Macrophage migration inhibitory factor promotes tumor aggressiveness of esophageal squamous cell carcinoma via activation of Akt and inactivation of GSK3ß.

The pleiotropic pro-inflammatory cytokine, macrophage migration inhibitory factor (MIF), represents an important link between chronic inflammation and tumorigenesis. Although accumulating evidence demonstrates that MIF overexpression is implicated in the development and progression of multiple cancers, including esophageal squamous cell carcinoma (ESCC), the molecular mechanisms underlying its tumor-promoting roles in ESCC remain unclear. In the present study, we observed that MIF is overexpressed in ESCC and correlated significantly with lymph node metastasis, advanced clinical stage, and poor survival of ESCC. MIF knockdown attenuated the proliferation, migration, and invasion of ESCC cells in vitro and in vivo. Moreover, blockage of MIF expression decreased the activation of the Akt, MEK/ERK, and NF-κB pathways and enhanced sensitivity to apoptosis. Meanwhile, repression of MIF expression resulted in activation of glycogen synthase kinase 3 beta (GSK3ß) and subsequent decrease of active ß-catenin, as well as its downstream targets including cyclin D1, matrix metalloproteinase (MMP)-7, c-myc, and c-Jun. Collectively, our results provided mechanistic insights into the tumor-promoting role of MIF in ESCC, and suggested that MIF represents a potential therapeutic target for treatment of ESCC.

Integrative topological analysis of mass spectrometry data reveals molecular features with clinical relevance in esophageal squamous cell carcinoma.

Combining MS-based proteomic data with network and topological features of such network would identify more clinically relevant molecules and meaningfully expand the repertoire of proteins derived from MS analysis. The integrative topological indexes representing 95.96% information of seven individual topological measures of node proteins were calculated within a protein-protein interaction (PPI) network, built using 244 differentially expressed proteins (DEPs) identified by iTRAQ 2D-LC-MS/MS. Compared with DEPs, differentially expressed genes (DEGs) and comprehensive features (CFs), structurally dominant nodes (SDNs) based on integrative topological index distribution produced comparable classification performance in three different clinical settings using five independent gene expression data sets. The signature molecules of SDN-based classifier for distinction of early from late clinical TNM stages were enriched in biological traits of protein synthesis, intracellular localization and ribosome biogenesis, which suggests that ribosome biogenesis represents a promising therapeutic target for treating ESCC. In addition, ITGB1 expression selected exclusively by integrative topological measures correlated with clinical stages and prognosis, which was further validated with two independent cohorts of ESCC samples. Thus the integrative topological analysis of PPI networks proposed in this study provides an alternative approach to identify potential biomarkers and therapeutic targets from MS/MS data with functional insights in ESCC.

MreBCD-associated Cytoskeleton is Required for Proper Segregation of the Chromosomal Terminus during the Division Cycle of Escherichia Coli.

BACKGROUND: In prokaryotic organisms, the mechanism responsible for the accurate partition of newly replicated chromosomes into daughter cells is incompletely understood. Segregation of the replication terminus of the circular prokaryotic chromosome poses special problems that have not previously been addressed. The aim of this study was to investigate the roles of several protein components (MreB, MreC, and MreD) of the prokaryotic cytoskeleton for the faithful transmission of the chromosomal terminus into daughter cells. METHODS: Strain LQ1 (mreB::cat), LQ2 (mreC::cat), and LQ3 (mreD::cat) were constructed using the Red recombination system. LQ11/pLAU53, LQ12/pLAU53, LQ13/pLAU53, LQ14/pLAU53, and LQ15/pLAU53 strains were generated by P1transduction of (tetO) 240 -Gm and (lacO) 240 -Km cassettes from strains IL2 and IL29. Fluorescence microscopy was performed to observe localization pattern of fluorescently-labeled origin and terminus foci in wild-type and mutant cells. SOS induction was monitored as gfp fluorescence from PsulA-gfp in log phase cells grown in Luria-Bertani medium at 37°C by measurement of emission at 525 nm with excitation at 470 nm in a microplate fluorescence reader. RESULTS: Mutational deletion of the mreB, mreC, or mreD genes was associated with selective loss of the terminus region in approximately 40% of the cells within growing cultures. This was accompanied by significant induction of the SOS DNA damage response, suggesting that deletion of terminus sequences may have occurred by chromosomal cleavage, presumably caused by ingrowth of the division septum prior to segregation of the replicated terminal. CONCLUSIONS: These results imply a role for the MreBCD cytoskeleton in the resolution of the final products of terminus replication and/or in the specific movement of newly replicated termini away from midcell prior to completion of septal ingrowth. This would identify a previously unrecognized stage in the overall process of chromosome segregation.

