Prognostic and predictive value of a five-molecule panel in resected pancreatic ductal adenocarcinoma: A multicentre study.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) has a devastating prognosis. The performance of clinicopathologic parameters and molecules as prognostic factors remains limited and inconsistent. The present study aimed to construct a multi-molecule biomarker panel to more accurately predict post-resectional prognosis of PDAC patients. METHODS: Firstly, a novel computational strategy integrating prognostic evidence from omics and literature on the basis of bioinformatics prediction (CIPHER) to generate the network, was designed to systematically identify potential high-confidence PDAC-related prognostic candidates. After specimens from 605 resected PDAC patients were retrospectively collected, 23 candidates were detected immunohistochemically in tissue-microarrays for the development cohort to construct a multi-molecule panel. Lastly, the panel was validated in two independent cohorts. FINDINGS: According to the constructed five-molecule panel, disease-specific survival (DSS) was significantly poorer in high-risk patients than in low-risk ones in development cohort (HR 2.15, 95%CI 1.51-3.05, P<0.0001; AUC 0.67). In two validation cohorts, similar significant differences between the two groups were also observed (HR 3.18 and 3.31, 95%CI 1.89-5.37 and 1.78-6.16, All P<0.0001; AUC 0.72 and 0.73). In multivariate analyses, this panel was the sole prognosticator that was significant in each cohort. Furthermore, its predictive power for long-term survival, higher than its individual constituents, could be largely enhanced by combination with traditional clinicopathological variables. Finally, adjuvant chemotherapy (ACT) correlated with better DSS only in high-risk patients, uni- and multi-variately, in all the cohorts. INTERPRETATION: The novel prognostic panel developed by a systematically network-based strategy presents strong ability in prediction of post-resectional survival of PDAC patients. Furthermore, panel-defined high-risk patients might benefit more from ACT.

Fucoidan affects oral squamous cell carcinoma cell functions in vitro by regulating FLNA-derived circular RNA.

Oral squamous cell carcinoma (OSCC) is one of the most common cancer types, with a high annual incidence. Although chemotherapy contributes to suppressing OSCC tumorigenesis, the available treatments result in poor prognosis because of local recurrence and regional lymph node metastasis. Thus, it is necessary to discover novel and safe drugs with greater effectiveness and fewer side effects. Fucoidan is a component of the cell wall of brown seaweed that has been shown to produce a wide range of biological activities. The present study aimed to investigate the effectiveness of fucoidan in treating OSCC. In in vitro studies, we found that fucoidan inhibited OSCC growth and suppressed migration and invasion of OSCC cells. In addition, the potential interaction between fucoidan and filamin A (FLNA)-derived circular RNA (circFLNA) was predicted using bioinformatics databases and then confirmed in OSCC samples and cell lines. Indeed, fucoidan increased the expression of circFLNA in OSCC cell lines. Furthermore, both fucoidan and circFLNA could mediate the expression of key proteins related to cell growth, apoptosis, migration, and invasion. In conclusion, our research demonstrated that fucoidan might be considered as a potential natural drug in the treatment of OSCC patients by targeting circFLNA.

The human allicin-proteome: S-thioallylation of proteins by the garlic defence substance allicin and its biological effects.

A single clove of edible garlic (Allium sativum L.) of about 10 g produces up to 5 mg of allicin (diallylthiosulfinate), a thiol-reactive sulfur-containing defence substance that gives injured garlic tissue its characteristic smell. Allicin induces apoptosis or necrosis in a dose-dependent manner but biocompatible doses influence cellular metabolism and signalling cascades. Oxidation of protein thiols and depletion of the glutathione pool are thought to be responsible for allicin's physiological effects. Here, we studied the effect of allicin on post-translational thiol-modification in human Jurkat T-cells using shotgun LC-MS/MS analyses. We identified 332 proteins that were modified by S-thioallylation in the Jurkat cell proteome which causes a mass shift of 72 Da on cysteines. Many S-thioallylated proteins are highly abundant proteins, including cytoskeletal proteins tubulin, actin, cofilin, filamin and plastin-2, the heat shock chaperones HSP90 and HSPA4, the glycolytic enzymes GAPDH, ALDOA, PKM as well the protein translation factor EEF2. Allicin disrupted the actin cytoskeleton in murine L929 fibroblasts. Allicin stimulated the immune response by causing Zn2+ release from proteins and increasing the Zn2+-dependent IL-1-triggered production of IL-2 in murine EL-4 T-cells. Furthermore, allicin caused inhibition of enolase activity, an enzyme considered a cancer therapy target. In conclusion, our study revealed the widespread extent of S-thioallylation in the human Jurkat cell proteome and showed effects of allicin exposure on essential cellular functions of selected targets, many of which are targets for cancer therapy.

