VDa, VLe and VLi were isolated and purified using in-house methods as previously described (16). Briefly, the dried aerial region of G. extensum (1.3 kg) was manually ground and extracted at room temperature successively, using CH₂Cl₂, a CH₂Cl₂/MeOH (1:1) mixture and MeOH. The extracts were filtered and then evaporated to yield CH₂Cl₂ (29.2 g), CH₂Cl₂/MeOH (5.4 g) and MeOH (50.8 g) extracts. The three active sesquiterpenoids, namely VDa, VLe and VLi, were separated from the CH₂Cl₂ extracts of G. extensum by a combination of chromatographic methods and semi-preparative high-performance liquid chromatography purification.

The A549 human lung adenocarcinoma cell line was purchased from the American Type Culture Collection (cat. no. CCL-185^™). The cells were cultured in RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) antibiotic-antimycotic (all from Gibco; Thermo Fisher Scientific, Inc.), and maintained at 37°C (5% CO₂) in a humidified atmosphere. The cells were passaged every 3–4 days and morphology was investigated by inverted phase-contrast microscopy. The G. extensum-derived compounds, VDa, VLe and VLi, were dissolved in DMSO, and diluted with culture media to the desired concentrations, while keeping DMSO concentration at 0.2% (v/v) throughout the study.

The effects of G. extensum-derived compounds on the viability of A549 lung cancer cells were assessed using an MTT assay. Various natural products, such as a resveratrol, bergapten and sotetsuflavone, have been reported to exert their biological effects, including cytotoxicity, cell cycle arrest and induction of apoptosis, at a concentration range of 10–100 µM (17–19). In the present study, a concentration range of 10–60 µM was selected for VDa, VLe and VLi treatment. Briefly, A549 cells were seeded into 96-well culture plates at a density of 1×10⁴ cells/well (100 µl). After 24 h, the cells were treated with 100 µl VDa, VLe and VLi at various concentrations (10, 30 and 60 µM), or with the vehicle (cell culture medium) for 24 h. The spent medium were replaced with 100 µl of fresh culture medium containing 0.5 mg/ml MTT (Sigma-Aldrich; Merck KGaA) and incubated for 2 h at 37°C. Finally, the MTT-containing medium was removed and 100 µl DMSO was added to dissolve the formazan crystals. The number of viable cells was determined by measuring the absorbance at 550 nm and subtracting the absorbance at the reference wavelength (650 nm), using a microplate reader. The absorbance of the untreated control was taken as 100%, and the decrease in the number of cells treated with the extracts was calculated. Assays were carried out three times with quadruplicate samples.

The apoptotic profiles of A549 cells treated with various concentrations of G. extensum-derived compounds were detected using the Muse^® Cell Analyzer (Luminex Corporation) as previously described (20). Briefly, cells were harvested after 24 h of treatment, stained with Muse Annexin V & Dead Cell kit (Luminex Corporation) for 20 min in the dark at room temperature, and immediately subjected to apoptosis detection. Results are presented as mean values of total apoptosis [the percentage of early (positive for Annexin V) + late (positive for both Annexin V and 7-AAD) apoptotic cell populations] from three independent experiments.

Cell cycle progression was investigated by flow cytometric analysis (Muse^® Cell Analyzer; Luminex Corporation) as previously described (21). Briefly, A549 cells were treated with various concentrations of G. extensum-derived compounds or a specific inhibitor of JAK2/STAT3, cucurbitacin-I (CBC-I), for 24 h. After treatment, the cells were collected, fixed with ice-cold 70% ethanol and stored at −20°C overnight prior to staining. Then, the cells were washed, stained with 200 µl Muse Cell Cycle reagent and subjected to cell cycle analysis. Results are presented as mean values of the percentage of the cells in each phase (G₀/G₁, S and G₂/M) from three independent experiments.

The protein structure of JAK2 (PDB:4Z32) was acquired from Protein Data Bank (http://www.rcsb.org/), while the molecular structures of VDa, VLe and VLi were obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov). iGEMDOCK v2.1 software (22) was used to perform molecular docking using the accurate docking function (slow docking). All images were generated using UCSF Chimera (23).

