Batatasin III Inhibits Migration of Human Lung Cancer Cells by Suppressing Epithelial to Mesenchymal Transition and FAK-AKT Signals

Materials and Methods

Materials. H460 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). Penicillin, streptomycin, glutamate, and phosphate-buffered saline (PBS) were purchased from GIBCO (Grand Island, NY, USA). Methanol, ethanol and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies to Focal Adhesion Kinase (FAK), phosphorylation of Focal Adhesion Kinase on Try397 (p-FAK (Try397)), Protein Kinase B (AKT), phosphorylation of Protein Kinase B on Ser473 (p-AKT (Ser473)), Cell Division Cycle 42 (CDC42), E-Cadherin, N-Cadherin, Vimentin, α-Tubulin and specific horseradish peroxidase (HRP) link secondary antibody were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Batatasin III was extracted from Dendrobium draconis Rchb.f. using methanol extraction (5).

Cell culture conditions. H460 cells was cultured with Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine and 100 units/ml penicillin and streptomycin at 37°C in a humidified atmosphere of 5% CO₂. Cells were seeded as recommended by the supplier and split at 80% confluence.

Cell viability assay. The 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyl tetrazoliumbromide (MTT) assay was performed to determine the effect of Batatasin III on the cellular viability of H460 cells. Briefly, approximately 1×10⁴ cells/well were plated in 96-well plates and kept in an incubator at 37°C under the previously described cell culture conditions. After overnight incubation to allow adherence, the cells were treated with different concentrations of Batatasin III (10 μM to 100 μM) in fresh medium for 24 h. Each treatment at this time point was assayed in at least in triplicate. At the stipulated time following treatment with Batatasin III, the medium was aspirated and MTT (4 mg/ml stock solution in 1×PBS) was added to each well of the 96-well culture plate. Incubation was continued at 37°C for additional 4 h, then supernatants were discarded, and purple-colored precipitates of formazan were dissolved in 100 ml of DMSO. The color absorbance of each aliquot was recorded at 570 nm with a microplate reader and repeated three times. The cytotoxic effect was expressed as the percentage cell viability compared to that of non-treated cells.

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Figure 1.

Structure of Batatasin III.

Cell proliferation assay. To evaluate proliferative effects, H460 cells were seeded at a density of 2×10³ cells/well in 96-well plates and treated with Batatasin III (10 μM to 100 μM) for 0, 24 and 48 h. Next, cell proliferation was determined through incubation with MTT (4 mg/ml stock solution in 1×PBS) for 4 h at 37°C, after which the optical densities (OD) of the formazan products were measured at 570 nm using a microplate reader (Anthros, Durham, NC, USA). The proliferation rates were determined using the following equation: OD at indicated time/OD at time 0. The relative proliferation rates were determined by comparing the proliferation rates of treated cells with those of untreated control cells.

Filopodia detection. Cells were washed with phosphate buffer saline (PBS) and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. Afterwards, cells were permeabilized by 0.1% Triton-X100 in PBS for 4 min, washed with PBS three times, and blocked with 0.2% bovine serum albumin (BSA) for 30 min as described elsewhere. After washing, cells were incubated with rhodamine-phalloidin in PBS for 30 min and rinsed three times. Images of cells stained with rhodamine-labeled phalloidin were taken under a fluorescence microscope (Olympus IX51 with DP70).

Wound scratch assay for cell motility determination. H460 cells (2×10⁵ cells/ml) were seeded in a 24-well tissue culture plate and grown to 90% confluence. After aspiration of the medium, the center of the cell monolayers was scraped with a sterile micro-pipet tip to create a denuded zone (gap) of constant width. Subsequently, cellular debris was washed with PBS, and H460 cells were exposed to various concentrations of Batatasin III (10 μM to 100 μM). Wound closure was monitored and photographed at 0, 24 and 48 h with a Nikon inverted microscope. To quantify the migrated cells, pictures of the initial wounded monolayers were compared with the corresponding pictures of cells at the end of the incubation. Artificial lines fitting the cutting edges were drawn on pictures of the original wounds and overlaid on the pictures of cultures after incubation. Cells that had migrated across the white lines were counted in four random fields from each triplicate treatment.

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Figure 2.

Viability of non-small cell lung cancer H460 cells in response to 10, 25, 50 and 100 μM Batatasin III treatment for 24 h (A) and proliferation of the cell at 24 and 48 h (B). Cell viability was evaluated using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazoliumbromide (MTT) assays. Data represent mean±SD (n=3). *p<0.05 versus non-treated control. After 24 h the cells were stained with Hoechst 33342 and visualized by florescence microscopy (C).

Cell invasion assay. Transwell invasion assay was performed using a Boyden chamber (BD Bioscience, MA, USA). To this end, the transwell membrane was coated with Matrigel and cells were seeded at a density of 2×10⁴ cells/well in the upper chamber supplemented with RPMI-1640 serum free medium, while RPMI-1640 complete medium containing 10% FBS was added to the lower chamber compartment. After incubation at 37°C, the non-invaded cells in the upper chamber were gently removed with a cotton swab, whereas the cells that had moved to the lower compartment of the membrane were fixed with cold absolute methanol for 10 min and stained with 10 μg/ml Hoechst 33342 for 10 min. Finally, these cells were visualized and captured using a fluorescence microscope (Olympus IX5; 20x).

Western blotting analysis. Migration proteins were detected by western blot analysis. H460 cells were treated with 50 and 100 μM of Batatasin III for 24 h and harvested. All cells were collected by scraping and were lysed with lysis buffer for 45 min. The protein concentrations were measured with the Pierce™ BCA Protein Assay Kit (Pierce, Rockford, IL, USA). The resultant lysates were boiled at 95°C for 5 min with Laemmli loading buffer and were subsequently loaded onto a 10% SDS-polyacrylamide gel. After separation, the proteins were transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The membranes were blocked for 1 h in 5% non-fat dry milk in TBST (25 mM Tris–HCl (pH 7.5), 125 mM NaCl and 0.05% Tween 20) and incubated overnight with the propriate primary antibodies at 4°C. The membranes were washed twice with TBST for 10 min and incubated with horseradish peroxidase-coupled isotype-specific secondary antibodies for 1 h at room temperature. The immune complexes were detected by enhancement with a chemiluminescence substrate (Pierce, Rockford, IL, USA) and quantified using analyst/PC densitometry software (Bio-Rad, Hercules, CA, USA).

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Figure 3.

Migration behavior of non-small cell lung cancer H460 cells. Cells were treated with 10, 25, 50 and 100 μM Batatasin III for 24 h and subjected to wound scratch assay. Representative photographs of migrating cells that received either control or Batatasin III treatment (A). The relative cell migration of non-small cell lung cancer H460 cells are shown in each Batatasin III concentration (B). Data represent mean±SD (n=3). *p<0.05 versus non-treated control.

Statistical analysis. Data from three or more independent experiments are presented as the mean±standard deviation (SD). Multiple comparisons for significant differences between multiple groups were performed using analysis of variance (ANOVA), followed by individual comparisons with Scheffe's post-hoc test. Statistical significance was considered at p<0.05.