Noninvasive Microsurgery Using Aptamer-Functionalized Magnetic Microdisks for Tumor Cell Eradication.

Magnetomechanical cell disruption using nano- and microsized structures is a promising biomedical technology used for noninvasive elimination of diseased cells. It applies alternating magnetic field (AMF) for ferromagnetic microdisks making them oscillate and causing cell membrane disruption with cell death followed by apoptosis. In this study, we functionalized the magnetic microdisks with cell-binding DNA aptamers and guided the microdisks to recognize cancerous cells in a mouse tumor in vivo. Only 10 min of the treatment with a 100 Hz AMF was enough to eliminate cancer cells from a malignant tumor. Our results demonstrate a good perspective of using aptamer-modified magnetic microdisks for noninvasive microsurgery for tumors.

Identification and expression of antioxidant and immune defense genes in the surf clam Mesodesma donacium challenged with Vibrio anguillarum.

The immune system in marine invertebrates is mediated through cellular and humoral components, which act together to address the action of potential pathogenic microorganisms. In bivalve mollusks biomolecules implicated in oxidative stress and recognition of pathogens have been involved in the innate immune response. To better understand the molecular basis of the immune response of surf clam Mesodesma donacium, qPCR approaches were used to identify genes related to its immune response against Vibrio anguillarum infection. Genes related to oxidative stress response and recognition of pathogens like superoxide dismutase (MdSOD), catalase (MdCAT), ferritin (MdFER) and filamin (MdFLMN) were identified from 454-pyrosequencing cDNA library of M. donacium and were evaluated in mantle, adductor muscle and gills. The results for transcripts expression indicated that MdSOD, MdFLMN and MdFER were primarily expressed in the muscle, while MdCAT was more expressed in gills. Challenge experiments with the pathogen V. anguillarum had showed that levels of transcript expression for MdSOD, MdCAT, MdFER, and MdFLMN were positively regulated by pathogen, following a time-dependent expression pattern with significant statistical differences between control and challenge group responses (p<0.05). These results suggest that superoxide dismutase, catalase, ferritin and filamin, could be contributing to the innate immune response of M. donacium against the pathogen V. anguillarum.

MicroRNA-155 inhibits proliferation and migration of human extravillous trophoblast derived HTR-8/SVneo cells via down-regulating cyclin D1.

MiR-155 is known to participate in various cellular processes by targeting gene expression. We previously revealed a link between miR-155 and perturbation of trophoblast invasion and differentiation. This study aimed to investigate the target molecule(s) of miR-155 on the influence on the proliferation and migration of trophoblast cells. Bioinformatics analysis showed that, at the 3' untranslated region (UTR) of cyclin D1, six bases are complementary to the seed region of miR-155. Luciferase assays and cyclin D1 3'UTR transfection assays validated that cyclin D1 3'UTR was the target of miR-155 in HTR-8/SVneo cells. Overexpression of miR-155 in HTR-8/SVneo cells reduced the level of cyclin D1 protein, decreased cell proliferation and invasion, and increased cell number at the G1 stage. Furthermore, the increased expression of miR-155 also regulated the protein levels of kinase inhibitory protein p27 and phosphorylated cytoskeletal protein filamin A. In conclusion, we found that cyclin D1 may be a target of miR-155 in HTR-8/SVneo cells, and demonstrated a negative regulatory role of miR-155 involved in cyclin D1/p27 pathway in proliferation and migration of the cells.

The calcium-sensing receptor-dependent regulation of cell-cell adhesion and keratinocyte differentiation requires Rho and filamin A.

