A Synergistic Approach Therapy for Colorectal Cancer Based on Exosomes and Exploitation of Metabolic Pathways.

In order to reduce the side effects of traditional chemotherapy in the treatment of colorectal cancer (CRC), a new drug delivery system has been developed in this work, based on exosomes that can host two drugs that act synergistically: farnesol (that stops the cell cycle) and paclitaxel (prevents microtubule system depolymerization). Firstly, exosomes were isolated from different cell cultures (from colorectal cancer and from fibroblast as example of normal cell line) by different methods and characterized by western blot, TEM and DLS, and results showed that they express classical protein markers such as CD9 and HSP-70 and they showed spherical morphology with sizes from 93 nm to 129 nm depending on the source. These exosomes were loaded with both drugs and its effect was studied in vitro. The efficacy was studied by comparing the viability of cell cultures with a colorectal cancer cell line (HCT-116) and a normal cell line (fibroblast HS-5). Results showed that exosomes present a specific effect with more reduction in cell viability in tumour cultures than healthy ones. In summary, exosomes are presented in this work as a promising strategy for colorectal cancer treatment.

Phosphatidylinositol-3-kinase-Akt pathway in negative-stranded RNA virus infection: a minireview.

The PI3K/Akt signalling pathway is a crucial signalling cascade that regulates transcription, protein translation, cell growth, proliferation, cell survival, and metabolism. During viral infection, viruses exploit a variety of cellular pathways, including the well-known PI3K/Akt signalling pathway. Conversely, cells rely on this pathway to stimulate an antiviral response. The PI3K/Akt pathway is manipulated by a number of viruses, including DNA and RNA viruses and retroviruses. The aim of this review is to provide up-to-date information about the role of the PI3K-Akt pathway in infection with members of five different families of negative-sense ssRNA viruses. This pathway is hijacked for viral entry, regulation of endocytosis, suppression of premature apoptosis, viral protein expression, and replication. Although less common, the PI3K/Akt pathway can be downregulated as an immunomodulatory strategy or as a mechanism for inducing autophagy. Moreover, the cell activates this pathway as an antiviral strategy for interferon and cytokine production, among other strategies. Here, we present new data concerning the role of this pathway in infection with the paramyxovirus Newcastle disease virus (NDV). Our data seem to indicate that NDV uses the PI3K/Akt pathway to delay cell death and increase cell survival as a means of improving its replication. The interference of negative-sense ssRNA viruses with this essential pathway might have implications for the development of antiviral therapies.

Identification of cellular proteins that interact with Newcastle Disease Virus and human Respiratory Syncytial Virus by a two-dimensional virus overlay protein binding assay (VOPBA).

Although it is well documented that the initial attachment receptors for Newcastle Disease Virus (NDV) and Respiratory Syncytial Virus (RSV) are sialic acid-containing molecules and glycosaminoglycans respectively, the exact nature of the receptors for both viruses remains to be deciphered. Moreover, additional molecules at the host cell surface might be involved in the entry mechanism. With the aim of identifying the cellular proteins that interact with NDV and RSV at the cell surface, we performed a virus overlay protein binding assay (VOPBA). Cell membrane lysates were separated by two dimensional (2D) gel electrophoresis and electrotransferred to PVDF membranes, after which they were probed with high viral concentrations. NDV interacted with a Protein Disulfide Isomerase from chicken fibroblasts. In the case of RSV, we detected 15 reactive spots, which were identified as six different proteins, of which nucleolin was outstanding. We discuss the possible role of PDI and nucleolin in NDV and RSV entry, respectively.

Entry of Newcastle Disease Virus into the host cell: role of acidic pH and endocytosis.

