ß-Catenin-treated peptides effectively inhibit the proliferation of colorectal cancer.

To verify the inhibitory mechanism of ß-catenin-designed peptides in colorectal cancer(CRC) tumors, the following experiments were performed. In vitro colony formation, Transwell assays, and flow cytometry were performed to assess the biological effects of designed peptides (F18KD, F20A4-7k, F20A4-10k, and F20A3-9k + F20A4-10k + F20A5-9k) in HT-29 cells. In vivo xenograft experiments were performed and treated with peptides. Next, tumors were subjected to Hematoxylin and eosin staining (HE), immunohistochemical, and terminal deoxynucleotidyl transferase dUTP nick end labeling staining assays to evaluate the inhibitory effect of peptides on tumors. ß-Catenin levels were quantified via western blotting (WB) and quantitative real-time polymerase chain reaction, and ß-catenin was located using confocal laser scanning microscopy. T-cell factor-4 (TCF-4), C-myc, and CCND1 levels were quantified via WB. Results were obtained as following. First, the peptides reduced viability, migration, and invasion; promoted apoptosis; and stabilized the S phase of HT-29 cells. Second, peptides suppressed tumor growth and downregulated the expression of CD34, vascular endothelial growth factor, and ß-catenin in tumors. Furthermore, we found that peptides downregulated ß-catenin expression in both the cytoplasm and nucleus; TCF-4, C-myc, and CCND1 expression was also downregulated. Notably, ß-catenin-targeting peptides had a better inhibitory effect on CRC than non-ß-catenin-target peptides, and a combination of peptides exerted a more potent inhibitory effect on CRC than single peptides. It suggested that ß-Catenin-targeting peptides promote apoptosis in CRC tumors by inhibiting activation of the Wnt/ß-catenin pathway.

Drug Sensitivity Screening and Targeted Pathway Analysis Reveal a Multi-Driver Proliferative Mechanism and Suggest a Strategy of Combination Targeted Therapy for Colorectal Cancer Cells.

Treatment of colorectal cancer mostly relies on traditional therapeutic approaches, such as surgery and chemotherapy. Limited options of targeted therapy for colorectal cancer narrowly focus on blocking cancer-generic targets VEGFR and EGFR. Identifying the oncogenic drivers, understanding their contribution to proliferation, and finding inhibitors to block such drivers are the keys to developing targeted therapy for colorectal cancer. In this study, ten colorectal cancer cell lines were screened against a panel of protein kinase inhibitors blocking key oncogenic signaling pathways. The results show that four of the 10 cell lines did not respond to any kinase inhibitors significantly, the other six were mildly inhibited by AZD-6244, BMS-754807, and/or dasatinib. Mechanistic analyses demonstrate that these inhibitors independently block the MAP kinase pathway, IR/IGF-1R/AKT pathway, and Src kinases, suggesting a multi-driver nature of proliferative signaling in these cells. Most of these cell lines were potently and synergistically inhibited by pair-wise combinations of these drugs. Furthermore, seven of the 10 cell lines were inhibited by the triple combination of AZD-6244/BMS-754807/dasatinib with IC50's between 10 and 84 nM. These results suggest that combination targeted therapy may be an effective strategy against colorectal cancer.

Construction of a cDNA library and preliminary analysis of the expressed sequence tags of the earthworm Eisenia fetida (Savigny, 1826).

