The A549 cell line was obtained from The Chinese Academy of Sciences Cell Bank. RPMI-1640 medium was purchased from GE Healthcare Life Sciences (Hyclone; Logan, UT, USA) and fetal bovine serum (FBS) was from Gemini Bio-Products, Inc. PQ and trypsin were purchased from Merck KGaA (Sigma-Aldrich), heparin and thapsigargin were obtained from Beijing Solarbio Science and Technology Co., Ltd. (Beijing, China), while Hoechst 33258 stain was from Beyotime Institute of Biotechnology (Shanghai, China). The Annexin V-FITC/PI kit was purchased from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan), Calcium detection kit and the Caspase activity kit was from Nanjing Keygen Biotech Co., Ltd. (Nanjing, China). Antibodies against GRP78 (cat. no. 66574-1-Ig), CHOP (cat. no. 15204-1-AP) and β-actin (cat. no. HRP-60008) were purchased from ProteinTech Group, Inc. Horseradish peroxidase-conjugated goat anti-rabbit (cat. no. ab6721) goat anti-mouse immunoglobulin G secondary antibodies (cat. no. ab97040) and ECL Substrate kit (cat. no. ab133406) were purchased from Abcam.

A549 cells were cultured in RPMI-1640 culture solution with 10% FBS in an incubator with 5% CO₂ at 37°C. The culture solution was changed every other day. At 70–80% confluence, cells were divided into six experimental groups, as follows: i) Control group, exposed to equal volume of PBS; ii) heparin group, preprocessed with heparin (200 µg/ml) for 2 h; iii) thapsigargin group, preprocessed with thapsigargin (4 µM) for 2 h; iv) PQ group, exposed to PQ (200 M); v) heparin + PQ group, preprocessed with heparin (200 µg/ml) for 2 h, followed by PQ (200 µM) treatment; vi) thapsigargin + PQ group, preprocessed with thapsigargin (4 µM) for 2 h, followed by PQ (200 µM) treatment. Cells in each group were cultured for 48 h, and a number of relevant indexes were then tested.

A total of 1×10⁵ cells/ml were seeded onto a 96-well plate, with each well containing 100 µl of solution, and cultured for 24 h. Next, the cells were treated accordingly in the different groups and continuously cultured for 48 h after treatment. A total of 20 µl MTT solution (5 mg/ml) was added into each well and mixed, and the cells were continuously cultured for 4 h in an incubator with 5% CO₂ at 37°C. Subsequently, the supernatant was discarded, and 150 µl DMSO was added to each well. The cells were shaken in the dark for 10 min to dissolve the formazan crystals. A microplate reader at 490 nm was used to determine the optical density (OD) of each well.

The culture solution was discarded after 48 h of incubation, and the cells were collected. Paraformaldehyde (4%) was added to fix for 10 min at room temperature. The fixation solution was then discarded, and the cells were washed three times with PBS. Next, Hoechst 33258 stain was added for 5 min and then discarded after washing three times with PBS. After mounting with an anti-fluorescence quenching agent the cell nuclei were observed under a microscope.

A549 cells were seeded onto a 6-well plate at a density of 1×10⁶ cells/well. When cells reached 70% confluence, they were processed accordingly in the different groups, and continuously cultured for 48 h. After the 48 h culture, cells in each group were collected, washed three times with PBS, and then incubated with a mixture containing 5 µl Annexin V-FITC and 5 µl PI for 15 min at room temperature in the dark. A flow cytometer was subsequently used for the detection of apoptosis rate.

Cells collected from each group were placed into culture solutions with 1.25 µM Fluo-3 AM probe of the Calcium Detection Kit (Nanjing Keygen Biotech Co., Ltd.) and incubated for 30 min at 37°C in the dark. Subsequent to incubation, the cells were washed three times with PBS and resuspended in 300 µl PBS in a flow tube. A flow cytometer was then used for the detection of Ca²⁺ concentration.

