RESUMEN
The RAS/RAF/MEK/ERK signaling pathway has been largely unexplored as a potential therapeutic target in lymphoma. The novel 2nd generation anti-MEK small molecule, AZD6244, down-regulated its direct downstream target, phospho-ERK (pERK) in germinal center and nongerminal center diffuse large B-cell lymphoma (DLBCL) cell lines and primary cells. Similar decreased pERK levels were noted despite constitutive activation (CA) of MEK. Consequently, several lymphoma-related ERK substrates were down-regulated by AZD6244 including MCT-1, c-Myc, Bcl-2, Mcl-1, and CDK1/2. AZD6244 induced time- and dose-dependent antiproliferation and apoptosis in all DLBCL cell lines and fresh/primary cells (IC(50) 100nM-300nM). Furthermore, AZD6244 resulted in significantly less tumor compared with control in an in vivo DLBCL SCID xenograft model. Cell death was associated with cleaved PARP, caspases-8, -9, and -3, and apoptosis was caspase-dependent. In addition, there was stabilization of FoxO3a, activation of BIM and PUMA, and a significant decrease in c-Myc transcripts. Moreover, siRNA knockdown of BIM abrogated AZD6244-related apoptosis, while shRNA knockdown of ERK minimally sensitized cells. Finally, manipulation of AKT with transfection of OCI-LY3 cells with CA-AKT or through chemical inhibition (LY294002) had minimal effect on AZD6244-induced cell death. Altogether, these findings show that the novel anti-MEK agent, AZD6244, induced apoptosis in DLBCL and that cell death was BIM-dependent.
Asunto(s)
Antineoplásicos/farmacología , Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis/efectos de los fármacos , Bencimidazoles/farmacología , Linfoma de Células B Grandes Difuso/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Proteína 11 Similar a Bcl2 , Western Blotting , Línea Celular Tumoral , Femenino , Humanos , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Ratones , Ratones SCID , Inhibidores de Proteínas Quinasas/farmacología , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Mantle cell lymphoma (MCL) is characterized by translocation t(11;14)(q13;q32), aggressive clinical behaviour, and poor patient outcomes following conventional chemotherapy. New treatment approaches are needed that target novel biological pathways. All trans retinoic acid (ATRA) is a key retinoid that acts through nuclear receptors that function as ligand-inducible transcription factors. The present study evaluated cell killing effects of ATRA-enriched nanoscale delivery particles, termed nanodisks (ND), on MCL cell lines. Results show that ATRA-ND induced cell death more effectively than naked ATRA (dimethyl sulphoxide) or empty ND. ATRA-ND induced reactive oxygen species (ROS) generation to a greater extent than naked ATRA. The antioxidant, N-acetylcysteine, inhibited ATRA-ND induced apoptosis. Compared to naked ATRA, ATRA-ND enhanced G1 growth arrest, up-regulated p21and p27, and down regulated cyclin D1. At ATRA concentrations that induced apoptosis, expression levels of retinoic acid receptor-alpha (RARalpha) and retinoid X receptor-gamma (RXRgamma) were increased. Compared to naked ATRA, ATRA-ND significantly stimulated transcriptional activity of RARA in a model carcinoma cell line. Furthermore, the RAR antagonist, Ro 41-5253, inhibited ATRA-ND induced ROS generation and prevented ATRA-ND induced cell growth arrest and apoptosis. In summary, incorporation of ATRA into ND enhanced the biological activity of this retinoid in cell culture models of MCL.
