Figure 1.
Basal and viral vector–mediated expression of AT2R in HCC line SMMC7721.
A, RQ comparison of AT2R mRNA expression following transduction with Ad-G-AT2R-EGFP gradient doses (1 ifu, 5 ifu, 10 ifu, 50 ifu, 100 ifu, 200 ifu, 300 infu, 500 ifu/cell). Columns, mean (n = 3); *P<0.05 and **P<0.01 versus 1 ifu of Ad-G-AT2R-EGFP–transduced cells. B, ethidium bromide-stained gels show AT2R and β-actin mRNA expression in transduced SMMC7721 cells.
Figure 2.
AT2R overexpression disturbs cell cycle in SMMC7721 but not LO2 cells.
A, the percentages of SMMC7721 cells transduced with 1–500 ifu/cell of either Ad-G-AT2R-EGFP or Ad-CMV-EGFP for 24 hrs in G1 phase, S phase and G2 phase. Columns, mean (n = 3); bars, SE. Data are derived from DNA histograms.*P<0.05 and **P<0.01 versus Ad-CMV-EGFP–transduced cells. B, fluorescence intensity of SMMC7721 and LO2 cells transduced with 300 ifu/cell of either Ad-G-AT2R-EGFP or Ad-CMV-EGFP for 24 hrs was measured by flow cytometry. C, data showing percentages of SMMC7721 and LO2 cells in G1 phase (left) and S phase (right) under each treatment condition. Columns, mean (n = 3); bars, SE. Data are derived from DNA histograms. **P<0.01 versus Ad-CMV-EGFP–transduced cells.
Figure 3.
AT2R overexpression inhibits cell proliferation in SMMC7721 cells.
A, growth curve of HCC SMMC7721 cells with 200/cell of Ad-G-AT2R-EGFP and Ad-CMV-EGFP–transduced. **P<0.01. B, representative Western blots showing the CDK4 and cyclin D1 in SMMC7721 cells induced for 24 hrs with Ad-G-AT2R-EGFP and Ad-CMV-EGFP (100, 200 and 300 ifu/cell, respectively). Data are representative of three experiments.
Figure 4.
AT2R overexpression induces apoptosis of SMMC7721 cells.
A, SMMC7721 cells were transduced with either Ad-CMV-EGFP or Ad-G-AT2R-EGFP (500 ifu/cell) for 24 hrs as described in materials and methods and apoptotic cells were detected using the DeadEnd Colorimetric TUNEL. Two representative phase-contrast micrographs from each treatment condition. Representative fluorescence micrographs from Ad-CMV-EGFP–transduced and Ad-G-AT2R-EGFP–transduced cells, showing EGFP fluorescence, TUNEL-positive (apoptotic) cell (brown-colored nuclei), and merged (G+A) EGFP/AT2R in each treatment condition. Scale bars, 50 µm. B, quantification of the TUNEL-positive cells as a percent of the total number of cells in the dish. Columns, mean of three experiments; bars, SE. **P<0.01 versus GFP group.
Figure 5.
Involvement of MAPK superfamily in AT2R-induced apoptosis in SMMC7721 HCC cells.
Representative Western blots show the p38 MAPK and pp38 MAPK in SMMC7721 cells transduced for 12-G-AT2R-EGFP and Ad-CMV-EGFP (500 ifu/cell) or mock-transduced and PP2A, JNK, pJNK, p42/44 MAPK(Erk1/2) and pp42/44 MAPK(pErk1/2) in SMMC7721 cells transduced for 24 hrs. Data are representative of three experiments.
Figure 6.
Role of caspases in AT2R-induced apoptosis in SMMC7721 HCC cells.
A, Western blot analysis of AT2R-induced increase in cleaved caspase-3 and caspase-8 generation in SMMC7721 cells. Cells were infected after 24 hrs with either 300 or 500 ifu/cell of Ad-CMV-EGFP, Ad-G-AT2R-EGFP or mock-transduced. Data are representative of three experiments. B, AT2R-induced increase in caspase-3 activity in SMMC7721 cells. Columns, mean A 405 nm from three experiments; bars, SE. *P<0.05 and **P<0.01 versus Ad-CMV-EGFP–transduced or mock-transduced cells.
Figure 7.
The effect of AT2R overexpression on the growth of HCC tumors.
A, CCCD signals of cellular orthotopic injection animal models post transfection with Ad-CMV-EGFP, Ad-G-AT2R-EGFP tail vein injection and PBS as negative control. B, liver tumor tissues were separated from human hepatocellular carcinoma nude mice models. C, the effect of AT2R on tumor size. Data are expressed as mean ± SD. (*P<0.05, n = 8). D, RQ comparison of AT2R mRNA expression in animal models (M-PBS; M-GFP; M-AT2R) and SMMC7721 cells. M-AT2R, M-GFP and M-PBS, AT2R mRNA expression in liver tumor tissues of the nude models injected with Ad-G-AT2R-EGFP, Ad-CMV-EGFP, PBS. C-AT2R1 and C-AT2R10, AT2R mRNA expression in SMMC7721 cells following transduction with gradient doses of Ad-G-AT2R-EGFP (1ifu, 10 ifu/cell). Columns, mean (n = 3); *P<0.05 versus 1 and 10 ifu/cell of Ad-G-AT2R-EGFP–transduced cells.
Figure 8.
The effect of AT2R overexpression on Ki67 expression in vivo.
A, the Ki67 immunohistochemistry staining of liver tumor tissues from human hepatocellular carcinoma nude mouse models injected with Ad-CMV-EGFP, Ad-G-AT2R-EGFP or PBS via tail vein. AT2R-N, GFP-N and PBS-N, PBS instead of Anti-Ki67 primary antibody as negative control (×400). B, the immunohistochemistry staining scoring from different groups. Data are expressed as mean ± SD. (*P<0.05, n = 8).