To evaluate Livin protein expression, paraffin-embedded tissue sections were collected from 18 patients who underwent definitive surgery for OSCC at Chonnam National University Hwasun Hospital (Jeonnam, Korea) between March 2006 and August 2009. All 18 patients underwent appropriate definitive surgery. In 6 patients, surgery was followed by radiation therapy alone or cisplatin-based concurrent chemoradiation therapy.

Patient characteristics including age, gender, tumor location, stage, depth of tumor invasion, lymph node metastasis, distant metastasis, treatment, recurrence, survival and follow-up information were obtained by review of the hospital records. All patients were staged according to the 7th edition of the American Joint Committee on Cancer (AJCC) staging system (33). Overall survival was calculated from the first surgical date to the date of death or the date of last follow-up.

Livin protein expression was investigated immunohistochemically in formalin-fixed, paraffin-embedded tissue blocks obtained from 18 OSCC patients. Tissue sections (5 μm) were cut from each paraffin block, mounted, and dried on glass slides. Tissues were deparaffinized, rehydrated, and retrieved with retrieval buffer. Endogenous peroxidase activity was blocked with peroxidase-blocking solution (Dako, Carpinteria, CA, USA), followed by incubation with polyclonal rabbit anti-human Livin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C. After washing in Tris-buffered saline-Tween-20 buffer (TBST), tissues were stained using the Dako Real™ Envision horseradish peroxidase (HRP)/3,3′-diaminobenzidine (DAB) detection system (Dako). Tissues were counterstained with hematoxylin and mounted with coverslips. Stained tissues were viewed and photographed using a light microscope. Assessment of staining was interpreted by two independent observers who were without knowledge regarding the clinical information. The percentage of positive immunostaining cells was graded according to three levels: 0, <5%; 1, 5–50%; and 2, ≥50%.

Cells of the human OSCC cell line, PCI 50, were cultured in RPMI-1640 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA), 50 U/ml penicillin, and 50 μg/ml streptomycin (Gibco, Grand Island, NY, USA) in a humidified atmosphere of 5% CO₂ at 37°C. The PCI 50 cell line originated from oral cavity cancer. For transfection, cells were prepared and maintained in culture dishes with medium, and cells were seeded on 6-well plates at 2×10⁵ cells per well at the time of transfection. To study the biological role of Livin in OSCC, small interfering RNA (siRNA) was used to knock down endogenous Livin gene expression in the PCI 50 cells. Cells were transfected with Livin-specific siRNA (Bioneer, Daejeon, Korea) and negative control siRNA (Qiagen, Valencia, CA, USA) using Lipofectamine™ 2000 (Invitrogen). Forty eight hours after transfection, the viable transfected cells were enumerated and re-seeded in the individual experimental apparatus.

Cell invasion was measured using a Transwell invasion apparatus (Costar, Cambridge, UK). Transwell filters (8.0-μm pore size) were coated overnight with 1% gelatin solution on both the top and bottom surfaces and dried at room temperature. Cells transfected with Livin siRNA or negative control siRNA were seeded at 3×10⁵ cells in 120 μl of a 0.2% bovine serum albumin (BSA) suspension in the upper chamber. Subsequently, 400 μl of 0.2% BSA containing 7 μg/ml fibronectin (Calbiochem, La Jolla, CA, USA) as the chemoattractant was loaded into the lower chamber. After incubation for 24 h, cells on the top surface of the filter were wiped off with cottonballs. Cells that had migrated to the bottom surface of the Transwell were stained with Diff Quik solution (Sysmex, Kobe, Japan) and counted in five random squares in the microscopic field of view. Results are expressed as the mean ± standard error of the number of cells/field in three individual experiments.

Cells transfected with Livin siRNA or negative control siRNA were seeded in each well of Culture-Inserts^® (Ibidi, Bonn, Germany) at 1.5×10⁵ cells/well. After incubation for 24 h, each insert was detached and the progression of cell migration was ascertained by photography at 0, 8, 12 and 24 h using an inverted microscope. The distance between gaps was normalized to 1 cm after capture of three random sites.

Cell viability was determined with the EZ-CyTox (tetrazolium salt, WST-1) cell viability assay kit (Daeil Lab Inc., Seoul, Korea). After applying the WST-1 reagent at 37°C, cell viability was measured using a microplate reader (Infinite M200; Tecan, Austria GmbH, Vienna, Austria) with Magellan V6 data analysis software (Tecan). Triplicate wells were used for each experiment, and all experiments were conducted at least in triplicate.

Apoptosis was determined by Annexin V-fluorescein isothiocyanate (FITC) assay. Cells transfected with Livin siRNA or negative control siRNA were collected using trypsin, washed twice in phosphate-buffered saline (PBS) twice and re-suspended in binding buffer (BD Biosciences^®, San Diego, CA, USA). Annexin V-FITC and 7-amino-actinomycin D (7-AAD; BD Biosciences) were added to the cells, which were then incubated in the dark for 15 min, then added and re-suspended in 400 ml of binding buffer. Cells were analyzed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA). Data analysis was performed using standard Cell Quest software (Becton Dickinson).

Cells and tissues were lysed in RIPA buffer (1 M Tris-HCl, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA) with 1 mM phenylmethanesulfonyl fluoride (PMSF), Halt™ phosphatase inhibitor and Halt™ protease inhibitor cocktail (Thermo, Rockford, IL, USA). Resolved proteins (10–20 μg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the resolved proteins were electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking with 5% BSA in TBST at room temperature for 1 h, the PVDF membranes were sequentially blotted with the primary antibodies: polyclonal anti-human Livin, cleaved caspase-3, cleaved caspase-7, cleaved poly-ADP ribose polymerase (PARP) and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and polyclonal anti-human glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Santa Cruz Biotechnology) at 4°C overnight. After rinsing in TBST, each membrane was incubated with the anti-rabbit or anti-mouse HRP-conjugated secondary antibody (Santa Cruz Biotechnology) at room temperature for 1 h. Blots were detected with chemiluminescence (ECL) HRP substrate (Millipore) and recorded by a Ras-4000 image reader (Fujifilm, Tokyo, Japan).

