Ursodeoxycholic Acid Modulates the Interaction of miR-21 and Farnesoid X Receptor and NF-κB Signaling.

(1) Background: This study investigates the effects of Ursodeoxycholic acid (UDCA) on NF-κB signaling, farnesoid X receptor (FXR) singling, and microRNA-21 in HepG2 cells. (2) Methods: HepG2 cells were treated with lipopolysaccharide (LPS) to simulate hepatic inflammation. The investigation focused on the expression of NF-κB activation, which was analyzed using Western blot, confocal microscopy, and Electrophoretic Mobility-shift Assays (EMSA). Additionally, NF-κB and farnesoid X receptor (FXR) singling expressions of micro-RNA-21, COX-2, TNF-α, IL-6, cyp7A1, and shp were assessed by RT-PCR. (3) Results: UDCA effectively downregulated LPS-induced expressions of NF-κB/65, p65 phosphorylation, and also downregulated FXR activity by Western blot. Confocal microscopy and EMSA results confirmed UDCA's role in modulating NF-κB signaling. UDCA reduced the expressions of LPS-induced COX-2, TNF-α, and IL-6, which were related to NF-κB signaling. UDCA downregulated LPS-induced cyp7A1 gene expression and upregulated shp gene expression, demonstrating selective gene regulation via FXR. UDCA also significantly decreased micro-RNA 21 levels. (4) Conclusions: This study demonstrates UDCA's potent anti-inflammatory effects on NF-κB and FXR signaling pathways, and thus its potential to modulate hepatic inflammation and carcinogenesis through interactions with NF-κB and FXR. The decrease in micro-RNA 21 expression further underscores its therapeutic potential.

Inhibition of Polyinosinic-Polycytidylic Acid-Induced Acute Pulmonary Inflammation and NF-κB Activation in Mice by a Banana Plant Extract.

NF-κB activation is pivotal for the excess inflammation causing the critical condition and mortality of respiratory viral infection patients. This study was aimed to evaluate the effect of a banana plant extract (BPE) on suppressing NF-κB activity and acute lung inflammatory responses in mice induced by a synthetic double-stranded RNA viral mimetic, polyinosinic-polycytidylic acid (poly (I:C)). The inflammatory responses were analyzed by immunohistochemistry and HE stains and ELISA. The NF-κB activities were detected by immunohistochemistry in vivo and immunofluorescence and Western blot in vitro. Results showed that BPE significantly decreased influx of immune cells (neutrophils, lymphocytes, and total WBC), markedly suppressed the elevation of pro-inflammatory cytokines and chemokines (IL-6, RANTES, IFN-γ, MCP-1, keratinocyte-derived chemokine, and IL-17), and restored the diminished anti-inflammatory IL-10 in the bronchoalveolar lavage fluid (BALF) of poly (I:C)-stimulated mice. Accordingly, HE staining revealed that BPE treatment alleviated poly (I:C)-induced inflammatory cell infiltration and histopathologic changes in mice lungs. Moreover, immunohistochemical analysis showed that BPE reduced the pulmonary IL-6, CD11b (macrophage marker), and nuclear NF-κB p65 staining intensities, whilst restored that of IL-10 in poly (I:C)-stimulated mice. In vitro, BPE antagonized poly(I:C)-induced elevation of IL-6, nitric oxide, reactive oxygen species, NF-κB p65 signaling, and transient activation of p38 MAPK in human lung epithelial-like A549 cells. Taken together, BPE ameliorated viral mimic poly(I:C)-induced acute pulmonary inflammation in mice, evidenced by reduced inflammatory cell infiltration and regulation of both pro- and anti-inflammatory cytokines. The mechanism of action might closely associate with NF-κB signaling inhibition.

Identification of Scoparone from Chinese Olive Fruit as a Modulator of Macrophage Polarization.

