RESUMO
Sirtuin 6 (SIRT6) is a member of the mammalian NAD+-dependent deac(et)ylase sirtuin family. SIRT6's anti-inflammatory roles are emerging increasingly often in different diseases and cell types, including endothelial cells. In this study, the role of SIRT6 in pro-inflammatory conditions was investigated by engineering human umbilical vein endothelial cells to overexpress SIRT6 (SIRT6+ HUVECs). Our results showed that SIRT6 overexpression affected the levels of adhesion molecules and sustained megakaryocyte proliferation and proplatelet formation. Interestingly, the pro-inflammatory activation of the ATP/purinergic axis was reduced in SIRT6+ HUVECs. Specifically, the TNFα-induced release of ATP in the extracellular space and the increase in pannexin-1 hemichannel expression, which mediates ATP efflux, were hampered in SIRT6+ cells. Instead, NAD+ release and Connexin43 expression were not modified by SIRT6 levels. Moreover, the Ca2+ influx in response to ATP and the expression of the purinergic receptor P2X7 were decreased in SIRT6+ HUVECs. Contrary to extracellular ATP, extracellular NAD+ did not evoke pro-inflammatory responses in HUVECs. Instead, NAD+ administration reduced endothelial cell proliferation and motility and counteracted the TNFα-induced angiogenesis. Altogether, our data reinforce the view of SIRT6 activation as an anti-inflammatory approach in vascular endothelium.
Assuntos
Células Endoteliais da Veia Umbilical Humana , Sirtuínas , Humanos , Trifosfato de Adenosina , Células Endoteliais da Veia Umbilical Humana/metabolismo , NAD , Sirtuínas/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaAssuntos
Mieloma Múltiplo , Sirtuínas , Humanos , Spliceossomos/genética , Mieloma Múltiplo/genéticaRESUMO
Cancer cells fuel growth and energy demands by increasing their NAD+ biosynthesis dependency, which therefore represents an exploitable vulnerability for anti-cancer strategies. CD38 is a NAD+-degrading enzyme that has become crucial for anti-MM therapies since anti-CD38 monoclonal antibodies represent the backbone for treatment of newly diagnosed and relapsed multiple myeloma patients. Nevertheless, further steps are needed to enable a full exploitation of these strategies, including deeper insights of the mechanisms by which CD38 promotes tumorigenesis and its metabolic additions that could be selectively targeted by therapeutic strategies. Here, we present evidence that CD38 upregulation produces a pervasive intracellular-NAD+ depletion, which impairs mitochondrial fitness and enhances oxidative stress; as result, genetic or pharmacologic approaches that aim to modify CD38 surface-level prime MM cells to NAD+-lowering agents. The molecular mechanism underlying this event is an alteration in mitochondrial dynamics, which decreases mitochondria efficiency and triggers energetic remodeling. Overall, we found that CD38 handling represents an innovative strategy to improve the outcomes of NAD+-lowering agents and provides the rationale for testing these very promising agents in clinical studies involving MM patients.
RESUMO
Recent investigations have improved our understanding of the molecular aberrations supporting Waldenström macroglobulinemia (WM) biology; however, whether the immune microenvironment contributes to WM pathogenesis remains unanswered. First, we showed how a transgenic murine model of human-like lymphoplasmacytic lymphoma/WM exhibits an increased number of regulatory T cells (Tregs) relative to control mice. These findings were translated into the WM clinical setting, in which the transcriptomic profiling of Tregs derived from patients with WM unveiled a peculiar WM-devoted messenger RNA signature, with significant enrichment for genes related to nuclear factor κB-mediated tumor necrosis factor α signaling, MAPK, and PI3K/AKT, which was paralleled by a different Treg functional phenotype. We demonstrated significantly higher Treg induction, expansion, and proliferation triggered by WM cells, compared with their normal cellular counterpart; with a more profound effect within the context of CXCR4C1013G-mutated WM cells. By investigating the B-cell-to-T-cell cross talk at single-cell level, we identified the CD40/CD40-ligand as a potentially relevant axis that supports WM cell-Tregs interaction. Our findings demonstrate the existence of a Treg-mediated immunosuppressive phenotype in WM, which can be therapeutically reversed by blocking the CD40L/CD40 axis to inhibit WM cell growth.
