Host–tumor interactions lead to a non-specific inflammatory response mediated in part by the chronic production and release of pro-inflammatory cytokines, including IFN-?, interleukins, and tumor necrosis factor. Cytokines are critical for tumor regression. IFN-? is a cofactor in the activation of macrophages, which kill tumor cells, and also enhances cytotoxicity by natural killer cells. Interferon inhibits the growth of melanoma, renal-cell carcinoma, acquired immune deficiency syndrome-related Kaposi’s sarcoma, and other malignant carcinoid tumors. Interferon therapy can induce remission in human malignancies and is established as a treatment of choice in several diseases. The effect of IFN-? on oral cancer cells was investigated here.

EPA is derived from linolenic acid (designated n-3); it forms trienoic prostaglandins and pentaene leukotriene derivatives within several cell systems. EPA can serve as a competitive inhibitor of the biosynthesis of dienoic prostaglandins and tetraene leukotrienes from arachidonic acid. Diets containing large amounts of n-3 polyunsaturated fatty acids inhibit tumorigenesis. Diets providing high concentrations of n-3 fatty acids suppress tumour progression. Inhibition of human breast-cancer cell growth by EPA may be by the suppression of tumour eicosanoid biosynthesis. EPA may also have a suppressive effect on other cancer cells, including oral. This report will examine the biosynthesis of eicosanoids by oral cancer cells.

A number of inhibitors of cyclo-oxygenase and lipoxygenase pathways of arachidonic acid are important tools for the study of the role of arachidonic acid metabolites in various biological or pathological processes. Indomethacin is an inhibitor of the cyclo- oxygenase pathway of arachidonic acid metabolism. NDGA is an inhibitor of the lipoxygenase pathways. ETYA, the tetraacetylenic analog of arachidonic acid, is recognized as a potent inhibitor of both pathways. Dexamethasone, an anti-inflammatory steroid, exerts its effect by inhibiting the release of arachidonic acid from phospholipids. Using these enzyme inhibitors can help to confirm that this eicosanoid biosynthesis is via enzymatic pathways, as can high-temperature incubation, which would inactivate the synthesizing enzymes and thereby suppress the biosynthesis.

The possibility that oral cancer cells may be affected by IFN-?, EPA, and inhibitors of cyclo- or lipoxygenase is worthy of in vitro examination. In our laboratory, we have established an OEC-M1 cell line from the gingival tissues of a Chinese patient with oral carcinoma. We have now investigated the effects of IFN-?, EPA, indemethacin, NDGA, ETYA, and dexamethasone on the biosynthesis of two eicosanoid-like substances in the OEC-M1 cell line in vitro. We have also tested the specificity of the results by comparing the above-mentioned effects with those of a normal fibroblast cell line derived from human buccal mucosa and another human oral cancer cell line (KB).

The OEC-M1 cell line was established in our laboratory from the gingival epidermal carcinoma of a Chinese patient. The study protocol was approved by the Ethics Committee of the Tri-Service General Hospital, and written informed consent was granted by the patient. Human buccal-mucosa fibroblast cell lines were obtained and cultured according to the method of Van Wyk et al. (1994). Another human oral epidermoid-carcinoma cell line (KB) was obtained from the American Type Culture Collection (Rockville, MD). The fibroblasts and KB cell line were cultured to confluence and harvested for the detection of eicosanoid synthesis as control cells. The culture flasks contained 25 ml of RPMI-1640 medium with 10% FBS, 2 mM -glutamine, 25 mM HEPES, 100 U/ml penicillin, 100 ?g/ml streptomycin, and 0.1% Fungizone (Gibco Labs., Grand Island, N.Y.). The cells were incubated in a humidified atmosphere of 5% CO₂ and 95% air at 37°C. The culture medium was changed twice per week. Confluent cells were trypsinized with 0.05% trypsin in 0.02% EDTA. The trypsinized cells were seeded into more flasks and used in the following experiments until confluent.

