Comparative Proteome Analysis Of Mitochondria Isolated From HCPT-Treated Hepatoma Cells And Study Function Of AIF

ObjectiveTo investigate changes in the protein spectrum of mitochondria isolated from hydroxycamptothecin (HCPT)-treated hepatoma cells with comparative proteome proteome research and to further elucidate the mechanism of HCPT-mediated cell apoptosis; In addition, to set up a technical platform of subcellular proteome research and to study function of apoptosis inducing factor.Methods1.Detection of mitochondrial alterations during apoptosis: The effects of HCPT on SMMC-7721 cells were measured by several methods including light microscopy, MTT assay, electron-microscopy, AnnexinⅤ-FITC and AO/EB staining; The apoptotic phase of mitochondria was ascertained by examination of changes in mitochondrial transmembrane potential, confocal microscopy and western blot analysis.2.Measurement of purity of isolated mitochondria: Mitochondria were isolated from hepatoma SMMC-7721 cells with mitochondria isolation kit and identified by Janus green B staining; Contaminations from cytosol, nucleus, and lysosomes were monitored by western blot.β-actin, Histone and Cathepsin-D protein were used as cytosol, nucleus, and lysosomes marker respectively.3.Analysis of differently expressed proteins in apoptotic mitochondria with comparative proteome: First, suitable conditions for two-dimensional electrophoresis were ascertained, including suitable loading quantity of sample, staining method and kind of glue strip. Mitochondrial proteins were separated into three different fractions based upon their differing solubility. Mitochondrial proteins in three different fractions were separated by two dimensional electrophoresis respectively. Image analysis was performed using PDQuest(7.0) image analysis software. Matrix assisted laser desorption/isonization time of flying mass spectrometry (MALDI-TOF-MS) was adopted to identify differentia protein spots and then mascot software was used to search for proteins in MSDB database.4.To examine whether AIF was involved in HCPT-mediated cell apoptosis: After cells treated with HCPT, RT-PCR and western blot analysis were used to detect the expression of AIF; AIF transcloation was assessed by confocal microscopy and western blot analysis, at the same time, DNA damage was monitored by DNA ladder analysis.5.Analysis of proteins which may interact with AIF: AIF gene (mitochondrial localization sequence, MLS, was deleted), which was amplified from the total mRNA extracted from hepatoma SMMC-7721 cells by RT-PCR, was inserted into pcDNA3.1 plasmid.The recombinant plasmids were then transfected into SMMC-7721 cells and expressions of AIF were detected by RT-PCR and western blot. Immunity precipitation(IP) and MALDI-TOF-MS were used to isolate and identify proteins which can interact with AIF respectively. Screened out proteins which may interact with AIF were then confirmed by CO-IP and western blot.6. Cloning and expression of recombinant AIF with biological activity: Following a strict step-by-step cloning procedure, AIF gene was subcloned into pET32a(+) vector from pcDNA3.1-AIF plasmid to construct the recombinant plasmid pET32a-AIF; The recombinant plasmid was transformed into E. coli BL-2l(DE3) and AIF expression was induced by isoprophylthio-β-D-galactoside(IPTG). AIF expression was analyzed by SDS-PAGE and western blot; AIF protein was purified by Ni affinity chromatography and the biological activity of AIF protein was confirmed by electrophoretic mobility shift assay(EMSA) and Hoechst stainingResults1.HCPT could noticeably inhibit the proliferation of SMMC-7721 cells and the IC50(50% inhibitory concentration)dose was about 80μg/ml; after cells treated by HCPT, phosphatidylserine(PS) was exposed from inner to outer leaflet of the plasma membrane; nucleus showed chromatin pyknosis and apoptosis; at the same time, mitochondria was swollen, mitochondrial transmembrane potential was reduced and cytoehrome c released from mitochondria to cytosol.2. After staining with Janus green B, separated mitochondria showed some blue granule and strip structures. Western blot analysis showed that noβ-actin, Histone or Cathepsin-D immunoreactivity had been detected in mitochondrial fraction.3.The loading quantity of sample, staining methods and category of glue strips were selected as 200μg, silver staining and IPG Strips (3–10 NL) respectively via the optimizing conditions. Compared with control, thirty-nine mitochondrial protein spots showed differentia expression in HCPT-treated cells. Among them, 25 protein spots were up-regulated while 14 were down-regulated.Twenty differentia proteins were successfully identified by MALDI-TOF-MS.4. mRNA and protein expression of AIF in HCPT-treated cells were not significantly changed compared with control. However, after cells were treated with HCPT for 6 h or longer, there was significant translocation of AIF to nucleus. At the same time, DNA damage was observed by agarose gel electrophoresis.5. Transfected AIF gene could express in SMMC-7721 cells.The same seven protein bands were observed both in control group and transfected group and one band was identified asβ-actin.6. The expression quantity of recombinant protein in recombinant E.coli strains amounted to 9% of total bacterial protein. Recombinant protein could be specially recognized by anti-AIF and anti-his antibody. Purity of purified protein reached over 95% and recombinant protein could bind DNA and induces nucleus apoptosis.Conclusions1.HCPT can induce SMMC-7721 cell apoptosis through the mitochondria apoptosis pathway.2. Mitochondria isolated with the methods mentioned above was pure enough to be used for the subsequent proteome analysis.3. The twenty differentialy expressed mitochondrial proteins identified in this study might play very important roles in HCPT-mediated cell apoptosis.4. AIF was involved in HCPT-mediated cell apoptosis.5.β-actin might have interactions with AIF.6.Recombinant AIF protein with high purity and biology activity was obtained to facilitate further studying function of AIF.