Effects of cyclooxygenase 2 inhibitors on biological traits of nasopharyngeal carcinoma cells

Pei-yi CHEN¹, Qi-cai LONG²

Institute of Clinical Pharmacology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510080, China

¹ Now in Inspection, Evaluation and Certification Committee for Drug, Guangdong Province Drug Administration, Guangzhou 510080, China. Phn 86-20-8731-8953. Fax 86-20-8769-2936. E-mail peiyichen@163.net

² Correspondence to Qi-cai LONG. Phn 86-20-8733-1782. Fax 86-20-8733-1782. E-mail qcli@gzsums.edu.cn

Received 2003-08-04 Accepted 2004-02-18

KEY WORDS cyclooxygenase inhibitors; nimesulide; celecoxib; nasopharyngeal neoplasms; apoptosis; cell cycle; angiogenesis inhibitors

ABSTRACT

AIM: to investigate effects of cyclooxygenase 2 (COX-2) inhibitors on nasopharyngeal carcinoma (NPC) cells and on angiogenesis in vitro. METHODS: Human NPC cell lines (CNE1, CNE2 and SUNE) were treated with nimesulide or celecoxib. MTT assay and colony formation assay were performed to observe antiproliferation activity of COX-2 inhibitors to NPC cell lines. Cell cycle arrest and apoptosis of NPC cell strains were tested by flow cytometry assay, microscopic morphology observation, and DNA fragmentation assay. The effect of COX-2 inhibitor on angiogenesis was tested by chick chorioallantoic membrane (CAM) model. RESULTS: Nimesulide (Nim) and celecoxib (Cel) could antiproliferate NPC cell lines with IC₅₀ 182 µmol/L_Nim-SUNE, 78 µmol/L_Nim-CNE1, 175 µmol/L_Nim-CNE2, 7.2 µmol/L_Cel-SUNE, 8.1 µmol/L_Cel-CNE1, and 7.6 µmol/L_Cel-CNE2. The antiproliferation presented dose-dependent (Nim 5-400 µmol/L, Cel 0.5-80 µmol/L) and time-dependent manner (Nim IC₅₀ 562 µmol/L_{24 h}, 316 µmol/L_{48 h}, 50.1 µmol/L_{240 h}). Nim and Cel arrested SUNE and CNE1 cell cycle at phase G₂/M (cell aggregation rate 28.9 %- 45.1 %_{Nim 25-200 µmol-12 h-SUNE}, 18.9 %-26.2 %_{Nim 25-200 µmol-24 h-SUNE}, 28.8 %-35.6 %_{Nim 25-200 µmol-48 h-SUNE}, 30.4 %-16.4 %_{Cel 25-100 µmol-12 h-SUNE}, 21.2 %-19.7 %_{Cel 25-100 µmol-24 h-SUNE}, 31.1 %-19.9 %_{Cel 25-100 µmol-24 h-SUNE}, 20.5 %-34.1 %_{Nim25-200 µmol-12 h-CNE1}, 25.2 %- 26.9 %_{Nim 25-200 µmol-24 h-CNE1}, 11.5 %-7.1 % _{Nim 25-200 µmol-48 h-CNE1}). Apoptosis shape and apoptosis strap displayed in NPC cells after treatment with Nim and Cel. Nim had a feature of anti-angiogenesis on cam model. CONCLUSION: Nim and Cel could suppress proliferation of squamous epithelium NPC cell (SUNE, CNE1 and CNE2) through blocking cell cycle and inducing cell apoptosis. Nim could apparently suppress CAM angiogenesis induced by

INTRODUCTION

The hypothesis that COX-2 was involved in the pathological process of cancer happening and development was supported by animal studies indicated that tumor genesis was inhibited in COX-2 knockout mice^[1,2]. Over expression of COX-2 led to phenotypic changes in intestinal epithelial cells that could enhance their tumorigenic potential^[3]. Selective inhibitors of COX-2 have been demonstrated to induce apoptosis in a variety of cancer cells, including those of colon, stomach and prostate and so on. Although COX-2 inhibitors have been reported as effective agents for many tumors, the effect of COX-2 inhibitor on nasopharyngeal carcinoma (NPC) had not been found yet. Our previous work confirmed that synergism or additivity of inhibition of CNE1 and SUNE lines in concomitance of celecoxib with bleomycin, cisplatin, or vincrestine in vitro^[4]. It seems to be necessary to further evaluate the effects of COX-2 inhibitors on NPC cells, to provide the evidences for using COX-2 inhibitors as chemoprevention agents and therapy agents for NPC, since COX-2 inhibitors are hopeful drugs for chemoprevention against cancer.

