The human NSCLC cell line A549 was purchased from Shanghai Bioleaf Biotech Co., Ltd. (Shanghai, China). GA of purity >98% was purchased from Shanghai Source Biological Technology Co., Ltd. (Shanghai, China). Primary antibodies against JAK1, STAT3, p-STAT3^Tyr705, Bax, Bcl-2, β-actin and GAPDH, and the secondary antibody anti-rabbit horseradish peroxidase (HRP)-immunoglobulin (Ig) G were acquired from Wuhan Boster Biological Technology Co., Ltd. (Wuhan, China). The antibody against phosphorylated (p)-JAK1^Y1022 was acquired from Elabscience Biotechnology Co., Ltd (Chengdu, China). An MTT Cell Proliferation and Cytotoxicity Assay kit, Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection kit, RPMI-1640 medium, penicillin-streptomycin liquid, trypsin-EDTA solution (0.25%) with phenol red and crystal violet were obtained from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Fetal bovine serum (FBS) was purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

The A549 cell line was maintained in RPMI-1640 medium containing 10% FBS, 100 U/ml penicillin and 0.1 mg/ml streptomycin, in a humidified incubator at 37°C with 5% CO₂. Cells at logarithmic phase were used in the following experiments.

An MTT assay was performed to evaluate the effects of cisplatin (Jiangsu Haosen Pharmaceutical Group, Co., Ltd., Jiangsu, China), GA and their combination on cell viability. A549 cells were seeded in 96-well plates at a density of 2×10⁴ cells/well. The cells were treated at 37°C with GA [varied dose (0–52 µg/ml) of GA for 24 h, or 12, 20 and 28 µg/ml GA for 6, 24 and 48 h], cisplatin [varied dose (0–32 µg/ml) of cisplatin for 24 h] or the two compounds combined (2.5 µg/ml of cisplatin, 28 µg/ml of GA or the two combined for 6, 12, 24 and 48 h) when the cells reached 80% confluency. The medium was removed following 6–24 h of incubation, 10 µl MTT (5 mg/ml) was added to each well, and the cells were incubated for a further 4 h. The medium of each well was then removed and replaced by 110 µl dimethylsulfoxide at the end of the incubation. Finally, the absorbance of each well at 492 nm was measured with a spectrophotometer (Thermo Fisher Scientific, Inc.) and cell viability was evaluated by analyzing the absorbance of each group.

A549 cells were seeded into 6-well plates at a density of 2×10⁵/well and divided into the following groups: i) Control group (Control), treated with normal medium; ii) GA group (GA), treated with 12–28 µg/ml GA; iii) cisplatin group (Pt 2.5), treated with 2.5 µg/ml cisplatin; and iv) GA + cisplatin group (Pt 2.5 + GA28), treated with 2.5 µg/ml cisplatin + 28 µg/ml GA. Cells from each group were treated at 37°C for 6 and 24 h, respectively. Cells were collected via centrifuged at 300 × g at 37°C for 5 min and washed 3 times with cold phosphate-buffered saline (PBS) and then suspended in binding buffer. The cells were then stained with Annexin V-FITC according to the instructions of the Apoptosis Detection kit (Beijing Solarbio Science & Technology Co., Ltd.). Finally, a flow cytometer (Sysmex Partec Gmbh, Görlitz, Germany) was used to determine the percentage of apoptotic cells in each group.

A549 cells were cultured in 96-well plates and treated as aforementioned for 24 h when cells reached a confluence of 70–80%. The medium was removed at the end of the treatment period and cells were washed with cold PBS and fixed in 4% paraformaldehyde for 30 min at room temperature. The cells were then washed again with cold PBS and stained at room temperature with crystal violet for a further 2 min. Finally, the cells were washed with PBS and dried naturally prior to being observed under an inverted phase contrast microscope (Olympus Corporation, Tokyo, Japan).