Earthworms are useful indicator organisms of soil health and Eisenia fetida have been extensively used as test organisms in ecotoxicological studies. In order to gain insight into the gene expression profiles associated with physiological functions of earthworms, a full­length enriched cDNA library of the Eisenia fetida genome was successfully constructed using Switching Mechanism at 5'End of RNA Template technology. Construction of a cDNA library and analysis of Expressed Sequence Tags (ESTs) are efficient approaches for collecting genomic information and identifying genes important for a given biological process. Furthermore, analysis of the expression abundance of ESTs was performed with the aim of providing genetic and transcriptomic information on the development and regenerative process of earthworms. Phrep and Crossmatch were used to process EST data and a total of 1,140 high­quality EST sequences were determined by sequencing random cDNA clones from the library. Clustering analysis of sequences revealed a total of 593 unique sequences including 225 contiguous and 368 singleton sequences. Basic Local Alignment Search Tool analysis against the Kyoto Encyclopedia of Genes and Genomes database resulted in 593 significant hits (P­value <1x10­8), of which 168 were annotated through Gene Ontology analysis. The STRING database was used to determine relationships among the 168 ESTs, identifying associated genes involved in protein­protein interactions and gene expression regulation. Based on nucleic acid and protein sequence homology, the mutual relationships between 287 genes could be obtained, which identified a portion of the ESTs as known genes. The present study reports on the construction of a high­quality cDNA library representative of adult earthworms, on a preliminary analysis of ESTs and on a putative functional analysis of ESTs. The present study is expected to enhance our understanding of the molecular basis underlying the biological development of earthworms.

TMPyP4-regulated cell proliferation and apoptosis through the Wnt/ß-catenin signaling pathway in SW480 cells.

BACKGROUND: The aim of this study was to investigate the potential effects of the 5, 10, 15, 20-tetrakis (1-methylpyridinium-4-yl) porphyrin (TMPyP4) on the proliferation and apoptosis of SW480 cells and the underlying mechanisms by which TMPyP4 exerted its actions. METHODS: After treated with different doses of TMPyP4, cell viability was determined by MTT method, the apoptosis was observed by flow cytometry (FCM) and the expression of Wnt, GSK-3ß, ß-catenin and cyclinD1 was measured by RT-PCR and Western blot analysis. RESULTS: The analysis revealed that TMPyP4 potently suppressed cell viability and induced the apoptosis of SW480 cells in a dose-dependent manner. In addition, the downregulation of Wnt, ß-catenin and cyclinD1 expression levels was detected in TMPyP4-treated SW480 cells. However, followed by the block of Wnt signaling pathway using siRNA methods, the effects of TMPyP4 on proliferation and apoptosis of SW480 cells were significantly reduced. CONCLUSION: It indicates that the TMPyP4-inhibited proliferation and -induced apoptosis in SW480 cells was accompanied by the suppression of Wnt/ß-catenin signaling pathway. Therefore, TMPyP4 may represent a potential therapeutic method for the treatment of colon carcinoma.

[Preparation and identification of DNA G-quadruplex antibody].

OBJECTIVE: To construct a prokaryotic expression plasmid of DNA G-quadruplex antibody, express it in E.coli BL21 (DE3) bacterial expression system, purify and identify the antibody. METHODS: Chemically synthesized BG4 gene of DNA G-quadruplex antibodies was inserted into pSANG10 plasmid to construct DNA G-quadruplex antibody expression vector pSANG10-BG4. BL21 (DE3) as the host strain was utilized for self-induced expression of the protein. Osmotic lysis method was used for collecting this protein. Thereafter, the protein was purified by histidine tag affinity chromatography and identified by SDS-PAGE and Western blotting. The function of this protein was verified in SW480 colon cancer cells. RESULTS: Double enzyme digestion and gene sequencing confirmed that DNA G-quadruplex antibody expression vector was successfully constructed. The relative molecular mass (Mr) of this protein was 30 000 to 37 000. The protein in a soluble form was expressed in the periplasm of BL21. The protein was of the same size as expected. CONCLUSION: The DNA G-quadruplex antibody has been successfully prepared.

Regenerating gene 1B silencing inhibits colon cancer cell HCT116 proliferation and invasion.

