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1.
Blood ; 123(14): 2199-203, 2014 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-24497531

RESUMO

Antigen receptor-mediated nuclear factor κB (NF-κB) activation relies on the formation of a large multi-protein complex that contains CARMA1, BCL10, and MALT1 (CBM complex). This signalosome is pirated in the activated B-cell-like subgroup of diffuse large B-cell lymphoma (ABC DLBCL) to drive aberrant NF-κB activation, thereby promoting cell survival and propagation. Using an unbiased proteomic approach, we screened for additional components of the CBM in lymphocytes. We found that the linear ubiquitin chain assembly complex (LUBAC), which was previously linked to cytokine-mediated NF-κB activation, dynamically integrates the CBM and marshals NF-κB optimal activation following antigen receptor ligation independently of its catalytic activity. The LUBAC also participates in preassembled CBM complex in cells derived from ABC DLBCL. Silencing the LUBAC reduced NF-κB activation and was toxic in ABC DLBCL cell lines. Thus, our findings reveal a role for the LUBAC during lymphocyte activation and in B-cell malignancy.


Assuntos
Linfoma/metabolismo , NF-kappa B/química , NF-kappa B/metabolismo , Domínios e Motivos de Interação entre Proteínas/fisiologia , Receptores de Antígenos de Linfócitos T/metabolismo , Ubiquitina/metabolismo , Catálise , Linhagem Celular Tumoral , Humanos , Células Jurkat , Ativação Linfocitária/fisiologia , Linfoma/patologia , Ligação Proteica , Ubiquitinação/fisiologia
2.
Cell Commun Signal ; 13: 11, 2015 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-25889342

RESUMO

BACKGROUND: The viral G protein-coupled receptor (vGPCR) is proposed to act as one of the predominant mediators of Kaposi's sarcoma (KS), a human herpes virus 8 (HHV8)-elicited disease. The actions of vGPCR manifest pathogenesis, in part, through increased permeability of endothelial cells. Endothelial cell-cell junctions have indeed emerged as an instrumental target involved in the vasculature defects observed within the tumor microenvironment. The pathway leading to adherens junction destabilization has been shown to involve the activation of the small GTPase Rac, in the context of either latent infection or the sole expression of vGPCR. However, the precise molecular mechanisms governed by vGPCR in vascular leakage require further elucidation. FINDINGS: Guanine exchange factors (GEFs) function as critical molecular switches that control the activation of small GTPases. We therefore screened the effects of 80 siRNAs targeting GEFs on vGPCR-driven endothelial permeability and identified switch-associated protein 70 (SWAP70) as necessary for its elevating effects. Pull-down experiments further showed that Rac activation by vGPCR was dependent on SWAP70. Examination of tissues and cells from HHV8-positive patients revealed that SWAP70 was ubiquitously expressed. Furthermore, SWAP70 was found to be crucial for vGPCR-driven endothelial tube formation and endothelial sprouting in vitro. CONCLUSIONS: SWAP70 appears to act as a molecular intermediate between vGPCR and endothelial activation. Because of the important role of vGPCR-mediated endothelial plasticity in KS pathogenesis, inhibition of SWAP70 function could be of interest for blocking vGPCR-driven activities in HHV8-defined diseases.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Herpesvirus Humano 8/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Proteínas Nucleares/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Virais/metabolismo , Proteínas de Ligação a DNA/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Herpesvirus Humano 8/genética , Humanos , Antígenos de Histocompatibilidade Menor , Proteínas Nucleares/genética , Receptores Acoplados a Proteínas G/genética , Proteínas Virais/genética
3.
Blood ; 118(15): 4120-8, 2011 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-21868573

RESUMO

Apoptosis is crucial for immune system homeostasis, including selection and survival of long-lived antibody-forming cells and memory cells. The interactions between proapoptotic and pro-survival proteins of the Bcl-2 family are critical for this process. In this report, we show that expression of the proapoptotic BH3-only Bcl-2 family member Puma was selectively up-regulated on in vitro activation with antigens or mitogens of both human and mouse B cells. Puma expression coincided in vivo, with the prosurvival Bcl-2 family member Mcl-1 within the germinal centers and its expression correlates with the germinal center like phenotype of Burkitt lymphoma. Experiments performed in Puma-deficient mice revealed that Puma is essential for apoptosis of mitogen-activated B cells in vitro and for the control of memory B-cell survival. In conclusion, using both human and murine models, our data show that Puma has a major role in the T cell- dependent B-cell immune response. These data demonstrate that Puma is a major regulator of memory B lymphocyte survival and therefore a key molecule in the control of the immune response.


