pRK5myc-MAP4K3 (M4K3) and AFG kinase-dead (KD) MAP4K3 were prepared as described previously (13). APS [2-(chlorom ethyl)-4-(4-nitrophenyl)-1,3-thiazole; ≥98%] was purchased from ShangHai YuanYe Biotechnology Co., Ltd. (Shanghai, China; CAS no. 89250-26-0; cat. no. C18M6Y1). HCPT (98%) was purchased from Shanghai Aladdin Biochemical Co., Ltd. (Shanghai, China; CAS no. 19685-09-7; cat. no. H1524105). 3-methyladenine (3-MA) and chloroquine (CQ) were obtained from Sigma-Aldrich, EMD Millipore (Billerica, MA, USA). Fetal bovine serum (FBS) was from HyClone, GE Healthcare Life Sciences (Logan, UT, USA) and penicillin G, streptomycin and Dulbecco’s modified Eagle’s medium (DMEM) were from Gibco, Thermo Fisher Scientific, Inc. (Waltham, MA, USA). p-nitrobenzyl mesylate (PNBM) was from Epitomics (Burlingame, CA, USA). Myelin basic protein (MBP), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and isopropyl alcohol were purchased from Sigma-Aldrich, EMD Millipore. β-actin mouse monoclonal antibody (mAb; cat. no. ab8226), Bax rabbit antibody (Ab; cat. no. 2772), Bcl-2 rabbit mAb (cat. no. 3498), caspase-3 rabbit Ab (cat. no. 9662), caspase-9 rabbit Ab (cat. no. 9502S), cytochrome c (D18C7) rabbit mAb (cat. no. 11940S), p70S6K mouse mAb (cat. no. 611261), phospho-p70 S6K (Thr389) rabbit Ab (cat. no. 9205), MAP4K3 rabbit Ab (cat. no. PAB3189), anti-myc 9E10 mouse mAb (cat. no. 05-419), thiophosphate ester rabbit mAb (cat. no. ab92570), microtubule-associated protein 1 light chain 3 (LC3) rabbit Ab (cat. no. 8025) and P62 rabbit mAb (cat. no. 11940) were purchased from Cell Signaling Technology, Inc. (Boston, MA, USA), Abcam (Cambridge, UK) or BD Biosciences (San Diego, CA, USA). Secondary horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (IgG; cat. no. 31460) and horseradish peroxidase-conjugated goat anti-mouse IgG (cat. no. 31430) antibodies were purchased from Thermo Fisher Scientific, Inc. All other chemicals were of analytical grade.

Human H1299 NSCLC cells (H1299), NCI H460 (H460) cells and 293T cells were obtained from the Chinese Academy of Sciences (Shanghai Institute of Cell Biology and Biochemistry, and the Chinese Type Culture Collection, Shanghai, China). The cell lines were cultured in DMEM supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin and 100 µg/ml streptomycin. The cells were maintained at 37°C in a humidified 5% CO₂ incubator.

For transfection, the cell lines were plated at 5 ×10⁵ cells/well in 6-well plates and incubated overnight prior to transfection with Lipofectamine^® 2000 (Invitrogen, Thermo Fisher Scientific, Inc.), according to the manufacturer’s protocol. Unless otherwise specified, H1299 cells or H460 cells were transfected with 0.5 µg pRK5myc Vec, 0.5 µg AFG MAP4K3-KD or 0.5 µg pRK5myc M4K3 and cultured for 24 h, after which, they were used in subsequent experiments.

Immunoprecipitated myc-tagged MAP4K3 from 293T cells, produced as previously described (13), were incubated with MBP as a substrate and the indicated agents. To screen for inhibitors of MAP4K3 by western blot analysis, ATPγS was used at a concentration of 1 mM in appropriate kinase buffers (21). The proteins were alkylated with 2.5 mM PNBM for 2 h at room temperature (18-22°C) and the products were analyzed by western blot analysis.

The H1299 cells were seeded in 96-well cell culture plates and incubated for 24 h. Following treatment with the agents at the indicated concentrations and times, cell viability was evaluated using an MTT assay, which is based on the reduction of a tetrazolium salt by mitochondrial dehydrogenase by viable cells. Following treatment, MTT solution (final concentration, 500 µg/ml) was added to each well and incubated for 4 h at 37°C. The formed formazan crystals were dissolved in DMSO and the absorbance was measured at 490 nm using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Cell cycles were analyzed using a propidium iodide (PI) cell cycle detection kit (BD Biosciences), according to the manufacturer’s protocol. The cells were trypsinized, washed with PBS, centrifuged at 190 × g for 5 min at room temperature, and the concentration was adjusted to 1×10⁶ cells/ml. The cells were resuspended in solution A at room temperature for 10 min. Solution B was added for 10 min and the cells were stained with fluorescein isothiocyanate (FITC)-labeled PI at room temperature for 15 min under light-protected conditions.