Most paramyxoviruses enter the cell by direct fusion of the viral envelope with the plasma membrane. Our previous studies have shown the colocalization of Newcastle Disease Virus (NDV) with the early endosome marker EEA1 and the inhibition of NDV fusion by the caveolin-phosphorylating drug phorbol 12-myristate 13-acetate (PMA) prompted us to propose that NDV enters the cells via endocytosis. Here we show that the virus-cell fusion and cell-cell fusion promoted by NDV-F are increased by about 30% after brief exposure to low pH in HeLa and ELL-0 cells but not in NDV receptor- deficient cell lines such as GM95 or Lec1. After a brief low-pH exposure, the percentage of NDV fusion at 29 °C was similar to that at 37 °C without acid-pH stimulation, meaning that acid pH would decrease the energetic barrier to enhance fusion. Furthermore, preincubation of cells with the protein kinase C inhibitor bisindolylmaleimide led to the inhibition of about 30% of NDV infectivity, suggesting that a population of virus enters cells through receptor-mediated endocytosis. Moreover, the involvement of the GTPase dynamin in NDV entry is shown as its specific inhibitor, dynasore, also impaired NDV fusion and infectivity. Optimal infection of the host cells was significantly affected by drugs that inhibit endosomal acidification such as concanamycin A, monensin and chloroquine. These results support our hypothesis that entry of NDV into ELL-0 and HeLa cells occurs through the plasma membrane as well as by dynamin- low pH- and receptor- dependent endocytosis.

α2-3- and α2-6- N-linked sialic acids allow efficient interaction of Newcastle Disease Virus with target cells.

Receptor recognition and binding is the first step in the viral cycle. It has been established that Newcastle Disease Virus (NDV) interacts with sialylated molecules such as gangliosides and glycoproteins at the cell surface. Nevertheless, the specific receptor(s) that mediate virus entry are not well known. We have analysed the role of the sialic acid linkage in the early steps of the viral infection cycle. Pretreatment of ELL-0 cells with both α2,3 and α2,6 specific sialidases led to the inhibition of NDV binding, fusion and infectivity, which were restored after α2,3(N)- and α2,6(N)-sialyltransferase incubation. Moreover, α2,6(N)-sialyltransferases also restored NDV activities in α2-6-linked sialic acid deficient cells. Competition with α2-6 sialic acid-binding lectins led to a reduction in the three NDV activities (binding, fusion and infectivity) suggesting a role for α2-6- linked sialic acid in NDV entry. We conclude that both α2-3- and α2-6- linked sialic acid containing glycoconjugates may be used for NDV infection. NDV was able to efficiently bind, fuse and infect the ganglioside-deficient cell line GM95 to a similar extent to that of its parental MEB4, suggesting that gangliosides are not essential for NDV binding, fusion and infectivity. Nevertheless, the fact that the interaction of NDV with cells deficient in N-glycoprotein expression such as Lec1 was less efficient prompted us to conclude that NDV requires N-linked glycoproteins for efficient attachment and entry into the host cell.

Cholesterol dependence of Newcastle Disease Virus entry.

Lipid rafts are membrane microdomains enriched in cholesterol, sphingolipids, and glycolipids that have been implicated in many biological processes. Since cholesterol is known to play a key role in the entry of some other viruses, we investigated the role of cholesterol and lipid rafts in the host cell plasma membrane in Newcastle Disease Virus (NDV) entry. We used methyl-ß-cyclodextrin (MßCD) to deplete cellular cholesterol and disrupt lipid rafts. Our results show that the removal of cellular cholesterol partially reduces viral binding, fusion and infectivity. MßCD had no effect on the expression of sialic acid containing molecule expression, the NDV receptors in the target cell. All the above-described effects were reversed by restoring cholesterol levels in the target cell membrane. The HN viral attachment protein partially localized to detergent-resistant membrane microdomains (DRMs) at 4°C and then shifted to detergent-soluble fractions at 37°C. These results indicate that cellular cholesterol may be required for optimal cell entry in NDV infection cycle.

Mutations in the ectodomain of newcastle disease virus fusion protein confer a hemagglutinin-neuraminidase-independent phenotype.

