Advanced 3D Through-Si-Via and Solder Bumping Technology: A Review.

Three-dimensional (3D) packaging using through-Si-via (TSV) is a key technique for achieving high-density integration, high-speed connectivity, and for downsizing of electronic devices. This paper describes recent developments in TSV fabrication and bonding methods in advanced 3D electronic packaging. In particular, the authors have overviewed the recent progress in the fabrication of TSV, various etching and functional layers, and conductive filling of TSVs, as well as bonding materials such as low-temperature nano-modified solders, transient liquid phase (TLP) bonding, Cu pillars, composite hybrids, and bump-free bonding, as well as the role of emerging high entropy alloy (HEA) solders in 3D microelectronic packaging. This paper serves as a guideline enumerating the current developments in 3D packaging that allow Si semiconductors to deliver improved performance and power efficiency.

Physiological and photochemical evaluation of pepper methionine sulfoxide reductase B2 (CaMsrB2) expressing transgenic rice in saline habitat.

Two pepper methionine sulfoxide reductase B2 (CaMsrB2) gene expressing transgenic rice lines (L-8 and L-23) were interrogated with respect to their physiological and photochemical attributes along with control (WT, Ilmi) as a standard against varying levels of salt concentration which are 75 mM, 150 mM and 225 mM. Against various levels of salt (NaCl) concentration, recurring detrimental effects of extreme salt stress was observed and more pronounced in the wild type plants as compared to our transgenic lines. As the exacerbated effects of salinity is responsible for pushing the plants to their ecological tolerance, our transgenic lines performed well uplifted in different realms of physiology and photochemistry such as relative water content (RWC = 60-75%), stomatal conductance (gs = 70-190 mmolm-2s-1), performance index (PIABS = 1.0-4.5), maximal photochemical yield of PSII (FV/FM = 0.48-0.72) and chlorophyll content index (CCI = 5-7.2 au) in comparison to the control. Relative gene expression, ion analysis and antioxidants activity were analyzed in all treatments to ensure the hypothesis obtained from data of physiology and photochemistry. Photosynthetic apparatus is known to lose energy in various forms such as NPQ, DIO/CS, damages of reaction center (FV/FO) which are the markers of poor health were clearly decreased in the L-23 line as compared to L-8 and WT. Present study revealed the protruding tolerance of L-23 and L-8 transgenic lines with L-23 line in the lead in comparison to control and L-8 transgenic lines.

Synthesis and properties of oligonucleotides bearing thymidine derivatives with 1,6-dioxaspiro[4.5]decane skeleton.

Thymidine derivatives bearing spiroacetal moieties on the C4'-position (5'R-spiro-thymidine and 5'S-spiro-thymidine) were synthesized and incorporated into oligonucleotides. The duplex- and triplex-forming abilities of both the oligonucleotides were evaluated from UV melting experiments. Oligonucleotides with the 5'S-spiro modifications could form thermally stable duplexes with complementary RNA and DNA; however, the 5'R-spiro modification significantly decreased the thermal stabilities of the duplexes and triplexes. Oligonucleotides with these spiro-thymidines showed significantly high resistance towards enzymatic degradation.

Transgenic Chickens Expressing the 3D8 Single Chain Variable Fragment Protein Suppress Avian Influenza Transmission.

The 3D8 single chain variable fragment (scFv) is a mini-antibody that causes unusual sequence-independent nuclease activity against all types of nucleic acids. We used recombinant lentiviruses to generate transgenic chickens expressing the 3D8 scFv gene under the control of the chicken ß-actin promoter. From 420 injected embryos, 200 chicks (G0) hatched and were screened for the 3D8 scFv using PCR, and 15 chicks were identified as transgenic birds expressing the transgene in their semen. The G0 founder birds were mated with wild-type hens to produce seven transgenic chicks (G1). 3D8 scFv expression in the chicken embryonic fibroblasts (CEFs) was verified by RT-PCR and Western blot analysis. Immunofluorescence staining for 3D8 scFv in the CEFs revealed that the 3D8 scFv protein was primarily cytosolic. To identify 3D8 scFv anti-viral activity, wild-type and two transgenic CEF lines were infected with H9N2 avian influenza virus (AIV). We selected one line of transgenic chickens that exhibited the lowest number of plaque-forming units to be challenged with H9N2 virus. The challenge experiment revealed that contact exposed transgenic chickens expressing 3D8 scFv exhibited suppressed viral shedding. This results suggest that the transgenic chickens developed in this study could be useful for controlling potential within-flock AIV transmission.

