The Tca8113 OSCC cell line was obtained from the American Type Culture Collection (Rockville, MD, USA). All cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 µg/ml streptomycin and 100 U/ml penicillin (Invitrogen; Thermo Fisher Scientific, Inc., Beijing, China). Cells were cultured in 10-cm tissue culture dishes and maintained in a 37°C incubator equilibrated with 5% CO₂ in humidified air.

The mfat-1 gene was created from the coding region of the C. elegans fat-1 gene (GenBank: NM_001028389) by optimizing for mammalian cell expression. The mfat-1 cDNA was synthesized (Tiangen Biotech Co., Ltd., Beijing, China) and constructed into the control vector, named pCB0, which is from pcDNA3.1(+), using restriction enzyme digestion and ligation (NEB, Beijing, China. The cytomegalovirus (CMV) promoter was replaced with the CMV immediate-early enhancer/chicken β-actin hybrid promoter, and the constructed mfat-1 expression vector was named pCB1.

Tca8113 cells were transfected with linearized pCB1 and pCB0 with Lipofectamine Plus reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The cells were then selected with G418 (Invitrogen; Thermo Fisher Scientific, Inc.) at 350 µg/ml. After 2 weeks of selection, the G418-resistant colonies were pooled to reduce potential variations amongst different clones. The mfat-1 transgene expression and function were confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and fatty acid profile analysis.

Total RNA was extracted using a SimplyP Total RNA Extraction kit (Bioer, Beijing, China). Reverse transcription was performed using random hexamer oligonucleotide primers using the BioRT Two-Step RT-PCR kit (Bioer) according to the manufacturer's protocol. Cells were then subjected to RT-PCR for the detection of mfat-1 mRNA expression. The sequences of the primers of mfat-1 were as follows: Sense 5′-CCGCCACCGCTCCCGTCAC-3′ and antisense 5′-TGCTTCCCAATCCTTAT-3′. The resulting amplification product was 527 bp in length. PCR primers for GAPDH were as follows: Sense 5′-CCATGGAGAAGGCTGGGG-3′ and antisense 5′-CAAAGTTGTCATGGATGACC-3′. GAPDH was used as the control, and the resulting amplification product was 194 bp. The PCR amplification was performed at 95°C for 30 sec, 60°C for 30 sec and 72°C for 30 sec for 30 cycles, with a final extension at 72°C for 6 min. The PCR products were separated on a 1.0% agarose gel (Tiangen Biotech Co., Ltd.) at 120 V and stained with ethidium bromide (Tiangen Biotech Co., Ltd.).

Lipids were extracted from the cells according to the technique of Bligh and Dyer (16). Briefly, the samples were homogenized in a mixture of methanol, chloroform, and water. After 15 min, chloroform was added and the samples were vortexed and centrifuged at 1,500 × g for 30 min. The lower phase was dried under nitrogen and resuspended in boron trifluoride methanol (Sigma-Aldrich, St. Louis, MO, USA). The samples were heated at 90°C for 30 min and extracted with 4.0 ml pentane (Beijing Chemical Works, Beijing, China) and 1.5 ml water. The mixtures were then vortexed and the upper phase was recovered. The extracts were dried, resuspended in heptane (Sigma-Aldrich), and injected into a capillary column (SP-2380, 105 m × 53 mm ID, 0.20-µm film thickness; Sigma-Aldrich). The gas chromatograph was performed on a Clarus 500 gas chromatograph (PerkinElmer, Inc., Waltham, MA, USA). The comparison of retention time to authentic standards (Sigma-Aldrich) were used to identify components.

Briefly, Tca8113 cells and the cells transfected with either pCB1 and pCB0 were plated in 96-well plates overnight. A density of 3,000 cells/well in 200 µl appropriate growth media was used, and these cells were incubated for 24, 48 and 72 h. Next, 20 µl/well MTT solution (Invitrogen; Thermo Fisher Scientific, Inc.) was added and the cells were incubated for another 3.5 h. The absorbance of each well at 570 nm, with the background subtraction at 650 nm, was read by a Multiskan GO Microplate Spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The average absorbance of 6 wells/experiment was used.

