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1.
Front Immunol ; 10: 1943, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31475004

RESUMO

Follicular lymphoma (FL) is the second most frequent subtype of B non-Hodgkin's lymphomas (NHL) for which the treatment is based on the use of anti-CD20 mAbs. NK cells play a crucial role in their mechanism of action and the number of these cells mediating antibody-dependent cell cycotoxicity (ADCC) in the peripheral blood of FL patients predict the outcome. However, their presence in FL biopsies, their activation and their role have been poorly investigated. Moreover, in vitro studies have not deciphered the exact signaling cascades triggered by NK cells in presence of anti-CD20 mAbs on both effector and target cells in a relevant FL model. We performed in silico analyses and ex vivo functional assays to determine the presence and the activation status of NK cells in FL biopsies. We modelized ADCC phenomenon by developing a co-culture model composed by 3D-cultured FL cells and NK cells. Thus, we investigated the biological effect of anti-CD20 mAbs by fluorescent microscopy and the phosphorylation status of survival pathways by cell bar coding phosphoflow in target cells. In parallel, we measured the status of activation of downstream FcγRIIIa signaling pathways in effector cells and their activation (CD69, perforin, granzyme B, IFNγ) by flow cytometry. We determined by in vivo experiments the effects of anti-CD20 mAbs in presence of NK cells in SCID-Beige engrafted FL mice. Here, we show that functional NK cells infiltrate FL biopsies, and that their presence tends to correlate with the survival of FL patients. Using our 3D co-culture model, we show that rituximab and GA101 are able to promote degranulation, CD69 expression, IFNγ production and activate FcγRIIIa signaling cascade in NK cells, and inhibit survival pathways and induce apoptosis in FL cells. The effect of GA101 seems to be more pronounced as observed in vivo in a xenograft FL model. This study strongly supports the role of NK cells in FL and highlights the application of the 3D co-culture model for in vitro validation.

2.
Haematologica ; 2019 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-31296574

RESUMO

CD38 is expressed in several types of non-Hodgkin lymphoma and constitutes a promising target for antibody-based therapy. Daratumumab (Darzalex) is a first-in-class anti-CD38 antibody approved for the treatment of relapsed/refractory multiple myeloma. It has also demonstrated clinical activity in Waldenstrom macroglobulinaemia and amyloidosis. Here, we have evaluated the activity and mechanism of action of daratumumab in preclinical in vitro and in vivo models of mantle cell lymphoma, follicular lymphoma and diffuse large B cell lymphoma, as monotherapy or in combination with standard chemo-immunotherapy. In vitro, daratumumab engages Fc-mediated cytotoxicity by antibody-dependent cell cytotoxicity and antibody-dependent cell phagocytosis in all lymphoma subtypes. In the presence of human serum, complement-dependent cell cytotoxicity was marginally engaged. We demonstrated by Selective Plane Illumination Microscopy that daratumumab fully penetrated a 3D lymphoma organoid and decreased organoid volume. In vivo, daratumumab completely prevents tumor outgrowth in models of mantle cell and follicular lymphoma, and shows comparable activity to rituximab in a disseminated in vivo model of blastic mantle cell lymphoma. Moreover, daratumumab improves overall survival in a mouse model of transformed CD20dim follicular lymphoma, where rituximab showed limited activity. Daratumumab potentiates the antitumor activity of CHOP and R-CHOP in mantle cell and follicular lymphoma xenografts. Furthermore, in a patient-derived diffuse large B cell lymphoma xenograft model, daratumumab anti-tumor activity was comparable to R-CHOP and the addition of daratumumab to either CHOP or R-CHOP led to full tumor regression. In summary, daratumumab constitutes a novel therapeutic opportunity in certain scenarios and these results warrant further clinical development.

