TPL (≥98%, L-004-130304) was purchased from Chengdu Herb Purity Co., Ltd. (Chengdu, China). It was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mmol/l to obtain the stock solution and kept below −20°C, then diluted to various concentrations with phosphate-buffered saline (PBS) prior to the assays. The final concentration of DMSO was less than 0.1%. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), RPMI-1640, a bicinchoninic acid (BCA) protein assay kit, inhibitor of ERK U0126, propidium iodide (PI) staining kit and Annexin V-fluorescein isothiocyanate (FITC)/PI apoptosis detection kit were purchased from Nanjing KeyGen Biotechnology Co. Ltd. (Nanjing, China). Anti-p-p38, anti-p-JNK, anti-p-ERK antibodies, inhibitor of p38 SB202190 and inhibitor of JNK SP600125 were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-p-Akt (Ser473), anti-p-GSK-3β (Ser9), cleaved caspase (c-caspase)-3 (1:200), c-caspase-9 (1:1,000), Bcl-2 (1:500) and COX IV (1:1,000) were supplied by Bioworld Technology (Nanjing, China). An enhanced chemiluminescence kit was purchased from Thermo Fisher (Shanghai, China). Bovine calf serum was purchased from Wisent corporation (Nanjing, China). All other chemicals and reagents used were of analytical grade.

The human lung cancer cell line A549 and the corresponding MDR cell line A549/Taxol were purchased from Nanjing KeyGen Biotechnology Co. Ltd., and cultured in RPMI-1640 medium supplemented with 10% bovine calf serum, 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in a humidified atmosphere of 5% CO₂. Paclitaxel (Taxol, 200 ng/ml) was added to the medium in order to maintain MDR in the A549/Taxol cells, and removed two weeks before the experiments. Cells were passaged every 2–3 days.

For the cell viability assay, A549 and A549/Taxol cells were seeded onto a 96-well plate at a density of 8×10³ cells per well. Following overnight incubation, the culture medium was aspirated, and the cells were incubated with various concentrations of TPL (the final concentrations were 0.01, 0.02, 0.04, 0.06 and 0.08 µmol/l) or co-treated with MAPK inhibitors in complete culture medium for 48 h. The same volume of complete culture medium served as the negative control. Then 20 µl MTT solution (5 mg/ml) was added to each well, and the plates were further incubated for 4 h. The medium was removed and 150 µl DMSO was added to solubilize the MTT formazan salt. The absorbance of the solution was measured on a microplate reader (Spectra MAX190, Molecular Devices LLC, Sunnyvale, CA, USA) at 490 nm and the results were expressed as a percentage of the control cells.

A549/Taxol cells were seeded onto six-well plates at a density of 3×10⁵ cells per well. Following overnight incubation, cells were treated with TPL at concentrations of 0.025 and 0.05 µM for 24 h. For the purpose of apoptosis analysis, following treatment with TPL, the cells were harvested and washed with PBS, and then stained with Annexin V/PI according to the manufacturer's recommendations. Stained samples were analyzed by flow cytometry (FACSCalibur, BD Biosciences, San Jose, CA, USA). For analysis of the cell cycle distribution, the supernatant was discarded, and attached cells were harvested and fixed in cold 70% ethanol overnight at −20°C. Cell cycle distribution was analyzed using a flow cytometer according to the manufacturer's instructions for the PI staining kit.

A549/Taxol cells (3×10⁵ cells/well) were treated with TPL at concentrations of 0.025 and 0.05 µM, or co-treated with MAPK inhibitors, for 24 h. Following the treatments, cells were collected, washed twice with pre-chilled PBS, lysed, and then centrifuged at 12,000 rpm for 10 min at 4°C. The supernatant of the lysate was boiled, and total protein was measured using the BCA protein assay kit. Proteins were separated by SDS-PAGE, and then transferred onto polyvinylidene fluoride (PVDF) membranes (0.45 µm; Millipore). Non-specific protein binding sites were blocked with 5% bovine serum albumin diluted in Tris-buffered saline buffer containing 0.1% Tween-20 (pH 7.4) for 2 h at room temperature, followed by incubation with the appropriate primary and secondary antibodies. The expression of proteins was detected using a Chemi Doc XRS+ system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The grey level ratio of the target protein to COX IV was used to represent the relative protein expression level.

Results are expressed as the means ± standard deviation. Statistical differences among groups were evaluated by the t-test using GraphPad Prism 6. P<0.05 and P<0.01 were considered to represent different levels of statistical significance.

A549 and A549/Taxol cells were treated with Taxol for 48 h and the cytotoxicity was assessed using MTT assay. The IC₅₀ of Taxol against A549 and A549/Taxol cells was 3.52±0.47 and 71.31±7.95 µM, respectively. A549/Taxol cells exhibited more than a 20-fold resistance to Taxol in comparison with the drug-sensitive A549 cell line.

