Cells (5×10⁵/ml) were exposed to 2 µM ADR, 5 µM Olaparib, or a combination of the two drugs at 37°C for 3 days. Subsequently, they were collected by centrifugation (30,000 × g for 5 min at 4°C), washed with cold PBS, and finally lysed with a cell lysis buffer (Cell Signaling Technology, Inc., Danvers, MA, USA). The protein concentrations were determined using a BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). Equal amounts of protein lysates were then mixed with SDS-PAGE protein loading buffer, boiled for 5 min, and then run on 10% SDS-PAGE. Proteins were then transferred onto polyvinylidene fluoride membranes, which were blocked with 5% skimmed milk powder in Tris-buffered saline with 0.1% Tween 20 (Beijing Solarbio Science & Technology Co., Ltd.) for 2 h at 4°C. Membranes were incubated with the following primary antibodies at 4°C overnight: PARP1 (1:1,000; cat. no. YT595; Immunoway Biotechnology Company, Plano, TX, USA); γ-H2AX (1:1,000; cat. no. pSer139; Novus Biologicals, LLC, Littleton, CO, USA); caspase 3 (1:1,000; cat. no. ab32351), cleaved-PARP (1:1,000; cat. no. ab4830; both Abcam, Cambridge, UK); cyclinB1 (1:1,000; cat. no. 4138); cleaved-caspase 3 (1:2,000; cat. no. 1177; both CST, Biological Reagents Co., Ltd., Shanghai, China); and β-actin (1:3,000; cat. no. 20536-1-AP; ProteinTech Group, Inc., Chicago, IL, USA). The next morning, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse or rabbit secondary antibody (cat. no. sc-2031; 1:10,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 2 h at room temperature. Following washing with PBS (Gibco; Thermo Fisher Scientific, Inc.) at room temperature for 5 minute, bands were captured with a Gel imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The relative expression of target proteins was calculated with ImageJ version 2× (National Institutes of Health, Bethesda, MD, USA) and normalized to β-actin.

For survival assays, stimulated K562/ADR cells were collected, centrifuged (10,000 × g for 5 min at 4°C), suspended in a fresh complete medium (time 0 on the graphics), and seeded at 5×10⁵ cells/ml in each well of 6-well culture plates. K562/ADR cells were divided into four treatment groups: i) a control group (medium only); ii) 2 µM ADR; iii) 5 µM Olaparib; iv) and a combination group of the two drugs at 37°C for 3 days. Cells were counted via a trypan blue dye exclusion assay in quadruplicate, once the cells were dispersed, 10 µl of cell suspension was transferred to a micro centrifuge tube,10 µl of trypan blue (Beijing Solarbio Science & Technology Co., Ltd.; C0040) was then added and mixed gently with a pipette; 10 µl of cell/trypan blue suspension was then loaded into a cell counting chamber and placed under an inverted microscope (Nikon Corporation, Tokyo, Japan) for counting. The assays were performed 3 days post-drug exposure. All of the experiments were performed three times each, and all three experiments were conducted independently.

Cell apoptosis was analyzed using an Annexin V/propidium iodide (PI) kit (Nanjing KeyGen Biotech, Co., Ltd.) according to the manufacturer's protocol. Cells were collected and analyzed on a FACS Canto II (BD Biosciences, San Jose, CA, USA) flow cytometer. To determine cell cycle distribution, 8×10⁴ K562/ADR cells were seeded in 6 cm dishes for a total of 3 days following lentiviral infection. Following the 3-day culture, the cells were washed twice with ice-cold PBS and were re-suspended in a PBS (ZSGB-BIO; OriGene Technologies, Inc., Beijing, China) containing 50 µg/ml RNaseA (Nanjing KeyGen Biotech, Co., Ltd.) and 50 µg/ml PI (Nanjing KeyGen Biotech, Co., Ltd.). Cells were incubated at 37°C in the dark for 1 h. The percentage of cells in each phase of the cell cycle was measured using FACScan (BD Biosciences) and subsequently the results were analyzed using ModFit software, version 3.2 (Verity Software House, Inc., Topsham, ME, USA).