Assuntos
Linfoma de Células B , Macroglobulinemia de Waldenstrom , Humanos , Animais , Camundongos , Macroglobulinemia de Waldenstrom/patologia , Ligante de CD40/genética , Fosfatidilinositol 3-Quinases , Ligantes , Transdução de Sinais , Linfoma de Células B/complicações , Microambiente TumoralRESUMO
Activating G protein-coupled estrogen receptor 1 (GPER1) is an attractive therapeutic strategy for treating a variety of human diseases including cancer. Here, we show that GPER1 is significantly upregulated in tumor cells from different cohorts of Waldenström Macroglobulinemia (WM) patients compared to normal B cells. Using the clinically applicable GPER1-selective small-molecule agonist G-1 (also named Tespria), we found that pharmacological activation of GPER1 leads to G2/M cell cycle arrest and apoptosis both in vitro and in vivo in animal models, even in the context of the protective bone marrow milieu. Activation of GPER1 triggered the TP53 pathway, which remains actionable during WM progression. Thus, this study identifies a novel therapeutic target in WM and paves the way for the clinical development of the GPER1 agonist G-1.
RESUMO
Identification of novel vulnerabilities in the context of therapeutic resistance is emerging as a key challenge for cancer treatment. Recent studies have detected pervasive aberrant splicing in cancer cells, supporting its targeting for novel therapeutic strategies. Here, we evaluated the expression of several spliceosome machinery components in multiple myeloma (MM) cells and the impact of splicing modulation on tumor cell growth and viability. A comprehensive gene expression analysis confirmed the reported deregulation of spliceosome machinery components in MM cells, compared to normal plasma cells from healthy donors, with its pharmacological and genetic modulation resulting in impaired growth and survival of MM cell lines and patient-derived malignant plasma cells. Consistent with this, transcriptomic analysis revealed deregulation of BCL2 family members, including decrease of anti-apoptotic long form of myeloid cell leukemia-1 (MCL1) expression, as crucial for "priming" MM cells for Venetoclax activity in vitro and in vivo, irrespective of t(11;14) status. Overall, our data provide a rationale for supporting the clinical use of splicing modulators as a strategy to reprogram apoptotic dependencies and make all MM patients more vulnerable to BCL2 inhibitors.
Assuntos
Antineoplásicos , Mieloma Múltiplo , Antineoplásicos/uso terapêutico , Apoptose , Compostos Bicíclicos Heterocíclicos com Pontes , Linhagem Celular Tumoral , Humanos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , SulfonamidasRESUMO
Tyrosine kinases have been implicated in promoting tumorigenesis of several human cancers. Exploiting these vulnerabilities has been shown to be an effective anti-tumor strategy as demonstrated for example by the Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib, for treatment of various blood cancers. Here, we characterize a new multiple kinase inhibitor, ARQ531, and evaluate its mechanism of action in preclinical models of acute myeloid leukemia. Treatment with ARQ531, by producing global signaling pathway deregulation, resulted in impaired cell cycle progression and survival in a large panel of leukemia cell lines and patient-derived tumor cells, regardless of the specific genetic background and/or the presence of bone marrow stromal cells. RNA-seq analysis revealed that ARQ531 constrained tumor cell proliferation and survival through Bruton's tyrosine kinase and transcriptional program dysregulation, with proteasome-mediated MYB degradation and depletion of short-lived proteins that are crucial for tumor growth and survival, including ERK, MYC and MCL1. Finally, ARQ531 treatment was effective in a patient-derived leukemia mouse model with significant impairment of tumor progression and survival, at tolerated doses. These data justify the clinical development of ARQ531 as a promising targeted agent for the treatment of patients with acute myeloid leukemia.