Assuntos
Proteínas Reguladoras de Apoptose/imunologia , Apoptose/fisiologia , Linfócitos B/imunologia , Memória Imunológica/fisiologia , Proteínas Proto-Oncogênicas/imunologia , Proteínas Supressoras de Tumor/imunologia , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/biossíntese , Proteínas Reguladoras de Apoptose/genética , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Memória Imunológica/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/fisiologia , Camundongos , Camundongos Mutantes , Mitógenos/farmacologia , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Proteínas Supressoras de Tumor/biossíntese , Proteínas Supressoras de Tumor/genética
4.
Cell Commun Signal ; 11(1): 25, 2013 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-23590831

RESUMO

BACKGROUND: NF-κB is a master gene regulator involved in plethora of biological processes, including lymphocyte activation and proliferation. Reversible ubiquitinylation of key adaptors is required to convey the optimal activation of NF-κB. However the deubiquitinylases (DUBs), which catalyze the removal of these post-translational modifications and participate to reset the system to basal level following T-Cell receptor (TCR) engagement continue to be elucidated. FINDINGS: Here, we performed an unbiased siRNA library screen targeting the DUBs encoded by the human genome to uncover new regulators of TCR-mediated NF-κB activation. We present evidence that knockdown of Ubiquitin-Specific Protease 34 (USP34) selectively enhanced NF-κB activation driven by TCR engagement, similarly to siRNA against the well-characterized DUB cylindromatosis (CYLD). From a molecular standpoint, USP34 silencing spared upstream signaling but led to a more pronounced degradation of the NF-κB inhibitor IκBα, and culminated with an increased DNA binding activity of the transcription factor. CONCLUSIONS: Collectively, our data unveils USP34 as a new player involved in the fine-tuning of NF-κB upon TCR stimulation.

5.
Front Immunol ; 14: 1199594, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37593736

RESUMO

The innate immune lymphocyte lineage natural killer (NK) cell infiltrates tumor environment where it can recognize and eliminate tumor cells. NK cell tumor infiltration is linked to patient prognosis. However, it is unknown if some of these antitumor NK cells leave the tumor environment. In blood-borne cancers, NK cells that have interacted with leukemic cells are recognized by the co-expression of two CD45 isoforms (CD45RARO cells) and/or the plasma membrane presence of tumor antigens (Ag), which NK cells acquire by trogocytosis. We evaluated solid tumor Ag uptake by trogocytosis on NK cells by performing co-cultures in vitro. We analyzed NK population subsets by unsupervised dimensional reduction techniques in blood samples from breast tumor (BC) patients and healthy donors (HD). We confirmed that NK cells perform trogocytosis from solid cancer cells in vitro. The extent of trogocytosis depends on the target cell and the antigen, but not on the amount of Ag expressed by the target cell or the sensitivity to NK cell killing. We identified by FlowSOM (Self-Organizing Maps) several NK cell clusters differentially abundant between BC patients and HD, including anti-tumor NK subsets with phenotype CD45RARO+CD107a+. These analyses showed that bona-fide NK cells that have degranulated were increased in patients and, additionally, these NK cells exhibit trogocytosis of solid tumor Ag on their surface. However, the frequency of NK cells that have trogocytosed is very low and much lower than that found in hematological cancer patients, suggesting that the number of NK cells that exit the tumor environment is scarce. To our knowledge, this is the first report describing the presence of solid tumor markers on circulating NK subsets from breast tumor patients. This NK cell immune profiling could lead to generate novel strategies to complement established therapies for BC patients or to the use of peripheral blood NK cells in the theranostic of solid cancer patients after treatment.


