Human head and neck squamous cell carcinoma cells are both targets and effectors for the angiogenic cytokine, VEGF

Abstract

Former vascular endothelial growth factor (VEGF)-head and neck squamous cell carcinoma (HNSCC) studies have focused on VEGF's contributions toward tumor-associated angiogenesis. Previously, we have shown that HNSCC cells produce high levels of VEGF. We therefore hypothesized that VEGF serves a biphasic role, that is, pro-angiogenic and pro-tumorigenic in HNSCC pathogenesis. Western blots confirmed the presence of VEGF's primary mitogenic receptors, VEGFR-2/KDR and VEGFR-1/Flt-1 in cultured HNSCC cells. Subsequent studies evaluated VEGF's effects on HNSCC intracellular signaling, mitogenesis, invasive capacities, and matrix metalloproteinases (MMPs) activities. Introduction of hrVEGF(165) initiated ROS-mediated intracellular signaling, resulting in kinase activation and phosphorylation of KDR and Erk1/2. As high endogenous VEGF production rendered HNSCC cells refractory to exogenous VEGF's mitogenic effects, siRNA was employed, inhibiting endogenous VEGF production for up to 96 h. Relative to transfection vector matched controls, siRNA treated HNSCC cells showed a significant decrease in proliferation at both 30 and 50 nM siRNA doses. Addition of exogenous hrVEGF(165) (30 and 50 ng/ml) to siRNA-silenced HNSCC cells resulted in dose-dependent increases in cell proliferation. Cell invasion assays showed VEGF is a potent HNSCC chemoattractant and demonstrated that VEGF pre-treatment enhanced invasiveness of HNSCC cells. Conditioned media from VEGF challenged HNSCC cells showed a moderate increase in gelatinase activity. Our results demonstrate, for the first time, that HNSCC cells are both targets and effectors for VEGF. These data introduce the prospect that VEGF targeted therapy has the potential to fulfill both anti-angiogenic and anti-tumorigenic functions.

Figures

Fig. 1

Cultured HNSCC cells retain possession of VEGFR-2/KDR and VEGFR-1/Flt-1. A and B: Three HNSCC cell lines, SCC4, SCC9, and SCC15, were screened for the cytoplasmic production of KDR and Flt-1 proteins by Western Blot. HUVECs were included as the positive control.

Fig. 2

VEGF induced phosphorylation of KDR and Erk1/2 in cultured HNSCC cells. A and C: HUVECs and 24h serum deprived HNSCC cells were challenged with PBS (negative control) or 100ng/ml VEGF for 20min in serum free media. Western Blot analyses were then conducted to determine phosphorylated and total levels of KDR and Erk1/2. hrVEGF165 activated intracellular kinases, resulting in increased phosphorylated ratios of KDR and Erk1/2. Densitometry analyses results were demonstrated in panels B, D and E, respectively.

Fig. 3

Introduction of hrVEGF165 initiated release of intracellular reactive oxygen species. HNSCC cells were seeded on chamber slides and incubated at 37°C, 5% CO2 for 24 h in serum-free medium. 0.2mM H2O2 (positive control) , 30ng/ml hrVEGF165 or equal volume of PBS (negative control) were then added to the chambers and incubated for 15 min, followed by 5 min staining of 10µM CM-H2 DCFDA. Increases in fluorescence intensities reflect intracellular probe oxidation.

Fig. 4

Endogenous VEGF renders HNSCC cells refractory to exogenous VEGF. A: Human umbilical vascular endothelial cells (HUVECs) served as positive control in this study to validate hrVEGF165‘s functional activity. Experimental groups were treated with hrVEGF165 containing medium versus control group received base medium alone. B: Serum deprived non-siRNA-silenced HNSCC cells were challenged with fresh hrVEGF165 every 24 h. Control group received serum free base medium only. C: HNSCC cells were transfected with different concentrations (0, 30nM and 50nM) of anti-VEGF siRNA and incubated at 37°C, 5% CO2. ELISA and MTT assay were performed every 24 h after transfection to determine the amount of VEGF production (presented as pictogram per 104 cells) in the conditioned medium. Values were then normalized to the percentage of negative control for comparisons. D: Impact of VEGF silencing on HNSCC cell proliferation. [Data collected from three HNSCC cell lines, mean ± SE, n=12. *=p<0.05; †=p<0.01; ‡=p<0.001, compared with the control group (Tukey-Kramer multiple comparisons test)]

Fig. 5

Human recombinant VEGF165 promoted HNSCC cell proliferation following suppression of endogenous VEGF production. A and B: HNSCC cells were successfully transfected with NeoFX/FAM-labeled siRNA (72 h after transfection, 200X). C: Serum deprived HNSCC cells were pre-treated with 30nM siRNA to inhibit the endogenous VEGF production 24 h prior to exogenous hrVEGF165 stimulation. D: HNSCC cells were pre-treated with 50nM siRNA. Cell proliferation was determined using the MTT assay. Data of cell numbers from three HNSCC cell lines were normalized to percentage of control group and combined for statistic analysis (mean ± SE, n=20). *=p<0.05. (Unpaired t test)

Fig. 6

hrVEGF165 is a potent chemoattractant for HNSCC cells. Five experimental groups for each cell line were included in the cell invasion assay: 1), Negative control, 72 h serum deprived HNSCC cells in the insert chamber with SF medium in the lower chamber; 2), Positive control, 72 h serum deprived cells with SF medium+ 10% FBS in the lower chamber serving as chemoattractant; 3), Positive control, 48 h serum deprived and 24 h 10% FBS pretreated cells with 10% FBS chemoattractant; 4), 72 h serum deprived cells with 100ng/ml VEGF chemoattractant; 5), 48 h serum deprived and 24 h 100ng/ml VEGF pretreated cells with 100ng/ml VEGF chemoattractant. Invaded cell numbers were determined after 48 h incubation relative to a cell line matched standard curve (mean ± SE, n=4 for each group. *=p<0.05, **=p<0.01, †=p<0.001, Tukey-Kramer multiple comparisons test).

Fig. 7

Treatment of HNSCC cells with hrVEGF165 modestly increased gelatinase release. A and C: HNSCC cells (SCC4 and SCC15) were serum deprived for 48 h followed by fresh treatment of serum-free medium with or without presence of 100ng/ml VEGF for 24 h. In selected groups, the conditioned media were pre-incubated with the pro-MMP activator, APMA, for 2 h. Gelatin zymography were then conducted to demonstrate released gelatinases (MMP2 and MMP9) levels in the conditioned media. B and D: Densitometry analyses were performed using Kodak 1D3 image analysis software.