Extracellular Ca(2+) (Ca(2+)(o)) functioning through the calcium-sensing receptor (CaR) induces E-cadherin-mediated cell-cell adhesion and cellular signals mediating cell differentiation in epidermal keratinocytes. Previous studies indicate that CaR regulates cell-cell adhesion through Fyn/Src tyrosine kinases. In this study, we investigate whether Rho GTPase is a part of the CaR-mediated signaling cascade regulating cell adhesion and differentiation. Suppressing endogenous Rho A expression by small interfering RNA (siRNA)-mediated gene silencing blocked the Ca(2+)(o)-induced association of Fyn with E-cadherin and suppressed the Ca(2+)(o)-induced tyrosine phosphorylation of ß-, γ-, and p120-catenin and formation of intercellular adherens junctions. Rho A silencing also decreased the Ca(2+)(o)-stimulated expression of terminal differentiation markers. Elevating the Ca(2+)(o) level induced interactions among CaR, Rho A, E-cadherin, and the scaffolding protein filamin A at the cell membrane. Inactivation of CaR expression by adenoviral expression of a CaR antisense complementary DNA inhibited Ca(2+)(o)-induced activation of endogenous Rho. Ca(2+)(o) activation of Rho required a direct interaction between CaR and filamin A. Interference of CaR-filamin interaction inhibited Ca(2+)(o)-induced Rho activation and the formation of cell-cell junctions. These results indicate that Rho is a downstream mediator of CaR in the regulation of Ca(2+)(o)-induced E-cadherin-mediated cell-cell adhesion and keratinocyte differentiation.

Amino acid-stimulated Ca2+ oscillations produced by the Ca2+-sensing receptor are mediated by a phospholipase C/inositol 1,4,5-trisphosphate-independent pathway that requires G12, Rho, filamin-A, and the actin cytoskeleton.

The G protein-coupled Ca(2+)-sensing receptor (CaR) is an allosteric protein that responds to two different agonists, Ca(2+) and aromatic amino acids, with the production of sinusoidal or transient oscillations in intracellular Ca(2+) concentration ([Ca(2+)](i)). Here, we examined whether these differing patterns of [Ca(2+)](i) oscillations produced by the CaR are mediated by separate signal transduction pathways. Using real time imaging of changes in phosphatidylinositol 4,5-biphosphate hydrolysis and generation of inositol 1,4,5-trisphosphate in single cells, we found that stimulation of CaR by an increase in the extracellular Ca(2+) concentration ([Ca(2+)](o)) leads to periodic synthesis of inositol 1,4,5-trisphosphate, whereas l-phenylalanine stimulation of the CaR does not induce any detectable change in the level this second messenger. Furthermore, we identified a novel pathway that mediates transient [Ca(2+)](i) oscillations produced by the CaR in response to l-phenylalanine, which requires the organization of the actin cytoskeleton and involves the small GTPase Rho, heterotrimeric proteins of the G(12) subfamily, the C-terminal region of the CaR, and the scaffolding protein filamin-A. Our model envisages that Ca(2+) or amino acids stabilize unique CaR conformations that favor coupling to different G proteins and subsequent activation of distinct downstream signaling pathways.

cDNA cloning and functional expression of a Ca2+-sensing receptor with truncated C-terminal tail from the Mozambique tilapia (Oreochromis mossambicus).

The complete cDNA sequence of the tilapia extracellular Ca(2+)-sensing receptor (CaR) was determined. The transcript length of tilapia CaR (tCaR) is 3.4 kbp and encodes a 940-amino acid, 7-transmembrane domain protein that is consistent in its structural features with known mammalian and piscine CaRs. The tCaR extracellular domain includes a characteristic hydrophobic segment, conserved cysteine residues that are implicated in receptor dimerization (Cys(129) and Cys(131)) and in coupling to the transmembrane domain (nine conserved cysteine residues), and conserved serine residues (Ser(147) and Ser(169-171)) that are linked to receptor binding of Ca(2+) and L-amino acid-mediated potentiation of function. mRNA expression of tCaR was strong in kidney, brain, and gill. Weaker expression was observed in pituitary, stomach, intestine, urinary bladder, and heart. This distribution is consistent with possible physiological roles in endocrine cells, excitable tissues, and ion-transporting barrier epithelia. Expression of tCaR mRNA in kidney and intestine was salinity-dependent, suggesting a role for the receptor in iono-/osmoregulation in this euryhaline teleost species. Human embryonic kidney-293 cells transiently transfected with tCaR cDNA demonstrated dose-dependent phospholipase C activation in response to elevations in the extracellular Ca(2+) concentration ([Ca(2+)](o)). Functional activation of the mitogen-activated protein kinase cascade by high [Ca(2+)](o) was also confirmed in these cells despite the naturally occurring truncation of the receptor's intracellular tail, which removes segments variably linked in mammalian CaRs to filamin-coupled activation of mitogen-activated protein kinase cascades. Sensitivity of phospholipase C activation to [Ca(2+)](o) was dependent on the ionic strength of the bathing medium, supporting a role in salinity sensing.

