RESUMEN
The compromised antioxidant defense system in chronic lymphocytic leukemia (CLL) suggested a potential use for reactive oxygen species (ROS) generating arsenic trioxide (ATO) and ascorbic acid. While both ATO and ascorbic acid mediate cytotoxicity in CLL B cells as single agents, the efficacy of ATO is enhanced by ascorbic acid. This effect is dependent on increased ROS accumulation, as pretreatment of B-CLL cells with a glutathione reducing buthionine sulfoximine or catalase inhibiting aminotriazole, enhanced ATO/ascorbic acid-mediated cytotoxicity. Pretreatment with reducing agents such as catalase, or thiol antioxidant, N-acetyl cysteine or GSH also abrogated ATO/ascorbic acid-mediated cytotoxicity. Furthermore, Hu1D10-mediated cell death was enhanced with ATO and ascorbic acid, thus justifying potential combination of ATO/arsenic trioxide therapy with antibodies such as Hu1D10 that also cause accumulation of ROS.
Asunto(s)
Arsenicales/farmacología , Ácido Ascórbico/farmacología , Leucemia Linfocítica Crónica de Células B/patología , Óxidos/farmacología , Clorometilcetonas de Aminoácidos/farmacología , Amitrol (Herbicida)/farmacología , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales Humanizados , Trióxido de Arsénico , Linfocitos B/efectos de los fármacos , Linfocitos B/patología , Butionina Sulfoximina/farmacología , Catalasa/antagonistas & inhibidores , Catalasa/farmacología , Línea Celular Tumoral/efectos de los fármacos , Proteasas de Cisteína/fisiología , Inhibidores de Cisteína Proteinasa/farmacología , Evaluación Preclínica de Medicamentos , Sinergismo Farmacológico , Activación Enzimática/efectos de los fármacos , Glutatión/metabolismo , Humanos , Proteínas de Neoplasias/fisiología , Oxidantes/farmacología , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The 1D10 antigen is the target for Hu1D10 (apolizumab), a humanized HLA-DR beta-chain-specific antibody that is currently in clinical trials for hematologic malignancies. We demonstrate that Hu1D10 induces caspase-independent apoptosis following secondary cross-linking in primary chronic lymphocytic leukemia (CLL) cells. Generation of reactive oxygen species (ROS) and signal transduction, as evidenced by phosphorylation of Syk and AKT, were noted. The source of the Hu1D10-induced ROS was examined using the Raji lymphoblastic cell line with engineered defects in the mitochondrial respiratory chain. Hu1D10 treatment of clones with deficient mitochondrial respiration produced ROS suggesting a cytoplasmic source. Administration of ROS scavengers to primary CLL cells prior to Hu1D10 treatment diminished AKT activation. Treatment with Hu1D10 and the phosphatidylinositol 3-kinase inhibitor LY294002 demonstrated in vitro synergy with enhanced apoptosis. In conjunction with an ongoing clinical trial, blood samples were collected following intravenous infusion of Hu1D10 and analyzed for phosphorylation of AKT. Two of 3 patient samples showed a sustained increase in AKT phosphorylation following Hu1D10 administration. These data suggest that Hu1D10 ligation in CLL cells induces death and survival signals for which combination therapies may be designed to greatly enhance efficiency of both Hu1D10 and other class II antibodies in development.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Leucemia Linfocítica Crónica de Células B/metabolismo , Acetilcisteína/farmacología , Anticuerpos Monoclonales Humanizados , Antineoplásicos/farmacología , Apoptosis , Western Blotting , Línea Celular , Línea Celular Tumoral , Supervivencia Celular , Cromonas/farmacología , Citoesqueleto/metabolismo , Transporte de Electrón , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Citometría de Flujo , Depuradores de Radicales Libres/farmacología , Humanos , Microdominios de Membrana , Mitocondrias/metabolismo , Morfolinas/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Especies Reactivas de Oxígeno , Transducción de Señal , Factores de TiempoRESUMEN
Chronic lymphocytic leukemia therapy has changed dramatically over the past decade, with recent studies supporting the use of fludarabine as the initial therapy for symptomatic disease. New findings relative to the use of fludarabine and complications arising from this therapy are reviewed. Exciting combination approaches using monoclonal antibodies such as rituximab or Campath-1H in combination with fludarabine offer the opportunity to improve the complete response rate further in chronic lymphocytic leukemia. Furthermore, the field of experimental therapeutics for chronic lymphocytic leukemia was advanced by the description of a new mouse model of human chronic lymphocytic leukemia and identification of several disrupted signal transduction pathways in this disease. The description of therapies that target AKT, protein kinase C, phosphodiesterase 4, mammalian target of rapamycin, histone deacetylase, and methyltransferase offer the opportunity to utilize molecularly targeted therapy for this disease. Such targeted approaches offer hope that we might be on the threshold to changing the natural history of chronic lymphocytic leukemia.