Flow cytometric analysis was performed using a BD FACSAria II Cell Sorter (BD Biosciences). The percentage of apoptotic cells was calculated using BD FACSDiva Software v7.0 (BD Biosciences, Franklin Lakes, NJ, USA). The apoptotic indices were determined using a FITC-labeled Annexin V/PI apoptosis detection kit (Sigma-Aldrich, EMD Millipore). Briefly, the cells were harvested, washed with PBS and centrifuged at 190 × g for 5 min at room temperature. The cells were adjusted to 1×10⁶ cells/ml, resuspended in binding buffer and stained with FITC-labeled Annexin V/PI at room temperature for 15 min under light-protected conditions. Flow cytometric analysis was performed using a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

Wound healing was assayed as previously described with modifications (22). Briefly, the H1299 cells and H460 cells were cultured to confluence in 6-well cell culture plates for 24 h in serum-free medium, respectively. The medium was replaced with serum-containing medium followed by the addition of APS or/and HCPT, and cell monolayers were disrupted by scraping with a 100-µl micropipette tip. At the indicated times (0 and 24 h without transfection; 0,19 and 60 h with transfection) following scraping, the cells were washed twice with PBS (pH 7.4) and images were captured using an optical microscope at ×40 magnification.

The chemotactic directional migration of H1299 cells was measured using 24-well Transwell inserts. Pore filters (8-µm; Corning Incorporated, Corning, NY, USA) were coated with gelatin (Sigma-Aldrich, EMD Millipore). Following culture for 24 h in serum-free DMEM, the cells were resuspended in serum-free medium, seeded in the upper chambers of the Transwell inserts (1×10⁵ cells/ml) and incubated with APS and/or HCPT. DMEM containing 10% FBS was placed in the lower chamber. The cells in each treatment group were incubated for 24 h at 37°C in a humidified atmosphere with 95% air and 5% CO₂. The filters were stained with 0.1% crystal violet. Cells that had migrated and adhered to the underside of the filters were counted and images were captured using an optical microscope at × 40 magnification. The invasiveness of the H1299 cells was measured using Matrigel-coated Transwell cell culture chambers (8-µm pore size) as previously described. The method was the same as for migration analysis. Cells that had penetrated through the Matrigel coating and adhered to the lower surface of the filters were counted and images were captured using an optical microscope at ×40 magnification.

The cells were washed in 1X PBS and lysed in lysis buffer as described (13). The total protein concentrations was determined using the Bradford method. The total protein or protein fractions (50 µg/lane) were loaded and separated by 10 or 12% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% skimmed milk powder for 45 min at room temperature. The membranes were then incubated with primary antibodies directed against β-actin (dilution 1:2,000), Bax (dilution 1:1,000), Bcl-2 (dilution 1:1,000), caspase-3 (dilution 1:1,000), caspase-9 (dilution 1:1,000), cytochrome c dilution 1:1,000), p70S6K (dilution 1:500), phospho-p70 S6K (Thr389) (dilution 1:1,000), MAP4K3 rabbit Ab (dilution 1:500), myc (dilution 1:2,000), thiophosphate ester (dilution 1:5,000), LC3 (dilution 1:1,000) and P62 (dilution 1:1,000) overnight at 4°C. The membranes were subsequently washed three times with PBS-0.1% Tween-20 for 10 min and were then incubated with goat anti-mouse (dilution 1:10,000) or goat anti-rabbit (dilution 1:5,000) secondary antibodies for 1 h at room temperature. The expression of individual proteins was detected with an enhanced chemiluminescence kit (Applygen Technologies, Inc., Beijing, China). The densitometric values of the bands were measured using ImageQuant TL software (version 8.1; GE Life Sciences, Chicago, IL, USA).

Data are reported as the mean ± standard error of the mean. Differences between groups were analyzed using Student’s t-test with SPSS 14.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

To identify an inhibitor specific for MAP4K3, the in vitro activity of MAP4K3 was measured by incubating the kinase with the generic substrate MBP. In the presence of ATPγS, MAP4K3 kinase activity was markedly inhibited by APS and HCPT (Fig. 1A).

Subsequently, it was demonstrated that APS and HCPT inhibited the phosphorylation of S6K. The endogenous expression of MAP4K3 in H1299 cells was markedly reduced by HCPT, although not by APS (Fig. 1B). HCPT and the combined agents inhibited S6K phosphorylation at Thr389 by 10.02±0.01 and 20.89±0.01-fold, respectively, which was increased compared with APS (1.33±0.02-fold). In addition, the overexpression of MAP4K3 reduced the inhibition of mTOR signaling in the presence of APS, although not when treated with HCPT or the combined agents (Fig. 1C). Unlike rapamycin, a specific inhibitor of mTOR, HCPT was observed to partially inhibit insulin-responsive mTOR activation (Fig. 1D). These results suggested that the two agents impaired the activation of MAP4K3 and act on the mTOR signaling pathway in a similar manner.