3.
Oncoimmunology ; 8(3): 1554175, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30723586

RESUMO

Follicular lymphoma (FL) is a common non Hodgkin's lymphoma subtype in which immune escape mechanisms are implicated in resistance to chemo-immunotherapy. Although molecular studies point to qualitative and quantitative deregulation of immune checkpoints, in depth cellular analysis of FL immune escape is lacking. Here, by functional assays and in silico analyses we show that a subset of FL patients displays a 'high' immune escape phenotype. These FL cases are characterized by abundant infiltration of PD1+ CD16+ TCRVγ9Vδ2 γδ T lymphocytes. In a 3D co-culture assay (MALC), γδ T cells mediate both direct and indirect (ADCC in the presence of anti-CD20 mAbs) cytolytic activity against FL cell aggregates. Importantly, PD-1, which is expressed by most FL-infiltrating γδ T lymphocytes with ADCC capacity, impairs these functions. In conclusion, we identify a PD1-regulated γδ T cell cytolytic immune component in FL. Our data provide a treatment rational by PD-1 blockade aimed at boosting γδ T cell anti-tumor functions in FL.

4.
Materials (Basel) ; 12(1)2019 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-30621089

RESUMO

In the fields of biology and medicine, nanoproducts such as nanoparticles (NPs) are specifically interesting as theranostic tools, since they offer the double capacity to locally deliver active drugs and to image exactly where the product is delivered. Among the many described possibilities, silica nanoparticles (SiNPs) represent a good choice because of their ease of synthesis, the possibility of their vast functionalization, and their good biocompatibility. However, SiNPs' passive cell internalization by endocytosis only distributes NPs into the cell cytoplasm and is unable to target the nucleus if SiNPs are larger than a few nanometers. In this study, we demonstrate that the cell penetration of SiNPs of 28⁻30 nm in diameter can be strongly enhanced using a physical method, called electroporation or electropermeabilization (EP). The uptake of fluorescently labelled silica nanoparticles was improved in two different cancer cell lines, namely, HCT-116 (human colon cancer) cells and RL (B-lymphoma) cells. First, we studied cells' capability for the regular passive uptake of SiNPs in vitro. Then, we set EP parameters in order to induce a more efficient and rapid cell loading, also comprising the nuclear compartment, while preserving the cell viability. In the final approach, we performed in vivo experiments, and evidenced that the labeling was long-lasting, as confirmed by fluorescence imaging of labeled tumors, which enabled a 30-day follow-up. This kind of SiNPs delivery, achieved by EP, could be employed to load extensive amounts of active ingredients into the cell nucleus, and concomitantly allow the monitoring of the long-term fate of nanoparticles.

5.
Cancers (Basel) ; 10(11)2018 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-30384489

RESUMO

Therapeutic blockade of PD-1/PD-L1 shows promising results in Hodgkin's lymphoma (HL) and in some diffuse large B-cell lymphoma (DLBCL) patients, but biomarkers predicting such responses are still lacking. To this end, we recently developed a transcriptional scoring of immune escape (IE) in cancer biopsies. Using this method in DLBCL, we identified four stages of IE correlated with overall survival, but whether Hodgkin's lymphomas (HL) also display this partition was unknown. Thus, we explored the transcriptomic profiles of ~1000 HL and DLBCL using a comparative meta-analysis of their bulk microarrays. Relative to DLBCL, the HL co-clustered at the advanced stage of immune escape, displaying significant enrichment of both IE and T-cell activation genes. Analyses via transcriptome deconvolution and immunohistochemistry showed more CD3⁺ and CD4⁺ tumor-infiltrating lymphocytes (TILs) in HL than DLBCL. Both HL and non-GCB DLBCL shared a high abundance of infiltrating CD8⁺ T-cells, but HL had less CD68⁺CD163⁺ macrophages. The same cellular distribution of PD-1 and TIM-3 was observed in HL and DLBCL, though HL had more PD-L1 tumor cells and LAG-3 ME cells. This study illuminates the advanced stage of immune activation and escape in HL, consistent with the response to checkpoint blockade therapies for this type of lymphoma.