Various doses (0.01, 0.02, 0.04, 0.06, 0.08 µM) of TPL were used to treat A549/Taxol cells for 48 h. The anti-proliferative effect of TPL on A549/Taxol was assessed by MTT assay, and the results revealed that TPL inhibited the proliferation of A549/Taxol cells efficiently in a dose-dependent manner, with an IC₅₀ value of 46.47±0.31 nM. It was far more efficient than the positive drug cisplatin, which demonstrated an IC₅₀ value of 8.87±0.98 uM. TPL exhibits potent cytotoxicity in vitro in A549/Taxol cells. Based on the primary findings, we used TPL at concentrations of 0.025 and 0.05 µM in subsequent experiments.

To investigate the modulatory effects of TPL on MAPKs in the course of inhibition of proliferation of A549/Taxol cells, we performed MTT assay using MAPK inhibitors (SB202190, SP600125 and U0126) coupled with TPL. SB202190 exerted little effect on the result, while SP600125 and U0126 significantly reinforced the inhibitory effects of TPL (Fig. 1). Since there was no significant effect observed with the addition of SB202190, it was suggested that the modulation of p38 contributed little to the inhibitory effect exhibited by TPL on A549/Taxol cells. SP600125 exhibited synergistic effects with TPL, while antagonism caused by U0126 was also noted. These findings suggested that TPL may exert its inhibitory effects by regulating the JNK and ERK signaling pathways.

To investigate the mechanism of TPL's involvement in the anti-proliferative activity of A549/Taxol cells, A549/Taxol cells were treated with TPL at concentrations of 0.025 and 0.05 µM. Twenty-four hours later, Annexin V-FITC/PI staining flow cytometry was used to assess the rate of cell apoptosis. Compared with the control group, we observed that the number of apoptotic cells increased significantly in the TPL group (Fig. 2A). In addition, we noted that TPL significantly increased the expression of caspase-3 and caspase-9 (Fig. 2B). These results indicated that TPL induced caspase family-dependent apoptosis in A549/Taxol cells. In addition, as shown in Fig. 2B, upregulation of the Bax/Bcl-2 ratio was observed following TPL treatment, which supported our previous hypothesis concerning the role of pro-apoptosis.

To elucidate the mechanism of TPL-induced proliferation inhibition, we examined the effect of TPL on cell phase distribution by flow cytometry. As shown in Fig. 3, concomitant with the growth inhibitory effect, treatment with TPL induced a significant S-phase arrest. The cell populations in the G₀/G₁, S and G₂/M phases were 41.97, 45.89 and 12.15% in the control group. However, after 24-h incubation with 0.025 and 0.05 µM TPL, the S-phase portion was notably enhanced by 27.2 and 6.11%, respectively. Contrarily, the G₂/M population of A549/Taxol was markedly reduced following treatment with 0.025 and 0.05 µM TPL, indicating the blockage of the S-G₂ transition in A549/Taxol cells. In addition, the sub-G₁ population was increased by 1.03 and 5.70%, respectively.

The inhibition of p38 usually promotes tumor formation, but in certain cases, the tumor-forming ability is suppressed by p38 activation. JNK plays two antagonistic roles: oncogenic and and pro-apoptotic. The oncogenic function of JNK pathways is highly expressed in cancer cells, which promotes cell proliferation (12,13). The ERK1/2 cascades are mainly involved in tumor cell survival and proliferation regulation, but increased levels of the phosphorylated form of ERK1/2 also induce cell apoptosis (14). The aberrant activation of Akt is one of the most frequent alterations observed in most types of cancer cells, and it has been observed to correlate with poor prognosis and resistance to various anti-cancer agents (15).

Preliminary observations in the current study shed light on the mechanism of action of TPL on the inhibition of proliferation of A549/Taxol cells. Based on the findings obtained, we proposed that modulation of JNK and ERK signaling by TPL contributed in a great manner to the anti-proliferative activity observed. To validate the conclusion, the expression of three MAPKs (p-p38, p-JNK and p-ERK) and PI3K/Akt (p-Akt and p-GSK-3β) was analyzed by western blot analysis. As shown in Fig. 4, TPL increased the expressions of p-p38, p-ERK and p-GSK-3β, but exerted the opposite effect on p-JNK and p-Akt. Compared with the TPL group, the expression of p-ERK, p-JNK and p-Akt was notably downregulated by MAPK inhibitors, while the p-GSK-3β expression level was significantly upregulated by SP600125. These results were consistent with conclusions deduced from the results of MTT assay.