[Regulatory effect of two component signal transduction system saeRS on the survival of Staphylococcus epidermidis].

OBJECTIVE: To investigate the survival ability of Staphylococcus epidermidis (S. epidermidis) wildtype (WT), saeRS mutant (SAE) and saeRS complementary (SAEC) strains under different concentrations of glucose. METHODS: We measured the survival ability, biofilm forming ability, medium acidity and antimicrobial susceptibility of S. epidermidis in medium containing different concentrations of glucose. RESULTS: Compared with WT, the survival ability, biofilm forming ability and resistance to antibiotics (such as penicillin, oxacillin, gentamicin, ciprofloxacin and amikacin) of saeRS mutant increased significantly in response to glucose. SAE and SAEC showed the strongest survival ability and biofilm forming ability when grown in medium containing 14 mmol/L glucose and 28 mmol/L, respectively. WT showed no significant different survival and biofilm forming abilities when cultured with various concentrations of glucose. The medium acidity of saeRS mutant (pH=8.07) was lower than the WT (pH=7.0) in the presence of 14 mmol/L glucose. CONCLUSION: SaeRS may influence the survival ability of S. epidermidis by affecting glucose utilization.

Downregulation of 14-3-3σ correlates with multistage carcinogenesis and poor prognosis of esophageal squamous cell carcinoma.

AIMS: The asymptomatic nature of early-stage esophageal squamous cell carcinoma (ESCC) results in late presentation and consequent dismal prognosis This study characterized 14-3-3σ protein expression in the multi-stage development of ESCC and determined its correlation with clinical features and prognosis. MATERIALS AND METHODS: Western blot was used to examine 14-3-3σ protein expression in normal esophageal epithelium (NEE), low grade intraepithelial neoplasia (LGIN), high grade intraepithelial neoplasia (HGIN), ESCC of TNM I to IV stage and various esophageal epithelial cell lines with different biological behavior. Immunohistochemistry was used to estimate 14-3-3σ protein in 110 biopsy samples of NEE, LGIN or HGIN and in 168 ESCC samples all of whom had follow-up data. Support vector machine (SVM) was used to develop a classifier for prognosis. RESULTS: 14-3-3σ decreased progressively from NEE to LGIN, to HGIN, and to ESCC. Chemoresistant sub-lines of EC9706/PTX and EC9706/CDDP showed high expression of 14-3-3σ protein compared with non-chemoresistant ESCC cell lines and immortalized NEC. Furthermore, the downregulation of 14-3-3σ correlated significantly with histological grade (P = 0.000) and worse prognosis (P = 0.004). Multivariate Cox regression analysis indicated that 14-3-3σ protein (P = 0.016) and T stage (P = 0.000) were independent prognostic factors for ESCC. The SVM ESCC classifier comprising sex, age, T stage, histological grade, lymph node metastasis, clinical stage and 14-3-3σ, distinguished significantly lower- and higher-risk ESCC patients (91.67% vs. 3.62%, P = 0.000). CONCLUSIONS: Downregulation of 14-3-3σ arises early in the development of ESCC and predicts poor survival, suggesting that 14-3-3σ may be a biomarker for early detection of high-risk subjects and diagnosis of ESCC. Our seven-feature SVM classifier for ESCC prognosis may help to inform clinical decisions and tailor individual therapy.

Proteomic profiling of N-linked glycoproteins identifies ConA-binding procathepsin D as a novel serum biomarker for hepatocellular carcinoma.