BACKGROUND: The human regenerating gene 1B (REG1B) is found to be frequently up-regulated in many types of human tumors. It is unclear whether REG1B expression may have therapeutic value in colorectal carcinoma. Additionally, how REG1B is associated with the clinical features of colorectal carcinoma is not known. To investigate the relationship between REG1B and colorectal cancer, we analyzed REG1B expression in clinical specimens and cell lines and the effect of down-regulation of REG1B by short hairpin RNA (shRNA) in HCT116 cells. METHODS: Paraffin-embedded specimens from 30 pairs of colorectal cancer tissues and adjacent colon tissues were used to investigate the expression of REG1B by immunohistochemistry. We also examined whether REG1B itself may be related to cell proliferation, cell cycle arrest, apoptosis, migration and invasion in colon cancer HCT116 cells. RESULTS: Our results showed that REG1B was highly expressed in colorectal carcinoma and was significantly associated with cell differentiation status. The results also illustrated that REG1B silencing with shRNA inhibited cell proliferation, migration and invasion but did not induce apoptosis. Furthermore, down-regulation of REG1B induces G1-phase cell cycle arrest in colon cancer cells. CONCLUSIONS: Knockdown of REG1B can inhibit cell proliferation, migration and invasion. It may act by a mechanism regulating cell cycle progression. Thus, REG1B may be a novel candidate therapeutic target for colorectal cancer.

Overexpression of Aurora-A enhances invasion and matrix metalloproteinase-2 expression in esophageal squamous cell carcinoma cells.

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive cancers, and metastasis is the principal cause of death in ESCC patients. It has been shown that amplification and overexpression of mitotic serine/threonine kinase Aurora-A occur in several types of human tumors, including ESCC. Moreover, increase in expression levels of Aurora-A has been predicted to correlate with the grades of tumor differentiation and invasive capability. However, the mechanisms by which Aurora-A mediates its invasive effects still remain elusive. In this article, we showed that Aurora-A overexpression significantly increased cell migration and invasion as well as secretion and expression of matrix metalloproteinase-2 (MMP-2). Conversely, siRNA-mediated knockdown of Aurora-A expression in human ESCC cells led to inhibition of cell invasiveness as well as secretion and expression of MMP-2. In addition, Aurora-A overexpression increased phosphorylation levels of p38 mitogen-activated protein kinase (MAPK) and Akt, and the knockdown of Aurora-A by siRNA decreased the activity of p38 MAPK and Akt. Moreover, the blocking of the activity of above kinases using chemical inhibitors suppressed the ability of Aurora-A to induce MMP-2 secretion and expression as well as cell invasion. These data show that overexpression of Aurora-A contributes to the malignancy development of ESCC by enhancing tumor cell invasion as well as MMP-2 activity and expression, which can occur through signaling pathways involving p38 MAPK and Akt protein kinases. Taken together, these studies provide a molecular basis for promoting the role of Aurora-A in malignancy development of ESCC.

[Significance of CD138 in immunohistochemical profiles and its correlation with prognosis in diffuse large B-cell lymphoma].

OBJECTIVE: The purpose of this study was to classify the diffuse large B-cell lymphoma (DLBCL) into different prognostic subgroups according to four different detection methods of the expression of CD138, CD10, bcl-6, and MUM1. In particular to investigate the significance of CD138 in immunohistochemical profiles and its correlation with prognosis in DLBCL. METHODS: Immunohistochemical EnVision method was used to detect the expression of CD138, CD10, bcl-6 and MUM1 in 106 cases of DLBCL and reconstructed into four different subtyping algorithms. Algorithm-1, according to the expression of CD10, bcl-6 and MUM1, the cases were assigned to GCB and non-GCB groups. Algorithm-2, according to the expression of CD138, CD10, bcl-6 and MUM1, the cases were assigned to A, B, C, D groups. Algorithm-3, according to the expression of CD10 and MUM1, the cases were assigned to GCB and non-GCB groups. Algorithm-4, according to the expression of CD138, CD10, bcl-6 and MUM1, the cases were assigned to GCB and non-GCB groups. Following up was included as well. Statistical analysis was performed using the SPSS 13.0 and differences were considered significant at P < 0.05. RESULTS: CD138, MUM1, CD10 and bcl-6 were positive in 15.1% (16/106), 56.6% (60/106), 21.7 (23/106) and 26.4% (28/106), respectively. The expression of CD10 and bcl-6 was associated with favorable OS (P = 0.001 and 0.041, respectively), whereas the expression of CD138 was associated with unfavorable OS (P = 0.003). Using multivariate Cox proportional hazards regression analysis, algorithm-1 and -4 were almost at the same level for prognosis of OS (OR = 0.259, 0.255) and PFS (OR = 0.248, 0.244). CONCLUSIONS: Both Hans's algorithm and Colombo's algorithm including CD138 detection are associated with the prognosis of DLBCL patients. The two algorithms have similar OR value according to Cox analysis. However, positive expression of CD138 is of minor significance in prediction of the prognosis in DLBCL patients.