Chinese olive (Canarium album L.) has been highlighted for its remarkable health benefits. We previously showed that the ethyl acetate fraction of Chinese olive (COE) is an effective anti-inflammatory agent. In this study, we used a luciferase-based RAW 264.7 cell platform to detect the transcriptional activity of NF-κB, a key mediator of inflammation, and the promoter activity of its downstream target, COX-2. Through functional-oriented screening using these platforms, we further divided COE into several subfractions. Subsequently, we used silica gel column chromatography for purification, and the active compounds were separated and isolated by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The structure of the resulting compound with high anti-inflammatory activity was then identified as scoparone. Our results showed that scoparone not only inhibited lipopolysaccharide (LPS)-induced secretion of nitric oxide and suppressed M1 macrophage markers (iNOS, Il-6, Ccl2, and Tnf-α) but also markedly decreased the production of pro-inflammatory cytokines (IL-6, CCL2, and TNF-α). Treatment with scoparone significantly reduced the protein level of TNF-α in LPS-treated bone-marrow-derived macrophages (BMDMs). In addition, scoparone promoted macrophages toward an M2 anti-inflammatory phenotype, as determined by the significantly increased gene expression of M2 macrophage markers (Arg1, Ym1, Mrc1, Il-10, and Cd206) and the protein level of Arg1. This study indicates that COE fruit has high therapeutic potential for various inflammatory diseases as a result of switching the macrophage phenotype from pro-inflammatory M1 to anti-inflammatory M2.

Nuclear-localized CTEN is a novel transcriptional regulator and promotes cancer cell migration through its downstream target CDC27.

C-terminal tensin-like (CTEN) is a tensin family protein typically localized to the cytoplasmic side of focal adhesions, and primarily contributes to cell adhesion and migration. Elevated expression and nuclear accumulation of CTEN have been reported in several types of cancers and found to be associated with malignant behaviors. However, the function of nuclear CTEN remains elusive. In this study, we report for the first time that nuclear CTEN associates with chromatin DNA and occupies the region proximal to the transcription start site in several genes. The mRNA expression level of CTEN positively correlates with that of one of its putative target genes, cell division cycle protein 27 (CDC27), in a clinical colorectal cancer dataset, suggesting that CTEN may play a role in the regulation of CDC27 gene expression. Furthermore, we demonstrated that CTEN is recruited to the promoter region of the CDC27 gene and that the mRNA expression and promoter activity of CDC27 are both reduced when CTEN is downregulated. In addition, we found that enhanced nuclear accumulation of CTEN in HCT116 cells by overexpression of CTEN fused with nuclear localization signals increases CDC27 transcript levels and promoter activity. The increased nuclear-localized CTEN also significantly promotes cell migration, and the migratory ability is suppressed when CDC27 is knocked down. These results demonstrate that nuclear CTEN regulates CDC27 expression transcriptionally and promotes cell migration through CDC27. Our findings provide new insights into CTEN moonlighting in the nucleus as a DNA-associated protein and transcriptional regulator involved in modulating cancer cell migration.

Analysis and identification of phenolic compounds with antiproliferative activity from Chinese olive (Canarium album L.) fruit extract by HPLC-DAD-SPE-TT-NMR.

Chinese olives (Canarium album L.) are rich in phenolic compounds, exhibiting a broad spectrum of potential clinical applications. This study is the first report on the isolation and elucidation of bioactive compounds with high antiproliferative activity from the ethyl acetate fraction of a Chinese olive fruit methanolic extract (CO-EtOAc). We used the WST-1 assay to determine which subfractions of CO-EtOAc had significant antiproliferative activity using the murine colon cancer cell line CT26. Subsequently, the functional compounds were characterized by the hyphenated technique and high-performance liquid chromatography-diode array detector-solid phase extraction-transfer tube-nuclear magnetic resonance (HPLC-DAD-SPE-TT-NMR). Thirteen phenolic constituents were identified from the antiproliferation-enhancing subfractions of CO-EtOAc, including two new compounds, 2,4-didehydrochebulic acid 1,7-dimethyl ester (5) and 1-hydroxybrevifolin (7), which were further purified and found to exhibit marked antiproliferative activity. Chebulic acid dimethyl ester (2), which was isolated from C. album for the first time, also possessed antiproliferative activity.