The aim of this study was to identify novel biomarkers for the diagnosis of, and potential therapeutic targets for, hepatocellular carcinoma (HCC). Multilectin affinity chromatography was used to enrich N-linked glycoproteins from nontumorous liver and HCC tissues followed by 2DE and protein identification by MS. Twenty-eight differentially expressed proteins were identified. Western blotting validated consistently lower concentrations of human liver carboxylesterase 1 and haptoglobin, and higher concentration of procathepsin D (pCD) in HCC tissues. Knockdown of cathepsin D (CD) expression mediated by siRNA significantly inhibited the in vitro invasion of two HCC cell lines, SNU449 and SNU473, which normally secrete high-levels of CD. Prefractionation using individual lectins demonstrated an elevation in ConA-binding glycoforms of proCD and CD in HCC tissues. In the serum of HCC patients, "ConA-binding proCD" (ConA-pCD) is significantly increased in concentration and this increase is comprised of several distinct upregulated acidic isoforms (pI 4.5-5.5). Receiver operating characteristic analysis showed that the sensitivity and specificity of serum ConA-pCD for HCC diagnosis were 85% and 80%, respectively. This is the first report that serum ConA-pCD is increased significantly in HCC and is potentially useful as a serological biomarker for diagnosis of HCC.

[Differential proteins in esophageal squamous cell line EC9706/CDDP identified by SILAC quantitative proteomic approach].

Multidrug resistance (MDR) is one of the main causes leading to the failure in cancer treatment. Differential proteins between esophageal squamous cell carcinoma (ESCC) cell line EC9706 and its cisdiamminedichloroplatinum (CDDP)-resistant subline EC9706/CDDP revealed by quantitative analysis may provide deeper insights into the molecular mechanisms of MDR implicated in ESCC. EC9706/CDDP was generated by exposure of its parental sensitive EC9706 to a step-wise increase of CDDP concentration during EC9706 cultivation. The stable isotope labeling with amino acids in cell culture (SILAC) was used to label EC9706 and EC9706/CDDP with heavy and light medium, separately. Mixed peptides derived from EC9706 and EC9706/CDDP were analyzed by high performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS/MS) and subsequently subjected to bioinformatics analysis to identify differential proteins between EC9706 and EC9706/CDDP. Compared to parental EC9706, EC9706/CDDP manifested phenotypes of slow proliferation, cell pleomorphology, atypia and increased resistant-index 3.23. Seventy-four differential proteins identified in the present study belongs to various families with multiple functions, such as cytoskeleton (20%), energy metabolism (11%), transcription regulation and DNA repair (11%), redox homeostasis (9.5%), protein biosynthesis and mRNA processing (12%), ribosome constituent (8.1%), molecular chaperone (8.1%), immunity/inflammation (5.4%), intracellular transport (5.4%) and nucleosome assembly (2.7%), which indicated that development of MDR is a complicated process involving dysregulation of multiple molecules and pathways. The data is of great value for in-depth elucidation of molecular mechanisms of the MDR implicated in ESCC and may represent potential molecular targets for future therapeutic development.

The genetic profiling of preferentially expressed genes in murine splenic CD8α+ dendritic cells.

In the murine splenocytes, CD8α+ dendritic cells (abbreviated as 8+DC) and CD8α- dendritic cells (abbreviated as 8-DC) are identified with some vague features for each of them. 8+DCs but not 8-DCs cross-prime cytotoxic T cells in vivo. We aim to distinguish the two subtypes of DC based on gene expression profiling. Suppressive subtractive hybridization was undertaken to get differentially expressed genes from such subtracted cDNA library specific to 8+DC. A total of 114 sequences from the subtracted cDNA library specific to 8+DC library were analyzed. Most of them are known proteins, but some of them were novel, either totally novel genes or homologs to known genes, but with novel exon. About 55 probably novel exons were discovered, and 11 exons had longer length than those in gene bank. The clones 12, 44, 79, and 110 have no match with known sequences in gene bank. Then, semi-quantitative PCR was done to compare the expression of the enriched sequences between 8+DC and 8-DC. About 14 genes are differentially expressed in 8+DC. Therefore, SSH is an effective method to clone differentially expressed genes for 8+DC compared to 8-DC.

A novel agonistic anti-human death receptor 5 monoclonal antibody with tumoricidal activity induces caspase- and mitochondrial-dependent apoptosis in human leukemia Jurkat cells.

