Isolated tumor endothelial cells maintain specific character during long-term culture

Tumor angiogenesis is necessary for solid tumor progression and metastasis. Increasing evidence indicates that tumor endothelial cells (TECs) are more relevant to the study of tumor angiogenesis than normal endothelial cells (NECs) because their morphologies and gene expression are different from NECs. However, it is challenging to isolate and culture large numbers of pure ECs from tumor tissue since the percentage of ECs is only about 1-2% and tumor cells and fibroblasts easily overgrow them. In addition, there has been concern that isolated TECs may lose their special phenotype once they are dissociated from tumor cells.In this study, we have successfully purified murine TECs from four different human tumor xenografts and NECs from murine dermal tissue. Isolated ECs expressed endothelial markers, such as CD31, VE-cadherin (CD144), and endoglin (CD105), for more than 3 months after isolation. TECs maintained tumor endothelial-specific markers, such as tumor endothelial marker 8 (TEM8) and aminopeptidase N (APN), as in tumor blood vessels in vivo. In addition, TECs were more proliferative and motile than NECs. TECs showed a higher response to VEGF and higher expression of VEGF receptors-1 and -2 than NECs did. Stem cell antigen-1 was up-regulated in all four TECs, suggesting that they have a kind of stemness. Cultured TECs maintain distinct biological differences from NECs as in vivo. In conclusion, it was suggested that TECs are relevant material for tumor angiogenesis research.     

The impact of miR‐34a on protein output in hepatocellular carcinoma HepG2 cells

MicroRNAs are small non‐coding RNA molecules that play essential roles in biological processes ranging from cell cycle to cell migration and invasion. Accumulating evidence suggests that miR‐34a, as a key mediator of p53 tumor suppression, is aberrantly expressed in human cancers. In the present study, we aimed to explore the precise biological role of miR‐34a and the global protein changes in HCC cell line HepG2 cells transiently transfected with miR‐34a. Transfection of miR‐34a into HepG2 cells caused suppression of cell proliferation, inhibition of cell migration and invasion. It also induced an accumulation of HepG2 cells in G1 phase. Among 116 protein spots with differential expression separated by 2‐DE method, 34 proteins were successfully identified by MALDI‐TOF/TOF analysis. Of these, 15 downregulated proteins may be downstream targets of miR‐34a. Bioinformatics analysis produced a protein–protein interaction network, which revealed that the p53 signaling pathway and cell cycle pathway were two major hubs containing most of the proteins regulated by miR‐34a. Cytoskeletal proteins such as LMNA, GFAP, MACF1, ALDH2, and LOC100129335 are potential targets of miR‐34a. In conclusion, abrogation of miR‐34a function could cause downstream molecules to switch on or off, leading to HCC development.     

Plasma and liver proteomic analysis of 3Z‐3‐[(1H‐pyrrol‐2‐yl)‐methylidene]‐1‐(1‐piperidinylmethyl)‐1,3‐2H‐indol‐2‐one‐induced hepatotoxicity in Wistar rats

3Z‐3‐[(1H‐pyrrol‐2‐yl)‐methylidene]‐1‐(1‐piperidinylmethyl)‐1,3‐2H‐indol‐2‐one (Z24), a synthetic anti‐angiogenic compound, inhibits the growth and metastasis of certain tumors. Previous works have shown that Z24 induces hepatotoxicity in rodents. We examined the hepatotoxic mechanism of Z24 at the protein level and looked for potential biomarkers. We used 2‐DE and MALDI‐TOF/TOF MS to analyze alternatively expressed proteins in rat liver and plasma after Z24 administration. We also examined apoptosis in rat liver and measured levels of intramitochondrial ROS and NAD(P)H redox in liver cells. We found that 22 nonredundant proteins in the liver and 11 in the plasma were differentially expressed. These proteins were involved in several important metabolic pathways, including carbohydrate, lipid, amino acid, and energy metabolism, biotransformation, apoptosis, etc. Apoptosis in rat liver was confirmed with the terminal deoxynucleotidyl transferase dUTP‐nick end labeling assay. In mitochondria, Z24 increased the ROS and decreased the NAD(P)H levels. Thus, inhibition of carbohydrate aerobic oxidation, fatty acid β‐oxidation, and oxidative phosphorylation is a potential mechanism of Z24‐induced hepatotoxicity, resulting in mitochondrial dysfunction and apoptosis‐mediated cell death. In addition, fetub protein and argininosuccinate synthase in plasma may be potential biomarkers of Z24‐induced hepatotoxicity.     

