Biphasic and Stage-Associated Expression of CPEB4 in Hepatocellular Carcinoma.

Cytoplasmic polyadenylation element binding protein 4 (CPEB4) is a sequence-specific RNA-binding protein and translational regulator, with expression associated with tumor progression. Nevertheless, CPEB4 seems to play paradoxical roles in cancers-an oncogenic promoter in pancreatic ductal adenocarcinoma (PDA) and glioblastomas but a tumor suppressor in hepatocellular carcinoma (HCC). To assess whether CPEB4-regulated carcinogenesis is tissue-specific, we reevaluated the role of CPEB4 in HCC. Although proliferation of hepatocytes appeared normal in CPEB4-knockout (KO) mice after partial hepatectomy, knockdown (KD) of CPEB4 in HepG2 liver cancer cells promoted colony formation in vitro. Moreover, the growth of CPEB4-KD cells was accelerated in an in vivo xenograft mouse model. In tumorous and adjacent non-tumorous paired liver specimens from 49 HCC patients, the protein level of CPEB4 was significantly upregulated in early-stage HCC but decreased toward late-stage HCC. This finding agrees with changes in CPEB4 mRNA level from analysis of two sets of HCC microarray data from the Gene Expression Omnibus (GEO) database. Taken together, downregulation of CPEB4 likely occurs at the late cancer stage to facilitate HCC progression. Biphasic alteration of CPEB4 expression during HCC progression suggests its complicated role in tumorigenesis.

Tissue-specific gene expression templates for accurate molecular characterization of the normal physiological states of multiple human tissues with implication in development and cancer studies.

BACKGROUND: To elucidate the molecular complications in many complex diseases, we argue for the priority to construct a model representing the normal physiological state of a cell/tissue. RESULTS: By analyzing three independent microarray datasets on normal human tissues, we established a quantitative molecular model GET, which consists of 24 tissue-specific Gene Expression Templates constructed from a set of 56 genes, for predicting 24 distinct tissue types under disease-free condition. 99.2% correctness was reached when a large-scale validation was performed on 61 new datasets to test the tissue-prediction power of GET. Network analysis based on molecular interactions suggests a potential role of these 56 genes in tissue differentiation and carcinogenesis.Applying GET to transcriptomic datasets produced from tissue development studies the results correlated well with developmental stages. Cancerous tissues and cell lines yielded significantly lower correlation with GET than the normal tissues. GET distinguished melanoma from normal skin tissue or benign skin tumor with 96% sensitivity and 89% specificity. CONCLUSIONS: These results strongly suggest that a normal tissue or cell may uphold its normal functioning and morphology by maintaining specific chemical stoichiometry among genes. The state of stoichiometry can be depicted by a compact set of representative genes such as the 56 genes obtained here. A significant deviation from normal stoichiometry may result in malfunction or abnormal growth of the cells.

Stimulatory effect of Echinacea purpurea extract on the trafficking activity of mouse dendritic cells: revealed by genomic and proteomic analyses.