MATERIALS AND METHODS

Reagents Nimesulide was provided by Haerbin Pharmaceutical Factory. Indomethacine was from National Institute for the Control of Pharmaceutical and Biological Products. Celecoxib was obtained from Pfizer Pharmaceutical Inc.

Cell culture CNE1 (human nasopharyngeal high differentiated squamous epithelium carcinoma cell), CNE2 and SUNE (human nasopharyngeal low differentiated squamous epithelium carcinomas cell) were obtained from Department of Oncopharmacology, Tumor Institute, Sun Yat-sen University. HT-29 (human colon carcinoma cell) and Hep-G2 (human hepatic carcinoma cell, as a control cell strain) were purchased from Experimental Animal Center of Sun Yat-sen University. The cell strains were maintained in RPMI-1640 supplemented with 10 % calf serum in a 5 % CO₂ atmosphere.

MTT assay To evaluate the antiproliferative activity of COX-2 inhibitors, a MTT assay was performed. Briefly, cells (CNE1, CNE2, SUNE, HT-29 and Hep-G2) were plated at 5000-6000/well in 200 µL volume in 96-well plates and recovered over night. The indicated amounts of tested drugs (nimesulide 5-400 µmol/L, indomethacin 5-400 µmol/L and celecoxib 0.5-80 µmol/L) or negative solvent (Me₂SO 0.25 %) were then added to each well. After incubation for 68 h, 20 µL of MTT (3 g/L) was added, and the cells were incubated at 37 ºC for 4 h. The tetrazolium crystal was solubilized by 100 µL Me₂SO and the absorbance was measured at 570 nm on spectrophotometric plate reader. IC₅₀ of antiproliferative activity of COX-2 inhibitors were calculated by interpolation method.

Colony formation assay Colony formation assay was done to evaluate time-dependent effect of COX-2 inhibitors on NPC cell strains. Following digestion by trypsin, CNE1 and SUNE cells were put in 6-well plate at the number of 500 cells in each well. Nimesulide 10-1000 µmol/L and negative fluid were added. Cells were exposed to COX-2 inhibitors for 24 h, 48 h, and 240 h,respectively. At the end of determination time, cells were fixed by 50 % methanol and 5 % acetic acid for 40 min, dyed by Giemsa for 20 min and then washed. The number of colony formation was observed through invert microscope.

Cell cycle analysis by flow cytometry assay following digestion by 0.25 % trypsin , CNE1 and SUNE cells in logarithmic growth phase were adjusted to a density of 5×10⁸-1×10⁹/L. A volume of 4 mL cells was placed into a cell culture bottle and incubated over night. Nimesulide 25-200 µmol/L, celecoxib 25-100 µmol/L and negative solvent were added to the bottles. Cells were harvested at several time points, centrifuged, vortexed in 0.3 mL PBS, fixed with 0.7 mL anhydrous alcohol at -20 ºC for 12 h, washed twice with PBS, dyed with 300 µL pyridine iodide (Coulter) at room temperature for 20 min. After incubation cells were analyzed by flow cytometry (Beckman-Coulter, USA) at a wavelength of 488 nm. Data were analyzed by software with the machine.

Apoptosis analysis by morphologic observation and DNA fragmentation assay CNE1 and SUNE cells were incubated at 2×10⁴-1×10⁵ /well in 6-well plate each well for 24 h. Celecoxib 10-50 µmol/L was added to wells and incubated at 37 ºC for 24 h. Cells were washed with PBS, fixed with 100 % methanol at room temperature for 45 min, dyed with Giemsa for 20 min, then washed and dried. Cells were observed morphologic change under a phase contrast microscope (LiCa, MPS30 Japan). Three sights were observed in each sample and taken a photograph.

After exposed to nimesulide 200 µmol/L or celecoxib 25 µmol/L for 24 h and 48 h, CNE1 and SUNE cells were harvested and re-suspended in 600 µL lysis buffer (Tris-HCl 10 mmol/L, EDTA 10 mmol/L, Triton X-100 2 g/L, pH 7.5) and incubated at 4 ºC for 20 min. Then cells were centrifuged at 1200 r/min for 10 min. Soluble DNAs were recovered from the supernatants by phenol/chloroform (1:1) extraction and isopropanol precipitation. These DNAs were mixed with TE (Tris-HCl 10 mmol/L, EDTA 1.0 mmol/L, pH 7.4) and Rnase A 0.6 g/L, incubated at 37 ºC for 30 min, separated by 1.0 % agarose gel electrophoresis for 1 h. Following dye by ethylingot bromide, the separated samples were analyzed by GDS7500 gel image system.