Proteomics analysis of liver samples from puffer fish Takifugu rubripes exposed to excessive fluoride: An insight into molecular response to fluorosis

Comparative proteomics was performed to identify proteins in the liver of Takifugu rubripes in response to excessive fluoride exposure. Sixteen fish were randomly divided into a control group and an experimental group. The control group was raised in soft water alone (F^? = 0.4 mg/L), and the experimental group was raised in the same water with sodium fluoride at a high concentration of 35 mg/L. After 3 days, proteins were extracted from the fish livers and then subjected to two‐dimensional polyacrylamide gel electrophoresis analysis. The matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) was applied to identify the proteins that were differentially expressed from the two groups of fish. Among an average of 816 and 918 proteins detected in the control and treated groups, respectively, 16 proteins were upregulated and 35 were downregulated (P < 0.01) in the fluoride‐treated group as compared with those in the control group. Twenty‐four highly differentially expressed proteins were further analyzed by MALDI‐TOF/TOF‐MS, and eight were identified by Mascot. These eight proteins include disulfide isomerase ER‐60, 4SNc‐Tudor domain protein, SMC3 protein, Cyclin D1, and mitogen‐activated protein kinase 10, as well as three unknown proteins. Consistent with their previously known functions, these identified proteins seem to be involved in apoptosis and other functions associated with fluorosis. These results will greatly contribute to our understanding of the effects of fluoride exposure on the physiological and biochemical functions of Takifugu and the toxicological mechanism of fluoride causing fluorosis in both fish and human. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:21–28, 2010; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20308     

Comparative proteome analysis of TGF‐β1‐induced fibrosis processes in normal rat kidney interstitial fibroblast cells in response to ascofuranone

Transforming growth factor‐β1 (TGF‐β1) has a wide range of biological functions such as the regulation of cell growth, differentiation, and immunological response in various types of cells. Particularly, TGF‐β1 induces plasminogen activator inhibitor‐1 (PAI‐1) as a major target protein. PAI‐1 is associated with fibrosis, thrombosis, and metabolic disorders. In this study, to identify proteins potentially involved in TGF‐β1‐induced fibrosis processes, we performed a proteomic analysis of TGF‐β1‐induced normal rat kidney cells exposed to ascofuranone (AF). In these cells, we detected 1500 proteins, with 74 differentially expressed proteins identified by MALDI‐TOF and reference to the NCBI and Swiss‐Prot databases, including PAI‐1, peroxisome prdifesator‐activated receptor, prohibitin, glutamate formyltransferase, LIM domain protein 1, LASP‐1, porphobilinogen deaminase, and peroxiredoxin 2. We also found that AF suppresses expression of profibrotic factors induced by TGF‐β in renal fibroblasts, including matrix proteins and PAI‐1. AF was also shown to inhibit selectively phosphorylation of epidermal growth factor receptor, and downstream kinases such as extracellular signal‐regulated kinase 1/2 (ERK‐1/2). Further ongoing analysis of fibrosis‐related proteins will determine AF's potential for application in fibrotic diseases and therapeutics.     

