374 27 4 2009 8 [ ] 1000-1182 2009 04-0374-04 Tca8113-2 1 2 2 (1. ; 2., 510515) [ ] -2 COX-2 Tca8113 COX-2 Tca8113 24 48 72 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 739.8 [ ] 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 2. 1. Dept. of Stomatology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China; 2. Dept. of Pathology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, 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-2 1.1 Tca8113 BK060127 Trizol Invitrogen [ ] 2008-08-15 [ ] 2008-11-10 [ ] 06024396 [ ] 1962 [ ] Tel 020-61642148
27 4 2009 8 375 5 10 20 40 80 μmol/l 24 48 72 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-3 10 80 μmol/l R 5 -CTGATGCGTGAAGTGCTG-3 PBS 48 h 238 bp β-actine F 5 -CACCCAGCAC- 37 95% 15 min 10% AATGAAGAT-3 R 5 -CAAATAAAG- 1 100 COX-2 4 CCATGCCAAT-3 255 bp PBS 37 58 MX3000p PCR 10~30 min PBS 50 μl PCR Ct Ct 37 10 min PBS, DAB 3~5 min β-actine Ct COX-2 Ct COX-2 Ct Ct Ct=Ct -Ct Ct Ct 1.4 Tca8113 2 - 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 Tca8113 1.6 PCR Tca8113 COX- 2 mrna Tca8113 10% Tca8113 200 μl 3 10 4 DMEM 4 10 3 96 96 DMEM 5%CO 2 24 h 10 20 40 80 μmol/l 24 48 h DMEM 2.5 Trizol RNA Tca8113 2 10 5 50 ml 24 h 40 80 μmol/l 3 3 24 h 1.7 PBS SPSS 13.0 x±s 1 10 7 90 μl acridine orange AO ethidium bromide LSD one-way ANOVA EB 5 μl 15 min 2 1.5 Annexin V-FITC/PI Tca8113 cdna mrna COX -2 mrna Primer 5 2.1 Tca8113 Tca- 8113 40 80 μmol/l Tca8113 2 10 5 Tca8113 1 6 ml 50 ml 24 h 2.2 COX-2 Tca8113 40 80 μmol/l COX-2 Tca8113 24 48 h 80 μmol/l PBS 5~10 1 000 g 5 min COX-2
376 27 4 2009 8 1 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 400 2.3 AO/EB Tca8113 Tca8113 24 h EB AO AO EB EB 3 2.4 Annexin V-FITC/PI Tca8113 Annexin V-FITC/PI 24 48 h 0% 0.93±0.11 % 40 80 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 0.000 80 μmol/l 40 24 48 h Tca8113 1 1 Tca8113 n=3 Tab 1 Early apoptotic rate of Tca8113 cell lines treated with Celecoxib n=3 /% 24 h 48 h 0 0.93±0.11 /40 μmol L -1 3.86±0.23 7.17±0.64 /80 μmol L -1 15.26±0.88 20.07±1.06 2.5 Tca8113 COX-2 mrna RNA cdna PCR Ct Tca8113 COX-2 mrna P>0.05 COX-2
27 4 2009 8 377 P>0.05 2 2 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-MB231 24 h 48 h Ct 2 - Ct Ct 2 - Ct MDA-MB231 5.80±0.32 1.00±0.00 5.76±0.39 1.00±0.00 10 μmol L -1 5.84±0.72 0.98±0.06 5.97±0.58 0.87±0.11 20 μmol L -1 5.89±0.65 0.94±0.02 5.99±0.51 0.85±0.10 COX-2 40 μmol L -1 5.92±0.74 0.92±0.08 6.02±0.34 0.84±0.13 -κb nuclear factor-κb NF-κB 80 μmol L -1 5.88±0.60 0.95±0.02 5.95±0.34 0.88±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 3 8 9 [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 40 80 μmol/l Tca8113 COX-2 Tca8113 208-219. 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 1927-2004 [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, 30 2 73-81. 385
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