Assuntos
Neoplasias da Mama , Neoplasias Hematológicas , Neoplasias Mamárias Animais , Animais , Humanos , Feminino , Antígenos de Neoplasias , Células Matadoras Naturais , Membrana Celular
6.
Sci Rep ; 12(1): 1341, 2022 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-35079096

RESUMO

Solid tumor cells have an altered metabolism that can protect them from cytotoxic lymphocytes. The anti-diabetic drug metformin modifies tumor cell metabolism and several clinical trials are testing its effectiveness for the treatment of solid cancers. The use of metformin in hematologic cancers has received much less attention, although allogeneic cytotoxic lymphocytes are very effective against these tumors. We show here that metformin induces expression of Natural Killer G2-D (NKG2D) ligands (NKG2DL) and intercellular adhesion molecule-1 (ICAM-1), a ligand of the lymphocyte function-associated antigen 1 (LFA-1). This leads to enhance sensitivity to cytotoxic lymphocytes. Overexpression of anti-apoptotic Bcl-2 family members decrease both metformin effects. The sensitization to activated cytotoxic lymphocytes is mainly mediated by the increase on ICAM-1 levels, which favors cytotoxic lymphocytes binding to tumor cells. Finally, metformin decreases the growth of human hematological tumor cells in xenograft models, mainly in presence of monoclonal antibodies that recognize tumor antigens. Our results suggest that metformin could improve cytotoxic lymphocyte-mediated therapy.


Assuntos
Molécula 1 de Adesão Intercelular/fisiologia , Metformina/farmacologia , Neoplasias/tratamento farmacológico , Animais , Humanos , Células Matadoras Naturais , Masculino , Camundongos , Camundongos Endogâmicos NOD , Células Tumorais Cultivadas
7.
Cells ; 11(9)2022 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-35563698

RESUMO

Cells have metabolic flexibility that allows them to adapt to changes in substrate availability. Two highly relevant metabolites are glucose and fatty acids (FA), and hence, glycolysis and fatty acid oxidation (FAO) are key metabolic pathways leading to energy production. Both pathways affect each other, and in the absence of one substrate, metabolic flexibility allows cells to maintain sufficient energy production. Here, we show that glucose starvation or sustained pyruvate dehydrogenase (PDH) activation by dichloroacetate (DCA) induce large genetic remodeling to propel FAO. The extracellular signal-regulated kinase 5 (ERK5) is a key effector of this multistep metabolic remodeling. First, there is an increase in the lipid transport by expression of low-density lipoprotein receptor-related proteins (LRP), e.g., CD36, LRP1 and others. Second, an increase in the expression of members of the acyl-CoA synthetase long-chain (ACSL) family activates FA. Finally, the expression of the enzymes that catalyze the initial step in each cycle of FAO, i.e., the acyl-CoA dehydrogenases (ACADs), is induced. All of these pathways lead to enhanced cellular FAO. In summary, we show here that different families of enzymes, which are essential to perform FAO, are regulated by the signaling pathway, i.e., MEK5/ERK5, which transduces changes from the environment to genetic adaptations.


Assuntos
Glucose , Proteína Quinase 7 Ativada por Mitógeno , Ácidos Graxos/metabolismo , Glucose/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Oxirredução , Oxirredutases/metabolismo , Piruvatos
8.
Sci Rep ; 12(1): 3234, 2022 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-35217717

RESUMO

Leukemic cells proliferate faster than non-transformed counterparts. This requires them to change their metabolism to adapt to their high growth. This change can stress cells and facilitate recognition by immune cells such as cytotoxic lymphocytes, which express the activating receptor Natural Killer G2-D (NKG2D). The tumor suppressor gene p53 regulates cell metabolism, but its role in the expression of metabolism-induced ligands, and subsequent recognition by cytotoxic lymphocytes, is unknown. We show here that dichloroacetate (DCA), which induces oxidative phosphorylation (OXPHOS) in tumor cells, induces the expression of such ligands, e.g. MICA/B, ULBP1 and ICAM-I, by a wtp53-dependent mechanism. Mutant or null p53 have the opposite effect. Conversely, DCA sensitizes only wtp53-expressing cells to cytotoxic lymphocytes, i.e. cytotoxic T lymphocytes and NK cells. In xenograft in vivo models, DCA slows down the growth of tumors with low proliferation. Treatment with DCA, monoclonal antibodies and NK cells also decreased tumors with high proliferation. Treatment of patients with DCA, or a biosimilar drug, could be a clinical option to increase the effectiveness of CAR T cell or allogeneic NK cell therapies.