Assuntos
Leucemia Mieloide Aguda , Inibidores de Proteínas Quinases , Tirosina Quinase da Agamaglobulinemia , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Tirosina Quinases , PirimidinasRESUMO
Metabolic reprogramming is emerging as a cancer vulnerability that could be therapeutically exploitable using different approaches, including amino acid depletion for those tumors that rely on exogenous amino acids for their maintenance. Ê-Asparaginase (ASNase) has contributed to a significant improvement in acute lymphoblastic leukemia outcomes; however, toxicity and resistance limit its clinical use in other tumors. Here, we report that, in multiple myeloma (MM) cells, the DNA methylation status is significantly associated with reduced expression of ASNase-related gene signatures, thus suggesting ASNase sensitivity for this tumor. Therefore, we tested the effects of ASNase purified from Erwinia chrysanthemi (Erw-ASNase), combined with the next-generation proteasome inhibitor (PI) carfilzomib. We observed an impressive synergistic effect on MM cells, whereas normal peripheral blood mononuclear cells were not affected. Importantly, this effect was associated with increased reactive oxygen species (ROS) generation, compounded mitochondrial damage, and Nrf2 upregulation, regardless of the c-Myc oncogenic-specific program. Furthermore, the cotreatment resulted in genomic instability and DNA repair mechanism impairment via increased mitochondrial oxidative stress, which further enhanced its antitumor activity. Interestingly, carfilzomib-resistant cells were found to be highly dependent on amino acid starvation, as reflected by their higher sensitivity to Erw-ASNase treatment compared with isogenic cells. Overall, by affecting several cellular programs, Erw-ASNase makes MM cells more vulnerable to carfilzomib, providing proof of concept for clinical use of this combination as a novel strategy to enhance PI sensitivity in MM patients.
Assuntos
Aminoácidos , Asparaginase , Asparaginase/farmacologia , Morte Celular , Humanos , Leucócitos Mononucleares , Mitocôndrias , Oligopeptídeos , Espécies Reativas de OxigênioRESUMO
The biological role and therapeutic potential of long non-coding RNAs (lncRNAs) in multiple myeloma (MM) are still open questions. Herein, we investigated the functional significance of the oncogenic lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) in MM. Our study demonstrates that NEAT1 expression level is higher in MM than in the majority of hematological malignancies. NEAT1 silencing by novel LNA-gapmeR antisense oligonucleotide inhibits MM cell proliferation and triggers apoptosis in vitro and in vivo murine MM model as well. By transcriptome analyses, we found that NEAT1 targeting downregulates genes involved in DNA repair processes including the Homologous Recombination pathway, which in turn results in massive DNA damage. These findings may explain the synergistic impact on apoptosis observed in MM cell lines co-treated with inhibitors of both NEAT1 and PARP. The translational significance of NEAT1 targeting is further underlined by its synergistic effects with the most common drugs administered for MM treatment, including bortezomib, carfilzomib, and melphalan. Overall, NEAT1 silencing is associated with a chemo-sensitizing effect of both conventional and novel therapies, and its targeting could therefore represent a promising strategy for novel anti-MM therapeutic options.
Assuntos
Reparo do DNA/fisiologia , Mieloma Múltiplo/patologia , RNA Longo não Codificante/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/fisiologia , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Oligonucleotídeos Antissenso/farmacologiaRESUMO
Genomic instability plays a pathological role in various malignancies, including acute myeloid leukemia (AML), and thus represents a potential therapeutic target. Recent studies demonstrate that SIRT6, a NAD+-dependent nuclear deacetylase, functions as genome-guardian by preserving DNA integrity in different tumor cells. Here, we demonstrate that also CD34+ blasts from AML patients show ongoing DNA damage and SIRT6 overexpression. Indeed, we identified a poor-prognostic subset of patients, with widespread instability, which relies on SIRT6 to compensate for DNA-replication stress. As a result, SIRT6 depletion compromises the ability of leukemia cells to repair DNA double-strand breaks that, in turn, increases their sensitivity to daunorubicin and Ara-C, both in vitro and in vivo In contrast, low SIRT6 levels observed in normal CD34+ hematopoietic progenitors explain their weaker sensitivity to genotoxic stress. Intriguingly, we have identified DNA-PKcs and CtIP deacetylation as crucial for SIRT6-mediated DNA repair. Together, our data suggest that inactivation of SIRT6 in leukemia cells leads to disruption of DNA-repair mechanisms, genomic instability and aggressive AML. This synthetic lethal approach, enhancing DNA damage while concomitantly blocking repair responses, provides the rationale for the clinical evaluation of SIRT6 modulators in the treatment of leukemia.