6.
Eur J Immunol ; 47(12): 2137-2141, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28741710

RESUMO

From several years, the anticancer effects of Vγ9 T lymphocytes make these cells good candidates for cancer immunotherapies. However, the proved efficacy of γδ Τ cell-based cancer immunotherapies in some clinical trials was minimized due to the inherent toxicity of IL-2, which is essential for the combination therapy with Phosphoantigen (PAg). Recently, we showed that IL-33, a γ chain receptor-independent cytokine, was able to induce the in vitro proliferation of PAg-activated Vγ9 T cells, which were fully functional expressing IFN-γ and TNF-α and showing in vitro anti-tumor cytotoxicity. We proposed IL-33 as an alternative to IL-2 for Vγ9 T cell-based cancer immunotherapies, and have therefore evaluated the efficacy of this cytokine in preclinical investigations. This study shows that human Vγ9 T cells are able to proliferate in a mouse model with the combination of PAg and rhIL-33, and that IL-33-expanded Vγ9 T cells can prevent tumor growth in a mouse lymphoma model.


Assuntos
Imunoterapia/métodos , Interleucina-33/farmacologia , Linfoma/tratamento farmacológico , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Linfócitos T/transplante , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Interleucina-33/genética , Linfoma/imunologia , Linfoma/metabolismo , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Proteínas Recombinantes/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/imunologia
7.
Oncotarget ; 8(27): 44960-44975, 2017 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-28402953

RESUMO

Immune checkpoint blockade therapeutics, notably antibodies targeting the programmed death 1 (PD-1) receptor and its PD-L1 and PD-L2 ligands, are currently revolutionizing the treatment of cancer. For a sizeable fraction of patients with melanoma, lung, kidney and several other solid cancers, monoclonal antibodies that neutralize the interactions of the PD-1/PD-L1 complex allow the reconstitution of long-lasting antitumor immunity. In hematological malignancies this novel therapeutic strategy is far less documented, although promising clinical responses have been seen in refractory and relapsed Hodgkin lymphoma patients. This review describes our current knowledge of PD-1 and PD-L1 expression, as reported by immunohistochemical staining in both non-Hodgkin lymphoma cells and their surrounding immune cells. Here, we discuss the multiple intrinsic and extrinsic mechanisms by which both T and B cell lymphomas up-regulate the PD-1/PD-L1 axis, and review current knowledge about the prognostic significance of its immunohistochemical detection. This body of literature establishes the cell surface expression of PD-1/PD-L1 as a critical determinant for the identification of non-Hodgkin lymphoma patients eligible for immune checkpoint blockade therapies.


Assuntos
Antígeno B7-H1/metabolismo , Biomarcadores Tumorais , Linfoma não Hodgkin/metabolismo , Linfoma não Hodgkin/mortalidade , Receptor de Morte Celular Programada 1/metabolismo , Animais , Antígeno B7-H1/genética , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Linfoma não Hodgkin/diagnóstico , Linfoma não Hodgkin/terapia , Terapia de Alvo Molecular , Prognóstico , Receptor de Morte Celular Programada 1/genética , Transdução de Sinais
8.
J Labelled Comp Radiopharm ; 58(7): 274-80, 2015 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-26017396

RESUMO

Lymphomas are the most frequent haematological malignancy. In non-Hodgkin's lymphomas (NHL), more than 90% of tumor cells express the cluster of differentiation (CD) 20 antigen. At the end of frontline therapy, the evaluation of remission is based on computed tomography (CT) and positron emission tomography coupled with computer tomography (PET/CT) with [(18)F]-fluorodeoxyglucose ([(18)F]FDG). Unfortunately, these techniques are not specific and cannot distinguish residual active tumor from inflammation. The aim of this study was to develop a specific radiotracer of NHL CD 20+ cells for clinical applications. The radiolabelling technique presented, based on the use of tricarbonyl compound, does not include an antibody reduction because this step could damage the protein. Actually, rituximab, an anti-CD 20 chimeric antibody used for the treatment of these NHL, was radiolabelled with Isolink® (99m)Tc-tricarbonyl compound in a three-step procedure without using a specific antibody reducer. Radiolabelling yield was greater than 97%. In vitro experiments showed a conservation of antibody integrity. In vivo experiments using Single-photon emission computed tomography/CT showed significant tumor targeting 24 h after injection of the radiotracer. It was consequently possible to develop an immunoradiolabelling method to specifically detect the residual disease. As this procedure is fast, reproducible and gentle, it will be possible to comply with Good Manufacturing Practices.


