The eukaryotic expression vector pEGFP-N1 and Escherichia coli DH5α competent cells are routinely maintained by the central laboratory at our hospital. The hepatoma cell line SMMC-7721 was obtained from the Chinese Academy of Sciences (Shanghai, China). Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM) culture medium was purchased from Gibco BRL (Gaithersburg, United States). Fetal bovine serum was obtained from Sijiqing Biological Engineering Material (Hangzhou, China). The following materials were used: RNeasy Protect Mini-kit (QIAGen Co., Germany), SMARTTM PCR cDNA synthesis kit (Clontech Co., United States), DNA gel extraction kit (Dalian TaKaRa Co., China), plasmid mini-preparation kit (Shanghai Huasun Biotechnology Co., China), KOD-Plus DNA polymerase (TOYOBO Co., United States), T4 DNA ligase and the HindIII and KpnI restriction enzymes (New England Biolabs, United States), Lipofectamine 2000 (Invitrogen, United States), anti-WWOX, anti-phospho-AKT(pThr308 and Ser473), cleaved caspasse-9 and caspase-3 monoclonal antibodies (Santa Cruz Co., United States), and horseradish peroxidase-conjugated goat anti-rabbit/mouse IgG (Zhongshan Co., China). Nucleic acid sequencing was performed by Shanghai Yingjun Bioengineering Co., China. The WWOX and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers were synthesized by Shanghai Yingjun Bioengineering Co., China.

SMMC-7721 cells were cultured in DMEM medium (HyClone Inc, United States) supplemented with 10% new calf bovine serum in a 37 °C and 5% CO₂ incubator.

The WWOX open reading frame was amplified from a cDNA clone using the forward primer 5’GGAAGCTTTTGGAGCGGGAGTGAG-3’ and the reverse primer 5’GGATCCCAGCAGTTGTTGAAGTACA-3’, which introduced KpnI and HinDIII restriction endonuclease sites. WWOX cDNA digested with KpnI and HindIII was cloned into a pEGFP-N1 eukaryotic expression vector. The resulting vector was transfected into SMMC-7721 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). An empty vector of pEGFP-N1 was used as a negative control. After 24-48 h, the transient transfection efficiency was determined under an Olympus fluorescence microscope. The cells were then passaged at appropriate ratios to six-well plates. The next day, the cells were cultured in the presence of 1000-2000 g/mL G418 (Life Technology, Paisley, Scotland), which was increased in concentration in a stepwise manner over 14 d. Cells highly expressing green fluorescent protein (GFP) were selected.

Protein was extracted from cultured cells using lysis buffer. After a 30-min incubation on ice, the lysates were heated at 100 °C for 15 min and centrifuged at 12 000 ×g for 15 min at 4 °C. Lysates containing an equal amount of protein (25 μg) were dissolved in sodium dodecyl sulphate (SDS) sample buffer, separated on 12% SDS slab gels, and transferred electrophoretically onto polyvinylidene difluoride membranes. Equal protein loading and transfer were confirmed by Ponceau S staining. After being blocked with 5% non-fat dry milk in Tris-Buffered Saline and Tween 20 (10 mmol Ttris-HCl, pH 8.0, 100 mmol/L NaCl and 0.05% Tween), the membrane was incubated at 4 °C overnight with the appropriate primary antibodies. Following washing, horseradish peroxidase conjugated secondary antibody was applied to the membrane. Proteins bound by the secondary antibody were visualized by electrochemiluminescence (Amersham Bioscience) according to the manufacturer’s instructions. The expression of GAPDH was measured as a control, and each experiment was performed in triplicate.

Cell growth was determined by the methyl thiazolyl tetrazolium (MTT) assay (Sigma, United States). Briefly, 1 × 10⁴ cells were seeded onto 96-well plates with four replicates for each condition. Approximately 72 h later, MTT reagent was added to each well at 5 mg/mL in a 20 μL volume, and the reaction was incubated for another 4 h. The formazan crystals formed by viable cells were subsequently solubilized in dimethyl sulfoxide, and the absorbance (A) at 490 nm was measured.

Approximately 100 cells were added to each well of a six-well culture plate. After incubation at 37 °C for 15 d, cells were washed twice with phosphate-buffered saline (PBS) and stained with Giemsa solution. The number of colonies containing ≥ 50 cells was counted under microscope [plate clone formation efficiency = (number of colonies/number of cells inoculated) × 100%]. Each experiment was performed in triplicate.

