Vasohibin 2 promotes malignant behaviors of pancreatic cancer cells by inducing epithelial-mesenchymal transition via Hedgehog signaling pathway

2.1 PC cell lines

The human pancreatic cancer cell lines PANC-1, SW1990, CFPAC, HPAC, Capan-1, Miapaca-2, and BxPc-3 from ATCC were used in the study. HPAC cells were cultured in DMEM/F12 (Hyclone, Logan, Utah, USA), CFPAC cells were cultured in IMDM (Hyclone), and other cell lines in DMEM (Hyclone), supplemented with 10% fetal bovine serum (FBS; Gibco, Waltham, MA, USA), 100 mg/mL penicillin and 100 mg/mL streptomycin at 37°C in a humidified chamber supplemented with 5% CO₂.

2.2 Western blot

Western blotting was performed using standard protocols. Briefly, cells were sonicated in RIPA buffer, and 30 μg proteins was resolved on 12% polyacrylamide SDS gels and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% BSA (bovine serum albumin) in TBS (tris-buffered saline), incubated overnight at 4°C with the appropriate primary antibodies. The antibodies used included those against VASH2 (ab116640, Abcam, Cambridge, UK), GAPDH (AG019-1, Beyotime, Shanghai, China), Vimentin (ab8069, Abcam), E-cadherin (ab11512, Abcam), ZEB1 (ab124512, Abcam), ZEB2 (ab25837, Abcam), Bcl-2 (ab32124, Abcam), SMO (ab38686,, Abcam), Gli-1 (ab49314, Abcam), and Gli-2 (ab167389, Abcam).

2.3 Plasmid construction and lentivirus packaging

The construction of plasmids for knockdown and overexpression of VASH2 (PANC-1-shVASH2, PANC-1-shcont, BxPc-3-VASH2, and BxPc-3-con) and lentivirus packaging were performed as described previously.14 PANC-1 and BxPc-3 cell lines were used to knock down and overexpress VASH2, respectively.

2.4 Morphology and immunofluorescence

Cells were plated into chamber slides (Falcon, Edison, NJ, USA) and fixed with 3% formaldehyde. Samples were then blocked with 5% BSA and incubated with anti-E-cadherin, anti-VASH2 and anti-Vimentin overnight at 4˚C. Rabbit IgGs were used as a negative control. After incubating with the primary antibodies, cells were washed three times in PBS (phosphate-buffered saline) followed by 60 minutes of incubation at room temperature with anti-rabbit FITC (fluorescein isothiocyanate) and secondary antibody. After washing with PBS, the cells were mounted using FLUORO-GEL II with DAPI (4′,6-diamidino-2-phenylindole) (Electron Microscopy Science, Pennsylvania). Fluorescence was analyzed using the fluorescence microscopy (Nikon, Japan).

2.5 MTT assay

PANC-1 and BxPC-3 cells were splited into 96 wells and cells were grown to 80%-90% subconfluence. Gemcitabine was then added to the cells at the indicated concentrations. After 48 hours of treatment, MTT (Thiazolyl Blue Tetrazolium Bromide) was added into the medium and the cell culture mixture was incubated for another 4 hours. The cell culture medium was then mixed with 150 μL DMSO (dimethyl sulfoxide) for 10 minutes before the measurement of optical absorbance at 490 nm.

2.6 Detection of cell apoptosis

Apoptosis was determined using FITC Annexin V Apoptosis Detection Kit (Beyotime, Shanghai, China) following the manufacturer's instructions. Briefly, cells were centrifuged, washed in PBS, resuspended in 100 μL of 1 ×  binding buffer and stained with 5 μL of FITC (fluorescein isothiocyanate)-labelled Annexin V and 5 μL of propidium iodine at room temperature for 15 minutes in the dark. The stained cells were then analyzed by flow cytometry.

2.7 In vitro invasion assay

The matrigel invasion assay was performed using 24-well transwell chambers (8.0 μm pore size with polycarbonate membrane; Corning Life Sciences, Lowell, MA, USA) following the manufacturer's instructions. Briefly, the lower compartment was seeded with 5 × 10⁴ cells in 600 μL complete medium and incubated for 12 hours, and then the medium was changed to serum-free medium. The upper chamber was coated with 100 μL of 1 mg/mL Matrigel (BD Biosciences, Bedford, MA) and incubated for 3 hours when 0.6 mL of 10% FBS-DMEM was added to the lower chamber. After incubation for 24 hours, noninvading cells were removed with cotton swabs. Cells migrating to the bottom of the membrane were stained with 0.1% crystal violet for 30 minutesat 37ºC, washed with PBS and photographed.

2.8 Flow cytometry

Flow cytometry was performed using the standard protocol. Briefly, cells were labeled with 5 μg/mL anti-CD44 (APC) and 2.5 μg/mL anti-CD24 (PE) antibodies for 10 minutes at 37ºC, using 2.5 μg/mL rat IgG2b (APC) and 5 μg/mL mouse IgG1 (PE) as isotype controls, respectively. All antibodies were purchased from eBioscience (San Diego, CA). Flow cytometry was performed using a BD FACSCalibar flow cytometer. Analysis of side population (SP) cells was performed as described previously.14

2.9 Statistical analysis

All experiments were repeated in triplicate. Statistical significance was determined by Student's t test. P-values < 0.05 were considered statistically significance.