The entry of enveloped viruses into host cells is preceded by membrane fusion, which in paramyxoviruses is triggered by the fusion (F) protein. Refolding of the F protein from a metastable conformation to a highly stable postfusion form is critical for the promotion of fusion, although the mechanism is still not well understood. Here we examined the effects of mutations of individual residues of the F protein of Newcastle disease virus, located at critical regions of the protein, such as the C terminus of the N-terminal heptad repeat (HRA) and the N terminus of the C-terminal heptad repeat (HRB). Seven of the mutants were expressed at the cell surface, showing differences in antibody reactivity in comparison with the F wild type. The N211A, L461A, I463A, and I463F mutants showed a hyperfusogenic phenotype both in syncytium and in dye transfer assays. The four mutants promoted fusion more efficiently at lower temperatures than the wild type did, meaning they probably had lower energy requirements for activation. Moreover, the N211A, I463A, and I463F mutants exhibited hemagglutinin-neuraminidase (HN)-independent activity when influenza virus hemagglutinin (HA) was coexpressed as an attachment protein. The data are discussed in terms of alterations of the refolding pathway and/or the stability of the prefusion and fusion conformations.

Over-expression of mammalian sialidase NEU3 reduces Newcastle disease virus entry and propagation in COS7 cells.

The paramyxovirus Newcastle Disease Virus (NDV) binds to sialic acid-containing glycoconjugates, sialoglycoproteins and sialoglycolipids (gangliosides) of host cell plasma membrane through its hemagglutinin-neuraminidase (sialidase) HN glycoprotein. We hypothesized that the modifications of the cell surface ganglioside pattern determined by over-expression of the mammalian plasma-membrane associated, ganglioside specific, sialidase NEU3 would affect the virus-host cell interactions. Using COS7 cells as a model system, we observed that over-expression of the murine MmNEU3 did not affect NDV binding but caused a marked reduction in NDV infection and virus propagation through cell-cell fusion. Moreover, since GD1a was greatly reduced in COS7 cells following NEU3-over-expression, we added [(3)H]-labelled GD1a to COS7 cells under conditions that block intralysosomal metabolic processing, and we observed a marked increase of GD1a cleavage to GM1 during NDV infection, indicating a direct involvement of the virus sialidase and host cell GD1a in NDV infectivity. Therefore, the decrease of GD1a in COS7 cell membrane upon MmNEU3 over-expression is likely to be instrumental to NDV reduced infection. Evidence was also provided for the preferential association of NDV-HN at 4 degrees C to detergent resistant microdomains (DRMs) of COS7 cells plasma membranes.

Newcastle disease virus may enter cells by caveolae-mediated endocytosis.

The entry into cells of Newcastle disease virus (NDV), a prototype member of the paramyxoviruses, is believed to occur by direct fusion at the plasma membrane through a pH-independent mechanism. In addition, NDV may enter host cells by an endocytic pathway. Treatment of cells with drugs that block caveolae-dependent endocytosis reduced NDV fusion and infectivity, the degree of inhibition being dependent on virus concentration. The inhibitory effect was reduced greatly when drugs were added after virus adsorption. Cells treated with methyl beta-cyclodextrin, a drug that sequesters cholesterol from membranes, reduced the extent of fusion, infectivity and virus-cell binding; this indicates that cholesterol plays a role in NDV entry. Double-labelling immunofluorescence assays performed with anti-NDV monoclonal antibodies and antibodies against the early endosome marker EEA1 revealed the localization of the virus in these intracellular structures. Using fluorescence microscopy, it was found that cell-cell fusion was enhanced at low pH. It is concluded that NDV may infect cells through a caveolae-dependent endocytic pathway, suggesting that this pathway could be an alternative route for virus entry into cells.

Gangliosides and N-glycoproteins function as Newcastle disease virus receptors.