COX-2 inhibitor suppressed angiogenesis on chick chorioallantoic membrane (CAM) model Sterilized filter paper slip at a diameter 5 mm was placed on 7-day chick chorioallantoic membrane. The slips contained 10 µL supernatant of SUNE cells culture medium, which was at a concentration of protein 1 g/L with nimesulide 100 µg, or nimesulide 100 µg only, or normal saline 10 µL. CAM method that Wu et al established was used to observe effect of COX-2 inhibitors on angiogenesis^[5].

RESULTS

Antiproliperative effect of COX-2 inhibitors to NPC cell lines The results showed that nimesulide, celecoxib, and indomethacin could inhibit growth of CNE1, CNE2 and SUNE cell lines in a concentration-dependent manner. IC₅₀ of COX-2 inhibitors to NPC cell lines was displayed in Tab 1.

Tab 1. The effect of COX-2 inhibitors on NPC cell survival after incubation for 72 h.

Cell	IC50/µmol·L-1
	Nimesulide	Celecoxib	Indomethacin
CNE1	175	8.1	208
CNE2	78	7.6	251
SUNE	182	7.2	183
HT-29	134	12.7	152

Antiproliperative effect of COX-2 inhibitor on NPC cell lines Colony formation assay showed that incubation of SUNE cells with nimesulide substantially inhibited the ability of cells to form colonies with the IC₅₀ value of 562 µmol/L for a 24-h incubation, 316 µmol/L for a 48-h incubation and 50.1 µmol/L for 240-h incubation (Tab 2).

Tab 2. Survival after incubation of SUNE cell with nimesulide for 24 h, 48 h and 240 h by colony formation assay. n=3. Mean±SD.

Time/h	Concentration/µmol·L-1
	Control	10	100	1000	IC50
24	1.0±0.11	0.98±007	0.94±0.06	0.35±0.12	562
48	1.0±0.05	0.98±0.08	0.85±0.13	0.15±0.08	316
240	1.0±0.04	0.88±0.06	0.34±0.10	0.04±0.05	50.1

Cell cycle alteration with treatment of COX-2 inhibitors by flow cytometry assay and apoptosis Phase G₂/M block of either SUNE cell or CEN1 cell was dose-dependent (nimesulide 25-200 µmol/L, celecoxib 25-100 µmol/L) and time-dependent at 12-48 h (Tab 3, 4). Aggregation percentage of SUNE cell in phase G₂/M obviously increased with treatment of nimesulide 25, 100, and 200 µmol/L. Celecoxib also could dose-dependently and time-dependently induce SUNE cells gathering in phase G₂/M. The aggregation percentage of SUNE cell in phase G₂/M also increased after treatment with celecoxib 25, 50, and 100 µmol/L. As shown in Tab 4, similar phenomena were also seen in the effects of nimesulide 25, 100, and 200 µmol/L on CNE1 cell line at 12 and 24 h but not at 48 h.

Tab 3. Distribution of the cell cycle (%) in SUNE cells following treatment with nimesulide (Nim) or celecoxib (Cel) at 12-48 h. n=5. Mean±SD.

Group	12 h			24 h			48 h
	G1	S	G2/M	G1	S	G2/M	G1	S	G2/M
Control	40.3±0.3	44.1±0.7	12.4±0.7	39.5±0.6	42.8±0.4	15.3±0.5	41.6±0.4	43.5±0.4	13.2±0.2
Nim 25 µmol·L-1	35.2±0.4	43.3±0.5	28.9±0.6	39.9±0.6	36.6±0.5	18.9±0.3	43.2±0.9	29.4±0.7	28.8±0.8
Nim 100 µmol·L-1	23.3±0.5	43.1±0.3	35.5±0.2	42.6±0.7	32.8±0.4	24.8±0.4	48.5±0.7	21.5±0.6	30.5±0.8
Nim 200 µmol·L-1	16.6±0.3	38.3±0.2	45.1±0.6	44.9±0.9	30.3±0.3	26.2±0.8	49.4±0.5	19.7±0.3	35.6±0.3
Cel 25 µmol·L-1	32.6±0.4	43.9±0.6	30.4±0.3	28.9±0.6	33.1±0.2	21.2±0.7	46.6±0.8	27.1±0.5	31.1±0.2
Cel 50 µmol·L-1	19.9±0.6	40.4±0.8	38.9±0.8	43.3±0.7	26.8±0.5	28.7±0.3	49.2±0.2	18.9±0.4	33.0±0.4
Cel 100 µmol·L-1	24.2±0.3	35.9±0.7	16.4±0.2	51.1±0.4	16.4±0.7	19.7±0.2	44.6±0.9	17.5±0.8	19.9±0.5