Asunto(s)
Antineoplásicos/farmacología , Linfoma de Células del Manto/patología , Receptores de Ácido Retinoico/efectos de los fármacos , Tretinoina/farmacología , Antineoplásicos/administración & dosificación , Apoptosis/efectos de los fármacos , Benzoatos/farmacología , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/biosíntesis , Proteínas de Ciclo Celular/efectos de los fármacos , Cromanos/farmacología , Sistemas de Liberación de Medicamentos , Evaluación Preclínica de Medicamentos , Factores de Intercambio de Guanina Nucleótido/biosíntesis , Factores de Intercambio de Guanina Nucleótido/efectos de los fármacos , Humanos , Linfoma de Células del Manto/metabolismo , Nanopartículas , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/efectos de los fármacos , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Receptores de Ácido Retinoico/antagonistas & inhibidores , Receptores de Ácido Retinoico/metabolismo , Receptores X Retinoide/efectos de los fármacos , Receptores X Retinoide/metabolismo , Transcripción Genética/efectos de los fármacos , Tretinoina/administración & dosificación , Células Tumorales CultivadasRESUMEN
PURPOSE: We investigated the cytotoxicity and mechanisms of cell death of the broad-spectrum histone deacetylase (HDAC) inhibitor PCI-24781, alone and combined with bortezomib in Hodgkin lymphoma and non-Hodgkin lymphoma cell lines and primary lymphoproliferative (CLL/SLL) cells. EXPERIMENTAL DESIGN: Apoptosis, mitochondrial membrane potential, cell cycle analysis, and reactive oxygen species (ROS) were measured by flow cytometry, whereas caspase activation was determined by Western blot. Nuclear factor kappaB (NF-kappaB)-related mRNAs were quantified by reverse transcription-PCR, NF-kappaB-related proteins by Western blotting, and NF-kappaB DNA-binding activity by electromobility shift assay. Finally, gene expression profiling was analyzed. RESULTS: PCI-24781 induced concentration-dependent apoptosis that was associated with prominent G(0)/G(1) arrest, decreased S-phase, increased p21 protein, and increased ROS in Hodgkin lymphoma and non-Hodgkin lymphoma cell lines. Dose-dependent apoptosis with PCI-24781 was also seen among primary CLL/SLL cells. PCI-24781-induced apoptosis was shown to be ROS- and caspase-dependent. Combined PCI-24781/bortezomib treatment resulted in strong synergistic apoptosis in all non-Hodgkin lymphoma lines (combination indices, 0.19-0.6) and was additive in Hodgkin lymphoma and primary CLL/SLL cells. Further, PCI-24781/bortezomib resulted in increased caspase cleavage, mitochondrial depolarization, and histone acetylation compared with either agent alone. Gene expression profiling showed that PCI-24781 alone significantly down-regulated several antioxidant genes, proteasome components, and NF-kappaB pathway genes, effects that were enhanced further with bortezomib. Reverse transcription-PCR confirmed down-regulation of NF-kappaB1 (p105), c-Myc, and IkappaB-kinase subunits, where NF-kappaB DNA binding activity was decreased. CONCLUSION: We show that PCI-24781 results in increased ROS and NF-kappaB inhibition, leading to caspase-dependent apoptosis. We also show that bortezomib is synergistic with PCI-24781. This combination or PCI-24781 alone has potential therapeutic value in lymphoma.
Asunto(s)
Apoptosis/efectos de los fármacos , Benzofuranos/farmacología , Ácidos Borónicos/farmacología , Ácidos Hidroxámicos/farmacología , FN-kappa B/metabolismo , Pirazinas/farmacología , Anciano , Antineoplásicos/farmacología , Apoptosis/genética , Western Blotting , Bortezomib , Caspasas/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Femenino , Citometría de Flujo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Inhibidores de Histona Desacetilasas , Humanos , Linfoma/genética , Linfoma/metabolismo , Linfoma/patología , Linfoma no Hodgkin/genética , Linfoma no Hodgkin/metabolismo , Linfoma no Hodgkin/patología , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Persona de Mediana Edad , Especies Reactivas de Oxígeno/metabolismo , Células Tumorales CultivadasRESUMEN
Sirtuins (SIRT) are nicotinamide adenine dinucleotide (NAD+) dependent deacetylases or ADP- ribosyl transferases (ARTs) that deacetylate lysine residues on various proteins regulating a variety of cellular and metabolic processes. These enzymes regulate metabolism, cell survival, differentiation and DNA repair. SIRT proteins play an important role in the survival and drug resistance of cancer cells. The purpose of the present study was to investigate the expression and role of SIRT in chronic lymphocytic leukemia (CLL). We analyzed the expression of SIRT1 and SIRT2 in CLL and normal B cells using the Oncomine database as well as by Western blotting of fresh CLL cells from patients and pro-lymphocytic leukemia (PLL) cell lines, JVM-3 and MEC-2. We showed that both primary CLL cells and JVM-3 and MEC-2 cell lines overexpress high levels of functional SIRT1 and SIRT2. SIRT inhibitors EX-527 and sirtinol impair cell growth, induce ROS production, loss of mitochondrial membrane potential and apoptosis in primary CLL cells and cell lines. Using shRNA knock down of SIRT1 and SIRT2 in JVM-3 and MEC-2 cell lines, we showed that expression of both proteins is crucial for the survival of these cells. Furthermore, studies in nutrient deprived conditions suggest a role of SIRT in metabolism in CLL. These results demonstrate that the inhibition of SIRT1 and SIRT2 activity may be a new therapeutic approach for CLL.