Clinical Impact of t(14;18) in Diffuse Large B-cell Lymphoma.

OBJECTIVE: Recent studies have suggested that t(14;18) is present in a significant proportion of diffuse large B-cell lymphomas (DLBCLs). However, the prognostic significance of this translocation and its relationship with BCL-2 protein expression remains controversial. Our study aimed to investigate the predictive power of t(14;18) and BCL-2 protein expression in the prognosis of DLBCLs. METHODS: Biopsy specimens from 106 DLBCLs were analyzed using interphase fluorescence in situ hybridization (FISH). Immunophenotypic analysis of CD20, CD3, CD10, BCL-6, MUM1 and BCL-2 was performed by immunohistochemistry. SPSS 13.0 software was used for statistical analysis. RESULTS: The t(14;18) was identified in 27 of 106 cases (25.5%). The percentages of tumor cells expressing CD10, BCL-6, MUM1 and BCL-2 were 21.7%, 26.4%, 56.6% and 73.6%, respectively. The presence of this translocation was significantly correlated with the expression of CD10 and immunophenotypic subtype (p<0.001). No association was observed between BCL-2 protein expression and the presence of t(14;18). Multivariate analysis confirmed that both t(14;18) and BCL-2 expression were significantly associated with survival. Moreover, patients with t(14;18) had worse prognosis, compared with those with BCL-2 expression (for overall survival: hazard ratio, 4.235; 95%CI, 2.153-8.329, p<0.001 vs. hazard ration, 2.743; 95%CI, 1.262-5.962, p=0.011). CONCLUSIONS: The t(14;18) is a useful prognostic tool for the evaluation of DLBCL immunophenotype and prognosis. The prognosis of GCB (germinal centre-like B cell) DLBCL patients should be made with the consideration of the presence of this translocation, and the detection of t(14;18) should be included as a routine diagnostic test in these cases.

Neither K+ channels nor PI3K/Akt mediates the vasodilative effect of nebivolol on different types of rat arteries.

PURPOSE: Nebivolol is a highly selective beta(1)-adrenoceptor blocker with additional vasodilating properties. It has been shown that the nebivolol-induced vasorelaxation is nitric oxide (NO) dependent. The serine/ threonine protein kinase Akt phosphorylates endothelial cell NO synthase (eNOS) and enhances the ability of eNOS to generate NO. Previous studies have shown that the release of NO from the endothelium may be ascribed to the modulation of different types of K(+) channels. The current study was designed to determine whether K(+) channels or phosphatidylinositol-3-kinase (PI3K)/Akt may affect vasorelaxation induced by nebivolol in different rat arteries. METHODS: Rings of the rat aorta, carotid artery, femoral artery, and renal artery were suspended for isometric force recording. During contraction by KCl (60 mmol/L) or phenylephrine (PE; 10(-6) mol/L; femoral artery and renal artery were precontracted by 10(-5) mol/L), the effect of nebivolol (10(-7)-10(- 5) mol/L) was obtained in the presence of different potassium channel, PI3K/Akt, or NOS inhibitors. RESULTS: Nebivolol (10(- 7)-10(-5) mol/L) relaxed precontractions induced by KCl and PE in different rat arteries, which was inhibited by the presence of the NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME; 100 micromol/ L). The effect of nebivolol was concentration dependent. The exposure of the vessel rings to a selective inhibitor of PI3K wortmannin (5 x 10(-7) mol/L) or a selective inhibitor of Akt (1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate, 10(-5) mol/L) did not influence nebivolol-induced vasorelaxation. Similarly, K(+) channels blockers, iberiotoxin (100 nmol/L), glibenclamide (0.1 mmol/L), 4-aminopyridine (1 mmol/L), or BaCl(2) (1 mmol/L) had no influence on the relaxation of nebivolol in arteries precontracted by PE. CONCLUSION: Nebivolol produced a concentration-dependent vasodilation in different rat arteries precontracted by PE or KCl. In the isolated rat aorta, carotid artery, femoral artery, and renal artery, neither K(+) channels nor PI3K/Akt pathway was involved in the relaxation induced by nebivolol.

