TP (>98% purity) was purchased from Dalian Meilun Co., Ltd. Human recombinant IL-6 (cat. no. 200-06; Peprotech, Inc.) was resolved and stored according to the manufacturer's instructions. 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and protease inhibitor cocktail (cat. no. 04693132001) were purchased from Sigma-Aldrich (Merck KGaA). Antibodies against anti-cleaved caspase-3 (cat. no. GB11009) were purchased from Servicebio. Antibodies against BCL-2 (cat. no. 182858) and MMP-9 (cat. no. 76003) were purchased from Abcam, MCL-1 (cat. no. 94296), cyclin D1 (cat. no. 2978S), C-myc (cat. no. 5605S), STAT3 (cat. no. 4904) and p-STAT3 (cat. no. 9145) were acquired from Cell Signaling Technology Inc., and antibodies against β-actin (cat. no. BB0710) were from Biossci Biotechnology Co., Ltd. DAPI, Triton X-100 and an anti-fade mounting medium were purchased from Servicebio. The Annexin V/PI Apoptosis Detection kit (cat. no. KGA105-KGA108) was from Nanjing KeyGen Biotech Co., Ltd. Propidium iodide (PI) and the JC-1 Staining kit were acquired from Beyotime Institute of Biotechnology. DyLight 488-conjugated goat anti-rabbit IgG antibody (cat. no. A24221) was from Abbkine Scientific Co., Ltd.

Human lung adenocarcinoma PC9 cells sensitive to epidermal growth factor receptor (EGFR) with low expression of IL-6 and human alveolar basal epithelial adenocarcinoma A549 cells with higher expression of IL-6 were provided by the Caner Laboratory, Tongji Medical College (Wuhan, China). The cells were cultured in RPMI-1640 medium (HyClone; GE Healthcare Life Sciences) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in a humidified incubator with 5% CO₂ and 95% air.

Cells were seeded in 96-well plates at a density of 5×10³ cells per well and cultured overnight. A series of concentrations ranging to 500 nM TP were added into the medium. After either 24 or 48 h, MTT was added to assay cell viability. Absorbance at 570 nm was recorded on a microplate reader (Synergy 2; BioTek Instruments, Inc.). The inhibition ratio was calculated as 1-optical density (OD)treated/ODcontrol. The IC₅₀ values were calculated by plotting the drug concentration versus the percent cell viability of the cells after TP intervention for 24 and 48 h.

At 3×10⁵ cells per well were exposed to TP in 12 wells for 24 h after overnight serum starvation. Thereafter, the cells were washed with PBS and fixed with 70% ethanol at 4°C overnight. Then, cell cycle analyses were performed through flow cytometry according to the manufacturer's instructions (Beyotime Institute of Biotechnology). Apoptosis was analyzed via pre-treatment with a selected concentration of TP for 24 h (PC9 cells) or 48 h (A549 cells) and then was detected with the Annexin V/PI Staining kit (Nanjing KeyGen Biotech Co., Ltd.) and assayed by flow cytometry (BD FACS Calibur; BD Biosciences). Cells undergoing early-stage apoptosis were categorized as Annexin V⁺/PI⁻, whereas Annexin V⁺/PI⁺ cells were assumed to be at a late stage of apoptosis. Subsequently, mitochondrial trans-membrane potential ΔΨ) was determined according to the manufacturer's instructions using the JC-1 Apoptosis Detection kit (Beyotime Institute of Biotechnology). Data analysis was conducted using the FlowJo software (oxs 10.6; FlowJo LLC).

Transwell migration assays were performed using 24-well Transwell inserts (8 μm pore size; Costar; Corning, Inc.), as described previously (23). A total of 1×10⁵ cells were pre-treated with TP for 24 h, then harvested and resuspended in RPMI-1640 (HyClone; GE Healthcare Life Sciences) at a concentration of 10⁵ cells/ml. Next, 200 μl of the cell suspension mixed with 750 μl RPMI-1640 medium supplemented with 10% FBS was added to the upper chamber, and incubated for another 24 h at 37°C. Non-migratory cells were gently wiped from the top surface of the membrane with a cotton swab. Migratory cells on the bottom side of the membrane were rinsed with PBS, fixed in 4% paraformaldehyde and stained with 0.1% (m/v) crystal violet for 10 min at room temperature. Images were captured under a ZEISS digital microscope (magnification ×100; Carl Zeiss AG). The migration rate was quantified by counting the stained cells in selected 3-5 fields of view.