Tensins - emerging insights into their domain functions, biological roles and disease relevance.

Tensins are a family of focal adhesion proteins consisting of four members in mammals (TNS1, TNS2, TNS3 and TNS4). Their multiple domains and activities contribute to the molecular linkage between the extracellular matrix and cytoskeletal networks, as well as mediating signal transduction pathways, leading to a variety of physiological processes, including cell proliferation, attachment, migration and mechanical sensing in a cell. Tensins are required for maintaining normal tissue structures and functions, especially in the kidney and heart, as well as in muscle regeneration, in animals. This Review discusses our current understanding of the domain functions and biological roles of tensins in cells and mice, as well as highlighting their relevance to human diseases.

Histone acetyltransferase p300 mediates the upregulation of CTEN induced by the activation of EGFR signaling in cancer cells.

Upregulation of C-terminal tensin-like (CTEN) is induced by the activation of epidermal growth factor receptor (EGFR) signaling and mainly contributes to cancer cell migration and invasion. CTEN is known as a downstream target of the EGFR-RAF-MEK-ERK pathway but the regulatory mechanism underlying EGFR signaling regulates the increased expression of CTEN is still incompletely understood. In this study, we investigated the epigenetic regulation of CTEN gene transcription upon EGFR activation. Analyses of chromatin accessibility revealed that the structure of CTEN promoter became more loosed and the acetylation state of the histone tails within the core promoter region was increased after EGF treatment. Moreover, activation of EGFR signaling facilitates histone acetyltransferase p300 to be recruited to CTEN promoter through MEK-ERK pathway. MEK-ERK activation also induces the phosphorylation of p300, thereby enhancing the levels of histone acetylation within CTEN promoter, which in turn upregulates CTEN gene expression. Our work provides new insights into the actions of EGFR signaling to upregulate CTEN, which may lead to the rational design of novel therapeutic approaches.

Downregulation of C-Terminal Tensin-Like Protein (CTEN) Suppresses Prostate Cell Proliferation and Contributes to Acinar Morphogenesis.

C-terminal tensin-like protein (CTEN) is a member of tensin family, which is crucial for the assembly of cell-matrix adhesome. Unlike other tensins, CTEN is selectively expressed only in a few tissues such as the prostate. However, the biological relevance of CTEN in normal prostate is poorly understood. In this study, we revealed that CTEN is selectively expressed in the prostate epithelial cells and enriched in the basal compartment. Knockdown of CTEN in RWPE-1 cells suppresses cell proliferation and results in G1/S cell cycle arrest as well as the accumulation of cyclin-dependent kinase (CDK) inhibitors, p21 and p27. Moreover, the expression of CTEN is decreased during acinar morphogenesis using Matrigel-based three-dimensional (3D) culture. In the course of acinar formation, induction of CTEN reactivates focal adhesion kinase (FAK) Y397 phosphorylation and disrupts the acini structure. This study, to our knowledge, is the first report demonstrating that downregulation of CTEN is required for luminal differentiation and acinar formation.

The methanol-ethyl acetate partitioned fraction from Chinese olive fruits inhibits cancer cell proliferation and tumor growth by promoting apoptosis through the suppression of the NF-κB signaling pathway.