An agonistic antibody against TNF-related apoptosis-inducing ligand death receptor 5 (DR5) is a practicable candidate drug for antitumor therapy. In this study, a novel murine anti-human DR5 monoclonal antibody, mDRA-6(IgG1-κ), has been generated. This study aimed to explore the caspase-dependent and mitochondrial mechanisms of mDRA-6 in inducing apoptosis in human leukemia Jurkat cells. The apoptotic effects of mDRA-6 on Jurkat cells, which express DR5 on the cell surface, were detected by flow cytometry and western blot after exposure to different doses of mDRA-6 and at fixed doses of mDRA-6 at different times. It was demonstrated that mDRA-6 can induce Jurkat cell apoptosis via caspase- and mitochondrial-dependent pathways. These results indicate that the novel antibody mDRA-6 against DR5 has an antitumor function and may provide a new reagent for tumor therapy.

[Hyperactivation of c-Jun NH(2)-terminal protein kinase contributes to the proliferation of B lymphoma cells].

This study was purposed to explore the effect of hyperactivation of c-Jun NH(2)-terminal protein kinase (JNK) on the proliferation of B lymphoma cells. The human B lymphoma cell lines Daudi and Raji were chosen as research objects. The expression of JNK protein was determined by Western blot. The subcellular localization of JNK protein was detected by immunofluorescence. The cell cycle was analyzed by flow cytometry. The suppressive effect of JNK inhibitor SP600125 on the proliferation of Daudi and Raji cells was assayed by ATPLite method. The results demonstrated that hyperactivation of JNK has been found in Daudi and Raji cells. Immunofluorescence confirmed the aberrant subcellular localization of JNK protein in Daudi and Raji cells. Cell cycle assay revealed that Daudi and Raji cells underwent G(2)-M arrest in the presence of SP600125. Furthermore, Daudi and Raji cells showed significant increase in sub-G(1) population, an indicator of apoptotic cells, with the treatment of JNK inhibitors. These data suggested that JNK inhibitors suppressed the growth of B lymphoma cells via cell cycle arrest and apoptosis. Daudi and Raji cells treated with different concentrations of JNK selective inhibitor SP600125 showed dose-dependent reduction in the growth of Daudi and Raji cells. It is concluded that hyperactivation of JNK enhance the proliferation of Daudi and Raji cells. The aberrant subcellular localization of JNK protein may facilitate the nuclear accumulation of basal JNK activity, which made JNK to be a potential target to treat human B lymphoma.

Relevance of serum estradiol and estrogen receptor beta expression from a high-incidence area for esophageal squamous cell carcinoma in China.

The striking 3-4:1 male predominance of esophageal squamous cell carcinoma (ESCC) has not yet been well explained. Our hypothesis is that the changes in level of estrogen and/or subtype of estrogen receptor (ER) may exert a protective factor in esophageal carcinogenesis and prognosis of ESCC. Radioimmunoassay (RIA) was used to determine the serum level of estradiol in healthy cohort from high-incidence area (HIA) and low-incidence area (LIA) for esophageal cancer as well as patients with ESCC from HIA in Henan, northern China. The ERß expression profiling during the multi-stage progression of ESCC pathogenesis was evaluated by immunohistochemistry (IHC). Both males and females from HIA had significant decreases of serum estradiol in high-risk subjects predisposing for ESCC compared to healthy counterparts from LIA (P < 0.01). Furthermore, patients with ESCC from HIA developed the lowest level of estradiol (P < 0.01). ERß expressed in precursor lesions of ESCC and changed quantitatively and qualitatively with disease progression during the multi-stages process of esophageal carcinogenesis. High frequency of ERß expression was correlated with less aggressive potential of clinical behavior (P = 0.012, 0.015 for lymph node metastasis and tumor stage, respectively). This study indicates that lower serum level of estradiol may represent higher predisposition for development of ESCC, and ERß expression and/or nuclear location may predict better outcome for patients with ESCC. The present results provide clues to explain the striking gender difference for ESCC, which warrants further investigations on potential applications of estrogen or analogs in prevention of ESCC.

Caspase-dependent molecular mechanisms of anti-human DR5 monoclonal antibody mDRA-6 inducing apoptosis of human leukemia Jurkat cells.