Assuntos
Compostos Radiofarmacêuticos/síntese química , Rituximab/química , Tecnécio/química , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Compostos Radiofarmacêuticos/farmacocinética , Distribuição Tecidual , Tomografia Computadorizada de Emissão de Fóton Único
9.
Immunol Lett ; 161(1): 133-7, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24925024

RESUMO

Human γδ cells expressing TCRVγ9 are T lymphocytes with great potential for cancer immunotherapy and unconventional pattern of antigen specificity. These HLA-unrestricted lymphocytes are specifically reactive to non-peptide metabolites (phosphoantigens) and to the butyrophilin 3A (BTN3A/CD277) protein. Whether recognition of such highly different structures trigger the same activation signaling pathway remains unclear, however. Here we combined fluorescent cell barcoding and phosphoflow analysis of TCRVγ9(+) T lymphocytes to compare simultaneously the level of several signaling phosphoproteins after activation by phosphoantigen (BrHPP) or by anti-BTN3A (monoclonal antibody 20.1). This approach shows that the same pathways involving ZAP70, PLCγ2, Akt, NFκB p65, MAPK p38 and Erk1, were induced by either of these stimuli. These data strongly suggest the TCRVγ9(+) T lymphocytes detect phosphoantigens and butyrophilin A3 by the same recognition process.


Assuntos
Antígenos CD/metabolismo , Ativação Linfocitária , Fosfoproteínas/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Transdução de Sinais , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Anticorpos Monoclonais/farmacologia , Antígenos de Superfície/metabolismo , Butirofilinas , Linhagem Celular , Humanos , Imunofenotipagem , Transdução de Sinais/efeitos dos fármacos , Subpopulações de Linfócitos T/efeitos dos fármacos
10.
Cell Mol Immunol ; 10(1): 35-41, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23241899

RESUMO

During the last several years, research has produced a significant amount of knowledge concerning the characteristics of human γδ T lymphocytes. Findings regarding the immune functions of these cells, particularly their natural killer cell-like lytic activity against tumor cells, have raised expectations for the therapeutic applications of these cells for cancer. Pharmaceutical companies have produced selective agonists for these lymphocytes, and several teams have launched clinical trials of γδ T cell-based cancer therapies. The findings from these studies include hematological malignancies (follicular lymphoma, multiple myeloma, acute and chronic myeloid leukemia), as well as solid tumors (renal cell, breast and prostate carcinomas), consisting of samples from more than 250 patients from Europe, Japan and the United States. The results of these pioneering studies are now available, and this short review summarizes the lessons learned and the role of γδ T cell-based strategies in the current landscape of cancer immunotherapies.


Assuntos
Imunoterapia/métodos , Neoplasias/imunologia , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Linfócitos T/imunologia , Ensaios Clínicos como Assunto , Humanos
11.
Mol Cancer Res ; 9(11): 1435-42, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21921050

RESUMO

The anti-CD20 monoclonal antibody rituximab is the backbone of treatment for the B-cell malignancies non-Hodgkin lymphoma and chronic lymphocytic leukemia. However, there is a wide variability in response to rituximab treatment, and some patients are refractory to current standard therapies. Rituximab kills B cells by multiple mechanisms of action, including complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity, which are immune-mediated mechanisms, as well as by direct effects on cell signaling pathways and cell membranes following CD20 binding. A large number of events that are affected by rituximab binding have been identified, including lipid raft modifications, kinase and caspase activation, and effects on transcription factors and apoptotic/antiapoptotic molecules. Studies on cell lines and isolated tumor cells have shown that by targeting these pathways, it may be possible to increase or decrease susceptibility to rituximab cell killing. An increased understanding of the direct effects of rituximab may therefore aid in the design of new, rational combinations to improve the outcome of CD20-based therapy for patients who currently have suboptimal outcome following standard treatments.


