Pris, Cis and 3-methyladenine (3-MA; an autophagy inhibitor) were obtained from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). LY294002, a phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) inhibitor were obtained from MedChem Express (Monmouth Junction, NJ, USA). The primary antibodies against microtubule-associated protein 1A/1B-light chain 3 (LC3B; cat. no. 4108; 1:2,000), beclin-1 (cat. no. 3738; 1:1,000), cyclin D1 (cat. no. 2922; 1:1,000), phosphorylated (p-)AKT (cat. no. 4058; 1:2,000), AKT (cat. no. 9272; 1:3,000), glycogen synthase kinase 3β (GSK-3β; cat. no. 12456; 1:1,000), p-GSK3β (Ser9; cat. no. 5558; 1:2,000), phosphatase and tensin homolog (PTEN; cat. no. 9559; 1:1,000), β-actin (cat. no. 4967; 1:4,000) and poly (ADP-ribose) polymerase (PARP; cat. no. 9542; 1:1,000) were purchased Cell Signaling Technology, Inc. (Danvers, MA, USA). The antibody against p21 (cat. no. 195720; 1:1,000) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

The A549 and NCI-H446 human lung carcinoma cell lines were purchased from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). The cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C with 5% CO₂.

The microRNA (miR)-23a inhibitor (5′-GGA AAU CCC UGG CAA UGU GAU-3′) and miR-23a negative control (NC, 5′-CAG UAC UUU UGU GUA GUA CAA-3′) were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). The A549 and NCI-H446 cells were seeded at 2×10⁵ cells/ml in 6-well plates and transfected with miR-23a inhibitor and miR-23a-NC using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.

Cell viability was detected using the Cell Counting kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). The A549 and NCI-H446 cells were seeded into 96-well plates at a density of 1×10⁴ cells/well and cultured for 24 h. The cells were treated with indicated concentrations of Pris (0, 10, 20, 40, 60 and 80 µM) and Cis (0, 0.1, 0.25, 0.5, 1 and 2 µM) for 24 h. A total of 10 µl of CCK-8 solution was then added to each well for an additional 4 h at 37°C. The absorbance at 450 nm was determined using an ELISA Reader (Tecan Group Ltd., Männedorf, Switzerland). This experiment was performed in triplicate.

The A549 and NCI-H446 cells were seeded at 2×10⁵ cells/ml in 6-well plates and treated with Pris and Cis for 24 h. The cell cycle phase distribution was detected using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and a Cell Cycle Detection kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). In brief, the cells were trypsinized and fixed with 75% ethanol overnight at 4°C. Propidium iodide (PI) was used to stain the DNA of samples for 15 min, and flow cytometry was used to determine cell cycle stage. All experiments were performed at least three times. Data was analyzed with ModFit LT 3.0 (Verity Software House, Topsham, ME, USA).

The A549 and NCI-H446 cells were seeded at 2×10⁵ cells/ml in 6-well plates and treated with Pris and Cis for 24 h. Cell apoptosis was assessed using an Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection kit (Nanjing KeyGen Biotech Co., Ltd.). Briefly, the cells were stained with Annexin V-FITC and PI for 15 min at room temperature in the dark. The apoptotic cells were then detected on a FACScalibur flow cytometer (BD Biosciences) and analyzed with FlowJo 7.6 software (FlowJo LLC, Ashland, OR, USA). Three independent experiments were performed.

Total RNA was extracted from the A549 and NCI-H446 cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA (2 µg) from the cell samples was reverse transcribed using the Mir-X™ miRNA First-Strand Synthesis kit (Takara Bio, Inc., Otsu, Japan) according to the manufacturer's protocol. The expression of miR-23a and U6 (Takara Bio, Inc.) was determined using Power SYBR Green PCR Master mix (2X; Applied Biosystems; Thermo Fisher Scientific, Inc.) on an ABI 7500 system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The forward primer of miR-23a was used (5′-ATC ACA TTG CCA GGG ATT TCC-3′). The primers of U6 were obtained from the Mir-X™ miRNA First-Strand Synthesis kit, and U6 was used as a control for normalization. The thermocycling conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 10 sec, 60°C for 20 sec and 72°C for 10 sec. The relative level of miR-23a was calculated using the 2^−ΔΔCq method (19).