Cells were treated with GA, cisplatin or the two compounds combined for 24 h, as aforementioned. The total proteins of cells in each group were then extracted using a Total Protein Extraction kit (Nanjing KeyGen Biotech, Co., Ltd. Nanjing, China) according to the manufacturer's instructions. The concentration of proteins was quantified with a BCA Protein Assay kit (Beijing Solarbio Science & Technology Co., Ltd.). The proteins were then mixed with sample buffer, separated on 10% SDS-polyacrylamide gels and transferred onto PVDF membranes. The membranes were blocked at room temperature for 2 h with 5% dried skimmed milk or 5% bovine serum albumin (Beijing Solarbio Science & Technology Co., Ltd.) dissolved in TBST (containing 0.02% Tween-20) and incubated with the primary antibodies at 4°C overnight: JAK1 (cat. no. BA1808), p-JAK1 (cat. no. ENP0154), STAT3 (cat. no. BA0621), p-STAT3 (cat. no. P00007), Bax (cat. no. BA0315), Bcl-2 (cat. no. BA0412), β-actin (cat. no. BM0627) or GAPDH (cat. no. BA2913) at the dilution of 1:1,000-1:5,000. Subsequently, the membranes were washed 3 times with TBST, incubated with anti-rabbit IgG secondary antibody conjugated with HRP (1:5,000; cat. no. BA1056) for 1 h at room temperature and then washed 3 times with TBST. Finally, the signals indicating expression levels of target proteins were detected using Chemiluminescent HRP Substrate (EMD Millipore, Billerica, MA, USA) and ImageJ 1.8.0 software (National Institutes of Health, Bethesda, MD, USA).

Cells were cultured in 24-well plates on sterile glass coverslips placed in each well and treated as aforementioned. The cells were then fixed at 4°C with 4% paraformaldehyde for 30 min and washed twice with PBS. Triton X-100 solution (0.1%) was used to disrupt the cytomembrane, then cells were blocked in 10% normal goat serum (Beijing Solarbio Science & Technology Co., Ltd.) at room temperature for 1 h, and incubated with p-STAT3 (dilution, 1:1,000; cat. no. P00007) at 4°C overnight. Subsequently, the cells were incubated with biotin-labeled secondary antibody (dilution, 1:64; cat. no. BA1090; Wuhan Boster Biological Technology Co., Ltd.) for 40 min at 37°C. Finally, nuclei were stained with DAPI at room temperature for 1 min and the slides were observed with a fluorescent microscope (Olympus Corporation; magnification, ×200).

The total RNA of cells treated with the various doses of GA (12–28 µg/ml) were isolated with TRIzol reagent (Thermo Fisher Scientific, Inc.), and then a NanoDrop ND-1000 spectrophotometer was used to measure the concentration and purity of RNA samples. Subsequently, 2 µg total RNA was reverse transcribed (37°C for 15 min and 85°C for 5 sec) into cDNA using a SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen; Thermo Fisher Scientific, Inc.). PCR was performed with a 25 µl reaction mixture including 2 µl cDNA. RT-qPCR was performed using SYBR Premix Ex Taq II supplied (Takara Bio, Inc., Otsu, Japan). Primers used for the RT-qPCR were as follows: GAPDH forward, 5′-ACTTTGGTATCGTGGAAGGACTCAT-3′ and reverse, 5′-GTTTTTCTAGACGGCAGGTCAGG-3′; Bax forward, 5′-TTTTGCTTCAGGGTTTCATCCA-3′ and reverse, 5′-TGCCACTCGGAAAAAGACCTC-3′; Bcl-2 forward, 5′-ATCGCCTGTGGATGACTGA-3′ and reverse, 5′-GAGACAGCCAGGAGAAATCAAAC-3′; STAT3 forward, 5′-ACCAAGCGAGGACTGAGCATC-3′ and reverse, 5′-CAGCCAGACCCAGAAGGAGAA-3′; and JAK1 forward, 5′-ACCAGGATGCGGATAAATAATG-3′ and reverse, 5′-GTTTCCAAGGTAGCCAAGTATTT-3′. qPCR amplification was performed in two steps: An initial step at 95°C for 30 sec, and then 40 cycles of 95°C for 5 sec and 60°C for 30 sec. Finally, the expression levels of target mRNAs were calculated according to 2^−ΔΔCq method (15).

