Epidermal growth factor receptor transactivation is involved in the induction of human hepatoma SMMC7721 cell proliferation by insufficient radiofrequency ablation

Abstract

Our previous study revealed that insufficient radiofrequency ablation (RFA) promotes the malignancy of human hepatocellular carcinoma (HCC) SMMC7721 cells via the Ca²⁺/calmodulin-dependent protein kinase II (CaMKII)/extracellular signal-regulated kinase (ERK)-induced overexpression of vascular endothelial growth factor (VEGF). The aims of the present study were to address the involvement of epidermal growth factor receptor (EGFR) transactivation in the enhanced SMMC7721 cell proliferation induced by insufficient RFA, in addition to its association with the CaMKII/ERK/VEGF signaling cascade. SMMC7721 cells were subjected to a 47°C treatment regimen to simulate insufficient RFA. Cell proliferation was determined using MTT and colony formation assays. The expression levels of VEGF, CaMKII, phosphorylated (phospho)-CaMKII, ERK, phospho-ERK, EGFR and phospho-EGFR were analyzed using western blotting. The results demonstrated that the enhancement of SMMC7721 cell proliferation by the 47°C treatment regimen was significantly inhibited by exposure of the cells to AG178 (a specific inhibitor of EGFR). Furthermore, AG1478 exposure prevented the overexpression of VEGF and phosphorylation of ERK, but had no significant effects on CaMKII phosphorylation. By contrast, 47°C treatment-induced EGFR phosphorylation was inhibited by treatment with KN93 (a specific inhibitor of CaMKII). Overall, the results of the present study have suggested a role for EGFR transactivation in the RFA-promoted growth of residual HCC. Thus, targeting EGFR may represent a useful preventive and therapeutic strategy for RFA-induced HCC progression and recurrence.

Keywords: Ca2+/calmodulin-dependent protein kinase II; SMMC7721; epidermal growth factor receptor; proliferation; radiofrequency ablation.

Figure 1.

The induction of SMMC7721 cell proliferation by the 47°C treatment regimen is dependent on EGFR activation. The effect of AG1478 (a specific EGFR inhibitor) on cell proliferation and colony formation rates are shown, as detected using (A) MTT and (B) colony formation assays, respectively. Data are presented as the mean ± standard error of the mean from five independent experiments. *P<0.05 vs. control; ^#P<0.05 vs. 47°C treatment group. EGFR, epidermal growth factor receptor.

Figure 2.

EGFR activation is involved in the induction of VEGF and ERK overexpression, but not in the activation of CaMKII, by 47°C treatment. (A) Representative blots are shown. (B) Quantified data from the western blot analysis are presented as the mean ± standard error of the mean from four independent experiments. *P<0.05 vs. control; ^#P<0.05 vs. 47°C treatment group. All data are normalized to β-actin. EGFR, epidermal growth factor receptor; VEGF, vascular endothelial growth factor; ERK, extracellular signal-regulated kinase; CaMKII, Ca²⁺/calmodulin-dependent protein kinase II; p-, phosphorylated.

Figure 3.

CaMKII activation occurs upstream of EGFR phosphorylation during 47°C treatment, as indicated by a significant decrease in p-EGFR expression following treatment with KN93, a specific inhibitor of CaMKII, and the 47°C treatment regimen. (A) Representative blots are shown. (B) Quantified data from the western blot analysis are presented as the mean ± standard error of the mean from four independent experiments. *P<0.05 vs. control; ^#P<0.05 vs. 47°C treatment group. All data are normalized to β-actin. CaMKII, Ca²⁺/calmodulin-dependent protein kinase II; EGFR, epidermal growth factor receptor; p-, phosphorylated.