The expression levels of selected signaling proteins were determined by western blot analysis. Since the aim was to evaluate the mechanisms by which VDa, VLe and VLi lead to cell death, western blot analysis was conducted 4 h after treatment. Preliminary results indicated that the changes in signaling pathways were detected 4 h after treatment. At 24 h post treatment, apoptosis had already taken place, so that the changes in signaling cascades and protein expression may have resulted from the apoptotic process, rather than changes that trigger apoptosis. Briefly, 4 h after treatment, A549 cells were treated with different concentrations of VDa, VLe and VLi (10, 30 or 60 µM), were harvested and lysed in cell lysis buffer (MilliporeSigma) supplemented with freshly added protease/phosphatase inhibitor cocktail (Cell Signaling Technology, Inc.). The total protein concentration was measured using a Bradford assay kit (Bio-Rad Laboratories, Inc.); 20 µg protein per lane was subjected to 10% SDS-PAGE and then electrophoretically-transferred to Immobilon-P Transfer Membranes (MilliporeSigma). Each membrane was incubated in 3% (w/v) BSA (Sigma-Aldrich; Merck KGaA) blocking buffer at room temperature for 1 h, and then probed with the primary antibodies against the following, at 4°C overnight: JAK2 (1:500; rabbit, monoclonal; cat. no. 3230), phospho-JAK2 (Y1008; 1:500; rabbit, monoclonal; cat. no. 8082), STAT3 (1:3,000; rabbit, polyclonal; cat. no. 9132), phospho-STAT3 (Y705; 1:250; rabbit, monoclonal; cat. no. 9145), p38 (1:1,000; rabbit, polyclonal; cat. no. 9212), phospho-p38 (T180/Y182; 1:250; rabbit, polyclonal; cat. no. 9211), CDK2 (1:1,000; rabbit, monoclonal; cat. no. 2546), p21 (1:500; rabbit, monoclonal; cat. no. 2947), cyclin B1 (1:1,000; rabbit, polyclonal; cat. no. 4138), Cdc2 (1:1,000; mouse, monoclonal; cat. no. 9116), Bax (1:5,000; rabbit, polyclonal; cat. no. 2772), survivin (1:250; rabbit, polyclonal; cat. no. 2803), myeloid cell leukemia-1 (Mcl-1; 1:250; rabbit, polyclonal; cat. no. 4572) and α-tubulin (1:20,000; mouse, monoclonal; cat. no. 3873) (all from Cell Signaling Technology, Inc.). The membrane was washed with TBS-T (Tris-buffered saline, 0.1% Tween-20) before incubating with the appropriate horseradish peroxidase-conjugated secondary antibody (1:5,000; goat anti-rabbit, monoclonal; cat. no. 7074; Cell Signaling Technology; or 1:5,000; rabbit anti-mouse, polyclonal; cat. no. P0260; Dako; Agilent Technologies, Inc.) for 1 h at room temperature. The relative protein expression levels were detected by chemiluminescence using the WesternBright ECL detection kit (Advansta, Inc.). Quantification of the bands was performed by densitometry using ImageJ version 1.53k (National Institutes of Health).

Experimental values from three independent experiments are presented as the mean ± standard deviation. The statistical significance between the control and treated groups was evaluated using one-way analysis of variance following by Tukey's multiple comparisons test (GraphPad Prism version 5.0; GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

The cytotoxic effects of VDa, VLe and VLi (Fig. 1a) on the A549 human lung cancer cell line were evaluated by MTT assay at three concentrations (10, 30 and 60 µM). After 24 h of incubation, VDa, VLe and VLi decreased cell viability in a dose-dependent manner, with VLi being the most potent (Fig. 1B). Comparison of cytotoxic activities indicated that treatments with 30 µM VDa, 30 µM VLe or 10 µM VLi were only marginally cytotoxic (~80% viability) and may therefore be considered as sub-cytotoxic doses, while high doses (60 µM for VDa and VLe, and 30 µM for VLi) were cytotoxic (~50% viability).

Morphological investigation by inverted phase-contrast microscopy revealed that treatment with VDa, VLe and VLi resulted in a decrease in cell numbers, induction of cell rounding and detachment of cells from the vessel surface (Fig. 2A). In addition, flow cytometric analysis was performed in A549 cells treated with G. extensum-derived compounds for 24 h. The results indicated that after treatment at sub-cytotoxic concentrations for 24 h, the total apoptotic cell population (early + late apoptosis) increased from 5.3±0.9% for the control group to 8.1±1.8, 7.6±1.2 and 8.5±2.6% for cells treated with VDa, VLe and VLi, respectively. Moreover, treatment of A549 cells with cytotoxic concentrations led to an increase in the total apoptotic cell population to 21.3±5.4, 22.2±1.6 and 10.1±3.6% for VDa, VLe and VLi, respectively (Fig. 2B and C). In addition, necrotic cell populations were <1% following all treatment types and concentrations, suggesting that VDa, VLe and VLi were dose-dependently cytotoxic towards A549 cells through the induction of apoptosis.

The experimental data demonstrated a decrease in A549 cell number after VDa, VLe and VLi treatment, suggesting that these compounds may exhibit cytostatic effects on A549 cells. To assess the anti-proliferative action of the three compounds, the cell cycle distribution of A549 cells treated with sub-cytotoxic and cytotoxic concentrations of VDa, VLe and VLi was investigated by flow cytometry. The results indicated alterations in cell cycle progression in all treatment groups. As shown in Fig. 3, treatment of A549 cells with sub-cytotoxic doses of VDa, VLe and VLi caused a significant increase in the G₀/G₁ population to 66.6±2.8, 68.7±2.5 and 66.2±2.6% for VDa, VLe and VLi, respectively, compared with untreated control cells (59.9±0.9%). Consistent with this result, a decrease in the proportion of cells in the S phase (8.2±1.3, 8.2±0.6 and 8.7±0.8% for VDa, VLe and VLi, respectively) and G₂/M phase (25.2±1.6, 23.1±2.9 and 24.8±2.2% for VDa, VLe and VLi, respectively) of the cell cycle was observed, compared with the control (9.6±0.1% for S and 30.5±0.8% for G₂/M phase, respectively).

