Regulation of cell growth by selective COX-2 inhibitors in oral carcinoma cell lines

Prostaglandins & other Lipid Mediators 72 (2003) 115–130

Regulation of cell growth by selective COX-2 inhibitors in oral carcinoma cell lines C.Y. Yang a,∗ , C.L. Meng a , C.L. Liao b , P.Y.-K. Wong c a

Department of Dentistry, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, ROC b Department of Microbiology and Immunology, National Defense Medical Center, Taipei, Taiwan, ROC c Department of Pharmacology, New York Medical College, Valhalla, NY, USA Received 10 June 2003; accepted 19 June 2003

Abstract Evidence indicates that NSAIDs that inhibit prostaglandin (PG) synthesis can reduce the incidence of colorectal cancers and that inhibition of cyclooxygenase-2 (COX-2) may be the underlying mechanism. The objective of this study was to investigate this putative mechanism by examining the effect of selective COX-2 inhibitors (Celebrex, DFU, NS-398) and COX-1 inhibitors (Aspirin) on the growth of two human oral carcinoma cell lines (OEC-M1 and KB) and one normal fibroblast cell line (NF). We found that the growth of OEC-M1 cells could be significantly inhibited by DFU concentrations above 30 ␮M (31%) after 4 days, and above 50 ␮M (35%) after 2 days in culture; by Celebrex at concentrations above 20 ␮M (52%) after 6 days, above 30 ␮M (36%) after 5 days, and above 40 ␮M (33%) after 4 days in culture; and by NS-398 above 1 ␮M (30%) after 6 days, and above 10 ␮M (35%) after 5 days in culture. The growth of KB cells could be significantly inhibited by DFU concentrations above 10 ␮M (33%) after 6 days, above 20 ␮M (35%) after 4 days in culture; and by Celebrex at concentrations above 10 ␮M (33%) after 5 days, and above 50 ␮M (30%) after 4 days in culture; and by NS-398 above 1 ␮M (45%) after 5 days, above 20 ␮M (36%) after 4 days in culture. The growth of NF cells could be significantly inhibited by DFU above 30 ␮M (45%) after 6 days, and above 40 ␮M (32%) after 3 days in culture, and by Celebrax at concentrations above 10 ␮M (42%) after 6 days, above 30 ␮M (31%) after 4 days, above 50 ␮M (32%) after 3 days in culture, and by NS-398 above 0.1 ␮M (35%) after 4 days, and above 1 ␮M (32%) after 3 days in culture. The growth-inhibitory concentration (IC50 ) values for DFU on OEC-M1, KB, and NF cells were about 39.1, 14.8, and 42.9 ␮M at 144 h, respectively, and on

Abbreviations: DFU, 5.5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulfonyl)phenyl-2(5H)-furanone; CLBX, Celebrex, Celecoxib, SC-58635, 4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzene-sulfonamide]; NS-398, N-{2-(cyclohexyloxy)-4-nitrophenyl}-methanesulfonamide; PHS, prostaglandin endoperoxide synthase; PGs, prostaglandins; OEC-M1 cell line, oral epidermal carcinoma cell line derived from gingiva of a Chinese patient; KB cell line, oral epidermal carcinoma cell line derived from the floor of mouth of a Caucasian patient; NF cell line, normal human buccal mucosa fibroblast cell line ∗ Corresponding author. Tel.: +886-2-8792-4922; fax: +886-2-2367-3114. 1098-8823/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S1098-8823(03)00053-4

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KB was about 45.2 ␮M at 120 h. The IC50 values for Celebrex on OEC-M1, KB, and NF cells were about 19.1, 8.6, and 15.8 ␮M at 144 h, respectively, and on KB and NF were about 27.7 and 35.3 ␮M, respectively, at 120 h. The IC50 values for NS-398 on OEC-M1, KB, and NF were about 18.9, and 0.7 1 ␮M, respectively, at 144 h; on KB and NF values were about 10.8 and 1.4 ␮M, respectively, at 120 h and on KB and NF were about 26.6 and 4.1 ␮M, respectively, at 96 h. The results show that the growth of these cell lines is inhibited by three COX-2 selective inhibitors but not by any COX-1 selective inhibitors. These findings suggest that COX-2 may play an important role in the generation of biochemical mediators that stimulate the growth of human oral cancer and normal fibroblast cell lines. © 2003 Elsevier Inc. All rights reserved. Keywords: GCOX-2; Cell growth; Oral cancer