Forty-eight hours after treatment, logarithmically growing cells were collected and washed with PBS three times and fixed with 75% ethanol at -20 °C for at least 1 h. After extensive washing with PBS, the cells were suspended in Hank’s balanced salt solution containing 50 mg/mL RNase A (Boehringer Mannheim) and 50 mg/mL propidium iodide (PI) (Sigma-Aldrich), incubated for 1 h at room temperature, and were analyzed by FACScan (Becton Dickinson).

Apoptosis was analyzed 48 h after treatment using the Annexin V-FITC Apoptosis Detection Kit (BD Biosciences) according to the manufacturer’s instructions.

Data were presented as the mean ± SD. Comparisons of experimental values between cisplatin-treated cells and untreated controls were conducted using analysis of variance or the Kruskal-Wallis rank test. Statistical significance was defined as P < 0.05.

To study the biological functions of WWOX, we introduced WWOX into SMMC-7721 cells using a pEGFP-N1 eukaryotic expression vector containing the WWOX gene. Seven stably transfected cell clones were obtained. Western blotting analysis with anti-GFP antibodies showed that WWOX-pGFP fusion protein in the SMMC-7721 cell clones was highly expressed compared with control cells and control-vector cells (Figure 1).

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

To analyze the function of WWOX, we studied the rate of cell growth in the WWOX-expressing SMMC-7721 cells. The results from the colony formation assay indicated that SMMC-7721 cells overexpressing WWOX formed significantly fewer colonies than did the control clone cells and the control-vector cells (P < 0.05) (Figure 2). Cells transfected with WWOX also showed significantly decreased cell proliferation compared with control cells and control-vector cells when examined by the MTT assay (Table 1, Figure 3).

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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).

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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.

To detect the effect of WWOX overexpression on the cell cycle, we measured the cell cycle distribution in WWOX-expressing SMMC-7721 cells. In these lines, there was a marked decrease in the S-phase population, while the G1 population was significantly increased compared with the control vector and wild type SMMC-7721 cells (P < 0.05). Neither cell lines showed significant changes in the G2 population (Figure 4, Table 2). Cells transfected with pEGFP-N1-WWOX demonstrated more apoptosis than did cells transfected with the mock plasmid or the parent cells (P < 0.05) (Figure 5).

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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).

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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.

Expression of cleaved caspase-9 and caspase-3 was upregulated, as measured by Western blotting in cells that were transfected with pEGFP-N1, compared with either the cells transfected with a control vector or parental wild-type cells (Figure 6).

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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.

To evaluate the effect of WWOX on Akt/PKB activity, the phosphorylation level at Akt Thr308 and Ser473 was examined with specific phospho-Akt antibodies. Western blot analysis showed that WWOX significantly reduced the level of Akt/PKB phosphorylation (Figure 7).

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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.

Several researches have revealed loss of heterozygosity of WWOX locus in gastric, pancreatic, esophageal and lung cancers. The role of WWOX in hepatic carcinoma is not well understood, and few studies have reported the effects of WWOX on hepatic carcinoma. In this study, the authors investigated the apoptotic effects of the WWOX gene on the human hepatic carcinoma cell line SMMC-7721.

This is the first report about WWOX gene relevant to human hepatoma cell line SMMC-7721.

By cloning the WWOX gene and transferring it into hepatocellular carcinoma cell line SMMC-7721, the authors investigated the growth-inhibiting and apoptosis-inducing effects of WWOX gene on human hepatoma cell line SMMC-7721 and concluded that the over-expression of WWOX gene could induce apoptosis and inhibit the growth of hepatic carcinoma cell line SMMC-7721.

WWOX may have a potential role in development of chemotherapeutic and chemopreventive strategies against liver cancer.

The tumor suppressor gene WWOX is localized in a common fragile site FRA16D (locus 16q23.3-24.1). Protein encoded by WWOX is an oxidoreductase containing two WW protein interaction domains.

The manuscript describes the results of studies on the effect of tumor suppressor gene, WWOX, expression in SMMC-7721. This is an interesting and well presented study, with thorough introduction and the succinct discussion of the topic.