Cells were treated with or without TP for 24 h, then cells were washed twice with ice-cold PBS and lysed in RIPA buffer containing a protease inhibitor cocktail (Thermo Fisher Scientific, Inc.). Soluble proteins were collected after centrifugation at 12,000 x g for 15 min at 4°C. Protein concentration was determined by the bicinchoninic acid (BCA) method. Subsequently, 50 μg of each protein sample was separated by SDS-PAGE on a 10-12% gel (120 V, 90 min) and transferred to a PVDF membrane (Bio-Rad Laboratories, Inc.) at 280 mA for 90 min. After blocking with 5% non-fat milk for 1 h at room temperature, the membranes were incubated with specific primary antibodies (1:1,000) at 4°C overnight. After three washes with TBS containing 0.1% Tween 20, the membranes were incubated with fluorescently-labeled secondary antibodies (1:20,000) for 1 h at room temperature. The bands were visualized using a near-infrared fluorescence imaging system (Odyssey; LI-COR Biosciences). Band densities were quantified in the ImageJ software (version 1.52 National Institutes of Health). In addition, cells were pretreated with TP or homoharringtonine (HHT) for 24 h, then 50 nM IL-6 (cat. no. 200-06; PeproTech, Inc.) was added for another 30 min, finally cells were harvested and lysed for western blotting.

Cells (10⁵ cells/well) were seeded on glass cover slides. After treatment with 60 nM TP or 4 μM STAT3 inhibitor for 24 h, the cells were fixed with 4% buffered formalin for 30 min at room temperature, permeabilized with 0.5% Triton X-100, and blocked with 10% goat serum in PBS (Servicebio) for 30 min at room temperature. After successive incubation with the primary antibody (1:100) at 4°C for 12 h, a secondary antibody (DyLight 488-conjugated goat anti-rabbit IgG; 1:8,000) was incubated for another 1 h at room temperature, at last cells were stained with DAPI (1:500) in the dark for 5 min. The fluorescence signals were captured using a fluorescence microscope (Nikon Eclipse CI; Nikon Corporation).

All experiments were conducted in triplicate and repeated three times. Representative results are shown as the mean ± SD and were plotted using Prism 6.0 (GraphPad Software, Inc.). Statistical calculations were performed via one-way ANOVA followed by the Student-Newman-Keuls test. P<0.05 was considered to indicate a statistically significant difference.

To evaluate the cytotoxic action of TP, PC9 and A549 cells were treated with a series of TP concentrations ranging from 0 to 500 nM for either 24 or 48 h. The IC₅₀ was calculated using log formula. The IC₅₀ of PC9 and was 1,238±243.8 nM and the IC₅₀ of A549 was 1,339±150.2 nM. After 48 h of treatment with TP, the corresponding values were 29.33±2.102 and 135.9±0.9328 nM. As shown in Fig. 1A, TP was cytotoxic after 48 h treatment even at a low concentrations (P<0.05). TP inhibited proliferation (according to an inhibition ratio) of PC9 and A549 cells at 48 h in a dose-dependent manner. During the 24 h treatment, TP at concentrations <15.625 nM was not cytotoxic at all toward PC9 and A549 cells. The inhibition of proliferation started to be noticeable via a sharp slope in the inhibition ratio (15.625-62.500 nM in PC9 cells and 15.625-125.000 nM in A549 cells) (24) followed by a relatively flat plateau at high TP concentrations. Therefore, these comparatively low doses were selected to conduct subsequent experiments.

Whether TP influences cell viability by affecting the cell cycle was examined (Fig. 1B). On the basis of the inhibition ratio, two concentrations within the sharp-slope region (30 and 60 nM) and one concentration in the plateau region (90 nM, PC-9; 120 nM, A549) were selected. As shown in Fig. 1C, TP at the concentration in the sharp-slope region (30 and 60 nM) strongly promoted the G1/S arrest in PC9 cells. There was a slight increase in the number of cells in the S phase at 90 nM TP as well, but the change was not statistically significant. Similar results were not seen in corresponding experiments on A549 cells after treatment with TP for 24 h. A slight decrease in the number of cells in the S phase at 120 nM TP was observed, though it appeared that TP in general did not affect the cell cycle in A549 cells (Fig. 1C).

