Large-Scale Proteome Quantification of Hepatocellular Carcinoma Tissues by a Three-Dimensional Liquid Chromatography Strategy Integrated with Sample Preparation

Hepatocellular carcinoma is one of the most fatal cancers worldwide. In this study, a reversed-phase–strong cation exchange–reversed-phase three-dimensional liquid chromatography strategy was established and coupled with mass spectrometry to investigate the differential proteome expression of HCC and normal liver tissues. In total, 2759 proteins were reliably quantified, of which, 648 proteins were dysregulated more than 3-fold in HCC liver tissues. Some important proteins that relate to HCC pathology were significantly dysregulated, such as NAT2 and AKR1B10. Furthermore, 2307 phosphorylation sites from 1264 phosphoproteins were obtained in our previous phosphoproteome quantification, and the nonphosphorylated counterparts of 445 phosphoproteins with 983 phosphorylation sites were reliably quantified in this work. It was observed that 337 (34%) phosphorylation sites exhibit significantly different expression trends from that of their corresponding nonphosphoproteins. Some novel phosphorylation sites with important biological functions in the progression of HCC were reliably quantified, such as the significant downregulation of pT185 for ERK2 and pY204 for ERK1

CD133/Prominin-1-Mediated Autophagy and Glucose Uptake Beneficial for Hepatoma Cell Survival

<div><p>CD133/Prominin-1 is a pentaspan transmembrane protein that has been frequently used as a biomarker for cancer stem cells, although its biological function is unclear. The aim of our study was to explore the intrinsic functions of CD133 membrane protein in hepatoma cells during autophagy, apoptosis, tumorigenesis and cell survival through expression or downregulation of CD133. In this study, CD133 was found to be dynamically released from plasma membrane into cytoplasm in both of complete medium(CM) and low glucose medium (LGM), and LGM promoted this translocation. Expression of CD133 enhanced autophagic activity in LGM, while silencing CD133 attenuated this activity in HCC LM3 and Huh-7 cells, suggesting that CD133 is associated with autophagy. Immunofluorescence and time-lapsed confocal techniques confirmed that CD133 was associated with autophagy marker, microtubule-associated protein light chain3 (LC3) and lysosome marker during the glucose starvation. We further found that Huh-7 cells with stable expression of shCD133 (Huh-7sh133) impaired the ability of cell proliferation and formation of xenograft tumors in the NOD/SCID mice. Although loss of CD133 did not affect the rates of glucose uptake in Huh-7con and Huh-7sh133 cells under the CM, Huh-7sh133 cells obviously died fast than Huh-7con cells in the LGM and decreased the rate of glucose uptake and ATP production. Furthermore, targeting CD133 by CD133mAb resulted in cell death in HepG2 cells, especially in the LGM, via inhibition of autophagic activity and increase of apoptosis. The results demonstrated that CD133 is involved in cell survival through regulation of autophagy and glucose uptake, which may be necessary for cancer stem cells to survive in tumor microenvironment.</p></div

CD133 underwent lysosome degradation.

<p><b>A.</b> LM3 cells on coverslips were transfected with CD133-GFP for 24 h and were labeled with Lysotracker for 1 h before observation. Localization of CD133 (green) and lysotracker (red) were observed under confocal microscope in the CM or LGM, respectively. The scale bars represent 10 µm. <b>B.</b> Inhibitory activity of lysosomes prevented degradation of CD133 in LGM. C: vector control; V: expression of CD133; CQ: Chloroquine. <b>C.</b> Movement of CD133 and lysotracker in LM3 cells. LM3 cells were transfected with CD133-GFP. Lysotracker(red) was added to cell medium for 1 h before observation with Leica confocal analytic system. Images showed here were selected from a series of records (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056878#pone.0056878.s003" target="_blank">movie S2</a>). The scale bar represents 10 µm. <b>D.</b> The stable shCD133-expressing Huh-7 cell (Hun-7sh133) and con8trol cells(Huh-7con) were established by lentivirus infection and puromycin selection. Autophagy was observed after transfection of LC3-GFP into these cells. Images represented the situation of autophagy at 4 h in the CM, EBSS and EBSS+CQ conditions. Right graph showed number of puncta per cells. CQ: Chloroquine. <b>E.</b> Levels of LC3-II in these two cell lines at the time indicated after incubating in the LGM in presence or absence of chloroquine(CQ). c: Huh-7con cells; sh: Huh-7sh133 cells.</p

CD133 was released into cytoplasm and participated in autophagy.