1. Introduction Several prostaglandins (PGs) have been detected in various malignant tumors [1–3] and in growth media from mouse fibrosarcoma cells in culture [4]. Both endogenous and exogenous PGs have been shown to inhibit, significantly, the rates of tumor cell proliferation in vitro [5–8]. COX (cyclooxygenase; prostaglandin endoperoxide synthase, EC 1.14.99.1) catalyzes the formation of PGH2 from arachidonic acid and further metabolizes it to various biologically active molecules including prostaglandins, prostacyclin, and thromboxane. Expression of cyclooxygenase-2 (COX-2) is abnormally increased in adenomatous polyps [9] and several published papers have suggested that COX-2 is associated with the pathogenesis of cancers [10–13]. Two COX genes, COX-1 and COX-2, have been identified which share over 60% identity at the amino acid level [14]. COX-1 and COX-2 are likely to have fundamentally different biological roles. COX-1 is constitutively expressed in most tissues and is involved in maintaining cellular function and homeostasis [15]. In contrast, COX-2 is frequently undetectable at baseline in normal tissues, but is readily induced and expressed in response to various inflammatory stimuli, including cytokines, LPS, mitogens, reactive oxygen intermediates, rheumatoid arthritis, ulcerative colitis, Crohn’s disease and infectious gastritis [16–19]. Recent studies have further demonstrated that COX-2 levels are increased in carcinomas of the colon [20–22], breast [23], stomach [24], esophagus [25], lung [26], liver [27], and pancreas [28]. There is a widespread idea in this field that the COX-2 enzyme will be found to be involved in the carcinogenesis of even more human tumors in the future. Experimental animal studies suggest that selective COX-2 inhibitors prevent carcinogenesis by inducing apoptosis and inhibiting growth of cancer cells [25,29–32]. Until now, however, the notion that the COX-2 enzyme is involved in the progression of oral cancers has not been examined. In our laboratory, we established and characterized an epidermoid carcinoma cell line derived from human gingiva (OEC-M1). Another human oral carcinoma cell line (KB) was obtained from the US ATCC. The objective of this investigation was to study whether COX-2 is involved in the growth of oral cancers. The effects of selective COX-2 inhibitors on cultured cancer cell lines and a normal fibroblast cell line (NF) were examined by determining their effects on cell growth pattern and their toxicity.