BACKGROUND AND OBJECTIVE: Both tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and some monoclonal agonistic antibodies against TRAIL receptors have antitumor activity. We have previously prepared a novel monoclonal agonistic antibody against human death receptor 5 (DR5) and designated it as mDRA-6. This study was to explore the Caspase-dependent molecular mechanisms of mDRA-6 inducing apoptosis of human leukemia Jurkat cells. METHODS: After exposure to different doses of mDRA-6, DNA fragmentation of Jurkat cells was detected by agarose gel electrophoresis, cell proliferation was detected by MTT assay, and cell apoptosis was detected by flow cytometry after Annexin V-FITC/PI double staining. Jurkat cells were further treated with the inhibitors for Caspase-10, -9, -8 and -3. The active cleavage products of Caspase-10, -9, -8, -3 and poly ADP-ribose polymerase (PARP), BH3 interacting domain death agonist (Bid), truncated Bid (tBid) and cytochrome c (Cyto c), were analyzed by western blot. RESULTS: After mDRA-6 treatment, DNA fragmentation was detected in Jurkat cells. mDRA-6 inhibited cell proliferation in a dose-dependent manner. When treated with 2.0 microg/mL mDRA-6, the apoptosis rates of Jurkat cells were 16.2% at 0.25 h, 28.3% at 0.5 h, 69.2% at 1 h and 78.2% at 2 h. Interestingly, the mDRA-6-induced apoptosis was repressed by 77.9% by Caspase-8 inhibitor ZIF, 54.2% by Caspase-3 inhibitor ZDF, and 8.7% by Caspase-9 inhibitor ZLF, but was not repressed by Caspase-10 inhibitor ZAF. After mDRA-6 exposure, the proenzymes of Caspase-8, -9 and -3 were reduced and their active cleavage products were increased along with the increase of exposure time, the cleavage products of PARP were also increased, Bid was degraded to tBid, and an abundance of Cyto c was released from mitochondria, but the proenzyme of Caspase-10 showed no change and no cleavage products of Caspase-10 were detectable. CONCLUSION: mDRA-6 can induce apoptosis of Jurkat cells via the Caspase-dependent and mitochondrial pathways.

Detachment of esophageal carcinoma cells from extracellular matrix causes relocalization of death receptor 5 and apoptosis.

AIM: To investigate the effect of detachment of esophageal cancer cells from extracellular matrix on the localization of death receptor 5 (DR5) and apoptosis. METHODS: Anchorage-dependent EC9706 cells of esophageal squamous cell carcinoma were pretreated or not treated with brefeldin A. Detached cells were harvested by ethylenediaminetetraacetic acid digestion. Expression and localization of DR5 in these cells were determined by immunocytochemical and immunofluorescence assays, as well as flow cytometry analysis. Apoptosis of EC9706 cells was detected by flow cytometry after stained with fluorescein isothiocyanate-labeled annexin V/propidium iodide. Activation of caspase 8 was detected by Western blot analysis. RESULTS: Immunocytochemical assay indicated that DR5 was predominantly perinuclear in adherent cells but was mainly localized in cell membrane in detached cells. In addition, immunofluorescence assay also confirmed the above-mentioned results, and further demonstrated that DR5 was present in the form of coarse granules in detached cells, but in the form of fine granules in adherent cells. Cytometry analysis revealed higher levels of DR5 expression on the surfaces of brefeldin-A-untreated cells than on the surfaces of brefeldin-A-treated cells, but brefeldin A treatment did not affect the total DR5 expression levels. Moreover, nocodazole did not influence the extracelluar DR5 expression levels in EC9706 cells. Apoptosis assay revealed that detached cells were more sensitive to DR5 antibody-induced apoptosis than adherent cells. Western blotting showed that caspase 8 was activated in temporarily detached cells 4 h earlier than in adherent cells. CONCLUSION: Progress from adhesion to detachment of EC9706 cells causes DR5 relocalization, and promotes cytoplasmic translocation of DR5 to cell surfaces via a Golgi-dependent pathway. Moreover, it might also result in DR5 aggregation to render apoptosis of detached cells.

Synthesis, cytotoxicity and apoptosis of naphthalimide polyamine conjugates as antitumor agents.