Assuntos
Antineoplásicos , Leucemia , Proteína Supressora de Tumor p53 , Antineoplásicos/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Leucemia/imunologia , Leucemia/metabolismo , Ligantes , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Proteína Supressora de Tumor p53/imunologia , Proteína Supressora de Tumor p53/metabolismo
9.
Apoptosis ; 15(12): 1529-39, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20640889

RESUMO

The protein Puma (p53-upregulated modulator of apoptosis) belongs to the BH3-only group of the Bcl-2 family and is a major regulator of apoptosis. Although the transcriptional regulation of Puma is clearly established, little is known about the regulation of its expression at the protein levels. We show here that various signals--including the cytokine TGFß, the death effector TRAIL or chemical drugs such as anisomycin--downregulate Puma protein levels via a novel pathway based on the sequential activation of caspase-3 and a protease inhibited by the serpase inhibitor N-tosyl-L-phenylalanine chloromethyl ketone. This pathway is specific for Puma because (1) the levels of other BH3-only proteins, such as Bim and Noxa were not modified by these stimuli and (2) this caspase-mediated degradation was dependent on both the BH3 and C-terminal domains of Puma. Our data also show that Puma is regulated during the caspase-3-dependent differentiation of murine embryonic stem cells and suggest that this pathway may be relevant and important during caspase-mediated cell differentiation not associated with apoptosis.


Assuntos
Proteínas Reguladoras de Apoptose , Caspase 3 , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Fragmentos de Peptídeos/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Serina Proteases , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Tosilfenilalanil Clorometil Cetona , Fator de Crescimento Transformador beta/farmacologia , Animais , Anisomicina/farmacologia , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Caspase 3/metabolismo , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Inativação Gênica/fisiologia , Humanos , Camundongos , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Serina Proteases/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/genética , Tosilfenilalanil Clorometil Cetona/farmacologia , Fator de Crescimento Transformador beta/genética
10.
Vaccines (Basel) ; 8(4)2020 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-33276644

RESUMO

The lymphocyte lineage natural killer (NK) cell is part of the innate immune system and protects against pathogens and tumor cells. NK cells are the main cell effectors of the monoclonal antibodies (mAbs) that mediates antibody-dependent cell cytotoxicity (ADCC). Hence, it is relevant to understand NK physiology and status to investigate the biological effect of mAbs in the clinic. NK cells are heterogeneous with multiple subsets that may have specific activity against different attacks. The presence of viral-sculpted NK cell populations has already been described, but the presence of cancer-sculpted NK cells remains unknown. Cancer induces a broad NK cell dysfunction, which has not been linked to a specific population. Here, we investigated the NK cell population by Uniform Manifold Approximation and Projection (UMAP) embed maps in Hodgkin lymphoma (HL) and acute myeloid leukemia (AML) patients at diagnosis and at least 30 days after treatment, which correlates with tumor cell clearance. We found that the NK lineage largely responded to the tumor by generating antitumor NK cells and renewing the population with a subset of immature NK cells. However, we failed to identify a specific "memory-like" subset with the NK cell markers used. Moreover, in patients in relapse, we found essentially the same NK populations as those found at diagnosis, suggesting that NK cells equally respond to the first or second tumor rise. Finally, we observed that previous cytomegalovirus (CMV) infection largely affects the tumor-associated changes in NK population, but the CMV-associated CD57+NKG2C+ NK cell population does not appear to play any role in tumor immunity.

11.
Front Immunol ; 10: 3026, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31998309

RESUMO

The innate lymphocyte lineage natural killer (NK) is now the target of multiple clinical applications, although none has received an agreement from any regulatory agency yet. Transplant of naïve NK cells has not proven efficient enough in the vast majority of clinical trials. Hence, new protocols wish to improve their medical use by producing them from stem cells and/or modifying them by genetic engineering. These techniques have given interesting results but these improvements often hide that natural killers are mainly that: natural. We discuss here different ways to take advantage of NK physiology to improve their clinical activity without the need of additional modifications except for in vitro activation and expansion and allograft in patients. Some of these tactics include combination with monoclonal antibodies (mAb), drugs that change metabolism and engraftment of specific NK subsets with particular activity. Finally, we propose to use specific NK cell subsets found in certain patients that show increase activity against a specific disease, including the use of NK cells derived from patients.


