RESUMO
Dissemination of HIV in the host involves transit of the virus and virus-infected cells across the lymphatic endothelium. HIV may alter lymphatic endothelial permeability to foster dissemination, but the mechanism is largely unexplored. Using a primary human lymphatic endothelial cell model, we found that HIV-1 envelope protein gp120 induced lymphatic hyperpermeability by disturbing the normal function of Robo4, a novel regulator of endothelial permeability. HIV-1 gp120 induced fibronectin expression and integrin α5ß1 phosphorylation, which led to the complexing of these three proteins, and their subsequent interaction with Robo4 through its fibronectin type III repeats. Moreover, pretreatment with an active N-terminus fragment of Slit2, a Robo4 agonist, protected lymphatic endothelial cells from HIV-1 gp120-induced hyperpermeability by inhibiting c-Src kinase activation. Our results indicate that targeting Slit2/Robo4 signaling may protect the integrity of the lymphatic barrier and limit the dissemination of HIV in the host.
Assuntos
Endotélio Linfático/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Ativação Enzimática , Genes src , Células HEK293 , Proteína gp120 do Envelope de HIV/genética , Infecções por HIV/genética , Humanos , Integrina alfa5beta1/genética , Integrina alfa5beta1/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas do Tecido Nervoso/genética , Permeabilidade , Receptores de Superfície Celular/genéticaRESUMO
BACKGROUND: Signaling through vascular endothelial growth factor C (VEGFC) and VEGF receptor 3 (VEGFR-3) plays a central role in lymphangiogenesis and the metastasis of several cancers via the lymphatics. Recently, the Slit2/Robo4 pathway has been recognized as a modulator of vascular permeability and integrity. Signaling via the Robo receptor inhibits VEGF-mediated effects; however, its effects on lymphatic endothelial cell function have not been well characterized. RESULTS: We found that pretreatment with Slit2N, an active fragment of Slit2, inhibited VEGF-C-mediated lung-derived lymphatic endothelial cell (L-LEC) proliferation, migration, and in vitro tube formation. Slit2N induced the internalization of VEGFR-3, which blocked its activation, and inhibited the activation of the PI3K/Akt pathway by VEGF-C in L-LECs. Moreover, we found that inhibition of VEGF-C-induced effects by Slit2N was Robo4-dependent. CONCLUSION: These results indicate that Slit2N/Robo4 modulates several key cellular functions, which contribute to lymphangiogenesis, and identify this ligand-receptor pair as a potential therapeutic target to inhibit lymphatic metastasis of VEGF-C-overexpressing cancers and manage lymphatic dysfunctions characterized by VEGF-C/VEGFR-3 activation.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Linfangiogênese , Proteínas do Tecido Nervoso/metabolismo , Receptores de Superfície Celular/metabolismo , Fator C de Crescimento do Endotélio Vascular/farmacologia , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Linhagem Celular , Movimento Celular , Proliferação de Células , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Vasos Linfáticos/citologia , Proteínas do Tecido Nervoso/genética , Fragmentos de Peptídeos/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Superfície Celular/genética , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Dendritic cells are among the first cells to encounter sexually acquired human immunodeficiency virus (HIV-1), in the mucosa, and they can transmit HIV-1 to CD4(+) T-cells via an infectious synapse. Recent studies reveal that actin-rich membrane extensions establish direct contact between cells at this synapse and facilitate virus transmission. Genesis of these contacts involves signaling through c-Src and Cdc42, which modulate actin polymerization and filopodia formation via the Arp2/3 complex and Diaphanous 2 (Diaph2). We found that Slit2N, a ligand for the Roundabout (Robo) receptors, blocked HIV-1-induced signaling through Arp2/3 and Diaph2, decreased filopodial extensions on dendritic cells, and inhibited cell-to-cell transmission of HIV-1 in a Robo1-dependent manner. Employing proteomic analysis, we identified Flightless-1 as a novel, Robo1-interacting protein. Treatment with shRNAs reduced levels of Flightless-1 and demonstrated its role in efficient cell-to-cell transfer of HIV-1. These results suggest a novel strategy to limit viral infection in the host by targeting the Slit/Robo pathway with modulation of cytoskeletal elements previously unrecognized in HIV-1 transmission.
