Quantitative Analysis Of Mitochondrial/Nuclear Proteome In HCPT-Induced Apoptosis Of Hepatoma Cells

ObjectiveTo quantify and qualify the mitochondrial proteome and nuclear proteome in hydroxycamptothecin(HCPT)-treated hepatoma cells with gel-based proteomics strategy and shotgun proteomics strategy, for further elucidating the mechanism of HCPT-mediated cell apoptosis at subcellular proteomics level. Moreover, to set up a technical platform based on cleavable isotope-coded affinity tag(c-ICAT) approach for subcellular quantitative proteomics research.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; Contaminations from endoplasmic reticulum and cell membrane were monitored by detecting specific enzyme activity. Succinate dehydrogenase(SDH) was used as indicator of mitochondria purity, NADPH cytochrome c reductase(CR) and 5′-nucleotidase(5′-NT) were used as endoplasmic reticulum and cell membrane marker respectively.3.Analysis of differentialy expressed proteins in apoptotic mitochondria with 2DE-MALDI-TOF/MS: First, suitable conditions for two-dimensional electrophoresis were ascertained, including suitable loading quantity of sample, staining method and category 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 and PeptIdent software were used to search for proteins in MSDB and SWISS-PROT database. Bioinformatics analysis was performed on the differentialy expressed proteins. 4.Quantitative analysis of the mitochondrial proteome in HCPT-treated hepatoma cells using cleavable isotope-coded affinity tag(c-ICAT) strategy combined with two-dimensional liquid chromatography/tandem mass spectrometry(2D-LC/MS/MS): Mitochondrial proteins of intermediate solubility and mitochondrial hydrophobic proteins were identified and quantified with c-ICAT proteomics strategy respectively. The protein sample were labeled with c-ICAT reagent and digested with trypsin. The labeled cysteine-containing peptides were fractionated using strong cation exchange(SCX) and purified on an avidin column, sequentially the affinity tags were cleaved with reagent before the peptides were analyzed byμRP-HPLC-MS/MS. Quantitate proteins by integration labeled peptides elution peak areas, identify proteins from sequence information from MS/MS. Bioinformatics tools were used to analyze the proteins identified and quantified.5.Detection of purity of isolated nucleus and quantitative analysis of the nuclear proteome in HCPT-treated hepatoma cells: Nucleus were isolated from hepatoma SMMC-7721 cells with kit. Contaminations from cytosol, mitochondria, and lysosomes were monitored by western blot.β-actin, HSP60 and Cathepsin-D protein were used as cytosol, mitochondria, and lysosomes marker respectively. The nuclear proteins were labeled with c-ICAT reagent, digested with trypsin. The labeled peptides were fractionated using strong cation exchange and purified on an avidin column, then the tags were cleaved with reagent before the peptides were analyzed byμRP-HPLC-MS/MS. Quantitate proteins by integration labeled peptides elution peak areas, identify proteins from sequence information from MS/MS. Bioinformatics tools were used to analyze proteins identified and quantified.Results1. HCPT could remarkably inhibit the proliferation of SMMC-7721 cells and the IC50(50% inhibitory concentration) dose was about 80μg/ml. Some morphologic changes occured after cells were treated by HCPT, including phosphatidylserine(PS) was exposed from inner to outer leaflet of the plasma membrane, nucleus showed chromatin pyknosis and apoptosis, mitochondria was swollen, mitochondrial transmembrane potential was reduced and cytoehrome c released from mitochondria to cytosol.2. Stained with Janus green B, separated mitochondria showed some blue granule and strip structures. Western blot analysis showed that littleβ-actin, Histone or Cathepsin-D immunoreactivity had been detected in mitochondrial fraction. Detection of specific enzyme activity showed the specific enzyme activity of SDH in mitochondria was 13.8 fold compared with the value in homogenate. The specific enzyme activity of both CR and 5′-NT in mitochondria were little.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. Ninety-one mitochondrial proteins of intermediate solubility were identified, among them, seventy-four proteins which were statistically significantly(P < 0.05) altered in HCPT-treated cells were quantified and identified. A total of forty-two proteins were significantly down-regulated, and thirty-two were up-regulated in response to apoptosis cells.5. 244 Mitochondrial hydrophobic proteins were identified statistically significantly(P < 0.05), a total of 154 proteins were quantified using shotgun proteomics method based on multiple dimensional liquid chromatography-linear ion trap /orbitrap mass spectrometer. Among them, compared with control cells, twelve proteins from apoptotic cells showed down-regulated, and 137 were up-regulated, five proteins showed no difference. In addition, thirteen alkali proteins, eleven proteins with Mr>200kDa and six proteins with Mr<10kDa were identified; fifty membrane proteins were identified. Moreover, there were forty-five proteins showed an elevation of more than 10-fold in apoptotic cells compared with control cells.6. Western blot analysis showed that littleβ-actin, HSP60 or Cathepsin-D immunoreactivity had been detected in nuclear fraction. Ninety-four nuclear proteins were analyzed statistically significantly(P < 0.05) using multiple dimensional liquid chromatography-linear ion trap /orbitrap mass spectrometer combined with c-ICAT strategy. A total of forty-three proteins were quantified, among them, twelve proteins showed down-regulated in HCPT-treated cells, thirty proteins showed up-regulated, and one showed no difference.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 proteomic analysis.3. The twenty differentialy expressed mitochondrial proteins identified in this study might play very important roles in HCPT-mediated cell apoptosis.4. The function of the quantitatively differentialy expressed mitochondrial proteins of intermediate solubility were likely involved in cell energy metabolism, nucleic acid translation and transcription, cytoskeleton and so on.5. The hydrophobic proteins altered quantitatively were likely involved in life processes of cells including energy metabolism; cell structure; nucleic acid synthesis and metabolism; ribosome; proliferation, differentiation, apoptosis; and signal transduction. The results of this study will provide experimental foundation for further investigating the pharmacological action of HCPT at quantitative proteomics level, and prove the research strategies established in our study are helpful to the researches of subcellular proteomics and pharmic proteomics.6. In this part, the quantitatively differentialy expressed proteins were likely involved in life processes of cells such as proliferation, apoptosis, differentiation, nucleic acid synthesis and metabolism, structure of cell skeleton, energy metabolism. Several proteins were observed with a trend of up-regulation in mitochondria and nucleus after cells apoptosis, which is a meaningful clue to study the pharmacological action of HCPT and molecular mechanisms of apoptosis.