Overexpression of cyclooxygenase-2 in human HepG2, Bel-7402 and SMMC-7721 hepatoma cell lines and mechanism of cyclooxygenase-2 selective inhibitor celecoxib-induced cell growth inhibition and apoptosis.
OBJECTIVE
To investigate the cyclooxygenase-2 (COX-2) expression level in human HepG2, Bel-7402 and SMMC-7721 hepatoma cell lines and the molecular mechanism of COX-2 selective inhibitor celecoxib-induced cell growth inhibition and cell apoptosis.


METHODS
Hepatoma cells were cultured and treated with celecoxib. Cell in situ hybridization (ISH) and immunocytochemistry were used to detect COX-2 mRNA and protein expression. Proliferating cell nuclear antigen and phosphorylated Akt were also detected by immunocytochemistry assay. Cell growth rates were assessed by 3-(4, 5-dimethylthiazol-2-yl-2, 5-diphenyltetrazolium (MTT) bromide colorimetric assay. Celecoxib-induced cell apoptosis was measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and flow cytometry (FCM). The phosphorylated Akt and activated fragments of caspase-9, caspase-3 were examined by Western blotting analysis.


RESULTS
Increased COX-2 mRNA and protein expression were detected in all three hepatoma cell lines. Celecoxib could significantly inhibit cell growth and the inhibitory effect was in a dose- and time-dependent manner evidenced by MTT assays and morphological changes. The apoptotic index measured by TUNEL increased correspondingly with the increased concentration of celecoxib and the reaction time. With 50 micromol/L celecoxib treatment for 24 h, the apoptotic index of HepG2, BEL-7402 and SMMC-7721 cells was 25.01+/-3.08%, 26.40+/-3.05%, and 30.60+/-2.89%, respectively. Western blotting analysis showed remarkable activation of caspase-9, caspase-3 and dephosphorylation of Akt (Thr(308)). Immunocytochemistry also showed the reduction of PCNA expression and phosphorylation Akt (Thr(308)) after treatment with celecoxib.


CONCLUSIONS
COX-2 mRNA and protein overexpression in HepG2, Bel-7402 and SMMC-7721 cell lines correlate with the increased cell growth rate. Celecoxib can inhibit proliferation and induce apoptosis of hepatoma cell strains in a dose- and time-dependent manner.
Liu
Ning-Bo
NB
Laboratory of Reproductive Medicine, Department of Pathology, Nanjing Medical University, Nanjing 210029, Jiangsu Province, China.
Peng
Tao
T
Pan
Chao
C
Yao
Yu-Yu
YY
Shen
Bo
B
Leng
Jing
J
D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D006528:Carcinoma, Hepatocellular; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D000068579:Celecoxib; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D051546:Cyclooxygenase 2 / Q000235:genetics / Q000378:metabolism*; D052246:Cyclooxygenase 2 Inhibitors / Q000494:pharmacology*; D004789:Enzyme Activation; D006801:Humans; D017403:In Situ Hybridization; D008113:Liver Neoplasms; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011720:Pyrazoles / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology*
D052246:Cyclooxygenase 2 Inhibitors; D011720:Pyrazoles; D012333:RNA, Messenger; D013449:Sulfonamides; D051546:Cyclooxygenase 2; D051057:Proto-Oncogene Proteins c-akt; C505407:CASP3 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases; D000068579:Celecoxib