The present study was approved by the ethics committee of Lanzhou University Second Hospital (Lanzhou, China). Leukemia cells were derived from routine blood samples collected from patients at the Third Affiliated Hospital of Xiangya (Changsha, Hunan, China). All patients provided written informed consent. The patients all underwent clinical hemograms and myelograms, and were diagnosed with leukemia (17). Peripheral blood (white blood cells >2×10¹⁰/l; 3–5 ml) or bone marrow fluid (1–1.5 ml) samples were extracted from the patients. The inner wall of a needle tube was wetted with 0.2 ml of aseptic heparin sodium anticoagulant (2 g/l) prior to being used to take bone marrow (or peripheral blood). Mononuclear cells were isolated using Hypaque-Ficoll solution. After being washed three times with serum-free RPMI 1640 (11875093; Thermo Fisher Scientific, Inc., Waltham, MA, USA), the cells were suspended in Iscove's modified Dulbecco's media (12440046; Thermo Fisher Scientific, Inc.) to obtain a 2×10⁷/ml cell suspension. Subsequently T and B lymphocytes were separated, nylon cotton was used to remove the T cells and a leukemia cell suspension was obtained. The survival rate of mononuclear cells was >95%. The obtained cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (11320082; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum, 100 µg/ml streptomycin and 100 U/ml penicillin in a 37° incubator containing 5% CO₂. Once cell density reached 85%, the cells were digested with 0.5% trypsin and 0.02% EDTA and were eluted with PBS. After 3 days, the medium was changed and subculture was performed at 1:3-1:6. The leukemia cells used in the subsequent experiments were in the logarithmic growth phase.

The cells in logarithmic growth phase were inoculated into a 96-well plate at 2×10⁵ cells/ml and 100 µl fresh DMEM was added to each well for overnight culture. Following treatment of human leukemia cells with 25, 50, 100, 200 or 500 ng/ml tunicamycin (Sigma-Aldrich; Merck Millipore KGaA, Darmstadt, Germany) for 24, 48, 72 and 96 h, various indicators were detected to determine the optimal concentration and duration of tunicamycin treatment. In the subsequent experiments, leukemia cells were divided into the following three groups: The ER activation group, in which cells were treated with the optimal concentration of tunicamycin (100 ng/ml tunicamycin for 72 h); the ER activation + TO901317 group, in which cells were treated with the optimal concentration of tunicamycin + 10 µmol/l PI3K activator TO901317; and the control group, which consisted of untreated cells.

The cells in logarithmic growth phase were inoculated into a 96-well plate at 2×10⁵ cells/ml and 100 µl fresh DMEM was added to each well for overnight culture. Following the removal of the medium, 100 µl fresh medium and 20 µl 0.5% MTT solution were added to each well for 3 h at 37°C. Subsequently, the medium was removed and 100 µl dimethyl sulfoxide was added to each well, and the plate was oscillated at a low speed for 15 min to fully dissolve the crystals. Finally, an enzyme-linked immunometric meter was used to measure the optical density value of each well at 490 nm; three duplicated wells were set up for each group. The MTT assay was used to measure and calculate cell proliferation in each group.

The leukemia cells were inoculated into a 96-well plate and 100 µl fresh DMEM was added to each well. The cells were incubated at 37°C in an atmosphere containing 5% CO₂ and saturated humidity for 24 h. The cells were collected and the cell concentration adjusted to 10⁶ cells/ml. They were washed twice with PBS. Subsequently, the cells were incubated with 0.05 mM MDC at 37°C for 1 h and were observed under an inverted fluorescence microscope (Leica DMI 4000B; Leica Microsystems GmbH, Wetzlar, Germany). The fluorescence intensity was calculated as follows: Fluorescence intensity (%)=(fluorescence intensity of treatment groups-control group)/control group ×100.

The leukemia cells were inoculated into a 96-well plate and 100 µl fresh DMEM was added to each well. The cells were incubated at 37°C in an atmosphere containing 5% CO₂ and saturated humidity for 24 h. The cells were collected and the cell concentration adjusted to 10⁶ cells/ml. They were washed twice with PBS and centrifuged at 1,000 × g for 3 min. Subsequently, 500 µl binding buffer was added and mixed gently, following which 5 µl Annexin V-FITC and 5 µl PI were added (K201-100; BioVision, Inc., Milpitas, CA, USA). The cells were incubated in the dark for 10 min and flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA) and CellQuest Pro software version 5.1 (BD Biosciences) were used to detect cell apoptotic rate. The results were analyzed as follows: The lower left quadrant consisted of normal cells, the lower right quadrant consisted of early apoptotic cells, the upper right quadrant consisted of late apoptotic cells and the upper left quadrant consisted of dead cells.

