Tumor-specific expression of αvβ3 integrin promotes spontaneous metastasis of breast cancer to bone

INTRODUCTION: Studies in xenograft models and experimental models of metastasis have implicated several β3 integrin-expressing cell populations, including endothelium, platelets and osteoclasts, in breast tumor progression. Since orthotopic human xenograft models of breast cancer are poorly metastatic to bone and experimental models bypass the formation of a primary tumor, however, the precise contribution of tumor-specific αvβ3 to the spontaneous metastasis of breast tumors from the mammary gland to bone remains unclear. METHODS: We used a syngeneic orthotopic model of spontaneous breast cancer metastasis to test whether exogenous expression of αvβ3 in a mammary carcinoma line (66cl4) that metastasizes to the lung, but not to bone, was sufficient to promote its spontaneous metastasis to bone from the mammary gland. The tumor burden in the spine and the lung following inoculation of αvβ3-expressing 66cl4 (66cl4beta3) tumor cells or control 66cl4pBabe into the mammary gland was analyzed by real-time quantitative PCR. The ability of these cells to grow and form osteolytic lesions in bone was determined by histology and tartrate-resistant acid phosphatase staining of bone sections following intratibial injection of tumor cells. The adhesive, migratory and invasive properties of 66cl4pBabe and 66cl4beta3 cells were evaluated in standard in vitro assays. RESULTS: The 66cl4beta3 tumors showed a 20-fold increase in metastatic burden in the spine compared with 66cl4pBabe. A similar trend in lung metastasis was observed. αvβ3 did not increase the proliferation of 66cl4 cells in vitro or in the mammary gland in vivo. Similarly, αvβ3 is not required for the proliferation of 66cl4 cells in bone as both 66cl4pBabe and 66cl4beta3 proliferated to the same extent when injected directly into the tibia. 66cl4beta3 tumor growth in the tibia, however, increased osteoclast recruitment and bone resorption compared with 66cl4 tumors. Moreover, αvβ3 increased 66cl4 tumor cell adhesion and αvβ3-dependent haptotactic migration towards bone matrix proteins, as well as their chemotactic response to bone-derived soluble factors in vitro. CONCLUSION: These results demonstrate for the first time that tumor-specific αvβ3 contributes to spontaneous metastasis of breast tumors to bone and suggest a critical role for this receptor in mediating chemotactic and haptotactic migration towards bone factors

Differential In Vitro Effects of Intravenous versus Oral Formulations of Silibinin on the HCV Life Cycle and Inflammation

Silymarin prevents liver disease in many experimental rodent models, and is the most popular botanical medicine consumed by patients with hepatitis C. Silibinin is a major component of silymarin, consisting of the flavonolignans silybin A and silybin B, which are insoluble in aqueous solution. A chemically modified and soluble version of silibinin, SIL, has been shown to potently reduce hepatitis C virus (HCV) RNA levels in vivo when administered intravenously. Silymarin and silibinin inhibit HCV infection in cell culture by targeting multiple steps in the virus lifecycle. We tested the hepatoprotective profiles of SIL and silibinin in assays that measure antiviral and anti-inflammatory functions. Both mixtures inhibited fusion of HCV pseudoparticles (HCVpp) with fluorescent liposomes in a dose-dependent fashion. SIL inhibited 5 clinical genotype 1b isolates of NS5B RNA dependent RNA polymerase (RdRp) activity better than silibinin, with IC50 values of 40–85 µM. The enhanced activity of SIL may have been in part due to inhibition of NS5B binding to RNA templates. However, inhibition of the RdRps by both mixtures plateaued at 43–73%, suggesting that the products are poor overall inhibitors of RdRp. Silibinin did not inhibit HCV replication in subgenomic genotype 1b or 2a replicon cell lines, but it did inhibit JFH-1 infection. In contrast, SIL inhibited 1b but not 2a subgenomic replicons and also inhibited JFH-1 infection. Both mixtures inhibited production of progeny virus particles. Silibinin but not SIL inhibited NF-κB- and IFN-B-dependent transcription in Huh7 cells. However, both mixtures inhibited T cell proliferation to similar degrees. These data underscore the differences and similarities between the intravenous and oral formulations of silibinin, which could influence the clinical effects of this mixture on patients with chronic liver diseases

Lipids as modulators of membrane fusion mediated by viral fusion proteins.

International audienceEnveloped viruses infect host cells by fusion of viral and target membranes. This fusion event is triggered by specific glycoproteins in the viral envelope. Fusion glycoproteins belong to either class I, class II or the newly described third class, depending upon their arrangement at the surface of the virion, their tri-dimensional structure and the location within the protein of a short stretch of hydrophobic amino acids called the fusion peptide, which is able to induce the initial lipid destabilization at the onset of fusion. Viral fusion occurs either with the plasma membrane for pH-independent viruses, or with the endosomal membranes for pH-dependent viruses. Although, viral fusion proteins are parted in three classes and the subcellular localization of fusion might vary, these proteins have to act, in common, on lipid assemblies. Lipids contribute to fusion through their physical, mechanical and/or chemical properties. Lipids can thus play a role as chemically defined entities, or through their preferential partitioning into membrane microdomains called "rafts", or by modulating the curvature of the membranes involved in the fusion process. The purpose of this review is to make a state of the art on recent findings on the contribution of cholesterol, sphingolipids and glycolipids in cell entry and membrane fusion of a number of viral families, whose members bear either class I or class II fusion proteins, or fusion proteins of the recently discovered third class.Enveloped viruses infect host cells by fusion of viral and target membranes. This fusion event is triggered by specific glycoproteins in the viral envelope. Fusion glycoproteins belong to either class I, class II or the newly described third class, depending upon their arrangement at the surface of the virion, their tri-dimensional structure and the location within the protein of a short stretch of hydrophobic amino acids called the fusion peptide, which is able to induce the initial lipid destabilization at the onset of fusion. Viral fusion occurs either with the plasma membrane for pH-independent viruses, or with the endosomal membranes for pH-dependent viruses. Although, viral fusion proteins are parted in three classes and the subcellular localization of fusion might vary, these proteins have to act, in common, on lipid assemblies. Lipids contribute to fusion through their physical, mechanical and/or chemical properties. Lipids can thus play a role as chemically defined entities, or through their preferential partitioning into membrane microdomains called "rafts", or by modulating the curvature of the membranes involved in the fusion process. The purpose of this review is to make a state of the art on recent findings on the contribution of cholesterol, sphingolipids and glycolipids in cell entry and membrane fusion of a number of viral families, whose members bear either class I or class II fusion proteins, or fusion proteins of the recently discovered third class