STAT5 plays a critical role in regulating the 5'-flanking region of the porcine whey acidic protein gene in transgenic mice.

The mammary gland serves as a valuable bioreactor system for the production of recombinant proteins in lactating animals. Pharmaceutical-grade recombinant protein can be harvested from the milk of transgenic animals that carry a protein of interest under the control of promoter regions genes encoding milk proteins. Whey acidic protein (WAP), for example, is predominantly expressed in the mammary gland and is regulated by lactating hormones during pregnancy. We cloned the 5'-flanking region of the porcine WAP gene (pWAP) to confirm the sequence elements in its promoter that are required for gene-expression activity. In the present study, we investigated how lactogenic hormones--including prolactin, hydrocortisone, and insulin--contribute to the transcriptional activation of the pWAP promoter region in mammalian cells, finding that these hormones activate STAT5 signaling, which in turn induce gene expression via STAT5 binding sites in its 5'-flanking region. To confirm the expression and hormonal regulation of the 5'-flanking region of pWAP in vivo, we generated transgenic mice expressing human recombinant granulocyte colony stimulating factor (hCSF2) in the mammary gland under the control of the pWAP promoter. These mice secreted hCSF2 protein in their milk at levels ranging from 242 to 1,274.8 ng/ml. Collectively, our findings show that the pWAP promoter may be useful for confining the expression of foreign proteins to the mammary gland, where they can be secreted along with milk.

A lactate-induced response to hypoxia.

Organisms must be able to respond to low oxygen in a number of homeostatic and pathological contexts. Regulation of hypoxic responses via the hypoxia-inducible factor (HIF) is well established, but evidence indicates that other, HIF-independent mechanisms are also involved. Here, we report a hypoxic response that depends on the accumulation of lactate, a metabolite whose production increases in hypoxic conditions. We find that the NDRG3 protein is degraded in a PHD2/VHL-dependent manner in normoxia but is protected from destruction by binding to lactate that accumulates under hypoxia. The stabilized NDRG3 protein binds c-Raf to mediate hypoxia-induced activation of Raf-ERK pathway, promoting angiogenesis and cell growth. Inhibiting cellular lactate production abolishes the NDRG3-mediated hypoxia responses. Our study, therefore, elucidates the molecular basis for lactate-induced hypoxia signaling, which can be exploited for the development of therapies targeting hypoxia-induced diseases.

Pin1 promotes neuronal death in stroke by stabilizing Notch intracellular domain.

OBJECTIVE: Stroke is a leading cause of mortality and disability. The peptidyl-prolyl cis/trans isomerase Pin1 regulates factors involved in cell growth. Recent evidence has shown that Pin1 plays a major role in apoptosis. However, the role of Pin1 in ischemic stroke remains to be investigated. METHODS: We used Pin1 overexpression and knockdown to manipulate Pin1 expression and explore the effects of Pin1 in cell death on ischemic stress in vitro and in a mouse stroke model. We also used Pin 1 inhibitor, γ-secretase inhibitor, Notch1 intracellular domain (NICD1)-deleted mutant cells, and Pin1 mutant cells to investigate the underlying mechanisms of Pin1-NICD1-mediated cell death. RESULTS: Our findings indicate that Pin1 facilitates NICD1 stability and its proapoptotic function following ischemic stroke. Thus, overexpression of Pin1 increased NICD1 levels and enhanced its potentiation of neuronal death in simulated ischemia. By contrast, depletion or knockout of Pin1 reduced the NICD1 level, which in turn desensitized neurons to ischemic conditions. Pin1 interacted with NICD1 and increased its stability by inhibiting FBW7-induced polyubiquitination. We also demonstrate that Pin1 and NICD1 levels increase following stroke. Pin1 heterozygous (+/-) and knockout (-/-) mice, and also wild-type mice treated with an inhibitor of Pin1, each showed reduced brain damage and improved functional outcomes in a model of focal ischemic stroke. INTERPRETATION: These results suggest that Pin1 contributes to the pathogenesis of ischemic stroke by promoting Notch signaling, and that inhibition of Pin1 is a novel approach for treating ischemic stroke.