All quantitative data were presented as the mean ± standard deviation, and statistical analysis was performed with SPSS 19.0 (IBM Corp., Armonk, NY, USA). One-way analysis of variance followed by the Least Significant Difference post hoc test was applied to analyze the differences among groups. P<0.05 was considered to indicate a statistically significant difference.

A549 cells were treated with GA (0–52 µg/ml) for 24 h and cell viability was detected by MTT assay. It was observed that GA decreased cell viability in a dose-dependent manner (Fig. 1A). Specifically, 12 µg/ml GA significantly decreased cell viability when compared with that of control cells (P<0.05); the greater concentration of 28 µg/ml in turn caused a greater inhibition of cell viability when compared with 12 µg/ml GA (P<0.05); cell viability was <10% when A549 cells were treated with GA at a dose of 36 µg/ml GA; and cell viability was further inhibited when cells were treated with 52 µg/ml GA. Based on these findings, 12–28 µg/ml GA was adopted for subsequent studies. It was further identified that the viability of cells was significantly inhibited by GA in dose- and time-dependent manners (Fig. 1B).

To investigate the influence of GA on apoptosis, A549 cells were treated with 12, 20 or 28 µg/ml GA for 24 h and the number of apoptotic cells was calculated by flow cytometry. The results demonstrated that 12 µg/ml GA significantly increased the percentage of early and total apoptotic cells when compared with the control group following 24 h of incubation (P<0.05). Notably, treatment with 28 µg/ml GA led to a more significant increase in apoptosis in A549 cells compared with 12 µg/ml GA (P<0.01), which indicated that GA induced A549 cell apoptosis in a dose-dependent manner (Fig. 2).

To further ascertain the antitumor effects of GA in A549 cells, the expression of Bax and Bcl-2 in cells from each group was measured by RT-qPCR and western blot analysis. The results revealed that GA upregulated Bax and downregulated Bcl-2 at the gene and protein levels (Fig. 3). Specifically, 12 µg/ml GA enhanced Bax protein (Fig. 3A) and gene (Fig. 3C) expression relative to the levels in control cells (P<0.05); and increased concentrations of GA (20 and 28 µg/ml) further enhanced Bax expression at the gene and protein levels when compared with 12 µg/ml GA (P<0.05). By contrast, 12 µg/ml GA decreased the expression of Bcl-2 protein (Fig. 3B) and mRNA (Fig. 3D) compared with control treatment (P<0.05), and further inhibition of Bcl-2 expression was observed when A549 cells were treated with 20 or 28 µg/ml GA (P<0.05 vs. 12 µg/ml GA; Fig. 3B and D).

It is widely accepted that the JAK/STAT3 signaling pathway is involved in various biological processes, including cell proliferation, survival and development (14). To determine whether the JAK/STAT3 signaling pathway was associated with the anticancer effects of GA, the expression of JAK1 and STAT3 was examined in A549 cells from each group by RT-qPCR and western blotting. The results revealed that 12 µg/ml GA reduced the levels of p-JAK1^Y1022 and p-STAT3^Tyr705 when compared with control treatment (P<0.05); greater reductions in p-JAK1^Y1022 and p-STAT3^Tyr705 expression were observed when cells were treated with 20 or 28 µg/ml GA (P<0.05 vs. 12 µg/ml; Fig. 4A and B), which indicated that GA reduced p-JAK1^Y1022 and p-STAT3^Tyr705 in a dose-dependent manner. By contrast, varied doses of GA exerted little influence on the gene expression of JAK1 and STAT3 (Fig. 4C and D), which indicated that GA interfered with the phosphorylation of JAK1 and STAT3, rather than expression.

