Fig. 5

Elafin induces EMT and metastasis though EGFR/AKT signaling independently and attenuated the effect of erlotinib in HCC. a and b Inhibitor of EGFR impaired migration (a) and invasion (b) of Elafin-overexpressing HCC cells. Cells were treated with Erlotinib (3 μM) for 24 h and then were subjected to trans-well assays. Scale bar, 100 μm. Statistical results are presented as mean ± SD (from triplicates), and significance is determined by Student t test (* P < 0.05; ** P < 0.01; *** P < 0.001). c and d Down-regulated of Elafin enhanced the effect of Erlotinib on suppressing migration (c) and invasion (d) in HCC cells. After treated with siRNA of Elafin, erlotinib (3 μM), and both of them, indicated cells were conducted trans-well assays. Statistical results are presented as mean ± SD (* P < 0.05; ** P < 0.01; *** P < 0.001). e Erlotinib inhibited phosphorylation of EGFR and downstream signaling. Indicated cells were treated with erlotinib (3 μM) for 24 h and then performed western blotting. f Representative immunofluorescence images illustrating cytoskeleton of control and Elafin overexpressing cells after treated with Erlotinib (3 μM) for 24 h. Scale bar, 20 μm. g Representative immunofluorescence images of EMT markers expression in Elafin overexpressing cells after treated with Erlotinib (3 μM) for 24 h. Scale bar, 50 μm. h The effects of inhibitors of AKT and ERK on the migration and invasion of Elafinoverexpressing HCC cells. Cells were treated with MK2206 (1 μM) and U0126 (15 μM) for 12 h and then were subjected to transwell assays. Statistical results are presented as mean ± SD (ns, no significance; * P < 0.05; ** P < 0.01; *** P < 0.001). i Erlotinib impaired lung metastases of Elafin-overexpressing HCC cells in vivo. Representative HE staining images are shown. Scale bar, 100 μm. Quantification of lung metastasis nodules is analyzed. ** P < 0.01