WWOX induces apoptosis and inhibits proliferation of human hepatoma cell line SMMC-7721

Figure 1 WWOX protein levels in SMMC-7721 cells. 1: Control group; 2: Empty vector transfection group; 3: pEGFP-WWOX transfection group.

Figure 2 Growth of SMMC-7721 cells examined by plate colony formation. Overexpression of WWOX (pEGFP-N1-WWOX/SMMC-7721) resulted in a decrease in the number of formed colonies compared with the SMMC-7721 and control vector (pEGFP-N1/SMMC-7721) cells (P < 0.05).

Figure 3 Overexpression of WWOX inhibits the proliferation of SMMC-7721 cells, as demonstrated by the methyl thiazolyl tetrazolium assay. There were no significant differences between the parental SMMC-7721 cell line and the control vector cell line based on the P values.

Figure 4 Cell cycle distribution of parental SMMC-7721, control vector (pEGFP-N1), and WWOX overexpressing (pEGFP-N1-WWOX) cells determined by fluorescently activated cell sorting caliber cytometry. The G1/S transition was inhibited in WWOX overexpressing cells compared with parental and control vector transformed cells (P < 0.05).

Figure 5 Cell apoptosis measured by flow cytometry using Annexin V/propidium iodide double staining. Cells transfected with pEGFP-N1-WWOX showed more apoptosis than parental cells or cells transfected with the mock plasmid (P < 0.05). PI: Propidium iodide; FITC: Fluorescein isothiocyanate.

Figure 6 Overexpression of WWOX activates the expression of caspase-9 and caspase 3 protein. A: Expression of cleaved caspase-9 was upregulated in pEGFP-WWOX cells compared with parental and control vector cells; B: Protein expression of cleaved caspase 3 was upregulated in pEGFP-WWOX cells compared with control-vector cells and parental SMMC-7721. Data are presented as mean ± SD (P < 0.05). 1: Control group; 2: Empty vector transfection group; 3: pEGFP-WWOX transfection group. GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Figure 7 Overexpression of WWOX suppresses the phosphorylation of p-AKT(Thr308) and p-AKT(Ser473). A: p-AKT(Thr308) levels were decreased in pEGFP-WWOX cells compared with parental and control vector transformed cells by Western blotting analysis; B: p-AKT(Ser473) levels were decreased in pEGFP-WWOX cells compared with parental and control vector transformed cells by Western blotting analysis. Data are presented as mean ± SD (P < 0.05). 1: Control group; 2: Empty vector transfection group; 3: pEGFP-WWOX transfection group. GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.