All-trans-retinoic acid alters Smads expression in embryonic neural tissue of mice.

Retinoic acid can cause malformations of the developing nervous system. Smad signaling is involved in embryonic development. The current study investigated all-trans-retinoic acid (ATRA)-induced alteration of Smad expression in the developing neural tubes of mice. Pregnant mice were treated with a single dose of 50 mg/kg ATRA by oral gavage on embryonic day E7. Western immunoblotting was used to examine Smads proteins, particularly phosphorylated (p-) Smad1, total Smad1 and Smad6 in the neural tissue of the embryos on E8-E11 following treatment. Results showed that ATRA treatment significantly increased expression of both p-Smad1 and total Smad1, while Smad6 was decreased in neural tissues of ATRA-exposed embryos in utero from E8 to E11, a critical period for neural tube formation. Data suggest that disruption of Smad signaling may be involved in ATRA-induced neural tube defects.

Suppression of invasive properties of colorectal carcinoma SW480 cells by 15-hydroxyprostaglandin dehydrogenase gene.

Colorectal carcinoma (CRC) is often lethal when invasion and/or metastasis occur. NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), an enzyme involved in prostaglandin (including PGE(2)) bio-inactivation, is down-expressed in several epithelial malignancies including CRC. Although its role in the suppression of colon tumorigenesis has been well learned, little is known about the role of 15-PGDH in the process of tumor metastasis. Here, we tested the hypothesis that 15-PGDH over-expression in CRC cells results in decreased cell motility and invasion. In this study, 15-PGDH was re-expressed in SW480 cells by the use of gene transient transfection with eukaryotic expression vector pcDNA3.1-PGDH. We confirmed the over-expression of 15-PGDH protein by Western blot and enzymatic activity assay. The cell motility was tested by counting the number of cells crossing an 8-micron pore size PET membrane and by measuring cells migration distance through wound healing assay. Furthermore, cell invasive activity was evaluated by counting the number of cells invading through a Matrigel-coated membrane simulating basement membrane. The effects of 15-PGDH on the adhesion were investigated by MTT assay. Ectopic expression of 15-PGDH in SW480 cancer cells significantly inhibited the cell migratory and invasive capacity in vitro by approximately 1.9- and 8.4-fold, respectively. To test the hypothesis that 15-PGDH affects proteases and inactivates extracellular matrix (ECM), Western blot and gelatin zymography were performed by using serum-free conditioned medium. The results showed that re-expression of 15-PGDH suppresed matrix metalloproteinase-2 (MMP2) synthesis and secretion. In addition, the analysis of the MMP2 activity indicated that re-expression of 15-PGDH could inhibit activation of MMP2. Furthermore, we found that 15-PGDH inhibited cell adhesion to ECM and reduced CD44 expression in SW480 cell. Taken together, these results suggest that induced 15-PGDH expression may contribute to the inhibition of the invasive and metastatic capacity of colon cancer cells in vitro.

Screening and evaluation of human single-chain fragment variable antibody against hepatitis B virus surface antigen.

