| Objective:To analysis GnRH inhibitor cetrorelix associated with the growth inhibitory effects of endometrial adenocarcinoma cell line ishikawa.Methods:1)Tissue microarray and immunohistochemistry were carried out to analysis the expression of GnRH-R protein in endometrial cancer tissue; 2) MTT assay was used to determine the inhibitory concentration of cetrorelix in treating ishikawa cell. The apoptosis percentage and cell cycle distribution were analyzed by flow cytometry(FCM); 3) Total cellular proteins of the control and cetrorelix treated ishikawa endometrial adenocarcinoma cell line was separated by two-dimensional gel electrophoresis. After colloide Coomassie G-250 staining and scanning, gel images were analyzed by PDQuest software. Matrix-assisted laser desorption ionization-time-of-fiight mass spectrometry (MALDI-TOF-MS)and Mascot software were used to identify differential expressed protein.Results: 1) Immunohistochemical positive staining for GnRH-R was expressed in the cytoplasm of normal endometrium and endometrial cancer cells. The positive expression rate of GnRH-R in endometrial cancer was 94% (59/63), The positive expression rate of GnRH-R in normal endometrium was 100% (7/7),There was no significant difference in the positive expression rate of GnRH-R between the normal endometrium and endometrial cancer cells (P>0.05). The positive expression rate of GnRH-R in endometrial cancer stage?was 97%(55/57), stage?was 80%(4/5), stage?was negative; The positive expression rate of GnRH-R in endometrial cancer grade?was 94%(33/35), grade?was 94%(17/18), grade?was 86% (7/8); There were no significant difference positive expression rates of GnRH-R among histological grade and clinical stages of endometrial cancer (P>0.05).2) cetrorelix could inhibit the cell proliferation in a time-and dose-dependent mode, and induced the increasing ratio of the S cells and decreasing ratio of G0/G1 cells, cetrorelix also induced the increasing ratio of the apoptosis rate. 3)Combined treatment of cetrorelix with paclitaxel liposome resulted in a synergistic effect of inhibiting growth.4)Well-resolved and reproducible 2-DE patterns of both control and cetrorelix treated ishikawa cell were obtained. fifty-three proteins displayed differential expression after cetrorelix treated, among which 12 were up-regulated, 33 were down-regulated. Peptide mass fingerprints(PMF)by MALDI-TOF-MS analysis and searched in MSDB enabled the identification of 21 proteins.3 proteins were only present in the cetrorelix-treated group,5 proteins were only present in the control group. The down-regulated proteins identified were heat shock 90kD (HSP90)?Protein disulfide-isomerase A6 (PDIA6), Prohibitin-2 (PHB2)?Dual specificity mitogen-activated protein kinase kinase 2 (MAP2K2)?Elongation factor 1-alpha 2(EEF-1 A2)?Annexin Al?Alpha Enolase.4) Western-blot analysis was used to prove the expression levels of Annexin Al and alpha enolase. The result of assay by western-blot techniques is the same as that of by proteomics techniques.Conclusions:GnRH-R is expressed in the majority of endometrial cancer patients, suggesting that GnRH-R maybe involve in tumor genesis of endometrial cancer. GnRH inhibitor cetrorelix can inhibit the proliferation of endometrial adenocarcinoma cell. The combined treatment of GnRH inhibitor cetrorelix with paclitaxel results in synergistic inhibiting effect on growth of endometrial cancer cell line, suggesting that GnRH inhibitor cetrorelix may play an important role in the tumor genesis and development of endometrial cancer. In this study, the well-resolved, reproducible 2-DE maps of cetrorelix treated and control ishikawa cells were established and the significantly different expressed proteins are preliminary identified. These will be helpful in understanding and further studying in the molecular mechanism of GnRH inhibitor cetrorelix. |
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