Characterization and miRNA-mediated posttranscriptional regulation of vitelline membrane outer layer protein I in the adult chicken oviduct.

The laying hen is the best model for oviduct growth and development. The chicken oviduct produces the egg components, including the egg white and eggshell. However, the mechanism of egg component production during oviduct development requires further investigation. Vitelline membrane outer layer protein 1 (VMO-1) is found in the outer layer of the vitelline membrane of avian eggs. Comparison of the chicken VMO-1 protein-coding sequence and the human, mouse, rat, and bovine VMO-1 proteins via multiple sequence alignment analysis revealed high degrees of homology of 55%, 53%, 48%, and 54%, respectively. Although the avian homologue of VMO-1 is highly expressed in the magnum of the oviduct, little is known about the transcriptional and posttranscriptional regulation of VMO-1 during oviduct development. The results of this study revealed that estrogen induces VMO-1 messenger RNA (mRNA) expression in oviduct cells in vitro. The expression of genes interacting with VMO-1 by RNA interference (RNAi) functional analysis revealed that ovomucin expression was decreased by VMO-1 silencing. In addition, gga-miR-1623, 1552-3p, and 1651-3p influenced VMO-1 expression via its 3'-UTR, suggesting the posttranscriptional regulation of VMO-1 expression in chickens. Collectively, these results suggest that VMO-1 is an estrogen-induced gene that is posttranscriptionally regulated by microRNAs (miRNAs). The present study may contribute to an understanding of egg component production during chicken oviduct development.

Synthesis and anti-influenza virus activity of 4-oxo- or thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-ones.

A target-free approach was applied to discover anti-influenza viral compounds, where influenza infected Madin-Darby canine kidney cells were treated 7500 different small organic chemicals individually and reduction of virus-induced cytopathic effect was measured. One of the hit compounds was (Z)-1-((5-fluoro-1H-indol-3-yl)methylene)-6-methyl-4-thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (15a) with half-maximal effective concentrations of 17.4-21.1µM against influenza A/H1N1, A/H3N2 and B viruses without any cellular toxicity at 900µM. To investigate the structure-activity relationships, two dozens of the hit analogs were synthesized. Among them, 15g, 15j, 15q, 15s, 15t and 15x had anti-influenza viral activity comparable or superior to that of the initial hit. The anti-influenza viral compounds efficiently suppressed not only viral protein level of the infected cells but also production of viral progeny in the culture supernatants in a dose-dependent manner. Based on a mode-of-action study, they did not affect virus entry or RNA replication. Instead, they suppressed viral neuraminidase activity. This study is the first to demonstrate that dihydrofuropyridinones could serve as lead compounds for the discovery of alternative influenza virus inhibitors.

Alleviation of salt stress by enterobacter sp. EJ01 in tomato and Arabidopsis is accompanied by up-regulation of conserved salinity responsive factors in plants.

Microbiota in the niches of the rhizosphere zones can affect plant growth and responses to environmental stress conditions via mutualistic interactions with host plants. Specifically, some beneficial bacteria, collectively referred to as Plant Growth Promoting Rhizobacteria (PGPRs), increase plant biomass and innate immunity potential. Here, we report that Enterobacter sp. EJ01, a bacterium isolated from sea china pink (Dianthus japonicus thunb) in reclaimed land of Gyehwa-do in Korea, improved the vegetative growth and alleviated salt stress in tomato and Arabidopsis. EJ01 was capable of producing 1-aminocy-clopropane-1-carboxylate (ACC) deaminase and also exhibited indole-3-acetic acid (IAA) production. The isolate EJ01 conferred increases in fresh weight, dry weight, and plant height of tomato and Arabidopsis under both normal and high salinity conditions. At the molecular level, short-term treatment with EJ01 increased the expression of salt stress responsive genes such as DREB2b, RD29A, RD29B, and RAB18 in Arabidopsis. The expression of proline biosynthetic genes (i.e. P5CS1 and P5CS2) and of genes related to priming processes (i.e. MPK3 and MPK6) were also up-regulated. In addition, reactive oxygen species scavenging activities were enhanced in tomatoes treated with EJ01 in stressed conditions. GFP-tagged EJ01 displayed colonization in the rhizosphere and endosphere in the roots of Arabidopsis. In conclusion, the newly isolated Enterobacter sp. EJ01 is a likely PGPR and alleviates salt stress in host plants through multiple mechanisms, including the rapid up-regulation of conserved plant salt stress responsive signaling pathways.

