BBR changed cell morphology and inhibited cell migration

We first analyzed the effect of BBR on cell morphology in A549 and H1299 cells. Treatment with BBR at 100 µM BBR effectively reduced cell-to-cell contact and led to a lower spreading with fewer formation of filopodia by comparison with the DMSO control groups (Figure 1A). We also tested the effect of BBR on cell migration by employing a wound-healing assay. As shown in Figure 1B, the part of the wounding space between cell layers after making a scratch was occupied completely by the migrating cells after 48 h in the control group. However, the empty space of the cells was not occupied by the migrating cells treated with 20 µM BBR. These results demonstrated the potentials of BBR in changing cell morphology and inhibiting cell migration in lung cancer cells

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 1. BBR changed cell morphology and inhibited migration.

(A) The changes in cell morphology and spreading in A549 and H1299 cells treated with 100 µM BBR for 48 h were observed and cells were photographed using a microscope fitted with digital camera. (B) Cell migration was analyzed by a wound-healing assay. Cells were grown to full confluency. The cell monolayers were wounded with a sterile pipette tip, and washed with medium to remove detached cells from the plates. Cells were left either untreated or treated with 20 µM BBR. After 48 h, the wound gap was observed and cells were photographed.

https://doi.org/10.1371/journal.pone.0069240.g001

BBR suppressed cell proliferation and colony formation

Berberine has been shown to induce growth inhibition and apoptosis of non-small cell human lung cancer cells via p53 signaling pathway [43]. We next quantitatively analyzed the effect of BBR on cell proliferation in lung cancer A549 and H1299 cells by MTT assay. Treatment with BBR at the dose of 10 µM to 200 µM inhibited cell viability in a dose-dependent manner. The IC₅₀ values of BBR for A549 and H1299 cells are 67.1 µM and 59.2 µM, respectively (Figure 2A). We also tested the effects of BBR on tumor cell clonogenicity in A549 cells (Figure 2B). Treatment with BBR markedly inhibited colony formation, resulting in a significant decrease at both colony formation ratio (Figure 2C) and colony size (Figure 2D).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 2. BBR suppressed lung cancer cell proliferation and colony formation.

(A–C) Human A549 and H1299 cells were treated with BBR at the indicated doses. At 48 hours after treatment, the cell viability was determined by a MTT assay. The IC50 values of BBR for A549 and H1299 cells were calculated (A). The tumor cell A549-induced colony formation was also analyzed (B), and the colony formation rate (C) and colony size (D) were calculated. Cells treated with DMSO were used as the referent group with cell viability set at 100%. The percent cell viability in each treatment group was calculated relative to cells treated with DMSO vehicle control. The data are presented as mean ± SD of three tests. *, P < 0.05, significant differences between treatment groups and DMSO control groups.

https://doi.org/10.1371/journal.pone.0069240.g002

BBR inhibited AP-2/hTERT signaling

hTERT has been regarded as the hallmark of carcinogenesis and the expression of hTERT is tightly controlled by transcriptional factor AP-2. To determine whether BBR targets the AP-2/hTERT signaling, we treated A549 cells with BBR (20 µM or 100 µM) and examined the expression of AP-2α, AP-2β and hTERT proteins and mRNAs by Western blot and RT-PCR, respectively. Treatment with BBR led to a marked inhibition of the expression of AP-2α, AP-2β and hTERT at protein (Figure 3A) and mRNA levels (Figure 3B).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 3. BBR inhibited AP-2/hTERT signaling.