In vivo chemoresistance of prostate cancer in metronomic cyclophosphamide therapy

A human prostate cancer (PC3) xenograft model was established which reflects acquired in vivo resistance towards metronomic cyclophosphamide (CPA) treatment. Cell cultures of two in vivo resistant PC3 tumors were established which maintain chemoresistant phenotypes upon xenografting into mice. A comparative proteome analysis of the two resistant cell lines PC3-D3 and -D4 versus the non-resistant parental PC3 cell line by 2D-DIGE approach followed by MALDI-TOF–TOF analysis revealed a total of 25 differently expressed proteins. Validation of protein candidates by Western blot analysis of the corresponding in vivo tumor xenografts identified three differentially expressed proteins (thioredoxin containing protein 5, cathepsin B, and annexin A3). Thioredoxin containing protein 5 was up-regulated in resistant xenografts only upon in vivo CPA therapy. A truncated version of cathepsin B translocated into mitochondria in the resistant clones whereas it stays cytoplasmic in corresponding parental PC3 cells. Annexin A3 (ANXA3) presents a very interesting candidate which was found to be up-regulated both in vitro and in xenografts, with protein levels further increased by metronomic CPA treatment in vivo. It is noteworthy that independent studies in other epithelial cancers recently identified ANXA3 as cancer progression and resistance marker.     

Alteration of nuclear protein profiling for NIH-3T3 cells exposed to H₂O₂

Oxidative stress is a threat to mammalian cells. To better understand the molecular response and mechanism underlying oxidative stress, we applied two‐dimensional polyacrylamide gel electrophoresis and matrix‐assisted laser desorption ionization time‐of‐fight mass spectrometry analysis to identify differential nuclear protein profiling of mouse fibroblast NIH‐3T3 cells exposed to mild‐H₂O₂. Thirteen differentially expressed proteins were identified by MS and two of them were further validated by Western blot. The results revealed that exposure to mild‐H₂O₂ for 12 h cause up‐regulated expression of DJ‐1, glutathione S‐transferase P 1, DNA ligase I, dynamin 2, nucleophosmin, and down‐regulated expression of nucleoside diphosphate kinase A, enolase‐α, barrier‐to‐autointegration factor 1, metastasis associated protein 1, glycosytransferase‐like domain containing protein 1, synaptonemal complex protein 1, alpha‐centractin, bromodomain, and PHD finger containing 1 (BRPF1). Most of the identified proteins are supported as nuclear proteins localized by previous research. The findings may provide some clues to elucidate cell responses to H₂O₂ and the potential mechanism underlying protection against oxidative stress in fibroblast cells. Copyright © 2010 John Wiley & Sons, Ltd.     

Structural insight into the interaction of proteins containing NPF,DPF, and GPF motifs with the C‐terminal EH‐domain of EHD1

Eps15 homology (EH)‐domain containing proteins are regulators of endocytic membrane trafficking. EH‐domain binding to proteins containing the tripeptide NPF has been well characterized, but recent studies have shown that EH‐domains are also able to interact with ligands containing DPF or GPF motifs. We demonstrate that the three motifs interact in a similar way with the EH‐domain of EHD1, with the NPF motif having the highest affinity due to the presence of an intermolecular hydrogen bond. The weaker affinity for the DPF and GPF motifs suggests that if complex formation occurs in vivo, they may require high ligand concentrations, the presence of successive motifs and/or specific flanking residues.     

Proteomic identification of molecular targets of gambogic acid: Role of stathmin in hepatocellular carcinoma

Gamboge has been developed as an injectable drug for cancer treatment in China. In this study, the inhibition ratio and their IC₅₀ values of two derivatives from Gamboge in hepatocellular carcinoma (HCC) were determined. Proteomic approach was employed to reveal the target proteins of these two derivatives, gambogic acid (GA), and gambogenic acid (GEA). HCC cells were cultured under varied conditions with the addition of either GA or GEA. Twenty differentially expressed proteins were identified and the four most distinctly expressed proteins were further validated by Western blotting. GA and GEA revealed inhibitory effects on HCC cell proliferation. The expression of cyclin‐dependent kinase 4 inhibitor A and guanine nucleotide‐binding protein β subunit 1 were upregulated by both xanthones, whilst the expression of 14‐3‐3 protein sigma and stathmin 1 (STMN1) were downregulated. Furthermore, overexpression of STMN1 in HCC cells decreased their sensitivity, whilst small interfering RNAs targeting STMN1 enhanced their sensitivity to GA and GEA. In conclusion, our study suggested for the first time that STMN1 might be a major target for GA and GEA in combating HCC. Further investigation may lead to a new generation of anticancer drugs exerting synergistic effect with conventional therapy, thus to promote treatment efficacy.     

