An Experimental Study On Proteomic Differential Expression Of EC304 Induced By Alphastatin

Gastric cancer is one of the most common disease of malignant tumors which are harmful to health.Surgical resection and postoperative chemotherapy are the main methods to treat this disease for decades.Previous studies proved that angiogenesis play important role in gastric cancer development and metastasis.Recently,angiogenesis inhibitors have become the focus of tumor treatment,and some agents are in clinical trial. Alphastatin is a potent agent inhibit endothelial cells tubular formation and murine CT26 tumor growth.On the base of this,we treated gastric cancer with alphastatin.The aims of this study was to find out the feasibility and efficiency of alphastatin in the treatment of malignant tumors,and furthermore investigate the mechanisms involved in these process. All of these were to provide experimental data to explain the biological functions of alphasatin. There were many reports about anti-angiogenic therapy against gastric cancer,but less studies on proteomic expression. Our study aims at seeking the protein which interacts with Alphastatin in anti-angiogenesis and exploring the mechanism of action of anti-angiogenic therapy by alphastatin through some thechniques such as immunofluorescence.Objective:To explore protein express tables changes in EC304 induced by Alphastatin.Methods:Cultured EC304 in exponential phase of growth were randomly divided into two groups:Alphastatin group and control group.After protein extraction, the protein two dimensional electrophoresis (2-DE) technique was applied to analyze the expression difference in EC304 induced by alphastatin. The total proteins were extracted and 2-DE was performed. The 2-DE gel images were analyzed by using Image Master 2D Platinum 5.0 software, so as to identify the differential protein expression. Analysis of amino acid sequence was conducted on significant protein differential spots by high performance liquid chromatography-electrospray tandem MS NanoUPLC-ESI-MS/MS. The proteins were searched and identified using the Swiss-prot database and MASCOT.Results: 1.1350±90 protein spots were detected by Imagemaster analysis software in the 2-DE gel images from normal and Alphastatin groups, respectively. The match rate is about 72-80% for spectrum profiles of normal group and Alphastatin group. Totally 29 significant protein spots were identified, including 10 up-regulated spots,17 down-regulated spots, and 2 new spots,and 1 lost. The pI of the differential spots are in 4.0-9.2, molecular weights rang from 14.0Kda to 100.0Kda, the up-regulated expressions are in 3-25 times, and the lowest down-regulated expression is 0.1 times.2.29 protein spots with lowwer abundance were analyzed by MS NanoUPLC-ESI-MS/MS technique, and 23 significant proteins were identified. The Mascot search score was 64～666, peptide matching number was 3～27 and sequence coverage was 8～62%.3.23 protein checked through Mass spectrometry, including Nm23 protein and profilin-2 isoform b. Nm23 protein associated with biology behavior, profilin-2 isoform b associated with adjusting cell framework.4. Nm23 protein and profilin-2 isoform b decreased significantly in cultured EC304 through the technique of western blot and immunofluorescence.Conclusion:1.The proteome in EC304 induced by Alphastatin alters dramatically.It can be concluding that throngh adjusting the protein and conducting access,Alphastatin plays important role in adusting EC304.2.Proteome plays an important role in investigating the mechanism of action of alphastatin and anti-angiogenic therapy to gastric cancer.3. Validating the results of Mass spectrometry through some thechniques such as immunofluorescence.Alphastatin may be concerned with anti-angiogenesis through some proteins such as Nm23 protein and profilin-2 isoform b. It provides a new way to study the mechanism of anti-angiogenic therapy to gatric cancer.