Tab 4. Distribution of the cell cycle (%) in CNE1 cells following treatment with nimesulide (Nim) at 12-48 h. n=5. Mean±SD.

Group	12 h			24 h			48 h
	G1	S	G2/M	G1	S	G2/M	G1	S	G2/M
Control	61.1±0.2	26.0±0.4	14.2±0.8	58.5±0.7	24.7±0.5	16.2±0.3	62.4±0.7	25.8±0.1	13.3±0.3
Nim 25 µmol·L-1	48.0±0.2	28.6±0.6	20.5±0.7	48.5±0.5	26.6±0.9	25.2±0.5	68.2±0.7	20.3±0.4	11.5±0.2
Nim 100 µmol·L-1	43.6±0.2	32.9±0.3	25.1±0.8	42.9±0.5	31.2±0.4	28.5±0.4	75.3±0.7	18.2±0.6	9.1±0.4
Nim 200 µmol·L-1	37.2±0.2	28.8±0.5	34.1±0.3	44.2±0.3	29.1±0.5	26.9±0.7	72.2±0.1	12.0±0.2	7.1±0.9

The apoptosis rate of SUSE and CNE1 after treatment with nimesulide and celecoxib were shown in Tab 5. Celecoxib could induce SUNE cells gathering in phase G₂/M, but a plenty of cells became apoptosis within 24 h after treatment with celecoxib 100 µmol/L.

Tab 5. Apoptosis rate (%) in SUNE and CNE1 cells following treatment with nimesulide (Nim) and celecoxib (Cel) at 12-48 h. n=5. Mean±SD.

Group	SUNE			CNE1
	12 h	24 h	48 h	12 h	24 h	48 h
Nim 25 µmol·L-1	3.6±0.2	4.3±0.3	5.9±0.4	4.1±0.3	6.6±0.2	10.6±0.7
Nim 100 µmol·L-1	2.5±0.1	7.0±0.5	11.2±0.6	5.7±0.2	8.9±0.4	24.0±0.5
Nim 200 µmol·L-1	1.7±0.2	3.8±0.6	4.0±0.7	2.9±0.3	4.6±0.5	6.4±0.2
Cel 25 µmol·L-1	3.8±0.3	5.2±0.3	7.1±0.5	-	-	-
Cel 50 µmol·L-1	4.4±0.2	5.1±0.6	6.7±0.4	-	-	-
Cel 100 µmol·L-1	51.0±0.6	69.4±0.7	9.2±0.9	-	-	-

Morphological change of NPC cells Apoptosis characteristics with nucleus condense, shrinkage could be observed in NPC cell incubated with COX-2 inhibitors under invert microscope (Fig 1, 2). Analysis of DNA trapezoid strap revealed that an apoptosis strap feature obviously appeared in NPC cells after treatment with celecoxib 25 µmol/L and nimesulide 200 µmol/L for 48 h (Fig 3).

Fig 1. Effect of nimesulide (Nim) on SUNE, CEN1, and CNE2 cells observed with phase-contrast microscope (×200). A1: SUNE, A2: SUNE with Nim 100 µmol/L; B1: CNE1, B2: CNE1 with Nim 100 µmol/L; C1: CNE2, C2: CNE2 with Nim 100 µmol/L.

Fig 2. Celecoxib (Cel)-induced morphological characteristics of apoptosis in SUNE and CNE1 cells. Photo was taken under phase-contrast microscope (×200). A1: SUNE; B1: SUNE with Cel 10 µmol/L; C1: SUNE with Cel 50 µmol/L. A2: CNE1; B2: CNE1 with Cel 10 µmol/L; C2: CNE1 with Cel 50 µmol/L.

Fig 3. Agarose gel electrophoresis of DNA fragmentation analysis on CEN1 and SUNE cells treated with nimesulide (Nim) 200 µmol/L and celecoxib (Cel) 25 µmol/L for 24 and 48 h. A: 1) Nim 24 h; 2) Nim 48 h; 3) Cel 24 h; 4) Cel 48 h. B: 1) Nim 24 h; 2) Nim 48 h; 3) Cel 24 h; 4) Cel 48 h.