However, increasing the concentration of VDa, VLe and VLi to cytotoxic doses resulted in a significant increase in the percentage of cells in the G₂/M phase (35.2±0.5, 36.4±1.7 and 35.8±2.1% for VDa, VLe and VLi, respectively) compared with untreated control cells. The increased number of cells in the G₂/M phase was accompanied by a proportional decrease in the G₀/G₁ cell populations (58.8±1.0, 55.3±0.6 and 56.1±1.4% for VDa, VLe and VLi, respectively) and S phase populations (5.7±0.5, 7.7±1.1 and 7.9±0.9% for VDa, VLe and VLi, respectively), compared with the control group. These data suggest that VDa, VLe and VLi exerted dose-related cytostatic effects on A549 cells, resulting in inhibition of cellular proliferation.

The chemical structures of the G. extensum-derived compounds are related to pathenolide, a sesquiterpene lactone which has been shown to inhibit JAK2/STAT3 signaling (24). Therefore, a molecular docking study was performed using iGEMDOCK v2.1 software to investigate whether JAK2 is a molecular target of VDa, VLe and VLi. As revealed in Fig. 4, the binding positions of VDa (purple) and VLe (deep blue) in JAK2 are located between the F1 and F3 lobes of the FERM domain, while VLi (sky blue) is bound between the F1 and F2 lobes of the FERM domain. The binding energies of VDa, VLe and VLi were −88.04, −82.81 and −89.54 kcal/mol, respectively (Table I). VDa formed hydrogen bonds with amino acids of FERM F1 domain (GLU65, ASN83 and PHE85) and linker (ASN395) (Fig. 5A) whereas VLe formed hydrogen bonds with amino acids of the FERM F1 domain (ASN83 and ALA86) and the FERM F3 domain (SER367 and ARG374) (Fig. 5B). However, VLi, which had the lowest binding energy, formed hydrogen bonds with amino acids of FERM F1 domain (SER50 and GLU51) and FERM F2 domain (ARG158 and HIS159) of JAK2 (Fig. 5C). These results suggested that the JAK2 may be the target of VDa, VLe and VLi.

To gain further insight into the mechanisms underlying the cytostatic and cytotoxic effects induced by VDa, VLe and VLi, the expression levels of signaling molecules associated with JAK2/STAT3 signaling, cellular stress response, cell cycle regulation and apoptosis were assessed. Western blot analysis indicated that treatment with VDa, VLe and VLi suppressed the levels of p-JAK2 and p-STAT3, and increased p-p38 levels in A549 cells, in a dose-dependent manner (Fig. 6A). These results are consistent with the ability of the three compounds to bind JAK2, as determined by the molecular docking study.

The levels of cell cycle regulatory proteins were also investigated. Treatment of A549 cells with sub-cytotoxic doses of VDa, VLe and VLi resulted in lower expression levels of CDK2, a G₁/S-specific CDK, and upregulated expression of a CDK inhibitor p21 (Fig. 6A). When cytotoxic doses of the three compounds were employed, the decreased levels of G₂/M-specific cyclin and CDK, cyclin B1 and Cdc2, were observed in the treated cells (Fig. 6A). Furthermore, Bax, the pro-apoptotic protein, was upregulated in cells treated with VDa, VLe and VLi compared with the untreated control, while the decreased levels of the anti-apoptotic proteins, survivin and Mcl-1, were detected in a dose-dependent manner (Fig. 6A). These results indicated that exposure of A549 cells to VDa, VLe and VLi induced changes in the expression levels of signaling molecules associated with the suppression of cell proliferation, increased stress and enhanced apoptosis.

A specific inhibitor of JAK2/STAT3, CBC-I, was used to confirm whether the effects of VDa, VLe and VLi on A549 cells were mediated through the inhibition of JAK2/STAT3 signaling. Cell cycle analysis indicated that the cell population tended to shift to G₀/G₁ and G₂/M phase arrest when treated with 10 and 50 µM CBC-I, respectively, similar to that observed with sub-cytotoxic and cytotoxic treatments of G. extensum-derived compounds (Table II). Western blot analysis revealed that treatment with CBC-I decreased the levels of p-JAK2, p-STAT3, survivin and Mcl-1, but caused increased phosphorylation of p38 (Fig. 6B). Treatment with 10 µM CBC-I resulted in a slight downregulation of CDK2 and an upregulation of p21. However, cells treated with 50 µM of CBC-I exhibited markedly decreased levels of cyclin B1 and Cdc2 (Fig. 6B). Collectively, these findings revealed similar tendencies in the effects of treatment with G. extensum-derived compounds and treatment with the JAK2/STAT3 inhibitor, CBC-I, suggesting the involvement of the JAK2/STAT3 pathway in actions of VDa, VLe and VLi.