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2. Materials and methods 2.1. Establishment of the normal fibroblast cell line (NF) The origin of the materials used for tissue cultures was described in a previous publication [33]. Biopsy specimens were obtained from the buccal mucosa of healthy individuals who neither chewed areca nuts nor smoked cigarettes [34]. The specimens were placed in RPMI-1640 medium supplemented with 10–12% FBS, penicillin (100 U/ml), streptomycin (100 ␮g/ml) and 0.2% amphotericin-B, and processed within 1 h for tissue culturing. Once the cell lines were established, the amphotericin-B was omitted from the medium. Cells were cultured under standard conditions (95% humidity, an atmosphere of 5% CO2 at 37 ◦ C) and passaged after detachment with 0.1% trypsin and 0.05% EDTA in Dulbecco’s phosphate buffered saline (PBS) for 5 min. Cells were stored in liquid nitrogen until required. 2.2. Primary culture and subculture of human epidermal carcinoma cell lines A human cancer cell line, established in our laboratory from gingiva epidermoid carcinoma (OEC-M1) was maintained in culture [35]. The study was approved by the Ethics Committee of the Tri-Service General Hospital, and written informed consent was granted by the patient. Another oral cancer cell line (KB, a human epidermoid carcinoma from the floor of the mouth) was obtained from the ATCC in the US. The culture flasks contained 25 ml of RPMI-1640 medium with 10% FBS, 2 mM l-glutamine, 25 mM Hepes, 100 U/ml penicillin, 100 ␮g/ml streptomycin, and 0.1% fungizone. The cells were incubated in a humidified atmosphere of 5% CO2 /95% air at 37 ◦ C. Culture medium was changed twice per week. Confluent cell monolayers were detached with 0.05% trypsin in 0.02% EDTA (Gibco Lab.) and suspended in fresh medium. The trypsinized cells were seeded into more flasks and utilized until the cell culture was confluent. 2.3. Preparation of the COX-2 inhibitors Two selective COX-2 inhibitors (DFU; 5.5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulfonyl)phenyl-2(5H)-furanone) were generous gifts from Dr. Denis Riendeau of Merck Frosst Canada, Kirland, Que. Other selective COX-2 inhibitors (Celebrex; Celecoxib; SC58635; 4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzene-sulfonamide]) were generous gifts from Dr. Peter Isakson of Pharmacia/UpJohn, Corp., NJ, USA. Another selective COX-2 inhibitor [NS-398; N-{2-(cyclohexyloxy)-4-nitrophenyl}-methanesulfonamide] was obtained from Caymen Chemicals (Ann Arbor, MI, USA) and was made up as a 30 mM stock solution in DMSO. A COX-1 inhibitor (Aspirin) was obtained from Sigma Chemical Co. (St. Louis, MO). Briefly, stock solutions were prepared in either DMSO (DFU, Celebrex, NS-398) or saline (Aspirin), filter-sterilized (0.45 ␮M Millipore), aliquoted and stored at −20 ◦ C. The required final concentration of each compound was prepared by diluting aliquots of the respective stock solutions in medium together with an appropriate volume of DMSO so that each control and treated flask received an equal final volume of DMSO that had a maximum concentration of 0.33% (v/v).

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2.4. Growth curves and cytotoxicity studies of COX-2 and COX-1 inhibitors on carcinoma and fibroblast cell lines The cells were plated out at 1.2 × 104 cells/ml into 24-well plates and counted randomly in duplicate for 6 consecutive days using a hemocytometer. Viability was determined by trypan blue exclusion. Growth curves of the cell lines were determined during incubation with or without the addition of different concentrations of COX-2 or COX-1 inhibitors. Cytotoxicities of various inhibitors were determined by counting the fraction of living cells that excluded the dye. The 50% growth-inhibitory concentration (IC50 ) values were calculated from dose–response curves obtained for each cell line. IC50 values represent the concentration of inhibitors required to reduce the living cells of a cell line to 50% of the control level over a given number of hours of treatment. 2.5. Statistical analysis The results were expressed as the means±S.D. from three replicate observations for each treatment group. All experiments were repeated three times with similar results. Two-way ANOVA was employed for the assessment of the significance of differences between experimental and control groups. P values less than 0.05 were considered statistically significant.

3. Results The effect of selective COX-2 (DFU, Celebrex, and NS-398) and COX-1 (Aspirin) inhibitors on two oral cancer cells (OEC-M1 and KB) and one human normal fibroblast cell line was determined by counting cell number during their growth periods. We found that the growth of the OEC-M1 cell line could be significantly inhibited by DFU above 30 ␮M (31%) after 4 days, 50 ␮M (35%) after 2 days in culture (Fig. 1A); by Celebrex at concentrations above 20 ␮M (52%) after 6 days, 30 ␮M (36%) after 5 days, and 40 ␮M (33%) after 4 days in culture (Fig. 1B); and by NS-398 above 1 ␮M (30%) after 6 days, 10 ␮M (35%) after 5 days in culture (Fig. 1C). The growth of OEC-M1 cell line was not significantly inhibited by Aspirin (Fig. 1D). The IC50 value of DFU on OEC-M1 was about 39 ± 2.1 ␮M at 144 h (Fig. 2A, Table 1), that of Celebrex was about 19.1 ± 1.1 ␮M at 144 h (Fig. 2B, Table 1) and that of NS-398 was about 18.9 ± 1.0 ␮M at 144 h (Fig. 2C, Table 1), and there were no earlier time points that reached half-maximal inhibition (Fig. 2, Table 1). At no time was half-inhibition reached for Aspirin (Fig. 2D, Table 1). The growth of the KB cell line was significantly inhibited by DFU above 10 ␮M (33%) after 6 days, 20 ␮M (35%) after 4 days in culture (Fig. 3A); by Celebrex at concentrations above 10 ␮M (33%) after 5 days, above 50 ␮M (30%) after 4 days in culture (Fig. 3B); and by NS-398 above 1 ␮M (45%) after 5 days, 20 ␮M (36%) after 4 days in culture (Fig. 3C). The growth of the KB cell line was not significantly inhibited by Aspirin (Fig. 3D). The IC50 values of DFU on KB was about 14.8 ± 0.7 ␮M at 144 h and 45.2 ± 2.3 ␮M at 120 h (Fig. 4A, Table 1); that of Celebrex on the KB cell line was about 8.6 ± 0.4 ␮M at 144 h and 27.7 ± 1.4 ␮M at 120 h (Fig. 4B, Table 1); that of NS-398 on KB was about 0.70 ± 0.04 ␮M at 144 h, 10.8 ± 0.5 ␮M at 120 h, and 26.6 ± 1.3 ␮M at 96 h (Fig. 4C, Table 1); there were