Isolation of differentially expressed genes in human heart tissues

We applied RNA arbitrarily primed-PCR (RAP-PCR) to screen the genes differentially expressed between common congenital heart defects (CHD) [atrial septal defect, ventricular septal defect, Tetrology of Fallot (TOF)] and normal human heart samples. Three of these differentially amplified fragments matched cDNA sequences coding for proteins of unknown function in humans: hCALO (human homologue of calossin), NP79 (coding for a nuclear protein of 79KD) and SUN2 (Sad-1 unc-84 domain protein 2). The other four fragments were from known human genes: apolipoprotein J, titin, dystrophin and protein kinase C-delta. Northern blot analysis confirmed that all of these genes are expressed in the human heart. The results of RAP-PCR were reconfirmed by quantitative RT-PCR in TOF and control heart samples. Both techniques showed the levels of expression of hCALO, NP79 and SUN2 to be comparable in TOF and control samples and the level of expression of dystrophin and titin, both coding for cytoskeletal proteins, to be significantly upregulated in TOF samples. In summary, we have shown that the RAP-PCR technique is useful in the identification of differentially expressed gene from biopsy samples of human CHD tissues. In this manner, we have identified three novel genes implicated in the normal function of the human heart and two known genes upregulated in TOF samples.     

The Neural Stem/Progenitor Cell Marker Nestin Is Expressed in Proliferative Endothelial Cells,but Not in Mature Vasculature

Nestin is an intermediate filament protein that is known as a neural stem/progenitor cell marker. It is expressed in undifferentiated central nervous system (CNS) cells during development, but also in normal adult CNS and in CNS tumor cells. Additionally, nestin is expressed in endothelial cells (ECs) of CNS tumor tissues and of adult tissues that replenish by angiogenesis. However, the regulation of nestin expression in vascular endothelium has not been analyzed in detail. This study showed that nestin expression was observed in proliferating endothelial progenitor cells (EPCs), but not in mature ECs. In adherent cultured cells derived from bone marrow cells, EPCs that highly expressed nestin also expressed the endothelial marker CD31 and the proliferation marker Ki67. ECs cultured without growth factors showed attenuated nestin immunoreactivity as they matured. Transgenic mice that carried the enhanced green fluorescent protein under the control of the CNS-specific second intronic enhancer of the nestin gene showed no reporter gene expression in EPCs. This indicated that the mechanisms of nestin gene expression were different in EPCs and CNS cells. Immunohistochemistry showed nestin expression in neovascular cells from two distinct murine models. Our results demonstrate that nestin can be used as a marker protein for neovascularization. (J Histochem Cytochem 58:721–730, 2010)     

Microenvironment of a tumor-organoid system enhances hepatocellular carcinoma malignancy-related hallmarks

Organ-like microenviroment and 3-dimensional (3D) cell culture conformations have been suggested as promising approaches to mimic in a micro-scale a whole organ cellular functions and interactions present in vivo. We have used this approach to examine biologic features of hepatocellular carcinoma (HCC) cells. In this study, we demonstrate that hepatocellular carcinoma (HCC) cells, fibroblasts, endothelial cells and extracellular matrix can generate organoid-like spheroids that enhanced numerous features of human HCC observed in vivo. We show that the addition of non-parenchymal cells such as fibroblast and endothelial cells is required for spheroid formation as well as the maintenance of the tissue-like structure. Furthermore, HCC cells cultured as spheroids with non-parenchymal cells express more neo-angiogenesis-related markers (VEGFR2, VEGF, HIF-α), tumor-related inflammatory factors (CXCR4, CXCL12, TNF-α) and molecules-related to induced epithelial-mesenchymal transition (TGFβ, Vimentin, MMP9) compared with organoids containing only HCC cells.