Donor-derived natural killer cells infused after human leukocyte antigen-haploidentical hematopoietic cell transplantation: a dose-escalation study.

The doses of donor-derived natural killer (NK) cells that can be given safely after human leukocyte antigen (HLA)-haploidentical hematopoietic cell transplantation (HCT) remain to be defined. Forty-one patients (ages 17 to 75 years) with hematologic malignancy underwent HLA-haploidentical HCT after reduced-intensity conditioning containing busulfan, fludarabine, and antithymocyte globulin. Cell donors (ages 7 to 62 years) underwent growth factor-mobilized leukapheresis for 3 to 4 days. Cells collected on the first 2 to 3 days were used for HCT, whereas those collected on the last day were CD3-depleted and cultured into NK cells using human interleukins-15 and -21. These NK cells were then infused into patients twice at 2 and 3 weeks after HCT at an escalating doses of .2 × 10(8) cells/kg of body weight (3 patients), .5 × 10(8) cells/kg (3 patients), 1.0 × 10(8) cells/kg (8 patients), and ≥ 1.0 × 10(8) cells/kg or available cells (27 patients). At all dose levels, no acute toxicity was observed after NK cell infusion. After HLA-haploidentical HCT and subsequent donor NK cell infusion, when referenced to 31 historical patients who had undergone HLA-haploidentical HCT after the same conditioning regimen but without high-dose NK cell infusion, there was no significant difference in the cumulative incidences of major HCT outcomes, including engraftment (absolute neutrophil count ≥ 500/µL, 85% versus 87%), grade 2 to 4 acute graft-versus-host disease (GVHD, 17% versus 16%), moderate to severe chronic GVHD (15% versus 10%), and transplantation-related mortality (27% versus 19%). There was, however, a significant reduction in leukemia progression (74% to 46%), with post-transplantation NK cell infusion being an independent predictor for less leukemia progression (hazard ratio, .527). Our findings showed that, when given 2 to 3 weeks after HLA-haploidentical HCT, donor-derived NK cells were well tolerated at a median total dose of 2.0 × 10(8) cells/kg. In addition, they may decrease post-transplantation progression of acute leukemia.

SH3RF2 functions as an oncogene by mediating PAK4 protein stability.

SH3RF (SH3-domain-containing RING finger protein) family members, SH3RF1-3, are multidomain scaffold proteins involved in promoting cell survival and apoptosis. In this report, we show that SH3RF2 is an oncogene product that is overexpressed in human cancers and regulates p21-activated kinase 4 (PAK4) protein stability. Immunohistochemical analysis of 159 colon cancer tissues showed that SH3RF2 expression levels are frequently elevated in cancer tissues and significantly correlate with poor prognostic indicators, including increased invasion, early recurrence and poor survival rates. We also demonstrated that PAK4 protein is degraded by the ubiquitin-proteasome system and that SH3RF2 inhibits PAK4 ubiquitination via physical interaction-mediated steric hindrance, which results in the upregulation of PAK4 protein. Moreover, ablation of SH3RF2 expression attenuates TRADD (TNFR-associated death domain) recruitment to tumor necrosis factor-α (TNF-α) receptor 1 and hinders downstream signals, thereby inhibiting NF-κB (nuclear factor-kappaB) activity and enhancing caspase-8 activity, in the context of TNF-α treatment. Notably, ectopic expression of SH3RF2 effectively prevents apoptosis in cancer cells and enhances cell migration, colony formation and tumor growth in vivo. Taken together, our results suggest that SH3RF2 is an oncogene that may be a definitive regulator of PAK4. Therefore, SH3RF2 may represent an effective therapeutic target for cancer treatment.

Human extracellular superoxide dismutase (EC-SOD) expression in transgenic chicken.