<p><b>A.</b> LGM condition promoted translocation of CD133 from membrane to cytoplasm. Huh-7 cells were stained by antibody for CD133 after culturing in CM and LGM for 3 h and counterstained by Hoechst. IF: Immunofluoresence. <b>B.</b> Autophagy in LM3 cells. Transient expression of plasmids as indicated together with GFP-LC3 in LM3 cells. Representative pictures were selected from observation at 1 h. <b>C.</b> Percentages of autophagic cells. Autohagic cells were measured by an individual cell with more than six puncta and expressed as mean±SD (n = 5). <b>D.</b> Levels of CD133 and LC3-II proteins in LM3 cells. p3xFlag-CD133 and pSuper-shCD133 were transfected into LM3 cells as indicated. Cells were harvested at 6 h after culturing in the LGM with or without 50 uM Chloroquine. <b>E.</b> CD133 levels were associated with LC3 levels. Under low glucose starvation for 3 h, silencing CD133 in Huh-7 cells reduced the level of LC3 (left). Isolated CD133⁺ huh-7 cells produced higher level of LC3-II than that in CD133⁻ Huh-7 cells (right). Densitometry analysis was done by normalization of CD133 and LC3-II levels with their own α-tubulin or β-actin and shown in graphs under immunoblots. <b>F.</b> Autophagy inhibitor abolished CD133-induced autophagy in LM3 cells. 3-MA (5 mM) inhibited CD133-induced increase of LC3-II in the LGM. Densitometry analysis was carried out by normalization of LC-II levels with GAPDH and shown under Western blotting panels.</p

CD133-mediated autophagy was beneficial for cell survival.

<p><b>A.</b> Silencing CD133 reduced proliferation of Huh-7 cells. Relative cell numbers were detected by CCK8 at the indicated time points. <b>B.</b> Huh-7con and Huh-7sh133 cells were cultured in the LGM for 3 days and cell numbers were detected by CCK8 at the time indicated (mean±SD, n = 3). <b>C.</b> ATP contents in Huh-7con and Huh-7sh133 cells in the LGM at during 2 days. <b>D.</b> 1×10⁶ cells from Huh-7con or Huh-7sh133 cells were subcutaneously injected into side back of NOD/SCID mice as indicated (3 mice). The significance of statistics is expressed as *p<0.05; **p<0.01; ***P<0.001.</p

CD133 promoted glucose uptake.

<p><b>A.</b> Levels of glucose uptake in Huh-7con and Huh-7sh133 cells. Mean fluorescence intensity of 2-NBDG uptake was measured by FASC analysis after incubating for 6 h. <b>B.</b> Time course of 2-NBDG uptake in these two cell lines. Statistical significance indicated as *p<0.05; **p<0.01. <b>C.</b> CD133 or empty vector was transfected respectively into LM3 cells. Glucose uptake was measured at the indicated times upon the medium added with 100 µM 2-NBDG. *p<0.05. <b>D.</b> Detection of relative activities of signaling pathways in Huh-7con and Huh-7sh133 cells by Western blotting. con: Huh-7con cells; sh: Huh-7sh133 cells. <b>E.</b> Detection of autophagic genes in Huh-7con and Huh-7sh133 cells by Western blotting.</p

Expression of CD133 promoted autophagosome formation.

<p><b>A.</b> LM3 cells were transfected with either empty vector or p3xFlag-CD133 and cultured in the LGM for 6 h and analyzed by transmission electron microscope. Arrows indicated autophagosomes. The number of autophagosomes per cell per cross-sectioned cells were counted (right graph) (mean±SD (n = 15). <b>B.</b> Huh-7 cells were placed on the coverslips overnight and cultured with fresh CM and LGM media for 3 h and subjected to confocal microscopic analysis. Magnified images (400X) are shown beside. Representative CD133-LC3 double-positive foci are indicated by arrows. CD133(red) and LC3(green). The scale bars represent 10 µm. <b>C.</b> CD133-Cherry and LC3-GFP were transfected into LM3 cells. Cells were observed with Leica confocal analytic system in EBSS. Images showed here were selected from a series of records (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056878#pone.0056878.s002" target="_blank">movie S1</a>). The scale bar represents 10 µm. D. Western blotting showed CD133 levels in fragmentation of membrane or cytoplasm of cells after culturing in the LGM for 8 h. E-cadherin was as membrane control; GADPH as cytosol control.</p