Assuntos
Citoesqueleto/metabolismo , Células Dendríticas/virologia , HIV-1/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Proteínas do Tecido Nervoso/farmacologia , Linfócitos T/virologia , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Proteínas de Transporte/metabolismo , Extensões da Superfície Celular/efeitos dos fármacos , Extensões da Superfície Celular/metabolismo , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/ultraestrutura , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/ultraestrutura , Ativação Enzimática/efeitos dos fármacos , Forminas , Células HEK293 , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/ultraestrutura , Humanos , Modelos Biológicos , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteína Proto-Oncogênica c-fli-1/metabolismo , Pseudópodes/efeitos dos fármacos , Pseudópodes/metabolismo , Receptores Imunológicos/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Vírion/metabolismo , Vírion/ultraestrutura , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Quinases da Família src/metabolismo , Proteínas RoundaboutRESUMO
Pulmonary complications are common in both AIDS patients and cocaine users. We addressed the cellular and molecular mechanisms by which HIV and cocaine may partner to induce their deleterious effects. Using primary lung lymphatic endothelial cells (L-LECs), we examined how cocaine and HIV-1 gp120, alone and together, modulate signaling and functional properties of L-LECs. We found that brief cocaine exposure activated paxillin and induced cytoskeletal rearrangement, while sustained exposure increased fibronectin (FN) expression, decreased Robo4 expression, and enhanced the permeability of L-LEC monolayers. Moreover, incubating L-LECs with both cocaine and HIV-1 gp120 exacerbated hyperpermeability, significantly enhanced apoptosis, and further impaired in vitro wound healing as compared with cocaine alone. Our studies also suggested that the sigma-1 receptor (Sigma-1R) and the dopamine-4 receptor (D4R) are involved in cocaine-induced pathology in L-LECs. Seeking clinical correlation, we found that FN levels in sera and lung tissue of HIV(+) donors were significantly elevated as compared to HIV(-) donors. Our in vitro data demonstrate that cocaine and HIV-1 gp120 induce dysfunction and damage of lung lymphatics, and suggest that cocaine use may exacerbate pulmonary edema and fibrosis associated with HIV infection. Continued exploration of the interplay between cocaine and HIV should assist the design of therapeutics to ameliorate HIV-induced pulmonary disorders within the drug using population.
RESUMO
PURPOSE: Mantle cell lymphoma (MCL) is an incurable B-cell lymphoma, and new therapeutic strategies are urgently needed. EXPERIMENTAL DESIGN: The effects of ON 013105, a novel benzylstyryl sulfone kinase inhibitor, alone or with doxorubicin or rituximab, were examined in Granta 519 and Z138C cells. For in vivo studies, CB17/SCID mice were implanted subcutaneously with Z138C cells and treated with various combinations of ON 013105, doxorubicin, and rituximab. Tumor burden and body weight were monitored for 28 days. RESULTS: ON 013105 induced mitochondria-mediated apoptosis in MCL cells. Death was preceded by translocation of tBid to the mitochondria and cytochrome c release. In addition, ON 013105-treated cells exhibited reduced levels of cyclin D1, c-Myc, Mcl-1, and Bcl-xL. Using nuclear magnetic resonance (NMR) spectroscopy, we showed specific binding of ON 013105 to eIF4E, a critical factor for the initiation of protein translation. We proffer that this drug-protein interaction preferentially prevents the translation of the aforementioned proteins and may be the mechanism by which ON 013105 induces apoptosis in MCL cells. Efficacy studies in a mouse xenograft model showed that ON 013105 inhibited MCL tumor growth and that combining ON 013105 with rituximab reduced tumor burden further with negligible unwanted effects. CONCLUSIONS: Our findings suggest that ON 013105, alone or in combination with rituximab, may be a potent therapeutic agent to treat MCLs.
Assuntos
Anticorpos Monoclonais Murinos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Linfoma de Célula do Manto/metabolismo , Linfoma de Célula do Manto/patologia , Estirenos/administração & dosagem , Sulfonas/administração & dosagem , Carga Tumoral/efeitos dos fármacos , Animais , Antibióticos Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Ciclina D1/genética , Ciclina D1/metabolismo , Modelos Animais de Doenças , Doxorrubicina/administração & dosagem , Fator de Iniciação 4E em Eucariotos/metabolismo , Feminino , Expressão Gênica , Linfoma de Célula do Manto/tratamento farmacológico , Linfoma de Célula do Manto/genética , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Rituximab , Estirenos/metabolismo , Estirenos/farmacocinética , Sulfonas/metabolismo , Sulfonas/farmacocinéticaRESUMO
Kaposi sarcoma is the most common neoplasm caused by Kaposi sarcoma-associated herpesvirus (KSHV). It is prevalent among the elderly in the Mediterranean, inhabitants of sub-Saharan Africa, and immunocompromised individuals such as organ transplant recipients and AIDS patients. Current treatments for Kaposi sarcoma can inhibit tumor growth but are not able to eliminate KSHV from the host. When the host's immune system weakens, KSHV begins to replicate again, and active tumor growth ensues. New therapeutic approaches are needed. Cannabidiol (CBD), a plant-derived cannabinoid, exhibits promising antitumor effects without inducing psychoactive side effects. CBD is emerging as a novel therapeutic for various disorders, including cancer. In this study, we investigated the effects of CBD both on the infection of endothelial cells (ECs) by KSHV and on the growth and apoptosis of KSHV-infected ECs, an in vitro model for the transformation of normal endothelium to Kaposi sarcoma. While CBD did not affect the efficiency with which KSHV infected ECs, it reduced proliferation and induced apoptosis in those infected by the virus. CBD inhibited the expression of KSHV viral G protein-coupled receptor (vGPCR), its agonist, the chemokine growth-regulated protein α (GRO-α), vascular endothelial growth factor receptor 3 (VEGFR-3), and the VEGFR-3 ligand, vascular endothelial growth factor C (VEGF-C). This suggests a potential mechanism by which CBD exerts its effects on KSHV-infected endothelium and supports the further examination of CBD as a novel targeted agent for the treatment of Kaposi sarcoma.