Extracellular superoxide dismutase (EC-SOD) is a metalloprotein and functions as an antioxidant enzyme. In this study, we used lentiviral vectors to generate transgenic chickens that express the human EC-SOD gene. The recombinant lentiviruses were injected into the subgerminal cavity of freshly laid eggs. Subsequently, the embryos were incubated to hatch using phases II and III of the surrogate shell ex vivo culture system. Of 158 injected embryos, 16 chicks (G0) hatched and were screened for the hEC-SOD by PCR. Only 1 chick was identified as a transgenic bird containing the transgene in its germline. This founder (G0) bird was mated with wild-type hens to produce transgenic progeny, and 2 transgenic chicks (G1) were produced. In the generated transgenic hens (G2), the hEC-SOD protein was expressed in the egg white and showed antioxidant activity. These results highlight the potential of the chicken for production of biologically active proteins in egg white.

Metabolic signature genes associated with susceptibility to pyruvate kinase, muscle type 2 gene ablation in cancer cells.

Pyruvate kinase, muscle type 2 (PKM2), is a key factor in the aerobic glycolysis of cancer cells. In our experiments, liver cancer cell lines exhibited a range of sensitivity to PKM2 knockdown-mediated growth inhibition. We speculated that this differential sensitivity is attributable to the variable dependency on glycolysis for the growth of different cell lines. Transcriptome data revealed overexpression of a glucose transporter (GLUT3) and a lactate transporter (MCT4) genes in PKM2 knockdown-sensitive cells. PKM2 knockdown-resistant cells expressed high levels of the lactate dehydrogenase B (LDHB) and glycine decarboxylase (GLDC) genes. Concordant with the gene expression results, PKM2 knockdown-sensitive cells generated high levels of lactate. In addition, ATP production was significantly reduced in the PKM2 knockdown-sensitive cells treated with a glucose analog, indicative of dependency of their cellular energetics on lactate-producing glycolysis. The PKM2 knockdown-resistant cells were further subdivided into less glycolytic and more (glycolysis branch pathway-dependent) glycolytic groups. Our findings collectively support the utility of PKM2 as a therapeutic target for high lactate-producing glycolytic hepatocellular carcinoma (HCC).

Dysregulation of overexpressed IL-32α in hepatocellular carcinoma suppresses cell growth and induces apoptosis through inactivation of NF-κB and Bcl-2.

IL-32 is a newly discovered cytokine. Recently, various reports suggest that it plays a role as a proinflammatory mediator and may be involved in several cancer carcinogenesis. However, IL-32 expression in hepatocellular carcinoma (HCC) remains unclear. In this study, we investigated the expression and role of IL-32α in hepatocellular carcinoma, because IL-32 was identified as an upregulated gene in hepatocellular carcinoma tissues compared to nontumorous regions using DNA microarray. IL-32α was overexpressed in tissue and serum from patients with HCC and localized in the cytoplasm and nucleus of hepatocellular carcinoma tumor cells. Moreover, secreted IL-32α concentration in the serum of patients with hepatocellular carcinoma was elevated as compared with those in the normal serum using a developed sandwich ELISA. Furthermore, IL-32α suppression in hepatocellular carcinoma decreased expression of phospho-p38 MAPK, NF-κB, and antiapoptotic protein Bcl-2 and induced expression of proapoptotic proteins as well as p53 and PUMA resulting in the suppression of cell growth and induction of intrinsic apoptosis. Based on our results, we suggest that IL-32α is involved in the progression of hepatocellular carcinoma and may be a useful biomarker for diagnosis and therapeutic target of hepatocellular carcinoma.

Liverome: a curated database of liver cancer-related gene signatures with self-contained context information.

