Dendrofalconerol A Suppresses Migrating Cancer Cells via EMT and Integrin Proteins

Materials and Methods

DF-A preparation. Dendrofalconerol A (>98%) was isolated from the aerial parts of Dendrobium falconeri. Briefly, the dried stems of this plant (800 g) were powdered and extracted with MeOH at room temperature. The MeOH extract was filtered and evaporated under reduced pressure to give a viscous mass (73 g). This material was subjected to vacuum-liquid chromatography on silica gel (EtOAc-hexane gradient) to give fractions A-K. Fraction I (1.51 g) was separated by CC (silica gel; EtOAc-haxane, gradient and EtOAc-hexane, 1:4) to give 35 fractions. Fraction 25 (216 mg) was further purified by Sephadex LH20 (MeOH) to give dendrofalconerol A (29 mg). The structure of DF-A was determined through analysis of its spectroscopic data.

Cell culture and DF-A treatment. The non-small cell lung cancer H460 cell line used in this study was obtained from American Type Culture Collection (Manassas, VA, USA). H460 cells were cultured in RPMI medium. Media were supplemented with 10% fetal bovine serum (FBS), 2 mmol/l L-glutamine and 100 units/ml penicillin/streptomycin. Cells were maintained in a humidified incubator containing 5% CO₂ at 37°C. DF-A was dissolved in DMSO and diluted with sterile PBS to achieve the working concentrations. The percentage of DMSO in the working solution was less than 0.1, which showed no toxicity in H460 cell.

Chemicals. Trypsin, Hoechst 33342, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), PBS, dimethysulfoxide (DMSO) were obtained from Sigma Chemical, Inc. (St. Louis, MO, USA). Integrin β₁, integrin α₄, vimentin, Snail, Slug, E-cadherin, N-cadherin and β-actin rabbit antibody were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Cell viability assay. To determine DF-A-mediated cytotoxicity, cell viability was determined by the MTT assay, which measures cellular capacity to reduce 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (yellow) to purple formazan crystals by the mitochondria dehydrogenase enzyme. After treatment, the medium was replaced by 100 μl/well of MTT solution (0.4 mg/ml) and cells were incubated for 4 h at 37°C. Subsequently, the MTT solution was removed and a 100 μl/well of DMSO were added to dissolve the formazan crystals. At the end of the assay period, the intensity was measured at 570 nm using a microplate reader (SpectraMax® M5, Molecular Devices, City, Ca, USA). All analyses were performed in at least three independent replicate cultures. The cell viability was calculated from the optical density (OD) ratio of treated to non-treated control cells and presented as a percentage of the non-treated controls.

Nuclear staining assay. Cells were treated with or without DF-A in RPMI serum medium under various concentrations for 24 h at 37°C. Apoptotic and necrotic cell death was determined by Hoechst 33342/propidium iodide (PI) co-staining. Then, cells were incubated with 10 μM of Hoechst 33342 dye and 5 μg/ml of PI for 30 min at 37°C in the dark. Finally, cells were visualized under a fluorescence microscope (Olympus IX51 with DP70, Olympus, Center Valley, PA, USA). The blue fluorescent Hoechst dye detected apoptotic cells, while the red fluorescent propidium iodide detected necrotic cells.

Western blot analysis. Cells were harvested and lysed on ice for 60 min. After the sample had been prepared, the protein content of cell lysate was determined using the BSA protein assay kit (Pierce, Rockford, IL, USA). An equal amount of protein of each sample was separated by size using SDS-PAGE and then transferred into nitrocellulose membranes. The membrane was blocked in 5% skim milk in TBST (25 mmol/l Tris-HCl, pH 7.4, 125 mmol/l NaCl, 0.1% Tween 20) for 1 h at room temperature and then probed with appropriate primary antibodies at 4°C overnight and washed three times with TBST for 8 min. Subsequently, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies according to the primary antibodies for 2 h at room temperature. Lastly, the signal of immunoreactive proteins was detected by enhanced chemiluminescence (Supersignal West Pico; Pierce). Protein expression (E-cadherin (E-cad), N-cadherin (N-cad), Snail, Slug, vimentin, integrin β₁, Integrin α₄) was investigated and β-actin was used as a loading of control in each treatment.

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Figure 1.

Structure of DF-A (C₃₂H₃₂O₈) MW. 544.

Statistical analysis. Mean data from at least three independent experiments were normalized to result in the non-treated control. All data were presented as the means±standard error of the mean (S.E.M.). Statistical differences between means were determined using an analysis of variance (ANOVA) and post-hoc test at a significance level of p<0.05.