Development of DNA-Designed Avian IgY Antibodies for Detection of Mycobacterium avium subsp. paratuberculosis Heat Shock Protein 70 (Hsp70) and Anti-Hsp70 Antibodies in the Serum of Normal Cattle

Mycobacterium avium subsp. paratuberculosis heat shock protein 70 (MAPHsp70) is an immunodominant antigen, which can be used as a subunit vaccine against bovine paratuberculosis. In the present study, we evaluated the immunogenic activities of MAPHsp70 expressed by DNA vaccine in chicken and the use of prepared specific avian IgY antibodies for western blotting and ELISA methods. The gene encoding MAP Hsp70 was subcloned into the eukaryotic expression vector, pcDNA3.1, and the recombinant plasmid (pcDNA3.1-MAP Hsp70) transfected into COS-7 cells. Chickens were also immunized with pcDNA3.1-MAP Hsp70, and egg yolk antibodies extracted from eggs were collected after immunization. DNA-designed IgY antibody was used in Western blotting analysis to detect the expression of MAPHsp70, and in a sandwich ELISA to assess the prevalence of anti-MAPHsp70 antibodies in cattle serum. Western blotting results indicate the expression of rMAP hsp70 in COS-7 cells and sandwich ELISA could detect anti-MAPHsp70 antibodies in 7.5% of cows. Chicken immunization with pcDNA3.1-MAPHsp70 could demonstrate the effective production of anti-MAPHsp70 IgY antibodies. Monospecific anti-MAPHsp70 antibody generated in chickens is useful for detection of MAPHsp70 peptide in cell culture and MAP lysate.     

Development and Characterization of Monoclonal Antibody Specifically Against TSP50

Testis-specific protease 50(TSP50) has been identified as a testis-specific protein that is expressed abnormally in most human breast cancer samples,which makes it an attractive molecular marker and a potential target for diagnosis and therapy.In the present study,we prepared a panel of monoclonal antibodies(mAbs) with high specificity and sensitivity against TSP50 by hybridoma method and characterized them by ELISA,Western blot,immunofluroescence and immunohistochemical analyses.The results show that all o...     

Development of DNA-Designed Avian IgY Antibodies for Quantitative Determination of Bovine Interferon-Gamma

Interferon-gamma (IFN-γ), a cytokine produced by sensitized T lymphocytes, is one of the key elements in defining T helper 1 lymphocyte immune responses. Quantitative evaluation of IFN-γ expression could provide an important analytical tool for measurement of cell-mediated immunity and investigating immune responses to infectious diseases. Method of DNA-designed avian IgY antibodies was used for production of monospecific polyclonal antibodies that allows quantification of the recombinant bovine IFN-γ protein. IFN-γ cDNA was subcloned and expressed in mammalian expression plasmid (pcDNA3.1(+)) under the control of the human cytomegalovirus promoter. Chickens were immunized by plasmid DNA, and eggyolk antibodies extracted from eggs were collected after immunization. IgY-specific antibodies were evaluated by an antigen capture enzyme-linked immunosorbent assay (ELISA) using recombinant IFN-γ. Based on the results, developed bovine IFN-γ capture ELISA could detect up to 1 ng/ml of IFN-γ by 64-fold diluted IgY. Monospecific anti-bovine IFN-γ antibodies generated in chickens are useful for quantifying different concentrations of recombinant bovine IFN-γ, which is expressed in cell culture.     

Cloned s-Lap Gene Coding Area, Expression and Localization of s-Lap／GFP Fusion Protein in Mammal Cells

s-Lap is a new gene sequence from pig retinal pigment epithelial (RPE) cells, which was found and cloned in the early period of apoptosis of RPE cells damaged with visible light. We cloned the coding area sequence of the novel gene of s-Lap and constructed its recombinant eukaryotic plasmid pcDNA3.1-GFP/s-lap with the recombinant DNA technique. The expression and localization of s-lap/GFP fusion protein in CHO and B16 cell lines were studied with the instantaneously transfected pcDNA3.1-GFP/s-lap recombinant plasmid. s-Lap/GFP fusion protein can be expressed in CHO and B16 cells with a high rate expression in the nuclei.     