Following a total of 3 days, the treatment groups were subjected to cell cycle analysis. Following centrifugation at 300 × g for 5 min at room temperature, cells (1×10⁶) were fixed with 70% ethanol on ice for 2 h, followed by centrifugation for 5 min at 300 × g at 4°C. Subsequently, 0.05 mg/ml PI (Nanjing KeyGen Biotech, Co., Ltd.) and 0.1 mg/ml RNAse A (Nanjing KeyGen Biotech, Co., Ltd.) were added to the samples at room temperature in the dark for 30 min. Cells were examined using a BD FACSCalibur flow cytometer (BD Biosciences) and Cell Quest software version 3.3 (BD Biosciences). The experiments were repeated three times.

The alkaline comet assay was used to detect ADR-induced DNA damage in K562/ADR cells. A total of 1×10⁵ cells were treated with either 2 µM ADR, 5 µM Olaparib, or a combination of the two drugs for at 37°C for 3 days. Then, cells were washed in PBS and resuspended in ice-cold PBS. A total of ~10 µl of the re-suspended cells were mixed with 65 µl of a low melting point agarose at 4°C. The slides were placed at 4°C in the dark until gelling occurred, at which point they were immersed in a pre-chilled lysis buffer at 4°C. Following a 1 h incubation period, the buffer was aspirated and replaced with pre-chilled alkaline solution for 90–120 min at 4°C. Following lysis and DNA double strand unwinding into a single strand, the slides were placed in a horizontal electrophoresis tank filled with freshly prepared alkaline electrophoresis buffer. Electrophoresis was run for 30 min at 25 V and 300 mA. Following this, the slides were transferred to a neutralizing buffer for 10 min, and then aspirated. This was repeated three times. Thereafter, the slides were allowed to air dry, and 50 µl/well of 5 mg/ml PI was added to each slide for 5 min in the dark at room temperature for DNA staining. DNA migration was observed using a fluorescence microscope (Olympus Corporation, Tokyo, Japan). For each sample, 200 cells were selected at random and were analyzed using CASP software version 1.2.2 (Comet Assay Software Project, Beijing, China).

To determine the potential clinical significance of the cell line studies, the present study isolated mononuclear cells from patients with chemoresistant leukemia, Each sample cells were divided into two parts; one part at 1×10⁵ cells/ml were seeded in duplicate in a 96-well culture plate. The cells were exposed to 0, 2, 4, 8, 16 and 32 µmol/l ADR at 37°C for 3 days. Then 10 µl CCK-8 solution was added to each well and plates were incubated at 37°C for 4 h. The absorbance at 570 nm was then measured using a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). The other part of the sample was exposed to 2 µM ADR, 5 µM Olaparib, or a combination of the two drugs at 37°C for 3 days. Subsequently, the cells were collected by centrifugation (30,000 × g for 5 min at 4°C), washed with cold PBS, and finally lysed with a cell lysis buffer (Cell Signaling Technology, Inc.). Western blotting, as above was then performed to analyze the levels of the selected proteins.

Statistical analyses was performed using GraphPad Prism 6.02 version software (GraphPad Software, Inc., La Jolla, CA, USA). The data were expressed as the mean ± standard deviation (SD), and statistical comparisons were made using the two-tailed Student's t-test and one-way analysis of variance followed by Fisher's Least Significant Difference for multiple comparisons. All of the experiments were repeated at least three times as independent experiments in order to be effective. P<0.05 was considered to indicate a statistically significant difference.

To investigate the sensitivity of K562/ADR and K562 cells to chemotherapeutic drugs, the two cell lines were treated with a gradually increasing concentration of ADR at different times as indicated. The CCK-8 assay revealed that ADR inhibited the growth of K562/ADR and K562 cells to varying degrees. ADR-induced cytotoxicity was dose- and time-dependent in the two cell lines (Fig. 1). The half-maximal inhibitory concentration (IC₅₀) values of ADR were calculated for K562 and K62/ADR cells. The IC₅₀ values of ADR for K562 cells were 1.50±0.03, 0.82±0.09 and 0.40±0.05 µmol/l at 24, 48 and 72 h, respectively. In addition, the corresponding values for the K562/ADR cells were 39.51±0.64, 28.93±0.74 and 24.31±0.78 µmol/l, respectively (Fig. 1D). The degree of ADR-resistance in K562/ADR cells was 26.34–60.70 fold greater when compared with K562 cells, which suggests that K562/ADR cells may have greater potential for resistance to ADR compared with K562 cells.