3.1 Expression of VASH2 in human pancreatic ductal adenocarcinoma tissues and pancreatic cancer cell lines

Our previous results have shown increased expression of VASH2 in pancreatic ductal adenocarcinoma tissues versus matched tumor-adjacent benign tissues by immunohistochemistry staining.13 We have confirmed this finding using qRT-PCR (data not shown). We further detected the expression of VASH2 protein in pancreatic cancer cell lines (Miapaca-2, Capan-1, CFPAC-1, PANC-1, SW1990, HPAC, and BxPc-3) by Western blot, which revealed that PANC-1 cells had higher expression of VASH2, while BxPc-3 cells displayed lower VASH2 expression (Figure 1). Therefore, we selected the PANC-1 cell line to knock down and the BxPc-3 cell line to overexpress VASH2 in subsequent experiments.

3.2 PC cells overexpressing VASH2 exhibit morphologic and molecular changes consistent with EMT

As shown in Figure 2A, VASH2-overexpressing BxPc-3 cells exhibited an elongated irregular fibroblastoid morphology. In contrast, PANC-1 cells with VASH2 knockdown had an epithelial-like appearance and grew in clusters. These changes suggest that VASH2 may be associated with EMT in pancreatic cancer cells. To further confirm this finding, we determined the expression of markers of epithelial and mesenchymal phenotypes by Western blot and immunofluorescence, which showed that the expression of E-cadherin, an epithelial marker, was reduced in VASH2-overexpressing BxPc-3 cells but increased in PANC-1 cells with VASH2 knockdown, while vimentin, a mesenchymal marker, showed an opposite expression pattern (Figure 2B,C and D). Of note, the expression levels of ZEB1 and ZEB2,14 which were previously found to mediate the function of VASH2, were increased in VASH2-overexpressing BxPc-3 cells and reduced in PANC-1 cells with VASH2 knockdown. Collectively, these results suggest that VASH2 may promote EMT in PC cells possibly by altering ZEB1/2 expression (Figure 2B).

3.3 VASH2 promotes gemcitabine resistance and invasion in PC cells

It is known that EMT can promote gemcitabine resistance and invasion of tumor cells.15 After measuring the IC50 of gemcitabine in BxPC-3 and PANC-1 cells (Figure 3), we examined the impact of VASH2 expression on the apoptosis of PC cells after treating VASH2-overexpressing BxPc-3 cells and control BxPc-3 cells with 1.0-20 μg/mL gemcitabine, and PANC-1 cells with VASH2 knockdown and control PANC-1 cells with 10-60 μg/mL gemcitabine, respectively. Flow cytometry analysis showed a significant increase in apoptotic cells in control BxPc-3 cells compared to VASH2-overexpressing BxPc-3 cells treated with 5.0, 10 and 20 μg/mL gemcitabine (Figure 4A), and a significant increase in apoptotic cells in PANC-1 cells with VASH2 knockdown compared to control PANC-1 cells treated with 40 and 60 μg/mL gemcitabine (Figure 5A). Furthermore, the expression of the antiapoptotic gene Bcl-2 was measured by Western blot, which showed that VASH2 overexpression increased Bcl-2 expression while VASH2 knockdown decreased Bcl-2 expression (Figure 4B and 5B). These findings suggest that VASH2 promotes the gemcitabine resistance of PC cells by increasing their antiapoptotic ability via upregulating Bcl-2.

To determine the impact of VASH2 expression on PC cell invasion, we evaluated the invasive ability of VASH2-overexpressing BxPc-3 cells and PANC-1 cells with VASH2 knockdown by in vitro invasion assay. The results showed that the invasive ability of VASH2-overexpressing BxPc-3 cells significantly increased compared to control BxPc-3 cells (Figure 6), while that of PANC-1 cells with VASH2 knockdown significantly decreased compared to control PANC-1 cells. Thus, these results suggest that VASH2 promotes PC cell invasion in vitro.

3.4 VASH2 increases the proportion of CD24⁺CD44⁺ cells and SP cells in PC cells

It is well documented that cancer cells positive for the cell surface antigens CD24, and CD44 exhibit increased self-renewal and tumor-initiating potential. Furthermore, cells in a side population (SP) exhibit distinguishing stem cell-like characteristics. Thus, the proportion of CD24⁺CD44⁺ cells and SP cells in cultured PC cells was detected by flow cytometry.16 As shown in Figure 7A, the proportion of CD24⁺CD44⁺ cells in PANC-1 cells with VASH2 knockdown significantly decreased compared to mock-transfected PANC-1 cells (P < 0.01). Interestingly, the proportion of CD44⁺ cells in VASH2-overexpressing BxPc-3 cells was significantly higher than that in mock-transfected BxPc-3 cells (P < 0.01), whereas the proportion of CD24⁺ cells in VASH2-overexpressing BxPc-3 cells was similar to that in control cells. We also found that VASH2 knockdown caused reduced expression of aldehyde dehydrogenase 1 (ALDH1) in PANC-1 cells and VASH2 overexpression led to increased expression of ALDH1, another well-known marker of cancer stem cells (Figure 7B).

The proportion of SP cells in PC cells was also detected by flow cytometry (Figure 7C), which revealed that the proportion of SP cells in PANC-1 cells with VASH2 knockdown was reduced compared to mock-transfected PANC-1 cells, and the proportion of SP cells in VASH2-overexpressing BxPc-3 cells was higher than that in mock-transfected BxPc-3 cells. Taken together, the above results suggest that VASH2 could influence the proportion of cancer stem-like cells in PC cells.

3.5 VASH2 upregulates the expression of molecules of the Hedgehog signaling pathway

Finally, we assessed the effect of VASH2 expression on the Hedgehog signaling pathway, which can alter the invasive and migratory capacity of PC cells and regulate EMT in PC,17 by detecting the expression of key molecules of this pathway by Western blot. We found that overexpression of VASH2 upregulated the expression of SMO, Gli-1, and Gli-2, while VASH2 knockdown downregulated the expression of these proteins (Figure 8A). These results suggest that VASH2 regulates the malignant behaviors of PC cells possibly via activation of the Hedgehog signaling pathway.