Asunto(s)
Antineoplásicos/uso terapéutico , Leucemia Linfocítica Crónica de Células B/terapia , Antineoplásicos/farmacología , Silenciador del Gen/efectos de los fármacos , Humanos , Transducción de Señal/efectos de los fármacosRESUMEN
Depsipeptide is in clinical trials for chronic lymphocytic leukemia (CLL) on the basis of earlier observations demonstrating selective in vitro activity in CLL. We sought to determine the relationship of histone H3 and H4 acetylation, inhibition of histone deacetylase, and apoptosis observed in CLL cells to justify a pharmacodynamic end point in these clinical trials. We demonstrate that in vitro depsipeptide induces histone H3 and H4 acetylation and histone deacetylase enzyme inhibition at concentrations corresponding to the LC50 (concentration producing 50% cell death) for cultured CLL cells (0.038 microM depsipeptide). The changes in histone acetylation are lysine specific, involving H4 K5, H4 K12, and H3 K9, and to a lesser extent H4 K8, but not H4 K16 or H3 K14. Depsipeptide-induced apoptosis is caspase dependent, selectively involving the tumor necrosis factor (TNF) receptor (extrinsic pathway) initiating caspase 8 and effector caspase 3. Activation of caspase 8 was accompanied by the down-regulation of cellular FLICE-inhibitory protein (c-FLIP, I-FLICE) without evidence of Fas (CD95) up-regulation. Changes in other apoptotic proteins, including Bcl-2, Bax, Mcl-1, and X-linked inhibitor of apoptosis (XIAP), were not observed. Our results demonstrate a relationship between target enzyme inhibition of histone deacetylase, histone H3 and H4 acetylation, and apoptosis involving the TNF-receptor pathway of apoptosis that is not used by other therapeutic agents in CLL. These data suggest use of histone H3 and H4 acetylation, inhibition of histone deacetylase, and down-regulation of FLIP as pharmacodynamic end points for further evaluation of this drug in patients.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteínas Portadoras/biosíntesis , Caspasas/fisiología , Depsipéptidos , Inhibidores Enzimáticos/farmacología , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Inhibidores de Histona Desacetilasas , Histonas/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Leucemia Linfocítica Crónica de Células B/patología , Proteínas de Neoplasias/antagonistas & inhibidores , Péptidos Cíclicos/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Acetilación/efectos de los fármacos , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD , Proteínas Portadoras/genética , Caspasa 8 , Caspasa 9 , Activación Enzimática/efectos de los fármacos , Humanos , Leucemia Linfocítica Crónica de Células B/enzimología , Lisina/metabolismo , Proteínas de Neoplasias/metabolismo , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismoRESUMEN
Therapy of B-cell chronic lymphocytic leukemia (CLL) is currently palliative, emphasizing the need for identification of new therapies for this disease. KRN5500 is a novel agent that has a unique sensitivity pattern in the National Cancer Institute cell line screening panel, suggesting a unique mechanism of action. To assess its in vitro activity in CLL, we exposed peripheral mononuclear cells from CLL patients (n = 11) to varying concentrations of this agent. Viability of the CLL cells was reduced by 50% (LC50) at 4 hours, 24 hours, and 4 days at KRN5500 concentrations of 2.50 microM, 0.276 microM, and 0.139 microM, respectively. KRN5500 induced cellular injury via caspase-dependent apoptosis involving the intrinsic mitochondrial (caspase-9) initiating caspase and caspase-3 effector caspase; however, expression of the antiapoptotic mitochondrial membrane protein Bcl-2 was unaffected. These data demonstrate KRN5500 has significant in vitro activity against human CLL cells, thus providing support for introduction of this agent into clinical trials for patients with CLL.