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1.
Mol Ther ; 30(4): 1553-1563, 2022 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-35038581

RESUMO

Toll-like receptors (TLRs) are key players in the innate immune system. Recent studies have suggested that they may affect the growth of pancreatic cancer, a disease with no cure. Among them, TLR7 shows promise for therapy but may also promotes tumor growth. Thus, we aimed to clarify the therapeutic potential of TLR7 ligands in experimental pancreatic cancer models, to open the door for clinical applications. In vitro, we found that TLR7 ligands strongly inhibit the proliferation of both human and murine pancreatic cancer cells, compared with TLR2 agonists. Hence, TLR7 treatment alters cancer cells' cell cycle and induces cell death by apoptosis. In vivo, TLR7 agonist therapy significantly delays the growth of murine pancreatic tumors engrafted in immunodeficient mice. Remarkably, TLR7 ligands administration instead increases tumor growth and accelerates animal death when tumors are engrafted in immunocompetent models. Further investigations revealed that TLR7 agonists modulate the intratumoral content and phenotype of macrophages and that depleting such tumor-associated macrophages strongly hampers TLR7 agonist-induced tumor growth. Collectively, our findings shine a light on the duality of action of TLR7 agonists in experimental cancer models and call into question their use for pancreatic cancer therapy.


Assuntos
Neoplasias Pancreáticas , Receptor 7 Toll-Like , Animais , Humanos , Ligantes , Macrófagos/metabolismo , Glicoproteínas de Membrana , Camundongos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/metabolismo , Microambiente Tumoral , Neoplasias Pancreáticas
2.
J Cell Sci ; 128(15): 2759-65, 2015 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-26065430

RESUMO

Gp78 (also known as AMFR), an endoplasmic-reticulum (ER)-associated protein degradation (ERAD) E3 ubiquitin ligase, localizes to mitochondria-associated ER and targets the mitofusin (Mfn1 and Mfn2) mitochondrial fusion proteins for degradation. Gp78 is also the cell surface receptor for autocrine motility factor (AMF), which prevents Gp78-dependent mitofusin degradation. Gp78 ubiquitin ligase activity promotes ER-mitochondria association and ER-mitochondria Ca(2+) coupling, processes that are reversed by AMF. Electron microscopy of HT-1080 fibrosarcoma cancer cells identified both smooth ER (SER; ∼8 nm) and wider (∼50-60 nm) rough ER (RER)-mitochondria contacts. Both short hairpin RNA (shRNA)-mediated knockdown of Gp78 (shGp78) and AMF treatment selectively reduced the extent of RER-mitochondria contacts without impacting on SER--mitochondria contacts. Concomitant small interfering RNA (siRNA)-mediated knockdown of Mfn1 increased SER-mitochondria contacts in both control and shGp78 cells, whereas knockdown of Mfn2 increased RER-mitochondria contacts selectively in shGp78 HT-1080 cells. The mitofusins therefore inhibit ER-mitochondria interaction. Regulation of close SER-mitochondria contacts by Mfn1 and of RER-mitochondria contacts by AMF-sensitive Gp78-mediated degradation of Mfn2 define new mechanisms that regulate ER-mitochondria interactions.


Assuntos
Retículo Endoplasmático Rugoso/genética , Retículo Endoplasmático Liso/genética , GTP Fosfo-Hidrolases/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas Mitocondriais/genética , Receptores do Fator Autócrino de Motilidade/genética , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Retículo Endoplasmático Rugoso/metabolismo , Retículo Endoplasmático Liso/metabolismo , Degradação Associada com o Retículo Endoplasmático/fisiologia , Humanos , Mitocôndrias , Interferência de RNA , RNA Interferente Pequeno
3.
Virology ; 482: 157-66, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25863880

RESUMO

The minute virus of mice, prototype strain (MVMp), is a non-enveloped, single-stranded DNA virus of the family Parvoviridae. Unlike other parvoviruses, the mechanism of cellular uptake of MVMp has not been studied in detail. We analyzed MVMp endocytosis in mouse LA9 fibroblasts and a tumor cell line derived from epithelial-mesenchymal transition through polyomavirus middle T antigen transformation in transgenic mice. By a combination of immunofluorescence and electron microscopy, we found that MVMp endocytosis occurs at the leading edge of migrating cells in proximity to focal adhesion sites. By using drug inhibitors of various endocytic pathways together with immunofluorescence microscopy and flow cytometry analysis, we discovered that MVMp can use a number of endocytic pathways, depending on the host cell type. At least three different mechanisms were identified: clathrin-, caveolin-, and clathrin-independent carrier-mediated endocytosis, with the latter occurring in transformed cells but not in LA9 fibroblasts.


Assuntos
Endocitose , Vírus Miúdo do Camundongo/fisiologia , Internalização do Vírus , Animais , Linhagem Celular , Células Epiteliais/virologia , Fibroblastos/virologia , Citometria de Fluxo , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica , Microscopia de Fluorescência
4.
Virology ; 481: 63-72, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25768892

RESUMO

Galectin-3 has previously been found to be required by the parvovirus minute virus of mice prototype strain (MVMp) for infection of mouse fibroblast cells. Since MVMp is an oncotropic virus, and galectin-3 is a multifunctional protein implicated in cancer metastasis, we hypothesized that galectin-3 and Mgat5, the Golgi enzyme that synthesizes high-affinity glycan ligands of galectin-3, might play a role in MVMp infection. Using siRNA-mediated knockdown of galectin-3 in mouse cells transformed with polyomavirus middle T antigen and Mgat5(-/-) mouse mammary tumor cells, we found that galectin-3 and Mgat5 are both necessary for efficient MVMp cell entry and infection, but not for cell binding. Moreover, we found that human cancer cells expressing higher levels of galectin-3 were more efficiently infected with MVMp than cell lines expressing lower galectin-3 levels. We conclude that galectin-3 and Mgat5 are involved in MVMp infection, and propose that galectin-3 is a determinant of MVMp oncotropism.