Assuntos
Anticorpos Monoclonais Murinos/uso terapêutico , Antineoplásicos/uso terapêutico , Linfoma de Células B/tratamento farmacológico , Animais , Anticorpos Monoclonais Murinos/farmacologia , Antineoplásicos/farmacologia , Humanos , Linfoma de Células B/patologia , Rituximab
12.
Curr Drug Targets ; 11(7): 790-800, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20370648

RESUMO

The development of therapeutic monoclonal antibodies (mAbs) has revolutionized the treatment of cancer along the last ten years. The best examples of their therapeutic efficacies have been obtained with rituximab for the treatment of CD20+ B-cell Non-Hodgkin Lymphoma (B-NHL), and several others antibodies with optimized bioactivities are now being developed for the treatment of various malignant hemopathies. We review here the main drugs developed in this field, and present some emerging concepts able to improve the bioactivities of the next generation of therapeutic mAbs.


Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Sistemas de Liberação de Medicamentos/métodos , Neoplasias Hematológicas/tratamento farmacológico , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Humanos , Modelos Biológicos , Modelos Moleculares , Proteínas Recombinantes de Fusão
13.
Blood ; 115(5): 985-94, 2010 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-19965664

RESUMO

Rituximab (RTX), a monoclonal antibody directed against the CD20 protein, is a drug commonly used in the treatment of B-cell-derived lymphoid neoplasias and of antibody-mediated autoimmune diseases. In addition to cell- and complement-mediated B-cell depletion, RTX is thought to inhibit B-cell survival and proliferation through negative regulation of canonical signaling pathways involving Akt, ERK, and mammalian target of rapamycin. However, surprisingly, although B-cell receptor (BCR) signaling has been considered critical for normal and more recently, for neoplastic B cells, the hypothesis that RTX could target BCR has never been investigated. Using follicular lymphoma cell lines as models, as well as normal B cells, we show here, for the first time, that pretreatment with RTX results in a time-dependent inhibition of the BCR-signaling cascade involving Lyn, Syk, PLC gamma 2, Akt, and ERK, and calcium mobilization. The inhibitory effect of RTX correlates with decrease of raft-associated cholesterol, complete inhibition of BCR relocalization into lipid raft microdomains, and down-regulation of BCR immunoglobulin expression. Thus, RTX-mediated alteration of BCR expression, dynamics, and signaling might contribute to the immunosuppressive activity of the drug.


Assuntos
Anticorpos Monoclonais/farmacologia , Colesterol/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Anticorpos Monoclonais Murinos , Western Blotting , Cálcio/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Citometria de Fluxo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Fosfolipase C gama/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Rituximab , Quinase Syk , Fatores de Tempo , Quinases da Família src/metabolismo
14.
Blood ; 113(20): 4875-84, 2009 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-19278954