Apoptosis was detected and quantified by the Cell Death Detection ELISA^PLUS kit (Roche Diagnostics, Shanghai, China). Briefly, cells were seeded in plates with a regular medium and allowed to grow until 70–80% confluent. Next, cells were washed twice with PBS and the regular media was changed to DMEM without serum. DNA fragmentation was calculated by the fold change and normalized with corresponding MTT results from identical culture conditions.

To establish xenografts in nude mice, Tca8113 cells that were transfected with either pCB1 or pCB0 in log-phase growth were washed twice with PBS and then counted. Subcutaneous tumors were established by injecting 1.2×10⁶ Tca8113 cells, which were resuspended in 200 µl PBS, onto each side of the dorsal surface on the BALB/c nude male mice. Six-week-old male BALB/c nude mice were obtained from Model Animal Research Center of Nanjing University (Nanjing, China). Mice were assigned to two groups (n=6 per group): Tca8113-pCB0 or Tca8113-pCB1, and were handled in accordance to institutional guidelines for animal care. The tumor size was measured once weekly for 5 weeks and the tumor volume (mm³) was calculated as follows: Tumor volume = LxW²×0.5. The tumor volume data were subjected to statistical analysis using either the Student's two-tailed t-test or the Mann-Whitney U test. The animal experiments were performed according to the protocol approved by the Jilin University Institutional Animal Care and Use Committee (#2012079; Changchun, China).

Xenograft tumors were harvested and placed in 10% formalin, embedded in paraffin, and sectioned at 5 µm thickness using an RM2255 microtome (Leica Microsystems, Inc., Buffalo Grove, IL, USA). Paraffin-embedded tumor blocks were cut at serial sections and the obtained slides were used for hematoxylin and eosin (H&E) staining and immunohistochemical analysis, as described by Lu et al (17). Tumor cell proliferation was studied by immunostaining with a Ki-67 antibody (#PA121520; Invitrogen; Thermo Fisher Scientific, Inc.). Similarly, apoptosis was detected using an Apoptag terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL) assay kit (EMD Millipore, Billerica, MA, USA), counterstained with methyl green (Sigma-Aldrich). The rates of proliferation and apoptosis were quantitatively determined by counting the percentage of positively stained cell bodies from three histologically similar fields viewed at ×400 magnification using a Labophot-2 microscope (Nikon Corporation, Tokyo, Japan), which were randomly selected from each slide. Slides of 3 fields per animal and 5 animals per group were used for comparison.

Cell samples were measured for total protein concentration using the bicinchoninic acid (BCA) assay (Pierce Biotechnology, Inc., Rockford, IL, USA). A total of 40 µg protein/samples were prepared and separated on 12% SDS-PAGE gels at 100 V prior to transfer to polyvinylidine difluoride membranes (Bio-Rad Laboratories, Inc., Beijing, China). The membranes were blocked and shaken at room temperature for 1 h and then incubated overnight with rabbit anti-mouse primary antibodies against caspase-3 (#9662), caspase-9 (#9504), GSK-3β (#9315), p-GSK-3β (#9322), β-catenin (#9562), Akt (#9272) and p-Akt (#9275; Cell Signaling Technology, Inc., Danvers, MA, USA), all at a dilution of 1:1,000. The next day, the blots were washed 3 times with PBS-Tween-20 (PBS-T), then incubated with the corresponding goat anti-rabbit polyclonal horseradish peroxidase-conjugated secondary antibodies (1:5,000: #12348; Sigma-Aldrich) for 1 h at room temperature. Following another 3 washes with PBS-T, the blots were subjected to the ECL Western blotting detection reagents and analysis system (GE Healthcare Life Sciences, Piscataway, NJ, USA) to visualize the protein bands.