(A–C) Human NSCLC A549 cells were treated with BBR at the indicated doses. At 48 hours after treatment, the AP-2 and hTERT proteins (A) and mRNA (B) were analyzed by Western blotting and RT-PCR, respectively. GAPDH were used as controls for sample loading. The binding of AP-2 to hTERT promoter probe (C) was analyzed by a streptavidin-agarose pulldown assay. (D) A549 cells were transfected with an AP-2 siRNA or an AP-2-expressing vector for 24 hours, and then treated with BBR (100 µM). At 48 hours after treatment, protein expression and cell viability were determined by Western blot and MTT assay, respectively. The percent cell viability in each treatment group was calculated relative to cells treated with the vehicle control. The data are presented as the mean ± SD of three separate experiments. *, P < 0.05, significant differences between treatment groups and DMSO control groups.

https://doi.org/10.1371/journal.pone.0069240.g003

Since hTERT expression is tightly controlled by the binding activity of AP-2 family on hTERT promoter, we next performed streptavidin-agarose pull-down assay to examine the effect of BBR on AP-2α and AP-2β binding activities at hTERT promoter. As shown in Figure 3C, BBR treatment effectively abrogated AP-2α and AP-2β binding to the biotin-labeled hTERT promoter DNA probe (Figure 3C).

To confirm the role of AP-2 signaling in BBR-mediated cell proliferation inhibition, we transfected A549 cells with AP-2α or AP-2β siRNA (100 nM) and then treated them with BBR (100 µM) for 48 h. The results showed that transfection with AP-2α or AP-2β siRNA effectively down-regulated the expression of AP-2α or AP-2β protein and considerably inhibited cell viability (Figure 3D). Knockdown of AP-2α or AP-2β by siRNA also markedly augmented the BBR-mediated inhibition of cell viability compared with the transfection with the control siRNA (si-NC) (Figure 3D). These results confirm that the effect of BBR on cell proliferation inhibition is mediated at least in part through AP-2/hTERT signaling pathway in lung cancer cells.

BBR inhibited NF-κB/COX-2 signaling

High expression of COX-2 is implicated in cancer cell growth, migration and angiogenesis. To determine the effects of BBR on COX-2 signaling in lung cancer cells, we next analyzed the expression of COX-2 protein in A549 cells treated with BBR by Western blot. Treatment with BBR at the dose of 20 µM did not significantly inhibited COX-2 protein expression, while the dose of 100 µM markedly decreased the COX-2 protein expression in A549 cells (Figure 4A).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 4. BBR inhibited NF-κB/COX-2 signaling.

(A) Human A549 cells were treated with BBR at the indicated doses. At 48 hours after treatment, the COX-2 protein was analyzed by Western blotting. GAPDH were used as controls for sample loading. (B) A549 cells were pretreated with the COX-2 selective inhibitor celecoxib (CB, 20 µM) for 24 hours, and then treated with BBR (20 µM). At 48 hours after treatment, cell viability was determined by MTT analysis. The percent cell viability in each treatment group was calculated relative to cells treated with the vehicle control. (C) A549 cells were treated with BBR at the indicated doses. At 48 hours after treatment, the binding of p50 and p65 to COX-2 promoter probe was analyzed by a streptavidin-agarose pulldown assay. (D) A549 cells were treated with BBR (100 nM). At 48 hours after treatment, the effect of BBR on NF-κB p65 and p50 translocation was analyzed by immunofluorescence assay. The data are presented as the mean ± SD of three separate experiments. *, P < 0.05, significant differences between treatment groups and DMSO control groups.

https://doi.org/10.1371/journal.pone.0069240.g004

The expression of COX-2 is tightly regulated by the binding activity of p50/p65 NF-κB on COX-2 promoter structure. We next determined whether the BBR-induced inhibition of tumor cell proliferation and COX-2 expression is mediated by inhibition of the binding of NF-κB to COX-2 promoter in A549 cells. Streptavidin-agarose pull-down assay showed that BBR at the dose of 100 µM markedly inhibited NF-κB p50 and p65 binding to COX-2 promoter probe as compared with the control group (Figure 4B). By contrast, BBR at the dose of 20 and 100 µM did not affect p50 and p65 protein levels in whole cell lysates (Figure 4A).