Chinese olives (Canarium album L.) have historically been used for medicinal purposes rather than commercially for oil. In this report, we reveal that the methanol-ethyl acetate partitioned fraction from Chinese olive fruits (MEO), of which ellagic acid accounted for 12%, exhibited profound anti-proliferative activities in the human colon cancer cell line, HCT116. Additionally, oral administration of MEO remarkably inhibited the tumor growth of subcutaneously implanted CT26 cells, a mouse colon carcinoma cell line, in BALB/c mice. Treatment with MEO induced a significant increase in the percentage of apoptotic cells and resulted in poly(ADP-ribose) polymerase (PARP) cleavage, suggesting that MEO inhibits cancer cell proliferation by promoting apoptosis. Our study also showed that MEO exerted the most potent effect on the inhibition of NF-κB-mediated signaling among the partitioned fractions from Chinese olives. This process employed the use of reporter-based bio-platforms that are capable of detecting the activation of NF-κB. In addition, phosphorylation of NF-κB signaling-associated proteins, IKKα/ß, IκBα, and p65, was reduced in MEO-incubated cancer cells, indicating that MEO suppresses NF-κB activation. Moreover, MEO treatment significantly suppressed lipopolysaccharide (LPS)-induced cancer cell proliferation, demonstrating that MEO promotes cancer cell apoptosis through the inhibition of the NF-κB signaling pathway. In summary, our findings demonstrate that the methanol-ethyl acetate partitioned fraction from Chinese olive fruits inhibits cancer cell proliferation and tumor growth by promoting apoptosis through the suppression of NF-κB signaling. Therefore, the Chinese olive fruit has promising potential in cancer treatment.

ΔNp63α Transcriptionally Regulates the Expression of CTEN That Is Associated with Prostate Cell Adhesion.

p63 is a member of the p53 transcription factor family and a linchpin of epithelial development and homeostasis. p63 drives the expression of many target genes involved in cell survival, adhesion, migration and cancer. In this study, we identify C-terminal tensin-like (CTEN) molecule as a downstream target of ΔNp63α, the predominant p63 isoform expressed in epithelium. CTEN belongs to the tensin family and is mainly localized to focal adhesions, which mediate many biological events such as cell adhesion, migration, proliferation and gene expression. Our study demonstrate that ΔNp63 and CTEN are both highly expressed in normal prostate epithelial cells and are down-regulated in prostate cancer. In addition, reduced expression of CTEN and ΔNp63 is correlated with prostate cancer progression from primary tumors to metastatic lesions. Silencing of ΔNp63 leads to decreased mRNA and protein levels of CTEN. ΔNp63α induces transcriptional activity of the CTEN promoter and a 140-bp fragment upstream of the transcription initiation site is the minimal promoter region required for activation. A putative binding site for p63 is located between -61 and -36 within the CTEN promoter and mutations of the critical nucleotides in this region abolish ΔNp63α-induced promoter activity. The direct interaction of ΔNp63α with the CTEN promoter was demonstrated using a chromatin immunoprecipitation (ChIP) assay. Moreover, impaired cell adhesion caused by ΔNp63α depletion is rescued by over-expression of CTEN, suggesting that CTEN is a downstream effector of ΔNp63α-mediated cell adhesion. In summary, our findings demonstrate that ΔNp63α functions as a trans-activation factor of CTEN promoter and regulates cell adhesion through modulating CTEN. Our study further contributes to the potential regulatory mechanisms of CTEN in prostate cancer progression.

Tracking and Finding Slow-Proliferating/Quiescent Cancer Stem Cells with Fluorescent Nanodiamonds.

Quiescent cancer stem cells (CSCs) have long been considered to be a source of tumor initiation. However, identification and isolation of these cells have been hampered by the fact that commonly used fluorescent markers are not sufficiently stable, both chemically and photophysically, to allow tracking over an extended period of time. Here, it is shown that fluorescent nanodiamonds (FNDs) are well suited for this application. Genotoxicity tests of FNDs with comet and micronucleus assays for human fibroblasts and breast cancer cells indicate that the nanoparticles neither cause DNA damage nor impair cell growth. Using AS-B145-1R breast cancer cells as the model cell line for CSC, it is found that the FND labeling outperforms 5-ethynyl-2'-deoxyuridine (EdU) and carboxyfluorescein diacetate succinimidyl ester (CFSE) in regards to its long-term tracking capability (>20 d). Moreover, through a quantification of their stem cell activity by measuring mammosphere-forming efficiencies (MFEs) and self-renewal rates, the FND-positive cells are identified to have an MFE twice as high as that of the FND-negative cells isolated from the same dissociated mammospheres. Thus, the nanoparticle-based labeling technique provides an effective new tool for tracking and finding slow-proliferating/quiescent CSCs in cancer research.