Several naphthalimide polyamine conjugates were synthesized and evaluated for in vitro cytotoxicity against human leukemia K562, murine melanoma B16, Chinese hamster ovary CHO cell lines. Both triamine moieties and the length of spacers were crucial in elevating the potency of 1,8-naphthalimide. The typical compounds 5a and 5d exhibited excellent cell selectivity to cancer cells through the human hepatoma BEL-7402 and human normal hepatocyte QSG-7701 screens. In addition, 5d could disturb the cell cycle in B16 cells. The research on caspase activity and cytochrome c indicated that 5d could induce B16 cell apoptosis via both the mitochondrial and membrane death receptor pathways, and the Bcl-2 family numbers were involved in the control of apoptosis.

catena-Poly[[[diaqua-thulium(III)]-µ-6-carboxy-nicotinato-µ-pyridine-2,5-di-carboxyl-ato] dihydrate].

The title compound, {[Tm(C(7)H(3)NO(4))(C(7)H(4)NO(4))(H(2)O)(2)]·2H(2)O}(n), is isotypic with the analogous Tb(III) compound [Li et al. (2009 ▶). Acta Cryst. E65, m410]. All interatomic distances and angles and the hydrogen-bond geometries are very similar for the two structures. The refined Flack parameter of 0.49 (2) suggests inversion twinning.

[Apoptosis induced by NNAMB, a novel polyamine conjugate, in human erythroleukemia K562 cells and its mechanism].

OBJECTIVE: To investigate the apoptosis-inducing effects of NNAMB, a novel polyamine conjugate, in erythroleukemia K562 cells and its molecular mechanism. METHODS: Cell viability was assessed by MTT assay and trypan blue dye exclusion method. The cell morphology was observed by fluorescence microscopy. The cell cycle distribution, apoptosis and mitochondrial membrane potential were measured by flow cytometry. The expression of caspase-3, -8, -9, cytochrome c in the K562 cells was detected by Western blot. RESULTS: NNAMB inhibited the proliferation of K562 cells. The cells treated with NNAMB showed a typical apoptotic morphology, Sub-G1 peak and loss of mitochondrial membrane potential. Western blot assay showed that NNAMB increased the expression of caspase-3, -9, cytochrome c but not caspase-8 in a dose-and time-dependent manner. CONCLUSION: NNAMB induces apoptosis via mitochondrial pathway in K562 cells.

[Immunoregulation effects in vitro of the xenoprotein in combination with recombinant human granulocyte-macrophage colony stimulating factor and bacillus Calmette-Guerin].

This study was aimed to investigate the effects of xenogeneic antigen neu-Fc in combination with the recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) and Bacillus Calmette-Guerin (BCG) on the regulation of Th1 and Th2 immune response in vitro. The rat neu L2-S2 domain was engineered as a chimeric protein with human IgG Fc. The eukaryotic expression vector was constructed. The recombinant protein was stably expressed in CHO cells and purified by rProtein A Sepharose Fast Flow column. The recombinant protein was identified by SDS-PAGE and Western blot. Peripheral blood mononuclear cells (PBMNCs) were obtained by means of standard Ficoll separation from the blood of healthy donors. Neu-Fc-induced PBMNC proliferation was tested by MTT. The production of IL-12 and IL-10 was measured by ELISA. The results showed that the level of IL-12 decreased and IL-10 increased after PBMNCs were incubated with MCF-7 cultural supernatant. 10 nmol/L neu-Fc strongly induced the cell proliferation. Compared with neu-Fc or GM-CSF or BCG treatment alone, neu-Fc in combination with GM-CSF and BCG significantly stimulated IL-12 production and inhibited IL-10 production (p < 0.01). It is concluded that the neu-Fc can stimulate the proliferation activity of PBMNCs. neu-Fc, GM-CSF and BCG costimulation efficiently induces Th1 immune response.

[Synergistic lethal effect of mDRA-6 and nimesulide on human hepatocellular cancer cell line SMMC-7721].