The translocation of NF-κB p65 and p50 in cell nuclei and cytoplasma plays a key role in regulate COX-2 gene expression. We next performed immunofluorescence assay to evaluate the effect of BBR on NF-κB p65 and p50 translocation in A549 cells. Constitutive translocation of NF-κB p65 and p50 to the cell nuclei was detected (Figure 4C). Treatment with BBR (100 µM) effectively promoted translocation of the NF-κB p65 and p50 from cell nuclei to cytoplasma. The results suggest that the inhibition of tumor cell growth by BBR might be also mediated by modulating the NF-κB/COX-2 signaling pathway in lung cancer cells.

BBR inhibited HIF-1α/VEGF, PI3K/AKT, Raf/MEK/ERK signaling

The HIF-1α/VEGF/PEDF signaling is closely associated with cancer cell growth, migration and angiogenesis. We next determined the effect of BBR on the expression of HIF-1α, VEGF and PEDF at protein and mRNA levels in lung cancer cells by RT-PCR and Western blot, respectively. Treatment of A549 cells with BBR significantly inhibited the expression of HIF-1α and VEGF, but not PEDF, at protein (Figure 5A) and mRNA levels (Figure 5B).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 5. BBR inhibited BBR inhibited HIF-1α/VEGF Raf/MEK/ERK and PI3K/AKT signaling.

A549 cells were treated with BBR at the indicated doses. At 48 hours after treatment, the expression of HIF-1α, VEGF and PEDF at protein (A) or mRNA levels (B), as well as the total and phosphorylated Akt and ERK1/2 proteins (D), were determined. A549 cells were treated with the HIF-1α-specific siRNA (si-HIF) (C) or the Akt-selective inhibitor LY294002 (LY,30 µM) or the ERK-selective inhibitor U0126 (U, 50 µM) (E) for 24 hours, respectively, and then treated with BBR. At 48 hours after treatment, cell viability was determined by MTT analysis. The percent cell viability in each treatment group was calculated relative to cells treated with the vehicle control. The data are presented as the mean ± SD of three separate experiments. *, P < 0.05, significant differences between treatment groups and DMSO control groups.

https://doi.org/10.1371/journal.pone.0069240.g005

To validate the effects of BBR on HIF-1α/VEGF/PEDF signaling, we transfected A549 cells with HIF-1α siRNA (100 nM) and then treated them with BBR (100 µM) for 48 h. As shown in Figure 5C, knockdown of HIF-1α by siRNA considerably increased the BBR-mediated inhibition of cell proliferation as compared with the transfection with the control siRNA. These results confirm that the effect of BBR on cell proliferation inhibition is also mediated partially through HIF-1α/VEGF/PEDF signaling pathway in lung cancer cells.

The PI3K/AKT and Raf/MEK/ERK signaling plays an important role in regulating tumor cell proliferation and survival. To determine whether the BBR-mediated cell growth inhibition is also through the inactivation of the PI3K/AKT and Raf/MEK/ERK signaling pathway, we analyzed the effect of BBR on the phosphorylation of Akt and ERK in A549 cells by Western blot. As shown in Figure 5D, treatment with BBR significantly decreased the levels of the phosphorylated Akt and ERK1/2 proteins, whereas the levels of total Akt and ERK1/2 protein did not change.

BBR activated caspase-dependent apoptotic pathway

We also determined whether the enhancement of cell growth inhibition induced by BBR is associated with the increase of apoptosis in lung cancer cells. Treatment with BBR at the doses of 20 µM and 100 µM induced 26% and 50% apoptotic cells in A549 and 28% and 60% apoptotic cells in H1299 cells (Figure 6A). To confirm the effect of BBR on apoptosis, we next detected the expression of some pro-apoptotic and anti-apoptotic proteins, caspase-3/7/9, PARP, BAX and Bcl-2 in A549 cells by Western blot analysis. BBR markedly increased the expression levels of cleaved caspase-3/7/9, cleaved PARP and BAX proteins, but decreased the Bcl-2 protein levels, as compared with the control group (Figure 6B).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 6. BBR activated caspase-dependet apoptotic pathway.