Effect of Regulation of HSV-tk Gene Expression and Tumor Killed Activity with a Single Tetracycline-regulatable Plasmid Vector on HeLa Cells

To construct a single tetracycline-regulatable plasmid vector based on the double tetracycline-regulatable plasmid vector system for regulating HSV-tk gene expression so as to effectively kill HeLa cells. Two tetracycline operator(TetO2) was cloned into pcDNA3.1 and a cassette was made for a cytomegalovirus-type 2 tetracycline operator(CMV-TetO2) promoter, and the obtained vector was named pcDNA3.1-CMV-TetO2. Herpes simplex virus thymidine kinase(HSV-tk) gene and tetracycline repressor(TR) gene were cloned ...     

PTEN/MMAC1 enhances the growth inhibition by anticancer drugs with downregulation of IGF-II expression in gastric cancer cells

PTEN/MMAC1 is a tumor suppressor gene that is mutated in a variety of advanced and metastatic cancers. Its major function is likely to be the phosphatase activity that regulates the phosphotidylinositol (PI)3-kinase/Akt pathway. On the other hand, IGF system plays an important role in cell proliferation and cell survival via PI3-kinase/Akt and mitogen-activated protein kinase pathways in many cancer cells. To evaluate effect of PTEN on cell growth and IGF system in gastric cancer, human gastric adenocarcinoma cells (SNU-5 & -216) were transfected with human PTEN cDNA. Those PTEN- transfected gastric cancer cells had a lower proliferation rate than the pcDNA3-transfected cells. PTEN overexpression induced a profound decrease in the IGF-II and IGF-IR expression levels, and downregulation of IGF-II expression by PTEN was mediated through the regulation of the IGF-II promoter. In addition, a PI3-kinase inhibitor, LY294002, induced the downregulation of IGF-II expression. The PTEN-overexpressing SUN-5 and -216 cells were more sensitive to death induced by etoposide and adriamycin that induce DNA damage than the pcDNA3-transfected cells. These findings suggest that PTEN suppresses the cell growth through modulation of IGF system and sensitizing cancer cells to cell death by anticancer drugs.     

A novel aptamer-based histochemistry assay for specific diagnosis of clinical breast cancer tissues

Pathological detection using immunohistochemistry (IHC) has become an indispensable process in the diagnosis confirmation of various cancers. However, the production of monoclonal antibodies is always very complex, expensive and time-consuming, and the batch differences are significant due to the corporeity and health statuses of animals may be different. In this work, an aptamer-based histochemistry (aptahistochemistry) assay was developed using a DNA aptamer for specific diagnosis of clinical breast cancer tissue sections. This aptahistochemistry assay can specifically distinguish Luminal A breast cancer molecular subtype from Luminal B (HER2+), HER2-enriched, and triple-negative breast cancer molecular subtypes, as well as para-carcinoma tissue, mastitis tissue and normal breast tissue. The accuracy of this aptahistochemistry assay for the diagnosis of Luminal A breast cancer was as high as 80%, which showed a great potential for clinical pathological diagnosis applications.     

p53 overexpression represses androgen-mediated induction of NKX3.1 in a prostate cancer cell line

Prostate cancer is a disease involving complicated multiple-gene alterations. Both NKX3.1 and p53 are related to prostate cancer and play crucial roles in prostate cancer progression. However, little is known about the relationships and interactions between p53 and NKX3.1 in prostate cancer. We found that NKX3.1 expression is down-regulated by over-expression of wild type (wt) p53 in prostate cancer LNCaP cells. NKX3.1 is down-regulated at both the mRNA and protein levels by p53 over- expression due to either transient transfection of exogenous p53 or induction of endogenous p53. p53 over-expression represses androgen-induced transactivation of NKX3.1 by inhibiting the promoter of the androgen acceptor (AR) gene and by blocking AR-DNA binding activity. In addition, transfection with the p21 expression vector (pPSA-p21) showed that p21 does not reduce NKX3.1 expression, indicating that NKX3.1 expression is not the result of nonspecific effects of cell growth arrest. Our results provide biochemical and cellular biologic evidence that NKX3.1 is down-regulated by p53 over-expression in prostate cancer cells.     