BACKGROUND: Phage display technology has become a vital tool in studies aimed at identifying molecules binding to a specific target. It enables the rapid generation and selection of high affinity, fully human antibody product candidates to essentially any disease target appropriate for antibody therapy. In this study, we prepared the recombinant single-chain fragment variable (ScFv) antibody to hepatitis B virus surface antigen (HBsAg) by the phage display technology for obtaining a virus-targeting mediator. METHODS: mRNA was isolated from B-lymphocytes from a healthy volunteer and converted into cDNA. The fragment variables of heavy and light chain were amplified separately and assembled into ScFv DNA with a specially constructed DNA linker by polymerase chain reaction. The ScFv DNA was ligated into the phagmid vector pCANTAB5E and the ligated sample was transformed into competent E.coli TG1. The transformed cells were infected with M13K07 helper phage to form a human recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by bacterial colony count and restriction analysis. After two rounds of panning with HBsAg, the phage clones displaying ScFv of the antibody were selected by enzyme-linked immunosorbant assay (ELISA) from the enriched phage clones. The antigen binding affinity of the positive clone was detected by competition ELISA. HB2151 E.coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the anti-HBsAg ScFv. ELISA assay was used to detect the antigen binding affinity of the soluble anti-HBsAg ScFv. Finally, the relative molecular mass of soluble anti-HBsAg ScFv was measured by SDS-PAGE. RESULTS: The variable heavy (VH) and variable light (VL) and ScFv DNAs were about 340 bp, 320 bp and 750 bp, respectively. The volume of the library was up to 2 x 10(6) and 8 of 10 random clones were recombinants. Two phage clones could strongly compete with the original HBsAb for binding to HBsAg. Within 2 strong positive phage clones, the soluble anti-HBsAg ScFv from one clone was found to have the binding activity with HBsAg. SDS-PAGE showed that the relative molecular weight of soluble anti-HBsAg ScFv was 32 kDa. CONCLUSION: The anti-HBsAg ScFv successfully produced by phage antibody technology may be useful for broadening the scope of application of the antibody.

[Fermentation and purification of Echistatin fusion protein expressed in Escherichia coli].

Lots of studies of Echistatin (Ecs) have proved its wide use in many aspects. However, the low yield of Ecs has impeded the relative researches of the protein. To establish the high-level expression system of Ecs, the fermentation and purification process of Ecs fusion protein expressed in E. coli were optimized. The Ecs gene was introduced into vector pTXB1 and placed under the control of highly efficient T7 promoter system. The cloned Ecs gene was expressed in E. coli BL21 (DE3) as soluble form. The Ecs production and biomass accumulation were optimized by examining medium composition, point of induction and induction time in fed-batch fermentation. Biomass accumulation was greatly affected by medium gradient, reaching 50.3g/L in 2 x YT medium. Ecs production was found to increase to 35% of total protein with 75g/L biomass accumulation after induced for 4h. Purification of Ecs from supernatant of sonication was done using one-step chromatographic procedure with chitin affinity chromatography and DTF cleavage, resulting in yields of 28mg/L and > 90% purity. The bioactivity of purified Ecs was determined and the result showed that purified Ecs could inhibit the aggregation of platelet in vitro with similar bioactivity to wild Ecs. This optimized method is readily scaled up for the expression and purification of Ecs in sufficient quantities for further structural and biological studies and applications.

Protein transduction domain of membrane penetrating peptide can efficiently deliver DNA and protein into mouse liver for gene therapy.

BACKGROUND: The development of a harmless and efficient nonviral gene delivery system that can facilitate the penetration of nucleic acids through the plasma membrane is a key to successful gene therapy. The aim of this study was to test a nonviral gene transferring vector's function of delivering DNA into liver cells to provide an important clue for gene transfer in liver gene therapy. METHODS: The complex of DNA and DNA delivering protein was injected into mice through their tail veins. Then the mice were killed and their liver tissue was sectioned. The gene transferring results were detected using a confocal laser scanning microscope. RESULTS: Fluorescence analysis indicated that both DNA-membrane penetrating peptide (MPP) complex and DNA- hepatocyte specific receptor binding domain (HSRBD)-MPP complex could go into liver cells. The fluorescence value of liver cells in the DNA- HSRBD-MPP group was higher than that in the DNA-MPP group. CONCLUSIONS: MPP can successfully deliver DNA and protein into cells, and MPP with a HSRBD can specifically deliver DNA into liver cells. These have laid a foundation for further study on the nonviral liver cell gene delivering system.