Increased susceptibility of H-Ras(G12V)-transformed human urothelial cells to the genotoxic effects of sodium arsenite.

Inorganic arsenite (iAs) is a human carcinogen. Numerous studies have shown that mutation-activated H-Ras is frequently observed in human urothelial carcinomas. The interaction between iAs, an environmental factor, and H-Ras, an oncogene, is not clear. In this study, we explored the genotoxic effects of iAs in human urothelial cells ectopically expressing H-Ras (G12V) an activated H-Ras oncogene. Our results showed that H-Ras(G12V)-transformed human urothelial cells (HUC-RAS) were more susceptible to arsenite-induced cell death, DNA damage, micronuclei formation and anchorage-independent growth than control cells (HUC-neo). Furthermore, iAs treatment induced higher intracellular levels of reactive oxygen species (ROS) in the HUC-RAS cells than in the HUC-neo cells. N-acetyl-L-cysteine could suppress the iAs-induced increases in ROS and genetic damage. We further demonstrated that the intracellular glutathione levels were significantly elevated by the iAs treatment of the HUC-neo cells, but that this effect was not observed in the HUC-RAS cells. The iAs treatment induced higher superoxide dismutase activity in the HUC-neo cells than in the HUC-RAS cells. Alternatively, catalase activity was higher in the HUC-RAS cells than in the HUC-neo cells, but this enzyme was significantly suppressed by iAs. Moreover, iAs activated the ERK and JNK signaling pathways, which are involved in iAs-induced ROS production and genetic damage. Taken together, our present results suggest that elevated catalase activity in H-Ras(G12V)-transformed cells is significantly suppressed by iAs via activation of ERK and JNK signaling pathways and hence attenuate the defense of the neoplastic transformed cells against iAs-induced oxidative injuries.

Phylogenetic analysis, expression patterns, and transcriptional regulation of human CTEN gene.

Cten is a focal adhesion molecule and a member of the tensin family. Its expression is highly enriched in the prostate and placenta, suggesting that cten gene might be closely associated with mammalian species. Recent studies have reported that cten expression is frequently up-regulated in a variety of cancers and its levels appear to correlate with tumorigenicity. Here, we have (1) analyzed cten sequences of various species to build a phylogenetic tree, (2) examined cten mRNA levels in human and mouse tissues to establish its expression profiles, and (3) determined the promoter region of human CTEN gene in cell lines and in a mouse model to understand its transcriptional regulation. Our analyses indicate that all currently known cten genes are present in mammals. The prostate and placenta are the two most cten abundant tissues in human and mouse, meanwhile brain and lung also express low levels of cten. Results from cell culture reporter assays demonstrate that a 327-bp fragment is the shortest functional promoter. All functional promoter constructs produce 40- to 160-fold increases in luciferase reporter activities in normal prostate cells, whereas lower activities (<40-fold) are detected in non-prostatic cell lines. To evaluate CTEN promoter activity in mice and develop a new tissue specific Cre recombinase mouse model, we have established pCTEN-Cre:R26R mice by crossing R26R ß-galactosidase reporter mice with pCTEN-Cre transgenic mice, in which the 327-bp cten promoter drives the expression of Cre recombinase. X-gal analysis has shown strong ß-galactosidase activities in the prostate, brain, and few other tissues in pCTEN-Cre:R26R mice. Altogether, we have identified the promoter region of human cten gene and provided a useful tool for investigating cell lineages and generating tissue-specific knockout or knockin mice.

DLC1 negatively regulates angiogenesis in a paracrine fashion.