A Comparative Study of Receptor-Targeted Magnetosome and HSA-Coated Iron Oxide Nanoparticles as MRI Contrast-Enhancing Agent in Animal Cancer Model

Magnetosomes are specialized organelles arranged in intracellular chains in magnetotactic bacteria. The superparamagnetic property of these magnetite crystals provides potential applications as contrast-enhancing agents for magnetic resonance imaging. In this study, we compared two different nanoparticles that are bacterial magnetosome and HSA-coated iron oxide nanoparticles for targeting breast cancer. Both magnetosomes and HSA-coated iron oxide nanoparticles were chemically conjugated to fluorescent-labeled anti-EGFR antibodies. Antibody-conjugated nanoparticles were able to bind the MDA-MB-231 cell line, as assessed by flow cytometry. To compare the cytotoxic effect of nanoparticles, MTT assay was used, and according to the results, HSA-coated iron oxide nanoparticles were less cytotoxic to breast cancer cells than magnetosomes. Magnetosomes were bound with higher rate to breast cancer cells than HSA-coated iron oxide nanoparticles. While 250 μg/ml of magnetosomes was bound 92 ± 0.2%, 250 μg/ml of HSA-coated iron oxide nanoparticles was bound with a rate of 65 ± 5%. In vivo efficiencies of these nanoparticles on breast cancer generated in nude mice were assessed by MRI imaging. Anti-EGFR-modified nanoparticles provide higher resolution images than unmodified nanoparticles. Also, magnetosome with anti-EGFR produced darker image of the tumor tissue in T2-weighted MRI than HSA-coated iron oxide nanoparticles with anti-EGFR. In vivo MR imaging in a mouse breast cancer model shows effective intratumoral distribution of both nanoparticles in the tumor tissue. However, magnetosome demonstrated higher distribution than HSA-coated iron oxide nanoparticles according to fluorescence microscopy evaluation. According to the results of in vitro and in vivo study results, magnetosomes are promising for targeting and therapy applications of the breast cancer cells.     

Transcriptional targeting of gene expression in breast cancer by the promoters of protein regulator of cytokinesis 1 and ribonuclease reductase 2

For cancer gene therapy, cancer-specific over- expression of a therapeutic gene is required to reduce side effects derived from expression of the gene in normal cells. To develop such an expression vector, we searched for genes over-expressed and/or specifically expressed in cancer cells using bioinformatics and have selected genes coding for protein regulator of cytokinesis 1 (PRC1) and ribonuclease reductase 2 (RRM2) as candidates. Their cancer-specific expressions were confirmed in both breast cancer cell lines and patient tissues. We compared each promoter's cancer-specific activity in the breast normal and cancer cell lines using the luciferase gene as a reporter and confirmed cancer-specific expression of both PRC1 and RRM2 promoters. To test activities of these promoters in viral vectors, the promoters were also cloned into an adeno-associated viral (AAV) vector containing green fluorescence protein (GFP) as the reporter. The GFP expression levels by these promoters were various depending on cell lines tested and, in MDA-MB-231 cells, GFP activities derived from the PRC1 and RRM2 promoters were as strong as that from the cytomegalovirus (CMV) promoter. Our result showed that a vector containing the PRC1 or RRM2 promoter could be used for breast cancer specific overexpression in gene therapy.     

2D-DIGE proteomic analysis of changes in estrogen/progesterone-induced rat breast hyperplasia upon treatment with the Mongolian remedy RuXian-I

RuXian-I has traditionally been used as a remedy for breast hyperplasia in the Inner Mongolia Autonomous Region of China. As a first step toward the investigation of biomarkers associated with RuXian-I treatment, a proteome-wide analysis of rat breast tissue was conducted. First, rat breast hyperplasia was induced by injection of estradiol and progesterone. After treatment with RuXian-I, there is a marked decrease in the hyperplasia, as can be shown by decreases in the nipple diameter and the pathological changes in breast. Subsequently, we used an approach that integrates size-based 2D-DIGE, MALDI-TOF/TOF-MS, and bioinformatics to analyze data from the control group, the model group and the RuXian-I treatment group. Using this approach, seventeen affected proteins were identified. Among these, 15 (including annexin A1, annexin A2, superoxide dismutase [Mn], peroxiredoxin-1, translationally-controlled tumor protein and a B-crystallin) were significantly up-regulated in the model group and down-regulated upon treatment with RuXian-I, and two (Tpil protein and myosin-4) have the opposite change trend. The expression of annexin A1 was confirmed using immunohistochemistry. The expression of superoxide dismutase (SOD) activity was confirmed biochemically. These results indicated that RuXian-I treats rat breast hyperplasia through regulation of cell cycle, immune system, metabolic, signal transduction, etc. The differential expressions of these proteins (annexin A1, superoxide dismutase [Mn], alpha B-crystallins and translationally controlled tumor protein, among others) were associated with occurrence and metastasis of breast cancer. These findings might provide not only far-reaching valuable insights into the mechanism of RuXian-I action, but also leads for prognosis and diagnosis of breast hyperplasia and breast cancer.     