To detect the cytotoxic effects of APS, the H1299 cells were exposed to HCPT and varying of doses of APS for 24 h and cell viability was examined using an MTT assay. As with HCPT, APS moderately inhibited the growth and proliferation of H1299 cells (Fig. 2A and B). The effects of APS and HCPT on apoptosis were assessed using an Annexin V-FITC/PI assay. APS significantly enhanced apoptosis in H1299 cells (early apoptosis, 6.48±1.27%; late apoptosis, 19.88±2.89%) and HCPT induced late apoptosis, up to 45.59±2.91% (Fig. 2C and D), indicating that APS and HCPT inhibited cell proliferation by promoting apoptosis in H1299 cells.

Flow cytometry was used to distinguish between cells in different phases of the cell cycle. As shown in Fig. 3A and B, following APS treatment, the percentages of cells in the S and G2/M phases increased to 42.94±3.09 and 11.12±1.32%, respectively, and following HCPT treatment these proportions were 66.57±2.93 and 9.73±1.39%, respectively, indicating that HCPT and APS treatment induced S phase and G2/M phase arrest. These results further suggested that APS and HCPT induced H1299 cell death by altering the cell cycle distribution.

The upregulation of caspase-3, cytochrome c and the Bax/Bcl-2 and caspase-9/pro-caspase-9 ratios were observed following treatment of the H1299 cells with APS (Fig. 4A-D). The results indicated that APS treatment induced H1299 cell death through the release of cytochrome c from the mitochondria.

As inhibition of the Akt/mTOR/S6K signaling pathway is linked to the induction of autophagy (23), the present study examined whether HCPT induces autophagy by inhibiting this pathway. Autophagy is activated by the increased expression of LC3-II, which controls the completion of autophagosome formation. P62 is selectively incorporated into autophagosomes through directly binding to LC3 and is efficiently degraded during autophagy. Therefore, the total cellular expression levels of P62 is inversely correlated with the autophagic activity. In the present study, it was demonstrated that HCPT induced the formation of LC3-II and decreased the expression of P62 following treatment with HCPT for 6, 12 and 24 h (Fig. 5A and B). To validate and interpret this result, western blot analysis was performed with the control extracts harvested from cells treated with 3-MA, a phosphoinositide 3-kinase inhibitor, which prevents the induction of autophagosomes and inhibits autophagic flux, and CQ, which inhibits the fusion of autophagosomes and lysosomes, inhibiting autophagy and leading to LC3 accumulation. Compared with the H1299 cells incubated with HCPT alone, pretreatment with 3-MA markedly decreased the levels of LC3-II and increased the expression of P62 (Fig. 5C and D), whereas pretreatment with CQ markedly increased the levels of LC3-II (Fig. 5E and F), suggesting that HCPT treatment induced autophagic flux.

To assess the effect of APS and HCPT on migration and invasion of H1299 cells, a gradual reduction in the area of wounding was observed in the control cells incubated with DMEM for 24 h. Treatment of the H1299 cells with APS resulted in significant inhibition of this migration, whereas HCPT treatment did not affect migration (Fig. 6A and B). The present study further assessed the effect of APS and HCPT on the migration of H460 cells. APS and HCPT affected the migration of H460 cells similar to that of the H1299 cells (Fig. 7A and B).

Transwell assays were also performed with polycarbonate filters to analyze the migration and invasion of H1299 (Fig. 8A-D). It was demonstrated that APS and the combination of APS and HCPT inhibited migration by 46.21±7.93 and 53.09±6.7%, respectively, whereas HCPT only had a moderate effect on migration, indicating that treatment with APS and the combined agents inhibited the migration of H1299 cells more effectively than HCPT (Fig. 8A and C). It was also observed that treatment with APS, the combined agents and HCPT inhibited the invasion of H1299 cells by 75.13±6.63, 87.22±3.01 and 42.52±16.36%, respectively, compared with the control, suggesting that the combination of the agents was more effective in inhibiting H1299 migration and invasion compared with APS treatment alone or HCPT alone (Fig. 8B and D).

The present study considered whether the overexpression of MAP4K3 stimulates the migration of H1299 cells, despite H1299 exhibit relatively high background expression of MAP4K3. The H1299 cells were transfected with wild-type MAP4K3 and KD MAP4K3, and it was demonstrated that cells transfected with wild-type MAP4K3 led to more rapid closure of the wounded area compared with the cells transfected with KD MAP4K3 or the vector control (Fig. 9A-C). It was also observed that cells transfected with wild-type MAP4K3 and the vector control almost fully reversed the inflicted wound within a 60-h incubation period, whereas the cells transfected with the KD MAP4K3 exhibited decreased migration, suggesting that the KD MAP4K3 behaves like a dominant negative mutation. The results also suggested that, when cells transfected with wild-type MAP4K3 were exposed to APS, their invasive properties were reduced (Fig. 9D and E). The overexpression of MAP4K3 also increased wound closure in the H460 cell line (Fig. 10A and B).