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Fig. 1. The growth curves of OEC-M1 cell line exposed to various concentrations of COX-2 selective inhibitors. (A) DFU. (B) CLBX. (C) NS-398. (D) Aspirin. Data are represented as the means±S.D. for triplicate experiments. ∗ Significantly different from control, as determined by Student’s paired t test (P < 0.05).

no earlier time points that reached half-maximal inhibition (Fig. 4, Table 1). There were no time points that reached half-maximal inhibition for Aspirin (Fig. 4D, Table 1). The growth of the NF cell line was significantly inhibited by DFU above 30 ␮M (45%) after 6 days, and above 40 ␮M (32%) after 3 days in culture (Fig. 5A); by Celebrex at

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Fig. 2. Cytotoxicity of various concentrations of COX-2 selective inhibitors on OEC-M1 cell line. (A) DFU. (B) CLBX. (C) NS-398. (D) Aspirin. Data are expressed as a percentage of control vs. concentrations of COX-2 inhibitors. Each point on the graph represented as the means ± S.D. for triplicate experiments. IC50 values were calculated from dose–response curves obtained for each cell line at 24, 48, 72, 96, 120, and 144 h time points. ∗ Significantly different from control, as determined by Student’s paired t test (P < 0.05).

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Table 1 IC50 value for DFU, CLBX, NS-398 and Aspirin inhibition in human oral carcinoma and normal fibroblasts cell lines Inhibitors

Incubation time (h)

Cell lines OEC-M1

KB

NF

DFU

120 144

None 39.1 ± 2.1

45.2 ± 2.3 14.8 ± 0.7

None 42.9 ± 2.2

CLBX

120 144

None 19.1 ± 1.1∗

27.7 ± 1.4∗ 8.6 ± 0.4∗

35.3 ± 1.8 15.8 ± 0.8∗

NS-398

96 120 144

None None 18.9 ± 1.0∗,∗∗

26.6 ± 1.3 10.8 ± 0.5∗∗ 0.70 ± 0.04∗,∗∗

Aspirin

At all times

None

None

4.1 ± 0.2 1.4 ± 0.1∗∗ 1.0 ± 0.1∗,∗∗ None

OEC-M1, KB, and NF cell lines were treated with three COX-2 inhibitors (DFU, CLBX, NS-398) and one COX-1 inhibitor (Aspirin) as described in Section 2. The cell number of each incubation was counted randomly in duplicate for 6 consecutive days using a hemocytometer and viability was determined by a trypan blue exclusion technique. Cytotoxicities of various inhibitors were determined by counting the living cells, which excluded the dye. The 50% growth-inhibitory concentration (IC50 ) values were calculated from dose–response curves obtained for each cell line. Data are expressed as the means ± S.D. for triplicate observations. None: no growth-inhibitory concentration values have reached half-inhibition. ∗ Significantly different from DFU, as determined by Student’s non-paired t test (P < 0.05). ∗∗ Significantly different from CLBX, as determined by Student’s non-paired t test (P < 0.05).