BACKGROUND: Several Echinacea species have been used as nutraceuticals or botanical drugs for "immunostimulation", but scientific evidence supporting their therapeutic use is still controversial. In this study, a phytocompound mixture extracted from the butanol fraction (BF) of a stem and leaf (S+L) extract of E. purpurea ([BF/S+L/Ep]) containing stringently defined bioactive phytocompounds was obtained using standardized and published procedures. The transcriptomic and proteomic effects of this phytoextract on mouse bone marrow-derived dendritic cells (BMDCs) were analyzed using primary cultures. RESULTS: Treatment of BMDCs with [BF/S+L/Ep] did not significantly influence the phenotypic maturation activity of dendritic cells (DCs). Affymetrix DNA microarray and bioinformatics analyses of genes differentially expressed in DCs treated with [BF/S+L/Ep] for 4 or 12 h revealed that the majority of responsive genes were related to cell adhesion or motility (Cdh10, Itga6, Cdh1, Gja1 and Mmp8), or were chemokines (Cxcl2, Cxcl7) or signaling molecules (Nrxn1, Pkce and Acss1). TRANSPATH database analyses of gene expression and related signaling pathways in treated-DCs predicted the JNK, PP2C-α, AKT, ERK1/2 or MAPKAPK pathways as the putative targets of [BF/S+L/Ep]. In parallel, proteomic analysis showed that the expressions of metabolic-, cytoskeleton- or NF-κB signaling-related proteins were regulated by treatment with [BF/S+L/Ep]. In vitro flow cytometry analysis of chemotaxis-related receptors and in vivo cell trafficking assay further showed that DCs treated with [BF/S+L/Ep] were able to migrate more effectively to peripheral lymph node and spleen tissues than DCs treated as control groups. CONCLUSION: Results from this study suggest that [BF/S+L/Ep] modulates DC mobility and related cellular physiology in the mouse immune system. Moreover, the signaling networks and molecules highlighted here are potential targets for nutritional or clinical application of Echinacea or other candidate medicinal plants.

Differential functional genomic effects of anti-inflammatory phytocompounds on immune signaling.

BACKGROUND: Functional comparative genomic analysis of the cellular immunological effects of different anti-inflammatory phytocompounds is considered as a helpful approach to distinguish the complex and specific bioactivities of candidate phytomedicines. Using LPS-stimulated THP-1 monocytes, we characterize here the immunomodulatory activities of three single phytocompounds (emodin, shikonin, and cytopiloyne) and a defined phytocompound mixture extracted from Echinacea plant (BF/S+L/Ep) by focused DNA microarray analysis of selected immune-related genes. RESULTS: Shikonin and emodin significantly inhibited the early expression (within 0.5 h) of approximately 50 genes, notably cytokines TNF-α, IL-1ß and IL-4, chemokines CCL4 and CCL8, and inflammatory modulators NFATC3 and PTGS2. In contrast, neither cytopiloyne nor BF/S+L/Ep inhibited the early expression of these 50 genes, but rather inhibited most late-stage expression (~12 h) of another immune gene subset. TRANSPATH database key node analysis identified the extracellular signal-regulated kinase (ERK) 1/2 activation pathway as the putative target of BF/S+L/Ep and cytopiloyne. Western blot confirmed that delayed inactivation of the ERK pathway was indeed demonstrable for these two preparations during the mid-stage (1 to 4 h) of LPS stimulation. We further identified ubiquitin pathway regulators, E6-AP and Rad23A, as possible key regulators for emodin and shikonin, respectively. CONCLUSION: The current focused DNA microarray approach rapidly identified important subgenomic differences in the pattern of immune cell-related gene expression in response to specific anti-inflammatory phytocompounds. These molecular targets and deduced networks may be employed as a guide for classifying, monitoring and manipulating the molecular and immunological specificities of different anti-inflammatory phytocompounds in key immune cell systems and for potential pharmacological application.

Blocking effect of an immuno-suppressive agent, cynarin, on CD28 of T-cell receptor.

PURPOSE: Cynarin, a potential immunosuppressant that blocks the interaction between the CD28 of T-cell receptor and CD80 of antigen presenting cells, was found in Echinacea purpurea by a new pharmaceutical screening method: After Flowing Through Immobilized Receptor (AFTIR; Dong et al., J Med Chem, 49: 1845-1854, 2006). This Echinacea component is the first small molecule that is able to specifically block "signal 2" of T-cell activation. METHODS: In this study, we used the AFTIR method to further confirm that cynarin effectively blocked the binding between CD80 of B-cells and CD28 of T-cells, and provide details of its mechanism of action. RESULTS: The experimental results showed that cynarin blocked about 87% of the CD28-dependent "signal 2" pathway of T-cell activation under the condition of one to one ratio of T-cell and B-cell in vitro. Theoretical structure modeling showed that cynarin binds to the "G-pocket" of CD28 (Evans et al., Nat Immunol, 6:271-279, 2005), and thus interrupts the site of interaction between CD28 and CD80. CONCLUSIONS: These results confirm both that AFTIR is a promising method for screening selective active compounds from herbal medicine and that cynarin has great potential as an immuno-suppressive agent.