On the mechanism of intracellular membrane fusion: In search of the genuine fusion factor

Intracellular membrane Fusion events require a general protein machinery that functions in vesicular traffic and in assembly and maintenance of organelles. An array of cytosolic and integral membrane proteins are currently identified, and in conjunction with ongoing detailed structural studies, rapid progress is made in understanding basic features of the overall mechanism of the fusion machinery, but above all a proper appreciation of its enormous complexity. Thus a highly sophisticated level of regulation of the different steps involved in tethering, docking and merging itself is apparent. Apart from the relevance of protein protein interactions, also a role of distinct lipids is gradually emerging, particularly in Fusion. However, although various suggestions have been made recently, largely based upon in vitro studies, the identity of the actual fusion factor(s) remains to be determined

Non-phospholipid fusogenic liposomes.

International audienceWe have demonstrated the capacity of non-phospholipid liposomes composed primarily of dioxyethylene acyl ethers and cholesterol to fuse with membranes composed primarily of phospholipid. Phase-contrast microscopy, freeze-fracture electron microscopy and a macromolecular probe indicate that these non-phospholipid liposomes can fuse with the plasma membranes of erythrocytes and fibroblasts. Furthermore, fluorescence probe experiments have demonstrated fusion between phosphatidylcholine liposomes and non-phospholipid liposomes. Mixing of internal contents was shown by a terbium/dipicolinate assay. Mixing of membrane lipid components was demonstrated by measuring (i) fluorescence resonance energy transfer between N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine and N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine, after phosphatidylcholine liposomes were mixed with non-phospholipid liposomes, and (ii) reduced concentration quenching of rhodaminephosphatidylethanolamine and octadecylrhodamine incorporated into phosphatidylcholine liposomes after mixing with the non-phospholipid liposomes. The degree of apparent fusion reported by the different probe techniques ranged from 25% to 64%.We have demonstrated the capacity of non-phospholipid liposomes composed primarily of dioxyethylene acyl ethers and cholesterol to fuse with membranes composed primarily of phospholipid. Phase-contrast microscopy, freeze-fracture electron microscopy and a macromolecular probe indicate that these non-phospholipid liposomes can fuse with the plasma membranes of erythrocytes and fibroblasts. Furthermore, fluorescence probe experiments have demonstrated fusion between phosphatidylcholine liposomes and non-phospholipid liposomes. Mixing of internal contents was shown by a terbium/dipicolinate assay. Mixing of membrane lipid components was demonstrated by measuring (i) fluorescence resonance energy transfer between N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine and N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine, after phosphatidylcholine liposomes were mixed with non-phospholipid liposomes, and (ii) reduced concentration quenching of rhodaminephosphatidylethanolamine and octadecylrhodamine incorporated into phosphatidylcholine liposomes after mixing with the non-phospholipid liposomes. The degree of apparent fusion reported by the different probe techniques ranged from 25% to 64%

Membrane fusion induced by a short fusogenic peptide is assessed by its insertion and orientation into target bilayers

To clarify the molecular mechanism by which an amphipathic negatively charged peptide consisting of 11 residues (WAE) induces fusion, and the relevance of these features for fusion, its mode of insertion and orientation into target bilayers were investigated. Using attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) in combination with techniques based on tryptophan fluorescence, the peptide was found to form an alpha-helix, shallowly inserted into the membrane to which it is anchored. Interestingly, in the presence of target membranes, WAE inserts into the target bilayer as an alpha-helix oriented almost parallel to the Lipid acyl chains. The accessibility of the peptide to either acrylamide (as an aqueous quencher of Trp fluorescence) or deuterium oxide ton the course of an FTIR deuteration kinetics) was lower in the presence than in the absence of target membranes, confirming that under those conditions, the peptide was shielded from the aqueous environment. Since fusion experiments have shown a temperature dependence, the effect of this later parameter on the structure and mode of insertion of the peptide was also analyzed. In the presence of target membrane, but not in their absence, the amount of alpha-helical structure increased with temperature, reflecting a similar temperature-dependent increase in the rate and extent of WAE-indnced fusion. Also, the extent of penetration of the helix into the target membrane was greater at 37 degrees C than at lower temperatures. This temperature-dependent distinction was revealed by a decreased accessibility of the peptide to deuterium oxide and acrylamide at 37 degrees C as compared to that at lower temperatures. These data underscore the role of peptide structure, peptide penetration, and orientation in the mechanism of protein-induced membrane fusion