West China Journal of Stomatology Vol.27 No.4 Aug Tca h MTT Annexin V-FITC/PI

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1 [ ] Tca (1. ; 2., ) [ ] -2 COX-2 Tca8113 COX-2 Tca h MTT COX-2 Tca8113 Annexin V-FITC/PI PCR Tca8113 COX-2 mrna COX-2 Tca8113 Tca8113 COX-2 Tca8113 COX-2 mrna Tca8113 COX-2 COX-2 [ ] -2 [ ] R [ ] A Effect of Celecoxib on cycloxygenase -2 expression and inducing apoptosis in Tca8113 cell lines LI Wei - zhong 1, WANG Xiao-yan 2, DING Yan-qing Dept. of Stomatology, Nanfang Hospital, Southern Medical University, Guangzhou , China; 2. Dept. of Pathology, Nanfang Hospital, Southern Medical University, Guangzhou , China [Abstract] Objective To observe whether Celecoxib could inhibit the growth, regulate the expression of COX-2 and induce apoptosis of Tca8113 cells. Methods Tca8113 cells were incubated with different concentrations of Celecoxib for 24, 48 and 72 h, and MTT was used to calculate growth inhibition rate. The expression of COX-2 protein and mrna in Tca8113 cells was detected with SP immunohistochemistry staining and fluorescent quantitative realtime RT-PCR. Morphology of apoptosis cells was observed by fluorescence microscopy, and Annexin V-FITC/PI double labeling method was employed to detect early stage cell apoptosis. Results COX-2 protein was strongly expressed in Tca8113 cells and was suppressed by Celecoxib. The growth and proliferation of Tca8113 cells treated with Celecoxib were inhibited in a dose-dependent manner. Celecoxib treatment resulted in significant increase in apoptosis and early apoptotic rate. Fluorescent quantitative real-time RT-PCR results showed no significant effet on regulating expression of COX-2 mrna. Conclusion Celecoxib shows a significant effect on inhibiting expression of COX-2 in Tca8113 cells, this is probably related to growth inhibition and inducing apoptosis of Tca8113 cells. [Key words] Celecoxib cycloxygenase-2 cell apoptosis squamous cell carcinoma -2 cycloxygenase-2 COX-2 Tca8113 COX-2 COX-2 1 COX-2 [1-3] COX Tca8113 BK Trizol Invitrogen [ ] [ ] [ ] [ ] 1962 [ ] Tel

2 μmol/l h 570 nm % =[1- A 570 nm ] 100% 1.3 Tca8113 A 570 nm PCR COX -2 mrna β -actine A 570 nm / COX-2 COX-2 β-actine COX-2 SP 1 F 5 -TTACAATGCTGACTATGGCTAC μmol/l R 5 -CTGATGCGTGAAGTGCTG-3 PBS 48 h 238 bp β-actine F 5 -CACCCAGCAC % 15 min 10% AATGAAGAT-3 R 5 -CAAATAAAG COX-2 4 CCATGCCAAT bp PBS MX3000p PCR 10~30 min PBS 50 μl PCR Ct Ct min PBS, DAB 3~5 min β-actine Ct COX-2 Ct COX-2 Ct Ct Ct=Ct -Ct Ct Ct 1.4 Tca Ct Ct= Ct - Ct 2 - Ct RT-PCR TaKaRa 195 μl Annexin V-FITC Annexin V-FITC COX- 5 μl Annexin V-FITC 2 10 min 10 μl COX-2 β-actine CellQuest Pro 1.2 MTT Tca PCR Tca8113 COX- 2 mrna Tca % Tca μl DMEM DMEM 5%CO 2 24 h μmol/l h DMEM 2.5 Trizol RNA Tca ml 24 h μmol/l h 1.7 PBS SPSS 13.0 x±s μl acridine orange AO ethidium bromide LSD one-way ANOVA EB 5 μl 15 min Annexin V-FITC/PI Tca8113 cdna mrna COX -2 mrna Primer Tca8113 Tca μmol/l Tca Tca ml 50 ml 24 h 2.2 COX-2 Tca μmol/l COX-2 Tca h 80 μmol/l PBS 5~ g 5 min COX-2

