Effects of epidermal growth factor on the invasive activity and cytoskeleton of oral squamous cell carcinoma cell lines

Abstract

Epidermal growth factor (EGF) is present at high concentrations in human saliva and modulates the growth and differentiation of various cancer cells. To elucidate the molecular mechanisms by which EGF affects oral cancer proliferation and invasion, the current study analyzed the Matrigel invasion activity of cultured oral cancer cell lines. Cell proliferation under the influence of EGF was subjected to Matrigel invasion assays, and cell proliferation in the absence of EGF was used as control. Northern blot analyses quantified the invasiveness and tumorigenicity. Chloramphenicol acetyltransferase assay determined the EGF stimulation of matrix metalloproteinase (MMP) 1 expression. EGF increased the number of cells penetrating the Matrigel membrane. Northern blot analysis revealed that MMP1 and cytokeratin 19 expression correlate with EGF. In addition, the morphology of HSC-3 and SAS cells changed following the addition of EGF to the culture medium. A transient transfection assay revealed that EGF increases the promoter activities of MMP1 in HSC-3 cells. These observations suggested that EGF increases the invasive activity of oral cancer cells, partly by increasing MMP1, and morphological changes may be induced by altering the composition of cytoskeletal proteins.

Keywords: cytokeratin 19; epidermal growth factor; matrix metalloproteinase 1; matrix metalloproteinases; squamous cell carcinoma.

Figure 1

Northern blot analysis of CK19 and EGFR mRNA in oral squamous cell carcinoma cell lines. In total, 10- and 5.6-kb EGFR mRNA and 1.5-kb CK19 mRNA were detected in HSC-3, SAS and Ca9-22 cells. CK19, cytokeratin 19; EGFR, epidermal growth factor receptor.

Figure 2

Morphological changes in HSC-3 and SAS cells. Cells were cultured in medium containing 0, 10 and 100 ng/ml EGF for six days. Images were captured using light microscopy at a magnification of ×40. EGF, epidermal growth factor.

Figure 3

EGF-enhanced invasive activity of oral cancer cells. The number of HSC-3 cells penetrating the Matrigel membrane was approximately three-fold higher in EGF-stimulated (10 ng/ml) cells than in unstimulated HSC-3 cells. The numbers of HSC-3 cells stimulated with 10 and 100 ng/ml EGF were almost the same. Data are presented as the mean ± standard errors of triplicate eperiments. EGF, epidermal growth factor.

Figure 4

Expression of MMP1 and CK19 mRNA in northern blot analysis. EGF increased the expression of MMP1. By contrast, the expression of CK19 mRNA was evidently reduced by EGF, even at the lowest concentration of 10 ng/ml. GAPDH mRNA was used as an internal marker of applied RNA amounts. EGF, epidermal growth factor; MMP1, matrix metalloproteinase 1; CK19, cytokeratin 19; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 5

MMP1 expression in western blot analysis. To confirm an increase in MMP1 protein levels, western blot analysis was performed using an anti-MMP1 monoclonal antibody. EGF increased the expression of MMP1 protein in an EGF concentration-dependent manner. EGF, epidermal growth factor; MMP1, matrix metalloproteinase 1.

Figure 6

Effect of EGF on MMP1 promoter activity. Reporter plasmids containing the MMP1 promoter (MMP1-CAT) were co-transfected with CH110 as an internal control into HSC-3 cells. Cells were placed in 10% Dulbecco’s modified Eagle’s medium with 10 ng/ml EGF 16 h following transfection. CAT activity, measured 48 h following transfection, was corrected for variations in the internal control and is presented as the relative activity. MMP1-CAT activity was increased 3.7-fold by EGF stimulation compared with that in controls. Data are presented as the mean ± standard errors of triplicate experiments. EGF, epidermal growth factor; MMP1, matrix metalloproteinase 1; CAT, chloramphenicol acetyltransferase.