Human adenocarcinoma A549 and H1299 cells (Sigma-Aldrich, St. Louis, MO, USA) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin and 100 µg/ml streptomycin. Cellular viability was measured by MTT reduction assay. MTT solution (5 mg/ml) was added to each well. After a 4-h incubation, MTT solution was removed and isopropanol-hydrochloric acid (25:1) was added to dissolve the formed formazan crystals. The absorbance was measured at 570 nm against a 690 nm reference with a microplate reader.

For adhesion assays, A549 and H1299 cells were treated with TGF-β1 (5 ng/ml) or/and nobiletin for 24 h. Cells were suspended in DMEM and plated at a density of 1×10⁴ cells/well onto 96-well plates coated with collagen IV. Cells were then fixed with methanol and stained with 0.1% crystal violet and absorbance was read at 595 nm spectrophotometrically.

For the scratch assays, A549 cells were plated in 24-well tissue culture plates and allowed to reach confluency. At this point a scratch was introduced into the monolayer using a sterile 200-µl pipette tip. The cells were then washed in warm phosphate-buffered saline (PBS) to remove the debris and the medium was replaced with either fresh medium (control samples) or medium containing TGF-β1 (5 ng/ml) or/and nobiletin (10 and 20 µM) for 24 h. Three independent experiments were carried out for each experimental condition.

The migration assay was performed using 24-well 8-µm pore Transwell inserts (Corning), with the upper face of polycarbonate filters coated with Matrigel. Cells (1×10⁵) were added to the upper chamber. The lower chambers were filled with 20% FBS/DMEM media to serve as a chemoattractant. The non-migrated cells in the upper chamber were removed gently by a cotton swab at 24 h following reseeding, whereas, the migrated cells in the lower side were stained with 0.1% crystal violet. The mean of six fields from three separate trials was used to calculate the average number of migratory cells.

Immunofluorescence assay was performed as previously described (16). In brief, cultured cells grown on plastic chamber slides were fixed in 4% formaldehyde for 30 min at room temperature. Then the cells were permeabilized in 0.2% Triton X-100 and blocked with 10% goat serum, and then incubated overnight at 4°C with the primary antibodies (diluted 1:100 in blocking buffer) against E-cadherin and N-cadherin. Afterwards, the sample was washed with PBS containing blocking buffer and incubated with the fluorophore-conjugated secondary antibody (Abcam). Cover glasses were counterstained with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) (5 mg/ml; Invitrogen Life Technologies). Fluorescence images were captured using a fluorescence microscope (Carl Zeiss, Göttingen, Germany).

PcDNA3.1-Flag-Smad3 plasmid was prepared using Qiagen plasmid DNA preparation kit. Plasmid or Smad2/3 small interfering RNA (siRNA) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) transient transfection of A549 cells was performed according to the protocol of the Lipofectamine 2000™ reagent (Invitrogen, Carlsbad, CA, USA).

A549 and H1299 cells were lysed at 4°C (30 min) in Tris-buffered saline. Whole lysates were analyzed by SDS-PAGE, and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% skim milk with Tween-20 and probed with primary antibodies against E-cadherin, N-cadherin, vimentin, Snail, Slug, ZEB1, Twist, Src, FAK and β-actin (Thermo Fisher Scientific). Bound antibodies were conjugated with horseradish peroxidase secondary antibodies, and the membranes were developed with chemoluminescence reagents. Protein expression was semi-quantified by densitometric analysis.

Total RNA was isolated from A549 cells using the total RNA extraction kit as described by the manufacturer (Life Technologies, Inc., Grand Island, NY, USA). RT-PCR was performed using the Qiagen RT-PCR kit. The appropriate primers (MMP-2, 5′-GGCCCTGTCACTCCTGAGAT-3′ and 5′-GGCATCCAGGTTATCGGGGA-3′; MMP-9, 5′-AGGCCTCTACAGAGTCTTTG-3′ and 5′-CAGTCCAACAAGAAAGGACG-3′; GADPH, 5′-GGCCTCCAAGGAGTAAGACC-3′ and 5′-AGGGGTCTACATGGCAACTG-3′) were used for PCR amplifications under the following conditions: 30 cycles at 95°C for 1 min, 60°C (MMP-2 and GAPDH) or 59°C (MMP-9) for 1 min, 72°C for 1 min and 72°C for 10 min. The PCR products were separated by 1.5% agarose gel and visualized by ethidium bromide staining.

