BACKGROUND:Highly selective cyclooxygenase-2(COX-2)inhibitors have recently been approved for the treatment of colon cancer,breast cancer,urinary bladder cancer,and skin cancer.For the highly selective COX-2 celecoxib,the mechanism of action for inhibiting neuroblastoma cells is still uncertain.OBJECTIVE:To observe the influence of different celecoxib concentrations on proliferation and cell cycle of SH-SY-5Y cells in vitro and to reveal potential COX-2-independent mechanisms of celecoxib on SH-SY-5Y cells.DESIGN:Controlled experiment. SETTING:Department of Hematology,Affiliated Hospital of the Qingdao Medical College,Qingdao University,Shandong Province.MATERIALS:The study was erformed at the Cerebrovascular Disease Institute of Shandong Province and the Laboratory of Molecular Biology,Affiliated Hospital of Qingdao Medical College,Qingdao University between September 2006 and June 2007.The SH-SY-5Y cell line was obtained from the Department of Molecular Biology,Qingdao Medical College,Qingdao University.Celecoxib was obtained from Pfizer Pharmaceuticals LLC,USA.Coulter DNA PREP reagent kit was purchased from Beckman Coulter,Inc.The antibodies against human CyclinD1,P21,and P16 were purchased from Santa Cruz Biotechnology. METHODS:SH-SY-5Y cells were treated with different concentrations of celecoxib(10,20,40,and 80 μ mol/L)for 48 hours and comprised the experimental groups.The same concentrations of DMSO (dimethyl sulphoxide)treatment for 48 hours served as the control group.MAIN OUTCOME MEASURES:①Cellular morphology of cells pre-treated and post-treated with celecoxib by inverted microscopy.②Methabenzthiazuron assay was used to measure cell proliferation. ③Cell cycle was measured by flow cytometry after incubation with different celecoxib concentrations for 48 hours.④CyclinD1,P16,and P21 protein expression was detected by Western blot analysis. RESULTS:①Cellular morphology:The shape of SH-SY-5Y cells pre-treated with celecoxib was a slender,fusiform shape.SH-SY-5Y cells became short or polygon after 48 hours of treatment with different celecoxib concentrations.②Cell proliferation:Cell absorbance decreased with increasing concentrations. The inhibitory effect of celecoxib on SH-SY-5Y cells was dose-dependent effect.③Cell cycle:Cell cycle analysis demonstrated a dose-dependent accumulation of cells in the G0-G1 phase.Heteroploid cells were significantly inhibited.④CyclinD1,P16,and P21 protein expression:CyclinD1 expression decreased with increasing celecoxib concentrations; P21 and P16 expression increased.CONCLUSION:Celecoxib inhibited SH-SY5Y cell growth.This effect was dose-dependent.The inhibitory mechanisms on SH-SY-5Y cell growth with Celecoxib resulted in the down-regulation of CyclinD1 expression,as well as the up-regulation of P16 and P21 expression in vitro.BACKGROUND:Highly selective cyclooxygenase-2(COX-2)inhibitors have recently been approved for the treatment of colon cancer,breast cancer,urinary bladder cancer,and skin cancer.For the highly selective COX-2 celecoxib,the mechanism of action for inhibiting neuroblastoma cells is still uncertain.OBJECTIVE:To observe the influence of different celecoxib concentrations on proliferation and cell cycle of SH-SY-5Y cells in vitro and to reveal potential COX-2-independent mechanisms of celecoxib on SH-SY-5Y cells.DESIGN:Controlled experiment. SETTING:Department of Hematology,Affiliated Hospital of the Qingdao Medical College,Qingdao University,Shandong Province.MATERIALS:The study was performed at the Cerebrovascular Disease Institute of Shandong Province and the Laboratory of Molecular Biology,Affiliated Hospital of Qingdao Medical College,Qingdao University between September 2006 and June 2007.The SH-SY-5Y cell line was obtained from the Department of Molecular Biology,Qingdao Medical College,Qingdao University.Celecoxib was obtained from Pfizer Pharmaceuticals LLC,USA.Coulter DNA PREP reagent kit was purchased from Beckman Coulter,Inc.The antibodies against human yclinD1,P21,and P16 were purchased from Santa Cruz Biotechnology. METHODS:SH-SY-5Y cells were treated with different concentrations of celecoxib(10,20,40,and 80 μ mol/L)for 48 hours and comprised the experimental groups.The same concentrations of DMSO (dimethyl sulphoxide)treatment for 48 hours served as the control group.MAIN OUTCOME MEASURES:①Cellular morphology of cells pre-treated and post-treated with celecoxib by inverted microscopy.②Methabenzthiazuron assay was used to measure cell proliferation. ③Cell cycle was measured by flow cytometry after incubation with different celecoxib concentrations for 48 hours.④CyclinD1,P16,and P21 protein expression was detected by Western blot analysis. RESULTS:①Cellular morphology:The shape of SH-SY-5Y cells pre-treated with celecoxib was a slender,fusiform shape.SH-SY-5Y cells became short or polygon after 48 hours of treatment with different celecoxib concentrations.②Cell proliferation:Cell absorbance decreased with increasing concentrations. The inhibitory effect of celecoxib on SH-SY-5Y cells was dose-dependent effect.③Cell cycle:Cell cycle analysis demonstrated a dose-dependent accumulation of cells in the G0-G1 phase.Heteroploid cells were significantly inhibited.④CyclinD1,P16,and P21 protein expression:CyclinD1 expression decreased with increasing celecoxib concentrations; P21 and P16 expression increased. CONCLUSION:Celecoxib inhibited SH-SY5Y cell growth.This effect was dose-dependent.The inhibitory mechanisms on SH-SY-5Y cell growth with Celecoxib resulted in the down-regulation of CyclinD1 expression,as well as the up-regulation of P16 and P21 expression in vitro.