Metabolomics Study of Hepatocellular Carcinoma: Discovery and Validation of Serum Potential Biomarkers by Using Capillary Electrophoresis–Mass Spectrometry

Hepatocellular carcinoma (HCC) is one of the most lethal malignancies. The lack of effective screening methods for early diagnosis has been a longstanding bottleneck to improve the survival rate. In the present study, a capillary electrophoresis–time-of-flight mass spectrometry (CE–TOF/MS)-based metabolomics method was employed to discover novel biomarkers for HCC. A total of 183 human serum specimens (77 sera in discovery set and 106 sera in external validation set) were enrolled in this study, and a “serum biomarker model” including tryptophan, glutamine, and 2-hydroxybutyric acid was finally established based on the comprehensive screening and validation workflow. This model was evaluated as an effective tool in that area under the receiver operating characteristic curve reached 0.969 in the discovery set and 0.99 in the validation set for diagnosing HCC from non-HCC (health and cirrhosis). Furthermore, this model enabled the discrimination of small HCC from precancer cirrhosis with an AUC of 0.976, highlighting the potential of early diagnosis. The biomarker model is effective for those <i>a</i>-fetoprotein (AFP) false-negative and false-postive subjects, indicating the complementary function to conventional tumor marker AFP. This study demonstrates the promising potential of CE–MS-based metabolomics approach in finding biomarkers for disease diagnosis and providing special insights into tumor metabolism

CD133mAb induced cell death through inhibition of autophagy and increase of apoptosis.

<p>A. HepG2 cells were treated with CD133mAb in the CM or LGM for three days. The images showed the situation at 48 h (magnification X200). Control: no treatment. IgG1∶1 µg/ml mouse IgG1; CD133mAb: 1 µg/ml AC133. <b>B.</b> Cell numbers were measured by CCK8 kit as described in Methods after treatment for 48 h(mean±SD, n = 3). **p<0.01. <b>C.</b> Effects of CD133mAb on autophagy and apoptosis of HepG2 cells. HepG2 cells were transfected with or without LC3-GFP for autophagy or apoptosis tests. The images (magnification X200) here showed the results of autophagy at 6 h after treatment (upper panels) and apoptosis (lower panels) at 48 h. <b>D.</b> Average percentages of autophagic or apoptotic cells were determined by five fields in each well and expressed as mean±SD. *p<0.05; **p<0.01. <b>E</b>. LC3-II levels in HepG2 cells after treatment for 48 h were analyzed by Western blotting. Normalization of LC3-II levels over β-actin was shown in right graph. The experiment was repeated at least three times. <b>F</b>. Apoptosis was detected in Huh-7 cells with flow cytometry. Histographs showed the distribution of DNA fragments in Huh-7 cells. Percentages were relatively number of cells with disrupted DNA.</p

Large-Scale Quantification of Single Amino-Acid Variations by a Variation-Associated Database Search Strategy

Global quantification of the single amino-acid variations (SAAVs) is essential to investigate the roles of SAAVs in disease progression. However, few efforts have been made on this issue due to the lack of high -throughput approach. Here we presented a strategy by integration of the stable isotope dimethyl labeling with variation-associated database search to globally quantify the SAAVs at the first time. A protein database containing 87 745 amino acid variant sequences and 73 910 UniProtKB/Swiss-Prot canonical protein entries was constructed for database search, and higher energy collisional dissociation combined with collision-induced dissociation fragmentation modes were applied to improve the quantification coverage of SAAVs. Compared with target proteomics in which only a few sites could be quantified, as many as 282 unique SAAVs sites were quantified between hepatocellular carcinoma (HCC) and normal human liver tissues by our strategy. The variation rates in different samples were evaluated, and some interesting SAAVs with significant increase normalized quantification ratios, such as T1406N in CPS1 and S197R in HTATIP2, were observed to highly associate with HCC progression. Therefore, the newly developed strategy enables the large-scale comparative analysis of variations at the protein level and holds a promising future in the research related to variations