Confluent cells were incubated with 0.4% FBS for 1–2 days until quiescence. After 1–2 days in the quiescent stage, the medium was removed and replaced with a serum-free medium containing different additives (IFN-?, Genzyme Corp., Cambridge, MA; EPA, NDGA, ETYA, Biomol Research Lab. Inc., Plymouth Meeting, PA; indomethacin, dexamethasone, Sigma Chemical Co., St. Louis, MO) for 12 h. Control groups were incubated in media containing no additives.

After this preincubation, cells from each group were harvested and resuspended in 4 ml of sodium phosphate buffer (10 mM NaH₂PO₄, 10 mM Na₂HPO₄, 100 ?M dithiothreitol, pH 7.4), and then homogenized with a Polytron operated at full speed for 2 min. The homogenates were centrifuged at 400×g for 5 min at 4°C to remove the unminced large fragments. The supernatant was removed and incubated with 10 ?M unlabelled arachidonic acid (Sigma) and 370 kBq [³H]-arachidonic acid (Amersham Corp., Arlington Heights, IL) in an equal volume of 0.1 M Tris–HCl buffer (pH 8.5), containing 2 mM glutathione, 1 mM hydroquinone, and 2?M hemin, at 37°C for 10 min. The reaction was terminated by the addition of 2 volumes of ethanol.

The precipitates from the cell homogenates were centrifuged at 800×g for 10 min at 4°C and the supernatant layer was evaporated to the aqueous phase. Methanol was added at a final concentration of 20%. An ODS–silica column (Sep-Pak C18 cartridge, Waters Associates, Milford, MA) was attached to a 20-ml polypropylene Luerlok syringe and washed successively with 5 ml of methanol and 5 ml of water, and the biological samples were then applied. The column was washed sequentially with 6 ml of 20% methanol and 6 ml of 80% methanol. The eicosanoids were collected and evaporated to dryness under a stream of nitrogen. The residues were then reconstituted in 1 ml of methanol and filtered through a 0.45 ?m filter (Millipore). The samples were again evaporated to dryness under nitrogen and reconstituted in 50 ?l of methanol/water (60:40, v/v), then separated by RP–HPLC.

The purified eicosanoids were separated by RP–HPLC in a Waters Dual Pump System equipped with a reverse-phase ultrasphere ODS column (Inertsil–ODS, 10?, 3.9 mm×30 cm; Vercotech Inc. Taipei, Taiwan). The products were eluted with an isocratic solvent system of methanol/water/acetic acid (60:40:0.01, v/v, pH 5.7) for 60 min at a flow rate of 1 ml/min. Column effluents were monitored with a Waters UV-VIS spectrophotometric detector (486 Turnable Absorbance Detector) at 280 nm. Tritium-labelled eicosanoid-like compounds in the eluate were simultaneously detected with a Radiomatic HPLC radioactivity monitor (Flo-one/Beta) attached to the Waters HPLC unit.

Samples eluted from the HPLC were simultaneously collected with an on-line fraction collector and ultraviolet absorbance spectra were analysed with a Hewlett-Packard 8450-A ultraviolet/visible spectrophotometer at 280 nm.

Statistical analyses used one-way ANOVA (Tukey posthoc test) by SPSS package. Probability values less than 0.05 were considered significant.

After incubation of OEC-M1 cells with different concentrations of IFN-?, EPA, indomethacin, NDGA, ETYA, and dexamethasone for 12 h, we observed the state of cell growth and detected the production of eicosanoids. There were two predominant peaks in the HPLC results which coincided with the radioactivity of [³H]-arachidonic acid. One compound (peak 1; P-1) eluted before prostaglandin B₂ (more polar than prostaglandin B₂) and the other compound (peak 2; P-2) eluted before leukotriene D₄ and just after leukotriene C₄ (more polar than D₄ and less polar than C₄). P-1 had ultraviolet absorption at a ?_max of 278 nm with shoulders at 272 and 284 nm. P-2 had ultraviolet absorption at a ?_max of 284 nm and shoulders at 278 and 290 nm. The front peak may contain a number of mixed polar metabolites and the last peak is arachidonic acid itself. These findings suggest that these compounds are possible leukotriene-like substances with conjugate triene-structure.