Direct interaction between the actin-binding protein filamin-A and the inwardly rectifying potassium channel, Kir2.1.

The role of filamins in actin cross-linking and membrane stabilization is well established, but recently their ability to interact with a variety of transmembrane receptors and signaling proteins has led to speculation of additional roles in scaffolding and signal transduction. Here we report a direct interaction between filamin-A and Kir2.1, an isoform of inwardly rectifying potassium channel expressed in vascular smooth muscle and an important regulator of vascular tone. Yeast two-hybrid screening of a porcine coronary artery cDNA library using the carboxyl terminus of Kir2.1 as bait yielded cDNA encoding a fragment of filamin-A (residues 2481-2647). Interaction between filamin-A and Kir2.1 was confirmed by in vitro overlay assay of membrane-bound Kir2.1 with glutathione S-transferase fusion protein of the isolated filamin clone. Additionally, antibodies directed against Kir2.1 coimmunoprecipitated filamin-A from arterial smooth muscle cell lysates, and immunocytochemical analysis of individual arterial smooth muscle cells showed that Kir2.1 and filamin co-localize in "hotspots" at the cell membrane. Interaction with filamin-A was found to have no effect on Kir2.1 channel behavior but, rather, increased the number of functional channels resident within the membrane. We conclude that filamin-A is potentially an important regulator of Kir2.1 surface expression and location within vascular smooth muscle.

Aluminum modifies the viscosity of filamentous actin solutions as measured by optical displacement microviscometry.

A microtechnique has been developed that is capable of measuring the viscosity of filamentous actin (F-actin) solutions. This method, called optical displacement microviscometry (ODM), was utilized to determine the changes in viscosity of solutions of rabbit muscle, human platelet, and maize pollen actin when measured in the absence and presence of aluminum. Measurements demonstrated that the viscosity of the different actin solutions decreased with aluminum concentration. In contrast, increases in viscosity were observed when aluminum was added to F-actin solutions containing filamin (chicken gizzard), a protein that bundles actin filaments. Confocal fluorescence imaging of pure actin solutions in the presence of aluminum showed a disrupted actin network composed of fragmented actin filaments in the form of small aggregates. In contrast, in the presence of filamin, aluminum promoted the formation of thicker actin filaments. These measurements demonstrate that aluminum can affect actin filaments differentially depending on the presence of an actin-binding protein. In addition, a strong correlation is observed between the changes in viscosity as measured by ODM and the thickness and assembled state of bundles of actin filaments.

Changes in the distribution of actin-associated proteins during epidermal wound healing.

We have examined the distribution of actin filaments and a number of actin-associated proteins during human epidermal wound healing, using a suction blister model in which the epidermis is detached from the dermis, leaving the basement membrane intact. Filamentous actin was found in all the living epidermal layers before, during and after wound healing. alpha-actinin was also present in all the living layers of normal epidermis, but diffuse cytoplasmic staining was observed at the leading edge of migrating epidermis. Vinculin and talin were concentrated at the basement membrane prior to wounding, but were absent from the leading edge during wound healing. In normal epidermis, filamin and gelsolin showed a complementary distribution, with filamin most abundant in the basal layer and gelsolin most abundant suprabasally. The abundance of both proteins was reduced at the leading edge of migrating epidermis. All of the changes were transient, as the expression patterns returned to normal by 1 week after wounding, when the epidermis had reformed. The relevance of these changes to the process of keratinocyte migration is discussed.