Assuntos
Citotoxicidade Imunológica , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Anticorpos Monoclonais/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Biomarcadores , Microambiente Celular/imunologia , Evolução Clonal , Citocinas/metabolismo , Humanos , Memória Imunológica , Antígenos Comuns de Leucócito/metabolismo , Ativação Linfocitária/imunologia
12.
Front Immunol ; 10: 2693, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31849934

RESUMO

Immunotherapy, which is seen as a major tool for cancer treatment, requires, in some cases, the presence of several agents to maximize its effects. Adjuvants can enhance the effect of other agents. However, despite their long-time use, only a few adjuvants are licensed today, and their use in cancer treatment is rare. Azoximer bromide, marketed under the trade name Polyoxidonium® (PO), is a copolymer of N-oxidized 1,4-ethylenepiperazine and (N-carboxyethyl)-1,4-ethylene piperazinium bromide. It has been described as an immune adjuvant and immunomodulator that is clinically used with excellent tolerance. PO is used in the treatment and prophylaxis of diseases connected with damage to the immune system, and there is interest in testing it in antitumor therapy. We show here that PO treatment for 1 week induced positive pathological changes in 6 out of 20 patients with breast cancer, including complete response in a triple-negative patient. This correlated with an increased tumor CD4+ T-lymphocyte infiltration. The immune effects of PO are associated with myeloid cell activation, and little is known about the action of PO on lymphocyte lineages, such as natural killer (NK) and T cells. We reveal that PO increases T-cell proliferation in vitro without negative effects on any activation marker. PO does not affect dendritic cell (DC) viability and increases the expansion of immature DC (iDC) and mature DC (mDC) at 100 µg/ml, and it stimulates expression of several DC co-stimulatory molecules, inducing the proliferation of allogeneic T cells. In contrast, PO decreases DC viability when added at day 5 post-expansion. PO is not toxic for NK cells at doses up to 100 µM and does not affect their activation, maturation, and cytotoxicity but tends to increase degranulation. This could be beneficial against target cells that show low sensitivity to NK cells, e.g., solid tumor cells. Finally, we have found great variability in PO response between donors. In summary, our in vitro results show that PO increases the number of costimulatory molecules on DC that prime T cells, favoring the production of effector T cells. This may support the future clinical development of PO in cancer treatment.


Assuntos
Adenocarcinoma/tratamento farmacológico , Adjuvantes Imunológicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Células Dendríticas/efeitos dos fármacos , Piperazinas/uso terapêutico , Polímeros/uso terapêutico , Adenocarcinoma/imunologia , Adulto , Idoso , Neoplasias da Mama/imunologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Quimioterapia Adjuvante/métodos , Células Dendríticas/imunologia , Feminino , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Pessoa de Meia-Idade , Terapia Neoadjuvante/métodos , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia
13.
Oncoimmunology ; 7(4): e1409322, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29632722

RESUMO

Obinutuzumab (OBZ) shows stronger antibody-dependent cell cytotoxicity (ADCC) compared to rituximab and improved clinical activity for treating certain CD20+ neoplasia. However, the efficacy of monoclonal antibody (mAb) as a monotherapy is limited. Natural Killer (NK) cells are mediators of ADCC. Hematological cancer patients possess antitumor NK cells that are unable to control disease, possibly because they are dysfunctional. The immunomodulatory drug lenalidomide (LEN) could be a treatment to restore exhausted NK cell cytotoxic functions. The clinical trial GALEN is a Phase Ib/II study of OBZ combined with LEN for the treatment of relapsed/refractory follicular and aggressive (DLBCL and MCL) B-cell Lymphoma. During treatment, we analyzed specific aspects of NK cell biology. Treatment reversed the immature NK phenotype of patients and increased expression of NK activating receptors. Inhibitory receptors were either unchanged or decreased. There was a strong NK response at the end of the 1st cycle: NK number and intracellular granzyme B (GrzB) expression decreased, degranulation increased and NK responded better to allogeneic target challenge. Moreover, the interaction of NK cells with B cell targets, measured by trogocytosis, decreased during treatment. At the end of treatment, when target cells had been wiped out, the proportion of reactive NK cells (CD69+, CD45RARO+, CD107a+, CD19+) strongly decreased. Because all patients received LEN and OBZ, it was uncertain which drug was responsible of our observations, or even if a combination of both products was necessary for the described effects on this lymphocyte lineage.