In the present study, 2 µM ADR and 5 µM Olaparib were selected for use in the experiments for the following reasons: i) ADR at 2 µM consistently enhanced toxicity in the K562 cell line, but not in the K562/ADR cell line (Fig. 1); and ii) Olaparib alone did not exhibit a cytotoxic effect on the viability of K562/ADR cells at doses up to 5 µM as determined by CCK-8 assays (Fig. 2A). The proliferation capability of K562/ADR cells was inhibited and cell growth was slower in the group treated with ADR+Olaparib for 3 days (Fig. 2B), which was significantly different compared with the other control group. This indicated that the cells treated with ADR+Olaparib had reduced proliferation rates when compared with cells treated with Olaparib alone, or in the untreated control cell group (P<0.01; Fig. 2B).

The percentage of apoptotic cells was measured by flow cytometry using Annexin V/PI staining. Notably, there was a significantly increased population of apoptotic cells in the combined treatment group (11.23±0.64%) when compared with the untreated control group (3.72±0.24%; P<0.001), the ADR group (4.30±0.31%; P<0.001) and the Olaparib group (4.42±0.66%; P<0.001). Therefore, combined treatment with Olaparib significantly improved the apoptotic percentage of K562/ADR cells (Fig. 2C and D).

To investigate whether cell cycle arrest contributed to growth inhibition, flow cytometry analysis was performed. Treatment with ADR+Olaparib significantly increased the percentage of K562/ADR cells in the G2/M phase. The percentage of cells at the G2/M phase were 38.23±0.86 for the control cells, 41.81±1.94 for the 2 µmol ADR-treated K562/ADR cells, 41.97±1.86 for the 5 µmol Olaparib-treated K562/ADR cells and 50.93±1.53 for the ADR+Olaparib-treated K562/ADR cells (Fig. 2E and F). K562/ADR cells treated with ADR+Olaparib had a greater number of G₂/M-phase cells than the cells treated with ADR alone, Olaparib alone or the untreated control cells (P<0.005, P<0.01 and P<0.005, respectively). Cyclin B1 was markedly downregulated when the K562/ADR cells were treated with ADR+Olaparib (Fig. 2G). Cyclin B1 is the regulatory subunit of Cyclin-dependent kinase 1, and a reduction in the expression of cyclin B1 can arrest cells in the G2/M phase of the cell cycle, triggering cell death (19). These results indicate that re-sensitization of ADR resistant leukemia cells may be partly mediated by cell cycle arrest at the G2/M phase (Fig. 2E and F).

The alkaline comet assay was conducted to investigate the genotoxic potential of DNA damage. Olaparib enhanced DNA damage in ADR treated cells as shown by the prevalence of distinct comet tails (Fig. 3A). The addition of Olaparib enhanced the average tail DNA by ~15-fold when compared with the control group (P<0.001; Fig. 3B). In addition, the tail moment was enhanced by ~150-fold P<0.01; Fig. 3C).

The expression of γ-H2AX was significantly increased in cells treated with Olaparib alone compared with the control cells (Fig. 4). In addition, it was revealed that PARP1 was significantly downregulated and cleaved-PARP, caspase 3 and cleaved caspase 3 were markedly upregulated in cells following Olaparib+ADR treatment, compared with the control group (Fig. 4C and D).

To determine the potential clinical significance of the aforementioned cell line studies, mononuclear cells were isolated from the bone marrow of patients with chemoresistant leukemia (their clinic pathological features are summarized in Table I). These cells were exposed to ADR, to test the degree of ADR resistance (Fig. 5A), as well as individual drugs or a combination of the two. Increased levels of γ-H2AX were observed in cells from the three leukemia patients exposed to ADR+Olaparib, which indicated that the combined treatment activated a DNA damage response. Cleaved PARP, caspase 3 and cleaved caspase 3 were markedly upregulated in cells exposed to ADR+Olaparib treatment, which indicated that apoptosis was activated (Fig. 5B). These results revealed that there was drug synergism in cells derived from patients with leukemia, involving mechanisms analogous to those observed in the cultured cell lines.