The LKB1-null human lung cell line A549 cells were plated in culture plates in Dulbecco’s minimal essential medium (DMEM, Gibco, Grand Island, NY, USA) containing 10% (v/v) fetal bovine serum (FBS, Gibco). The cultures were maintained at 37°C in a humidified atmosphere of 5% CO₂. After culture to 70% confluence on 6-well plates, cells were transfected with pcDNA/LKB1 or control (vehicle) pcDNA3.1 (the vectors containing Neomicin/G418 resistance gene were kindly provided by Dr Qingzhi Xu) in the presence of Lipofectamine 2000 (Invitrogen Corp., Grand Island, NY, USA) following the manufacturer’s protocol. Transfection medium was replaced with growth medium containing 10% FBS after cells were incubated with transfection reagents for 6 h. Then, day 16 after selection with complete DMEM containing 600 μg/ml G418 (Gibco), all untransfected cells died and discrete clones were visible in transfected cells. These clones were expanded in the presence of 300 μg/ ml G418 to be used for the further study. Cells transfected with pcDNA/LKB1 and pcDNA3.1 vectors were termed A549/LKB1 and A549/vec, respectively. Both transfection and G418 selection were conducted under sterile conditions and duplicate plates were tested for each condition. The full-length human Sp1 AN cDNA was made by cloning EcoRI restriction fragments from the plasmid pBS-Sp1-AN (a kind gift from Dr J. Marvel and Dr Y. Leverrier, France) and was inserted into EcoRI sites of pcDNA3.1.

Total RNA was isolated from the cells using TRIzol reagent (Invitrogen) according to the procedure provided by the manufacturer. Reverse transcription was performed from total RNA at 42°C for 60 min by M-MLV Reverse Transcriptase (RNase-free, Takara, Shiga, Japan). PCR amplification was performed to synthesize these gene products, and the products were analyzed by electrophoresis on a 1.5% agarose gel. The ratio of the yield of mRNA to the yield of the relevant internal standard [glyceraldehyde-3-phosphate dehydrogenase (GAPDH)] was calculated and compared. The specific primers and their annealing temperatures are listed in Table I.

Total cell lysates were prepared using the cell lysis buffer (Pierce, Rockford, IL, USA) following the manufacturer’s instructions. Total protein was estimated using BCA analysis (Pierce). The samples were re-suspended in SDS-PAGE loading buffer and heated at 100°C for 5 min. Equal amounts of protein were loaded on gel and then transferred to nitrocellulose membrane (Mini-Protean and Trans-Blot systems, Bio-Rad Laboratories, Hercules, CA, USA). The nitrocellulose membrane was blocked with 5% non-fat dry milk and separately incubated with corresponding specific primary antibodies, including 1:800 rabbit anti-LKB1 (Cell Signaling Technology, Danvers, MA, USA), 1:1,500 mouse anti-actin, 1:750 mouse anti-TF (AbD Serotec, Oxford, UK), 1:500 goat anti-VEGF (Santa Cruz Biotechnology, Santa Cruz, CA, USA), 1:1000 mouse anti-SP1 (Santa Cruz Biotechnology). After washing and incubating with corresponding secondary antibodies conjugated horseradish peroxidase (1:2,000 dilution of goat anti-rabbit IgG antibody, 1:1,500 dilution of rabbit anti-goat IgG antibody, 1:2,000 dilution of goat anti-mouse IgG antibody, respectively). The proteins were detected by chemiluminescence and exposure to light-sensitive film.

Cell adhesiveness to the extracellular matrices was measured by an adhesion assay. The 96-well culture plates were precoated with 50 μl Matrigel (diluted 1:8 in complete DMEM cell culture medium) and dried overnight at 4°C. The wells were then rehydrated and blocked for 1 h at 37°C with 1% bovine serum albumin (BSA) in serum-free DMEM cell culture medium. Cells were seeded at a density of 1×10⁵ cells/well on the Matrigel-coated 96-well plates and allowed to adhere for 1 h in a 37°C humid chamber. After incubation, non-adherent cells were carefully removed by washing gently with PBS. After stained for 4 h at 37°C with 10 μl MTT (5 mg/ml), adherent cells were diluted in 150 μl of DMSO and measured at 570 nm using a microtiter plate reader (Wako, Shanghai, China). All experiments were performed in triplicate. The adhesion index was calculated as the percentage ratio between the number of adherent test cells and the number of control cells.

