RESUMO
NK-cell leukemia is a clonal expansion of NK cells. The illness can occur in an aggressive or chronic form. We studied cell lines from human and rat NK-cell leukemias (aggressive NK-cell leukemia) as well as samples from patients with chronic NK-cell leukemia to investigate pathogenic mechanisms. Here we report that Mcl-1 was overexpressed in leukemic NK cells and that knockdown of Mcl-1 induced apoptosis in these leukemic cells. In vitro treatment of human and rat NK leukemia cells with FTY720 led to caspase-dependent apoptosis and decreased Mcl-1 expression in a time- and-dose-dependent manner. These biologic effects could be inhibited by blockade of reactive oxygen species generation and the lysosomal degradation pathway. Lipidomic analyses after FTY720 treatment demonstrated elevated levels of sphingosine, which mediated apoptosis of leukemic NK cells in vitro. Importantly, systemic administration of FTY720 induced complete remission in the syngeneic Fischer rat model of NK-cell leukemia. Therapeutic efficacy was associated with decreased expression of Mcl-1 in vivo. These data demonstrate that therapeutic benefit of FTY720 may result from both altered sphingolipid metabolism as well as enhanced degradation of a key component of survival signaling.
Assuntos
Apoptose/efeitos dos fármacos , Imunossupressores/uso terapêutico , Células Matadoras Naturais/efeitos dos fármacos , Leucemia/tratamento farmacológico , Propilenoglicóis/uso terapêutico , Esfingosina/análogos & derivados , Animais , Western Blotting , Proliferação de Células/efeitos dos fármacos , Doença Crônica , Cloridrato de Fingolimode , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Leucemia/imunologia , Leucemia/patologia , Masculino , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Interferente Pequeno/genética , Ratos , Ratos Endogâmicos F344 , Esfingosina/uso terapêuticoRESUMO
Large granular lymphocyte (LGL) leukemia results from chronic expansion of cytotoxic T cells or natural killer (NK) cells. Apoptotic resistance resulting from constitutive activation of survival signaling pathways is a fundamental pathogenic mechanism. Recent network modeling analyses identified platelet-derived growth factor (PDGF) as a key master switch in controlling these survival pathways in T-cell LGL leukemia. Here we show that an autocrine PDGF regulatory loop mediates survival of leukemic LGLs of both T- and NK-cell origin. We found high levels of circulating PDGF-BB in platelet-poor plasma samples from LGL leukemia patients. Production of PDGF-BB by leukemic LGLs was demonstrated by immunocytochemical staining. Leukemic cells expressed much higher levels of PDGFR-beta transcripts than purified normal CD8(+) T cells or NK cells. We observed that phosphatidylinositol-3-kinase (PI3 kinase), Src family kinase (SFK), and downstream protein kinase B (PKB)/AKT pathways were constitutively activated in both T- and NK-LGL leukemia. Pharmacologic blockade of these pathways led to apoptosis of leukemic LGLs. Neutralizing antibody to PDGF-BB inhibited PKB/AKT phosphorylation induced by LGL leukemia sera. These results suggest that targeting of PDGF-BB, a pivotal regulator for the long-term survival of leukemic LGLs, may be an important therapeutic strategy.
Assuntos
Comunicação Autócrina , Leucemia Linfocítica Granular Grande/patologia , Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais , Anticorpos Neutralizantes/farmacologia , Apoptose/efeitos dos fármacos , Comunicação Autócrina/efeitos dos fármacos , Becaplermina , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Leucemia Linfocítica Granular Grande/sangue , Leucemia Linfocítica Granular Grande/enzimologia , Leucemia Linfocítica Granular Grande/genética , Linfócitos/efeitos dos fármacos , Linfócitos/enzimologia , Linfócitos/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/genética , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-sis , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Coloração e Rotulagem , Quinases da Família src/antagonistas & inibidoresRESUMO
The natural killer (NK) type of aggressive large granular lymphocytic (LGL) leukemia is a fatal illness that pursues a rapid clinical course. There are no effective therapies for this illness, and pathogenetic mechanisms remain undefined. Here we report that the survivin was highly expressed in both aggressive and chronic leukemic NK cells but not in normal NK cells. In vitro treatment of human and rat NK-LGL leukemia cells with cell-permeable, short-chain C6-ceramide (C6) in nanoliposomal formulation led to caspase-dependent apoptosis and diminished survivin protein expression, in a time- and dose-dependent manner. Importantly, systemic intravenous delivery of nanoliposomal ceramide induced complete remission in the syngeneic Fischer F344 rat model of aggressive NK-LGL leukemia. Therapeutic efficacy was associated with decreased expression of survivin in vivo. These data suggest that in vivo targeting of survivin through delivery of nanoliposomal C6-ceramide may be a promising therapeutic approach for a fatal leukemia.