The RNA from cells was isolated using TRIzol reagent (Invitrogen), reverse transcribed, and amplified using specific primers for Livin and GAPDH. Primer sequences were: Livin 5′-CACACAGGCCATCAGGAC AAG-3′/5′-ACGGCACAAAGACGATGGAC-3′ and GAPDH 5′-ACCACAGTCCATGCCATCAC-3′/5′-TCCACCACCCTG TTGCTGTA-3′. The size of the amplified products was 456 bp for Livin α and 403 bp for Livin β. For cDNA synthesis, 1 μg of mRNA was mixed with 50 ng/μl oligo-dT (Promega, Madison, WI, USA), MMLV reverse transcriptase (Invitrogen) and RNAsin (Takara Bio, Shiga, Japan) and incubation was continued at 42°C for 1 h and at 72°C for 15 min. PCR amplification of cDNA was performed using specific primers and GoTaq^® DNA polymerase (Promega). PCR products were separated by electrophoresis on a 1% agarose gel containing ethidium bromide.

Experimental differences between the Livin knockdown group and the control group were tested with the Student’s t-test. The statistical software program SigmaPlot Software (version 6.0; Systat Software, San Jose, CA, USA) was used. Survival curves were calculated using the Kaplan-Meier method, and comparison of the curves was performed using the log-rank test. The statistical software program used was Statistical Package for the Social Sciences (SPSS) version 18.0 (Microcal Software Inc., Chicago, IL, USA) was used. A p-value <0.05 was considered to indicate a statistically significant result.

The patient data are summarized in Table I. The study group of patients included 12 males and 6 females. The mean age was 59.8±10.8 years (mean ± standard deviation), with a range from 41 to 79 years. The mean follow-up period was 50.5 months, with a range from 11.2 to 84.7 months. Immunohistochemical analysis revealed that 8 (44.4%) of the 18 OSCC tissues overexpressed Livin relative to the adjacent normal mucosa. A pattern of strong nuclear staining was evident (Fig. 1A). In contrast, there was no staining in another OSCC tissue sample as negative control (Fig. 1B). The number of positive tumor cells for each tissue was recorded. Three tissues (16.7%) showed 2+ staining and five tissues (27.8%) had 1+ staining. Ten tissues (55.6%) had negative staining.

Correlations between Livin protein expression and clinicopathological characteristics in human OSCC are shown in Table II. Livin protein expression was not associated with age, gender, tumor location, stage, T stage, N stage, distant metastasis and recurrence (p>0.05) (Table II).

For the 18 patients with OSCC enrolled in this study, the 3-year overall survival rate was 72%. The 3-year overall survival was 80% in the negative Livin group, and 63% in the positive Livin group. Livin expression was not associated with patient overall survival (p=0.54) (Fig. 2).

In the cell invasion assay, the number of invasive Livin siRNA-transfected PCI 50 cells was 1029.0±28.7, whereas the number of invasive cells in the negative control siRNA-transfected PCI 50 cells was 511.0±24.1. The difference between the two groups was statistically significant (p<0.01) (Fig. 4). Livin knockdown resulted in significantly reduced cell invasiveness in the human OSCC cells.

In the cell migration assay, the artificial wound gap became significantly narrower in the plates of the negative control siRNA-transfected PCI 50 cells when compared with that of the Livin siRNA-transfected PCI 50 cells at 12 and 24 h (p<0.01) (Fig. 5). After 24 h, the wound gaps were filled in the plates of the negative control siRNA-transfected OSCC cells. In contrast, the wound gaps were wide open in the plates of Livin siRNA-transfected OSCC cells after 24 h. The results clearly showed that Livin knockdown resulted in significantly diminished cell migration in the human OSCC cells.

The cell viability was assessed at 1, 2, 3 and 4 days in the negative control siRNA-transfected PCI 50 cells and Livin siRNA-transfected PCI 50 cells to assessed the effect of Livin on cell proliferation (Fig. 6). The number of proliferating cells was significantly decreased in the Livin siRNA-transfected PCI 50 cells when compared to this number in the negative control siRNA-transfected PCI 50 cells at 2, 3 and 4 days (p=0.03, p=0.01, and p=0.003, respectively). The results clearly showed that Livin knockdown resulted in significantly decreased cell proliferation in human OSCC cells.

To determine the impact of Livin on cell apoptosis, responses of Livin siRNA-transfected OSCC cells and negative control siRNA-transfected OSCC cells were compared by an Annexin V apoptosis assay. Livin-knockdown OSCC cells displayed increased apoptosis, compared with the control cells (Fig. 7A). The proportions of early and late apoptotic PCI 50 cells induced by transfection of Livin siRNA (37.4 and 7.1%, respectively) were greater than that induced following transfection with negative control siRNA (11.6 and 1.2%, respectively) (Fig. 7A). Additionally, an increase in cleaved caspase-3, cleaved caspase-7 and cleaved PARP, which are key enzymes of apoptosis, was noted in the Livin-knockdown OSCC cells (Fig. 7B). These results suggest that Livin knockdown-induced apoptosis was associated with the modulation of apoptosis regulatory proteins such as caspase-3, caspase-7 and PARP.