Fig 1.
Effects of tamoxifen, raloxifene and bazedoxifene on the TGF-α-induced migration of HuH7 cells.
The cells were pretreated with the indicated concentrations of tamoxifen (A), raloxifene (B) or bazedoxifene (C) for 60 min, and then stimulated by 3 ng/ml of TGF-α or vehicle for 23 h. The migrated cells were stained with DAPI for the nuclei. The cells were photographed by fluorescent microscopy at a magnification of 20× (upper panel) and counted (bar graph). Each value represents the mean ± SD of triplicate determinations from three independent cell preparations. *p<0.05, compared to the value of the control cells without TGF-α stimulation (panel 1). **p<0.05, compared to the value of TGF-α stimulation alone (panel 2). N.S. designates no significant difference between the indicated pairs. Scale bar: 100 μm.
Fig 2.
Effects of PPT, ERB041, DPN and PHTPP on the TGF-α-induced migration of HuH7 cells.
The cells were pretreated with the indicated concentrations of PPT (A), ERB041 (B and D) or DPN (C) for 60 min, and then stimulated with 3 ng/ml of TGF-α or vehicle for 23 h. When indicated (D), the cells were exposed with 3 μM of PHTPP 60 min prior to the ERB041 treatment. The migrated cells were stained with DAPI for the nuclei. The cells were photographed by fluorescent microscopy at a magnification of 20× (upper panel) and counted (bar graph). Each value represents the mean ± SD of triplicate determinations from three independent cell preparations. *p<0.05, compared to the value of the control cells without TGF-α stimulation (panel 1). **p<0.05, compared to the value of TGF-α stimulation alone (panel 2). ***p<0.05, compared to the value of TGF-α stimulation with ERB041 pretreatment (panel 4). N.S. designates no significant difference between the indicated pairs. Scale bar: 100 μm.
Fig 3.
Combined effects of PPT and ERB041 on the TGF-α-induced migration of HuH7 cells.
The cells were pretreated with PPT (3 μM) and ERB041 (1 μM) for 60 min, and then stimulated by 3 ng/ml of TGF-α or vehicle for 23 h. The migrated cells were stained with DAPI for the nuclei. The cells were photographed by fluorescent microscopy at a magnification of 20× (upper panel) and counted (bar graph). Each value represents the mean ± SD of triplicate determinations from three independent cell preparations. *p<0.05, compared to the value of the control cells without TGF-α stimulation (panel 1). **p<0.05, compared to the value of TGF-α stimulation alone (panel 2). ***p<0.05, compared to the value of TGF-α stimulation with PPT pretreatment (panel 6). N.S. designates no significant difference between the indicated pairs. Scale bar: 100 μm.
Fig 4.
Effects of ERB041 on the TGF-α-induced phosphorylation of EGFR (A), p38 MAPK (B), 54 kDa JNK (C) and AKT (D) in HuH7 cells. The cells were pretreated with the indicated concentrations of ERB041 for 8 h, and then stimulated by 30 ng/ml of TGF-α or vehicle for 1 min for EGFR (A) and AKT (D), 5 min for p38 MAPK (B), or 20 min for JNK (C). The histogram shows the quantitative representation of the phosphorylated levels after normalization with respect to GAPDH obtained from a densitometric analysis. The average of density levels of the TGF-α stimulation alone (lane 2) was expressed as 1.0. Each value represents the mean ± SD of triplicate determinations from three independent cell preparations. *p<0.05, compared to the value of the control cells with TGF-α stimulation alone (lane 2). N.S. designates no significant difference between the indicated pairs.
Fig 5.
Effects of DPN on the TGF-α-induced phosphorylation of EGFR (A) and AKT (B) in HuH7 cells. The cells were pretreated with the indicated concentrations of DPN for 8 h, and then stimulated by 30 ng/ml of TGF-α or vehicle for 1 min. The histogram shows the quantitative representation of the phosphorylated levels after normalization with respect to GAPDH obtained from a densitometric analysis. The average of density levels of the TGF-α stimulation alone (lane 2) was expressed as 1.0. Each value represents the mean ± SD of triplicate determinations from three independent cell preparations. *p<0.05, compared to the value of the control cells with TGF-α stimulation alone (lane 2). N.S. designates a significant difference between the indicated pairs.
Fig 6.
Effects of raloxifene on the TGF-α-induced phosphorylation of EGFR (A) and AKT (B) in HuH7 cells. The cells were pretreated with the indicated concentrations of raloxifene for 8 h, and then stimulated by 30 ng/ml of TGF-α or vehicle for 1 min. The histogram shows the quantitative representation of the phosphorylated levels after normalization with respect to GAPDH obtained from a densitometric analysis. The average of density levels of the TGF-α stimulation alone (lane 2) was expressed as 1.0. Each value represents the mean ± SD of triplicate determinations from three independent cell preparations. *p<0.05, compared to the value of the control cells with TGF-α stimulation alone (lane 2). N.S. designates no significant difference between the indicated pairs.