Assuntos
Antineoplásicos/farmacologia , Dano ao DNA/efeitos dos fármacos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Sirtuínas/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Biomarcadores Tumorais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quinase do Ponto de Checagem 2/metabolismo , Reparo do DNA , Modelos Animais de Doenças , Ativação Enzimática , Expressão Gênica , Instabilidade Genômica , Humanos , Imunofenotipagem , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/patologia , Camundongos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Prognóstico , Ligação Proteica , Sirtuínas/genéticaRESUMO
The NAD+-dependent deacetylase SIRT6 is an emerging cancer drug target, whose inhibition sensitizes cancer cells to chemo-radiotherapy and has pro-differentiating effects. Here we report on the identification of novel SIRT6 inhibitors with a salicylate-based structure. The new SIRT6 inhibitors show improved potency and specificity compared to the hit inhibitor identified in an in silico compound screen. As predicted based on SIRT6 biological roles, the new leads increase histone 3 lysine 9 acetylation and glucose uptake in cultured cells, while blocking TNF-α production and T lymphocyte proliferation. Notably, the new SIRT6 inhibitors effectively sensitize pancreatic cancer cells to gemcitabine. Finally, studies of compound fingerprinting and pharmacokinetics defined the drug-like properties of one of the new SIRT6 inhibitors, potentially allowing for subsequent in vivo proof-of-concept studies. In conclusion, new SIRT6 inhibitors with a salicylate-like structure were identified, which are active in cells and could potentially find applications in disease conditions, including cancer and immune-mediated disorders.
Assuntos
Sistemas de Liberação de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Salicilatos/química , Sirtuínas/antagonistas & inibidores , Acetilação/efeitos dos fármacos , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Humanos , Imunossupressores/química , Imunossupressores/farmacologia , Concentração Inibidora 50 , Camundongos , Estrutura Molecular , Salicilatos/farmacologia , Sirtuínas/metabolismo , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacosAssuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Neoplasias Hematológicas/tratamento farmacológico , NAD/metabolismo , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Citocinas/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos , Neoplasias Hematológicas/patologia , Humanos , Nicotinamida Fosforribosiltransferase/antagonistas & inibidoresRESUMO
PURPOSE: Nicotinamide phosphoribosyltransferase (Nampt) regulates intracellular NAD+ pool and is highly expressed in a number of malignancies. FK866, a selective inhibitor of Nampt, depletes intracellular NAD+ levels, thereby blocking cellular metabolism and triggering sensitization to other drugs and cell death. Here we characterized the antitumor effects of Nampt inhibition in Waldenström macroglobulinemia. EXPERIMENTAL DESIGN: We investigated Nampt role in MW cells using both mRNA and protein expression analyses. We have also used loss-of-function approaches to investigate the growth and survival effects of Nampt on MW cells and further tested the anti-MW activity of dual Nampt and BTK inhibition in vitro and in vivo RESULTS: We found that Waldenström macroglobulinemia cells exhibit high levels of Nampt compared with normal B cells. Loss of function studies suggested a potential oncogenic role of Nampt in Waldenström macroglobulinemia cells, and BTK-inhibitor ibrutinib and FK866 resulted in a significant and synergistic anti-Waldenström macroglobulinemia cell death, regardless of MYD88 and CXCR4 mutational status. Cell death was associated with: (i) activation of caspase-3, PARP and downregulation of Mcl-1, (ii) enhanced intracellular ATP and NAD+ depletion, (iii) inhibition of NF-κB signaling, and (iv) inhibition of multiple prosurvival signaling pathways. In a murine xenograft Waldenström macroglobulinemia model, low-dose combination FK866 and ibrutinib is well tolerated, significantly inhibits tumor growth, and prolongs host survival. CONCLUSIONS: Our results show intracellular NAD+ level as crucial for proliferation and survival of Waldenström macroglobulinemia cells, and provides the mechanistic preclinical rationale for targeting Nampt, either alone or with Ibrutinib, to overcome drug resistance and improve patient outcome in Waldenström macroglobulinemia. Clin Cancer Res; 22(24); 6099-109. ©2016 AACR.