All mice were obtained from the SLAC Laboratory Animal Center of Shanghai. Ethical approval was obtained for the use of the animals, and all experiments were performed in accordance with the Guidelines for Animal Care of the Institutional Animal Care and Use Committee of the Tumor Hospital of Xinjiang Medical University. i) Five-week-old nude mice were injected with A549 cells via the tail vein (4×10⁵ cells in 0.2 ml PBS). Nobiletin (20 and 40 mg/kg) was orally administered once daily. Cyclophosphamide (20 mg/kg) was used as the positive control, injected intraperitoneally every three days. Saline was administered orally every day for the control group (n=8 mice/group). After 42 days, the mice were sacrificed. After fixation in Bouin's solution, the number of lung metastases were confirmed under a dissecting micro-scope. The lungs were further subjected to hematoxylin and eosin (H&E) staining. ii) C57BL/6 mice were injected with Lewis cells via the tail vein (8×10⁵ cells in PBS) to establish a metastatic model. Administration was the same as the first model. After 15 days, the mice were sacrificed and lung meta-static nodules were confirmed under a microscope. iii) Nude mice were implanted subcutaneously with A549-Luc cells. Administration was the same as the first model. Thirty days after cell implantation, imaging was performed with an IVIS imaging system (Caliper Life Sciences, Inc., Hopkinton, MA, USA), and nude mice were sacrificed and tumors were excised and weighed.

Immunohistochemistry of lung tissue was performed as previously reported (17). Formalin-fixed, paraffin-embedded tissue sections were cut at a thickness of 4 µm. Antigen recovery was performed by heating in 0.01 M sodium citrate buffer at 80°C for 30 min using a microwave oven. Endogenous peroxidase activity and non-specific staining were blocked with the kit reagents. The tissues were incubated overnight with the primary E-cadherin and N-cadherin anti-bodies. Tissues were then incubated with peroxidase-bound IgG. The slides were counterstained with hematoxylin. Mounted tissues were imaged on an Olympus microscope. The numbers of E-cadherin- or N-cadherin-positive cells were determined at a magnification of ×400 in three segments/slide.

Statistical analysis was performed using ANOVA, χ² or Student's t-test using SPSS 16.0 software. Statistical significance was assigned at P<0.05.

An MTT assay was used to determine the non-cytotoxic concentrations of nobiletin (0–100 µM) in the presence of TGF-β1 (5 ng/ml) for 24 h. According to the MTT assays (Fig. 1B), concentrations of 10 and 20 µM were selected for further study, which exhibited no significant toxicity on cell viability. EMT is a crucial step in tumor metastasis, characterized by polarity and epithelial markers, as well as motile mesenchymal cells. Specifically, E-cadherin is a negative EMT biomarker, while N-cadherin is a mesenchymal biomarker. As shown in the immunofluorescence assay, TGF-β1-stimulated A549 and H1299 cells exhibited characteristic N-cadherin increase and E-cadherin decrease (Fig. 1C). However, nobiletin rebalances EMT-related genes disturbed by TGF-β1. In addition, similar results were obtained from the protein assays (Fig. 1D). The most prominent E-cadherin regulators are Snail-related zinc-finger transcription factors (Snail and Slug), ZEB1 and Twist. TGF-β1 treatment increased the expression of Snail, Slug, ZEB1 and Twist, and the increase was reversed by nobiletin treatment (Fig. 1D).

Nobiletin suppressed both A549 and H1299 cell invasion in the transmembrane migration assay induced by TGF-β1 (Fig. 2A). Moreover, nobiletin inhibited the capacity of A549 and H1299 cells to adhere to wells coated with human collagen type IV. After TGF-β1 stimulation, A549 cells displayed enhanced wound closure activity and almost filled the scratched area, whereas the wound still displayed an obvious gap in the nobiletin-pretreated group.

RT-PCR and western blot assays were applied to examine the mRNA and protein levels of MMP-2/-9. An increase in the mRNA and protein levels of MMP-2/-9 was observed following TGF-β1 incubation, while nobiletin treatment abrogated the increase (Fig. 2B). Activation of FAK is normally induced by various growth factors, including TGF-β. Therefore, we further explored the effect of nobiletin on the activation of focal adhesion-related signaling molecules, FAK, Src and paxillin. As shown in Fig. 2C, TGF-β1 induced the phosphorylation of FAK, Src and paxillin and these phosphorylation events were inhibited by nobiletin.

