The Study Of The Remedial Mechanism Of Cyclooxygenase-2 Inhibitor NS-398 In Endometrial Carcinoma

Objective: To analysis the relationship between cyclooxygenase-2 and endometrial cancer, determining proteins associated with the growth inhibitory effects of selective cyclooxygexmse-2 (COX-2) inhibitor NS-398 in endmometrial adenocarcina cell.Methods: 1) Tissue microarray and immunohistochemistry were carried out to analysis the expression of COX-2 and aromatase protein in endometrial cancer tissue; 2) MTT assay was used to determin the Inhibitory concentration of NS-398 in treating RL95-2 cell. The apoptosis percentage and intracellular calcium concentration was examined by flow cytometry (FCM); 3 ) Total RNA was extracted for RT-PCR used to determine the VEGF mRNA expression after RL95-2 cells were treated with NS-398; 4) Total cellular proteins of the control and NS-398 treated RL95-2 endometrial adenocarcinoma cell line was separated by two-dimensional gel electrophoresis. After colloide Coomassie G-250 staining and scanning, gel images were analysied by PDQuest software. Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) and Mascot software were used to identify differential expressed protein.Results: 1) Immunohistochemical staining for COX-2 and aromatase was positive for 67.24% (39/58) and 65.52% (38/58) patients in cancer epithelial cells respectively. The immunoreactivity of aromatase correlated positively with COX-2 positive staining in cancer epithelial cells. 2) NS-398 could inhibit the cell proliferation in a time-and dose-dependent mode, and induced the increasing ratio of the Go / G₁ cells. 3) Combined treatment of NS-398 with low dose cisplatin resulted in a synergistic effect of inhibiting growth . 4) Treatment of the RL95-2 cell with NS-398 can significantly reduce the expression of VEGF mRNA. 4) Well-resolved and reproducible 2-DE patterns of both control and NS-398 treated RL95-2 cell were obtained. Eighteen proteins displayed differential expression after NS-398 treated, among which 4 were up-regulated, 14 were down-regulated. Peptide mass fingerprints (PMF) by MALDI-TOF-MS analysis and searched in MSDB enabled the identification of 5 proteins. The down-regulated proteins identified were heat shock 70kDa protein 8 isoform 2 variant, Tropomyosin, protein disulfide-isomerase, Alpha enolase. The up-regulated protein was heterogeneous nuclear ribonucleoprotein K.Conclusions: COX-2 and aromatase are expressed in the majority of...