Statview software, version 4.5 (Abacus Concepts, Berkley, CA, USA) was used for the statistical analysis. Analysis of variance was used for initial analysis, followed by Fisher's protected least significant difference for post hoc analyses. All experiments were performed at least in triplicate and the results are presented as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

Following transfection of the Tca8113 cells with the mfat-1 gene (pCB1), RT-PCR with mfat-1-specific primers indicated that the cells transfected with pCB1 expressed high levels of the mfat-1 gene (Fig. 1A). As expected, the mfat-1 gene was not expressed in the parental cells or the cells that were transfected with the control vector (pCB0). To analyze the function of the mfat-1 gene, gas chromatography was performed to assess the composition of PUFAs in the cells. The level of ω-3 PUFAs was significantly increased in cells transfected with pCB1, compared with cells transfected with pCB0 (Table I). The level of PUFAs in cells transfected with pCB1 and pCB0 are presented in Table I as a percentage of the total cellular lipids. The expression of the mfat-1 gene led to a significant increase in ω-3 PUFAs, accompanied by a decrease in ω-6 PUFAs (Table I). The ratio of ω-6/ω-3 PUFAs with or without exogenous mfat-1 were 0.381 and 1.275, respectively (P<0.001; Fig. 1B). This indicates that the expression of mfat-1 can convert ω-6 PUFAs into the corresponding ω-3 forms. It can be predicted that the change in the ω-6/ω-3 PUFA ratio affects the endogenous ω-3 PUFA production mechanism, while reducing the level of ω-6 PUFA production.

To determine whether the change in the ratio of ω-6/ω-3 PUFAs in Tca8113 cells inhibits tumor cell proliferation in vitro, cells were cultured for different time periods and MTT cell proliferation assays were performed. Expression of the mfat-1 gene significantly inhibited the Tca8113 cell proliferation in cells that were transfected with pCB1, compared with the cells that transfected with pCB0 at 48 and 72 h (P<0.001; Fig. 2A). To evaluate whether mfat-1 gene expression in Tca8113 cells promotes programmed cell death, a DNA fragmentation ELISA assay was performed. The results demonstrated that mfat-1 gene expression enhances apoptosis in Tca8113 cells at 24 and 48 h, compared with cells transfected with the control vector (P<0.001; Fig. 2B).

To determine the antitumor effect of the mfat-1 gene in vivo, Tca8113 cells transfected with either pCB1 or pCB0 were implanted into nude mice. The cells were allowed 5 weeks for tumor growth. The results in Fig. 3 demonstrate that mfat-1 gene expression significantly reduces Tca8113 cell tumor volume at 4 and 5 weeks subsequent to implantation, compared with cells that were transfected with the control vector (P<0.05; Fig. 3A).

To determine the mechanism of tumor growth inhibition by the mfat-1 gene, histological analyses were performed. H&E staining demonstrated that the overexpression of the mfat-1 gene was associated with a reduced number of viable cells and an increased area of necrosis (Fig. 3B). The number of proliferating cells, identified by Ki-67-positive nuclei, was significantly reduced in the tumor cells transfected with the mfat-1 gene compared with the control-transfected cells (P<0.001; Fig. 3C). It was also observed, using a TUNEL assay, that the introduction of the mfat-1 gene to Tca8113 cells was associated with an increased number of positively stained apoptotic cells. Cell counts indicated that 50–60% of cells transfected with the mfat-1 gene were apoptotic, whereas 10–20% of cells were apoptotic in the control group, and this difference was demonstrated to be statistically significant (P<0.001; Fig. 3D).

To further investigate the mechanism of mfat-1 gene expression on the inhibition of tumor cell growth, the protein expression of caspase-3, caspase-9, p-GSK-3β, GSK-3β, β-catenin, p-Akt and Akt was examined (Fig. 4). It was demonstrated that the cleavage of caspase-9 and caspase-3 was enhanced in cells transfected with the mfat-1 gene, and mfat-1-transfected cells presented a reduced level of GSK-3β phosphorylation and β-catenin expression. No alterations were observed in Akt expression or phosphorylation.