Male BALB/c nude mice (8 weeks old; 18-20 g) were obtained from the Animal Experiment Center of Xi'an Jiaotong University (Xi'an, China). All mice were housed in a specific pathogen free (SPF) animal at 20-26°C and 40-70% humidity with a 12 h light/dark cycle. Food and water were available ad libitum. The experiments were approved by the Laboratory Animal Care Committee of Xi'an Jiaotong University (approval no. XJTULAC2018-527). The xenograft tumor model was performed as previously described (20-22). Briefly, A549 cells (5×10⁶ cells/ml in 0.2 ml) were subcutaneously injected into the right flanks of BALB/c nude mice. The mice were divided into four groups (n=3 per group): Saline control group, Pris (0.8 mg/kg) treatment group, Cis (2 mg/kg) treatment group, and combined Pris + Cis treatment group. The xenograft tumors were developed for 14 days post-injection. Following this, the nude mice were treated with Pris (0.8 mg/kg) and Cis (2 mg/kg) for 14 days. Tumor volume was calculated as follows: Tumor volume (mm³) = long diameter of the tumor × short diameter of the tumor²/2. On the last day of the experiment (day 28), the tumor samples were collected and weighed. Hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL) assays were used to examine morphology and cell apoptosis in the xenografted lung tumors. Images were captured using a Olympus BX53 microscope (Olympus Corporation, Tokyo, Japan).

Following treatment with Pris and Cis, the cells were lysed in lysis buffer for western blot detection, as described previously (23). Briefly, the cell samples were lysed on ice with radioimmunoprecipitation assay buffer containing protease inhibitors and the proteins were quantified with Bicinchoninic Acid Protein Assay kit (Thermo Fisher Scientific, Inc.). Proteins (40 µg/well) were separated on 6-12% gels using SDS-PAGE and protein was transferred onto nitrocellulose membranes (Pall Life Sciences, Port Washington, NY, USA) and the membranes were blocked with 5% nonfat milk for 2 h, followed by incubation with anti-cyclin D1, anti-P21, anti-beclin1, anti-LC3B, anti-p-AKT anti-AKT, anti-PTEN, anti-p-GSK3β, anti-GSK-3β, anti-PARP and anti-β-actin antibodies at 4°C overnight. Membranes were then incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (cat. no. 7076; 1:20,000) and HRP-conjugated anti-rabbit IgG (cat. no. 7074; 1:20,000; both Cell Signaling Technology, Inc.) secondary antibodies for 2 h at 25°C. The proteins were visualized using SuperSignal West Pico Chemiluminescent Substrate (Pierce; Thermo Fisher Scientific, Inc.) and exposed to X-ray film. Densitometry was performed using ImageJ version 1.38× software (National Institutes of Health, Bethesda, MD, USA) and the resulting data analyzed using GraphPad Prism 6.0 (GraphPad Software, Inc., La Jolla, CA, USA).

All data are presented as the mean ± standard deviation. Data were analyzed using GraphPad Prism 6.0 (GraphPad Software, Inc., La Jolla, CA, USA). Differences between groups were determined by one-way analysis of variance followed by Dunnett's or Tukey's post hoc tests. P<0.05 was considered to indicate a statistically significant difference.

To investigate the effects of Pris and Cis on cell proliferation, the A549 and NCI-H446 cells were exposed to different concentrations of Pris or Cis for 24 h, and cell viability was investigated using a CCK-8 assay. As shown in Fig. 1A and B, Pris or Cis significantly inhibited the growth of A549 and NCI-H446 cells in a concentration-dependent manner. According to these results, experiments were performed to investigate whether the combined treatment of Pris + Cis significantly promoted the inhibition of A549 and NCI-H446 cell viability in comparison with Pris or Cis treatment alone. As shown in Fig. 1C, the combination treatment significantly enhanced the inhibitory effect on cell viability compared with Pris or Cis treatment alone in the A549 and NCI-H446 cell lines. These results indicated that Pris may significantly increase the sensitivity to Cis by inhibiting cell viability in A549 and NCI-H446 cells.

It is well known that cell cycle arrest may lead to cell growth inhibition. Therefore, the effect of Pris and Cis on cell cycle arrest in A549 and NCI-H446 cells was detected using flow cytometry. As shown in Fig. 2A and B, the percentage of cells at G₀/G₁ phase significantly increased with Pris treatment and the percentage of cells at S phase significantly increased with Cis treatment in A549 and NCI-H446 cells. Furthermore, it was observed that the combination treatment significantly elevated the percentage of cells in the G₀/G₁ phase compared with Cis treatment alone in the A549 and NCI-H446 cells (Fig. 2A and B). These data indicated that G₀/G₁ phase arrest may contribute to enhancing cell growth inhibition induced by Cis.

The G₀/G₁-related proteins were examined by western blotting. As shown in Fig. 2C, Pris or Cis treatment alone markedly upregulated the expression level of p21 but downregulated the expression of cyclin D1 compared with the control group. The combination treatment markedly upregulated the expression level of p21 but downregulated the expression of cyclin D1 compared with either Pris or Cis treatment alone in A549 and NCI-H446 cells. These results indicated that Pris increased the antiproliferative effect of Cis in A549 and NCI-H446 cells via upregulating p21 and downregulating cyclin D1.