Fig 7.
T Effects of bazedoxifene on the TGF-α-induced phosphorylation of EGFR (A) and AKT (B) in HuH7 cells. The cells were pretreated with the indicated concentrations of bazedoxifene for 8 h, and then stimulated by 30 ng/ml of TGF-α or vehicle for 1 min. The histogram shows the quantitative representation of the phosphorylated levels after normalization with respect to GAPDH obtained from a densitometric analysis. The average of density levels of the TGF-α stimulation alone (lane 2) was expressed as 1.0. Each value represents the mean ± SD of triplicate determinations from three independent cell preparations. *p<0.05, compared to the value of the control cells with TGF-α stimulation alone (lane 2). N.S. designates no significant difference between the indicated pairs.
Fig 8.
Effects of lactacystin on the amont of total AKT in the presence of ERB041 or raloxifene in HuH7 cells.
The cells were treated with the indicated concentrations of lactacystin immediately prior to the pretreatment of 10 μM of ERB041 (A) or 20 μM of raloxifene (B) for 8h, and then stimulated by 30 ng/ml of TGF-α or vehicle for 1 min. The histogram shows the quantitative representation of the total AKT levels after normalization with respect to GAPDH obtained from a densitometric analysis. The average of density levels of the TGF-α stimulation alone (lane 2) was expressed as 1.0. Each value represents the mean ± SD of triplicate determinations from three independent cell preparations. N.S. designates no significant difference between the indicated pairs.
Fig 9.
Effects of TGF-α and NSC23766 on the levels of GTP-binding Rac in HuH7 cells (A, C) and effect of NSC23766 on the TGF-α-induced migration of HuH7 cells (B). The cells were stimulated with 30 ng/ml of TGF-α for indicated times (A), or pretreated with 300 μM of NSC23766 or vehicle for 60 min, and then stimulated with 30 ng/ml of TGF-α for 5 min (C). The GTP-binding Rac in the cell extracts was immunoprecipitated using Rac1 activation assay kit. The immunoprecipitted GTP-binding Rac and pre-immunoprecipitated lysates (total Rac) were subjected to Western blot analysis using antibodies against Rac. (B), The cells were pretreated with indicated concentrations of NSC23766 for 60 min, and then stimulated by 3 ng/ml of TGF-α or vehicle for 23 h. The migrated cells were stained with DAPI for the nuclei. The cells were photographed by fluorescent microscopy at a magnification of 20× (upper panel) and counted (bar graph). Each value represents the mean ± SD of triplicate determinations from three independent cell preparations. *p<0.05, compared to the value of the control cells without TGF-α stimulation (panel 1). N.S. designates no significant difference between the indicated pairs. Scale bar: 100 μm.
Fig 10.
Effect of G-1 on the TGF-α-induced phosphorylation of AKT in HuH7 cells.
The cells were pretreated with 1 μM of G-1 or vehicle for 1h and then stimulated by 30 ng/ml of TGF-α or vehicle for 1 min. The histogram shows the quantitative representation of the phospho-AKT levels after normalization with respect to GAPDH obtained from a densitometric analysis. The average of density levels of the TGF-α stimulation alone (lane 2) was expressed as 1.0. Each value represents the mean ± SD of triplicate determinations from three independent cell preparations. N.S. designates no significant difference between the indicated pairs.
Fig 11.
Effects of PHTPP on the suppression by raloxifene or bazedoxifene of the TGF-α-induced migration of HuH7 cells.
The cells were exposed with 3 μM of PHTPP or vehicle for 60 min, and then pretreated with 5 μM of raloxifene (A), 5 μM of bazedoxifene (B) or vehicle for another 60 min. After the pretreatment, the cells were stimulated by 3 ng/ml of TGF-α or vehicle for 23 h. The migrated cells were stained with DAPI for the nuclei. The cells were photographed by fluorescent microscopy at a magnification of 20× (upper panel) and counted (bar graph). Each value represents the mean ± SD of triplicate determinations from three independent cell preparations. *p<0.05, compared to the value of the control cells without TGF-α stimulation (panel 1). **p<0.05, compared to the value of TGF-α stimulation alone (panel 2). ***p<0.05, compared to the value of TGF-α stimulation with the pretreatment of raloxifene or bazedoxifene (panel 4). N.S. designates no significant difference between the indicated pairs. Scale bar: 100 μm.
Fig 12.
Schematic illustration of the mechanism behind the inhibition of the TGF-α-induced HCC cell migration by raloxifene and bazedoxifene.
TGF, transforming growth factor; EGFR, epidermal growth factor receptor; ER, estrogen receptor; GPR30, G protein-coupled ER; MAPK, mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; HCC, hepatocellular carcinoma.