Genomics and proteomics of immune modulatory effects of a butanol fraction of echinacea purpurea in human dendritic cells.

BACKGROUND: Echinacea spp. extracts and the derived phytocompounds have been shown to induce specific immune cell activities and are popularly used as food supplements or nutraceuticals for immuno-modulatory functions. Dendritic cells (DCs), the most potent antigen presenting cells, play an important role in both innate and adaptive immunities. In this study, we investigated the specific and differential gene expression in human immature DCs (iDCs) in response to treatment with a butanol fraction containing defined bioactive phytocompounds extracted from stems and leaves of Echinacea purpurea, that we denoted [BF/S+L/Ep]. RESULTS: Affymetrix DNA microarray results showed significant up regulation of specific genes for cytokines (IL-8, IL-1beta, and IL-18) and chemokines (CXCL 2, CCL 5, and CCL 2) within 4 h after [BF/S+L/Ep] treatment of iDCs. Bioinformatics analysis of genes expressed in [BF/S+L/Ep]-treated DCs revealed a key-signaling network involving a number of immune-modulatory molecules leading to the activation of a downstream molecule, adenylate cyclase 8. Proteomic analysis showed increased expression of antioxidant and cytoskeletal proteins after treatment with [BF/S+L/Ep] and cichoric acid. CONCLUSION: This study provides information on candidate target molecules and molecular signaling mechanisms for future systematic research into the immune-modulatory activities of an important traditional medicinal herb and its derived phytocompounds.

An optimized procedure greatly improves EST vector contamination removal.

BACKGROUND: The enormous amount of sequence data available in the public domain database has been a gold mine for researchers exploring various themes in life sciences, and hence the quality of such data is of serious concern to researchers. Removal of vector contamination is one of the most significant operations to obtain accurate sequence data containing only a cDNA insert from the basecalls output by an automatic DNA sequencer. Popular bioinformatics programs to accomplish vector trimming include LUCY, cross_match and SeqClean. RESULTS: In a recent study, where the program SeqClean was used to remove vector contamination from our test set of EST data compiled through various library construction systems, however, a significant number of errors remained after preliminary trimming. These errors were later almost completely corrected by simply using a re-linearized form of the cloning vector to compare against the target ESTs. The modified trimming procedure for SeqClean was also compared with the trimming efficiency of the other two popular programs, LUCY2, and cross_match. Using SeqClean with a re-linearized form of the cloning vector significantly surpassed the other two programs in all tested conditions, while the performance of the other two programs was not influenced by the modified procedure. Vector contamination in dbEST was also investigated in this study: 2203 out of the 48212 ESTs sampled from dbEST (2007-04-18 freeze) were found to match sequences in UNIVEC. CONCLUSION: Vector contamination remains a serious concern to the data quality in the public sequence database nowadays. Based on the results presented here, we feel that our modified procedure with SeqClean should be recommended to all researchers for the task of vector removal from EST or genomic sequences.

Super pairwise alignment (SPA): an efficient approach to global alignment for homologous sequences.

Sequence analysis is the basis of bioinformatics, while sequence alignment is a fundamental task for sequence analysis. The widely used alignment algorithm, Dynamic Programming, though generating optimal alignment, takes too much time due to its high computation complexity O(N(2)). In order to reduce computation complexity without sacrificing too much accuracy, we have developed a new approach to align two homologous sequences. The new approach presented here, adopting our novel algorithm which combines the methods of probabilistic and combinatorial analysis, reduces the computation complexity to as low as O(N). The computation speed by our program is at least 15 times faster than traditional pairwise alignment algorithms without a loss of much accuracy. We hence named the algorithm Super Pairwise Alignment (SPA). The pairwise alignment execution program based on SPA and the detailed results of the aligned sequences discussed in this article are available upon request.