It was subsequently analyzed whether TP treatment has any impact on apoptosis. Starting from the concentration of 60 nM, treatment of PC9 cells with TP for 24 h caused a significant increase in total apoptosis (Fig. 2A and B). In contrast, after 48 h treatment of A549 cells with TP, total apoptosis increased strongly, and this change was attributed to a late-apoptosis population (Fig. 2A and B). Then, ΔΨ was measured as disruption of mitochondria is regarded as a distinctive feature of the intrinsic pathway of apoptosis. As demonstrated by the JC-1 blue ratio, treatment with TP for 24 h slightly decreased ΔΨ (Fig. 2C and D). These results suggested that TP induced apoptosis at least partially at an early stage by disrupting active mitochondria.

Considering the anti-metastasis effect of TP, we an in vitro chamber migration assay was performed to test whether TP affects NSCLC cell migration. As presented in Fig. 3, the number of migratory PC9 cells was decreased by TP treatment and dropped dramatically at 60 and 90 nM; whereas the number of migratory A549 cells stayed largely unchanged upon TP treatment; however, a statistically significant decrease in the number of migratory cells was observed after treatment with 60 and 120 nM TP, but this was less dramatic than the effect in PC9 cells.

Aberrant and constitutive activation of STAT3 in NSCLC has been reported (9) and is associated with tumor progression and a poor prognosis (14). After treatment with TP for 24 h, p-STAT3 was decreased in PC9 (Fig. 4A) and A549 cells (Fig. 4B). The mRNA expression of PC9 (Fig. S1) and A549 (Fig. S2) was consistent with protein expression. As shown in Fig. S3, the ratio of p-STAT3/total STAT3 was marginally decreased by TP in A549 cells, with statistical significance at 120 nM (P<0.05).

Expression levels of target genes of STAT3 were also examined. C-myc expression was found to greatly decreased upon TP treatment in PC9 (Fig. 4A) and A549 cells (Fig. 4B). Although cyclin D1 was downregulated by TP in PC9 cells (Fig. 4B), cyclin D1 was upregulated by TP treatment in A549 cells (Fig. 4B). The overexpression of cyclin D1 may contribute to resistance to TP (25), as well as cell DNA damage (24). Caspase-3 is a critical executioner of apoptosis, as it is either partially or totally responsible for the proteolytic cleavage of many key proteins, and cleaved caspase-3 is the active form of the protein. As shown in PC9 cells (Fig. 4A), TP increased the level of cleaved caspase-3. Expression of anti-apoptotic proteins MCL-1 and BCL-2 was decreased after TP treatment in a dose-dependent manner in PC9 (Fig. 4A) and A549 cells (Fig. 4B). The two bands of BCL-2 seen in Fig. 4A may be homedimers or poor antibody specificity (26). TP also downregulated MMP-9 expression in a dose-dependent manner in PC9 (Fig. 4A) and A549 cells (Fig. 4B). The above results suggested that TP treatment inhibited cell proliferation, induced apoptosis and inhibited cell migration by suppressing the activation of STAT3.

Signal transduction in the STAT3 pathway, as visualized by STAT3 translocation into the nucleus, was inhibited by TP treatment. Less prevalent and weaker STAT3 staining was observed in cell nuclei in the TP treatment groups (Fig. 5).

Elevated IL-6 expression has been observed in many patients with lung cancer (27). A549 cells appear to be much less sensitive to TP compared with PC9 cells, thus it was speculated that the expression of IL-6 in these cells is associated with the results. The effect of TP in the presence of exogenous IL-6 in PC9 cells was analyzed, with a non-specific IL-6/STAT3 inhibitor HHT serving as a positive control, which prevents the initial elongation step of protein synthesis (Fig. 6). Simlar to Ham et al (28), IL-6 seems not significantly increase the phosphorylation of STAT3, but did impair the ability of HHT to inhibit STAT3 phosphorylation. IL-6 appeared to induce expression of the STAT3 downstream target gene, BCL-2, but there was no statistical difference (P>0.05). At the same time, TP and HHT could reduce the expression of BCL-2 compared with IL-6-stimulated cells. Compared with the control group, the expression of MCL-1 was significantly increased by exogenous IL-6 (P<0.05). Similar to HHT, pretreated TP can almost completely block MCL-1 expression. Briefly, TP might enhance the effects of chemotherapy by inhibiting IL-6-induced MCL-1 activation.