A549 and H1299 cells were treated with BBR (20 µM or 100 µM). At 48 hours after treatment, the apoptosis was determined by a FACS analysis (A). The levels of the cleaved caspase-3, 7, 9, cleaved PARP, BAX and Bcl-2 proteins in A549 cells were analyzed by Western blot (B). The release of Cyt-c in A549 cells was analyzed by immunofluorescence imaging analysis to monitor Cyt-c release from the inter-mitochondrial space into the cytosol (C). The apoptosis are represented by relative percentages of apoptotic cells versus that in DMSO-treated cells. *, P<0.05, significant differences between the BBR-treated groups and the DMSO-treated groups.

https://doi.org/10.1371/journal.pone.0069240.g006

Cytochrome-c (Cyto-c) release is an important event in the caspase-dependent apoptosis pathway. We next performed immunofluorescence imaging (IF) analysis to monitor changes in the subcellular localization of Cyto-c in A549 cells to determine whether BBR could induce Cyto-c release. Treatment with BBR (20 µM or 100 µM) markedly triggered the release of Cyto-c from the inter-mitochondrial space into the cytosol (Figure 6C). These results demonstrate that BBR may facilitate the downstream Cyto-c-dependent apoptosome assembly and caspase activation in the cytosol in lung cancer cells.

Cell culture

The human lung cancer cell lines A549 and H1299 were obtained from American Type Culture Collection (ATCC, Manassas, VA). Cells were cultured as monolayers in RPMI 1640 culture media supplemented with 10% heat-inactivated fetal bovine serum, 100 µg/ml penicillin, and 100 µg/ml streptomycin and maintained in an incubator with a humidified atmosphere of 95% air and 5% CO₂ at 37 °C.

Reagents and antibodies

Berberine (BBR), U0126, LY294002 and celecoxib were purchased from Sigma (St. Louis, MO) and dissolved in a small amount of DMSO before addition to the complete cell culture medium. Streptavidin-agarose was purchased from Sigma (St. Louis, MO). Antibodies to GAPDH, VEGF, PEDF, HIF-1α, p50, p65, COX-2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against AP-2α, AP-2β, hTERT, cytochrome-c, PARP, caspase-3/7/9, BAX, Bcl-2, Akt, ERK1/2, phosphrylated Akt or ERK1/2 were purchased from Cell Signaling (Beverly, MA).

Wound-healing assay

The wound-healing assay was performed to detect cell migration. The cells were grown to full confluency in six-well plates and incubated overnight in starvation medium. Cell monolayers were wounded with a sterile 100 µL pipettetip, washed with starvation medium to remove detached cells from the plates. Cells were treated with indicated doses of BBR in full medium and kept in a CO₂ incubator. After 48 h, medium was replaced with PBS, the wound gap was observed and cells were photographed using an Olympus microscope fitted with digital camera.

Cell viability assay

Cell viability was determined using MTT assay (Roche Diagnosis, Indianapolis, IN). Briefly, lung cancer cell lines were seeded at 4×103 cells/well in 96-well plates. Cells were cultured for overnight, and then the cells were changed to fresh medium containing various concentrations of BBR dissolved in DMSO (final concentration, 0.1%). After the cells were incubated for 48 h, the growth of cells was measured. The effect on cell viability was assessed as the percent cell viability compared with vehicle-treated control cells, which were arbitrarily assigned 100% viability. The BBR concentration required to cause 50% cell growth inhibition (IC50) was determined by interpolation from dose–response curves.

Anchorage independent colony formation assay

A549 cells plated in 6-well plates were treated with BBR. After 24h, cells were washed with PBS and trypsinized. Then cells (8×10³/ml) were mixed in 1.0 ml of 0.3% McCoy’s 5a agar containing 10% FBS. The cultures were maintained in a 37°C, 5% CO₂ incubator for 14 days. The medium was discarded and the cells were carefully washed with PBS twice. After being fixed with 4% paraformaldehyde for 15min, the cells were stained with 0.1% crystal violet for 15 min before washing with tap water and air-drying. The colonies with more than 50 cells were counted with an ordinary optical microscope. The colony formation rate was calculated with the following formula: Plate colony formation inhibitory ratio = (number of colonies treated with BBR/number of cells inoculated) ×100%.

