Experimental Study On Inhibition Of Human Neuroblastoma Cell Lines SK-N-SH Growth By A Highly Selective COX-2 Inhibitor CAY10404 In Vitro

Neuroblastoma(NB) is the most common malignant noumenal tumors of infants, and poor prognosis has been sustained constantly because of its high malignancy, insidious location, earlier metastasis, insensitivity and tolerance to both radiotherapies and chemotherapies, especially in advanced stages. Cyclooxygenase (COX) is also known as prostaglandin synthetase,which catalyzes the biosynthesis of prostaglandins(PGs) from arachidonic acid. Recent years,COX-2 has been detected in a variety of tumor tissues,in addition,selective inhibitors of COX-2 have been shown to suppress the growth of many established tumors. In our study, we evaluated the efficacy and the possible mechanism of cell growth suppression induced by CAY10404,a selective COX-2 inhibitor in human neuroblastoma cell lines SK-N-SH.Objective To investigate the efficacy and the possible mechanism of cell growth suppression induced by CAY10404,a highly selective COX-2 inhibitor in human neuroblastoma cell lines SK-N-SH.Methods Human neuroblastoma cell lines SK-N-SH were cultured in vitro,and morphological variety was observed by invert phase-contrast microscope every day. Treated with CAY10404 of 30,60,90,120μmol/l,cells were harvested in 24,48,72hours respectively.The dose-response curve and inhibition rate were gotten by means of MTT assay. The population doubling time was observed by trypan blue dyeexclusion test.Cell morphological variety was observed by invert phase-contrast microscope.Cell cycle analysis and apoptosis rate were evaluated by flow cytometer(FCM). The expression of bcl-2 , bax , survivin was detected with reverse transcription polymerase chain reaction(RT-PCR) .The expressions of COX-2,PCNA,Bcl-2,Bax,Survivin were analyzed with immunocytochemistry.Results(1)Exposed to CAY10404 of various concentrations,the proliferation of human neuroblastoma cell lines SK-N-SH was decreased in time- and dose-dependent manners.(2)Treated with CAY10404, COX-2 and PCNA in cell lines SK-N-SH were decreased in contrast with control detected by immunocytochemistry.(3) Cell apoptosis rate increase had been detected by flow cytometer(FCM) contrasted with control after being treated with CAY10404 48 hours.The significance of difference had been shown between experimental groups and control(p<0.05).Otherwise,cell cycle redistribution had not performed in low concentration groups(30,60μmol/l), nevertheless,performed in high concentration groups(90,120μmol/l).(4)The expression of bcl-2,surviving decreased and bax increased with concentration of CAY10404 risen,which were detected by immunocytochemistry and reverse transcription polymerase chain reaction(RT-PCR).Conclusions(1)High expression of COX-2 presented in human neuroblastoma cell lines SK-N-SH. Proliferation of SK-N-SH was suppressed by highly selective COX-2 inhibitor CAY10404 in time- and dose-pependent manners, which could be through COX-2 dependent path.(2)Apoptosis and cell cycle redistribution of SK-N-SH were induced by CAY10404, and apoptosis related genes including bcl-2,survivin,bax reacted in these process.