The Boyden chamber procedure was used to evaluate cell migration activity in a 24-microwell chamber (Millipore, Billerica, MA, USA). The upper and lower wells were separated by a polyvinyl-pyrolidone-free poly-carbonate filter (8-μm pore size). The lower wells contained DMEM with 10% FBS. Cell suspension (2.5×10⁴ cells in 100 μl of serum-free medium) of A549 cells and A549/LKB1 cells (transfected with LKB1), was added to the upper wells, respectively. The chamber was incubated at 37°C for 24 h. Non-migrating cells on the upper surface of the filters were removed with a cotton swab. The filters were then removed and fixed in 95% ethanol for 10 min. Cells were stained with 0.5% crystal violet in 20% ethanol and counted using a light microscope in 10 random fields per each well. Migration was assayed by measuring the number of cells moving across the filter. The number of cells that migrated is given as the mean ± SD. Each experiment was performed in triplicate.

The Matrigel invasion assay (BD Biosciences, Heidelberg, Germany) was used to assess the invasive potential of A549 lung cancer cells. Briefly, BioCoat Matrigel invasion chambers were rehydrated according to the manufacturer’s instructions. Following rehydration, the medium was carefully removed. DMEM cell culture medium (500 μl) supplemented with 10% FBS was added to the bottom of 24-well plates. Cells were seeded at a density of 1×10⁵ cells/well into upper inserts and incubated at 37°C for 24 h in 5% CO₂. After 24 h, the non-invading cells were removed from the upper surface of the separating membrane by gentle scrubbing with a cotton swab. Invading cells were fixed in 4% paraformaldehyde for 5 min and stained with Giemsa for 9 min. The membranes were mounted on glass slides and manually counted in five random fields using a light microscope. All assays were performed in triplicate.

We previously constructed adenovirus mediated shRNA against TF (Ad-shRNA-TF) and identified its silencing effect on TF in our previous study (15). To determine the effect of TF expression on A549 cells, we knocked down the TF expression by using Ad-shRNA-TF. Briefly, the A549 cells were plated in 6-well plates and allowed to grow in 2% FBS media. At 70–80% confluence, the cells were infected with Ad-shRNA-TF or control at a MOI of 100 vp/cell. Three wells were left uninfected to serve as a negative control. All infections were performed in triplicate. After 12 h, the viral containing media was removed and replaced with fresh media containing 10% FBS. The silencing effect against TF of the recombinant adenovirus was detected by using RT-PCR and western blot analysis as previously described.

The chemiluminescent secreted alkaline phosphatase (SEAP) analysis kit (Clontech, Palo Alto, CA, USA) was used to detect the transcriptional activity of Sp1 in the transfected cells. pTALSEAP vector (negative control), pSEAG2-SEAP vector (positive control) and pSP-SEAP vector, in which SEAP expression is under the control of the Sp1 E-box binding element, were used to transfect the cells, respectively, with Lipofectamine 2000 (Gibco) as described above, except that cells were cultured in 24-well culture plates instead of 60-mm dishes. The culture medium was collected at 24 h after transfection, was then subjected to the chemiluminescent assay of SEAP activity using the great EscAPe SEAP kit (Clontech) according to the manufacturer’s instructions. The activity data were calculated as: (Sp1 driving vector activity - the negative control activity)/the positive control activity. Each vector was assayed in triplicate and three independent experiments were performed.

Results are expressed as the mean ± SD unless indicated otherwise. ANOVA and Student’s t-test were used to determine the statistical significance of differences among experimental groups. Significance was defined as p<0.05.