To confirm the optimum concentration of cisplatin for subsequent investigations, an MTT assay was performed. As presented in Fig. 1C, treatment with cisplatin (0–32 µg/ml) for 24 h significantly decreased the viability of A549 cells in a dose-dependent manner (P<0.05). It was determined that the half-maximal inhibitory concentration of cisplatin was between 2 and 4 µg/ml, and thus 2.5 µg/ml cisplatin was adopted for subsequent assays.

To evaluate the effects of combined treatment with GA and cisplatin (Pt 2.5 + GA28 group), cells were treated with the established doses of GA and/or cisplatin for 6–48 h and evaluated by MTT assay. The results demonstrated that GA, cisplatin and their combined treatment decreased cell viability in a time-dependent manner (P<0.01). Furthermore, cotreatment with GA markedly strengthened the effects of cisplatin at different time points (P<0.05; Fig. 1D).

To confirm whether GA influenced the stimulatory effects of cisplatin on apoptosis, A549 cells were treated with GA, cisplatin or the two combined for 6 or 24 h, and cell apoptosis was measured by cytometry. The results indicated that cisplatin treatment for 6 or 24 h increased the apoptosis of A549 cells by varying extents (P<0.05 vs. Control), while GA significantly increased the percentage of apoptotic cells at 24 h but not at 6 h following incubation (P<0.05 vs. Control at 24 h). Notably, no combined effect was observed when A549 cells were treated with cisplatin and GA for 6 h, which may be due to 28 µg/ml GA not exhibiting any apoptosis-inducing effects on A549 cells following 6 h of incubation. However, combined treatment with GA and cisplatin induced a significant increase in apoptosis when compared with single treatment with either of the two agents following 24 h of incubation (P<0.01 vs. single treatments), which indicated that GA increased the apoptosis-inducing effects of cisplatin on A549 cells (Fig. 5C and D).

The morphological changes of cells from different groups were observed by crystal violet staining assay (Fig. 6). Cellular structure was intact in the control group, while cells exhibited apparent apoptotic changes such as cell shrinkage, nuclear chromatin condensation and fragmentation when treated with GA, cisplatin, or a combination of the two agents for 24 h. Additionally, cells in the experimental groups exhibited an evident decrease in the number of cells; notably, combined treatment with GA and cisplatin together lead to the greatest decrease in cell number. These results revealed that GA or cisplatin treatment alone resulted in morphological changes in A549 cells, though their combination could lead to more evident changes.

To determine whether GA could enhance the effects of cisplatin on the regulation of the JAK/STAT3 signaling pathway, several major molecules associated with apoptosis, anti-apoptosis and proliferation in cells were examined by western blot and immunofluorescent staining assays. The results demonstrated that GA or cisplatin treatment alone increased Bax protein expression and decreased Bcl-2 protein expression (P<0.05 vs. Control); while combined treatment with the two agents significantly enhanced these effects on the expression of Bax and Bcl-2 (P<0.05 vs. single treatments; Fig. 7A and B).

The expression of the JAK/STAT3 signaling pathway in cells treated with GA, cisplatin or the two agents combined was also evaluated. The results demonstrated that the levels of p-STAT3^Tyr705 and p-JAK1^Y1022 were significantly decreased in cells treated with GA or cisplatin alone (P<0.05 vs. Control); however, more marked decreases were observed in the combined treatment group (P<0.05 vs. single treatments; Fig. 7C and D). Additionally, the results of immunofluorescent staining revealed that GA, cisplatin or the combination of the two agents suppressed the phosphorylation of STAT3 and the translocation of p-STAT3 from the cytoplasm to the nucleus (Fig. 8), which was consistent with the results of western blotting. Notably, these results also revealed that combined treatment with GA and cisplatin lead to markedly stronger suppression of the phosphorylation of STAT3 and translocation of p-STAT3.