The Rho GTPase-activating protein DLC1 is a tumor suppressor that is often deleted in liver cancer and downregulated in other cancers. DLC1 regulates the actin cytoskeleton, cell shape, adhesion, migration, and proliferation through its Rho GTPase-activating protein activity and focal adhesion localization. In this study, we silenced DLC1 in nonmalignant prostate epithelial cells to explore its tumor suppression functions. Small hairpin RNA-mediated silencing of DLC1 was insufficient to promote more aggressive phenotypes associated with tumor cell growth. In contrast, DLC1 silencing promoted pro-angiogenic responses through vascular endothelial growth factor (VEGF) upregulation, accompanied by the accumulation of hypoxia-inducible factor 1α and its nuclear localization. Notably, modulation of VEGF expression by DLC1 was dependent on epidermal growth factor receptor-MAP/ERK kinase-hypoxia-inducible factor 1 signaling but on RhoA pathways. Clinically, VEGF upregulation is a highly significant event in prostate cancers in which DLC1 is downregulated. Thus, our results strongly suggest that loss of DLC1 may serve as a "second hit" in promoting angiogenesis in a paracrine fashion during tumorigenesis.

Up-regulation of C-terminal tensin-like molecule promotes the tumorigenicity of colon cancer through beta-catenin.

C-terminal tensin-like (cten) is a focal adhesion molecule belonging to the tensin family. Previous studies have suggested that cten may function as a prostate-specific tumor suppressor. Here, we show that although cten is expressed at a very low level in normal colon, its expression is significantly up-regulated in colon cancer. Furthermore, a high population of cten is found in the nucleus, where it interacts with beta-catenin, a critical player in the canonical Wnt pathway. This interaction may contribute to the role of cten in enhancing the colony formation, anchorage-independent growth, and invasiveness of colon cancer cells. Our studies have identified cten as a novel nuclear partner of beta-catenin, showed an oncogenic activity of cten in colon cancers, and revealed cten as a potential biomarker and target for colon cancers.

Mechanism of patulin-induced apoptosis in human leukemia cells (HL-60).

Patulin (PAT) is a fungal secondary metabolite that exhibits potential cellular and animal toxicities. In this study, human promyelocytic leukemia (HL-60) cells were used to elucidate the mechanism and death mode associated with PAT. Morphological evidence of apoptosis, including membrane blebbing, nuclei fragmentation and DNA laddering formation was clearly observed 6h after exposure to PAT. The results of Western blotting indicated that PAT activated various processed caspases, and cleaved DFF45 and poly (ADP-ribose) polymerase (PARP) in a dose-dependent manner; it also induced a time-dependent increase in caspase 3 and 9 catalytic activities. The apoptosis mediated by PAT in HL-60 was accompanied with cytochrome c release from mitochondria and Bcl-2 expression decrease. The presence of thiol-containing compounds with PAT dramatically reduced the caspase 3 activity that was triggered by PAT; the addition of antioxidants, including mannitol and Tiron, had a similar effect. However, the suppression of p53 protein expression by RNA interference (RNAi) in human embryonic kidney (HEK293) cells did not significantly modify PAT-elicited caspase 3 activity. These findings suggest that PAT-induced apoptosis is mediated through the mitochondrial pathway without the involvement of p53; the interaction with sulfhydryl groups of macromolecules by PAT and the subsequent generation of reactive oxygen species (ROS) plays a primary role in the apoptotic process.

Mutations in the focal adhesion targeting region of deleted in liver cancer-1 attenuate their expression and function.

Deleted in liver cancer-1 (DLC-1) is a RhoGTPase-activating protein (RhoGAP) domain containing tumor suppressor that is often down-regulated in various cancer types. Previously, we have shown that DLC-1 is recruited to focal adhesions by binding to the Src homology 2 domains of tensins and the focal adhesion localization is critical for the tumor suppression activity of DLC-1. To investigate whether mutations in the focal adhesion targeting (FAT) region might occur and attenuate the expression, localization, and function of DLC-1, we have first mapped the FAT region to the amino acid residues from 201 to 500, and then sequenced cDNAs and genomic DNAs encoding the FAT region from cancer patients. Several missense and nonsense mutations were detected. All missense mutations were further examined for the potential effect on the function of DLC-1. Although these mutations did not seem to affect the focal adhesion localization of DLC-1, the activities of suppressing tumor cell growth were impaired in two mutants: T301K and S308I. Consistent with the fact that the RhoGAP activity of DLC-1 is essential for inhibiting tumor cell growth, the RhoGAP activities were significantly reduced in these mutants, suggesting that the FAT region also contains a regulatory element for its COOH-terminal RhoGAP domain. Our studies have shown that mutations in DLC-1 may lead to loss of function and contribute to the tumorigenesis, and have revealed an allosteric regulation site for its RhoGAP activity.