Asunto(s)
Leucemia Linfocítica Crónica de Células B/enzimología , Sirtuina 1/metabolismo , Sirtuina 2/metabolismo , Acetilación , Apoptosis/efectos de los fármacos , Carbazoles/farmacología , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen , Humanos , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Potencial de la Membrana Mitocondrial , NAD/metabolismo , ARN Interferente Pequeño/genética , Especies Reactivas de Oxígeno/metabolismo , Sirtuina 1/genética , Sirtuina 2/genética , Superóxidos/metabolismo , Tubulina (Proteína)/metabolismo , Regulación hacia ArribaRESUMEN
PURPOSE: Darinaparsin (Zio-101) is a novel organic arsenical compound with encouraging clinical activity in relapsed/refractory T-cell lymphoma (TCL) and Hodgkin lymphoma (HL); however, little is known about its mechanism of action. EXPERIMENTAL DESIGN: TCL cell lines (Jurkat, Hut78, and HH) and HL cell lines (L428, L540, and L1236) were examined for in vitro cell death by MTT assay and Annexin V-based flow cytometry. Jurkat and L540-derived xenografts in SCID mice were examined for in vivo tumor inhibition and survival. Biologic effects of darinaparsin on the MAPK pathway were investigated using pharmacologic inhibitors, RNAi and transient transfection for overexpression for SHP1 and MEK. RESULTS: Darinaparsin treatment resulted in time- and dose-dependent cytotoxicity and apoptosis in all TCL and HL cell lines. In addition, darinaparsin had more rapid, higher, and sustained intracellular arsenic levels compared with arsenic trioxide via mass spectrometry. In vivo experiments with Jurkat (TCL) and L540 (HL)-derived lymphoma xenografts showed significant inhibition of tumor growth and improved survival in darinaparsin-treated SCID mice. Biologically, darinaparsin caused phosphorylation of ERK (and relevant downstream substrates) primarily by decreasing the inhibitory SHP1 phosphatase and coimmunoprecipitation showed significant ERK/SHP1 interaction. Furthermore, ERK shRNA knockdown or constitutive overexpression of SHP1 resulted in increased apoptosis, whereas cotreatment with pharmacologic MEK inhibitors resulted in synergistic cell death. Conversely, SHP1 blockade (via pharmacologic inhibition or RNAi) and MEK constitutive activation decreased darinaparsin-related cell death. CONCLUSIONS: Altogether, these data show that darinaparsin is highly active in HL and TCL and its activity is dependent primarily on MAPK mechanisms.
Asunto(s)
Apoptosis/efectos de los fármacos , Arsenicales/farmacología , Glutatión/análogos & derivados , Enfermedad de Hodgkin/metabolismo , Linfoma de Células T/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Animales , Arsénico/metabolismo , Arsenicales/administración & dosificación , Ciclo Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Glutatión/administración & dosificación , Glutatión/farmacología , Enfermedad de Hodgkin/tratamiento farmacológico , Enfermedad de Hodgkin/mortalidad , Enfermedad de Hodgkin/patología , Humanos , Espacio Intracelular/metabolismo , Linfoma de Células T/tratamiento farmacológico , Linfoma de Células T/mortalidad , Linfoma de Células T/patología , Ratones , Transducción de Señal/efectos de los fármacos , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Hypoxia inducible factor (HIF) is important in cancer, as it regulates various oncogenic genes as well as genes involved in cell survival, proliferation, and migration. Elevated HIF-1 protein promotes a more aggressive tumor phenotype, and greater HIF-1 expression has been demonstrated to correlate with poorer prognosis, increased risk of metastasis and increased mortality. Recent reports suggest that HIF-1 activates autophagy, a lysosomal degradation pathway which may promote tumor cell survival. We show here that HIF-1α expression is constitutively active in multiple diffuse large B cell lymphoma (DLBCL) cell lines under normoxia and it is regulated by the PI3K/AKT pathway. PCI-24781, a pan histone deacetylase inhibitor (HDACI), enhanced accumulation of HIF-1α and induced autophagy initially, while extended incubation with the drug resulted in inhibition of HIF-1α. We tested the hypothesis that PCI-24781- induced autophagy is mediated by HIF-1α and that inhibition of HIF-1α in these cells results in attenuation of autophagy and decreased survival. We also provide evidence that autophagy serves as a survival pathway in DLBCL cells treated with PCI-24781 which suggests that the use of autophagy inhibitors such as chloroquine or 3-methyl adenine in combination with PCI-24781 may enhance apoptosis in lymphoma cells.