Cells collected from each group were washed three times with PBS and lysed with a lysis buffer consisting of dichloroacetic acid (0.1%), PMSF (1 mM), protease inhibitor cocktail (10 µM), Na₃PO₄ (1 mM), Triton X-100 (1%), NaCl (150 mM), Tris-HCl (10 mM), EDTA (1 mM) and EGTA (1 mM). Next, centrifugation was performed for 30 min at 14,000 × g at 4°C, and the supernatant was collected and transferred into a new EP tube. The BCA method was then used to measure the protein concentration. Subsequently, 30 µg of the sample was loaded and separated by 12% SDS-PAGE at 4°C at 70 V for 60 min. The protein was then transferred to a PVDF membrane and incubated for 2 h at room temperature with blocking solution that consisted of 5% nonfat milk powder dissolved in Tris-buffered saline/Tween-20 (TBST). Antibodies targeting GRP78 (1:500), CHOP (1:1,000) and β-actin (1:1,000) were added, and the membrane was fixed overnight at 4°C and maintained at room temperature for 2 h. Subsequent to washing three times with TBST, secondary antibodies (1:5,000) were added to the PVDF membrane, maintained at room temperature for 2 h and washed three times with TBST. An ECL kit was used to visualize the protein bands. Finally, the image was developed and fixed, while ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to perform gray analysis.

Cells collected from each group were washed three times with PBS. Lysis solution was added in moderation for 2 h on ice, and centrifugation was performed for 1 min at 11,000 × g at 4°C. The supernatant was collected and transferred into a new EP tube, and the BCA method was used to measure the protein concentration. Next, 50 µl of 2X buffer solution and 5 µl caspase substrate was added to 50 µl cell lysis product (containing 150 µg of protein), and incubated for 4 h at room temperature in the dark. The OD at 405 nm was measured using a microplate reader.

SPSS software (version 20.0; IBM Corporation, Armonk, NY, USA) was used to perform the statistical analysis. The data in each group are expressed as the mean ± standard deviation. Intergroup differences were assessed by one-way analysis of variance. If equal variance was assumed, Dunnett's test was used for comparisons between the two groups, whereas if unequal variance was assumed, Dunnett's T3 test was used. P<0.05 was considered to indicate a statistically significant difference.

Compared with the control group, there were no evident changes in the heparin and thapsigargin groups, which indicated that the concentrations of heparin and thapsigargin used in the present study exerted no cytotoxicity (Fig. 1). However, the cell activity in the PQ group was significantly decreased, which indicated that the concentration of PQ used in the present study had a strong toxic effect on A549 cells. Compared with the PQ group, the cell activity was significantly increased in the heparin + PQ group, while cell activity was significantly decreased in the thapsigargin + PQ group. These results suggested that the inhibitionof ER Ca²⁺ release was able to alleviate the cytotoxicity exerted by PQ, while the inhibition of ER Ca²⁺ absorption resulted in aggravation of cytotoxicity (Fig. 1). Taken together, these findings indicated that the cytotoxicity of PQ is correlated with the Ca²⁺ signal.

Compared with the control group, there were no evident changes in the nuclear form in the heparin and thapsigargin groups (Fig. 2). By contrast, characteristic changes, such as karyopyknosis, karyorrhexis and nuclear apoptosis, appeared at different degrees in the PQ, heparin + PQ and thapsigargin + PQ groups. Compared with the PQ group, fewer changes were observed in the nuclear form in the heparin + PQ group, while the nuclear form changes in the thapsigargin + PQ group were more prominent (Fig. 2). These findings indicated that Ca²⁺ signal had an effect on the A549 cell apoptosis induced by PQ.