To study the effects of restoring LKB1 expression on A549 lung cancer cells that lack functional LKB1 expression. As described previously, cells were transfected with a mammalian expression vector alone (pcDNA3.1), with pcDNA/Neo carrying human LKB1 cDNA. We selected transfected cells using the Neomicin/G418 resistance gene contained in the pcDNA/Neo expression vector. Sixteen days after selection with G418, there was a clear reduction in the number of colonies expressing wild-type LKB1 compared with those carrying the empty vector. To confirm whether exogenous LKB1 is expressed in A549 transfected cells, we performed RT-PCR and western blot analysis on these transfectants, including clones 1, 2 and 3. As shown in lane 5 in Fig. 1, the clone 3 had a higher yield of both LKB1 mRNA and protein. Therefore, the third clone was used to make further experiments in this study.

Compared to the controls, the LKB1 reduced adhesion of A549 cells by 30.44% (p<0.05). The effects of LKB1 expression on cell migration in vitro are shown in Fig. 3. LKB1-expressing cells exhibited decreased migration. The number of A549/LKB1 cells that migrated through the filter was 54.2±13.77/HPF, which was significantly lower than that in A549 cells (97.4±10.98/HPF, p<0.05) (Fig. 2). The effect of LKB1 in regulating the invasive ability of A549 lung cancer cell was examined by using Matrigel invasion assays. The number of A549/LKB1 cells that migrated through the filter was 95.4±13.68/HPF. The transfected A549 cells with LKB1 reduced their invasive ability compared to untreated cells (177.2±16.43/HPF, p<0.05) (Fig. 3). Analysis on the invasive capacity of the analyzed lung cancer cells demonstrated that there was a correlation between LKB1 and invasiveness.

To further study the potential correlation between LKB1 and the relevant effectors, we examined the expression of TF and VEGF by using RT-PCR and western blot analysis. Compared with A549 control cells, we showed that LKB1-transfected A549 cells constitutively expressed significantly lower levels of TF and VEGF in both mRNA and protein levels (p<0.05; Fig. 4).

Specific gene inhibition in mammalian cells can be achieved by the use of small interfering RNA molecules (siRNA). The silencing effect against TF of the recombinant adenovirus pAd-shRNA-TF was detected by using RT-PCR and western blot analysis. Our results showed that specific reduction in the target protein level was observed after adenoviral infection, and that the reduction in the protein level was correlated with a specific reduction in the mRNA level (Fig. 5). We show that siRNA delivery significantly decreased levels of TF in the lung cancer cell line A549. After transfected with adenoviral vector expressing siRNA against TF, A549 cells showed significantly decreased invasion activity through Matrigel (Fig. 6).

Sp1 is an essential transcription factor for many genes that are key to the regulation of multiple aspects of tumor cell growth and metastasis. To determine whether LKB1 represses Sp1 expression, we examined the effects of LKB1 overexpression on Sp1 expression by using RT-PCR and western blot analysis. We found that overexpressing LKB1 mediated transcriptional repression of Sp1 gene expression at both the mRNA and protein levels (Fig. 7A and B). To verify this decreased expression of Sp1 protein after overexpressing LKB1, we then investigated the transcriptional activity of Sp1 by means of Sp1 driving the SEAP reporter expression strategy. In this reporter system, expression of SEAP (a secreted form of human placental alkaline phosphatase) in the pSP-SEAP vector is under the control of the E-box enhancer, an Sp1 binding element. Thus, the level of SEAP can well reflect the Sp1 activity in the tested cells. The culture medium was collected at 24 h after transfecting the cells with the pSP-SEAP vector or the control vectors, and the activities of SEAP in the medium were detected. As shown in Fig. 7E, the transcriptional activity of Sp1 was significantly decreased in the cells overexpressed LKB1.

We transfected increasing amounts of Sp1 recombinant plasmid into A549/LKB1 cells and measured the expression of mRNA and protein levels of TF and VEGF gene. Increasing expression of Sp1 enhanced the TF and VEGF mRNA and protein levels in A549/LKB1 cells (Fig. 8). These results supported the positive role of Sp1 in the regulation of TF and VEGF.