RESUMO

In human blood, 1% to 5% of lymphocytes are gammadelta T cells; they mostly express the gammadelta T-cell receptor (TCR)Vgamma9, recognize nonpeptide phosphoantigens (PAgs) produced by microbes and tumor cells, and mediate different modes of lytic activities directed against tumor target cells. Antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by cytolytic lymphoid cells is essential for the clinical activity of anticancer monoclonal antibodies (mAbs), but whether PAgs affect ADCC by gammadelta T cells is unknown. Here we report that, in association with the CD20(+)-specific mAb rituximab (RTX), the synthetic PAg bromohydrin pyrophosphate (BrHPP) increased TCRVgamma9(+) cell binding to CD20(+) lymphoma cells in vitro. This combination activated phospho-ZAP70 and phospho-ERK1/2 signaling in TCRVgamma9(+) cells and strongly enhanced their ADCC activity. We obtained similar results with BrHPP in the context of the mAbs alemtuzumab and trastuzumab. Furthermore, BrHPP enhanced RTX-mediated depletion of CD20(+) cells in vitro from peripheral blood mononuclear cells of healthy subjects and enhanced ADCC by gammadelta T cells from patients with chronic lymphocytic leukemia. In cynomolgus macaques, a regimen combining RTX, BrHPP, and IL2 activated TCRVgamma9(+) lymphocytes and enhanced B-cell depletion from blood and lymph nodes. Thus, the combination with BrHPP PAg is able to improve the efficacy of cancer immunotherapy by therapeutic mAbs.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Difosfatos/farmacologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Adulto , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Murinos , Protocolos de Quimioterapia Combinada Antineoplásica/química , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Células Cultivadas , Difosfatos/administração & dosagem , Difosfatos/imunologia , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Interleucina-2/administração & dosagem , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/imunologia , Macaca fascicularis , Masculino , Fosfatos/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Rituximab , Linfócitos T/imunologia , Linfócitos T/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologia
15.
Blood ; 111(1): 285-91, 2008 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-17855629

RESUMO

Previous studies have documented that, in malignant B cells, rituximab elicits a complex and not yet totally understood signaling network contributing to its antitumor effect. In this context, we investigated the role of protein kinase C zeta (PKCzeta), an atypical PKC isoform, in the cellular response to rituximab. We found that follicular lymphoma cells displayed an increase in PKCzeta expression and activity levels, compared with nonmalignant B cells, and that this enzyme was a critical regulator of the classical MAPK module by stimulating Raf-1 kinase activity. PKCzeta appeared to be a significant contributor of abnormal mTOR regulation in follicular lymphoma cells through a MAPK-dependent mechanism. Rituximab was found to inhibit the PKCzeta/MAPK/mTOR module in these cells but not in other B-cell lymphomas. Importantly, the expression of a constitutively active form of PKCzeta resulted in an efficient protection of these cells toward rituximab. Altogether, our study describes a new regulatory component of mTOR pathway in follicular cell lymphoma and demonstrates that PKCzeta is a target for rituximab. Therefore, PKCzeta could represent an important parameter for rituximab efficacy and a promising target for future targeted therapy in follicular lymphoma.


Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Linfoma Folicular/tratamento farmacológico , Linfoma Folicular/metabolismo , Proteína Quinase C/metabolismo , Proteínas Quinases/metabolismo , Anticorpos Monoclonais Murinos , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/enzimologia , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Rituximab , Serina-Treonina Quinases TOR
16.
Biochem J ; 405(1): 77-83, 2007 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-17346242

RESUMO

In a recent study, we described that UV-C irradiation resulted in redox-dependent activation and relocalization of A-SMase (acid sphingomyelinase) to the external surface of raft membrane microdomains, hydrolysis of SM (sphingomyelin) associated with the plasma membrane outer leaflet, ceramide generation and apoptosis. In the present study, we have investigated the influence of PKCzeta (protein kinase Czeta), an atypical form of PKC on this pathway. This study shows that PKCzeta overexpression resulted in the abrogation of UV-C-induced A-SMase translocation and activation into the raft microdomains, lack of ceramide generation and apoptosis inhibition. Moreover, PKCzeta overexpression resulted in a decrease in UV-C-induced ROS (reactive oxygen species) production, which correlated with increased gene expression level of various antioxidant enzymes, including TRx (thioredoxin), TR (thioredoxin reductase) 1, TR2 and peroxiredoxin 1/TPx2 (thioredoxin peroxidase 2). Importantly, enforced TPx2 gene expression inhibited UV-C-induced A-SMase translocation. Finally, PKCzeta inhibition led to a significant reduction in TPx2 protein expression. Altogether, these results suggest that PKCzeta interferes with the UV-activated sphingolipid signalling pathway by regulating the TRx system. These findings may have important consequences for UV-induced carcinogenesis and resistance to phototherapy.