The interaction of enveloped viruses with cell surface receptors is the first step in the viral cycle and an important determinant of viral host range. Although it is established that the paramyxovirus Newcastle Disease Virus binds to sialic acid-containing glycoconjugates the exact nature of the receptors has not yet been determined. Accordingly, here we attempted to characterize the cellular receptors for Newcastle disease virus. Treatment of cells with tunicamycin, an inhibitor of protein N-glycosylation, blocked fusion and infectivity, while the inhibitor of O-glycosylation benzyl-N-acetyl-alpha-D-galactosamide had no effect. Additionally, the inhibitor of glycolipid biosynthesis 1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol blocked viral fusion and infectivity. These results suggest that N-linked glycoproteins and glycolipids would be involved in viral entry but not O-linked glycoproteins. The ganglioside content of COS-7 cells was analyzed showing that GD1a was the major ganglioside component; the presence of GM1, GM2 and GM3 was also established. In a thin-layer chromatographic binding assay, we analyzed the binding of the virus to different gangliosides, detecting the interaction with monosialogangliosides such as GM3, GM2 and GM1; disialogangliosides such as GD1a and GD1b, and trisialogangliosides such as GT1b. Unlike with other viruses, our results seem to point to the absence of a specific pattern of gangliosides that interact with Newcastle disease virus. In conclusion, our results suggest that Newcastle disease virus requires different sialic acid-containing compounds, gangliosides and glycoproteins for entry into the target cell. We propose that gangliosides would act as primary receptors while N-linked glycoproteins would function as the second receptor critical for viral entry.

Sialidase, receptor-binding and fusion-promotion activities of Newcastle disease virus haemagglutinin-neuraminidase glycoprotein: a mutational and kinetic study.

Mutations were generated in residues at the putative catalytic site of the haemagglutinin-neuraminidase (HN) protein of Newcastle disease virus Clone 30 strain (Arg498, Glu258, Tyr262, Tyr317 and Ser418) and their effects on its three associated activities were studied. Expression of the mutant proteins at the surface of HeLa cells was similar to that of the wild-type. Sialidase, receptor-binding and fusion-promotion activities were affected to different degrees for all mutants studied. Mutant Arg498Lys lost most of its sialidase activity, although it retained most of the receptor-binding activity, suggesting that, for the former activity, besides the presence of a basic residue, the proximity to the substrate molecule is also important, as Lys is shorter than Arg. Proximity also seems to be important in substrate recognition, since Tyr262Phe retained most of its sialidase activity while Tyr262Ser lost most of it. Also, Ser418Ala displayed most of the wild-type sialidase activity. However, a kinetic and thermodynamic study of the sialidase activity of the Tyr262Ser and Ser418Ala mutants was performed and revealed that the hydroxyl group of these residues also plays an important role in catalysis, since such activity was much less effective than that of the wild-type and these mutations modified their activation energy for sialidase catalysis. The discrepancy of the modifications in sialidase and receptor-binding activities in the mutants analysed does not account for the topological coincidence of the two sites. These results also suggest that the globular head of HN protein may play a role in fusion-promotion activity.

Conformational changes of Newcastle disease virus envelope glycoproteins triggered by gangliosides.

We have investigated the conformational changes of Newcastle disease virus (NDV) glycoproteins in response to receptor binding, using 1,1-bis(4-anilino)naphthalene-5,5-disulfonic acid (bis-ANS) as a hydrophobicity-sensitive probe. Temperature- and pH-dependent conformational changes were detected in the presence of free bovine gangliosides. The fluorescence of bis-ANS was maximal at pH 5. The binding of bis-ANS to NDV was not affected by chemicals that denature the fusion glycoprotein, such as reducing agents, nor by the presence of neuraminidase inhibitors such as N-acetyl neuramicic acid. Gangliosides partially inhibited fusion and hemadsorption, but not neuraminidase hemagglutinin-neuraminidase glycoprotein (HN) activity. A conformational intermediate of HN, triggered by the presence of gangliosides acting as receptor mimics, was detected. Our results indicate that, upon binding to free gangliosides, HN undergoes a certain conformational change that does not affect the fusion glycoprotein.