3 Tca8113 Fig 1 The growth curve of Tca8113 cell lines treated with Celecoxib 80 μmol/l 48 h 2 COX-2 Tca8113 SP 400 Fig 2 Expression of COX-2 protein in Tca8113 cell lines SP AO/EB Tca8113 Tca h EB AO AO EB EB Annexin V-FITC/PI Tca8113 Annexin V-FITC/PI h 0% 0.93±0.11 % μmol/l 24 h 3.86±0.23 % 15.26± 0.88 % 48 h 7.17 ± 0.64 % 20.07±1.06 % 80 μmol/l 40 P μmol/l h Tca Tca8113 n=3 Tab 1 Early apoptotic rate of Tca8113 cell lines treated with Celecoxib n=3 /% 24 h 48 h ±0.11 /40 μmol L ± ±0.64 /80 μmol L ± ± Tca8113 COX-2 mrna RNA cdna PCR Ct Tca8113 COX-2 mrna P>0.05 COX-2

4 P> COX-2 Ct n=3 Tab 2 The Ct value and 2 - Ct value of COX-2 van Wijngaarden [4] mrna expression in Tca8113 cell lines in control and Celecoxib treated group n=3 MDA-MB h 48 h Ct 2 - Ct Ct 2 - Ct MDA-MB ± ± ± ± μmol L ± ± ± ± μmol L ± ± ± ±0.10 COX-2 40 μmol L ± ± ± ±0.13 -κb nuclear factor-κb NF-κB 80 μmol L ± ± ± ±0.10 κb NF-κB 3 NF-κB COX-2 COX-2 Bcl-2 Bcl-xL Mcl-1 Survivin [4-7] Bad C [8-9] Apaf-1 Caspases [10] COX-2 [11-13] COX-2 - [2-3] Ca 2+ COX-2 Ca 2+ C Ca 2+ - COX-2 Tca8113 Tca8113 AO/EB COX-2 Annexin V- FITC/PI μmol/l Tca8113 COX-2 Tca COX-2 PCR Ct 2 - Ct COX-2 mrna COX-2 COX-2 COX-2 COX-2 [ ] [1] Botting RM. Cyclooxygenase Past, present and future. A tribute to John R. Vane [J]. J Therm Biol, 2006, 31 1/2 [2] Hosomi Y, Yokose T, Hirose Y, et al. Increased cyclooxygenase 2 COX-2 expression occurs frequently in precursor lesions of human adenocarcinoma of the lung[j]. Lung Cancer, 2000,