14.
Oncotarget ; 9(1): 1114-1129, 2018 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-29416681

RESUMO

Changes in metabolism require the efflux and influx of a diverse variety of metabolites. The ABC superfamily of transporters regulates the exchange of hundreds of substrates through the impermeable cell membrane. We show here that a metabolic switch to oxidative phosphorylation (OXPHOS), either by treating cells with dichloroacetate (DCA) or by changing the available substrates, reduced expression of ABCB1, ABCC1, ABCC5 and ABCG2 in wild-type p53-expressing cells. This metabolic change reduced histone changes associated to active promoters. Notably, DCA also inhibited expression of these genes in two animal models in vivo. In contrast, OXPHOS increased the expression of the same transporters in mutated (mut) or null p53-expressing cells. ABC transporters control the export of drugs from cancer cells and render tumors resistant to chemotherapy, playing an important role in multiple drug resistance (MDR). Wtp53 cells forced to perform OXPHOS showed impaired drug clearance. In contrast mutp53 cells increased drug clearance when performing OXPHOS. ABC transporter promoters contain binding sites for the transcription factors MEF2, NRF1 and NRF2 that are targets of the MAPK ERK5. OXPHOS induced expression of the MAPK ERK5. Decreasing ERK5 levels in wtp53 cells increased ABC expression whereas it inhibited expression in mutp53 cells. Our results showed that the ERK5/MEF2 pathway controlled ABC expression depending on p53 status.

15.
FEBS Lett ; 580(11): 2547-52, 2006 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-16631755

RESUMO

Doxorubicin, cis-diamminedichloroplatinum (II) and 5-fluorouracil used in chemotherapy induce apoptosis in Hep3B cells in the absence of p53, p73, and functional Fas. Since mediators remain unknown, the requirement of PKC delta (PKCdelta) and c-Abl was investigated. Suppression of c-Abl or PKCdelta expression using SiRNAs impaired PARP cleavage, Gleevec and/or rottlerin inhibited the induction of the subG1 phase and the increase of reactive oxygen species level. Co-precipitations and phosphorylations to mitochondria of c-Abl, PKCdelta and Bcl-X(L/s) were induced. A depolarization of the mitochondrial membrane and activations of caspase-2 and -9 were observed. We propose that, in the absence of p53, p73 and Fas, genotoxic drugs could require both PKCdelta and c-Abl to induce apoptosis through the mitochondrial pathway.


Assuntos
Apoptose/efeitos dos fármacos , Fluoruracila/toxicidade , Mitocôndrias/metabolismo , Mutagênicos/toxicidade , Proteína Quinase C-delta/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Caspase 2 , Caspase 9 , Caspases/metabolismo , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/efeitos dos fármacos , Cisplatino/toxicidade , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/metabolismo , Doxorrubicina/toxicidade , Humanos , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Proteínas Nucleares/deficiência , Proteínas Nucleares/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica , Proteína Quinase C-delta/genética , Proteínas Proto-Oncogênicas c-abl/genética , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/metabolismo , Proteína bcl-X/metabolismo , Receptor fas/metabolismo
16.
Ann N Y Acad Sci ; 1090: 1-17, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17384242

RESUMO

IGF-II and type I-IGF receptor (IGF-IR) gene expression is increased in primary liver tumors, and transgenic mice overexpressing IGF-II in the liver develop hepatocellular carcinoma (HCC) spontaneously, suggesting that alterations of IGF-IR signaling in vivo may play a role in the auto/paracrine control of hepatocarcinogenesis. We have addressed the contribution of PI-3'K/Akt signaling on the proliferation of HepG2 human hepatoma cells and on their protection against doxorubicin-induced apoptosis. Both basal HepG2 cell DNA replication and that stimulated by IGF-IR signaling were inhibited by the specific PI-3'K inhibitor Ly294002 (Ly). In the former case, PI-3'K signaling overcame cell cycle arrest in G1 via increased cyclin D1 protein and decreased p27kip1 gene expression. Doxorubicin treatment induced apoptosis in HepG2 cells and was concomitant with the proteolytic cleavage of Akt-1 and -2. Drug-induced apoptosis was reversed by IGF-I and this effect was (i) dependent on Akt-1 and -2 phosphorylation and (ii) accompanied by the inhibition of initiator caspase-9 activity, suggesting that IGF-IR signaling interferes with mitochondria-dependent apoptosis. Accordingly, Ly enhanced doxorubicin-induced apoptosis and suppressed its reversal by IGF-I. Altogether, the data emphasize the crucial role of PI-3'K/Akt signaling (i) in basal as well as IGF-IR-stimulated HepG2 cell proliferation and (ii) in controlling both doxorubicin-induced apoptosis (e.g., drug-induced cleavage of Akt) and its reversal by IGF-I (protection against apoptosis parallels the extent of Akt phosphorylation). They suggest that targeting Akt activity or downstream Akt effectors (e.g., GSK3-beta, FOXO transcription factors) may help define novel therapeutic strategies of increased efficacy in the treatment of HCC-bearing patients.