Assuntos
Apoptose/efeitos da radiação , Ceramidas/biossíntese , Proteína Quinase C/metabolismo , Esfingomielina Fosfodiesterase , Animais , Linhagem Celular , Humanos , Peróxido de Hidrogênio/metabolismo , Microdomínios da Membrana/metabolismo , Oxidantes/metabolismo , Proteína Quinase C/genética , Ratos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologia , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Esfingomielina Fosfodiesterase/metabolismo , Tiorredoxinas/metabolismo , Raios Ultravioleta
17.
Blood ; 108(13): 4156-62, 2006 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-16912221

RESUMO

The mammalian target of rapamycin (mTOR) is emerging as a promising target for antitumor therapy. However, the mechanism that contributes to its regulation in B lymphomas remains unknown. This study shows that in follicular lymphoma (FL) cells, mTOR is active because the cells displayed rapamycin-sensitive phosphorylation of p70S6 kinase and 4E-BP1. Moreover, immunohistochemistry applied on lymph node tissue sections obtained from patients with FL revealed that, in most cases, p70S6 kinase was highly phosphorylated compared to normal tonsillar tissue. In FL cells, mTOR was under control of both phospholipase D (PLD) and phosphatidylinositol 3-kinase (PI3K). Moreover, we demonstrated that Syk plays a central role in mTOR activation because we found that both expression and activity are elevated compared to normal or chronic lymphocytic leukemia B cells. We also provide evidence that Syk operates through PLD- and PI3K-independent pathways. Finally, Syk inhibition by piceatannol or by siRNA plasmids resulted in a potent inhibition of mTOR activity in FL cells, as well as in mantle cell lymphoma, Burkitt lymphoma, and diffuse large B-cell lymphoma. These findings suggest that the Syk-mTOR pathway has a critical function in FL survival, and therefore, that Syk could be a promising new target for B-lymphoma therapy.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Linfoma Folicular/enzimologia , Proteínas de Neoplasias/metabolismo , Proteínas Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Linfoma de Burkitt/tratamento farmacológico , Linfoma de Burkitt/enzimologia , Linfoma de Burkitt/patologia , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/enzimologia , Leucemia Linfocítica Crônica de Células B/patologia , Linfonodos/metabolismo , Linfonodos/patologia , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/enzimologia , Linfoma de Células B/patologia , Linfoma Folicular/tratamento farmacológico , Linfoma Folicular/patologia , Linfoma de Célula do Manto/tratamento farmacológico , Linfoma de Célula do Manto/enzimologia , Linfoma de Célula do Manto/patologia , Tonsila Palatina/enzimologia , Tonsila Palatina/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfolipase D/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estilbenos/farmacologia , Quinase Syk , Serina-Treonina Quinases TOR
18.
J Biol Chem ; 280(19): 19196-204, 2005 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-15769735

RESUMO

The initiation of UV light-induced signaling in mammalian cells is largely considered to be subsequent to DNA damage. Several studies have also described ceramide (CER), a lipid second messenger, as a major contributor in mediating UV light-induced c-Jun N-terminal kinase (JNK) activation and cell death. It is demonstrated here that UV-C light irradiation of U937 cells results in the activation and translocation of a Zn2+-independent acid sphingomyelinase, leading to CER accumulation in raft microdomains. These CER-enriched rafts aggregate and play a functional role in JNK activation. The observation that UV-C light also induced CER generation and the externalization of acid sphingomyelinase and JNK in human platelets conclusively rules out the involvement of a nuclear signal generated by DNA damage in the initiation of a UV light response, which is generated at the plasma membrane.


