Proteomics Analysis Of Creative New Antitumor Drug Of TNBG

Cancer is still a great threat to the health of human beings, but few forward steps have been made in the way of fighting for it. The major problem of clinical antitumor drugs is: low selectivity, little effect on solid tumors, crossed drug-resistances, etc.Tetrazanbigen(TNBG) is a novel synthesized antitumor drug with azagonane structure. Many experiments, both in vivo and in vitro, have proved its significant antitumor effects. Moreover, TNBG could prolong the lifetime of tumor-loading animals, and have no crossed drug-resistance. In hepatocellular carcinoma cell line QGY-7701 exposed to TNBG, a large quantity of lipid droplets was observed, and in cDNA microarray analysis on QGY-7701 cell, a set of genes involved in lipid metabolism were proved to be differentially expressed. So we proposed that tetrazanbigen may kill cancer cells by interfering lipid metabolism. but it need to do some further research to elucidate its antitumor mechanism.As a result, using comparative proteomics, we compared the protein profiling of human hepatocellular carcinoma cell line QGY-7701 from control and TNBG-treated cells. It is drawn up to elucidate the antitumor mechanism of interfering lipid metabolism and explore the target, which would provide new insight into antitumor drugs.The main experiments and results are as follows:1.We established two-dimensional(2-DE) gel electrophoresis of cytoplasma, membrane and nucleus proteins from human hepatocellular carcinoma cell line QGY-7701. Standardized subcellular proteomes of QGY-7701 cells were extracted using the ProteoExtract Subcellular Proteome Extraction Kit from Merk. Different methods were used to remove saline matter, and two different buffer were used to resuspend the protein.As a result, the combination of Acetone and Trichloroacetic acid can acquire good desalination and reduce the loss of protein; bufferⅡ(7mol/L Urea + 2mol/L Thiourea + 4% CHAPS + 40mmol/L Tris + 65mmol/L DTT + 2% ampyolyte)can increase the solubility of membrane protein and acquire more information. The cytoplasma protein were detected about 995±53, The membrane spots were about 1035±58, and the nucleus spots were about 893±45.2.The subcellular proteome of human hepatocellular carcinoma cell line QGY-7701 induced by TNBG were isolated using established 2-DE technique. Gel images were processed and compared by the 2-DE PDQuest software(7.4.0). As a result, after administration of TNBG at a final concentration of 4μg/mL for 72h, of total 155 proteins, 56 cytoplasma protein spots, 65 membrane protein spots and 34 nucleus protein spots were detected differentially expressed.3.In regard with cDNA microarray analysis, 14 cytoplasma protein spots, 25 membrane protein spots and 9 nucleus protein spots were choosed to cut from the gel, digested with trypsin, and tryptic protein hydrolysates were analyzed in a Voyager-DE Pro MALDI-TOF-MS (ABI,USA). To identify proteins, the measured monoisotopic masses of peptide were analyzed using World Wide Web search programs MS-Fit. As a result, 33 protrein spots were successfully identified, of which 11 proteins related with lipid synthesis, 4 proteins related with lipid degradation and 3 proteins related with lipid transport. Proteome analysis were consistent with cDNA microarray results.4.Western blot were used to further verify candidate proteins in order to ensure the reliability of the proteome results.The results were: In QGY-7701 cells exposed to TNGB at 4μg/mL for 72h, the expression of SREBP-1 increased, the expression of MTTP decreased. Western blot results were consistent with proteomics analysis. It further proved that TNBG exert its antitumor action by interference of lipid metabolism of cancer cells.5.By analysis of the differentially expressed proteins, we conclude that TNBG may exert the antitumor action through the following pathways to produce a large quantity of lipid droplets.①Stimulation the SREBP-2 pathway related to cholesterol synthesis, ②Stimulation the SREBP-1 pathway related to triglyceride synthesis,③Inhibition of lipid degradation pathway,④Inhibition of MTTP pathway related to lipid efflux.So it was assumed that in addition to increase synthesis of triglyceride and cholesterol via SREBPs pathway, the main cause of lots of lipid droplets in cell may be inhibition of MTTP pathway related to lipid efflux.In conclusion, we selected the human hepatocellular carcinoma cell line QGY-7701 as a model to investigate the antitumor mechanism of TNBG. By comparative proteomics analysis, we proposed that the inhibition of MTTP is the main cause of TNBG producing lots of lipid droplets accumulated in cell.MTTP may be a new target of TNBG.