A Bibenzyl from Dendrobium ellipsophyllum Inhibits Epithelial-to-Mesenchymal Transition and Sensitizes Lung Cancer Cells to Anoikis

Materials and Methods

Cell culture. Human H292 lung cancer cells were obtained from the American Type Culture Collection (Manassas, VA, USA). They were cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS), 2 mmol/l L-glutamine, and 100 units/ml penicillin/ streptomycin (Gibco, Gaithersburg, MA, USA). Cells were maintained in a humidified incubator containing 5% CO2 at 37°C.

Chemical reagents. TDB with approximately 98% w/w was isolated from D. ellipsophyllum. Botanical identification was performed in comparison with herbarium specimens at the Department of National Park, Wildlife and Plant Conservation, Ministry of National Resources and Environment. A voucher specimen (BS-DE-052555) is on deposit at the Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Chulalongkorn University. Briefly, the whole plant of D. ellipsophyllum was dried and ground into powder. This dry powder (4.8 kg) was then extracted with methanol (3×10 l) at room temperature. The methanolic extract was filtered and evaporated under reduced pressure to give a viscous mass (400 g). This material (200 g) was subjected to vacuum-liquid chromatography (VLC) (Kieselgel 60, 70-320 mesh; Merck, Darmstadt, Germany) on silica gel (n-hexane–ethyl acetate gradient) to give five fractions (A-E). Fraction D (63 g) was separated by VLC over silica gel, eluted with n-hexane–ethyl acetate gradient to give seven fractions (D1-D7). Fraction D5 (5.4 g) was subjected to medium pressure liquid chromatography (MPLC) over silica gel, eluted with n-hexane–ethyl acetate gradient to give 14 fractions (D5A-D5N). Fraction D5G (954.3 mg) was separated by column chromatography (Kieselgel 60, 230-400 mesh, Merck; Darmstadt, Germany) over silica gel (n-hexane–ethyl acetate, gradient), and further purified on Sephadex LH20 (25-100 μm; Pharmacia Fine Chemical Co. Ltd., Uppsala, Sweden) with acetone to obtain TDB. For structural confirmation, the compound was analyzed through mass spectrophotometer and nuclear magnetic resonance spectroscopy (NMR) as described. Mass spectra were recorded on a UPLC mass spectrophotometry (Waters 2996-2695, ESI-MS). NMR spectra were recorded on a Bruker Avance DPX-300 FT-NMR spectrometer or a Varian Unity INOVA-500 NMR spectrometer.

Trypsin, Hoechst33342, 2,3-b-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide salt (XTT), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI), dimethylsulfoxide (DMSO) and agarose were obtained from Sigma Chemical, Inc. (St. Louis, MO, USA). Antibody to E-cadherin, vimentin, transcription factor SLUG, transcription factor SNAIL, ERK, pERK (Thr202/Thr204), AKT, pAKT (Ser 473), β-actin and specific horseradish peroxidase (HRP)-link secondary antibody were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Cell viability assay. H292 lung cancer cells were treated with TDB at various concentrations (0.5-20 μM) for 24 h or left untreated as a control. After indicated time, cell culture medium was replaced by 100 μl/well of MTT solution (0.4 mg/ml) and left in the dark at 37°C for 4 h. After that, MTT solution was removed and 100 μl/well of DMSO was added to dissolve the formazan crystals. The intensity of formazan color was measured at 570 nm using microplate reader (Anthros, Durham, NC, USA). All analyses were performed in at least three independent replicate cultures. The cell viability was calculated from optical density (OD) ratio of treated to non-treated control cells and presented as a percentage to that of non-treated controls.

Anoikis assay. H292 cells that had been treated with non-toxic concentrations of TDB for 24 h were detached. A single-cell suspension in culture medium was seeded into an ultra-low attachment plate (Corning, Acton, MA, USA) at a density of 1.5×10⁵ cells/ml. The cells were harvested and incubated with 20 μM of XTT for detection of cell viability at 0, 6, 12, and 24 h. After light protection at 37°C for 4 h, the intensity of the formazan product from XTT was measured at 450 nm using a microplate reader.

Nuclear staining assay. Apoptotic and necrotic cell death was determined by the Hoechst33342/PI co-staining assay. Treated cells were incubated with 10 μM of the Hoechst33342 dye and 5 μg/ml of the PI dye for 30 min at 37°C in the dark. Modes of cell death were visualized under a fluorescence microscope (Olympus IX51 with DP70). Blue fluorescent Hoechst33342 was detected as apoptotic cells, while the red fluorescence of PI represented necrotic cells.

Anchorage-independent growth assay. Anchorage-independent cell growth was carried out in two-layer soft agar. Briefly, 1% agarose was melted and kept at 55°C in a water-bath. The bottom layer was a mixture of 1% agarose and RPMI culture medium at a ratio of 1:1. After adding 500 μl/well of the mixture into a 24 well-plate, the agar was solidified at 4°C for 5 min. The upper layer was prepared form 1% agarose and a single-cell suspension of TDB-treated H292 cells to give the final concentration of 0.33% agarose and provide 1,500 cells/well (250 μl). The upper layer was allowed to set in an incubator at 37°C for 4 h, then the culture medium was added on top. Fresh complete RPMI medium (250 μl/well) was added every three days. After two weeks, colonies were photographed in order to count and measure colony number and size, respectively.

Western blot analysis. After treatment with TDB at non-toxic concentrations for 24 h or left untreat as a control. H292 cells were harvested and lysed on ice for 60 min. The protein content of cell lysate was determined using BSA protein assay kit (Pierce, Rockford, IL, USA). An equal amount of protein of each sample was separated by size using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to 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. Then the membrane was probed with primary antibody of E-cadherin, vimentin, SLUG, SNAIL, ERK, pERK (Thr202/Thr204), AKT, pAKT (Ser 473) and β-actin (Cell Signaling, Danvers, MA, USA) at 4°C overnight and washed three times with TBST for 8 min. After that, the membrane was incubated with HRP-conjugated secondary antibody (Cell Signaling, Danvers, MA, USA) for 2 h at room temperature. The signal of immunoreactive proteins was detected by enhanced chemiluminescence (Supersignal West Pico, Pierce). The quantitative analysis was performed using analyst/PC densitometric software (Bio-Rad Laboratory, Hercules, CA, USA).

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Figure 1.

Effect of 4,5,4’-trihydroxy-3,3’-dimethoxybibenzyl (TDB) on cell viability and proliferation. A: H292 cells were treated with different concentrations of TDB (0-20 μM) for 24 h, and viable cells were examined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. B: H292 cells were treated with 0.5-5 μM of TDB or left untreated as control for different time points (24-72 h). Cell proliferation was evaluated by MTT assay. The viability of untreated cells is represented as 100%. C: H292 cells were similarly treated with TDB for 24 h. Apoptotic and necrotic cell death was evaluated using Hoechst33342/propidium iodide (PI) co-staining. Data represent the means±SD (n=3). *p<0.05 versus untreated control cells.

Statistical analysis. Mean data from at least three independent experiments were normalized to the non-treated contros. Statistical analysis was performed using one-way ANOVA. A p-value of less than 0.05 was considered as statistically significant.