Asunto(s)
Inhibidores de Histona Desacetilasas/farmacología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Linfoma de Células B Grandes Difuso/patología , Proteínas Quinasas Activadas por AMP/metabolismo , Autofagia/efectos de los fármacos , Benzofuranos/farmacología , Supervivencia Celular/efectos de los fármacos , Sinergismo Farmacológico , Activación Enzimática/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ácidos Hidroxámicos/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacosAsunto(s)
Carcinoma Papilar/tratamiento farmacológico , Carcinoma de Células Renales/tratamiento farmacológico , Neoplasias Renales/tratamiento farmacológico , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Anciano , Ataxia Telangiectasia/tratamiento farmacológico , Humanos , MasculinoRESUMEN
PURPOSE: Andrographolide is a diterpenoid lactone isolated from Andrographis paniculata (King of Bitters), an herbal medicine used in Asia. It has been reported to have anti-inflammatory, antihypertensive, antiviral, and immune-stimulant properties. Furthermore, it has been shown to inhibit cancer cell proliferation and induce apoptosis in leukemia and solid tumor cell lines. EXPERIMENTAL DESIGN: We studied the Burkitt p53-mutated Ramos cell line, the mantle cell lymphoma (MCL) line Granta, the follicular lymphoma (FL) cell line HF-1, and the diffuse large B-cell lymphoma (DLBCL) cell line SUDHL4, as well as primary cells from patients with FL, DLBCL, and MCL. RESULTS: We found that andrographolide resulted in dose- and time-dependent cell death as measured by MTT. Andrographolide significantly increased reactive oxygen species (ROS) production in all cell lines. To determine mechanism of cell death, we measured apoptosis by Annexin V/propidium iodide in the presence and absence of the antioxidant N-acetyl-l-cysteine (NAC), the glutathione (GSH)-depleting agent buthionine sulfoxamine (BSO), or caspase inhibitors. We found that apoptosis was greatly enhanced by BSO, blocked by NAC, and accompanied by poly(ADP-ribose) polymerase cleavage and activation of caspase-3, caspase-8, and caspase-9. We measured BAX conformational change and mitochondrial membrane potential, and using mouse embryonic fibroblast (MEF) Bax/Bak double knockouts (MEF(Bax-/-/Bak-/-)), we found that apoptosis was mediated through mitochondrial pathways, but dependent on caspases in both cell lines and patient samples. CONCLUSIONS: Andrographolide caused ROS-dependent apoptosis in lymphoma cell lines and in primary tumor samples, which was enhanced by depletion of GSH and inhibited by NAC or the pan-caspase inhibitor Z-VAD-FMK. Further studies of diterpenoid lactones in lymphoma are warranted.
Asunto(s)
Andrographis/química , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Diterpenos/farmacología , Linfoma/tratamiento farmacológico , Linfoma/patología , Mitocondrias/efectos de los fármacos , Animales , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Diterpenos/química , Diterpenos/aislamiento & purificación , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Humanos , Linfoma/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factores de TiempoRESUMEN
The E2A gene encodes two E protein/class I basic helix-loop-helix transcription factors, E12 and E47, that are essential for B lymphopoiesis. In addition to the DNA-binding and protein dimerization domain, the E proteins share two highly conserved transcription activation domains. In this study, we show that both activation domains are required for optimal E2A-dependent transcription. Surprisingly, however, neither activation domain is required for E2A to rescue B lymphopoiesis from E2A(-/-) hemopoietic progenitors, although the N terminus of E2A, which harbors some transcription capacity, is required. Therefore, the E protein activation domains function redundantly in promoting B cell development. In contrast, the N-terminal activation domain, AD1, is required for a newly described ability of E2A to suppress macrophage development in vitro. Our findings demonstrate distinct functionalities for the E protein activation domains in B lymphocytes and macrophages.