concentrations above 10 ␮M (42%) after 6 days, above 30 ␮M (31%) after 4 days, and above 50 ␮M (32%) after 3 days in culture (Fig. 5B); and by NS-398 above 0.1 ␮M (35%) after 4 days, 1 ␮M (32%) after 3 days in culture (Fig. 5C). The growth of the NF cell line was not significantly inhibited by Aspirin (Fig. 5D). The IC50 of DFU on NF was about 42.9±2.2 ␮M at 144 h (Fig. 6A, Table 1); that of Celebrex was about 15.8±0.8 ␮M at 144 h, and 35.3 ± 1.8 ␮M at 120 h (Fig. 6B, Table 1); and that of NS-398 was about 1.0 ± 0.1 ␮M at 144 h, 1.4 ± 0.1 ␮M at 120 h, and 4.1 ± 0.2 ␮M at 96 h (Fig. 6C, Table 1); there were no earlier time points that reached half-maximal inhibition (Fig. 6, Table 1). There were no time points that reached half-maximal inhibition for Aspirin (Fig. 6D, Table 1). The results show that the growth of these cell lines are inhibited by COX-2 selective inhibitors but not by COX-1 inhibitors.

4. Discussion It is well known that people who ingest Aspirin regularly may not develop colon cancer [36] as readily and patients with coronary artery disease receiving regular treatment of NSAIDs and HRIs also show a reduction in the incidence of colon cancer [37]. Other epidemiological studies also suggest that NSAIDs are effective agents in reducing mortality from colorectal cancer [38]. Studies have shown that the mechanism by which NSAIDs inhibit tumor growth is to block the COX-2 enzyme [31]. Our study was designed to determine if three newly developed COX-2 selective inhibitors reduce the growth of oral

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Fig. 3. The growth curves of KB cell line exposed to various concentrations of COX-2 selective inhibitors. (A) DFU. (B) CLBX. (C) NS-398. (D) Aspirin. Data are represented as the means ± S.D. for triplicate experiments. ∗ Significantly different from control, as determined by Student’s paired t test (P < 0.05).

cancer cell lines. We demonstrated that COX-2 inhibitors—DFU, Celebrex, and NS-398— significantly inhibit the growth of two oral cancer cell lines and one fibroblast cell line and that this inhibition is concentration- and time-dependent. In contrast, a COX-1 inhibitor (Aspirin) showed no significant inhibiting effect on any of these cell lines.

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Fig. 4. Cytotoxicity of various concentrations of COX-2 selective inhibitors on KB cell line. (A) DFU. (B) CLBX. (C) NS-398. (D) Aspirin. Data are expressed as a percentage of control vs. concentrations of COX-2 inhibitors. Each point on the graph represented as the means ± S.D. for triplicate experiments. IC50 values were calculated from dose–response curves obtained for each cell line at 24, 48, 72, 96, 120, and 144 h time points. ∗ Significantly different from control, as determined by Student’s paired t test (P < 0.05).

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Fig. 5. The growth curves of NF cell line exposed to various concentrations of COX-2 selective inhibitors. (A) DFU. (B) CLBX. (C) NS-398. (D) Aspirin. Data are represented as the means ± S.D. for triplicate experiments. ∗ Significantly different from control, as determined by Student’s paired t test (P < 0.05).

In order to compare the potency between these three COX-2 selective inhibitors, we examined the IC50 ratio between the inhibitors (Table 1). CLBX was approximately 1.6–2 times more effective than DFU in the OEC-M1 cell line at 144 h (39.1 ± 2.1 ␮M:19.1 ± 1.1 ␮M), and in the KB cell line at 120 h (45.2 ± 2.3 ␮M:27.7 ± 1.4 ␮M), and at 144 h (14.8 ±

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Fig. 6. Cytotoxicity of various concentrations of COX-2 selective inhibitors on NF cell line. (A) DFU. (B) CLBX. (C) NS-398. (D) Aspirin. Data are expressed as a percentage of control vs. concentrations of COX-2 inhibitors. Each point on the graph represented as the means ± S.D. for triplicate experiments. IC50 values were calculated from dose–response curves obtained for each cell line at 24, 48, 72, 96, 120, and 144 h time points. ∗ Significantly different from control, as determined by Student’s paired t test (P < 0.05).