These results demonstrate the importance of non-parenchymal cells in the cellular composition of HCC organoids. The novelty of the multicellular-based organotypic culture system strongly supports the integration of this approach in a high throughput approach to identified patient-specific HCC malignancy and accurate anti-tumor therapy screening after surgery.     

Madin–Darby canine kidney cells are increased in aerobic glycolysis when cultured on flat and stiff collagen‐coated surfaces rather than in physiological 3‐D cultures

We investigate the influence of the dimensionality and the biochemistry of the culture system on the cellular functionality by analyzing the protein expression levels in Madin–Darby canine kidney (MDCK) cells grown in 3‐D and 2‐D substrates. We cultured MDCK cells on a hard and flat 2‐D uncoated plastic surface, on a 2‐D collagen‐coated plastic surface and in 3‐D collagen gel and employed 2‐D gel electrophoresis, MALDI‐TOF‐MS, and LC‐MS/MS analysis to identify the differentially regulated proteins. We found significant differences in the expression of antioxidant proteins, actin‐binding proteins, glycolytic enzymes, and heat‐shock proteins/chaperons among the three types of cultures. While MDCK cells cultured in 3‐D collagen up‐regulate antioxidant proteins and proteins involved in the dynamic remodeling of the actin cytoskeleton, 2‐D collagen‐coated plastic surfaces induce the up‐regulation of glycolytic enzymes. Our data shows that the culture conditions have profound effects on the physiology of the cell. Culture in 3‐D collagen induces a differentiated polarized phenotype. In contrast, collagen‐coated 2‐D substrates favor a tumor‐like phenotype with increased glycolysis. Thus, the suitability of 2‐D cultures to study the physiological behavior of cells, especially in drug discovery, bioprocessing, and toxicology, should be carefully reconsidered.     

Galectin‐3, a marker for vacuole lysis by invasive pathogens

Shigella bacteria invade macrophages and epithelial cells and following internalization lyse the phagosome and escape to the cytoplasm. Galectin‐3, an abundant protein in macrophages and epithelial cells, belongs to a family of beta‐galactoside‐binding proteins, the galectins, with many proposed functions in immune response, development, differentiation, cancer and infection. Galectins are synthesized as cytosolic proteins and following non‐classical secretion bind extracellular beta‐galactosides. Here we analysed the localization of galectin‐3 following entry of Shigella into the cytosol and detected a striking phenomenon. Very shortly after bacterial invasion, intracellular galectin‐3 accumulated in structures in vicinity to internalized bacteria. By using immuno‐electron microscopy analysis we identified galectin‐3 in membranes localized in the phagosome and in tubules and vesicles that derive from the endocytic pathway. We also demonstrated that the binding of galectin‐3 to host N‐acetyllactosamine‐containing glycans, was required for forming the structures. Accumulation of the structures was a type three secretion system‐dependent process. More specifically, existence of structures was strictly dependent upon lysis of the phagocytic vacuole and could be shown also by Gram‐positive Listeria and Salmonella sifA mutant. We suggest that galectin‐3‐containing structures may serve as a potential novel tool to spot vacuole lysis.     

Grb2 dominantly associates with dynamin II in human hepatocellular carcinoma HepG2 cells