Assuntos
Citocinas/metabolismo , Mutação/genética , Fator 88 de Diferenciação Mieloide/genética , Nicotinamida Fosforribosiltransferase/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores CXCR4/genética , Macroglobulinemia de Waldenstrom/tratamento farmacológico , Acrilamidas/farmacologia , Adenina/análogos & derivados , Tirosina Quinase da Agamaglobulinemia , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Caspase 3/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Humanos , Camundongos , Camundongos SCID , Mutação/efeitos dos fármacos , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , NF-kappa B/metabolismo , Piperidinas/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Macroglobulinemia de Waldenstrom/genética , Macroglobulinemia de Waldenstrom/metabolismoRESUMO
The most frequently used treatment for hormone receptor positive breast cancer in post-menopausal women are aromatase inhibitors. In order to develop new aromatase inhibitors, we designed and synthesized new imidazolylmethylpiperidine sulfonamides using the structure of the previously identified aromatase inhibitor SYN 20028567 as starting lead. By this approach, three new aromatase inhibitors with IC50 values that are similar to that of letrozole and SYN 20028567 were identified.
Assuntos
Inibidores da Aromatase/farmacologia , Aromatase/metabolismo , Piperidinas/farmacologia , Sulfonamidas/farmacologia , Inibidores da Aromatase/síntese química , Inibidores da Aromatase/química , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Piperidinas/síntese química , Piperidinas/química , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/químicaRESUMO
BACKGROUND: Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in NAD(+) biosynthesis from nicotinamide, is one of the major factors regulating cancer cells metabolism and is considered a promising target for treating cancer. The prototypical NAMPT inhibitor FK866 effectively lowers NAD(+) levels in cancer cells, reducing the activity of NAD(+)-dependent enzymes, lowering intracellular ATP, and promoting cell death. RESULTS: We show that FK866 induces a translational arrest in leukemia cells through inhibition of MTOR/4EBP1 signaling and of the initiation factors EIF4E and EIF2A. Specifically, treatment with FK866 is shown to induce 5'AMP-activated protein kinase (AMPK) activation, which, together with EIF2A phosphorylation, is responsible for the inhibition of protein synthesis. Notably, such an effect was also observed in patients' derived primary leukemia cells including T-cell Acute Lymphoblastic Leukemia. Jurkat cells in which AMPK or LKB1 expression was silenced or in which a non-phosphorylatable EIF2A mutant was ectopically expressed showed enhanced sensitivity to the NAMPT inhibitor, confirming a key role for the LKB1-AMPK-EIF2A axis in cell fate determination in response to energetic stress via NAD(+) depletion. CONCLUSIONS: We identified EIF2A phosphorylation as a novel early molecular event occurring in response to NAMPT inhibition and mediating protein synthesis arrest. In addition, our data suggest that tumors exhibiting an impaired LBK1- AMPK- EIF2A response may be especially susceptible to NAMPT inhibitors and thus become an elective indication for this type of agents.
Assuntos
Citocinas/antagonistas & inibidores , Fator de Iniciação 2 em Eucariotos/metabolismo , Leucemia/genética , Leucemia/metabolismo , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Biossíntese de Proteínas , Estresse Fisiológico/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/metabolismo , Acrilamidas/farmacologia , Trifosfato de Adenosina/metabolismo , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Fator de Iniciação 4E em Eucariotos/metabolismo , Humanos , Células Jurkat , NAD/metabolismo , Fosforilação , Piperidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Transcrição GênicaRESUMO
PURPOSE: The nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, APO866, has been previously shown to have antileukemic activity in preclinical models, but its cytotoxicity in primary leukemia cells is frequently limited. The success of current antileukemic treatments is reduced by the occurrence of multidrug resistance, which, in turn, is mediated by membrane transport proteins, such as P-glycoprotein-1 (Pgp). Here, we evaluated the antileukemic effects of APO866 in combination with Pgp inhibitors and studied the mechanisms underlying the interaction between these two types of agents. EXPERIMENTAL DESIGN: The effects of APO866 with or without Pgp inhibitors were tested on the viability of leukemia cell lines, primary leukemia cells (AML, n = 6; B-CLL, n = 19), and healthy leukocytes. Intracellular nicotinamide adenine dinucleotide (NAD(+)) and ATP levels, mitochondrial transmembrane potential (ΔΨ(m)), markers of apoptosis and of endoplasmic reticulum (ER) stress were evaluated. RESULTS: The combination of APO866 with Pgp inhibitors resulted in a synergistic cytotoxic effect in leukemia cells, while sparing normal CD34(+) progenitor cells and peripheral blood mononuclear cells. Combining Pgp inhibitors with APO866 led to increased intracellular APO866 levels, compounded NAD(+) and ATP shortage, and induced ΔΨ(m) dissipation. Notably, APO866, Pgp inhibitors and, to a much higher extent, their combination induced ER stress and ER stress inhibition strongly reduced the activity of these treatments. CONCLUSIONS: APO866 and Pgp inhibitors show a strong synergistic cooperation in leukemia cells, including acute myelogenous leukemia (AML) and B-cell chronic lymphocytic leukemia (B-CLL) samples. Further evaluations of the combination of these agents in clinical setting should be considered.