Assuntos
Núcleo Celular/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Microdomínios da Membrana/química , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Western Blotting , Membrana Celular/metabolismo , Separação Celular , Ceramidas/metabolismo , Dano ao DNA , Fibroblastos/metabolismo , Citometria de Fluxo , Humanos , Hidrólise , Linfócitos/metabolismo , MAP Quinase Quinase 4 , Microscopia Confocal , Transporte Proteico , Espécies Reativas de Oxigênio , Esfingomielina Fosfodiesterase/metabolismo , Esfingomielinas/metabolismo , Fatores de Tempo , Células U937 , Raios Ultravioleta , Zinco/química
19.
Blood ; 104(4): 1166-73, 2004 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-15126316

RESUMO

Rituximab is a chimeric human immunoglobulin G1 (IgG1) anti-CD20 monoclonal antibody with significant activity against CD20+ malignant B cells. Rituximab is currently used with success in the treatment of B-cell-derived lymphoid neoplasias either alone or in combination with chemotherapy. However, the predominant mechanism by which rituximab exerts its antitumor properties in vivo remains unknown. In the present study, we demonstrate that in Daudi and RL B-lymphoma cells, rituximab (without cross-linking) used at the saturating dose of 10 microg/mL induced moderate accumulation in G1 phase, growth inhibition, and significant loss in clonogenic potential. However, in these cells, rituximab induced no apoptosis. Furthermore, we observed that treatment with rituximab resulted in a rapid and transient increase in acid-sphingomyelinase (A-SMase) activity and concomitant cellular ceramide (CER) generation in raft microdomains. We also observed that rituximab-treated cells externalized both A-SMase and CER that colocalized with the CD20 receptor. Finally, we present evidence that rituximab-induced growth inhibition may be mediated through a CER-triggered signaling pathway, leading to the induction of cell cycle-dependent kinase inhibitors such as p27Kip1 through a mitogen-activated protein kinase (MAPK)-dependent mechanism.


Assuntos
Anticorpos Monoclonais/farmacologia , Linfoma de Células B/patologia , Microdomínios da Membrana/enzimologia , Esfingomielina Fosfodiesterase/metabolismo , Anticorpos Monoclonais Murinos , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ceramidas/metabolismo , Ceramidas/fisiologia , Ativação Enzimática , Fase G1 , Humanos , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/enzimologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Transporte Proteico/efeitos dos fármacos , Rituximab , Transdução de Sinais
20.
J Exp Ther Oncol ; 3(1): 36-46, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-12724857

RESUMO

Taxanes are known to activate several cellular signals including mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-kappa B), tyrosine phosphorylation of Shc, and serine phosphorylation of Bcl-2. However, the mediators of these signaling pathways are unknown. Using U937 leukemic cells, we evaluated the effect of docetaxel on phosphatidylcholine (PC) and its metabolites, phosphatidic acid (PA) and diacylglycerol (DAG), and their impact on MAPK and NF-kappa B activation, as well as on Raf-1 and Bcl-2 phosphorylation. Metabolic labeling studies showed that docetaxel (10 nM) induced two waves of PA production (130-140%), which were detected at 1 and 10 min. Docetaxel also stimulated DAG production (130%), which followed the first PA wave. The initial PA burst was due to phospholipase D (PLD)-mediated PC hydrolysis. Subsequent DAG production was inhibited by the phosphatidate phosphohydrolase (PAP) inhibitor, propranolol. R59949, a DAG kinase inhibitor, increased DAG accumulation and blocked the second PA wave. These results suggest that docetaxel triggers a metabolic cascade consisting in PLD-mediated PC hydrolysis, PA release, PAP-dependent DAG production, and DAG kinase stimulation, leading to DAG conversion back to PA. Neither R59949 nor propranolol influenced docetaxel-induced Raf-1/ERK activation. However, R59949 abrogated both NF-kappa B activation and Bcl-2 phosphorylation, suggesting that DAG and/or DAG-derived PA contribute in regulating these events.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Diglicerídeos/biossíntese , Ácidos Fosfatídicos/biossíntese , Fosfatidilcolinas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Taxoides/farmacologia , Diacilglicerol Quinase/metabolismo , Docetaxel , Ensaio de Desvio de Mobilidade Eletroforética , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Humanos , Hidrólise , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Fosforilação , Piperidinas/farmacologia , Propranolol/farmacologia , Proteínas Proto-Oncogênicas c-raf/metabolismo , Quinazolinas/farmacologia , Quinazolinonas , Células U937
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