Detection and expression analysis of recombinant proteins in plant-derived complex mixtures using nanoUPLC-MS(E)

The use of mass spectrometry to identify recombinant proteins that are expressed in total soluble proteins (TSPs) from plant extracts is necessary to accelerate further processing steps. For example, the method consists of TSP sample preparation and trypsin digestion prior to the preliminary characterization using nanoUPLC-MS(E) analysis of the recombinant proteins that are expressed in TSP samples of transgenic soybean seeds. A TSP sample as small as 50 μg can be effectively analyzed. In this study, transgenic soybean seeds that expressed recombinant cancer testis antigen (CTAG) were used. The procedure covered 30% of the protein sequence and was quantified at 0.26 ng, which corresponded to 0.1% of the TSP sample. A comparative proteomic profile was generated by the comparison of a negative control and sample that showed a unique expression pattern of CTAG in a transgenic line. The experimental data from the TSP extraction, sample preparation and data analysis are discussed herein.     

Expression of GD3 synthase modifies ganglioside profile and increases migration of MCF-7 breast cancer cells

B- and c-series of gangliosides are over-expressed in neuro-ectoderm-related cancers, including breast cancer. It has been shown that G_D3 ganglioside is over-expressed in about 50% of invasive ductal breast carcinoma and the G_D3 synthase (GD3S) gene displays higher expression among estrogen receptor (ER) negative breast tumors. We previously showed that GD3S expression in MDA-MB-231 breast cancer cells induces the expression of G_D2 and increased cell proliferation and migration via a G_D2-dependent activation of c-Met receptor. Here, we show that in ER-positive MCF-7 breast cancer cells, GD3S expression resulted in an increase of G_D1b, which was associated with a decrease of G_M1a and G_M2. Meanwhile, GD3S expressing MCF-7 cells exhibited an increased migration without any modification of proliferation rate. Therefore, GD3S expression can result in different modifications of both ganglioside profiles and cell phenotypes depending on breast cell types.     

Multifunctional polymeric nanoparticles from diverse bioactive agents

We present a rational approach for assembling diverse bioactive agents, such as DNA, proteins, and drug molecules, into core-shell multifunctional polymeric nanoparticles (PNPs) that can be internalized in human breast cancer cells. Using ring-opening metathesis polymerization (ROMP), block copolymers containing small-molecule drug segments (>50% w/w) and tosylated hexaethylene glycol segments were prepared and assembled into PNPs that allowed for the surface conjugation of single-stranded DNA sequences and/or tumor-targeting antibodies. The resulting antibody-functionalized particles were readily uptaken by breast cancer cells that overexpressed the corresponding antigens.     

A proteomics study on human breast cancer cell lines by fluorogenic derivatization-liquid chromatography/tandem mass spectrometry

Although several molecular markers for human breast cancer exist, their versatility is limited. Here we demonstrate, through a differential proteome analysis utilizing the fluorogenic derivatization-liquid chromatography/tandem mass spectrometry (FD-LC-MS/MS) method between seven cancer cells and one normal cell, that the presence of cooperatively expressed annexin-2 and galectin-1 without tropomyosin-1 in a tissue could be used to diagnose metastatic breast cancer. Interestingly, in a metastatic cancer cell, the expression of the former two together with highly expressed cofilin-1 activates the Rho signal pathway to aggressively form disorganized actin filaments. Despite the excess expression of annexin-2 and galectin-1 in the normal cell, the highly expressed tropomyosin-1 counteracted the activity of cofilin-1 and stabilized the filaments, resulting in the restoration of the disorganization. This phenomenon suggests that enhancement of tropomyosin-1 should be used as therapy for metastatic breast cancer.     