To further evaluate the effects of Pris and Cis on cell growth inhibition, cell apoptosis was detected in A549 and NCI-H446 cells using flow cytometry. As shown in Fig. 3A and B, Pris, Cis and the combination treatment significantly induced cell apoptosis in the A549 and NCI-H446 cells. Combination treatment with Pris and Cis significantly increased the number of apoptotic cells compared with either drug alone in the A549 and NCI-H446 cells (Fig. 3A and B). To further evaluate the effect of apoptosis induced by Pris and Cis, the apoptosis-related protein PARP was analyzed using western blotting. As shown in Fig. 3C, Pris, Cis and the combination treatment markedly upregulated the expression level of cleaved PARP. Therefore, these results showed that Pris markedly enhanced Cis-induced apoptosis in the A549 and NCI-H446 cells.

To further elucidate the molecular mechanism of Pris and Cis-induced cell apoptosis in A549 and NCI-H446 cells, the expression of proteins related to the Akt/GSK3 signaling pathway were detected in A549 and NCI-H446 cells using western blotting. As shown in Fig. 4A, the levels of p-Akt and p-GSK3β were markedly inhibited by Pris, Cis and the combination treatments. Furthermore, the combination treatment of Pris + Cis markedly inhibited the phosphorylation of Akt and GSK3β compared with either drug alone in the A549 and NCI-H446 cells (Fig. 4A). LY294002, a type of PI3K inhibitor, markedly inhibited the levels of p-GSK3β and p-Akt in A549 and NCI-H446 cells (Fig. 4B). In addition, as shown in Fig. 4C, LY294002 in combination with Cis enhanced the inhibitory effect on cell viability compared with Cis alone in the A549 and NCI-H446 cell lines. These results indicated that Pris enhanced the sensitivity of A549 and NCI-H446 cells to Cis via suppressing Akt/GSK3β signaling.

miR-23a has been indicated to be as an important regulator of PTEN/Akt signaling (24,25). Therefore, the present study investigated whether miR-23a contributed to the sensitivity of A549 and NCI-H446 cells to Cis via the PTEN/Akt signaling pathway. As shown in Fig. 5A, Pris significantly downregulated the expression level of miR-23a in the A549 and NCI-H446 cells. To further examine the effect of miR-23a on the PTEN/Akt signaling pathway, the protein expression of GSK3β, Akt and PTEN in A549 and NCI-H446 cells were examined using western blotting. As shown in Fig. 5B, the miR-23a inhibitor markedly upregulated the expression level of PTEN and downregulated the phosphorylation of Akt and GSK3β compared with that in the blank control cells. In addition, the results of the flow cytometry revealed that combination treatment with miR-23a inhibitor and Cis significantly increased the number of apoptotic A549 and NCI-H446 cells compared with the Cis treatment alone (Fig. 5C and D). These results indicated that Pris enhanced the sensitivity of A549 and NCI-H446 cells to Cis via suppressing the miR-23a/PTEN/Akt signaling pathway.

Numerous studies have demonstrated that autophagy is associated with the regulation of cell apoptosis induced by antitumor drugs (26-28). To evaluate whether autophagy is involved in increasing the apoptosis of Cis-treated A549 and NCI-H446 cells, the protein expression levels of LC3B and beclin-1 were detected by western blotting. As shown in Fig. 6A, the expression levels of LC3BII and beclin-1 were markedly downregulated by Pris treatment with the blank control cells. In addition, flow cytometry revealed that combination treatment with 3-MA (an autophagy inhibitor) and Cis significantly increased the number of apoptotic cells compared with Cis treatment alone in the A549 and NCI-H446 cells (Fig. 6B and C). These results indicated that Pris enhanced the sensitivity of A549 and NCI-H446 cells to Cis via suppressing autophagy.

To evaluate the synergistic effect of Pris and Cis, an in vivo xenograft model was established. A549 cells were injected into BALB/c nude mice. The xenograft tumors were developed for 14 days post-injection and the nude mice were then treated with Pris (0.8 mg/kg) and Cis (2 mg/kg) for a further 14 days. As shown in Fig. 7A-D, the tumor volumes and weights in the Pris treatment group, Cis treatment group and combination treatment group were lower compared with those in the control group. Furthermore, combination treatment significantly attenuated tumor volume and weight compared with either drug alone. However, no significant changes in body weight were observed among the four experimental groups (Fig. 7E). The H&E staining and TUNEL analysis showed that apoptotic cells in the tumor tissues were markedly increased following Pris and Cis combination treatment compared with treatment with either drug alone (Fig. 7F). In addition, western blotting revealed that combination treatment with Pris and Cis markedly inhibited the phosphorylation of Akt and GSK3β compared with treatment with either drug alone in A549 tumor tissues (Fig. 7G-I). Taken together, the results suggested that Pris and Cis acted synergistically against lung cancer in vivo.