title: Enhanced sensitivity of celecoxib in human glioblastoma cells: Induction of DNA damage leading to p53-dependent G1 cell cycle arrest and autophagy.
abstract: BACKGROUND
  Selective cyclooxygenase (COX)-2 inhibitors elicit anti-proliferative responses in various tumours, however the underlying anti-tumour mechanisms are unclear. Mutational inactivation of the tumour suppressor p53 gene is frequent in malignant gliomas. The role of p53 mutation in the anti-tumour responses of the selective COX-2 inhibitor celecoxib in human glioblastoma cells is unknown. In this study, we used human glioblastoma cells with various p53 status; U87MG (with high and low p53 functional levels), LN229 (functional p53) and U373MG (mutant p53) cells. Inhibition of p53 was achieved in U87MG cells transfected with E6 oncoprotein (U87MG-E6) and treated with pifithrin-alpha, a reversible inhibitor of p53 (U87MG-PFT). We investigated whether the anti-glioblastoma responses of celecoxib were p53-dependent, and whether celecoxib induced DNA damage leading to p53-dependent G1 cell cycle arrest, followed by autophagy or apoptosis.
  
  
  RESULTS
  Our findings demonstrated that celecoxib concentration-dependently reduced glioblastoma cell viability, following 24 and 72 hours of treatment. Inhibition of functional p53 in glioblastoma cells significantly reduced the anti-proliferative effect of celecoxib. In U87MG cells, celecoxib (8 and 30 muM) significantly induced DNA damage and inhibited DNA synthesis, corresponding with p53 activation. Celecoxib induced G1-phase cell cycle arrest, accompanied with p21 activation in U87MG cells. Cell cycle progression of U87MG-E6 and U87MG-PFT cells was not affected by celecoxib. In parallel, celecoxib induced G1 cell cycle arrest in LN229 cells, but not in U373MG cells. Autophagy was induced by celecoxib in U87MG and LN229 cells, as shown by the significantly greater population of acridine orange-stained cells and increased levels of LC3-II protein (in comparison with non-treated controls). Celecoxib did not induce significant autophagy in U87MG-PFT, U87MG-E6 and U373MG cells, which lack functional p53. Regardless of p53 status, celecoxib caused no significant difference in apoptosis level of U87MG, U87MG-PFT, U87MG-E6 and U373MG cells.
  
  
  CONCLUSIONS
  Our findings reveal that p53 increases human glioblastoma sensitivity to celecoxib. Celecoxib inhibits glioblastoma cell viability by induction of DNA damage, leading to p53-dependent G1 cell cycle arrest and p53-dependent autophagy, but not apoptosis.
authors.affiliation: Brain Tumour Research Laboratory, Division of Medical Sciences, Humphrey Oei Institute of Cancer Research, National Cancer Centre, 11 Hospital Drive, 169610, Singapore. dmskkb@nccs.com.sg
authors.forename: Khong Bee
authors.initials: KB
authors.lastname: Kang
authors.forename: Congju
authors.initials: C
authors.lastname: Zhu
authors.forename: Sook Kwin
authors.initials: SK
authors.lastname: Yong
authors.forename: Qiuhan
authors.initials: Q
authors.lastname: Gao
authors.forename: Meng Cheong
authors.initials: MC
authors.lastname: Wong
mesh_terms: D000704:Analysis of Variance; D001343:Autophagy / Q000187:drug effects*; D052160:Benzothiazoles / Q000494:pharmacology; D015153:Blotting, Western; D000068579:Celecoxib; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D020552:Comet Assay; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D016193:G1 Phase / Q000187:drug effects*; D006801:Humans; D007150:Immunohistochemistry; D011720:Pyrazoles / Q000494:pharmacology*; D016192:Resting Phase, Cell Cycle / Q000187:drug effects; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013449:Sulfonamides / Q000494:pharmacology*; D014050:Toluene / Q000031:analogs & derivatives / Q000494:pharmacology; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
chemical_list: D052160:Benzothiazoles; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016861:Cyclooxygenase Inhibitors; D011720:Pyrazoles; D013449:Sulfonamides; D016159:Tumor Suppressor Protein p53; D014050:Toluene; C121565:pifithrin; D000068579:Celecoxib
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