Apoptosis assay

Apoptosis were measured by flow cytometry using Annexin-V staining-based fluorescence-activated cell sorter (FACS). In brief, cells plated in 6-well plates were treated with BBR. At 48 h after treatment, cells were collected and washed once with cold PBS, and subsequently stained with Annexin V (Invitrogen, Carlsbad, CA). Stained cells were analyzed by flow cytometry.

Western blot analysis

Cell lysate proteins were separated by electrophoresis in a 10% sodium dodecyl sulfate-polyacrylamide minigel (SDS-PAGE) and electrophoretically transferred to a PVDF membrane. Western blots were probed with the specific antibodies. The protein bands were detected by enhanced chemiluminescence.

Reverse transcription-polymerase chain reaction (RT-PCR)

Total cellular RNA was extracted with Tri-Zol reagent (Life Technologies, Glasgow, UK) according to the manufacturer’s instructions. Total RNA was reverse-transcribed by using the Superscript™-III kit (Invitrogen, Carlsbad, CA). PCR analysis was performed on aliquots of the cDNA preparations to detect gene expression. The amplified products were visualized on 1% agarose gels. PCR conditions were 4 min at 94°C followed by 35 cycles (25 for GAPDH): 30 seconds at 94°C, 30 seconds at 60°C, and 1 min at 72°C.

Transfection

The transfections of siRNAs or expressing vectors were performed by Lipofectamine 2000 reagent according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA).

DNA-protein binding by streptavidin-agarose pulldown assay

Binding of AP-2α, AP-2β or p65, p50 NF-κB to hTERT core promoter or COX-2 core promoter probes were determined by a streptavidin-agarose pulldown assay. A biotin-labeled double-stranded probe corresponding to hTERT or COX-2 promoter sequence was synthesized. The binding assay was performed by mixing 400 µg of nuclear extract proteins, 4 µg of the biotinylated DNA probe and 40 µl of 4% streptavidin-conjugated agarose beads at room temperature for 1 h in a rotating shaker. Beads were pelleted by centrifugation to pull down the DNA-protein complex. After washing, proteins in the complex were analyzed by immunoblotting using antibodies (1 µg/ml each) specific for AP-2α, AP-2β or p65, p50 NF-κB. The mixture was incubated at room temperature for 1 h with shaking, and centrifuged to pull down the DNA-protein complex. DNA-bound AP-2α, AP-2β or p65, p50 NF-κB protein was dissociated and analyzed by Western blotting. A non-immune rabbit IgG (1 µg/ml) was also used as negative controls.

Confocal immunofluorescence

For confocal microscopy analysis, cells grown on chamber slides were washed in PBS, and fixed and got partial permeabilisation with 4% para formaldehyde for 15 min at room temperature. The samples were pretreated with 10% bovine serum albumin (BSA) in PBS for 30 min. Antibodies against cytochrome-c, p65, p50 in the blocking solution were added to the sample and incubated for overnight at 4°C. Nonimmune IgG was included as controls. Following five 5-min washes with PBS, fluorescein isothiocyanate- and rhodamineconjugated secondary antibodies were added in blocking solutions and incubated for 1 hour. After five additional 5-min washes, samples were examined with a Zeiss LSM700 confocal microscope, and images were processed with Zen2010 software. More than 100 cells were inspected per experiment, and cells with typical morphology were presented.

Statistical analysis

Analysis of variance and Student’s t test were used to compare the values of the test and control samples. P<0.05 was considered to a statistically significant difference. SPSS6.0 software was used for all statistical analysis. The significance was analyzed by the paired t test. All the experiments were done three times, and mean values and standard deviation were calculated.