In the TGF-β1/Smad signaling pathway, TGF-βR2 induces recruitment and phosphorylation of TGF-βR1 after binding to TGF-β1 (18). Both TGFβR1 and TGFβR2 can be detected in A549 cells. As expected, the expression of TGFβR1 and TGFβR1 did not change following either TGF-β1 or nobiletin treatment. TGF-β1-triggered EMT is Smad-dependent. The activated TGF-βR1 subsequently induces phosphorylation of Smad2/3. Therefore, western blot analysis was carried out to examine whether nobiletin influences Smad phosphorylation. TGF-β1-treated cells showed aggravated phosphorylation of Smad2/3 after a 60-min incubation, and the increase in phosphorylation was not affected after nobiletin exposure (Fig. 3A). Phosphorylated Smad2 and Smad3 form heteromeric complexes with the mediator Smad4. These Smad2/3/4 complexes translocate to the nucleus, and bind to the Smad binding elements (SBEs). Nobiletin failed to prevent Smads from translocating into the nucleus, as induced by TGF-β (Fig. 3B). Transcriptional activity of Smad was also examined. A SBE-luciferase reporter plasmid was stably transfected into A549 cells to evaluate the luciferase activity. Consistent with previous studies, TGF-β1 considerably augmented the luciferase-reporter activity of Smad, which was greatly abolished by nobiletin treatment (Fig. 3C). These data infer that nobiletin prohibited EMT by eliminating the transcriptional activity of Smads, rather than influencing the translocation or phosphorylation.

In order to explore the detailed mechanism of nobiletin in regards to EMT, we transfected Smad2 siRNA and Smad3 siRNA into A549, and then detected the EMT-related protein levels. The protein expression of E-cadherin was significantly upregulated while N-cadherin, Snail and ZEB1 were downregulated due to Smad3 knockdown. In contrast, Smad2 siRNA had little or negligible effect on the expression of EMT-related proteins. In the nobiletin-treated group, these protein expression levels exhibited a similar tendency compared with the Smad3 siRNA-transfected A549 cells (Fig. 4A). To further confirm the interaction between nobiletin and Smad3, A549 cells were transfected with PcDNA3.1-Flag-Smad3. Accordingly, the expression of E-cadherin was decreased, while N-cadherin, Snail and ZEB1 were increased. Smad3 overexpression considerably reduced the ability of nobiletin to inhibit the TGF-β1-induced increase in N-cadherin and to reverse the TGF-β-stimulated decrease in E-cadherin. These data reveal the essential role of Smad3 in EMT progression and propose that Smad3 is involved in the inhibitory effects of nobiletin on EMT. We further examined the promoter expression of E-cadherin and N-cadherin. Nobiletin treatment resulted in the reversal of the E-cadherin promoter decrease as well as the suppression of N-cadherin promoter increase (Fig. 4B). Since the promoters of E-cadherin and N-cadherin are dependent on Smad3, we propose that nobiletin obstructs EMT by blocking Smad3-dependent transcriptional activity.

To further explore the antimetastatic potential of nobiletin in vivo, two experimental metastatic models of lung cancer were applied. One model was established by injecting A549 cells in nude mice via the tail vein. Nobiletin (20 and 40 mg/kg) was administered orally once daily. Cyclophosphamide was used as the positive control. As shown in Fig. 5A, the number of lung metastatic nodules was 65±15 in the control group on day 42, while there were only 43±11 and 29±9 nodules in the 20 and 40 mg/kg group, respectively (P<0.05, vs. the control group). As depicted in the H&E staining, tumor cells were organized as nests, with necrosis in the center of the metastatic area. Metastasis was less severe in the nobiletin-treated group, suggesting that nobiletin markedly inhibited metastasis in vivo. Similar results were observed in another experimental metastatic model, which was established by injecting Lewis cells into C57BL/6 mice via the tail vein. Representative lung tissue images are shown in Fig. 5B.

Tumor xenografts transplanted by A549-Luc cells were applied to assess the antitumor effect of nobiletin in vivo. IVIS imaging results revealed strong suppression of tumor volume in the nobiletin-treated nude mice (P<0.01 vs. the control group) (Fig. 5C). In the IHC analysis, nobiletin facilitated E-cadherin protein expression, and inhibited N-cadherin protein. All these results suggest that nobiletin suppressed EMT and the growth of lung cancer cells in vivo.