COX-2 inhibitor suppressed angiogenesis on chick chorioallantoic membrane model (CAM) As shown inFig 4, blood vessels in CAM manifested nervure shape in natural condition. Blood vessels formed as radiation with paper slip as the center, some vessels ran through area of paper slip, and vessels far from slip attracted each other after normal saline was added. Vessel growth of CAM was mildly suppressed under the slip with nimesulide 100 µmol/L and area around the slip compared with blank slip. Diameter of free vessel area was about 4-6 mm as a center of slip. Normal nervure shape vessels were seen in free drug area. Counting number of vessels showed that nimesulide (66.7±12.5, n=7, P<0.05) could mildly depress blood vessel growth compared with that of control (90.0±18.7, n=4). A great deal vessels with radiation in CAM formed outward with the position of SUNE cell culture liquid as the center. Diameter of the outward vessels was more than 3 cm. Density of the vessel increased. Vessel number intersected circle was 142±45 (n=4). CAM angiogenesis was clearly depressed after supernatant of SUNE culture solution and nimesulide 100 µmol/L were placed to slip. Number of vessels was 70±14 (n=5).

Fig 4. Effect of nimesulide (Nim) 100 µmol/L on angiogenesis of chick chorioallantoic membrane. A: Normal saline, B: Nim, C: Supernatant of SUNE culture solution, D: Supernatant of SUNE culture solution and Nim.

DISCUSSION

Both nimesulide and celecoxib are COX-2 inhibitors. The former is a benzenesulfonyl structure NSAID to be considered for predominant inhibiting COX-2 (IC₅₀ 7.1 µmol/L) and light COX-1 inhibitor (IC₅₀ 985 µmol/L), while the later is a selective COX-2 inhibitor (IC₅₀ 0.04 µmol/L, for COX-1 IC₅₀ 15 µmol/L) with a structure of tricycle.

SUNE and CNE2 are human low differentiated NPC squamous epithelium cell. SUNE was certified as positive LMP1 (latent membrane protein 1). CNE1 is a human high differentiated NPC squamous epithelium cell. HT-29 is a human colon carcinoma strain with positive COX-2, and often used to study effect of COX-2 inhibiter on cancer of colon. HT-29 was a positive line of sensitive COX-2 inhibitor in our experiment.

NPC cells proliferation was evidently suppressed by nimesulide, celecoxib, and indomethacin from MTT assay. IC₅₀ (7-8 µmol/L) of celecoxib to depress NPC cells approached IC₅₀ of clinical concentration (3 µmol/L). IC₅₀ of celecoxib and nimsulide to depress NPC was much more than that to inhibit COX-2, but less than that to inhibit COX-1. Compared with positive control HT-29 (IC_{50 nimesulide} 134 µmol/L, IC_{50 indomethacin} 152 µmol/L) and Hep-G2 (IC_{50 celecoxib} 12.7 µmol/L), it was valuable for nimesulide, celecoxib and indomethacin to present antiproliferation to NPC cells. Because the three drugs did not balance in the fields of depressing NPC cells and inhibiting of COX-2, other pathways to be function might exist except for depression of NPC through inhibiting COX-2.

Results of colony formation analysis clearly displayed that antiproliferation of COX-2 inhibitors to SUNE was time- and dose-dependent. The longer time and the more dose, the more evident antiproliferating action was (n=3, P<0.05).

COX-2 inhibitor inhibited NPC cells mainly due to cell cycle arrest and apoptosis demonstrated by flow cytometry assay, cell morphological change and DNA fragmentation agarose gel electrophoresis. There was a temporary block phase G₂/M for about 24 h in nimesulide and celecoxib acting to NPC cell. The cell number in phase G₀/G₁ relatively increased as drug action time prolongation and the cell number in phase G₂/M decreased. NPC cell eventually gathered in phase G₀/G1 and apoptosis started to display. NPC cell in phase G₂/M increasingly gathered with early time in apoptosis and apoptosis rate increasing when COX-2 inhibitor concentration was higher. Our experimental results differed from that NSAID influenced cell cycle chiefly by blocking G₀/G₁ in references^[6,7]. Causes of the difference probably were as a result of different cell strain with different biological characteristics in experiment and different observation time. The observed time reported in references was often more than 24 h that was the beginning of G₂/M decrease and G₀/G₁ increase.