BACKGROUND: Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. A number of molecular profiling studies have investigated the changes in gene and protein expression that are associated with various clinicopathological characteristics of HCC and generated a wealth of scattered information, usually in the form of gene signature tables. A database of the published HCC gene signatures would be useful to liver cancer researchers seeking to retrieve existing differential expression information on a candidate gene and to make comparisons between signatures for prioritization of common genes. A challenge in constructing such database is that a direct import of the signatures as appeared in articles would lead to a loss or ambiguity of their context information that is essential for a correct biological interpretation of a gene's expression change. This challenge arises because designation of compared sample groups is most often abbreviated, ad hoc, or even missing from published signature tables. Without manual curation, the context information becomes lost, leading to uninformative database contents. Although several databases of gene signatures are available, none of them contains informative form of signatures nor shows comprehensive coverage on liver cancer. Thus we constructed Liverome, a curated database of liver cancer-related gene signatures with self-contained context information. DESCRIPTION: Liverome's data coverage is more than three times larger than any other signature database, consisting of 143 signatures taken from 98 HCC studies, mostly microarray and proteome, and involving 6,927 genes. The signatures were post-processed into an informative and uniform representation and annotated with an itemized summary so that all context information is unambiguously self-contained within the database. The signatures were further informatively named and meaningfully organized according to ten functional categories for guided browsing. Its web interface enables a straightforward retrieval of known differential expression information on a query gene and a comparison of signatures to prioritize common genes. The utility of Liverome-collected data is shown by case studies in which useful biological insights on HCC are produced. CONCLUSION: Liverome database provides a comprehensive collection of well-curated HCC gene signatures and straightforward interfaces for gene search and signature comparison as well. Liverome is available at http://liverome.kobic.re.kr.

Identification of endothelial cell-specific molecule-1 as a potential serum marker for colorectal cancer.

No ideal serum markers for screening colorectal cancer (CRC) have been identified. The aim of this study was to determine the usefulness of endothelial cell-specific molecule-1 (ESM-1) as a serum marker for CRC. Illumina microarray was carried out to search CRC-related biomarkers. cDNA microarray detected that ESM-1 was one of the overexpressed genes in CRC. Overexpression of ESM-1 mRNA was confirmed in tissues of CRC by RT-PCR and real-time PCR. Immunohistochemical staining showed strong expression of ESM-1 in the cytoplasm of tumor cells. Overexpression of ESM-1 in human serum with CRC was found by Western blot analysis. For quantitative analysis of ESM-1 in serum, we determined the ESM-1 levels in serum specimens using an ELISA kit. We showed that the ESM-1 levels in the serum of patients with CRC were significantly elevated (70.1 ± 29.7 pg/mL) compared to healthy subjects (29.7 ± 14.9 pg/mL). The accuracy, sensitivity, and specificity of ESM-1 for CRC were 0.94, 99%, and 73%, respectively, by receiver operating characteristics curve analysis. The positive predictive value and negative predictive value were 63% and 95%, respectively. The likelihood ratios of a positive or negative test result were 73 and 0.27, respectively. When analyzed with a Cox regression model, a higher serum ESM-1 level (≥76.0 pg/mL) was correlated with poor prognosis. This study suggests that expression of ESM-1 is increased in tissue and serum of CRC patients and that ESM-1 can be used as a potential serum marker for the early detection of CRC.

Functional switching of TGF-beta1 signaling in liver cancer via epigenetic modulation of a single CpG site in TTP promoter.

BACKGROUND & AIMS: Acquisition of resistance to the antiproliferative effect of transforming growth factor (TGF)-beta1 is crucial for the malignant progression of cancers. In this study, we sought to determine whether deregulated expression of tristetrapolin (TTP), a negative posttranscriptional regulator of c-Myc, confers resistance to the antiproliferative effects of TGF-beta1 on liver cancer cells. METHODS: The epigenetics of TTP promoter regulation and its effects on TGF-beta1 signaling were examined in hepatocellular carcinoma (HCC) cell lines and patient tissues. RESULTS: TTP was down-regulated in HCC cell lines (10/11), compared with normal liver, as well as in tumor tissues (19/24) from paired HCC specimens. Methylation of a specific single CpG site located within the TGF-beta1-responsive region (TRR) of the TTP promoter was significantly associated with TTP down-regulation in both HCC cell lines and tumor tissues (r = -0.606383, P < .001). The singly methylated CpG site was specifically bound by a transcriptional repressor complex consisting of MECP2/c-Ski/DNMT3A and abolished the TGF-beta1-induced as well as basal-level expression of TTP. The epigenetic inactivation of TTP led to an increased half-life of c-Myc mRNA and blocked the cytostatic effect of TGF-beta1. Statistically significant correlations were observed between the single CpG site methylation and expression levels of TTP or c-Myc in clinical samples of HCC. CONCLUSIONS: Abrogation of the post-transcriptional regulation of c-Myc via methylation of a specific single CpG site in the TTP promoter presents a novel mechanism for the gain of selective resistance to the antiproliferative signaling of TGF-beta1 in HCC.