Assuntos
Galectina 3/metabolismo , Vírus Miúdo do Camundongo/fisiologia , Infecções por Parvoviridae/veterinária , Doenças dos Roedores/metabolismo , Animais , Linhagem Celular , Galectina 3/genética , Humanos , Camundongos , Vírus Miúdo do Camundongo/genética , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Infecções por Parvoviridae/genética , Infecções por Parvoviridae/metabolismo , Infecções por Parvoviridae/virologia , Doenças dos Roedores/genética , Doenças dos Roedores/virologia
5.
Virology ; 468-470: 150-159, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25173091

RESUMO

The parvovirus minute virus of mice, prototype strain (MVMp), preferentially infects and kills cancer cells. This intrinsic MVMp oncotropism may depend in part on the early stages of MVMp infection. To test this hypothesis, we investigated the early events of MVMp infection in mouse LA9 fibroblasts and a highly invasive mouse mammary tumor cell line derived from polyomavirus middle T antigen-mediated transformation. Using a combination of fluorescence and electron microscopy, we found that various parameters of the cell migration process affect MVMp infection. We show that, after binding to the plasma membrane, MVMp particles rapidly cluster at the leading edge of migrating cells, which exhibit higher levels of MVMp uptake than non-motile cells. Moreover, promoting cell migration on a fibronectin matrix increased MVMp infection, and induction of epithelial-mesenchymal transition allowed MVMp replication in non-permissive epithelial cells. Hence, we propose that cell migration influences the early stages of MVMp infection.


Assuntos
Movimento Celular/fisiologia , Vírus Miúdo do Camundongo/fisiologia , Animais , Linhagem Celular , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Células Epiteliais/virologia , Transição Epitelial-Mesenquimal , Fibronectinas , Camundongos
6.
J Proteomics ; 79: 123-32, 2013 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-23268121

RESUMO

Cellular factors associated with the parvovirus minute virus of mice (MVM) during infection are thought to play important roles in the MVM life cycle but only a few of these have been identified. Here we used a proteomic-based approach in order to identify host-binding partners of MVM. Using purified MVM as bait for immunoprecipitation assays, a total of 150 proteins were identified in MVM immunoprecipitates by quantitative liquid chromatography-tandem mass spectrometry. Galectin-3 was one of six proteins showing a statistically significant enrichment across replicates. Small interfering RNA depletion studies revealed an important role for galectin-3 in MVM endocytosis and infectivity in LA9 mouse fibroblast cells. Galectin-3-depleted cells were less susceptible to MVM infection than control cells and showed a significant reduction of MVM cellular uptake, but not of MVM binding to the cell surface. Our results indicate an important role for galectin-3 in the cellular uptake of MVM. We propose that galectin-3 facilitates the access of MVM to its receptor(s) at the plasma membrane and in this way promotes MVM endocytosis.


Assuntos
Galectina 3/fisiologia , Vírus Miúdo do Camundongo/fisiologia , Receptores Virais/fisiologia , Internalização do Vírus , Animais , Endocitose/fisiologia , Camundongos , Vírus Miúdo do Camundongo/genética , Vírus Miúdo do Camundongo/patogenicidade , Proteômica , Replicação Viral/genética
7.
J Virol ; 85(10): 4863-74, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21367902

RESUMO

Parvoviruses are small, nonenveloped, single-stranded DNA viruses which replicate in the nucleus of the host cell. We have previously found that early during infection the parvovirus minute virus of mice (MVM) causes small, transient disruptions of the nuclear envelope (NE). We have now investigated the mechanism used by MVM to disrupt the NE. Here we show that the viral phospholipase A2, the only known enzymatic domain on the parvovirus capsid, is not involved in causing NE disruption. Instead, the virus utilizes host cell caspases, which are proteases involved in causing NE breakdown during apoptosis, to facilitate these nuclear membrane disruptions. Studies with pharmacological inhibitors indicate that caspase-3 in particular is involved. A caspase-3 inhibitor prevents nuclear lamin cleavage and NE disruption in MVM-infected mouse fibroblast cells and reduces nuclear entry of MVM capsids and viral gene expression. Caspase-3 is, however, not activated above basal levels in MVM-infected cells, and other aspects of apoptosis are not triggered during early MVM infection. Instead, basally active caspase-3 is relocalized to the nuclei of infected cells. We propose that NE disruption involving caspases plays a role in (i) parvovirus entry into the nucleus and (ii) alteration of the compartmentalization of host proteins in a way that is favorable for the virus.


Assuntos
Caspase 3/metabolismo , Vírus Miúdo do Camundongo/patogenicidade , Membrana Nuclear/patologia , Animais , Linhagem Celular , Fibroblastos/virologia , Humanos , Camundongos , Fosfolipases A2/metabolismo , Proteínas Virais/metabolismo
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