Deleted in liver cancer-1 (DLC-1): a tumor suppressor not just for liver.

Deleted in liver cancer 1 (DLC-1), as its name implied, was originally isolated as a potential tumor suppressor gene often deleted in hepatocellular carcinoma. Further studies have indicated that down-expression of DLC-1 either by genomic deletion or DNA methylation is associated with a variety of cancer types including lung, breast, prostate, kidney, colon, uterus, ovary, and stomach. Re-expression of DLC-1 in cancer cells regulates the structure of actin cytoskeleton and focal adhesions and significantly inhibits cell growth, supporting its role as a tumor suppressor. This tumor suppressive function relies on DLC-1's RhoGTPase activating protein (RhoGAP) activity and steroidogenic acute regulatory (StAR)-related lipid transfer (START) domain, as well as its focal adhesion localization, which is recruited by the Src Homology 2 (SH2) domains of tensins in a phosphotyrosine-independent fashion. Therefore, the expression and subcellular localization of DLC-1 could be a useful molecular marker for cancer prognosis, whereas DLC-1 and its downstream signaling molecules might be therapeutic targets for the treatment of cancer.

A reciprocal tensin-3-cten switch mediates EGF-driven mammary cell migration.

Cell migration driven by the epidermal growth factor receptor (EGFR) propels morphogenesis and involves reorganization of the actin cytoskeleton. Although de novo transcription precedes migration, transcript identity remains largely unknown. Through their actin-binding domains, tensins link the cytoskeleton to integrin-based adhesion sites. Here we report that EGF downregulates tensin-3 expression, and concomitantly upregulates cten, a tensin family member that lacks the actin-binding domain. Knockdown of cten or tensin-3, respectively, impairs or enhances mammary cell migration. Furthermore, cten displaces tensin-3 from the cytoplasmic tail of integrin beta1, thereby instigating actin fibre disassembly. In invasive breast cancer, cten expression correlates not only with high EGFR and HER2, but also with metastasis to lymph nodes. Moreover, treatment of inflammatory breast cancer patients with an EGFR/HER2 dual-specificity kinase inhibitor significantly downregulated cten expression. In conclusion, a transcriptional tensin-3-cten switch may contribute to the metastasis of mammary cancer.

Development of a monoclonal antibody against domoic acid and its application in enzyme-linked immunosorbent assay and colloidal gold immunostrip.

A monoclonal antibody (mAb) specific to domoic acid was produced from a stable hybridoma cell line, 9F1F11, generated by the fusion of P3/NS1/1-AG4-1 myeloma cells with spleen cells isolated from a Balb/c mouse immunized with domoic acid--keyhole limpet hemocyanin. The 9F1F11 mAb belongs to the immunoglobulin G1 (kappa-chain) isotype. A competitive direct enzyme-linked immunosorbent assay (cdELISA) and a competitive indirect ELISA were established for antibody characterization. In the cdELISA, the concentration causing 50% inhibition (IC50) of binding of domoic acid-horseradish peroxidase to the antibody by domoic acid was found to be 0.58 ng/mL. A sensitive and rapid mAb-based colloidal gold immunostrip was also developed. The immunostrip assay, which has a detection limit of 5 ng/mL for domoic acid, can be completed in 10 min. Analysis of domoic acid in blue mussel samples revealed that data obtained from immunostrip were in a good agreement with those obtained from cdELISA. The mAb-based cdELISA and immunostrip assay established in this study were sensitive and accurate for rapid screening of domoic acid in shellfish samples.