BACKGROUND & OBJECTIVE: Both mDRA-6, a monoclonal antibody of death receptor 5 (DR5) in human cells prepared by our key laboratory, and nimesulide, a specific cyclooxygenase-2 (COX-2) inhibitor, can induce apoptosis of some malignant tumor cells. This study was to investigate the lethal effects of mDRA-6 and nimesulide on human hepatocellular cancer cell line SMMC-7721, and explore the possible mechanism. METHODS: The expression of DR5 on SMMC-7721 cells was detected by flow cytometry (FCM). SMMC-7721 cells were treated with mDRA-6 and nimesulide alone or in combination. Cell morphology was observed under microscope with Hoechst33258 staining. Cytotoxicity was examined by MTT assay. Cell apoptosis was detected by FCM. RESULTS: The positive rate of DR5 on SMMC-7721 cells was 95.0%. The apoptosis of SMMC-7721 cells could be induced by both mDRA-6 and nimesulide: the apoptosis rates were 10.5% when treated with 25 ng/mL mDRA-6 for 12 h, 35.0% when treated with 1 600 ng/mL mDRA-6, 5.0% when treated with 200 micromol/L nimesulide, and 34.0% when treated with 800 micromol/L nimesulide. The combination of mDRA-6 and nimesulide exhibited synergistic effect on the apoptosis of SMMC-7721 cells (q=1.23): the apoptosis rates were 31.2% when treated with 200 micromol/L nimesulide and 25 ng/mL mDRA-6 for 12 h, and 91.1% when treated with 200 micromol/L nimesulide and 1 600 ng/mL mDRA-6 for 12 h. CONCLUSIONS: Both mDRA-6 and nimesulide can induce the apoptosis of SMMC-7721 cells. The combination of mDRA-6 and nimesulide exhibits synergistic lethal effect on SMMC-7721 cells.

Proteomic identification of malignant transformation-related proteins in esophageal squamous cell carcinoma.

Esophageal cancer (EC) persists to be a leading cancer-related death in northern China. Clinical outcome of EC is the most dismal among many types of digestive tumors because EC at early stage is asymptomatic. The current study used 2-DE-based proteomics to identify differentially expressed proteins between esophageal cancer cell lines and immortal cell line. Fifteen proteins were identified with differences of more than five folds, comprising the down-regulation of annexin A2, histone deacetylase 10 isoform beta and protein disulfide-isomerase ER-60 precursor, and the up-regulation of heat shock 70 kDa protein 9B precursor, solute carrier family 44 Member 3, heterogeneous nuclear ribonucleoprotein L (hnRNP L), eukaryotic translation initiation factor 4A isoform 2, triosephosphate isomerase1 (TPI), peroxiredoxin1 (PRX1), forminotransferase cyclodeaminase form (FTCD), fibrinogen gamma-A chain precursor, kinesin-like DNA binding protein, lamin A/C, cyclophilin A (CypA), and transcription factor MTSG1. Expression pattern of annexin A2 was verified by Western blotting, immunocytochemistry and immunohistochemistry analysis. The implication of these protein alterations correlated to the esophageal malignant transformation is discussed.

Autoantibody profiling of Chinese patients with autoimmune hepatitis using immunoproteomic analysis.

In the present study, immunoproteomic analysis was utilized to systemically characterize global autoantibody profiles in autoimmune hepatitis (AIH). Sera from 21 patients with AIH and 15 healthy controls were analyzed for the antibody reactivity against the protein antigens of HepG2, a human hepatoma cell line. The lysates of HepG2 cells were separated by two-dimensional electrophoresis and then immunoblotted with each serum sample. Matrix-assisted laser desorption/ionization mass spectrometry or/and nanoelectrospray ionization MS/MS were then used to identify antigens, among which a bifunctional enzyme in mitochondrial, fumarate hydratase (FH), was further analyzed by ELISA using recombinant FH as a coating antigen. A total of 18 immunoreactive spots were identified as 13 proteins, 8 of which have not been reported in AIH. Immune reactivity to FH was detected in 66.67% of patients with AIH, 19.35% of patients with primary biliary cirrhosis (PBC), 12.31% of patients with chronic hepatitis B (CHB), 6.35% of patients with chronic hepatitis C (CHC), 11.32% of patients with systemic lupus erythematosus (SLE), and 3.57% of normal individuals. The differences of prevalence between AIH patients and healthy controls as well as other diseases were of statistical significance (P<0.001). These data demonstrate the serological heterogeneity in AIH and suggest the diversity of the mechanisms underlying AIH. FH, recognized mainly in AIH rather than in viral hepatitis and other autoimmune diseases, may have utility in improved diagnosis of AIH.