The two SH3 domains and one SH2 domain containing adaptor protein Grb2 is an essential element of the Ras signaling pathway in multiple systems. The SH2 domain of Grb2 recognizes and interacts with phosphotyrosine residues on activated tyrosine kinases, whereas the SH3 domains bind to several proline‐rich domain‐containing proteins such as Sos1. To define the difference in Grb2‐associated proteins in hepatocarcinoma cells, we performed coprecipitation analysis using recombinant GST‐Grb2 fusion proteins and found that several protein components (p170, p125, p100, and p80) differently associated with GST‐Grb2 proteins in human Chang liver and hepatocarcinoma HepG2 cells. Sos1 and p80 proteins dominantly bind to Grb2 fusion proteins in Chang liver, whereas p100 remarkably associate with Grb2 in HepG2 cells. Also GST‐Grb2 SH2 proteins exclusively bound to the p46^Shc, p52^Shc, and p66^Shc are important adaptors of the Ras pathway in HepG2 cells. The p100 protein has been identified as dynamin II. We observed that the N‐SH3 and C‐SH3 domains of Grb2 fusion proteins coprecipitated with dynamin II besides Sos1. These results suggest that dynamin II may be a functional molecule involved in Grb2‐mediated signaling pathway on Ras activation for tumor progression and differentiation of hepatocarcinoma cells. J. Cell. Biochem. 84: 150–155, 2002. © 2001 Wiley‐Liss, Inc.     

Surface proteomic analysis of differentiated versus stem‐like osteosarcoma human cells

Cancer stem cell characterization represents a breakthrough in cancer research. Despite evidence showing the existence and the role of cancer stem cells in osteosarcoma (OS) onset and progression, little is known about their specific surface phenotype. To address this issue, we carried out a cytometric analysis with an antibody‐array comprising 245 membrane proteins comparing the stem and differentiated OS cells. As experimental model, we chose the stem‐like cell line 3aminobenzamide‐OS and its parental, differentiated, cell line MG63. We identified 50 differentially expressed, 23 homogeneously expressed, and 172 not expressed proteins in the two cell line models, thus defining a surface protein signature specific for each of them. Furthermore, we selected ERK1/2 (p44/42 mitogen‐activated protein kinases) as a potential pathway correlated with processes that characterize tumorigenic potential and stemness of 3aminobenzamide‐OS cells.     

Identification of complement C3a as a candidate biomarker in human chronic hepatitis C and HCV-related hepatocellular carcinoma using a proteomics approach

Although the significant risk factors for hepatocellular carcinoma (HCC) are well known from epidemiological studies, diagnosis of this disease at an early stage is difficult, and HCC remains one of the leading causes of cancer death worldwide. Thus, to identify any useful HCC-related biomarkers is still a need. We performed SELDI-TOF MS to identify differentially expressed proteins in HCC serum using weak cation exchange protein chips. Protein characterization was performed by 2-DE separation and nano flow LC-MS/MS. A total of 55 sera were collected from HCC patients and compared with those from 48 patients with chronic hepatitis and 9 healthy individuals. A candidate marker of about 8900 Da was detected as differentially expressed in patients with chronic hepatitis C and hepatitis C virus (HCV)-related HCC. We identified this differentially expressed protein as complement C3a. The expression of C3a in HCC sera was further validated by PS20 chip immunoassay and Western blotting. Complement C3a was found to be elevated in patients with chronic hepatitis C and HCV-related HCC. The combination of SELDI-TOF MS and 2-DE provides a solution to identify disease-associated serum biomarkers.     

Proteomic analysis of sera from hepatocellular carcinoma patients after radiofrequency ablation treatment

Comparative proteomic analysis was used to search for characteristic alterations in the sera of hepatocellular carcinoma (HCC) patients who had undergone curative radiofrequency ablation treatment. Serum samples collected from eight patients before and after treatment were subjected to 2-DE. Eighty-eight protein spots differentially expressed with the treatment were selected by clustering analysis, and the proteins were identified by MS based on MALDI-TOF/TOF analysis and public database searches. The statistical analysis suggested that four proteins decreased after treatment (pro-apolipoprotein, alpha2-HS glycoprotein, apolipoprotein A-IV precursor, and PRO1708/PRO2044, which is the carboxy terminal fragment of albumin) and that seven proteins were increased after treatment, including leucine-rich alpha2-glycoprotein and alpha1-antitrypsin. These data facilitate the identification of differentially expressed proteins that are involved in HCC carcinogenesis and provide candidate biomarkers for the development of diagnostic and therapeutic tools.