Assuntos
Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Replicação do DNA/efeitos dos fármacos , Doxorrubicina/farmacologia , Fase G1 , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , Receptor IGF Tipo 1
17.
Biochem Pharmacol ; 68(6): 1003-15, 2004 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-15313394

RESUMO

Strong evidence emphasizes the role of the insulin-like growth factor (IGF) system and of type-I IGF receptor (IGF-IR) signalling in tumourigenesis. In this connection: (i) changes in the expression pattern of components of the IGF system (autocrine/paracrine expression of IGF-I and -II, overexpression of IGF-IR, decreased expression of IGF-binding proteins (IGFBPs) and of type-II IGF receptor/cation-independent mannose-6-phosphate receptor (IGF-II/M6PR) and (ii) increased serum concentrations of proteases that cleave the IGFBPs (e.g., cathepsin D) were observed in patients with hepatocellular carcinomas (HCC), in human hepatoma cell lines and in their conditioned culture medium, as well as in rodent models of hepatocarcinogenesis. Accordingly, studies carried out with animal models do suggest that the IGF system and IGF-IR signalling may play a role in hepatocarcinogenesis and in deregulated proliferation and apoptosis of HCC cells. Finally the instrumental role of Raf/MEK/ERK, one of the signalling cascades stimulated by IGF-IR, in anthracycline-induced apoptosis of HepG2 and Huh-7 human hepatoma cell lines emphasizes that care must be taken when designing combinations of antitumoural molecules for antineoplastic treatment. This review addresses the putative roles of the IGF system in primary HCC, with a special focus on the underlying molecular mechanisms. In a second part it emphasizes the putative interference of IGF-IR signalling with chemotherapeutic drug-induced apoptosis in human hepatoma cells.


Assuntos
Antineoplásicos/farmacologia , Apoptose , Fator de Crescimento Insulin-Like II/fisiologia , Fator de Crescimento Insulin-Like I/fisiologia , Receptor IGF Tipo 1/fisiologia , Carcinoma Hepatocelular/patologia , Transformação Celular Neoplásica , Resistencia a Medicamentos Antineoplásicos/fisiologia , Humanos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/fisiologia , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Neoplasias Hepáticas/patologia , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais/fisiologia , Células Tumorais Cultivadas
18.
Ann N Y Acad Sci ; 1030: 219-29, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15659801

RESUMO

Enhanced insulin-like growth factor II (IGF-II) and type I IGF receptor (IGF-IR) gene expression in liver tumors and the development of liver tumors in transgenic mice overexpressing IGF-II in the liver suggest that the IGFs and underlying signaling cascades may play auto/paracrine roles in the control of hepatocarcinoma (HCC) cell proliferation and in their protection against apoptosis. We have focused on the role of mitogen-activated protein kinase (ERK1/2) signaling on human HepG2 and Huh-7 hepatoma cell proliferation and on the protection of these cells against drug-induced apoptosis. Physiological concentrations of IGF-I stimulated DNA replication in HepG2 cells (1.5-fold) but not in Huh-7 cells, and this effect was abolished by PD98059 (MEK-1 inhibitor). Doxorubicin or cisplatin treatment induced apoptosis (caspase-dependent poly[ADP-ribose]polymerase cleavage) in both cell lines, but dose-dependent reversion of drug-induced apoptosis (57-84%) by IGF-I was only observed in HepG2 cells. The very low level of IGF-IR at the plasma membrane of Huh-7 cells may account for their unresponsiveness to IGF-I. We have shown that drug treatment enhanced (17-fold) or did not modify constitutive ERK1/2 activity in cultured HepG2 or Huh-7 cells, respectively. In both cell lines, inhibition of constitutive and drug-induced ERK1/2 activity by PD98059 yielded a complete inhibition of drug-induced apoptosis. Altogether, our data demonstrate the heterogeneous response of human hepatoma cells to an IGF stimulus and suggest (1) that auto/paracrine effects of IGF-I/-II might contribute to the proliferation of HCC cells and to their protection against apoptosis in vivo and (2) that drug-induced activation of ERK1/2 plays a role in drug-induced apoptosis in human hepatoma cells.