Evaluation of annexin II as a potential serum marker for hepatocellular carcinoma using a developed sandwich ELISA method.

Annexin II (Annexin A2, ANXA2) is a 36 kDa calcium-dependent phospholipid-binding protein that is located on the surface of most eukaryotic cells. ANXA2 is involved in several biological processes, including anti-inflammatory effects, Ca27+-dependent exocytosis, immune responses, Ca2+ transport and phospholipase A2 regulation. In our previous study, ANXA2 was identified as an up-regulated gene in hepatocellular carcinoma (HCC) tissue by cDNA microarray. In the present study, we have evaluated ANXA2 as a tumor-associated marker of HCC. We determined the ANXA2 levels in human liver tissues with HCC using real-time RT-PCR and Western blot analysis. For quantitative analysis of the ANXA2 protein in body fluids, we developed a sandwich ELISA system in which a polyclonal antibody and a monoclonal antibody specific to ANXA2 were employed as a capture antibody and a probe antibody, respectively. We detected the ANXA2 concentration in human serum using our newly developed system and evaluated its usefulness as a tumor marker. Overexpression of ANXA2 in human liver tissue was confirmed by real-time RT-PCR and Western blot analysis. The sandwich ELISA system for ANXA2 was developed for the detection of ANXA2 in human samples. The dose-response relationship between ANXA2 and optical density was linear in the range of 0-10 microg/ml and the sensitivity was 0.02 microg/ml. We determined the ANXA2 concentration in serum specimens using the newly developed sandwich ELISA. The serum ANXA2 concentrations of the patients with HCC (53.38+/-36.23 microg/ml) were significantly elevated when compared with those of normal individuals (28.81+/-24.94 microg/ml). These results suggest that expression of ANXA2 may be increased in HCC patients and may play an important role in liver cancer progression. This new ELISA method can be used as a tool for the detection of ANXA2 in human serum, particularly for cancer diagnostics.

Functional and clinical evidence for NDRG2 as a candidate suppressor of liver cancer metastasis.

We searched for potential suppressors of tumor metastasis by identifying the genes that are frequently down-regulated in hepatocellular carcinomas (HCC) while being negatively correlated with clinical parameters relevant to tumor metastasis, and we report here on the identification of N-myc downstream regulated gene 2 (NDRG2) as a promising candidate. NDRG2 expression was significantly reduced in HCC compared with nontumor or normal liver tissues [87.5% (35 of 40) and 62% (62 of 100) at RNA and protein levels, respectively]. Reduction of NDRG2 expression was intimately associated with promoter hypermethylation because its promoter region was found to carry extensively methylated CpG sites in HCC cell lines and primary tumors. Immunohistochemical analysis of NDRG2 protein in 100 HCC patient tissues indicated that NDRG2 expression loss is significantly correlated with aggressive tumor behaviors such as late tumor-node-metastasis (TNM) stage (P = 0.012), differentiation grade (P = 0.024), portal vein thrombi (P = 0.011), infiltrative growth pattern (P = 0.015), nodal/distant metastasis (P = 0.027), and recurrent tumor (P = 0.021), as well as shorter patient survival rates. Ectopically expressed NDRG2 suppressed invasion and migration of a highly invasive cell line, SK-Hep-1, and experimental tumor metastasis in vivo, whereas small interfering RNA-mediated knockdown resulted in increased invasion and migration of a weakly invasive cell line, PLC/PRF/5. In addition, NDRG2 could antagonize transforming growth factor beta1-mediated tumor cell invasion by specifically down-regulating the expression of matrix metalloproteinase 2 and laminin 332 pathway components, with concomitant suppression of Rho GTPase activity. These results suggest that NDRG2 can inhibit extracellular matrix-based, Rho-driven tumor cell invasion and migration and thereby play important roles in suppressing tumor metastasis in HCC.