<p>B-cell chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world. Although treatment of this disease has advanced considerably over the past decade, CLL remains incurable with current chemotherapeutics. In addition, available drug regimens for CLL are associated with frequent cytopenia-related complications, such as infection and fatigue. Thus, the major challenge in CLL treatment today is the need for alternative therapeutics with decreased toxicity and improved efficacy for disease refractory to currently available drugs.</p><p> </p><p>CLL is characterized by slow accumulation of malignant cells, which are supported in the microenvironment by cell-cell interactions and soluble cytokines such as tumor necrosis factor (TNF). We evaluated the effect of the small molecule TNF inhibitor LMP-420 on primary CLL cells. LMP-420 exhibited cytotoxic activity against these cells in the MTS assay, with similar potency to the front-line CLL drug fludarabine. LMP-420 induced time- and dose-dependent apoptosis in CLL cells, as demonstrated by annexin V staining, caspase activation, and DNA fragmentation. These changes were associated with decreased expression of the anti-apoptotic proteins Mcl-1, Bcl-xL, Bcl-2, and XIAP. CLL cells from patients with poor prognostic indicators exhibited LMP-420 sensitivity equal to that for cells from patients with favorable characteristics. In addition, LMP-420 potentiated the cytotoxic effect of fludarabine and inhibited in vitro proliferation of CLL cells. In contrast to other CLL therapeutics, LMP-420 exhibited minimal effects on normal peripheral blood mononuclear cell viability, mitogen-stimulated B- and T-cell proliferation, and hematopoietic colony formation. Our data suggest that LMP-420 may be a useful treatment for CLL with negligible hematologic toxicities. </p><p> </p><p>The effect profile of this compound in normal immune cells and the microarray studies in CLL cells indicate that the mechanism of action of LMP-420 likely involves modulation of the NF-kB pathway. Our initial studies demonstrate moderate but significant inhibitory activity against p65, a key member of the NF-kB transcription factor family. Research is ongoing to gain a better understanding of the specific cytotoxicity of LMP-420 for CLL cells and to elucidate other components of its mechanism of action. Regardless of the ultimate mechanistic findings with LMP-420, our studies support this molecule as a promising new CLL therapeutic that warrants further preclinical evaluation.</p> / Dissertation

Submitted by Ronildo Prado (ronisp@ufscar.br) on 2017-08-22T17:21:37Z No. of bitstreams: 1 TeseRNS.pdf: 3515617 bytes, checksum: 251005e22df508ed87d98457bd4a5d3d (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2017-08-22T17:21:47Z (GMT) No. of bitstreams: 1 TeseRNS.pdf: 3515617 bytes, checksum: 251005e22df508ed87d98457bd4a5d3d (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2017-08-22T17:21:54Z (GMT) No. of bitstreams: 1 TeseRNS.pdf: 3515617 bytes, checksum: 251005e22df508ed87d98457bd4a5d3d (MD5) / Made available in DSpace on 2017-08-22T17:22:00Z (GMT). No. of bitstreams: 1 TeseRNS.pdf: 3515617 bytes, checksum: 251005e22df508ed87d98457bd4a5d3d (MD5) Previous issue date: 2016-09-15 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Hyperammonemia impairs skeletal muscle protein synthesis and induces autophagy by upregulating myostatin via nuclear factor-kappaB (NF-kB). Skeletal muscle ammonia metabolism occurs via synthesis of glutamate and glutamine via critical TCA intermediate α-KG, that regulates increase expression of hypoxia inducible factor 1α (HIF-1α). Furthermore, there is a interaction between HIF-1α and NF-kB. Objective: This study evaluated the effects of hyperammonemia in NF-kB and HIF-1α cross-talking. Methods: To examine the effects of ammonium acetate intervention under HIF-1α signaling through NF-kB pathway it has generated a stable knockdown cell line for NFkB and the subunits α and β of the I kappa B kinase (IKK) complex (IKKα and IKKβ) evaluating the HIF-1α and myostatin activities. Results: The protein expression of HIF- 1α was significantly higher in C2C12 murine myotubes under ammonium acetate intervention compared to control. Once the deletion of NF-kB, IKKα and IKKβ occurs, the HIF-1α is not expressed, suggesting a cross-talking between them. The protein expression of myostatin was significantly higher in C2C12 IKKα deletion under ammonium acetate intervention compared to C2C12 random suggesting that myostatin is not IKKα dependent. Conclusion: We conclude that hyperammonemia is a normoxemic activator of HIF-1α through NF-kB pathway that results in sarcopenia via up-regulation of myostatin expression. / Um dos grandes mediadores de sarcopenia em cirrose é a hiperamonemia. Esta anormalidade metabólica é frequente em doenças hepáticas promovendo conversão danificada de amônia em uréia prejudicando a síntese protéica e induzindo autofagia do músculo esquelético pela up-regulação de miostatina via fator nuclear kappa B (NFkB). O metabolismo de amônia no músculo esquelético ocorre via síntese de glutamato e glutamina através do intermediário metabólico α-cetoglutarato do ciclo de Krebs, que regula o Fator Induzido por Hipóxia (HIF-1α). Além disso, existe um interação entre os dois fatores de transcrição HIF-1α and NF-kB. Objetivo: Este estudo avalia os efeitos da hiperamonemia no cross-talking entre NF-kB and HIF-1α. Métodos: Para examinar os efeitos do aumento da concentração de amônia na expressão de HIF-1α através da via NF-kB foi realizado um knockdown estável de NF-kB, e as subunidades IKKα e IKKβ do complexo I kappa B quinase (IKK) em células C2C12 avaliando as atividades de HIF- 1α e miostatina. Resultados: A expressão protéica de HIF-1α foi significativamente alta em células C2C12 sob tratamento com acetato de amônia comparado com controle. Uma vez que ocorre o silenciamento de NF-kB, IKKα and IKKβ em células C2C12, não é observada a expressão protéica de HIF-1α, sugerindo um cross-talking entre eles. A expressão protéica de miostatina foi significativamente alta em células C2C12 silenciadas para IKKα sob tratamento com acetato de amônia comparado com células C2C12 random (controle), sugerindo que miostatina não é dependente da quinase IKKα. Conclusão: Foi concluído que hiperamonemia é um ativador normóxico de HIF-1α através da via de sinalização NF-kB resultando em sarcopenia via up-regulação de miostatina.