Tumogenesis, on the one hand, is considered to be out of control of cell proliferation and abnormal differentiation by gene regulation and control. On the other hand, cell apoptosis process might be inhibited. Tumor cell apoptosis and alteration of cell cycle play an important role in the treatment of tumor. Our experimental results displayed that nimesulide, celecoxib and indomethacin could all inhibit NPC cell proliferation, induce apoptosis and change cell cycle in cell culture in vitro. These suggested that COX-2 inhibitors could probably play a certain part in caner therapy. Because expression of COX-2 gene in tumor cell was much more than that in normal cell, COX-2 inhibitors could selectively act on cancer cell, but relatively little effect on normal cell function^[8]. It was inferred that COX-2 inhibitors might potentiate the action of anticancer agents^[4].

Filter paper slip could induce blood vessel formation as an exogenous irritant in our experiments. It seems to be more suitable for a carrier of drug to depress anginogenesis than cellothyl. Supernatant from SUNE cell culture solution strongly induced CAM angiogenesis. Nimesulide 100 µmol/L, however, could not only depress CAM angiogenesis, but also depress angiogensis that induced by treatment with supernatant of SUNE cell culture solution (P=0.01). The results implied that COX-2 inhibitors could possibly inhibit angiogenesis-induced materials synthesized by SUNS cell. SUNE used in our experiment was a NPC cell with LMP1 gene. Murono et al found that LMP1 gene expression in NPC cell might induce the expression of COX-2 gene, whereas activity increase of COX-2 would further induce the expression of vascular endothelial growth factor (VEGF)^[9]. The study results suggested that SUNE cell might induce angiogenesis through a pathway of LMP1-COX-2-VEGF. If COX-2 inhibitors will become anti-angiogenesis agents is supposed to be further investigated.

In conclusion, COX-2 inhibitors (nimesulide and celecoxib) could suppress proliferation of squamous epithelium NPC cell (SUNE, CNE1 and CNE2) through cell cycle block and inducing cell apoptosis. The antiproliferaion presented dose- and time-dependent features. And nimesulide could apparently suppress CAM angiogenesis induced by SUNE cell, although did not obviously suppress normal angiogenesis in CAM.

REFERENCES

• 1 Oshima M, Dinchuk JE, Kargaman SL, Oshima H, Hancok B, Kwong E, et al. Suppression of intestinal polyposis in Apc delta716 knokout mice by inhibition of cyclooxygenase 2 (COX-2). Cell 1996; 87: 803-9.
• 2 Takako MM. COX-2 and colon cancer. Inflamm Res 1998; 47 (Suppl 2): S112-6.
• 3 Tsujii M, Dubois RN. Alterations in cellular adhesion and apoptosis in epithelial cells overexpressing prostaglandin endoperoxide synthase 2. Cell 1995; 83: 493-501.
• 4 Chen PY, Long QC. Interaction of inhibiting effect of cyclooxygenase-2 and anticancer drugs on nasopharyngeal carcinoma strain. Chin J Clin Pharmacol 2002; 18: 425-30.
• 5 Wu LC, Sun GJ, Zhang WD. Study on model of endothelial cell migration and proliferation using chick chorioallantoic membrane. Chin J Pathophysiol 2000; 16: 572-3.
• 6 Schober W, Kehlbach R, Gebert R, Wiskirchen J, Rodegerdts E, Claussen CD, et al. Meclofenamic acid for inhibition of human vascular smooth muscle cell proliferation and migration: an in vitro study. Cardiovasc Intervent Radiol 2002; 25: 57-63.
• 7 Nikitakis NG, Hamburger AW, Sauk JJ. The nonsteroidal anti-inflammatory drug sulindac causes down-regulation of signal transducer and activator of transcription 3 in human oral squamous cell carcinoma cells. Cancer Res 2002; 62:1004-7.
• 8 Grosch S, Tegeder I, Niederberger E, Brautigam L, Geisslinger G. COX-2 independent induction of cell cycle arrest and apoptosis in colon cancer cells by the selective COX-2 inhibitor celecoxib. FASEB J 2001; 15: 2742-4.
• 9 Murono S, Inoue H, Tanabe T, Joab I, Yoshizaki T, Furukawa M, et al. Induction of cyclooxygenase 2 by Epstein-Barr virus latent membrane protein 1 is involved in vascular endothelial growth factor production in nasopharyngeal carcinoma cells. Proc Natl Acad Sci USA 2001; 98: 6905-10.