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0.7 ␮M:8.6 ± 0.4 ␮M). NS-398 was almost as effective as CLBX in the OEC-M1 cell line (19.1 ± 1.1 ␮M:18.9 ± 1.0 ␮M). NS-398 was approximately 4.2 times more effective than DFU in the KB cell line at 120 h (45.2 ± 2.3 ␮M:10.8 ± 0.5 ␮M), and three times more effective than that of CLBX at 120 h (27.7±1.4 ␮M:10.8±0.5 ␮M), and 12 times more than that of CLBX at 144 h (8.6 ± 0.4 ␮M:0.70 ± 0.04 ␮M). NS-398 was approximately 25.2 times more effective than CLBX in the NF cell line at 120 h (35.3 ± 1.8 ␮M:1.4 ± 0.1 ␮M), and 16 times more than that of CLBX at 144 h (15.8 ± 0.8 ␮M:1.0 ± 0.1 ␮M). NS-398 was approximately two times more effective than DFU in the OEC-M1 cell line at 144 h (39.1 ± 2.1 ␮M:18.9 ± 1.0 ␮M), 21 times more than that of DFU in the KB cell line at 144 h (14.8 ± 0.7 ␮M:0.70 ± 0.04 ␮M), and 43 times more than that of DFU in the NF cell line at 144 h (42.9 ± 2.2 ␮M:1.0 ± 0.1 ␮M). It appears that NS-398 has the highest potency among these three drugs in the KB and NF cell lines; CLBX is the second most potent drug; and DFU is the least potent. Both NS-398 and CLBX had potencies that were two-fold higher than DFU in the OEC-M1 cell line. Our results are consistent with some of the findings of Warner et al. [39], namely that the IC50 of NS-398 and CLBX in the human whole blood assay for COX-2 (WBA-COX-2) are 0.35 and 0.83 ␮M, respectively, and that of NS-398 and CLBX in the modified human whole blood assay for COX-2 (WHMA-COX-2) are 0.042 and 0.34 ␮M, respectively. In contrast, our results may not fully be in accord with the findings reported by Riendeau et al. [40], that the order of potency of COX-2 selective inhibitors against COX-1 was CLBX > NS-398 > DFU. It is unclear whether this difference is due to the nature of the cell line or the drug itself. DFU is a furanone derivative that competitively and tightly binds COX-2 with arachidonic acid, thereby reducing the activity of COX-2 and the consequent production of PGE2 . In contrast, its effect on COX-1 is very weak [41]. DFU has been demonstrated to have antipyretic activity [41,42] and can block the febrile response of WT mice to LPS and rmIL-1␤ [43]. No fever developed in COX-2 knockout mice in response to LPS [42]. These studies indicate that the antipyretic action of DFU is indeed due to its COX-2 blocking ability [43]. We report here that DFU exhibits a time- and concentration-dependent inhibition of the growth of OEC-M1, KB and NF cell lines, but there was no inhibition by COX-1 inhibitor (Aspirin). These findings support the hypothesis that the antiproliferation action of DFU on oral cancer cells may be due to its COX-2 inhibiting effect. Celebrex (SC-58635) is a novel, specific inhibitor of COX-2 with significant anti-inflammatory and analgesic properties [44]. Kawamori and his coworkers demonstrated that tumor size and number are both decreased after prolonged treatment with Celebrex in the AOM rat [32,45]. Mice fed Celebrex in utero and as weanlings up to the time of promotion, showed a significant resistance to tumor development [46]. Moreover, combined therapy with Celebrex and Zyflo (selective 5-lipoxygenase inhibitor) in a solid model of pancreatic adenocarcinoma in Syrian hamster decreases tumor growth in liver metastases [47]. The anti-proliferative and proapoptotic effects of COX-2 inhibitors on cancer cells was demonstrated by Arico et al. He showed that Celebrex induces apoptosis in colon cancer cells by inhibiting PDK1 signaling [48]. That the action of Celebrex on the growth of oral cancer cell lines is compatible with previous studies urges us to continue with more sophisticated indepth research projects. Tucker et al. observed that the levels of COX-2 mRNA and protein expression are upregulated in human pancreatic cancer [49]. Ding et al. demonstrated that NS-398 inhibited