Leukemia cells were treated with the optimal concentration of tunicamycin and were then inoculated into a 6-well plate at 1.5×10⁶ cells/well for 24 h. Subsequently, the cells were washed three times with ice-cold PBS and protein was extracted from the cleaved cells using RIPA cell lysis solution (BB-3209; BestBio, Co., Shanghai, China). Protein concentration was measured using a Bradford kit (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The proteins (20–30 µg) were separated by 10% SDS-PAGE and were then transferred to a nitrocellulose membrane. The membrane was washed with Tris-buffered saline containing 0.1% Tween (TBST) and was blocked with 5% skim milk powder at room temperature for 1 h. Subsequently, the membrane was incubated overnight at 4°C with the following primary antibodies: Rabbit polyclonal antibodies: Anti-caspase-3 (ab13847; 1:500; Abcam, Cambridge, UK), anti-mTOR (ab2732; 1:2,000; Abcam), anti-AKT (ab8805; 1:500; Abcam), anti-phosphorylated (p)-protein kinase R-like endoplasmic reticulum kinase (PERK; E19-7579-1; 1:1,000, EnoGene Biotech Co., Ltd., New York, NY, USA), anti-p-α subunit of eukaryotic initiation factor 2 (elF2α; ab227593; 1:1,000; Abcam), anti-microtubule-associated protein 1A/1B-light chain 3 (LC3; ab128025; 1:2,000; Abcam) and anti-78-kDa glucose-regulated protein (GRP78; ab21685; 1:1,000; Abcam), rabbit antibody Anti-PI3K (ab40776; 1:2,000; Abcam). Following washing with TBST, the membrane was incubated with a secondary antibody sheep anti-rabbit IgG (Cell Signaling Technology, Inc., Danvers, MA, USA) for 1 h at 37°C. Finally, the membrane was visualized using electrochemiluminescence (ECL) (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) for 3 min. Then the membrane was put into the Gel imaging system instrument (Bio-Rad Laboratories, Inc., Hercules, CA, USA), and 400 µl ECL hypersensitive luminescence (Beijing Solarbio Science & Technology Co., Ltd.) added and developed for 2 min. The relative expression of the target protein=gray value of target protein band/gray value of internal reference band (β-actin). The experiment was repeated 3 times).

All experiments were repeated at least three times under the same conditions. Statistical analyses were conducted using SPSS 21.0 (IBM Corp., Armonk, NY, USA). Measurement data were expressed as the mean ± standard deviation, enumeration data were expressed as a percentage. Differences between two groups were analyzed using Student's t-test, and differences among multiple groups were analyzed by one-way analysis of variance (ANOVA) and LSD analysis. Cell growth conditions following treatment with various concentrations for various time points were assessed by repeated measures ANOVA. P<0.05 was considered to indicate a statistically significant different.

The results of an MTT assay and flow cytometry demonstrated that cell survival rate was decreased as tunicamycin concentration and treatment duration increased. Specifically, when cells were treated with 100 ng/ml tunicamycin for 72 h, the survival rate of human leukemia cells was 57.68±3.12%. This concentration and duration of tunicamycin treatment was used in subsequent experiments (Fig. 1A).

After the leukemia cells were treated with 100 ng/ml tunicamycin for 72 h, the results of a western blot analysis demonstrated that the expression levels of p-PERK, p-eIF2α and the ER molecular chaperone GRP78 were significantly increased compared with in cells prior to treatment (P<0.05; Fig. 1B). These findings indicated that ER activation was induced in response to treatment with 100 ng/ml tunicamycin for 72 h.

Following treatment of the leukemia cells with 100 ng/ml tunicamycin for 72 h, the results of a western blot analysis demonstrated that compared with in the control group, the expression levels of key proteins in the PI3K/AKT/mTOR signaling pathway exhibited no significant difference in the ER activation + TO901317 group (P>0.05). However, compared with in the control and ER activation + TO901317 groups, the expression levels of mTOR, AKT and PI3K were significantly decreased in the ER activation group (P<0.05; Fig. 2), which indicated that ERS may inhibit the PI3K/AKT/mTOR signaling pathway.

Observation under a microscope demonstrated that, following treatment of leukemia cells with 100 ng/ml tunicamycin for 72 h, cells in the ER activation and ER activation + TO901317 groups exhibited shrinkage and cracking, and the number of cells markedly decreased while no obvious difference between the ER activation + TO901317 group and control group was observed (Fig. 3A). The results of an MTT assay also revealed that compared with in the control group, cell viability was significantly inhibited in the ER activation group (P<0.05). Although cell viability was decreased in the ER activation + TO901317 group, there was no significant difference compared with in the control group (P>0.05; Fig. 3B), which suggested that activation of PI3K may reduce the inhibitory effects of ERS on the viability of human leukemia cells.

Increased MDC fluorescence intensity indicates that cell autophagy is increased. Compared with in the control group, MDC intensity in the ER activation + TO901317 group was slightly increased; however, there was no significant difference (P>0.05). MDC fluorescence intensity in was significantly increased the ER activation group (P<0.05; Fig. 4A). The formation of autophagosomes is positively associated with the expression of LC3-II. The results of a western blot analysis demonstrated that compared with in the control and ER activation + TO901317 groups, the expression levels of LC3-II were significantly increased in the ER activation group (P<0.05; Fig. 4B), which indicated that PI3K activation inhibited the occurrence of autophagy, suggesting that ERS-induced autophagy of leukemia cells may be caused by inhibiting the PI3K/AKT/mTOR signaling pathway.

The results of a flow cytometric analysis demonstrated that compared with in the control group, the apoptotic rates of the other two groups were increased; the apoptotic rate in the ER activation group was significantly increased (P<0.05; Fig. 5A). The results of a western blot analysis indicated that in the ER activation group, the apoptosis-associated key factor caspase-3 was activated and the expression levels of CHOP were significantly increased compared with in the control group (P<0.05), thus suggesting that apoptosis occurred in this cell group. No significant differences were observed in the expression of caspase-3 and CHOP between the ER activation + TO901317 group and the control group (P>0.05; Fig. 5B), thus suggesting that activation of PI3K could inhibit the apoptosis of human leukemia cells. In addition, these results indicated that ERS may induce apoptosis of human leukemia cells by inhibiting the PI3K/AKT/mTOR signaling pathway.