A quantitative analysis of mitochondrial protein differential expressions in hydroxycamptothecin-treated hepatoma cells / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To investigate the differentially expressed mitochondrial proteins in hydroxycamptothecin (HCPT)-treated SMMC-7721 cells by using quantitative proteome.</p><p><b>METHODS</b>SMMC-7721 cell apoptosis was induced by HCPT and the mitochondria were isolated with a mitochondria isolation kit. Mitochondrial proteins labeled with a cleavable isotope-coded affinity tag were identified and quantified using two-dimensional liquid chromatography/tandem mass spectrometry.</p><p><b>RESULTS</b>Highly purified mitochondria were obtained. Seventy-four mitochondrial proteins, which were statistically significantly altered (P less than 0.05) in HCPT-treated cells, were identified and analyzed. A total of 42 proteins were significantly down-regulated, and 32 were up-regulated in the cells that responded to apoptosis. The functions of these proteins were likely involved in cell energy metabolism, nucleic acid translation and transcription, cytoskeleton, etc.</p><p><b>CONCLUSION</b>Our results about the information of differentially expressed mitochondrial proteins in HCPT-treated cells and the control cells will help to understand the mechanism by which HCPT induces cell apoptosis. The integrated techniques we used in this study will be helpful to the investigation of subcellular quantitative proteomics.</p>

Proteomic analysis of mitochondrial proteins in hydroxycamptothecin-treated SMMC-7721 cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the differentially expressed mitochondrial proteins in hydroxycamptothecin (HCPT)-treated SMMC-7721 cells by comparative proteomic analysis.</p><p><b>METHODS</b>Apoptosis of SMMC-7721 cells were induced by using HCPT and their mitochondria were isolated with a mitochondria isolation kit for cultured cells. Three different solubility protein fractions were extracted with ReadyPrep Sequential Extraction Kit and were separated by two-dimensional gel electrophoresis (2-DE). PDQuest software was used to differentiate mitochondrial proteins between control cells and HCPT-treated cells. Matrix assisted laser desorption/ionization time of flying mass spectrometry (MALDI-TOF-MS) was used to identify some of the different proteins.</p><p><b>RESULTS</b>Highly purified mitochondria and high resolution 2-DE patterns of the proteins were obtained. Forty-four mitochondrial protein spots from the HCPT-treated cells showed different expressions compared to those of the control cells. Twenty of the different protein spots were analyzed by MALDI-TOF-MS.</p><p><b>CONCLUSION</b>Differently expressed mitochondrial proteins in HCPT-treated cells and control cells were obtained in this study. This will be of help to understand the mechanism by which HCPT induces cell apoptosis.</p>

Effect of hydroxycamptothecin on apoptosis-inducing factor (AIF) expression and on AIF translocation in human hepatocellular cancer cell SMMC-7721 / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the effect of hydroxycamptothecin (HCPT) on apoptosis-inducing factor (AIF) expression and AIF translocation from mitochondria to the nucleus in human hepatocellular cancer cell SMMC-7721 during apoptosis.</p><p><b>METHODS</b>After treatment with 80 mg/ml of HCPT, the cancer cells were stained with A0/EB to monitor their apoptosis. Their mitochondria was examined with electronmicroscopy and the AIF expression of the cells was tested by RT-PCR and Western blot. The translocation of AIF from mitochondria to the nucleus during apoptosis was analyzed by confocal microscopy.</p><p><b>RESULTS</b>SMMC-7721 cells treated with HCPT showed chromatin condensation, nuclear fragmentation and mitochondria swelling. The mRNA and protein expression of AIF in treated and untreated SMMC-7721 cells were not significantly different. However, cells treated with 80 mg/ml HCPT for 6 h or 12 h showed massive translocation of AIF into the nuclei.</p><p><b>CONCLUSION</b>These results show the important role the mitochondrial pathway of apoptosis plays in HCPT-induced tumor cell death, at least in SMMC-7721 cells.</p>