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proliferation and induced apoptosis in human pancreatic cancer cells [50]. Furthermore, Elder et al. [51] also found that NS-398 revealed anti-proliferative and apoptosis-inducing effects in three pre-malignant human colorectal adenoma cell lines and three colorectal carcinoma cell lines. NS-398 upregulated COX-2 protein expression in one carcinoma cell line and three adenoma cell lines. A recent study done by Lee et al. [52] demonstrated that treatment of head and neck cancer cells with NS-398 and indomethacin for 72 h elicits a significant cell growth inhibition and cell cycle arrest. Chang and Weng [53] further observed that NS-398 induced apoptosis of high COX-2 expressing A549 lung cancer cells under serum-free condition, whereas NS-398 induces G1 growth arrest of A549 cells maintained in 10% serum medium. Conversely, low COX-2-expressing H226 lung cancer cells were resistant to NS-398-induced apoptosis under both serum-free and serum-containing conditions. Abiru et al. [54] further showed that NS-398 and Aspirin inhibited HGF-induced invasiveness of HepG2 human hepatoma cells through ERK1/2. These results are in accordance with our findings that NS-398 also inhibits proliferation in oral cancer cells. In contrast, the action of Aspirin in hepatoma cells is not compatible with its action in oral cancer cells. It remains to be seen whether COX-2 protein is regulated by NS-398 in oral cancer cells. Studies have demonstrated that salicylates can induce apoptosis in normal human fibroblasts [55] and inhibit growth and induce apoptosis in human colorectal tumor cells [56]. Epidemiological studies have shown that salicylates have a chemopreventive effect against colorectal cancer and patients who took Aspirin had up to 50% reduction in the incidence of or mortality from colorectal cancer [57,58]. Sodium salicylate can enhance TNF-␣-induced apoptosis in one human pancreatic cancer cell line (BxPC-3), but showed no increase in apoptosis in another pancreatic cancer cell line (PANC-1) [59]. Furthermore, Aspirin can inhibit HGF-induced invasiveness of HepG2 human hepatoma cells [54]. In this report, we found that Aspirin has no significant effect on the growth of normal human oral fibroblasts and two oral cancer cell lines. Our results, though, are in contrast to previous reports, and may not preclude a role for COX-1 in oral cancer biology because indomethacin (a non-selective COX inhibitor) showed even more potent growth inhibition in our culture system (data not shown). Furthermore, Tiano et al. also demonstrated that both COX-1 and COX-2 have roles in skin tumorigenesis [60]. The expression of both COX-1 and COX-2 proteins in our culture cell lines is undergoing further investigation to resolve questions raised by these studies. On the other hand, COX-1 activity had been thought to be the pharmacological target of Aspirin [61], and prostaglandins produced through the activation of COX-2 may be specifically involved in cellular differentiation and proliferation [62]. Furthermore, meloxicam (a COX-2 inhibitor) can significantly inhibit colony size and tumor growth in a COX-2 expressing cell line (HCA-7), but did not effect the growth of the COX-2 negative HCT-116 cells [63]. In this investigation we report that the growth of OEC-M1, KB, and NF cell lines was not significantly inhibited by a COX-1 inhibitor (Aspirin), but was significantly inhibited by COX-2 inhibitors (DFU, CLBX, and NS-398). These findings support the hypothesis that the COX-2 enzyme may participate in the differentiation and proliferation of two human oral cancer cell lines (OEC-M1, and KB) and one human normal fibroblast cell line. COX-2 selective inhibitors have anti-inflammatory effects with less effect on COX-1 activity and without gastric toxicity [64]. It is important to conduct further laboratory and

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