Assuntos
Acrilamidas/farmacologia , Antineoplásicos/farmacologia , Ciclosporina/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Leucemia/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Piperidinas/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Idoso , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Aberrações Cromossômicas , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Feminino , Expressão Gênica , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Leucemia/genética , Leucemia/mortalidade , Leucemia/patologia , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Pessoa de Meia-Idade , Mutação , NAD/metabolismo , Estadiamento de Neoplasias , Niacina/farmacologia , Niacinamida/farmacologia , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Nicotinamida Fosforribosiltransferase/genética , Nicotinamida Fosforribosiltransferase/metabolismo , Cultura Primária de Células , Prognóstico , Células Tumorais Cultivadas , Resposta a Proteínas não Dobradas/efeitos dos fármacosRESUMO
Tyrosine kinase inhibitors (TKIs) are now the mainstay of treatment in many types of cancer. However, their benefit is frequently short-lived, mandating the search for safe potentiation strategies. Cycles of fasting enhance the activity of chemo-radiotherapy in preclinical cancer models and dietary approaches based on fasting are currently explored in clinical trials. Whether combining fasting with TKIs is going to be potentially beneficial remains unknown. Here we report that starvation conditions increase the ability of commonly administered TKIs, including erlotinib, gefitinib, lapatinib, crizotinib and regorafenib, to block cancer cell growth, to inhibit the mitogen-activated protein kinase (MAPK) signaling pathway and to strengthen E2F-dependent transcription inhibition. In cancer xenografts models, both TKIs and cycles of fasting slowed tumor growth, but, when combined, these interventions were significantly more effective than either type of treatment alone. In conclusion, cycles of fasting or of specifically designed fasting-mimicking diets should be evaluated in clinical studies as a means to potentiate the activity of TKIs in clinical use.
Assuntos
Antineoplásicos/farmacologia , Jejum/fisiologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neoplasias/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Xenoenxertos , Humanos , Immunoblotting , Camundongos , Camundongos Endogâmicos BALB C , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Boosting NAD(+) biosynthesis with NAD(+) intermediates has been proposed as a strategy for preventing and treating age-associated diseases, including cancer. However, concerns in this area were raised by observations that nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme in mammalian NAD(+) biosynthesis, is frequently up-regulated in human malignancies, including breast cancer, suggesting possible protumorigenic effects for this protein. We addressed this issue by studying NAMPT expression and function in human breast cancer in vivo and in vitro. Our data indicate that high NAMPT levels are associated with aggressive pathological and molecular features, such as estrogen receptor negativity as well as HER2-enriched and basal-like PAM50 phenotypes. Consistent with these findings, we found that NAMPT overexpression in mammary epithelial cells induced epithelial-to-mesenchymal transition, a morphological and functional switch that confers cancer cells an increased metastatic potential. However, importantly, NAMPT-induced epithelial-to-mesenchymal transition was found to be independent of NAMPT enzymatic activity and of the NAMPT product nicotinamide mononucleotide. Instead, it was mediated by secreted NAMPT through its ability to activate the TGFß signaling pathway via increased TGFß1 production. These findings have implications for the design of therapeutic strategies exploiting NAD(+) biosynthesis via NAMPT in aging and cancer and also suggest the potential of anticancer agents designed to specifically neutralize extracellular NAMPT. Notably, because high levels of circulating NAMPT are found in obese and diabetic patients, our data could also explain the increased predisposition to cancer of these subjects.