Inhibitory effect and mechanism on antiproliferation of isoatriplicolide tiglate (PCAC) from Paulownia Coreana

Paulownia coreana has traditionally been used as the medicine and health food in the treatment of cancer and infectious diseases. In the present study, a new antiproliferation agent, isoatriplicolide tiglate (PCAC) was isolated from the chloroform soluble fraction of the leaves of Paulownia coreana. The antiproliferation activities of PCAC plant extract was examined in breast and cervical cancer cell lines in a time-and dose-dependent manners. Our in vitro experiments showed that PCAC suppresses the cell growth and proliferation of cancer cells at a relatively low concentration (< 10 μg/mL) and induces apoptosis at a high concentration (> 50 μg/mL). Western blot analysis showed that concentration higher than 50 μg/mL induces a time-dependent increase in the percentage of apoptotic cells. In this case, PCAC uses both extrinsic and intrinsic pathways for the apoptosis. PCAC treatment decreased the expression of pro-caspase 8, 9, and 3, the main regulators of apoptotic cell death, in MDA-MB-231 cells, accompanied by the activation of caspase 8, 9, and 3. More importantly, PCAC inhibited the in vitro proliferation of six other human breast and cervical cancer cell lines. In conclusion, our data strongly suggest that PCAC acts as an antiproliferation agents particularly against breast and cervical cancers by inducing cell cycle arrest in the S/G2 phase and caspase dependent apoptosis at relatively low (< 10 μg/mL) and high (> 50 μg/mL) concentrations, respectively.     

Cowpea mosaic virus capsid: a promising carrier for the development of carbohydrate based antitumor vaccines

Immunotherapy targeting tumor cell surface carbohydrates is a promising approach for cancer treatment. However, the low immunogenecity of carbohydrates presents a formidable challenge. We describe here the enhancement of carbohydrate immunogenicity by an ordered display on the surface of the cowpea mosaic virus (CPMV) capsid. The Tn glycan, which is overexpressed on numerous cancer cell surfaces, was selected as the model antigen for our study. Previously it has been shown that it is difficult to induce a strong T cell-dependent immune response against the monomeric form of Tn presented in several ways on different carriers. In this study, we first synthesized Tn antigens derivatized with either a maleimide or a bromoacetamide moiety that was conjugated selectively to a cysteine mutant of CPMV. The glycoconjugate was then injected into mice and pre- and post-immune antibody levels in the mice sera were measured by enzyme-linked immunosorbant assays. High total antibody titers and, more importantly, high IgG titers specific for Tn were obtained in the post-immune day 35 serum, suggesting the induction of T cell-dependent antibody isotype switching by the glycoconjugate. The antibodies generated were able to recognize Tn antigens presented in their native conformations on the surfaces of both MCF-7 breast cancer cells and the multidrug resistant breast cancer cell line NCI-ADR RES. These results suggest that the CPMV capsid can greatly enhance the immunogenicity of weak antigens such as Tn and this can provide a promising tool for the development of carbohydrate based anti-cancer vaccines.     

Triptolide-induced suppression of phospholipase D expression inhibits proliferation of MDA-MB-231 breast cancer cells

In spite of the importance of phospholipase D (PLD) in cell proliferation and tumorigenesis, little is known about the molecules regulating PLD expression. Thus, identification of small molecules inhibiting PLD expression would be an important advance for PLD-mediated physiology. We examined one such here, denoted "Triptolide", which was identified in a chemical screen for inhibitors of PLD expression using cell assay system based on measurement of PLD promoter activity. Triptolide significantly suppressed the expression of both PLD1 and PLD2 with sub-µM potency in MDA-MB-231 breast cancer cells as analyzed by promoter assay and RT-PCR. Moreover, triptolide abolished the protein level of PLD in a time and dose-dependent manner. Triptolide-induced PLD1 downregulation was also observed in all the cancer cells examined, suggesting a general phenomenon detected in various cancer cells. Decrease of PLD expression by triptolide suppressed both basal and PMA-induced PLD activity. In addition, triptolide inhibited activation of NFκB which increased PLD1 expression. Ultimately, downregulation of PLD by triptolide inhibited proliferation of breast cancer cells. Taken together, we demonstrate that triptolide suppresses the expression of PLD via inhibition of NFκB activation and then decreases cell proliferation.