RESUMO
Cell-mediated transmission and dissemination of sexually-acquired human immunodeficiency virus 1 (HIV-1) in the host involves the migration of immature dendritic cells (iDCs). iDCs migrate in response to the HIV-1 envelope protein, gp120, and inhibiting such migration may limit the mucosal transmission of HIV-1. In this study, we elucidated the mechanism of HIV-1-gp120-induced transendothelial migration of iDCs. We found that gp120 enhanced the binding of Wiskott-Aldrich Syndrome protein (WASp) and the Actin-Related Protein 2/3 (Arp2/3) complex with ß-actin, an interaction essential for the proper formation of podosomes, specialized adhesion structures required for the migration of iDCs through different tissues. We further identified Leukocyte-Specific Protein 1 (LSP1) as a novel component of the WASp-Arp2/3-ß-actin complex. Pretreating iDCs with an active fragment of the secretory glycoprotein Slit2 (Slit2N) inhibited HIV-1-gp120-mediated migration and podosome formation, by inducing the cognate receptor Roundabout 1 (Robo1) to bind to and sequester WASp and LSP1 from ß-actin. Slit2N treatment also inhibited Src signaling and the activation of several downstream molecules, including Rac1, Pyk2, paxillin, and CDC42, a major regulator of podosome formation. Taken together, our results support a novel mechanism by which Slit2/Robo1 may inhibit the HIV-1-gp120-induced migration of iDCs, thereby restricting dissemination of HIV-1 from mucosal surfaces in the host.
Assuntos
Movimento Celular/imunologia , Células Dendríticas/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Proteínas dos Microfilamentos/imunologia , Proteínas do Tecido Nervoso/imunologia , Receptores Imunológicos/imunologia , Proteína da Síndrome de Wiskott-Aldrich/imunologia , Complexo 2-3 de Proteínas Relacionadas à Actina/imunologia , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/imunologia , Actinas/metabolismo , Western Blotting , Células Cultivadas , Células Dendríticas/metabolismo , Quinase 2 de Adesão Focal/imunologia , Quinase 2 de Adesão Focal/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , Células Endoteliais da Veia Umbilical Humana/imunologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Microscopia Confocal , Proteínas do Tecido Nervoso/metabolismo , Paxilina/imunologia , Paxilina/metabolismo , Ligação Proteica/imunologia , Proteínas Proto-Oncogênicas pp60(c-src)/imunologia , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Pseudópodes/imunologia , Interferência de RNA , Receptores Imunológicos/metabolismo , Transdução de Sinais/imunologia , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Proteína cdc42 de Ligação ao GTP/imunologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/imunologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas RoundaboutRESUMO
Cannabidiol (CBD), a major nonpsychoactive constituent of cannabis, is considered an antineoplastic agent on the basis of its in vitro and in vivo activity against tumor cells. However, the exact molecular mechanism through which CBD mediates this activity is yet to be elucidated. Here, we have shown CBD-induced cell death of breast cancer cells, independent of cannabinoid and vallinoid receptor activation. Electron microscopy revealed morphologies consistent with the coexistence of autophagy and apoptosis. Western blot analysis confirmed these findings. We showed that CBD induces endoplasmic reticulum stress and, subsequently, inhibits AKT and mTOR signaling as shown by decreased levels of phosphorylated mTOR and 4EBP1, and cyclin D1. Analyzing further the cross-talk between the autophagic and apoptotic signaling pathways, we found that beclin1 plays a central role in the induction of CBD-mediated apoptosis in MDA-MB-231 breast cancer cells. Although CBD enhances the interaction between beclin1 and Vps34, it inhibits the association between beclin1 and Bcl-2. In addition, we showed that CBD reduces mitochondrial membrane potential, triggers the translocation of BID to the mitochondria, the release of cytochrome c to the cytosol, and, ultimately, the activation of the intrinsic apoptotic pathway in breast cancer cells. CBD increased the generation of reactive oxygen species (ROS), and ROS inhibition blocked the induction of apoptosis and autophagy. Our study revealed an intricate interplay between apoptosis and autophagy in CBD-treated breast cancer cells and highlighted the value of continued investigation into the potential use of CBD as an antineoplastic agent.