Assuntos
Ceramidas/farmacologia , Leucemia Linfocítica Granular Grande/terapia , Lipossomos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Nanopartículas/química , Animais , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Ceramidas/uso terapêutico , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Humanos , Leucemia Linfocítica Granular Grande/tratamento farmacológico , Leucemia Linfocítica Granular Grande/enzimologia , Leucemia Linfocítica Granular Grande/patologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/patologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Ratos , Ratos Endogâmicos F344 , Indução de Remissão , Survivina , Resultado do TratamentoRESUMO
Abstract Natural killer (NK) cell leukemia is characterized by clonal expansion of CD3 - NK cells and comprises both chronic and aggressive forms. Currently no effective treatment exists, thus providing a need for identification of novel therapeutics. Lipidomic studies revealed a dysregulated sphingolipid metabolism as evidenced by decreased levels of overall ceramide species and increased levels of cerebrosides in leukemic NK cells, concomitant with increased glucosylceramide synthase (GCS) expression. GCS, a key enzyme of this pathway, neutralizes pro-apoptotic ceramide by transfer of a uridine diphosphate (UDP)-glucose. Thus, we treated both rat and human leukemic NK cells in combination with: (1) exogenous C6-ceramide nanoliposomes in order to target mitochondria and increase physiological pro-apoptotic levels of long chain ceramide, and (2) 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP), an inhibitor of GCS. Co-administration of C6-ceramide nanoliposomes and PPMP elicited an increase in endogenous long-chain ceramide species, which led to cellular apoptosis in a synergistic manner via the mitochondrial intrinsic cell death pathway in leukemic NK cells.
Assuntos
Antineoplásicos/administração & dosagem , Apoptose/efeitos dos fármacos , Ceramidas/administração & dosagem , Glucosiltransferases/metabolismo , Leucemia Linfocítica Granular Grande/metabolismo , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ceramidas/metabolismo , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Glucosiltransferases/antagonistas & inibidores , Glucosiltransferases/genética , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Leucemia Linfocítica Granular Grande/genética , Lipossomos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Morfolinas/farmacologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Esfingolipídeos/farmacologia , SurvivinaRESUMO
Ceramide is a sphingolipid metabolite that induces cancer cell death. When C6-ceramide is encapsulated in a nanoliposome bilayer formulation, cell death is selectively induced in tumor models. However, the mechanism underlying this selectivity is unknown. As most tumors exhibit a preferential switch to glycolysis, as described in the "Warburg effect", we hypothesize that ceramide nanoliposomes selectively target this glycolytic pathway in cancer. We utilize chronic lymphocytic leukemia (CLL) as a cancer model, which has an increased dependency on glycolysis. In CLL cells, we demonstrate that C6-ceramide nanoliposomes, but not control nanoliposomes, induce caspase 3/7-independent necrotic cell death. Nanoliposomal ceramide inhibits both the RNA and protein expression of GAPDH, an enzyme in the glycolytic pathway, which is overexpressed in CLL. To confirm that ceramide targets GAPDH, we demonstrate that downregulation of GAPDH potentiates the decrease in ATP after ceramide treatment and exogenous pyruvate treatment as well as GAPDH overexpression partially rescues ceramide-induced necrosis. Finally, an in vivo murine model of CLL shows that nanoliposomal C6-ceramide treatment elicits tumor regression, concomitant with GAPDH downregulation. We conclude that selective inhibition of the glycolytic pathway in CLL cells with nanoliposomal C6-ceramide could potentially be an effective therapy for leukemia by targeting the Warburg effect.