Assuntos
Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Divisão Celular , Fator de Crescimento Insulin-Like II/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Neoplasias Hepáticas/patologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Carcinoma Hepatocelular/enzimologia , Linhagem Celular Tumoral , Replicação do DNA/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/enzimologia
19.
Sci Signal ; 6(291): ra79, 2013 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-24003256

RESUMO

The innate and adaptive immune responses involve the stimulation of nuclear factor κB (NF-κB) transcription factors through the Lys(63) (K(63))-linked ubiquitylation of specific components of NF-κB signaling pathways. We found that ubiquitylated components of the NF-κB pathway accumulated on the cytosolic leaflet of the endoplasmic reticulum (ER) membrane after the engagement of cell-surface, proinflammatory cytokine receptors or antigen receptors. Through mass spectrometric analysis, we found that the ER-anchored protein metadherin (MTDH) was a partner for these ubiquitylated activators of NF-κB and that it directly bound to K(63)-linked polyubiquitin chains. Knockdown of MTDH inhibited the accumulation of ubiquitylated NF-κB signaling components at the ER, reduced the extent of NF-κB activation, and decreased the amount of proinflammatory cytokines produced. Our observations highlight an unexpected facet of the ER as a key subcellular gateway for NF-κB activation.


Assuntos
Moléculas de Adesão Celular/imunologia , Retículo Endoplasmático/imunologia , NF-kappa B/imunologia , Poliubiquitina/imunologia , Transdução de Sinais/imunologia , Ubiquitinação/imunologia , Imunidade Adaptativa/fisiologia , Moléculas de Adesão Celular/genética , Citocinas/genética , Citocinas/imunologia , Retículo Endoplasmático/genética , Células HEK293 , Células HeLa , Humanos , Imunidade Inata/fisiologia , Células Jurkat , Proteínas de Membrana , NF-kappa B/genética , Poliubiquitina/genética , Proteínas de Ligação a RNA , Transdução de Sinais/genética , Ubiquitinação/genética
20.
J Biol Chem ; 283(37): 25514-25523, 2008 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-18579528

RESUMO

Engagement of the IgE receptor (FcepsilonRI) on mast cells leads to the release of preformed and newly formed mediators as well as of cytokines. The signaling pathways responsible for these responses involve tyrosine phosphorylation of multiple proteins. We previously reported the phosphorylation on tyrosine of phospholipid scramblase 1 (PLSCR1) after FcepsilonRI aggregation. Here, PLSCR1 expression was knocked down in the RBL-2H3 mast cell line using short hairpin RNA. Knocking down PLSCR1 expression resulted in significantly impaired degranulation responses after FcepsilonRI aggregation and release of vascular endothelial growth factor, whereas release of MCP-1 was minimally affected. The release of neither leukotriene C4 nor prostaglandin D2 was altered by knocking down of PLSCR1. Analysis of FcepsilonRI-dependent signaling pathways revealed that whereas tyrosine phosphorylation of ERK and Akt was unaffected, tyrosine phosphorylation of LAT was significantly reduced in PLSCR1 knocked down cells. Tyrosine phosphorylation of phospholipase Cgamma1 and consequently the mobilization of calcium were also significantly reduced in these cells. In nonactivated mast cells, PLSCR1 was found in part in lipid rafts where it was further recruited after cell activation and was constitutively associated with Lyn and Syk but not with LAT or Fyn. Altogether, these data identify PLSCR1 as a novel amplifier of FcepsilonRI signaling that acts selectively on the Lyn-initiated LAT/phospholipase Cgamma1/calcium axis, resulting in potentiation of a selected set of mast cell responses.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cálcio/metabolismo , Mastócitos/metabolismo , Proteínas de Membrana/metabolismo , Fosfolipase C gama/metabolismo , Proteínas de Transferência de Fosfolipídeos/fisiologia , Fosfoproteínas/metabolismo , Receptores de IgE/química , Receptores de IgE/metabolismo , Quinases da Família src/metabolismo , Animais , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Proteínas de Transferência de Fosfolipídeos/metabolismo , Prostaglandina D2/metabolismo , Ratos , Transdução de Sinais
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