5 [4],,, 2005, MSCs 40 min με MSCs ALP TGF-β IGF- MSCs MSCs Suppl 1S-12S. [ ] growth factor beta -1 and osteocalcin gene expression [J]. Plast [1] Jiang Y, Jahagirdar BN, Reinhar dt RL, et al. Pluripotency of Reconstr Surg, 1999, mesenchymal stem cells derived from adult marrow [J]. Nature, 2002, [8] Robey PG, Young MF, Flanders KC, et al. Osteoblasts synthesize and respond to transforming growth factor-type beta TGF-beta [2] Qi MC, Hu J, Zou SJ, et al. Mechanical strain induces osteogenic in vitro[j]. J Cell Biol, 1987, differentiation Cbfa1 and Ets-1 expression in stretched rat mesenchymal stem cells[j]. Int J Oral Maxillofac Surg, 2008, [9] Farhadieh RD, Dickinson R, Yu Y, et al. The role of transforming growth factor-beta, insulin-like growth factor, and basic fibroblast growth factor in distraction osteogenesis of the mandible [3],,,. MSCs [J]. J Craniofac Surg, 1999, [J]., 2004, HAN Li-chi, HU Jing, QI Meng-chun, et al. Isolation and multidifferentiation of rat bone marrow mesenchymal stem cells in vitro [10] Butt RP, Bishop JE. Mechanical load enhances the stimulatory effect of serum growth factors on cardiac fibroblast procollagen synthesis[j]. J Mol Cell Cardiol, 1997, [J]. J Oral Sci Res, 2004, [3] Shiotani H, Denda A, Yamamoto K, et al. Increased expression of cyclooxygenase-2 protein in 4-nitroquinoline-1-oxide-induced rat tongue carcinomas and chemopreventive efficacy of a specific inhibitor, nimesulide[j]. Cancer Res, 2001, [4] van Wijngaarden J, van Beek E, van Rossum G, et al. Celecoxib enhances doxorubicin-induced cytotoxicity in MDA-MB231 cells by NF -kappab - mediated increase of intracellular doxorubicin accumulation[j]. Eur J Cancer, 2007, [5] Nam DH, Park K, Park C, et al. Intracranial inhibition of glioma cell growth by cyclooxygenase -2 inhibitor celecoxib [J]. Oncol Rep, 2004, [6] Wun T, McKnight H, Tuscano JM. Increased cyclooxygenase -2 COX-2 A potential role in the pathogenesis of lymphoma[j]. Leuk Res, 2004, [7] Dandekar DS, Lopez M, Carey RI, et al. Cyclooxygenase -2 inhibitor celecoxib augments chemotherapeutic drug-induced apoptosis by enhancing activation of caspase-3 and -9 in prostate cancer cells[j]. Int J Cancer, 2005, [8] Kern MA, Schubert D, Sahi D, et al. Proapoptotic and antiproliferative potential of selective cyclooxygenase-2 inhibitors in human liver tumor cells [J]. Hepatology, 2002, 36 4 Pt [9] Kim SH, Song SH, Kim SG, et al. Celecoxib induces apoptosis in cervical cancer cells independent of cyclooxygenase using,. [J]. HAN Li -chi, HU Jing, QI Meng -chun, et al. Comparison of proliferation ability and osteogenic potential of rat bone marrow mesenchymal stem cells isolated by two different methods [J]. J Modern Stomatol, 2005, [5] Rubin JP, Bennett JM, Doctor JS, et al. Collagenous microbeads as a scaffold for tissue engineering with adipose -derived stem cells[j]. Plast Reconstr Surg, 2007, [6] Linkhart TA, Mohan S, Baylink DJ. Growth factors for bone growth and repair IGF, TGF-β and BMP[J]. Bone, 1996, 19 1 [7] Mehrara BJ, Rowe NM, Steinbrech DS, et al. Rat mandibular distraction osteogenesis.. Molecular analysis of transforming NF -kappab as a possible target [J]. J Cancer Res Clin Oncol, 2004, [10] Ruvolo PP. Intracellular signal transduction pathways activated by ceramide and its metabolites[j]. Pharmacol Res, 2003, [11] Stoica BA, Movsesyan VA, Knoblach SM, et al. Ceramide induces neuronal apoptosis through mitogen-activated protein kinases and causes release of multiple mitochondrial proteins [J]. Mol Cell Neurosci, 2005, [12] Gomez Del Pulgar T, Velasco G, Sanchez C, et al. De novosynthesized ceramide is involved in cannabinoid-induced apoptosis [J]. Biochem J, 2002, 363 Pt [13] Siskind LJ. Mitochondrial ceramide and the induction of apoptosis [J]. J Bioenerg Biomembr, 2005, [14] Johnson AJ, Hsu AL, Lin HP, et al. The cyclo -oxygenase -2 inhibitor celecoxib perturbs intracellular calcium by inhibiting endoplasmic reticulum Ca 2 + -ATPases A plausible link with its anti-tumour effect and cardiovascular risks[j]. Biochem J, 2002, 366 Pt [15] Wang S, El-Deiry WS. Cytochrome C Acrosslink between the mitochondria and the endoplasmic reticulum in calcium-dependent apoptosis[j]. Cancer Biol Ther, 2004,

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