Compared with the control group, there were no evident changes in the heparin and thapsigargin groups, which indicated that the concentrations of heparin and thapsigargin used in the experiments of the present study had no significant cytotoxic effect. However, in the PQ group, the cell apoptosis rate was significantly enhanced, which suggested that the concentration of PQ used in the present study was able to strongly induce A549 cell apoptosis (Fig. 3). In order to determine whether the Ca²⁺ signals of ER affect the rate of cell apoptosis induced by PQ, the apoptosis rate was measured in PQ-treated cells that were preprocessed with thapsigargin or heparin. As presented in Fig. 3, the cells apoptosis rates in the PQ, heparin + PQ and thapsigargin + PQ groups were increased by different levels compared with the control group. When compared with the PQ group, the cell apoptosis rate was significantly decreased in the heparin + PQ group, but was significantly increased in the thapsigargin + PQ group. These results further revealed that the Ca²⁺ signal of ER participated in the A549 cell apoptosis induced by PQ.

Ca²⁺ is known to be an important signal transduction factor in eukaryotic cells, and maintaining its homeostasis serves an important role in the normal physiological activity of cells (10). Ca²⁺ overload in cells is usually an early manifestation of cell apoptosis and death. The Ca²⁺ influx from outside the cell, the activities of Ca²⁺ stores in the cells, and changes in Ca²⁺ spatial distribution may cause Ca²⁺ overload in cells (11). In order to investigate the influence of PQ on Ca²⁺ concentration in cells, a Fluo-3 AM fluorescent probe was used to detect the changes in Ca²⁺ concentration. The results revealed that, compared with the control group, the Ca²⁺ concentration in cells treated with PQ was significantly increased, indicating that PQ induced Ca²⁺ overload in cells (Fig. 4). Heparin, an inhibitor of IP3R in the ER, and thapsigargin, an inhibitor of sarco-ER Ca²⁺-ATPases in the ER, were used to determine the association between Ca²⁺ overload in the cells and Ca²⁺ in the ER. The results indicated that, compared with the PQ group, Ca²⁺ fluorescence intensity was significantly decreased in the heparin + PQ group, indicating that heparin prevented the release of Ca²⁺ in the ER and significantly reversed the increase in Ca²⁺ concentration induced by PQ. By contrast, a marked increase in Ca²⁺ fluorescence intensity was observed in the thapsigargin + PQ group as compared with the PQ group (Fig. 4). These aforementioned results suggested that Ca²⁺ overload in the cells existed during the A549 cell apoptosis induced by PQ, and that the ER served an important role in the imbalance of Ca²⁺ and outflow of Ca²⁺.

In order to determine whether PQ induced ERS in A549 cells, the expression levels of two relevant proteins, GRP78 and CHOP, were determined. The results demonstrated that, compared with the control group, there were no evident changes in the heparin and thapsigargin groups, whereas the levels of the two proteins were significantly increased in the PQ group (Fig. 5). This suggested that PQ significantly induced ERS during the induction of A549 cell apoptosis. Compared with the PQ group, the expression levels of GRP78 and CHOP were significantly decreased in the heparin + PQ group and significantly increased in the thapsigargin + PQ group (Fig. 5). These results indicated that the intervention of Ca²⁺ release and absorption in the ER influenced the ERS.

caspase-7 and caspase-12 are members of the Caspase protein family, and are defined as the executive proteins in cell apoptosis. The activation of caspase-7 and caspase-12 is necessary for the ERS-induced cell apoptosis. In the present study, the expression levels of caspase-7 and caspase-12 in each group was detected to determine whether PQ was able to promote Ca²⁺ release and reduce Ca²⁺ absorption, and influence and trigger A549 cell apoptosis mediated by ERS. As shown in the Fig. 6, the activities of caspase-7 and caspase-12 were increased by different degrees in the PQ, heparin + PQ and thapsigargin + PQ groups as compared with the control group. However, compared with the PQ group, the activities of caspase-7 and caspase-12 were significantly decreased in the heparin + PQ group, but significantly increased in the thapsigargin + PQ group (Fig. 6). These results revealed that intervention of Ca²⁺ signals in the ER has an effect on A549 cell apoptosis mediated by the ERS induced by PQ.