Assuntos
Neoplasias da Mama/genética , Citocinas/genética , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/genética , Nicotinamida Fosforribosiltransferase/genética , Fator de Crescimento Transformador beta1/genética , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Citocinas/antagonistas & inibidores , Citocinas/metabolismo , Feminino , Humanos , NAD/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Estadiamento de Neoplasias , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Nicotinamida Fosforribosiltransferase/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptores de Estrogênio/deficiência , Receptores de Estrogênio/genética , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Chemokines trigger leukocyte trafficking and are implicated in cardiovascular disease pathophysiology. Chemokine-binding proteins, called "Evasins" have been shown to inhibit both CC and CXC chemokine-mediated bioactivities. Here, we investigated whether treatment with Evasin-3 (CXC chemokine inhibitor) and Evasin-4 (CC chemokine inhibitor) could influence post-infarction myocardial injury and remodelling. C57Bl/6 mice were submitted in vivo to left coronary artery permanent ligature and followed up for different times (up to 21 days). After coronary occlusion, three intraperitoneal injections of 10 µg Evasin-3, 1 µg Evasin-4 or equal volume of vehicle (PBS) were performed at 5 minutes, 24 hours (h) and 48 h after ischaemia onset. Both anti-chemokine treatments were associated with the beneficial reduction in infarct size as compared to controls. This effect was accompanied by a decrease in post-infarction myocardial leukocyte infiltration, reactive oxygen species release, and circulating levels of CXCL1 and CCL2. Treatment with Evasin-4 induced a more potent effect, abrogating the inflammation already at one day after ischaemia onset. At days 1 and 21 after ischaemia onset, both anti-chemokine treatments failed to significantly improve cardiac function, remodelling and scar formation. At 21-day follow-up, mouse survival was exclusively improved by Evasin-4 treatment when compared to control vehicle. In conclusion, we showed that the selective inhibition of CC chemokines (i.e. CCL5) with Evasin-4 reduced cardiac injury/inflammation and improved survival. Despite the inhibition of CXC chemokine bioactivities, Evasin-3 did not affect mouse survival. Therefore, early inhibition of CC chemokines might represent a promising therapeutic approach to reduce the development of post-infarction heart failure in mice.
Assuntos
Anti-Inflamatórios/administração & dosagem , Infarto do Miocárdio/tratamento farmacológico , Miocárdio/metabolismo , Receptores CXCR/administração & dosagem , Receptores de Quimiocinas/administração & dosagem , Animais , Proteínas de Artrópodes , Movimento Celular/efeitos dos fármacos , Quimiocina CXCL1/sangue , Vasos Coronários/cirurgia , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/diagnóstico , Miocárdio/patologia , Estresse Oxidativo/efeitos dos fármacos , Proteínas e Peptídeos SalivaresRESUMO
NAD(+) is mainly synthesized in human cells via the "salvage" pathways starting from nicotinamide, nicotinic acid, or nicotinamide riboside (NR). The inhibition with FK866 of the enzyme nicotinamide phosphoribosyltransferase (NAMPT), catalyzing the first reaction in the "salvage" pathway from nicotinamide, showed potent antitumor activity in several preclinical models of solid and hematologic cancers. In the clinical studies performed with FK866, however, no tumor remission was observed. Here we demonstrate that low micromolar concentrations of extracellular NAD(+) or NAD(+) precursors, nicotinamide mononucleotide (NMN) and NR, can reverse the FK866-induced cell death, this representing a plausible explanation for the failure of NAMPT inhibition as an anti-cancer therapy. NMN is a substrate of both ectoenzymes CD38 and CD73, with generation of NAM and NR, respectively. In this study, we investigated the roles of CD38 and CD73 in providing ectocellular NAD(+) precursors for NAD(+) biosynthesis and in modulating cell susceptibility to FK866. By specifically silencing or overexpressing CD38 and CD73, we demonstrated that endogenous CD73 enables, whereas CD38 impairs, the conversion of extracellular NMN to NR as a precursor for intracellular NAD(+) biosynthesis in human cells. Moreover, cell viability in FK866-treated cells supplemented with extracellular NMN was strongly reduced in tumor cells, upon pharmacological inhibition or specific down-regulation of CD73. Thus, our study suggests that genetic or pharmacologic interventions interfering with CD73 activity may prove useful to increase cancer cell sensitivity to NAMPT inhibitors.